sauerkraut and pickle fermentations. This organism initiates the desirable lactic acid fermentation in these products. L. mesenteroides produces carbon dioxide ...
CONTENTS
Chapter 1 Introduction
Page
No. 1.1)
SIGNIFICANCE OF THE STUDY
1
1.2)
RESEARCH OBJECTIVES
2
1.3)
RESEARCH HYPOTHESIS
2
1.4)
SCOPE AND LIMITATION OF THE STUDY
2
1.5)
EXPECTED RESULTS
3
Chapter 2 Literature Review 2.1) MICROBES AND FERMENTATION
4
2.1.1) Industrial microbiology
4
2.1.2) Fermentation Technology & Microbes
6
2.2) LACTIC ACID BACTERIA
13
2.2.1) Classification of Lactic acid bacteria
15
2.2.2) Biochemistry and genetics of lactic acid bacteria
18
2.2.3) Natural Habitat of Lactic acid bacteria
25
2.2.4) Lactic acid bacteria in food products
26
2.2.5) Lactic acid bacteria in agricultural products
31
2.2.6) Lactic acid bacteria as Probiotics
32
2.3) STREPTOCOCCUS THERMOPHILUS
42
2.3.1) Physiology of S.thermophilus
43
2.3.2) Characterization of S. thermophilus
45
2.3.3) Factors affecting the growth of lactic acid bacterial fermentations
57
2.4) Media formulations and nutrient requirement of S.thermophilus
59
2.5) Starter Culture and its role in Dairy Industry
61
2.5.1) Functions of Starter Culture
64
2.5.2) Types of Starter Culture
65
2.5.3) Fermented Milk Products
66
2.5.4) Bacteriophage
69
2.5.5) Fermented milk products in India
69
Chapter 3 Material & Methods 3.1) CHEMICAL AGENTS, NUTRIENTS AND CULTURES
78
3.2) SCREENING AND IDENTIFICATION
80
3.2.1) Sample Collection
80
3.2.2) Selective Enumeration and isolation methods
80
3.2.3) Morphological Characterization
81
3.2.4) Biochemical Characterization
82
3.2.5) Molecular Characterization
86
3.3) Media Formulations and effects of different sources of nutrition 3.3.1) Effect of carbon sources on mass production of S.thermophilus
88 88
3.3.2) Effect of nitrogen sources on mass production of S.thermophilus 89 3.4) EFFECT OF OTHER FACTORS AFFECTING THE GROWTH
89
3.4.1) Temperature Optimization
90
3.4.2) pH optimization
90
3.5) UP-SCALING PROCESS STANDARDIZATION
90
3.5.1) Inocolum Preparation
90
3.5.2) Process standardization for incubating conditions
91
3.5.3) Commercial viability of the standardized process
92
3.6) VIABILITY AND CFU STANDARDIZATION
92
3.7) DOWN-STREAM PROCESS STANDARDIZATION
93
3.7.1) Centrifugation and micro-filtration
93
3.7.2) Cryoprotectant preparation and standardization
94
3.7.3) Freezing temperature and incubation
95
3.7.4) Lyophilization standardization
95
3.8) APPLICATION TESTING OF ISOLATED STARTER CULTURE
96
3.8.1) Curd Preparation
96
3.8.2) Comparative testing
98
3.9) COMMERCIAL UP-SCALING TRIALS
99
3.10) PROTOCOL FOLLOWED AT TBI
100
Chapter 4 Results & Discussion 4.1) SCREENING AND ISOLATION OF LACTIC ACID BACTERIA
101
4.1.1) Isolation of Lactic acid bacteria
101
4.1.2) Selection of LAB for curd trials and lactic acid production
101
4.1.3) Morphological and Phenotypic characterization of Lactic acid bacteria
`
4.2) Biochemical Characterization of Isolates
105 113
Chapter 5 Molecular Characterization 5.1) Molecular Characterization
123
5.1) 16s rDNA sequencing
125
5.1.1) 16s rDNA sequencing results for ST-100
126
5.1.2) 16s rDNA sequencing results for ST-200
127
5.1.3) 16s rDNA sequencing results for ST-300
128
5.1.4) 16s rDNA sequencing results for ST-400
130
5.1.5) 16s rDNA sequencing results for ST-500
131
5.1.6) 16s rDNA sequencing results for ST-600
132
5.1.6) 16s rDNA sequencing results for ST-700
133
5.1.7) 16s rDNA sequencing results for ST-800
134
5.1.8) 16s rDNA sequencing results for LB-100
135
5.1.9) 16s rDNA sequencing results for LB-200
136
5.2) Whole Genome Sequencing of ST-500
140
Chapter 6 Media Standardization 6.1) Nutritional Requirements of Bacteria
147
6.2) Effect of different sources of nutrition on ST-500
148
6.2.1) Effect of Carbon Sources
148
6.2.2) Effect of Nitrogen Sources
152
6.2.3) Effect of Vegetable protein and Whey protein sources
155
6.2.4) Effect of Yeast Extract, Buffers and other salts used
157
6.3) Influence of factors on lactic acid bacteria fermentation
159
6.3.1) Effect of various temperatures on cell mass production 6.3.2) Effect of various initial pH on cell mass production
160 161
6.3.3) Effect of various agitation speeds on cell mass production
163
6.4) Media Costing
165
6.5) Batch Fermentation of ST-500 and process standardization
171
6.6) Down Stream processing of ST-500
181
6.6.1) Lyophilization process standardization
181
Chapter 7 Dairy Applications 7.0) Dairy Applications
189
7.1) Selected Parameters for application testing
190
7.1.1) Texture of Curd
190
7.1.2) Sensory Evaluation
191
7.1.3) Syneresis
191
7.1.4) Acidity
191
7.1.5) pH
191
7.1.6) Rheological
191
7.2) Application testing for cheese production from ST-500
197
7.3) Chemical & Physiological Parameters for Fermented Milk (set curd)
202
7.4) Testing with market Cultures
202
Chapter 8 Summary & Conclusion
205
References
208
Appendix
227
FIGURE CONTENT FIGURE NUMBER
LIST & TITLE OF FIGURES
1
Figure 2.1
Consensus tree, based on comparative sequence analysis of 16S rRNA, showing major phylogenetic groups of lactic acid bacteria
2
Figure 2.2
Characterization of Lactic acid bacteria
3
Figure 2.3
Homo-fermentative pathway illustrating the production of lactic acid from glucose
4
Figure 2.4
Hetero-fermentative pathway showing the production of lactic acid, carbon-dioxide and either ethanol or acetic acid
5
Figure 2.5
Phylogenetic tree and important commercial importance of Lactic acid bacteria
6
Figure 2.6
Silent features of lactic acid bacteria
7
Figure 2.7
Various applications of Lactic acid bacteria modulating human health
8
Figure 2.8
Electron microscopic image of S. thermophilus (Source: Durso and Hotkins 2003)
9
Figure 2.9
Phylogenetic tree of Lactobacillales based concatenated alignments of ribosomal proteins
10
Figure: 2.10
Flow diagram of the main steps involved in the production of Stirred yogurt
11
Figure: 2.11
Flow sheet for the preparation of acidophilus milk
12
Figure: 2.12
Flow sheet for the preparation of Curd
13
Figure: 3.1
14
Figure: 3.2
15
Figure: 4.1
16
Figure: 4.2
17
Figure: 4.3
Step elaboration of Inocolum preparation from 1st. 2nd and 3rd generation Chart for preparation of Indain traditional curd from starter culture Sample colonies of ST-500(A) and ST-600(B) growing on Streptococcus thermophilus isolation agar Sample colonies of LB-100(C) and LB-200(D) growing on MRS agar Microscopic view of ST-200 (A), ST-300(B), ST-400(C), ST-500(D), LB-100(E) and LB-200(F)
S.N.
on
18
Figure: 4.4
19
Figure: 4.5
20
Figure: 4.6
21
Figure: 5.1
22
Figure 5.2
23
Figure 5.3
24
Figure 5.4
25
Figure 5.5
26
27
Analysis of biochemical results of all selected 10 strains by Kit menthod Results of MR test with occurrence of a Ruby pink color in all tubes except BLANK which indicates that the organism produces a large amount of organic acid. Change in color indicates MR positive Results of VP test with no change in color in all tubes except BLANK which indicates that the organism is not capable of converting pyruvate into acetonin. No acetonin in broth indicates negative test. Digest results of the 16s rDNA genes of representative isolates along with the reference. Sample IDs of the different lanes are mentioned on the top of the lane along with the last lane of Ladder (L) Hind III digested lambda ladder. Results of phylogenetic analysis of ST-100, ST-200 and ST-300 in the form of Phylogenetic tree Results of phylogenetic analysis of ST-400, ST-500 and ST-700 in the form of Phylogenetic tree Results of phylogenetic analysis of LB-100 and LB-200 in the form of Phylogenetic tree Figure presents the library preparation report with peak indicating the ST-500 library optimal of sequencing
Figure: 5.6
Circular genomic map of ST-500 (Streptococcus thermophilus) in comparison with Streptococcus thermophilus MN-ZLW-002
Figure 6.1 (A)
Influence of carbon sources on lactic acid fermentation for mass cell production supplied with modified MRS broth and 1% of the selected carbon sources. All the carbon sources were tested indiviually with their 10gm/L content in media.
28
Figure: 6.1 (B)
29
Figure: 6.1 (C)
30
Figure: 6.1 (D)
Influence of carbon sources on lactic acid fermentation for mass cell production supplied with modified MRS broth and 2% of the selected carbon sources. All the carbon sources were tested indiviually with their 20gm/L content in media. Influence of carbon sources on lactic acid fermentation for mass cell production supplied with modified MRS broth and 3% of the selected carbon sources. All the carbon sources were tested indiviually with their 30gm/L content in media Influence of carbon sources on lactic acid fermentation for mass cell production supplied with modified MRS broth and 4% of the selected carbon sources. All the
31
32
33
34
35
36
37
38
Figure: 6.2 (A)
carbon sources were tested indiviually with their 40gm/L content in media Effect of nitrogen sources on lactic acid fermentation for mass cell production supplied with modified MRS broth and 1% of the selected nitrogen sources. All the nitrogen sources were tested indiviually with their 10gm/L content in media
Figure: 6.2 (B)
Effect of nitrogen sources on lactic acid fermentation for mass cell production of supplied with modified MRS broth and 2% of the selected nitrogen sources. All the nitrogen sources were tested indiviually with their 20gm/L content in media
Figure: 6.2 (C)
Effect of nitrogen sources on lactic acid fermentation for mass cell production supplied with modified MRS broth and 3% of the selected nitrogen sources. All the nitrogen sources were tested indiviually with their 30gm/L content in media
Figure: 6.2 (D)
Effect of nitrogen sources on lactic acid fermentation for mass cell production supplied with modified MRS broth and 4% of the selected nitrogen sources. All the nitrogen sources were tested indiviually with their 40gm/L content in media
Figure: 6.3 (A)
Effect of vegetable protein and milk protein sources on lactic acid fermentation for mass cell production supplied with modified MRS broth and 2% of the selected sources. All the sources were tested indiviually with their 20gm/L content in media
Figure: 6.3 (B)
Effect of vegetable protein and milk protein sources on lactic acid fermentation for mass cell production supplied with modified MRS broth and 4% of the selected sources. All the sources were tested indiviually with their 40gm/L content in media
Figure: 6.4
Influence of sodium, potassium, ammonium and magnesium salts on lactic acid fermentation for mass cell production supplied with modified MRS broth and were tested indiviually with their mentioned content in media.
Figure: 6.5
Influence of yeast extract (amino acid source) on lactic acid fermentation for mass cell production supplied with modified MRS broth and were tested indiviually with 1gm/L to 5gm/L content in media.
The influence of temperature on cell mass yield on self designed Media 5 by Streptococcus thermophilus NCIM 5539 The influence of initial set point of pH on cell mass yield on self designed Media 5 by Streptococcus thermophilus NCIM 5539 Reducing sugar concentrations (Sucrose) during batch fermentation of Streptococcus thermophilus NCIM 5539 Effect of agitation speed on the cell growth, reducing sugar concentration and mass production by Streptococcus thermophilus NCIM 5539: 100 rpm (A), 200 rpm (B), and 300 rpm (C). Price & Fresh cell mass weight gm/L comparison of media from different vendors Price & Fresh cell mass weight gm/L comparison of media M17, MRS and Media 5 from same vendor
39
Figure: 6.6
40
Figure: 6.7
41
Figure: 6.8
42
Figure: 6.9
43
Figure 6.10
44
Figure 6.11
45
Figure: 6.12
Prepared media poured in 2L Fermenter
46
Figure: 6.13
Media after autoclaving for 15 minutes
47
Figure: 6.14
Assembling of fermenter for inoculation
48
Figure: 6.15
Completely assembled fermenter ready for inoculation
49
Figure: 6.16
Turbidity observed after 16 hours of incubation
50
Figure: 6.17
Batch closed after 24 hours of incubation
51
Figure: 6.18
Collection of turbid broth in sterile flasks for further processing
52
Figure: 6.19
Collected material with cell mass for further processing
53
Figure: 6.20
54
Figure: 6.21
55
Figure: 6.22
56
Figure: 6.23
History plot of the 5 L batch of Streptococcus thermophilus showing the change in pH and consumption of acid and base for maintaining the set pH balance. Fermentation profile of ST-500 recorded on the basis of OD at 660nm Batch fermentation profile of ST-500 in 5L fermenter showing cell mass concentration, lactic acid percentage and reducing sugar concentration Media preparation and mixing in sterile buckets [Fig. 6.23 (A, B)], Media addition in 300 L fermenter (Fig. 6.23 (C), Seed inoculation in fermenter with heat sterile conditions (Fig. 6.23 (C), Adjustment of standardized parameters through manual mode of operation (Fig. 6.23 (D).
57
Figure: 6.24
58
Figure: 6.25
59
Figure: 6.26
60
Figure: 6.27
Technology Based incubator facility at Delhi University. (A) Visible turbidity, (B) Peristatic pumps and control panel showing the flow of base to maintain pH, (C, D) Overall view of the facility with 50 L fermenter on extreme left, 200 L in middle and 300 L fermenter on the right Fermentation profile of ST-500 recorded on 300L fermenter on the basis of OD at 660nm Effect of different lyoprotectants on the viability after lyophilization. If not other indicated 10% (w/w) solution were used. The error bars show the standard deviation Front view of BioSata A plus lyophilizer, (B, C, D) Lyophilization of cell mass mixed with lyophilization media in plates and vials at -47° C and vacuum pressure of 0.04mbar, (E) Final lyophilized product (starter culture) after complete drying
61
(A) presents the commercial model lyophilizer for large scale lyophilization. (B, C) presents the lyophilized culture in trays after complete lyophilization cycle of 16 hours
62
Figure: 6.29
Effect of different Lyophilization media on the viability after lyophilization. The error bars show the standard deviation
Figure: 7.1
A, B, C presenting the texture, cut ability, thickness, water binding and robustness of the curd prepared from ST-500 starter culture
64
Figure: 7.2
A and C presenting the texture, cut ability, thickness, water binding and robustness of the flavored yogurt prepared by mixture of ST-500 and LB-200. Images 7.2 B and D presents stirred yogurt with different flavors.
65
Figure 7.3
Graphical presentation of pH drop rate after inoculation of ST-500 with different fruit and sugar percentages on incubation at 42°C
66
Figure: 7.4
Application testing results of ST-500 (Streptococcus thermophilus NCIM 5539 for cheese preparation after 30 days of aging and processing incubation period
67
Figure 7.5
Graphical presentation of pH drop rate after inoculation of ST-500 with different percentage of milk fat on incubation at 42°
Figure: 6.28
63
TABLE CONTENT S.N.
TABLE NUMBER
1
Table 2.1
2
Table 2.2
3
Table 2.3
4
Table 2.4
5
Table 2.5
6
Table 2.6
7
Table 2.7
8 9
Table 2.8 Table 2.9
10
Table 2.10
11
Table 2.11
12
Table 2.12
13 14 15
Table 2.13 Table 3.1 Table 3.2
16
Table 4.1
17
Table 4.2
18
Table 4.3
19
Table 4.4
20
Table 4.5
21
Table 4.6
22
Table 5.1
23
Table 5.2
LIST & TITLE OF TABLES Examples of industrial fermentation products and their producer microorganisms Examples of microorganisms classified as GRAS (Generally regarded as safe) Examples of aseptic and non-aseptic fermentations Differentiating Lactic acid bacteria and a comparison with current taxonomic classification Some of the major fermented food products along with the microbe involved around the world Differences between Probiotics, Prebiotics and Synbiotics Health benefits along with the proposed mechanism of action of lactic acid bacteria Classification of cocci lactic acid bacteria Characterizing features of S. thermophilus Genomic details of Streptococcus thermophilus strains with whole genome sequencing available data till Feb 2014 Taxonomy of dairy Starter Cultures with old and new names and their products Major starter culture producer companies all over the world Characteristics of sweet and sour Dahi pH indicators for carbohydrate fermentation media Details of the analytical tools and reference genome used Percentage lactic acid yield and pH drop results of the 128 isolates after first selective enumeration. Phenotypic characterization, colony observation and gas production results of the selected 32 isolates. Results of complete morphological and phenotypical analysis of 10 selected strains Fermentation results of selected isolates from different sugars Results of enzyme assays conducted on selected isolates Biochemical characterization results on the basis of different biochemical test analysis Quality Control reports of all of sample isolates. DNA Concentration and purity of samples estimated using nanodrop spectrophotometer 16s rDNA sequencing results of selected 10 isolates with best hit similarities at data in NCBI
24
Table 5.3
25
Table 5.4
26
Table 5.5
27
Table 5.6
28
Table 6.1
29
Table 6.2
30
Table 6.3
31
Table 6.4
32
Table 6.5
33
Table 6.6
34
Table 6.7
35
Table 6.8
36
Table 7.1
Qubit values of prepared library Summary of the platforms and alignments of WGS of ST-500 Details of alignments and Raw data sequences obtained from WGS Alignment statics of ST-500 in comparison with S.thermophilus MN-ZLW-002 and S. thermophilus MTCC_5461 Results of different media compositions concluded after individual studies of media ingredients for mass cell production of Streptococcus thermophilus NCIM 5539 Media per litre cost comparison between MRS and M17 on individual ingredient basis from Himedia, India catalogue. Costing of the designed media on the basis of ingredient prices available with Himedia Costing of the designed media on the basis of commercial prices provided by Himedia Comparison of Batch results with complete up-stream analysis of ST-500 Cell growth rate with respect to incubation hours Combinations of protectants along with the ratio of addition Viability loss during up-downstream bio-processing Results of application testing done on the basis of certain parameters
37
Table 7.2
Selected Parameters and standards for comparative analysis
38
Table 7.3
Results of comparative testing done on selected parameters
List of abbreviations
AAD
:
Antibiotic associated diarrhea
ASA
:
Associated chronic gastritis
ATCC
:
American Type Culture Collection
BIS
:
Bureau of Indian Standards
BLAST
:
Basic local alignment search tool
bp
:
Base pair
CFU
:
Colony Forming Unit
DGGE
:
Denaturing gradient gel electrophoresis
DNA
:
Deoxyribonucleic acid
EDTA
:
Ethylenediaminetetraacetic acid
EMP
:
Embden–Meyerhof–Parnas pathway
EPS
:
Extracellular polysaccharides
g GC
: :
Gram
Guanine Cytosine
GRAS
:
Generally recognized as safe
h
:
hours
H2SO4
:
Sulphuric acid
HCl
:
Hydrochloric acid
HGT
:
Horizontal gene transfer
IBD
:
Inflammatory bowel disease
IL
:
Interleukin
K2HPO4
:
dipotassium hydrogen phosphate
Kg
:
Kilogram
KH2PO4
:
monopotassium phosphate
L
:
Liter
LAB
:
Lactic acid bacteria
LDH
:
Lactate dehydrogenases
LPS
:
Lipopolysaccharides
mbar
:
milli bar
mL
:
Milliliter
MRS
:
De Man, Rogosa and Sharpe
MR-VP
:
Methyl red and Voges-Proskauer
MTCC
:
Microbial type culture collection
N
:
Normality (Normal)
NaCl
:
Sodium Chloride
NaOH
:
Sodium Hydroxide
NCBI
:
National center for Biotechnology information
NCIM
:
National Collection of Industrial Microorganisms
NSLAB
:
Non-starter lactic acid bacteria
ºC
:
Degree Centigrade
OD
:
Optical Density
PCR
:
Polymerase chain reaction
PFGE
:
Pulse-field gel electrophoresis
psi
:
Pound per square inch
PTS
:
Phosphotransferase
PYR
:
Pyrrolidonyl Arylamidase
RFLP
:
Restriction fragment length polymorphism
RNA
:
Ribonucleic acid
Rpm
:
Revolution per minute
SMP
:
Skim Milk powder
SNF
:
Solid not Fat
SWP
:
Sweet whey powder
TGGE
:
Temperature gradient gel electrophoresis
TNF
:
Tumor necrosis factor
WGS
:
Whole Genome sequencing
WPC
:
Whey protein concentrate
Chapter 1 Introduction 1.1
SIGNIFICANCE OF THE STUDY
Milk and milk products provide wealth of nutrition benefits with their healthy contents along with the micro-flora these products carry. The major group present in milk and dairy products with tremendous potential and most desirable in domestic and commercial food fermentation is lactic acid bacteria (LAB) group. Some bacteria in this group are described as psychrophilic, which means that they grow best at cold temperatures, while others are severely retarded by being in the refrigerator and grow rapidly only at warmer temperatures. Fermentation was invented long before microorganisms were discovered, and therefore the process seemed mysterious. The need for an inocolum was understood and usually satisfied by keeping a sample from the previous production. But with advancement in science and technology over years, the capability of microorganisms to perform fermentation leading to various useful end products is well documented now. LAB are widely used in the production of fermented food, and they constitute the majority of the volume and the value of the commercial starter cultures. The demand of a cost economic starter culture is also swelling with modernization in fermented food industries. It is; therefore, very attractive to develop a low cost single strain starter culture for Indian food fermentation industries for manufacturing of Indian traditional curd, as in India, food industries are still using imported starter cultures due to the unavailability of any domestic starter culture producer.
1.2
RESEARCH OBJECTIVES This research work focuses on isolation of Streptococcus thermophilus and
development of a complete up-stream down-stream fermentation process for the isolated strain including a cost efficient media development with complete process optimization. This involves bacterial enumeration, morphological, biochemical and molecular
characterization of the isolated strain followed by standardization of the lyophilization process and finally making sure the commercial viability of the end product. The objectives are: •
To isolate and identify potential Streptococcus thermophilus strain capable of fast acid production.
•
Complete morphological and biochemical analysis of the isolated strain.
•
To study the molecular behavior of the strain including 16s rDNA sequencing followed by whole genomic sequencing on Ion torrent platform.
•
To optimize fermentation conditions best suitable for high yield basis cell mass production.
•
Low cost high yield media designing and development for economic production.
•
Commercial trails of the isolated strain on large scale fermenters (200-300L).
•
Standardization of the commercial viability of the strain by comparing with the existing imported cultures available in the market
1.3
RESEARCH HYPOTHESIS Isolated lactic acid bacteria could produce lactic acid as end product leading to
production of mild flavored Indian traditional curd and capable of multiplying fast on low cost medium with high yield. The behavior of the strain could be analyzed on complete characterization and positive approach will lead this type of study to the development of an economical starter culture with commercial viability.
1.4
SCOPE AND LIMITATION OF THE STUDY Present work involves investigation about production of starter culture by
isolating a potential strain of lactic acid bacteria with special interest on Streptococcus thermophilus for production of Indian traditional curd. The ultimate objective is to develop a cost efficient starter culture especially for Indian market by economically standardizing the required media along with process development. Designing of media includes changes in the nutritional requirements of the isolated strain by using inexpensive sources of carbon and nitrogen to obtain optimum yield of viable cell mass.
In addition, fermentations were carried out from laboratory scale fermenters to pilot scale commercial bioreactor facilities. Only a few strains (18-20) of Streptococcus thermophilus are openly available with their complete genomic study by sequencing the whole genome with NCBI (National Center for Biotechnology Information). The isolated strain in current study has been used as starter but its whole genome sequence has also been studied on Ion torrent platform. Commercial trails of the product for its use as starter culture were also conducted with several big dairies in the country to check the performance of strain against the cultures already available in the market.
1.5
EXPECTED RESULTS •
Low cost high yield viable cell mass from the fermentation process under the optimum conditions.
•
Standardized nutritional and lyophilization parameters with minimum cell loss during agitation and freezing shocks.
•
Successful continuous recovery of cell mass from both laboratory scale and commercial scale pilot plant bioreactors.
•
A final lyophilized product with all potentials of a good starter culture, to resist competition with the available imported cultures in the market.
Chapter 2 Literature Review 2.1
MICROBES AND FERMENTATION The microorganisms are the most successful group of all living species occupying
each habitat in water, soil, plants and animals including humans with enormous success. This leads to a fundamental impact on all research areas in modern biology and medicine. Microorganisms are capable of growing on a wide range of substrates and can produce a remarkable spectrum of products.
The advent of in vitro genetic manipulation has
extended the range of products that may be produced by microorganisms and has provided new methods for increasing the yields of existing ones. The commercial exploitation of the biochemical diversity of microorganisms has resulted in the development of the fermentation industry and the techniques of genetic manipulation have given this well-established industry the opportunity to develop new processes and to improve existing ones. The term fermentation is derived from the Latin verb fervere, to boil, which describes the appearance of the action of yeast on extracts of fruit or malted grain during the production of alcoholic beverages. However, fermentation is interpreted differently by microbiologists and bio-chemists. To a microbiologist the word means any process for the production of a product by the mass culture of microorganisms. To a biochemist, however, the word means an energy-generating process in which organic compounds act as both electron donors and acceptors, that is, an anaerobic process where energy is produced without the participation of oxygen or other inorganic electron acceptors. 2.1.1
Industrial microbiology Surprisingly enough an estimated 99% of all living microorganisms have not even
been discovered although (or maybe because) they colonies any habitat with great success. From an evolutionary point of view the microbes show a large degree of biodiversity,
commonly being unified
by the feature of their small
size.
Biotechnologically designed and employed microorganisms significantly increase the importance of microbes for applications in food industry, chemistry and pharmacy. Industrial microbiology is primarily associated with the commercial exploitation of microorganisms, and involves processes and products that are of major economic, environmental and social importance throughout the world. There are two key aspects of industrial microbiology, the first relating to production of valuable microbial products via fermentation processes. These include traditional fermented foods and beverages, such as bread, beer, cheese and wine, which have been produced for thousands of years. In addition, over the last hundred years or so, microorganisms have been further employed in the production of numerous chemical feed stocks, energy sources, enzymes, food ingredients and pharmaceuticals. The second aspect is the role of microorganisms in providing services, particularly for waste treatment and pollution control, which utilizes their abilities to degrade virtually all natural and man-made products. However, such activities must be controlled while these materials are in use, otherwise consequent biodeterioration leads to major economic losses. Over the last twenty years, many traditional and established industrial fermentation processes have been advanced through the contribution of genetic engineering (in vitro recombinant DNA technology) which has also facilitated the development of many novel processes and products. It not only accelerates strain development, leading to improvement in the production of microorganisms, but can aid in downstream processing and other elements of the process. It involved the manipulation of bacteria, cloning in yeasts, filamentous fungi, and plant and animal cells. Microbial biosurfactants with high ability to reduce surface and interfacial surface tension and conferring important properties such as emulsification, detergency, solubilization, lubrication and phase dispersion have a wide range of potential applications in many industries. Significant interest in these compounds has been demonstrated by environment, bioremediation, oil, petroleum, food, beverage, cosmetic and pharmaceutical industries with their low toxicity, biodegradability and sustainable production technologies. Despite having significant potentials associated with emulsion formation, stabilization, antiadhesive and antimicrobial activities, significantly less output and applications have been reported in food industry (Campos et al., 2013). Enzymes are considered as a potential biocatalyst for a large number of reactions. Particularly, the microbial enzymes have widespread uses in industries and medicine. In
addition, the microorganisms represent an alternative source of enzymes because they can be cultured in large quantities in a short time by fermentation and owing to their biochemical diversity and susceptibility to gene manipulation. Industries are looking for new microbial strains in order to produce different enzymes to fulfill the current enzyme requirements (Anbu et al., 2013). 2.1.1.1 Industrial Microbes The development of industrial microbial processes is gaining unprecedented momentum. Increased concern for environmental issues and the prospect of declining petroleum resources has shifted the industrial focus increasingly to microorganisms as biocatalysts. At the same time systems biology and synthetic biology supply industry and academia with new tools to design optimal microbial cell factories (Sauer and Mattanovich, 2012). Microorganisms are used extensively to provide a vast range of products and services (Table 2.1). They have proved to be particularly useful because of the ease of their mass cultivation, speed of growth, use of cheap substrates (which in many cases are wastes) and the diversity of potential products. Their ability to readily undergo genetic manipulation has also opened up almost limitless further possibilities for new products and services from the fermentation industries. Most of these microorganisms, irrespective of their origins, were subsequently modified by conventional strain improvement strategies, using mutagenesis or breeding programmes, to improve their properties for industrial use. Several processes developed in the last 20 years have involved recombinant microorganisms and genetic engineering technology to improve established industrial strains. In most cases, regulatory considerations are of major importance when choosing microorganisms for industrial use. Fermentation industries often prefer to use established GRAS (generally regarded as safe) microorganisms (Table 2.2), particularly for the manufacture of food products and ingredients.
Table: 2.1 Examples of industrial fermentation products and their producer microorganisms
Products
Bacteria
Yeast and Filamentous fungi
Traditional Products Bread, beer, wine and spirits Cheeses, other dairy products Ripening of blue and Camembert-type cheeses Fermented meats and vegetables, Mushrooms, Soy sauce Sufu (soya bean curd) Vinegar Agricultural Products Gibberellins, Fungicides Insecticides, Silage
Amino acids l-Glutamine, l-Lysine l-Tryptophan Enzymes Carbohydrates a-amylase, b-amylase amyloglucosidase, glucose isomerase, invertase, lactase (b-galactosidase) Cellulases Lipases, Pectinases
Lactic acid bacteria
Mainly Saccharomyces cerevisiae
Mostly lactic acid bacteria
Agaricus bisporus, Lentinula edodes, Aspergillus oryzae, Zygosaccharomyces rouxii
Acetobacter species
Mucor sp.
Bacillus thuringiensis, Lactic acid bacteria
Fusarium moniliforme, Coniothyrium minitans
Corynebacterium glutamicum, Brevibacterium lactofermentum, Klebsiella aerogenes
Bacillus subtilis Streptomyces olivaceus
Fuels and chemical feedstock Acetone, Butanol, Ethanol Glycerol, Methane
Clostridium species Clostridium acetobutylicum Zymomonas mobilis Methanogenic archaeans
Nucleotides
Bacillus subtilis,
Aspergillus niger Kluyveromyces species Kluyveromyces lactis Trichoderma viride Candida cylindraceae Aspergillus wentii
Saccharomyces cerevisiae Zygosaccharomyces rouxii
5¢-Inosine monophosphate 5¢-Guanosine monophosphate Organic acids Acetic Citric
Brevibacterium ammoniagenes
Acetobacter xylinum
Aspergillus niger Yarrowia lipolytica
Fumaric, Gluconic , Itaconic Kojic , Lactic
Acetobacter suboxydans,
Rhizopus species,
Lactobacillus delbrueckii
Aspergillus itaconicus, Aspergillus flavus Claviceps purpurea, Claviceps fusiformis, Claviceps paspali
Pharmaceuticals and related compounds Alkaloids Ergotamine, ergometrine, d-lysergic acid Antibiotics Aminoglycosides streptomycin b-Lactams penicillins, cephalosporins clavulanic acid Lantibiotics Nisin, Macrolides Erythromycin, Peptides bacitracin gramicidin Tetracyclines, chlortetracycline
Streptomyces griseus, Streptomyces clavuligerus
Penicillium chrysogenum Acremonium chrysogenum
Lactococcus lactis Saccharapolyopora erythraea Bacillus licheniformis Bacillus brevis Streptomyces aureofasciens Recombinant Saccharomyces cerevisiae
Hormones Human growth hormone Insulin
Recombinant Escherichia coli
Vitamins B12 (cyanocobalamin) b-Carotene (provitamin A) Ascorbic acid (vitamin C) Riboflavin Recombinant
Pseudomonas denitrificans Acetobacter suboxydans Bacillus subtilis
Blakeslea trispora Ashbya gossypii
Polymers Alginates Cellulose
Azotobacter vinelandii
Aureobasidium pullulans
Dextran, Gellan Polyhydroxybutyrate Pullulan Scleroglucan Xanthan
Acetobacter xylinum Leuconostoc mesenteroides Sphingomonas paucimobilis Ralstonia eutropha
Sclerotium rolfsii Xanthomonas campestris
Single cell protein
Methylococcus capsulatus Methylophilus methylotrophus
Immunosuppressants Cyclosporin Interferon Steroids
Recombinant Escherichia coli Arthrobacter species Rhizopus species
Candida utilis Fusarium venenatum Kluyveromyces marxianus Paecilomyces variotii Saccharomyces cerevisiae Trichoderma polysporum Recombinant Saccharomyces cerevisiae
Table: 2.2 Examples of microorganisms classified as GRAS (Generally regarded as safe)
BACTERIA
Streptococcus thermophilus Bacillus subtilis Lactobacillus bulgaricus Lactococcus lactis Leuconostoc oenos
YEASTS
Candida utilis Kluyveromyces marxianus Kluyveromyces lactis Saccharomyces cerevisiae
FILAMENTOUS FUNGI
Aspergillus niger Aspergillus oryzae Mucor javanicus (Mucor circinelloides f. circinelloides) Penicillium roqueforti
2.1.2
Fermentation Technology and Microbes Industrial microbiology came into existence, primarily, based on a naturally
occurring microbiological process called fermentation. Fermentation is a metabolic process that converts sugar to acids, gases and/or alcohol. Commercially, fermentation is the process of controlling bacteria, yeast, and moulds to modify food, producing a desired product. Microbiologists use the term fermentation in two different contexts. First, in metabolism, fermentation refers to energy-generating processes where organic compounds act as both electron donor and acceptor. Second, in the context of industrial microbiology, the term also refers to the growth of large quantities of cells under aerobic or anaerobic conditions, within a vessel referred to as a fermenter or bioreactor. Apart from their use for cell cultivation, and for live vegetative cell and spore biotransformations, similar vessels are used in processes involving cell-free and immobilized enzyme transformations.
Lactic acid bacteria are the most important bacteria in desirable food fermentations, being responsible for the fermentation of sour dough bread, sorghum beer, all fermented milks, and most "pickled" (fermented) vegetables. L. acidophilus, L. bulgaricus, L. plantarum, L. pentoaceticus, L. brevis and S. thermophilus are examples of lactic acidproducing bacteria involved in food fermentations. Some of the species are homofermentative, because they produce lactic acid only, while others are heterofermentative and produce lactic acid plus other volatile compounds and small amounts of alcohol. Leuconostoc mesenteroides is a bacterium associated with the sauerkraut and pickle fermentations. This organism initiates the desirable lactic acid fermentation in these products. L. mesenteroides produces carbon dioxide and acids which rapidly lower the pH and inhibits the development of undesirable microorganisms. The carbon dioxide produced replaces the oxygen, making the environment anaerobic and suitable for the growth of subsequent species of lactobacillus. Removal of oxygen also helps to preserve the colour of vegetables and stabilises any ascorbic acid that is present. Organisms from the Gram-positive Propionibacteriaceae family are responsible for the flavor and texture of some fermented foods, especially Swiss cheese, where they are responsible for the formation of 'eyes' or holes in the cheese. These bacteria break
down lactic acid into acetic, propionic acids and carbon dioxide. Several other bacteria, for instance Leuconostoc citrovorum, Streptococcus lactis and Brevibacterium species are important in the fermentation of dairy products (Table 2.3). Microorganisms vary in their optimal pH requirements for growth. Most bacteria favour conditions with a near neutral pH. The varying pH requirements of different groups of microorganisms are beneficial for the fermented foods where successions of microorganisms take over from each other as the pH of the environment changes. Certain bacteria are acid tolerant and will survive at reduced pH levels. Notable acid-tolerant bacteria include the Lactobacillus and Streptococcus species playing a key role in the fermentation of dairy and vegetable products. Most lactic acid bacteria work best at temperatures of 18 to 22 ºC and tolerate high salt concentrations. The salt tolerance gives them an advantage over other less tolerant species and allows the lactic acid fermenters to begin metabolism, which produces acid that further inhibits the growth of non-desirable organisms. In general, bacteria require a fairly high water activity (0.9 or higher) to survive. There are a few species which can tolerate water activities lower than this, but usually the yeasts and fungi will predominate on foods with a lower water activity. Nearly all food fermentations are the result of more than one microorganism, either working together or in a sequence. Bacteria from different species and the various microorganisms – yeast and moulds - all have their own preferences for growing conditions, which are set within narrow limits. There are very few pure culture fermentations. An organism that initiates fermentation will grow there until its byproducts inhibit further growth and activity. During this initial growth period, other organisms develop which are ready to take over when the conditions become intolerable for the former ones. In general, growth will be initiated by bacteria, followed by yeasts and then moulds. The advantages of the use of starter cultures against spontaneous fermentation are well known and widely spread especially for dairy and meat products, but are not often used in the vegetable fermentations. The spontaneous fermentation of sauerkraut can result in the formation of biogenic amines. The utilization of starter culture enables producers to make food products with a standard quality in a shorter time. Selection of
starter culture, however, should not be only done considering the lactic acid production of the strains but also their activity for biogenic amine synthesis. Although numerous studies have been carried out on cabbage, olive and pickle fermentation, little is known on the lactic fermentation of other vegetables. Usually, lactofermented vegetables are pasteurized and there is no information on the behavior of lactobacilli during storage of unpasteurized fermented vegetables. With fermentation of beetroot by appropriately selected lactobacilli a juice could be produced which combines benefits of betalains and lactobacilli (Halász et al., 1999).
Non-starter lactic acid bacteria (NSLAB) can be commonly isolated from Cheddar cheese. The majority of NSLAB are Lactobacillus sp., although Pediococcus and Leuconostoc sp. also can be present (Peterson and Marshall 1990). According to their proteolytic activity lactobacilli can contribute to the development of desirable flavours in cheese but can also cause the accumulation of bitter peptides that result in off-flavours. In addition NSLAB can cause defects such as gas formation and calcium lactate crystallization on the surface of the cheese, which forms a white haze. These crystals are the end result of lactate racemization by some NSLAB that convert L(+) -lactic acid to the less soluble D(-) isomer. The source of NSLAB contamination is believed to be originated from post-pasteurization, usually through contact with equipment surfaces or from the air.
Table: 2.3 Examples of aseptic and non-aseptic fermentations ASEPTIC
NON ASEPTIC
AEROBIC
ANAEROBIC
AEROBIC
ANAEROBIC
Animal and plant cell cultures, Alkaloids, Amino acids, Most antibiotics, Most biomass (SCP) production, Most enzymes, Most organic acids rDNA, proteins, Steroid biotransformations Some toxins Most vaccines Most vitamins Xanthan gum.
Acetone
Acetification of
Alcoholic
ethanol in vinegar
beverages; beer,
production,
wine etc, Primary
Ripening of some
dairy fermentations,
cheeses, Mushroom
Silage production,
production, Aerobic
Anaerobic waste-
waste-water
water treatment
2.2
Butanol Ethanol Glycerol Lactic acid Some toxins
treatment
LACTIC ACID BACTERIA (LAB) LAB are heterogeneous group of bacteria contributing to various sectors of food,
beverage, health tonics and pharmaceutical industries worldwide. All the member of the group shares a common property which is their gram-positive appearance and can be differentiated on their physiology and the mode of metabolic pathway they choose; either homo-fermentative or hetero-fermentative. This group on fermentation produces various anti-microbial substances that promote their activity of health modulation and many organic compounds producing flavors and aromas in the fermented food products. Lactic acid bacteria (LAB) are regarded as a major group of probiotics (Sharma et al., 2012; Schrezenmeir and de Vrese 2001). These lactic acid bacteria are industrially important organisms recognised for their fermentative ability as their health and nutritional benefits. They are comprised of an ecologically diverse group of microorganisms united by formation of lactic acid as the primary metabolite of sugar
metabolism (Carr et al. 2002). These bacteria are basically Gram-positive non-spore forming cocci, cocci-bacilli or rods, non-rispiring, catalase-negative bacteria that are devoid of cytochromes and are of non-aerobic habit but are aero-tolerant, fastidious, acid tolerant and strictly fermentative; lactic acid is the major end-product of sugar fermentation. They are chemo-organotrophic and grow in complex media and generally are non pathogenic to man and animals. LAB consist of several genera, which include Carnobacterium, Enterococcus, Lactobacillus, Lactococcus, Lactosphaera, Leuconostoc, Melissococcus, Oenococcus, Pediococcus, Streptococcus, Tetragenococcus, Vagococcus and Weissella (Ercolini et al. 2001; Holzapfel et al. 2001). Based on similarities in physiology, metabolism and nutritional needs, these genera are grouped together. A primary similarity is that all members produce lactic acid as a major or virtually sole end product of the fermentation of sugars. LAB were first isolated from milk (Carr et al. 2002) and have since been found in such foods and fermented products as meat, milk products, vegetables, beverages and bakery products (Liu 2003; O’Sullivan et al. 2002). These bacteria occur naturally in fermented food and have been detected in soil, water, manure and sewage (Holzapfel et al. 2001). LAB exist in human (Martin et al. 2003; Schrezenmeir and de Vrese 2001) and in animal. However, some lactic acid bacteria are part of the oral flora which can cause dental caries (Sbordone and Bortolaia 2003). LAB can work as spoilage organisms in foods such as meat, fish and beverages (Liu 2003). Several lactobacilli, lactococci and bifidobacteria are held to be health-benefiting bacteria (Rolfe 2000; Tuohy et al. 2003), but little is known about the probiotic mechanisms of gut microbiota (Gibson and Fuller 2000). LAB constitute an integral part of the healthy gastrointestinal (GI) microecology and are involved in the host metabolism and Streptococcus thermophilus, inhibit food spoilage and pathogenic bacteria and preserve the nutritive qualities of raw food material for an extended shelf life (O’Sullivan et al. 2002; Heller 2001). The taxonomy of LAB based on comperative 16S ribosomal RNA (rRNA) sequencing analysis has revealed that some taxa generated on the basis on phenotypic features do not correspond with the phylogenetic relations. Molecular techniques, especially polymerase chain reaction (PCR) based methods, such as rep-PCR fingerprinting and restriction fragment length polymorphism (RFLP) as well as pulse-field gel electrophoresis (PFGE), are regarded
important for specific characterization and detection of LAB strains (Gevers et al., 2001; Holzapfel et al., 2001). Recently, culture-independent approaches have been applied for the detection of intestinal microbiota (Zoetendal et al., 2002). Denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) analysis of faecal 16S rDNA gene and its rRNA amplicons have shown to be powerful approaches in determining and monitoring the bacterial community. 2.2.1
Classification of Lactic acid bacteria The term Lactic Acid Bacteria (LAB) was gradually accepted in the beginning of
the 20th century (Carol et. al., 2010). In earlier times some other terms were used for lactic acid bacteria like milk sourcing and lactic acid producing which created confusion. All the confusion came to an end in 1919 when Orla-Jensen proposed a monograph about lactic acid bacteria having great impact over systematic lactic acid bacteria (Axelsson, 1989). Classification of LAB genera was based on morphology, mode of glucose fermentation, growth at certain temperatures, and range of sugar utilization and configuration of the lactic acid produced, ability to grow at high salt concentrations, and acid or alkaline tolerance. Even though the taxonomy has been revised since then, characters used by Orla-Jensen are still very important in current classification of LAB. Lactic acid bacteria constitute a group of bacteria that have morphological, metabolic and physiological similarities with relatively closely related phylogeny. For some of the newly described genera (Pilar et. al., 2008), additional characteristics such as fatty acid composition and motility are used in classification. The measurements of true phylogenetic relationship with rRNA sequencing have aided the classification of lactic acid bacteria and clarified the phylogeny of the group (Figure 2.1). Most genera in the group form phylogenetically distinct group, but some, in particular Lactobacillus and Leuconostoc are heterogeneous and the phylogenetic cluster do not correlate with the current classification based on phenotypic characters. New tools for classification and identification of lactic acid bacteria are underway (Sascha and Magdalena 2010). The most promising for routine used are nucleic acid probing techniques, partial rRNA gene sequencing using the polymerase chain reaction, and soluble protein patterns. The growth is optimum at pH 5.5-5.8 and the organisms have
complex nutritional requirements for amino acids, peptides, nucleotide bases, vitamins, minerals, fatty acids and carbohydrates. Orla-Jensen used morphology (cocci or rods, tetrad formation), mode of glucose fermentation (homo- or heterofermentation), growth at certain “cardinal” temperatures (e.g., 10ºC and 45ºC), and form of lactic acid produced (D, L, or both) (Kenji et al., 2009), as the basis for classifying them. Table: 2.4 Differentiating Lactic acid bacteria and a comparison with current taxonomic classification
These characters are still very important in current lactic acid bacterial classification (Figure 2.2). The general description of the bacteria within the group is gram-positive, nonsporulating, non-respiring cocci or rods, which do, through fermentation of carbohydrates, produce lactic acid as their major end product. The common agreement is that there is a core group consisting of four genera; Lactobacillus, Leuconostoc, Pediococcus and Streptococcus (Table 2.4). The taxonomic revisions have proposed several new genera and the remaining group now comprises the following: Aerococcus,
Alloiococcus,
Carnobacterium,
Dolosigranulum,
Enterococcus,
Globicatella, Lactococcus, Oenococcus, Tetragenococcus, Vagococcus, and Weissella.
Lactobacilli, Carnobacteria and some Weissella are rods while the remaining genera are cocci (Jin et al., 2009).
Figure 2.1:
Consensus tree, based on comparative sequence analysis of 16S rRNA, showing major phylogenetic groups of lactic acid bacteria with low mol% guanine plus cytosine in the DNA and the nonrelated grampositive genera Bifidobacterium and Propionibacterium. (Schleifer and Ludwig, 1995).
Lactic acid bacteria lack the ability to synthesize cytochromes and porphyrins (components of respiratory chains) and therefore cannot generate ATP by creation of a proton gradient. The LAB can only obtain ATP by fermentation, usually of sugars. Since they do not use oxygen in their energy production, lactic acid bacteria happily grow under anaerobic conditions.
Figure 2.2:
2.2.2
Characterization of Lactic acid bacteria
Biochemistry and genetics of lactic acid bacteria Lactic acid bacteria are chemotrophic; they find the energy required for their
entire metabolism from the oxidation of chemical compounds. The oxidation of sugars constitutes the principle energy producing pathway. Lactic acid bacteria of the genera Lactobacillus, Leuconostoc and Pediococcus, the important bacteria to winemaking, assimilate sugars by either a homofermentative or heterofermentative pathway. 2.2.2.1 Homo-fermentative metabolism Homo-fermentative bacteria ferment glucose with lactic acid as the primary byproduct. Homofermentative LAB includes Lactococcus sp., used in dairy starter culture
applications where the rapid development of lactic acid and reduced pH is desirable. Other homofermentative LAB include yogurt strains consisting of rods (Lactobacillus delbruckii subsp. bulgaricus, L. acidophilus) and cocci (Streptococcus salivarius subsp. thermophilus) and thermophilic strains that might be used in cheese (L. helveticus). Other homofermentative cocci that might be found in milk and dairy products, but are rarely used as starter cultures include other Streptococcus sp., Enterococcus, Pediococcus and Aerococcus.Lactic acid bacteria utilize sugars (glucose) to form lactic acid by either the homo- or hetero-fermentative pathway. The homo-fermentative pathway (Figure 2.3) results in the transformation of glucose to pyruvate through the Embden–Meyerhof– Parnas pathway (EMP, or glycolysis), eventually yielding lactic acid. NADH produced by the oxidation of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate is reoxidized to NAD+ in the formation of lactate from pyruvate through the action of lactate dehydrogenases (LDHs). The LDH enzymes vary in their stereospecificity and can yield d- or l-lactic acid or the racemic mixture. 2.2.2.2 Hetero-fermentative metabolism Hetero-fermentors, ferment glucose with lactic acid, ethanol/acetic acid and carbon dioxide (CO2) as by-products. Testing for heterofermentative fermentation generally involves the detection of gas (CO2). With the exception of certain fermented milk products, hetero-fermentative LAB are rarely used as dairy starter cultures, although they are not uncommon in milk and dairy products. If allowed to grow to significant numbers, they can cause defects related to their acid and CO2 production, such as slits in hard cheeses or bloated packaging in other dairy products. Hetero-fermentative LAB includes Leuconostoc sp. (Gram-positive cocci) and Gram-positive rods such as Lactobacillus brevis, L. fermentum, and L. reuteri.
Other Lactobacillus species are
considered “facultatively” hetero-fermentative, meaning they will produce CO2 and other by-products only under certain conditions or from specific substrates. These strains would include L. plantarum, L. casei and L. curvatus. Other hetero-fermentors like Oenococcus oeni (known as Leuconostoc oeni until 1995) L. brevis, L. hilgardii, L.fructivorans, and L. kunkeei) lack aldolase and must divert the flow of carbon through a different series of reactions, the pentose phosphate, or phosphoketolase, pathway (Figure 2.4).
Figure 2.3:
Homo-fermentative pathway illustrating the production of lactic acid from glucose.
Figure 2.4:
Hetero-fermentative pathway showing the production of lactic acid, carbon-dioxide and either ethanol or acetic acid.
From 1 mole of glucose, hetero-fermentative bacteria produce 1 mole each of lactate, CO2, and either acetic acid or ethanol. In reality, these bacteria produce 0.8 mole lactate from glucose. Unlike homofermentative microorganisms, these bacteria do not have aldolase but possess phosphoketolase, the enzyme responsible for the cleavage of xylulose-5-phosphate to form glyceraldehyde-3-phosphate and acetyl phosphate. Due to the biosynthesis of five-carbon sugars in this pathway (ribulose-5-phosphate and xylulose-5-phosphate), some strains can utilize the pentose present in wine such as ribose, xylose, and arabinose. An important consequence of only half of the carbon from glucose going to glyceraldehyde-3-phosphate is formation of only 1 mole of ATP per mole glucose. However, hetero-fermentative bacteria can gain additional energy though conversion of acetyl-phosphate to acetate.
2.2.2.3 Genetics Currently, more than 19 complete genomes of streptococci are available, covering different strains of five species. A program aimed at extensive sequencing of the genomes of non-pathogenic LAB was announced in 2002 by the Lactic Acid Bacteria Genome Sequencing Consortium (Klaenhammer et al., 2002), but the actual breakthrough occurred only in the last 6 years (2005 and 2006). The Lactobacillales have relatively small genomes for non-obligatory bacterial parasites or symbionts (characteristic genome size, ~2 megabases, with ~ 2,000 genes), with the number of genes in different species spanning the range from ~1,600 to ~3,000. This variation in the number of genes suggests that the evolution of LAB involved active processes of gene loss, duplication, and acquisition. The current collection of LAB genomes is a unique data set that includes multiple related genomes with a gradient of divergence in sequences and genome organizations. This set of related genomes is amenable to detailed reconstruction of genome evolution, which is not yet attainable with other groups of bacteria. Divergence of Lactobacillales from their ancestor in the Bacilli was marked by the loss of 600–1200 genes, including many genes encoding biosynthetic enzymes. Other losses include genes related to sporulation, a function that is seemingly unnecessary in nutrient-rich food environments (Makarova and Koonin, 2007). Besides gene losses occurring early in the lineage of the LAB, more recent events have contributed to shaping these species, including parallel losses in genes involved in various metabolic processes. The most notable example of gene loss occurred in Streptococcus thermophilus, which diverged from pathogenic Streptococcus species through the loss and decay of virulenceassociated genes, such as those involved in antibiotic resistance and adhesion. This genomic record has thus far provided solid evidence supporting the ‘generally recognized as safe’ status for use of S. thermophilus in foods. Gene gains in the LAB also reflected a shift toward a nutrient-rich lifestyle during specific niche adaptations. Soon after the divergence of the Lactobacillales, there occurred duplication of genes involved in the transport and metabolism of carbohydrates, including genes for enolases and phosphotransferase (PTS) systems. Genes involved in amino acid transport and peptidases were also duplicated, further enhancing the ability of
these species to exploit protein-rich environments (Makarova and Koonin, 2007). Horizontal gene transfer (HGT) has also shaped these genomes. For example, many sugar transport and metabolism genes in Lactobacillus plantarum are clustered in a lower GC content area of the genome, and it is possible that many of these genes were acquired as a result of HGT (Kleerebezem et al. 2003). HGT has also shaped the genome of S. thermophilus, which possesses a 17-kb region that contains extensive identity with genes in L. lactis and L. bulgaricus subsp. delbrueckii, two species that are also associated with growth in milk. The genes from L. bulgaricus enable S. thermophilus to synthesize methionine, which is rare in milk. Genome sequencing has shown that the metabolic capabilities of these two organisms make them reliant on each other for maximum growth. For example, L. bulgaricus encodes a complete folate biosynthesis pathway, but lacks the ability to produce p-aminobenzoic acid (PABA), a key intermediate that is supplied by S. thermophilus (van de Guchte et al. 2006). In addition, an exchange of polyamines might occur between these organisms that could have a role in their oxidative stress tolerance. A major difference in the genomes of L. bulgaricus and S. thermophilus is reflected in their biosynthetic capabilities. The presence of an extracellular protease in L. bulgaricus, but the absence of many amino acid biosynthesis pathways, reflects the adaptation of this species to the protein-rich milk environment. By contrast, S. thermophilus has retained the pathways to synthesize all amino acids except histidine. It is unclear if S. thermophilus exploits the proteolytic capabilities of L. bulgaricus or retains some advantage in synthesizing its own amino acids (van de Guchte et al. 2006). Application of molecular genetic techniques to determine the relatedness of foodassociated lactic acid bacteria has resulted in significant changes in their taxonomic classification. During the 1980s the genus Streptococcus was separated into the three genera Enterococcus, Lactococcus and Streptococcus.
Figure 2.5:
Phylogenetic tree and important commercial importance of Lactic acid bacteria
The lactic acid bacteria associated with foods now include species of the genera Carnobacterium, Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Oenococcus, Pediococcus, Streptococcus, Tetragenococcus, Vagococcus and Weissella. The genus Lactobacillus remains heterogeneous with over 60 species (ymol% G+C content ranging from 33 to 55), of which about one-third are strictly heterofermentative. However, many changes have been made and reorganization of the genus along lines that do not follow previous morphological or phenotypic differentiation from Leuconostoc and Pediococcus is being studied (Figure 2.5). Phylogenetically belonging to the Actinomyces branch of the bacteria, Lactobacillus bifidus has been moved to the genus Bifidobacterium also on account of its greater than 50 mol% G+C content. It is therefore no longer considered one of the lactic acid bacteria senso strictu, which form part of the Clostridium branch of the bacteria. The new genus Weissella has been established to include one member of the genus Leuconostoc (L. paramesenteroides) and heterofermentative lactobacilli with unusual interpeptide bridges in the peptidoglycan. Contrary to the clear-cut division of the streptococci, morphological and physiological features of Weissella do not directly support this grouping which now incorporates species that produce D(-)- as well as DL-
lactate. The new genus Carnobacterium is morphologically similar to the lactobacilli, but it shares some physiological similarities (growth at pH 9.5) and a common phylogenetic branch with the genus Enterococcus (Stiles and Holzapfel, 1997). 2.2.3
Natural Habitat of Lactic acid bacteria LAB can be isolated from various natural sources. For example, Lactococci,
Streptococci, Lactobacillus can be found in milk and milk products. Studies revealed the presence of lactic acid bacteria in goat, cow, sheep, buffalo and camel milk (Sharma et al., 2013). Recent studies suggest that composite bio-waste is a preferred habitat of lactic acid bacteria, suggesting that the unsterilized bio-waste and its natural flora could be used in a fermentation process for lactic acid production (Probst et al., 2013). The sequencing of multiple complete genomes has created unprecedented opportunities for evolutionary genomics of LAB. Some of the genes acquired by Lactobacillales are clearly adaptations to existence in the nutrient-rich habitats of these bacteria (Makarova and Koonin, 2007). Barley is also a natural habitat for lactic acid bacteria. The numbers of LAB on barley have been shown to increase as a result of the malting process during brewing, possibly because the steeping conditions create a favorable environment for their growth. However, LAB counts start to decrease during barley germination and continue to decline throughout the kilning process (Rouse et al., 2008). Diversity and density of lactic acid isolated from Algerian raw goats' milk in arid zones were studied by determination of morphological, cultural, physiological and biochemical characteristics. Two hundred and six lactic acid bacterial strains were isolated, 115 of them belonging to lactic acid cocci and others to the genus Lactobacillus. The representative species of the total cocci were Lactococcus sp. (76.16%), S. thermophilus (14.78%) and Leuconostoc sp. (8.6%), respectively. The dominating species is L. lactis subsp. lactis. Lactobacilli species found in local raw goats' milk and their proportion were: L. curvatus (25.25%), L. helviticus (10.98%), L. plantarum (9.89%), L. reuteri (9.89%), L. casei (7.69%), L. brevis (5.49%), L. bulgaricus (5.49%), L. paracasei (4.39%) and L. acidophilus (2.19%) (Badis et al., 2004). It has also been reported that some lactic acid bacteria isolated from the gastrointestinal tract of fish can act as probiotic (Olympia et al., 1995). The variation of the ecological parameters acting on the microbial association such as the nature of cereal,
temperature, size of inoculum, and length of propagation intervals leads in each case to a characteristic species association. Cereals are suitable fermentable substrates for the growth of potentially probiotic microorganisms. Previous studies showed that four potentially probiotic strains (Lactobacillus fermentum, L. reuteri, L. acidophilus and L. plantarum) were cultured in malt, barley and wheat media. All strains attained high cell populations (8.1-10.1
log10
cfu/g). The malt medium supported the growth of all strains
more than barley and wheat media due to its chemical composition, while L. plantarum and L. fermentum appeared to be less fastidious and more resistant to acidic conditions than L. acidophilus and L. reuteri (Talamond et al., 2002). Another study showed that the natural sour cassava starch fermentation was mainly due to the action of lactic acid bacteria. Fermentation temperature and duration as well as the composition of the microflora influenced the expansion properties of the final cassava sour starch. However, some LAB strains (such as L. lactis, Streptococcus sp., Enterococcus saccharolyticu, L. plantarum, and L. mesenteroides) involved in the natural sour cassava starch fermentation were isolated, identified and characterized using classical microbiological techniques (Ampe et al., 2001). 2.2.4
Lactic acid bacteria in food products LAB has a long tradition of use in the food industry, and the number and diversity
of their applications has increased considerably over the years. These are the most important group of microorganisms used in the food industry for the production of various fermented products, such as yogurts, cheese, and pickled vegetables (Figure 2.6). In addition, LAB can inhibit the growth of spoilage microbes and/or pathogens in their environment by lowering the pH and/or through the production of antimicrobial peptides, called bacteriocins. Both LAB and Bifidobacteria are also thought to have healthpromoting abilities and many are used as probiotics for the prevention, alleviation, and treatment of intestinal disorders in humans and animals. LAB is very important in the food and dairy industries because lactic acid and other organic acids produced by these bacteria act as natural preservatives as well as flavor enhancers. LAB find increasing acceptance as probiotic which aid in stimulating immune responses, preventing infection by enteropathogenic bacteria, and treating and preventing diarrhea. Fermented foods
constitute a substantial part of the diet in many African countries are considered as an important means of preserving and introducing variety into the diet, which often consists of staple foods such as milk, cassava, fish and cereals. For example, Ben saalga is a traditional Burkinabè gruel obtained by cooking a diluted fermented paste of pearl millet (Pennisetum glaucum). This fermented food is widely accepted and consumed by the population, particularly by young children. The processing of pearl millet into ben saalga comprises the following successive main steps: soaking the grains (first fermentation), grinding and 13 filtration of humid flour, decanting (second fermentation) and cooking (Sanni et al., 2002). The production of this fermented food is still largely a traditional art associated with poor hygiene, inconsistent quality presentation and short shelf life. The preparation of this indigenous food generally depends on a spontaneous or chance inoculation by naturally occurring lactic acid bacteria and the use of starter cultures is still at very early development stages. LAB play an essential role in the majority of food fermentations, and a wide variety of strains are routinely employed as starter cultures in the manufacture of dairy, meat, and vegetable and bakery products. One of the most important contributions of these microorganisms is the extended shelf life of the fermented product by comparison to the raw substrate.
Figure: 2.6 Silent features of lactic acid bacteria Growth of spoilage and pathogenic bacteria in these foods is inhibited due to competition for nutrients and the presence of starter-derived inhibitors such as lactic acid, hydrogen peroxide and bacteriocins. Bacteriocins are heterogeneous group of antibacterial proteins that vary in spectrum of activity, mode of action, molecular weight, genetic origin and biochemical properties (Lee, 2005). Currently, artificial chemical preservatives are employed to limit the number of microorganisms capable of growing within foods, but increasing consumer awareness of potential health risks associated with some of these substances has led researchers to examine the possibility of using bacteriocins produced by LAB as biopreservatives as well as the application of bacteriocinogenic LAB in starter cultures. According to the generally poor sanitary conditions of ben saalga and other traditional fermented foods, the use of selected bacteriocinogenic LAB with antimicrobial activity against the most frequent foodborne pathogenic bacteria could be an affordable way to improve the safety of these fermented foods. For examples, a total of 14,020 lactic acid bacteria (LAB) are isolated from Nham and two traditional Indonesian fermented foods “Tapai” (fermented tapioca), and “Tempoyak” (fermented durian flesh). Chilli puree and fresh goat’s milk are used as sources 14 for the isolation of lactic acid bacteria (LAB), and the total amount of 126 isolates are obtained (Visessanguan et al., 2006).
In many societies including our own where yogurt has been heralded as a health food since the 19th century, fermented food has gained a reputation for its beneficial effects on immunity, intestinal health and general well-being. Modern researchers are just beginning to understand what the sages of old were tuned in to: fermented food conveys clear and calculable health benefits to the human diet. Lactic acid fermentation in and of itself enhances the micronutrient profile of several foods (Table 2.5). For example, milk that undergoes lactic acid fermentation either in the wild as in the case of clabbered milk or inoculated by a starter culture as in the case of yogurt, piima, matsoni and other fermented dairy products conveys more vitamins to the eater in comparison to raw milk and, particularly, pasteurized and ultra-high-temperature pasteurized milk. Table: 2.5 Some of the major fermented food products along with the microbe involved around the world. NAME OF FOOD Beer
INGREDIENT Barley
Cheese Dahi Dawadawa Kefir
Milk Milk Locust beans Milk
Koko Mahewu Gari
Maize, Shorgum Maize Cassava
I-sushi Kaanga piro Kimchi
Fish Maize Vegetables
Idli/Doas
Rice & Black gram
Salami Yogurt
Meat Milk
Soy sauce Sauerkraut
Soy beans Cabbage
MICROBE INVOLVED Saccharomyces cerevisiae, Leuconostoc, Lactobacillus, Lactococcus etc Lactic acid bacteria Lactic acid bacteria Bacillus, Staphylococcus Streptococcus, Lactobacillus and Leuconostoc sp, Candida kefyr, Kluyveromyces fragilis. Lactic acid bacteria L lactis, Lactobacillus sp Leuconostoc, Alcaligenes, Corynebacterium, Lactobacillus Lactic acid Bacteria Lactic acid bacteria L. mesenteroides, L brevis, L plantarum L. mesenteroides, E. faecalis, yeast Lactic acid bacteria L.bulgaricus and S. thermophilus Lactic acid bacteria Lactic acid bacteria
COUNTRY Worldwide
Worldwide India Africa Eastern Europe Ghana South Africa Nigeria Japan New Zealand Korea India Worldwide Worldwide South Asia Europe, North
Trahanas Tempeh Palm Wine Nono Ogi
Milk & wheat Soy Bean Palm Sap Milk Maize
Nam
Pork, Garlic, rice
Lambic Beer Poi Injera
Barley Taro Tef
2.2.5
Lactic acid Bacteria Lactic acid Bacteria Lactic acid Bacteria Lactic acid Bacteria L. plantarum, Corynebacterium sp,Yeast P. cerevisiae, L plantarum, L brevis Yeast and Lactic acid bacteria Lactic acid bacteria L. mesenteroides, P. cerevisiae, L. plantarum, S. cerevisiae
America Greece Indonesia Worldwide Nigeria Nigeria Thailand Belgium Hawaii Euthopia
Lactic acid bacteria in agricultural products The major genera found in the microflora of fermented or sour cassava-starch
were Streptococcus, Bacillus, Lactobacillus and Saccharomyces with amylase activity (Lacerda et al., 2005). Lactic acid bacteria predominated whereas the presence of moulds was not significant. Traditional fermentation of cassava is dominated by a lactic acid bacteria population. A total of 139 predominant strains isolated from fermenting cassava were identified using phenotypic tests and genotypic methods. Moreover, fermented tapioca was used as sources for the isolation of lactic acid bacteria. A total of 126 isolates were obtained. In addition, by sequential screening for catalase activity and Gramstaining, 55 were determined to be LAB, out of which 16 were established to be homofermentative. Moreover, Thailand is the world’s largest exporter of tapioca starch and starch derivatives with annual production of over 2 million tons of starch. Development of lactic acid production using cassava as the main substrate is very attractive because it is a cheap source, contains high starch content with low quantity of impurities, and also abundant in Nakhon Ratchasima province (Tonukari, 2004). 2.2.6
Lactic acid bacteria as Probiotics Probiotics can be defined as a food (feed) or drug containing live microbes, when
ingested, is expected to confer beneficial physiologic effects to the host animal through microbial actions. Microbial components and metabolites are essentially excluded from the definition of probiotics. Microbes used in probiotics should be able to express their
activities in the host body. The first consideration is the bacteria that normally inhabit the intestinal tract, and ingestion of these bacteria may affect the intestinal microbial balance. The human digestive tract is inhabited by numerous microbes. For bacteria to exert any probiotic effect, they have to be able to survive both the stomach acids (pH as low as 1.5) and the bile acids (pH as low as 2). This is true of most lactobacilli. Secondly, the bacteria must arrive in the intestines in sufficient quantities to have an effect. The amount required depends on the strain and the health benefit being studied. The minimum effective level for individual bacteria and specific health benefits is not yet established. The bacteria may need to adhere to the wall of the intestine (“implant”) and colonize in order for there to be an effect. Sherwood Gorbach, one of the discoverers of Lactobacillus GG, states, “Our research over the previous 20 years had established beyond doubt that implantation in the gut was the critical feature that a strain must possess to influence the intestinal mili” (Gorbach, 1996). However, others contend that continuous transit (continually eating a prebiotic food) is an alternative to the organism implanting and colonizing (Saloff-Coste, 1997). Finally, the bacteria must show some beneficial effects on human health. Some examples of beneficial effects under investigation include alleviation of lactose intolerance, prevention and treatment of diarrhea, maintenance of normal intestinal flora, antagonism against pathogens, and stimulation of the immune system, anti-carcinogenic activity, and reduction of serum cholesterol levels.
Table 2.6: Differences between Probiotics, Prebiotics and Synbiotics
PROBIOTICS
PREBIOTICS
Live microbial food supplement having beneficial effects on host through microbial actions It should be nonpathogenic, non-toxic, capable of surviving in the gut, and should contain a large number of viable cells. Should confer various postulated health advantages like improved digestion, anti-cancerous, lactose intolerance and various nutritional benefits. Example: Lactic acid bacteria
SYNBIOTICS
A non-digestible food A combination a probiotic and ingredient stimulating the probiotic again with beneficial available microbes in colon effects resulting beneficial effects It should neither be hydrolyzed nor absorbed in the upper part of gastrointestinal tract.
This combination should have the capability to increase the survival chances of probiotic,
Prebiotics should be able as a consequence to alter the colonic micro-flora towards a healthier composition.
Synbiotics show a synergistic effect, as probiotics acts in small intestine and prebiotics acts in large intestine, so synbiotics acts symbiosis.
Example: FOS (oligofructose and neosugar), Inulin, Lactulose, Lactitol.
Example: Bifidobacteria + FOS, Lactobacilli + Lactitol, Bifidobacteria + GOS.
The balance of this microbial flora greatly influences the intestinal environment. Among the numerous intestinal microbes, those that are expected to beneficially affect the host by improving the intestinal microbial balance, and hence are selected as probiotics, include species of the genera Lactobacillus, Bifidobacterium, and Enterococcus.
The
representative
species
include
Lactobacillus
acidophilus,
Lactobacillus johnsonii, Lactobacillus gasseri, Lactobacillus casei, Lactobacillus rhamnosus, Lactobacillus plantarum, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium
bifidum,
Bifidobacterium
infantis,
Enterococcus
faecalis,
and
Enterocuccus faecium. Bifidobacterium species that specifically inhabit the intestinal tracts of animals, such as Bifidobacterium thermophilum and Bifidobacterium pseudolongum, are used in animal probiotics. Some bacteria that do not normally inhabit the intestinal tract may also come under the category of probiotics. They are used as
starters in dairy products and include mainly Lactobacillus bulgaricus, Streptococcus thermophilus, Leuconostoc and Lactococcus species (Sharma et al., 2012). Lactic Acid bacteria are beneficial for both human and animals because of various properties which directly or indirectly enhance the immune system and help us to fight against various infectious diseases. This group of bacteria have been placed among the best human friendly microbe which not only reduce the effect of infections and kill pathogens; they even modulate our immune system and reduce hypertension. In some countries the use of Enterococcus sp. as a probiotic has been questioned because of safety aspects with regard to transfer of genes conferring antibiotic resistance. Most scientists agree that probiotic strains shall be able to survive transit through the gastric acid environment as well as exposure to bile and pancreatic juice in the upper small intestine to be able to exert beneficial effects in the lower small intestine and the colon, although there are convincing data on beneficial immunological effects also from dead cells (Mottet and Michetti, 2005). Best effect is achieved if the microorganisms colonise the intestinal surface mucus layer since they then can affect the intestinal immune system, displace enteric pathogens, provide antioxidants and antimutagens, and possibly other effects by cell signalling. The intake of LAB influences multiple systems was elegantly shown for Lactobacillus GG using microarray analysis (Di Caro et al., 2005). One month treatment resulted in up-regulation of 334 genes and down-regulation of 92 genes involved in inflammation, apoptosis, cell-cell signalling, cell adhesion and differentiation and signal transcription and transduction. In recent years, multiple reports have described beneficial effects from various aspects on important diseases, like intestinal infections, inflammatory bowel disease (IBD), and allergy by addition of selected strains to food products, often together with fiber or a prebiotic substance. In many countries, there are now several probiotic products on the market but the documentation is often based upon case reports, animal studies or uncontrolled small clinical trials. Furthermore, there is no general acceptance on how to characterize prebiotic microorganisms, and few products declare the actual content of live microorganisms.
There are various health modulating properties of the bacteria some most effective and commonly known are:•
Immunomodulation
•
Anti-cancerous
•
Bio-therapeutic
•
Anti-inflammatory
•
Prevention from diarrhoea
•
Hypocholesterolemic effects
•
As Live Vaccine
•
Anti-viral properties. An impressive number of reviews and books have reported the constantly
evolving knowledge and state of the art in lactic acid bacteria. The various health benefits are summarized in short are as under: 2.2.6.1
Immunomodulation The lactic acid bacteria produces some extracellular polysaccharides
(EPS) associated with rheology, mouthfeel and texture of fermented milk products. These extracellular polysaccharides from Lactobacillus bulgaricus purified from the supernatant have certain immuomodulatory activities shown in case of mice. The yoghurt containing immunostimulative EPS would have an immunomodulatory effect on human body (Makino et al. 2006). A halophilic lactic acid bacterium, Tetragenococcus halophilus, was found to possess an immunomodulatory activity that promotes T helper type 1 (Th1) immunity in addition to its important roles in soy sauce brewing (Masuda et al. 2008). Oncococcus oeni and Pediococcus parvulus also found to have immune-stimulatory activities as the strains found to stimulate cytokines released by peripheral blood mononuclear cells (Foligne et al. 2010). Studies on the relationship between nutritional food and immune-modulation have been increased based on the hypothesis that consumption of some foods create a barrier for immunological diseases (Sandre et al. 2001). Gut microflora participates in immune exclusion. It prevents other bacteria from adhering by competition for nutrients and places of adhesion, it produces anti-bacterial agents, and it stimulates the production of specific antibodies (Premalatha and Dhasarathan 2011). There are many reports on the immunomodulatory activities of lactic
acid bacteria (Wells 2011; Izumo et al. 2011; Foligne et al. 2010). Probiotics can influence the microflora composition by increasing the number of Lactobacilli and other anaerobes (Salminen et al. 1998). Dietary supply of probiotic bacteria stimulates IgA immune response (Kaila et al. 1992) and the transport of target antigens through Peyer’s patches (Isolauri et al. 1993). 2.2.6.2
Anti-Cancerous Lactic acid bacteria play an important role in retarding colon
carcinogenesis by possibly influencing metabolic, immunologic, and protective functions in the colon (Roberfroid et al. 1995). Concentrations of LAB may increase in the colon after the consumption of foods containing probiotics; however, probiotic ingestion also increases the number and metabolic activity of LAB in the colon of humans and animals (Salminen and Salminen, 1997). In animals, LAB ingestion was shown to prevent carcinogen-induced preneoplastic lesions and tumors (Rowland et al. 1998). A reduced activity of pro-carcinogenic enzymes in humans also was shown as a consequence of prebiotic intake. However, in humans, there is no evidence available on whether probiotics and prebiotics can prevent the initiation of colon cancer. Epidemiologic studies are contradictory; some studies could not find an association between the consumption of fermented-milk products and the risk of colon cancer, whereas other studies showed a lower incidence of colon cancer in persons consuming fermented- milk products or yogurt. In spite of various controversies, lactic acid bacteria have been shown to affect intestinal barrier interfering the metabolic activity of tumor cells preventing and treating a variety of cancers (Qi-Wei et al. 2011). 2.2.6.3
Bio therapeutic Lactic acid bacteria supplements are becoming more and more popular all
over the world in which a live food microbial food ingredient is ingested found to be very beneficial to human health. Streptococcus thermophilus is found to be potentially therapeutic against the associated chronic gastritis (ASA) (Rodriguez et al. 2010). Gastritis is a common disorder in which discontinuity of the gastric mucosa is observed. It is caused by various factors like excess alcohol, infection, intensive consumption of anti-inflammatory drugs with Helicobacter pylori or may be stress. Also, ASA affects
various mucosal defense lines such as bicarbonate secretion, mucus synthesis, and decrease of mucosal blood flow. The first therapeutic effect of the fermented milk with the polymer producing strain of S. thermophilus on chronic gastritis induced by ASA was experimented in mice. It was able to generate immune response in mice and increased the thickness of the gastric mucus gel layer. Studies suggest that recombinant lactic acid bacteria are the excellent candidates for the production of various bio therapeutic proteins and also their delivery to specific places of requirement within the gastrointestinal tract (Daniel et al. 2011).
Figure 2.7:
2.2.6.4
Various applications of Lactic acid bacteria modulating human health
Anti-inflammatory
The genus Lactobacillus includes a restricted set of intestine-indigenous species from a pool of more than 100 Lactobacillus species and studies suggest that some strains of the species have potent anti-inflammatory effects (Versalovic et al. 2008). The strains have the capability to down regulate the production of TNF-• by macrophage cell lines and successfully suppressed inflammation in mouse colitis models. Human-derived Lactobacillus reuteri strains have been identified with potent human TNF-• -inhibitory
effects on lipopolysaccharide (LPS)-activated human myeloid cell lines and primary monocyte-derived macrophages from children with Crohn's disease (Versalovic et al. 2008). Some Lactic acid bacteria have shown to regulate mucosal immune response by modulating the production and liberation of regulatory agents such as cytokines by the host.Some of these cytokines, such as the anti-inflammatory interleukin-10 (IL-10), modulate the inflammatory immune response, thus immunomodulation is a mechanism by which LAB can prevent certain inflammatory bowel diseases (IBD). Extracts from soymilk fermented with lactic acid bacteria and Bifidobacteria showed the inhibitory effect on LPS-induced pro- inflammatory cytokines including tumor necrosis factor (TNF)-• , interleukin (IL)-6, IL-1β and prostaglandin E2 (PGE2) produced by RAW 264.7 macrophages (Liao et al. 2010). 2.2.6.5
Prevention from diarrhoea Intestinal microflora maintains a barrier against the development of
pathogenic bacteria in the digestive tract and is mandatory to the establishment of oral tolerance to food antigens. Lactic acid bacteria may be useful in preventing and shortening the duration of several types of diarrhea. It is believed that lactic acid bacterium competes with the pathogenic bacteria in terms of nutrition and space in the intestines shortening its chances of survivability. Undergoing a strong metabolism process, the lactic acid bacteria produce various metabolites like acidophilin and bulgarican inhibiting the growth of other bacteria preventing and shortening the duration of diarrhoea. Recent studies suggest that children in India who receive probiotic demonstrated protective efficacy of 14% prevention from diarrhoea (Hajela et al. 2010). Approximately 20% of the patients treated with antibiotics will develop AAD because their intestinal flora, responsible for the natural colonization resistance, is disturbed or reduced. The intestinal flora modification (in particular in the LAB population) could be the cause of diarrhea, dehydration and electrolytic imbalance. Also, the fermentation in the colon can be reduced. Many preparations have been tested for their preventive efficacy against AAD (Contardi, 1991). However, more studies need to be performed using well controlled conditions and strains, before one can finally understand which prophylactic probiotics should be taken against secondary effects of specific antibiotics, applied at a specific dose in a specific type of patient.
2.2.6.6
Hypocholesterolemic effects Hyperlipidemia is a leading death cause in many countries of the world
with the risk of cardiovascular disease. A number of scientific studies suggest that lactic acid bacterial fermented food shows hypolipidimic effects by reducing the cholesterol biosynthesis and decreasing low density lipoproteins (Gao et al. 2011). The hypocholesterolemic effects of lactic acid bacteria (LAB) have now become an area of great interest and controversy for many scientists. Some strains of L. acidophilus can take up cholesterol in the presence of bile, suggest that lactic acid bacteria can reduce cholesterol level up to 50% in presence of bile salt (Lavanya et al. 2011). Some lactic acid bacteria could adjust blood lipid and lower cholesterol which can also prevent some of the diseases by stimulating antioxidant enzymes (Koiche and Dilmi 2010). These strains have the ability to tolerate both acid and bile concentrations typically found in the upper gastrointestinal tract of humans. In a study milk fermented with lactic acid bacteria was feeded to rats and it was observed that it reduces the serum total cholesterol and LDL cholesterol levels after 12 days of feeding (Pato et al. 2004). Hypocholesterolemic effect of Lb. lactis subsp. lactis was attributed to its ability to suppress the reabsorption of bile acids into the enterohepatic circulation and to modulate the excretion of bile acids in feces of hypercholesterolemic rats (Pato et al. 2004).
Table: 2.7 Health benefits along with the proposed mechanism of action of lactic acid bacteria Health Benefit by Lactic acid Bacteria Immuno-modulation
Intestinal Tract Infections
Proposed Mechanism
Lactose Intolerance Anti-inflammatory and Anti-allergic.
Anti- Colon cancer
Urogenital infections
2.2.6.7
Increase in IgA production. Non-specific defence against infection. Increased phagocytic activity of WBC. Proliferation of intra-epithelial lymphocyte. Alteration of toxic binding sites. Stimulation of the systemic or secretory immune response. Adjuvant effect increasing antibody production. Adherence to intestinal mucosa, preventing pathogen. Competition for nutrients. Bacterial β -galactosidase acts on lactose. Restoration of the homeostasis of the immune system Regulation of cytokine synthesis Prevention of antigen translocation into blood stream Mutagen binding Carcinogen deactivation Alteration of activity of colonic microbes Immune response Influence on secondary bile salt concentration Adhesion to urinary and vaginal tract cells Competitive exclusion Inhibitor production (H2O2, biosurfactants)
Lactic acid bacteria as live vaccine The most promising new application for LAB is their use as live delivery
vectors for antigenic or therapeutic protein delivery to mucosal surfaces. Such engineered lactic acid bacteria are able to elicit both mucosal and systemic immune responses. The delivery of vaccine in the body through mucosal routes is much beneficial compared to direct systemic inoculation. But human mucosal surface is a site in which the host encounters a large variety of micro organisms initiating infections. Lactic acid bacteria have proved to be effective delivery vehicles for functional proteins to mucosal tissues. Oral administration of Lactococcus lactis has shown to induce antigen-specific oral
tolerance (OT) to secreted recombinant antigens (Wells 2011). The food manufacturing sector has developed the so called functional food containing ingredients for promoting health. Non pathogenic food grade bacteria such as lactic acid bacteria (LAB) are being tested for their efficacy as live antigen carriers (Mercenier et al. 2000). The advantages of using live bacterial vaccines include their mimicry of a natural infection, intrinsic adjuvant properties and their possibility to be administered orally. Derivatives of pathogenic and non-pathogenic food related bacteria are currently being evaluated as live vaccines (Detmer and Glenting, 2006).
The first evidence that recombinant commensal bacterium can be used as a live vaccine vector was obtained with S. gordonii. There are two major approaches being followed to achieve efficient mucosal delivery of antigens. A variety of synthetic (nonliving) delivery systems, in which purified antigens are entrapped in microspheres, liposomes, nanoparticules, or ISCOMS, are presently being investigated. An attractive alternative consists in the use of live viral or bacterial vectors for the production of replicative particulate antigens in vivo. This technology, which alleviates the drawbacks of subunit vaccine development, was first described in the early 1980s. Most of the lactic acid bacteria are quite acid resistant and various strains are able to effectively survive passage through stomach. The bacterial group has today created a vaccine vehicle system depending upon their immunization routes and models of antigens they carry. It is found that the lactic acid bacteria have a low innate antigenicity even though several strains clearly exhibit immunoadjuvant properties (Pouwels et al. 1998). The ideal GMM for use in humans should therefore contain the minimal amount of foreign DNA and must not include an antibiotic resistance marker (Lee 2010). 2.2.6.8
Anti-viral activities Lactic acid bacteria were found to be effective against the ‘Transmissible
Gastroenteritis Coronavirus (TGEV) and Rotavirus RF strain (RV)’. A selected number of strains of lactic acid bacteria were used in the study along with the CLAB cell line from pig. However, the co-incubation of the CLAB cell line with specific LAB strains resulted in increased survival percentages, from 40% up to 80%. Various literature and clinical studies have confirmed the beneficial and alleviating effects of probiotic bacteria,
such as Lactobacillus rhamnosus GG, on the infection and symptoms of rotavirus diarrhea. Also probiotics have been reported on their antiviral activities on animals (Zhang et al. 2008). L. acidophilus NCFM has a significant immune-stimulating effect on the intestinal and systemic HRV-specific T and B cell immune responses induced by the AttHRV vaccine and is safe in neonates; therefore it may have the potential to be used as an adjuvant for rotavirus vaccines (Versalovic et al. 2008). 2.3
STREPTOCOCCUS THERMOPHILUS Streptococcus thermophilus was of major importance for the food industry since it
was massively used for the manufacture of dairy products and it was considered as the second most important industrial dairy starter after Lactococcus lactis. Nevertheless, over 1021 live cells of Streptococcus thermophilus ingested annually by the human population (Trevan et al., 2004). Streptoccocus thermophilus (S. thermophilus) belongs to the group of the thermophilic lactic acid bacteria and is traditionally and widely used as a starter in manufacturing dairy products (Emmental, Gruyere, Parmigiano, Mozarella, Yoghurt etc). Yoghurt results from the fermentation of milk by S. thermophilus and Lactobacillus delbrueckii sp. bulgaricus (L. bulgaricus), fulfils the current specifications required to be recognized as a probiotic product (Guarner et al., 2005). The health beneficial effect of yoghurt consumption is linked to the metabolic properties of S. thermophilus and L. bulgaricus. Streptococcus thermophilus is an important bacterium that is extensively used in starter cultures in the dairy industry. It is also found growing spontaneously in traditional products around the world, and is believed to persist in the farm environment. As such, it improves lactose digestion in the gastro-intestinal tract (GIT) through their lactose hydrolyzing activity present in yoghurt and in the GIT, thus reducing symptoms of lactose intolerance (Lomer et al., 2008; Rabot et al., 2010). Yoghurt cultures were shown to induce other health benefits such as reduction of diarrhoea or allergic disorders as well as modulation of the immune system (Guarner et al., 2005; Higashikawa et al., 2010). S. thermophilus is also present at high concentration in VSL#3, a probiotic mixture of eight different bacterial strains that possesses beneficial effects in several intestinal conditions (Pagnini et al., 2010; Preidis et al., 2009). Recent data indicate that strains related to S. thermophilus LMD-9 are among the 57 bacteria species found in 90%
of 124 European individuals intestinal microbiota (Qin et al., 2010). In comparison with the overall human intestinal microbiota, S. thermophilus is numerically non dominant species with variable levels (Mater et al., 2005; Elli et al., 2006; Firmesse et al., 2008; Qin et al., 2010). At birth, Streptococcus genus -with in some studies a precision at the level of S. thermophilus species- is among the first coloniser of the GIT, since it has been detected in infant faeces and breast milk (Palmer et al., 2007; Perez et al., 2007). Thus, Streptococcus, as pioneer bacteria colonising a yet immature GIT, may impact the maturation and homeostasis of intestinal epithelium after birth. 2.3.1
Physiology of S. thermophilus S. thermophilus is closely related to Lactococcus lactis, but it is even more closely
related to other streptococcal species including several pathogens (Mitchell, 2003). S. thermophilus is highly adapted to grow on lactose, the main carbon source in milk and rapidly converts it into lactate during growth. Lactose is transported into the cell by a lactose permease (LacS), which operates as a galactosideproton symport system or as a lactose-galactose antiporter. Lactose is efficiently transported into the cell and subsequently hydrolyzed by an intracellular β -galactosidase. The vast majority of S. thermophilus strains only metabolized the glucose moiety of lactose, while galactose is excreted into the medium. The milk is poor in free amino acids (AA) and short peptides, therefore for optimal growth; S. thermophilus requires either hydrolysis of caseins followed by the internalization and the degradation of the resulting peptides or de novo AA biosynthesis (Hols et al., 2005). For many LAB including S. thermophilus, the hydrolysis of milk caseins (AA supply) mostly depends on the activity of a cell-wallanchored proteinase (Herve-Jimenez et al., 2008). Fortunately, nature has provided us with different kind of nucleic acids for different kind of taxonomic studies. Close relations (at species and subspecies level) can be determined with DNA-DNA homology studies. For determining phylogenetic positions of species and genera, ribosomal RNA (rRNA) is more suitable, since the sequence contains both well-conserved and lessconserved regions. It is now possible to determine the sequence of long stretch of rRNA (~1500 bases of 16S rRNA) from bacteria (Huili et al., 2011) comparisons of these sequences are currently the most
powerful and accurate technique for determining phylogenetic relationships of microorganisms (Philippe et. al., 2009). With this technique, a clearer picture of phylogeny of lactic acid bacteria is emerging, and the ideas of Orla-Jensen can be examined with some accuracy. The genera that, in most respects, fit the general description of the typical lactic acid bacteria are (as they appear in the latest edition of Bergey’s Manual from 1986) Aerococcus, Lactobacillus, Leuconostoc, Pediococcus, and Streptococcus. Major revision of taxonomy of lactic acid bacteria, in particular of streptococci, was anticipated in Bergey’s Manual of 1986 (Barry 2009) and to some extent already realized by the year of that issue. Thus, the former genus Streptococcus was first divided into three: Enterococcus, Lactococcus, and Streptococcus sensu stricto (Alexander et al., 2001; Zongzhi et. al., 2008). Later, some motile lactic acid bacteria, otherwise resembling lactococci, were suggested to form separate genus, Vagococcus (Aly et al., 2004). The genera Lactobacillus, Leuconostoc, and Pediococcus have largely remained unchanged, but some rod-shaped lactic acid bacteria, previously included in Lactobacills, are now forming the genus Carnobacterium (Elliot et al., 1991), and the former species Pediococcus halophilus has been raised to genus level, forming the genus Tetragenococcus (Facklam et al., 1995). Revisions after 1986 are supported by extensive chemotaxonomic and genetic data (Khalid, 2011).
2.3.2
Characterization of Streptococcus thermophilus
2.3.2.1 Phenotypic Characterization
Streptococcus thermophilus is the only starter dairy streptococci in Streptococcus genus. According to DNA-DNA homology and membrane fatty acid profile studies, researchers have thought that S. thermophilus should be reclassified as S. salivarius sub sp. thermophilus. However, S. thermophilus and S. salivarius are not found in the same ecological niche possessing large number of physiological differences. In 1987, S. thermophilus has been restored to species level again as a result of more sophisticated homology studies with huge phenotypic differences (Zirnstein and Hutkins 1999).
Figure: 2.8
Electron microscopic image of S. thermophilus (Source: Durso and Hotkins 2003)
S. thermophilus prefers the disaccharides lactose and sucrose, and its growth on the constituent monosaccharides, glucose, fructose and galactose is slower than the disaccharides
suggesting
that
the
transport
systems
required
to
accumulate
monosaccharides might be absent or has low activity. It appears that it is dependent on the availability of the necessary transport system. (Hutkins and Ponne 1991). This phenomenon has been explained with the findings that the enzymes of the Leloir pathway for galactose metabolism are present in S. thermophilus, however their activities are very low and the activity of the first enzyme galactokinase is undetectable under normal growth conditions (Grossiord et al. 1998). General characters are:
Gram positive, non-motile coccus
Spherical/ovoid cells of 0.7-0.9µm diameter
Occurs in pairs or in long chains of 10-20 cells
Homo-fermentative, L(+) lactic acid as the major end product
Facultative anaerobe
Catalase negative
Lacks cytochromes
Optimum growth temperature is between 40-45°C, a minimum at 20-25°C and maximum at 50-52°C.
Thermo tolerant, survives at 60°C for 30 min
Weak or no growth at 2 % NaCl
Does not utilize arginine
Lacks group specific antigen
G-C mol ratio is 37-40%
Above 10g of lactic acid/kg of yoghurt, the growth reaches exp8-9 and, the metabolism of bacterium ceases. The final pH in broth culture is 4-4.5
S. thermophilus has limited proteolytic activity and requires free amino acids for its growth. These are glutamic acid, histidine, cysteine, methionine, valine, leucine, isoleucine, tryptophan, arginine and tyrosine. However, free aminoacids naturally found in milk are not sufficient. The free amino acids are supplemented during heat treatment in milk or the absorption of short-chain peptides released by the breakdown of milk proteins by Lactobacillus delbrueckii sub sp. bulgaricus (Pearce and Flint 1999). 2.3.2.2 Biochemical Characterization
In traditional identification, most commonly used characteristics are morphology, staining reactions, nutritional requirements, cell wall chemistry, ability to use different energy sources, fermentation byproducts, gas requirements, temperature and pH tolerance, antibiotic sensitivity, pathogenicity, immunological characteristics and habitat (Morata et al. 1999). The genus Streptococcus includes Gram positive bacteria with similar metabolic properties but they live in different habitats and have many physiological differences. In the past two decades, several important Streptococcus species have been reclassified as members of recently named genera Enterococcus and Lactococcus. The only dairy Streptococcus remained is S. thermophilus. Streptococci grouped as “oral”, “pyogenic” and “other streptococci”. “Oral” streptococci are also subdivided into four groups; S. mutans, S. mitis, S. anginosus and S. thermophilus groups (Gobbetti and Corsetti 1999). Although S. thermophilus is a member of “S. thermophilus group” phylogenetically, it is the only bacterium in Streptococci with dairy origin. The
Gram positive and cocci genera sharing the same habitat with S. thermophilus includes enterococci, lactococci, pediococci and leuconostocs (Table 2.8; 2.9). The pediococci is readily distinguished from other genera by the tetrad morphology in broth media. Some of the physiological differences which are helpful for the first grouping at the genus level are given in the table below:
Table: 2.8 Classification of cocci lactic acid bacteria Microrganism
Growth at 10°C
Growth at 45°C
Growth in 6.5% NaCl
Type Lactate formed
Gas from Glucose
Growth in broth at pH 9.6
Enterococcus
+
+
+
ND
-
+
Argin ine Hydr olysis +
Lactococcus
+
-
-
L
-
-
V
Streptococcus
-
+
-
L
-
-
V
Leuconostoc
+
+
-
D
+
ND
-
* ND indicates no data available, V indicates variable: some produce (+) results and some (-), L indicates levo-lactic acid and D indicates dextro-lactic acid S. thermophilus is highly adapted to the dairy environment, and in the wild. It can only be isolated from dairy products. S. waius is a recently identified thermophilic Streptococcus isolated from stainless still pasteurization machinery of milk. It shares many phenotypic characteristics with S. thermophilus but can be distinguished by the fermentation of galactose, salicin, cellobiose, maltose, melibiose and D-raffinose. S.waius is also tolerant up to 7% NaCl (Pearce and Flint 1999). Unlike other streptococci, S. thermophilus does not possess a group- specific antigen. The peptidoglycan structure is identical to Enterococcus faecalis; however it is distinguished from enterococci and lactococci by its sensitivity to salt. It does not grow in the presence of 4% salt, and some strains will not grow in as little as 2% salt (Zirnstein and Hutkins 1999). The mechanism of lactose transport in S thermophilus differs from lactococci, which possess a specific system
for
lactose
transport,
the
phosphoenolpyruvate
(PEP)-dependent
phosphotransferase system (PTS). Lactose-6-phosphate formed during transport is hydrolyzed by an intracellular phospho-β -galactosidase into glucose and galactose-6phosphate which are then metabolized into lactic acid. S. thermophilus does not possess
lactose PTS or phospho-û- galactosidase, but has a lactose permease system including a proton dependent membrane-located permease.
Table: 2.9 Characterizing features of S. thermophilus Characteristics of Streptococcus thermophilus B4 isolated from Goat Milk (Sharma et al 2013) Gram
Cell
Spore
Staining Morphology Formation
Catalase
Fermentation
Glucose
Nitrate
Activity
Type
Fermentation
Reduction
Negative
Homo
Positive
Negative
Cocci in Positive
chains
Negative
Sugar Fermentation Streptococcus thermophilus B4 isolated from Goat Milk (Sharma et al 2013) Sucrose Lactose Maltose Dextrose Ribose Sorbitol Mannose Positive
Positive
Positive
Positive
Negative
Negative
Positive
2.3.2.3 Genotypic Characterization DNA hybridization assays are not without their shortcomings, however, being time-consuming, labor-intensive, and expensive to perform. Today, fewer and fewer laboratories worldwide perform such assays, and many studies describing new species are solely based upon small subunit (SSU) sequences or other polyphasic data. In the early 1990s the availability DNA sequencers in terms of cost, methodologies, and technology improved dramatically, such that many centers can now afford such instrumentation. In 1994, Stackebrandt and Goebel summarized the emergence of SSU sequence technology and its potential usefulness in the definition of a species. Although it has been demonstrated that 16S rRNA gene sequence data on an individual strain with a nearest neighbor exhibiting a similarity score of 97% is not as clear (Petti, 2007). This latter value can represent a new species or, alternatively, indicate clustering within a previously defined taxon. DNADNA hybridization studies have traditionally been required to provide definitive answers for such questions. Whereas 16S rRNA gene sequence data can be used for a multiplicity
of purposes, unlike DNA hybridization (>70% reassociation) there are no defined “threshold values” (e.g., 98.5% similarity) above which there is universal agreement of what constitutes definitive and conclusive identification to the rank of species. One of the most attractive potential uses of 16S rRNA gene sequence informatics is to provide genus and species identification for isolates that do not fit any recognized biochemical profiles, for strains generating only a “low likelihood” or “acceptable” identification according to commercial systems, or for taxa that are rarely associated with human infectious diseases. The cumulative results from a limited number of studies to date suggest that 16S rRNA gene sequencing provides genus identification in most cases (>90%) but less so with regard to species (65 to 83%), with from 1 to 14% of the isolates remaining unidentified after testing (Drancourt et al, 2000).
Bioinformatics is now an established and vital resource for molecular biology research and is also a mainstay of routine analysis of DNA. The use of bioinformatics has been driven by the increase in genetic sequence information and the need to store, analyse and manipulate the data. There are now a huge number of sequences stored in genetic databases from a variety of organisms, including the human genome. Indeed the genetic information from various organisms is now an indispensable starting point for molecular biology research. The main primary databases include GenBank at the National Institutes of Health (NIH) in the USA, EMBL at the European Bioinformatics Institute (EBI) at Cambridge, UK and the DNA Database of Japan (DDBJ) at Mishima in Japan. These databases contain the nucleotide sequences which are annotated to allow easy identification. There are also many other databases such as secondary databases that contain information relating to sequence motifs, such as core sequences found in cytochrome P450 domains, or DNA-binding domains. Importantly all of the databases may be freely accessed over the internet. Consequently the new expanding and exciting areas of bioscience research are those that analyse genome and cDNA sequence databases (genomics) and also their protein counterparts (protomics).
This is sometimes referred to as in silico research. Sequencing the genomes of many species in a class of bacteria enables the examination of their evolution and
divergence. Although the phenotypic techniques have proven to be useful, there is a general awareness that strains with similar phenotypes do not necessarily have closely related genotypes. Phenotypic methods have also poor reproducibility, ambiguous, and poor discriminatory power. Wild type strains isolated from natural habitats show phenotypic variability and are often classified as “atypical”. Genotypic techniques have different levels of discrimination, from species level to individual strain level. Most of them are based on Polymerase Chain Reaction (PCR), which enables the amplification of targeted DNA fragments by the use of designed primers under controlled reaction conditions. The most powerful and most extensively used phylogenetic marker is 16S ribosomal RNA and the genes code for it. The advantages result from the fact that the ribosomes must have been present in the earliest prokaryotic cells, the components of the ribosomes have not changed their function and the presence of multiple genes coding for rRNA makes horizontal gene transfer unlikely. That is why there is a high degree of conservation within the tRNA genes sequences. More than 12.000 sequences of 16S rDNA are available for prokaryotic strains in gene banks (Morata et al. 1999). PCRRFLP (Restriction Fragment Length Polymorphism) is the most commonly used method to identify isolates at intra-species level. Method is based on the digestion of amplicons resulting from PCR with restriction enzymes. When RFLP is applied to ribosomal genes, the method is called amplified ribosomal DNA restriction analysis (ARDRA). PCRARDRA with EcoR I has been to be a reliable and rapid method for identifying L. delbrueckii isolates at the subspecies level and for differentiating this species from L. helveticus and L. acidophilus (Bouton et al. 2002, Coeuret et al. 2003). This was also justified by another study, which differentiated the subspecies of L. delbrueckii and also reclassified some of the ATCC type strains known to be L. delbrueckii sub sp. bulgaricus as L. delbrueckii sub sp. lactis (Delley and Germond 2002). The use of 16S rRNA gene sequences to study bacterial phylogeny and taxonomy has been by far the most common housekeeping genetic marker used for a number of reasons. These reasons include (i) its presence in almost all bacteria, often existing as a multigene family, or operons; (ii) the function of the 16S rRNA gene over time has not changed, suggesting that random sequence changes are a more accurate measure of time (evolution); and (iii) the 16S rRNA gene (1,500 bp) is large enough for informatics
purposes. In 1980 in the Approved Lists, 1,791 valid names were recognized at the rank of species. Today, this number has ballooned to 8,168 species, a 456% increase. In the early 1990s the availability DNA sequencers in terms of cost, methodologies, and technology improved dramatically, such that many centers can now afford such instrumentation. In 1994, Stackebrandt and Goebel summarized the emergence of SSU sequence technology and its potential usefulness in the definition of a species. Although it has been demonstrated that 16S rRNA gene sequence data on an individual strain with a nearest neighbor exhibiting a similarity score of 97% is not as clear. This latter value can represent a new species or, alternatively, indicate clustering within a previously defined taxon. DNA-DNA hybridization studies have traditionally been required to provide definitive answers for such questions. Whereas 16S rRNA gene sequence data can be used for a multiplicity of purposes, unlike DNA hybridization (>70% reassociation) there are no defined “threshold values” (e.g., 98.5% similarity) above which there is universal agreement of what constitutes definitive and conclusive identification to the rank of species. DNA fingerprinting methods that solely relie on PCR include Randomly Amplified Polymorphic DNA (RAPD). This analysis makes use of short arbitrary primers and lowstringency conditions to amplify DNA randomly. Fragments obtained are then separated electrophoretically to produce a fingerprint. The great flexibility in primer choice enables it to be used to differentiate LAB at different taxonomic levels, from genus to intraspecific level. However, since the primers are random and not directed to a specific region, reproducibility is major problem. RAPD-PCR was shown to be superior in distinguishing individual L. delbrueckii strains. On the other hand, S. thermophilus strains showing phenotypic anomalisms were not easy to locate within S. thermophilus clusters using this method (Moschetti et al. 1998). Species-specific PCR is another method which relie on PCR for rapid and accurate identification for S. thermophilus isolates. Differentiation of S. thermophilus strains from other Streptococcus species such as S. mutans, S. salivarius, and other bacteria such as L. delbrueckii subspecies, Lactococcus lactis subspecies,
L.acidophilus, L. brevis, L. fermentum, L. helveticus, L. plantarum, Enterococcus subspecies and E. coli was accomplished clearly using primers homologous within the lacZ gene. Phylogenetic tree of Lactobacillales based on concatenated alignments of ribosomal proteins of Lactobacillaceae, Leuconostocaceae and Streptococcaceae is presented in Figure 2.9. This method was also justified by a study which makes use of the polyphasic approach to show the genotypic and phenotypic heterogeneity of S. thermophilus strains (Giraffa et al. 2001) and also with the study, which investigates the species composition of commercial dairy starters (Giraffa and Rossetti 2004). Pulsed Field Gel Electrophoresis (PFGE) is another molecular typing method. It has an alternating field of electrophoresis to separate large DNA fragments resulting from restriction with rare cutting enzymes. The crucial point in PFGE is the extraction of intact chromosomal DNA, which is more time consuming than other fingerprinting techniques. Since large DNA fragments, representing the whole genome, are analyzed with PFGE, it has a superior discriminatory power at subspecies and strain levels. Ribotyping combines an enzymatic restriction digestion and the detection of the restriction fragments by means of rDNA probes. Fluorescent or radioactively labeled probes can be used for hybridization. Discriminatory power of this method is dependent on the number and type of endonucleases and probes. In a study, where ribotyping has been applied to 30 different L. delbrueckii strains, it has been found that only ribotyping with EcoR I allowed the differentiation of three subspecies on the basis of a typical hybridization pattern (Miteva 2001). Differentiation at strain level has also been achieved for S. thermophilus strains both by restriction endonucleases digestion combined with pulsed field gel electrophoresis and by ribotyping (Salzano et al 1994). 16S or 23S rDNA sequencing is another useful method, when unknown isolates are to be identified. Obtained sequences are compared with the sequences previously deposited in a database. Stackobrandt and Goobel stated that strains that are more than 3% divergent in 16S rRNA are nearly always members of different species, as determined by DNA-DNA hybridization studies. Whereas, the strains with less than 3% divergency are generally members of the same species. A cutoff of 3% is a recommended limit as a conservative criterion (Cohan 2002). DNA-DNA hybridization has a higher discrimination power than 16S sequencing. Various approaches such as nitrocellulose filter methods, free-solution methods, and recently the microarray technology have been used. As a general rule,
strains, which have a DNA-DNA relatedness of more than 70% and 5°C or less difference in melting point, belong to the same species. However, use of isotopes and lack of database affect the popularity of method negatively. As was observed in Archaea and Proteobacteria (Snel et al., 2002), genome reduction was an overall trend during the evolution of the LAB. Divergence of Lactobacillales from their ancestor in the Bacilli was marked by the loss of 600–1200 genes, including many genes encoding biosynthetic enzymes (Makarova and Koonin, 2007). Other losses include genes related to sporulation, a function that is seemingly unnecessary in nutrient-rich food environments (Hufner et al., 2007). Besides gene losses occurring early in the lineage of the LAB, more recent events have contributed to shaping these species, including parallel losses in genes involved in various metabolic processes. The most notable example of gene loss occurred in Streptococcus thermophilus, which diverged from pathogenic Streptococcus species through the loss and decay of virulence-associated genes, such as those involved in antibiotic resistance and adhesion. This genomic record has thus far provided solid evidence supporting the ‘generally recognized as safe’ status for use of S. thermophilus in foods.
Figure 2.9:
Phylogenetic tree of Lactobacillales based on concatenated alignments of ribosomal proteins. Colors represent current taxonomy, with Lactobacillaceae in blue, Leuconostocaceae in red and Streptococcaceae in green
Table: 2.10 Genomic details of Streptococcus thermophilus strains with whole genome sequencing available data till Feb 2014
Strain Streptococcus thermophilus CNRZ1066 Streptococcus thermophilus LMD-9 Streptococcus thermophilus LMG 18311
Primary application
NCBI / Gene bank accession
Genomic Size
Starter culture
NC_006449
Starter culture
NC_008532
Protein s
Reference
1.8 Mb
1915
Bolotin et al., 2004
1.8 Mb
1710
Makarova et al., 2006 Bolotin et al., 2004
Starter culture
NC_006448
1.8 Mb
1889
Streptococcus thermophilus ND03 Streptococcus thermophilus JIM8232 Streptococcus thermophilus MN-ZLW-002
Starter culture
CP002340.
N/A
3840
Non StarterMulti locus
FR875178.
N/A
4305
Starter culture
CP003499
N/A
3822
Kang et al., 2012
Streptococcus thermophilus TH1435
Starter culture
AYSG0000000 0
N/A
1724
Treu et al., 2014
Starter culture
AYTT0000000 0
N/A
1698
Streptococcus thermophilus TH1436
Zhihong Sun et al., 2011 Delorme et al., 2011
Treu et al., 2014
To date, the complete genomes at the chromosome level of S. thermophilus strains CNRZ1066, JIM8232, LMD-9, LMG18311, MN-ZLW-002, and ND03 have been published. However, S. thermophilus TH1435 and TH1436 represent the first cases of S. thermophilus strains isolated in Italy and the only ones from goat milk (Table 2.10). S. thermophilus TH1435 and TH1436, collected from two alpine huts (malghe) from raw goat milk used for the artisanal production of Italian cheese, are capable of rapidly lowering the pH of milk, which represents a key technological feature for the dairy industry. Moreover, strain TH1436 is capable of utilizing galactose, while TH1435
lacks this feature. Whole-genome sequencing of strains TH1435 and TH1436 was performed with an Illumina MiSeq sequencer at the Ramaciotti Centre, Sydney, Australia. Genomic libraries were prepared using the Nextera XT kit Illumina (Illumina, Inc., San Diego, CA), which produced a mean insert size between 800 and 1,200 bp. A total of 776,373 and 1,527,857 paired-end reads (2 × 250 bp) were generated and gave 134- and 159-fold coverages of the TH1435 and TH1436 genomes, respectively. Approximately 85% of these reads were assembled into 36 and 28 large scaffolds, respectively, using a manually curated consensus of assemblies obtained using version 1.2.10 of the Velvet software (Zerbino and Birney, 2008) and version 2.8 of the 454 Newbler Assembler (454 Life Sciences, Branford, CT). The draft genome of S. thermophilusTH1435 is a single circular chromosome of 1,750,348 bases in length, with a mean G+C content of 38.9% and two putative plasmids individuated by BLAST match (scaffolds 33 and 35). The draft genome of strain TH1436 is a single circular chromosome of 1,780,473 bases, with a mean G+C content of 39.0%; no plasmid sequences were detected for this strain. S. thermophilus MN-ZLW-002 was originally isolated from a traditional fermented dairy food called Yogurt Block, originating from the Gannan region of Gansu province, China by Kang et al., 2012. MN-ZLW-002 has many good fermentation characteristics, but the most prominent is the ability to produce exopolysaccharides (EPSs). EPSs produced by LAB in fermented milk or yogurt improve the viscosity, body, texture, and taste of thefinal product (De Vuyst et al., 2001). In addition, LAB -derived EPSs contribute to human health through their potential antitumor, antiulcer, immunomodulating, and cholesterol-lowering properties (Duboc et al., 2001; RuasMadiedo et al., 2002). Whole-genome sequencing of S. thermophilus strain MN-ZLW002 was performed with a combined strategy of 454 sequencing (Margulies et al., 2005) and Solexa paired-end sequencing technologies (Bentley et al., 2008). Genomic libraries containing 8-kb inserts were constructed, and 188,861 paired-end reads and 64,276 single-end reads were generated using the GS FLX system, giving 45-fold coverage of the genome. The majority (97.4%) of reads were assembled into three large scaffolds, including 50 nonredundant contigs, using the 454 Newbler assembler (454 Life Sciences, Branford, CT).
A total of 339,700,400 reads (500 bp library) were generated to reach a depth of 180-fold coverage with an Illumina Solexa GA IIx (Illumina, San Diego, CA) and mapped to the scaffolds using Burrows-Wheeler alignment (BWA) (Li and Durbin, 2009). The gaps between scaffolds were filled by sequencing PCR products using an ABI 3730 capillary sequencer. With decreasing costs for sequencing and annotation, it is likely that most industrial strains will be sequenced, which could aid in strain selection and performance in an industrial setting. Genome sequences alone, however, do not provide a full understanding of a microorganism. In the post-sequencing era, scientists are taking the first steps to integrating sequence data with transcriptional and functional studies so as to better define complex traits. New methods of analysis, such as metabolomics and metagenomics, can also aid in characterization and should be added to the repertoire of tools for investigation of complex microbial ecosystems. Humans have relied on the LAB for thousands of years for food preservation. Our understanding of LAB has increased exponentially with the applications of genomics and biotechnology, opening up new horizons in bioprocessing, human health and food production. 2.3.3
Factors affecting the growth of lactic acid bacterial fermentations However, there are still several researches that need to be addressed in order to
produce lactic acid within the targeted cost, development of high performance lactic acid producing microorganisms and lowering the cost of the raw material. Many factors affected in lactic acid fermentation have been investigated. The optimization of fermentation processes requires profound knowledge of the factors determining microbial metabolism, and the influence of process parameters 2.3.3.1 Temperature Temperature and pH are the key environmental parameters that affect the fermentation process (Yuwono and Kokugan, 2008). Low temperature has been reported to positively influence the outgrowth of contaminating microorganism, thereby influencing the performance of the lactic acid production (Neysens and Vuyst, 2005; Hujanen and Linko, 1996). For Lactobacillus amylophilus, which is known to grow at 15
°C but not at 45 °C, the optimal temperatures were 25 °C and 35 °C for maximum productivity and yield, respectively (Yumoto and Ikeda, 1995). The results from measuring the residual starch and reducing sugar in 4 h and 8 h indicated that there was increased in starch hydrolysis and reducing sugar accumulation as the temperature increased from 22-30 °C, and a further increase from 30-40 °C resulted in a slight improvement for the saccharification in both Rhizopus oryzae 2062 and Rhizopus oryzae 36017 cultures (Huang et al., 2005). Naturally occurring lactic acid bacteria (LAB) load was found to vary between 1.97×105 cfu/g to 4×105cfu/g at10˚C. The yeast and mold counts decrease from 1.04 ×105cfu/g to 0cfu/gr at 10˚C. Lactic acid bacteria load was found to vary between1.97×105cfu/g to 4.3×105cfu/g at 20˚C. The yeast and mold counts decrease from 1.04 ×105cfu/g to 3×104 cfu/g at 20˚C and salt content 0.5%. Lactic acid bacteria load was found to vary between1.97×105cfu/g to 1.1×106cfu/g at 37˚C. The yeast and mold counts decrease from 1.04 ×105cfu/g to 3×104 cfu/g at 37˚C and salt content 0.5%. The largest increase in the numbers of LAB was noted during the first 24 h of fermentation and further incubation led to decrease (Tabatabaei-Yazdi et al., 2013). 2.3.3.2 Incubation temperature An increase in lactose utilization and subsequent lactic acid production was found up to 36 h of incubation and thereafter no improvement in both the functions was observed (Panesar et al., 2010). This could be attributed to the growth of the culture reached to the stationary phase and as a consequence of metabolism, microorganisms continuously change the characteristics of the medium and the environment. The incubation period of 48 h has been generally used for lactic acid production using different lactobacilli cultures (Gandhi et al., 2000). In addition, different optimal conditions reported by various workers for maximum lactic acid production could be explained by the differences in the nature of the strains and medium composition used in their studies.
2.3.3.3 pH The fermentation pH is either set at the beginning and then left to decrease due to acid production or it is controlled by an addition of alkaline solutions. The optimal pH for
lactic acid production varies between 5.0 and 7.0. A pH below 5.7 was optimal for Lactobacillus strains, which are known to tolerate lower pH than lactococci. The optimum pH for cell growth of Enterococcus faecalis RKY1 was seen to be 8.0, the lactic acid fermentation at pH 7.0 was completed faster than that at pH 8.0. The cell growth at pH 5.0 almost ceased after 10 h of fermentation (Wee et al., 2004). At initial pH 6.5, cell started to utilize glucose earlier and at a faster rate than at other initial pH. Maximum lactic acid concentration was attained at initial pH 6.5. Further increase in initial pH beyond 6.5 does not improve the lactic acid production (Idris and Suzana, 2006). It is possible that the higher initial pH brought too much stress on the microorganism metabolic abilities (Vijayakumar et al., 2008). 2.3.3.4 Agitation Different lactic acid bacterial strains differed in their requirement for growth conditions. The maximum lactic acid concentrations from Lactobacillus rhamnosus strain, could be achieved when fermentation was carried out at pH 6, temperature of 40°C and agitation speed of 150 rpm, in accordance with a previous report (Hofvendahl and Hagerdal, 2000) the optimal condition for lactic acid is pH 5.0-6.8, temperature 30- 45°C with continuously agitating at 100-200 rpm (Timbuntam et al., 2008).
2.4
MEDIA FORMULATIONS AND NUTRIENT REQUIREMENT OF S. THERMOPHILUS Lactic Acid Bacteria (LAB) are characterized by fastidious nutritional
requirements leading to an important biosynthesis deficiency (Djeghri-Hocine et al., 2010; Cogan et al., 1997; Loubiere et al., 1996; Monnet and Grippon, 1994). LAB growth required complex and rich media, containing complex nitrogen sources (peptides), carbon sources, vitamins and minerals (to supply for trace elements) (Amouzou et al.,1985; Juillard et al., 1995). These nutrients should be supplied at optimal concentrations (Benthin and Villadsen, 1996; Cocaign-Bousquet et al., 1995; Desmazeaud, 1994). Several specific and selective culture media are available for the isolation and selection of LAB (De Mann et al., 1960; Rogosa et al., 1951; Talwalkar and
Kailasapathy, 2004; Terzaghi and Sandine, 1975; Vinderola and Reinheimer, 1999; Chamba et al., 1994). The variability in the nutritional requirements varied significantly with the strain used (Chamba et al., 1994). Consequently, a selective media cannot allow the numeration of all LAB genera and species. The available selective media are based on the tolerance of LAB to acidity, to inhibitory compounds or the replacement of glucose by other sugars (Coeuret et al., 2003; Dave and Shah, 1996; Roy, 2001). M17 media is found to be critical for the growth of Streptococcus thermophilus. Therefore, a cheaper media is necessary and the development of economical medium requires selection of carbon, nitrogen, phosphorous, potassium and trace element sources (Naveena et al., 2005). Prebiotics are non-digestible carbon sources that stimulate the growth of beneficial microbial populations (Pinheiro et al., 2012; Mishra and Mishra, 2013), and the growth rates of Streptococcus strains increased when an amino acid mixture was added. Carbon sources and amino acids have a huge influence on viable bacteria of S. thermophilus. Xylooligosaccharides, lactose, isomaltooligosaccharide, glutamate, lysine and valine out of selected substances affect the growth of S. thermophilus
significantly.
In
addition,
xylooligosaccharides,
lactose,
isomaltooligosaccharide, glutamate and lysine showed positive effect, but valine had negative trend. Whatever, these positive effectors can be used in the cultivation of S. thermophilus and are also conducive to the accumulation of bacterial cell. In addition, the negative trend of valine for growth of S. thermophilus suggested that this kind of amino acid must be limited in the medium (Chen et al., 2013). A culture medium for LAB, based on deproteinated whey supplemented with yeast autolysis and de-lapidated egg yolk was developed (Djeghri-Hocine et al., 2007). Egg yolk, an economically attractive local resource (Algeria), contains about 160 g kg-1 proteins, mainly lipoproteins along with high number of vitamins and was found to be efficient for LAB supplementation (Djeghri-Hocine et al., 2007). The protein efficiency ratio, namely the ratio of body weight gain to net protein intake, was higher than that of milk casein for rat nutrition (Sakanaka et al., 2000). Pure cultures of some LAB strains were considered to evaluate its potential for LAB growth.
Deproteinated whey supplemented with yeast autolysate and de-lipidated egg yolk showed an interesting potential for growth of the bacterial flora of the various tested dairy products (Djeghri-Hocine et al., 2007). The addition of sodium azide, to inhibit the contamination flora, including Gram negative flora, as well as purple bromocresol allowed a direct selection of the Gram + and lactose + flora, characteristics of the majority of the LAB species. An acidic pH (5.0) appeared is also helpful, but should be considered after protein hydrolysis to avoid protein denaturation (or flocculation) and to favor the release of amino acids and especially small peptides, essential metabolites for LAB growth. 2.5
STARTER CULTURE AND ITS ROLE IN DAIRY INDUSTRY Starter
cultures
are
preparations
to
assist
the
beginning
of
the fermentation process in preparation of various foods and fermented drinks. A starter culture is a microbiological culture which actually performs fermentation. These starters usually consist of a cultivation medium, such as grains, seeds, or nutrient liquids that have been well colonized by the microorganisms used for the fermentation. Dairy starter cultures are carefully selected microorganisms, which are deliberately added to milk to initiate and carry out desired fermentation under controlled conditions in the production of fermented milk products. Most of them belong to lactic acid bacteria (Lactococcus, Lactobacillus, Streptococcus and Leuconostocs). The taxanomy of the wide range of starters is explained in Table 2.11. In some cases, few non-lactic starters (bacteria, yeast and mold) are also used along with lactic acid bacteria during manufacturing of specific fermented milk products, such as kefir, kumiss and mold ripened cheeses. The preservation of food by fermentation is one of the oldest methods known to mankind. A typical example is lactic acid fermentation, which is widely used for the preparation of several fermented milk products, such as dahi (curd), yoghurt, acidophilus milk, shrikhand and various varieties of cheeses. In the modern dairy industry, dairy starter cultures are prerequisite for the production of safe products of uniform quality. Lactic acid bacteria are often called dairy starter cultures, which are used for the production of various fermented milk products. There are 13 major starter producing industries in the world covering the whole dairy sector across the globe for production of fermented food products (Table 2.12)
Dairy starter cultures are microorganisms that are intentionally added to milk in order to create a desired outcome in the final product, most often through their growth and “fermentation” processes. The most common use of starter cultures is for the production of lactic acid from lactose (milk sugar), which in most cases causes or assists in the coagulation of milk protein by lowering its pH value. Cultures that produce lactic acid are generally referred to as “lactic acid bacteria” (LAB). Certain starter organisms are added specifically for their ability to produce flavor compounds such as diacetyl, although lactic acid and other culture created compounds contribute to flavor as well. Starter organisms can also influence flavor and texture of cultured and/or aged products through the breakdown of proteins, fats and other milk constituents in addition to the pH effect. The lower pH of cultured products can be inhibitory to certain spoilage organisms, although inhibition is also associated with other by-products of growth with some starters. More recently, probiotic cultures are finding their way into cultured milk products. These are organisms that have some claimed health benefit for those that consume them, e.g., better digestion, anti-cancer compounds, and prevention of heart disease. Probiotic cultures may be added as adjuncts or they may be directly involved in the fermentation process. Slow acid development by cultures can result in an inferior product or the loss of a vat full of milk. Starter activity can be influenced by a number of factors including the age of the culture, handling and storage practices, incubation temperature, the quality of the raw milk, bacteriophage, and the presence of inhibitors such as drugs or sanitizers. Penicillin and related antibiotics can inhibit cultures at levels as low as 1-2 parts per billion. Sanitizers can cause inhibition, especially those that leave residues, such as quaternary ammonia compounds. Natural inhibitors associated with high somatic cells and late lactation can also slow growth.
Table: 2.11
Taxonomy of dairy Starter Cultures with old and new names and their products
OLD NAME
NEW NAME
Mesophilic Starter Streptococcus lactis Streptococcus cremoris
Lactococcus sub-sp. lactis
Streptococcus diacetylactis
Lactococcus lactis Flavor & Acid sub-sp. lactis biovar diacetylactis Leuconostoc Flavor mesenteroides subsp. cremoris
Leuconostoc cremoris
MAJOR FUNCTION lactis Acid Production
Lactococcus lactis Acid Production sub-sp. cremoris
Leuconostoc lactis
Unchanged
Flavor
Thermophilic Starter Streptococcus thermophilus Lactobacillus bulgaricus
Unchanged
Acid (& Flavor)
Lactobacillus lactis Lactobacillus helveticus
Lactobacillus Acid & Flavor delbrueckii sub-sp. bulgaricus Lactobacillus Acid & Flavor delbrueckii sub-sp. lactis Unchanged Acid & Flavor
PRODUCT USE Buttermilk, sour cream, many types of cheese Buttermilk, sour cream, many types of cheese Sour cream, ripened butter,cheese, buttermilk Buttermilk, sour cream cottage cheese, ripened butter Buttermilk, sour cream cottage cheese, ripened butter Yogurts, fermented milks, Italian cheese, emmenta Yogurts, fermented milks, Italian cheese, emmental Yogurts, fermented milks, Italian cheese, emmental Yogurts, fermented milks, Italian cheese, emmental
Table: 2.12
Major starter culture producer companies all over the world
S.N.
Company
Country
1
Alce
Italy
2
ASCRC
Australia
3
Centro Sperimentale del Latte
Italy
4
Chr. Hansen
Denmark
5
CSK
The Nederlands
6
Danisco
Denmark
7
Degussa
Germany
8
DSM
The Nederlands
9
Gewu¨rzmu¨ller
Germany
10
Lallemand
Canada
11
NZDRI
New Zealand
12
Quest International
13
2.5.1
Rhodia
The Nederlands France
Functions of Starter Culture
Starter cultures can be used as single strain, mixed strain and multiple strains depending upon the type of products to be prepared. The ability of starter culture to perform its functions efficiently during manufacture of fermented dairy foods depends primarily on purity and activity of starter cultures. The major roles of starter culture during fermentation of milk are:
a) Production of primarily lactic acid and few other organic acids, such as formic acid and acetic acid. b) Coagulation of milk and changes in body and texture in final products. c) Production of flavouring compounds, e.g., diacetyl, acetoin and acetaldehyde. d) Help in ripening of cheeses by their enzymatic activities. e) Produce antibacterial substances in the finished product. f) In addition, they may possess functional properties. Thus, an ideal starter culture should be selected for the preparation of various fermented milks with the following characteristics. •
It should be quick and steady in acid production.
•
It should produce product with fine and clean lactic flavour.
•
It should not produce any pigments, gas, off-flavour and bitterness in the finished products.
• 2.5.2
Should be associative in nature in product development. Types of Starter Culture There are two major groups of starter cultures which are used in the preparation of
fermented milk products classified on the basis of their physiological, biochemical and growth characteristics: 2.5.2.1 Mesophillic starter culture These cultures have optimum temperature for growth between 20 to 30°C and include Lactococcus and Leuconostoc. These mesophillic lactic cultures are used in the production of many cheese varieties where important characteristics are: •
Acid producing activity
•
Gas production, and
•
Production of enzymatic activity for cheese ripening, e.g., proteases and peptidases enzymes.
2.5.2.2 Thermohillic starter culture These cultures have optimum temperature for growth between 37 to 45°C. Thermophilic cultures are generally employed in the production of yoghurt, acidophilus milk, swiss type cheese. Thermophilic cultures include species of Streptococcus and Lactobacillus. These cultures grow in association with milk and form the typical yoghurt starter culture. This growth is considered symbiotic because the rate of acid development is greater when two bacteria are grown together as compared to single strains. Thermophilic starter cultures are microaerophillic and fresh heated milk should be used to achieve a better growth of the culture since heat treatment reduces amount of oxygen in the product. The important metabolic activities of thermophilic cultures in development of fermented milk products are: •
Acid production, e.g. lactic acid
•
Flavour compounds, e.g., acetaldehyde
•
Ropiness and consistency, e.g., polysaccharides
•
Proteolytic and lipolytic activities, e.g., peptides, amino acids, fatty acids
•
Possesses therapeutic significance, such as Improvement of intestinal organisms, Produce antibacterial substances, and immunity improvement.
2.5.2.3 Homo-fermentative lactic starter
These lactic acid bacteria are characterized for their ability to ferment lactose almost exclusively to lactic acid while pentoses and gluconate are not fermented. The examples of these cultures are: L. acidophilus, L. bulgaricus.
2.5.2.4 Hetero-fermentative lactic starter
Main characteristics of these bacteria are the ability to ferment hexoses and pentoses to lactic acid, acetic acid, alcohol and CO . The examples of these cultures are 2
Lb. brevis, Lb. fermentum.
2.5.3
Fermented Milk Products Fermented milks are sour milk products prepared from milk, whole, partially or
fully skimmed concentrated milk or milk substituted from partially or fully skimmed dried milk, homogenized or pasteurized or sterilized and fermented by means of specific dairy starter cultures. The origin of cultured dairy product is obscure and it is difficult to be precise about the date when they were first made. In the early part of the century, Metchnikoff (1845-1916) claimed that owing to lactic acid and other products present in sour milks, fermented by lactic acid bacteria, the growth and toxicity of anaerobic, sporeforming bacteria in the large intestine are inhibited. Lactic acid is biologically active and capable of suppressing harmful microorganisms, especially putrefactive ones and so has a favorable effect on human vital activities. Metchnikoff’s theory of longevity considerably influenced the spread of fermented milk products to many countries, particularly in Europe. He also promoted extensive studies concerning biochemical and physiological properties of fermented milks. Milk fermentation for processing of milk into fermented milk products for increasing the shelf-life and having different flavour and texture characteristics have been practiced in different parts of the world. Milk has been processed into cheese, yoghurt, acidophilus milk, kefir, dahi, kumiss and various other fermented products. In the preparation of various fermented milk products, lactic starters occupy the key position as the success or failure of such products is directly related to the types of starter used. Spoilage of fermented milk products on storage also takes place due to non-lactic contaminants, such as sporeformers, micrococci, coliform, yeast and molds. These undesirable organisms rapidly increase in number when the starters are weak and the ratio of non-lactic to lactic organisms is high. Containers having a large surface of air in contact with the fermented milk accelerate the process of spoilage. Fermented milk products are generally spoiled by yeasts and molds and also by lactic acid bacteria which may cause sour, bitter and cheesy flavor. From dietary point of view, sour milk products, such as yoghurt, dahi, acidophilus milk, kumiss and other fermented milks are far more valuable than milk. During fermentation of milk, the composition of the minerals remains unchanged, while those of proteins, carbohydrates, and vitamins and to some extent fat
constituents change which produce special physiological effects. Dietary and therapeutic qualities of sour milk products are determined by microorganisms and substances formed as a result of biochemical process accompanying milk souring. These substances are lactic acid, alcohol, carbon dioxide, antibiotics and vitamins. Following biochemical processes make fermented milk products more nutritive than milk: •
Milk Proteolysis Proteolysis in milk takes place by exo- or endo-peptides of lactic acid bacteria. The biological value of protein increases significantly from a value of 85.4 to 90 per cent. This increase is due to breakdown of protein into peptones, peptides and amino acids. The contents of essential amino acids such as leucine, isoleucine, methionine, phenylalanine, tyrosine, threonine, tryptophane and valine increase considerably to offer special advantages not only to healthy people but also particularly to the physically weak persons. Fermented milks (yoghurt, kefir, dahi) are having higher protein digestibility due to precipitating into fine curd particle by lactic acid that contributes to its higher nutritional value and capacity to regenerate liver tissue. During fermentation and storage, the amount of free amino acids increases, particularly lysine, proline, cystine, isoleucine, phenylalanine, and arginine. Due to these biochemical changes in milk protein during fermentation make these products dietetic in nature.
•
Lactose Hydrolysis Lactose in milk is hydrolysed by metabolic activity of bacteria. Approximately 4550% lactose; 16–20% galactose and 0.6-0.8% glucose are obtained from lactose hydrolysis on the basis of on average 5% lactose in milk. Lactose hydrolysis takes place due to β -galactosidase production by lactic acid bacteria. The importance of lactose is due to the lactic acid produced from the hydrolysis of lactose leading to a pH range in the bowel inhibiting the growth of putrefactants. In addition to this, lactic acid is important for organoleptic properties and calcium absorption.
•
Lipolysis The homogenization process reduces the size of fat globules, which become digestible. The production of free fatty acids as a consequence of lipolytic activity
increases due to lactic acid bacteria as compared to milk. This leads to some physiological effects. •
Changes in vitamins There is more than two fold increase in vitamins of B-group especially thiamine (B ), 1
riboflavin (B ) and nicotinamide as a result of biosynthetic process during milk 2
fermentation. Subsequently, vitamin B ascorbic acid and vitamin B decrease by 2
1
approximately one half as they are utilized by the bacteria present in milk. However, the increase or decrease in vitamin content depends on the type of culture. •
Antibacterial activities The bactericidal properties of fermented milk products are determined by antibiotic activity of bacteria growing in the product. The antibiotic properties are generally associated with lactobacilli in yoghurt and materials responsible for such antibacterial actions are described as lactic acid, hydrogen peroxide and other substances such as antibiotics and bacteriocins.
•
Changes in Minerals Infact there is not any significant changes in minerals in milk after or during fermentation process by lactic acid bacteria and the nutritional values of fermented milk products remain intact.
2.5.4
Bacteriophage Bacteriophages (phage) are viruses that attack and destroy bacteria. They are very
small and cannot be seen with an ordinary microscope. Phage requires a host cell to reproduce; one phage per bacterial infection can result in up to 200 phage being released, each of which can infect a new bacterial cell. Phages are very strain specific, which is why culture rotation and resistance are used as control mechanisms. Phage can enter the dairy plant through the raw milk supply although some culture strains are “carriers.” Problems with “dead vats” due to phage can often be linked to phage in the plant
environment (poor plant hygiene, residual culture). Stringent culture handling and plant sanitation programs are essential in preventing phage problems. 2.5.5
Fermented milk products in India
2.5.5.1 Yogurt Yogurt (also spelled jugurt or yoghurt) is a semisolid fermented milk product, which originated centuries ago in Bulgaria. Its popularity has grown and is now consumed in almost all parts of the world. Although the consistency, flavour and aroma may vary from one region to another, the basic ingredients and manufacturing processes are consistent. Yogurt is strictly defined as a milk product produced by the action of two bacteria – Streptococcus thermophilus and Lactobacillus delbrueckii sub sp. bulgaricus. In addition, yogurt may contain bifidobacteria and supplementary flora like Lactobacillus acidophilus for improving its therapeutic significance. Although milk of various animals has been used for yogurt production in various parts of the world, most of the industrialized yogurt production uses cow’s milk, whole milk, partially skimmed milk, skim milk or cream. Good quality milk is clarified and then standardized to achieve the desired fat content. The various ingredients are then blended together in a mix tank equipped with a powder funnel and an agitation system. The mixture is then pasteurized for 30 min at 85°C or 10 min at 95°C. These heat treatments, which are much more severe than fluid milk pasteurization, are necessary to: • Produce a relatively sterile and conducive environment for the starter culture • Denature and coagulate whey proteins to enhance the viscosity and texture The mix is homogenized using pressures of 2000 to 2500 psi before final heat treatment. Besides thoroughly mixing the ingredients, homogenization also prevents creaming and wheying off during incubation and storage (Figure 2.10). Stability, consistency, body and texture are enhanced by homogenization. After the final heat treatment, the mix is cooled to an optimum growth temperature and inoculated with the yoghurt starter culture. A ratio of 1:1 of Streptococcus thermophilus and Lb. bulgaricus inoculation is added to the jacketed fermentation tank. A temperature of 42°C is maintained for about 4 h without agitation, till the milk sets. This temperature is suitable for the two microorganisms. The titratable acidity is carefully monitored until the titrable acidity is 0.85 to 0.90 per cent.
At this time, chilled water is circulated in the jacket and agitation begins, both of which slow down the fermentation. The coagulated product is cooled to 5-22°C, depending on the product. Fruit and flavour may be incorporated at this time, and then packaged. The product is now cooled and stored at refrigeration temperatures (5°C) to slow down the physical, chemical and microbiological degradation. There are two types of plain yogurt: stirred yogurt and set yogurt. In set style, the yogurt is packaged immediately after inoculation with the starter and is incubated in the packages. Other yogurt products include fruit and flavored yoghurt, frozen yoghurt and liquid yoghurt.
Figure 2.10: Flow diagram of the main steps involved in the production of Stirred yogurt
2.5.5.2 Acidophilus Milk Acidophilus milks are sour milk products in which milk is allowed to ferment under conditions that favour the growth and development of larger number of Lactobacillus acidophilus alone or in combination with other lactic acid bacteria or lactose fermenting yeasts. •
Types of acidophilus milks Organisms
Acidophilus sour milk
Lb. acidophilus
Acidophilus yoghurt
Lb. acidophilus + S. thermophilus
Bioghurt
Lb. acidophilus, L. bulgaricus, S. thermophilus
Acidophilus yeast milk
Lb. acidophilus + Lactose fermenting yeast
Acidophilin
Lb. acidophilus, Lc. lactis, Kefir fungi
•
Acidophilus concentrates
Acidophilus paste
Lb. acidophilus
Dried acidophilus
Lb. acidophilus
Lyophilized form of milk
Lb. acidophilus
Therapeutic importance of acidophilus milk products •
Possesses significant nutritional and prophylactic properties.
•
Control gastrointestinal disorders, such as diarrhoea, constipation, dyspepsia, flatulence and colitis.
•
Acidophilus yeast milk, which is rich in alcohol and CO2, excite respiratory and central nervous system.
•
Induction of L. acidophilus into the intestine return to normalcy in the intestinal microflora and body comforts.
•
Possible lowering of blood cholesterol.
•
Possible improvement in immune status.
•
Lower proliferation of cancerous cells. Preparation of acidophilus milk The milk for this product can be skimmed from full cream milk but because L.
acidophilus does not grow well in milk and would be easily overgrown by usual
microflora, the base milk has to be virtually sterile when the culture is added. The milk is then left to incubate at 37°C for 12-16 h or till the acidity of the product reaches around 0.8 to 0.9 per cent (as lactic acid). Consequently, the optimum acidity is achieved by cooling the milk to 5°C or less and halting any further activity by the culture (Figure 2.11). The culture could generate up to 1.0 to 2.0 per cent lactic acid, but the impact of such levels on cell viability over 2-3 weeks can be devasting in a low solid product. After cooling, the acidophilus milk is bottled and consumed under chilled conditions. Acidophilus milk has shelf life of two weeks under refrigeration.
Figure: 2.11 Flow sheet for the preparation of acidophilus milk
2.5.5.3 Curd Dahi or curd is an Indian fermented milk product which is equally known for its palatability, refreshing taste and therapeutic importance as claimed in the ayurvedic literature. Some of its characteristics are similar to other fermented milk products such as yoghurt and acidophilus milk but it differs with regard to heat treatment of milk, starter culture, chemical composition and taste. In addition, dahi also has antibacterial properties against pathogenic and non-pathogenic organisms. Types of Curd Some of the fermented milks and different types of dahi consumed throughout India have been categorized as follows: •
North Zone
:
Dahi, Lassi
•
South Zone
:
Dahi, Buttermilk (Mattha)
•
East Zone
:
Payodhi or Lal dahi or Mishti dahi
•
West Zone
:
Shrikhand, Chakka, Chhash, Dahi
Based on the acidity level (% lactic acid), dahi has been classified into categories such as sweet dahi with a maximum acidity of 0.7 per cent and sour dahi with 1.0 per cent acidity. Starter culture used in the preparation of dahi is normally dahi left over from previous day. The composition of microflora varies from one household to another and from one place to another. In general, it has been found that dahi culture is dominated by streptococci and lactobacilli. In sour dahi, however, lactobacilli predominate. For commercial manufacture by organized dairy, single starter culture (Lactococcus lactis subsp. diacetylactis) or mixed culture is used. The raw materials used are cow and/or buffalo milk, standardized milk, skim milk and reconstituted skim milk powder. The traditional method for preparation of dahi invariably involves a small scale, either in consumers’ household or in the sweet makers shop in urban areas. In the household, milk is boiled, cooled to about 37°C and inoculated with 0.5 – 1 per cent of starter (previous day’s dahi or butter milk) and allowed to set overnight (Figure 2.12). It is then stored under refrigeration and consumed
Figure: 2.12 Flow sheet for the preparation of Curd
According to Bureau of Indian Standards (1978), specifications for fermented milk, dahi, should have a pleasing flavour and a clean acid taste, devoid of undesirable flavour, should have firm, solid body and texture and be uniform with negligible whey separation. Some important characteristics have been defined in Table 2.13.
Table 2.13: Characteristics of sweet and sour Dahi Characteristics
Sweet dahi
Sour dahi
Acidity (% lactic acid)
0.7
1.0
Yeast and molds (per gram) Max.
100
100
Coliforms (per gram) Max.
10
10
Negative
Negative
Phosphatase test
A good quality dahi made from whole milk has a cream layer on the top, the rest being made up of a homogenous body of curd and the surface being smooth and glossy, while the cut surface should be firm and free from cracks of gas bubbles and it should have a pleasant acid taste with sweetish aroma. Composition and quality of dahi vary widely from one locality to another as it is being prepared under different domestic conditions as well as milk, with variable chemical and bacteriological quality used for the preparation. However, the chemical composition of dahi has been reported as fat ranging from 5 to 8 per cent, protein 3.3 to 3.4 per cent, ash 0.75 to 0.79 per cent and lactic acid 0.5 to 1.1 per cent. Quality of dahi can be improved with regard to increase in riboflavin and folic acid by incorporating propionic acid bacteria such as Propionibacterium shermani along with dahi starter culture. Regarding palatability and therapeutic importance of dahi, it has been known to create relish for food, promote the appetite, increases strength and leads to longevity. 2.5.5.4 Cultured Butter Milk Buttermilk is really the liquid left from butter making. However, cultured buttermilk is a fermented milk product made from pasteurized skim milk low fat milk in which mesophilic lactic acid bacteria is added as starter. •
Starter culture for cultured buttermilk
Starter cultures are typically mixtures of flavour and acid producers Leuconostoc sp. and Lactococcus lactis sub sp. diacetylactis produces diacetyl, the flavour most commonly associated with flavored butter and Lactococcus lactis is used to produce
lactic acid which contributes to the acidic flavour typically associated with cultured butter milk. •
Preparation of cultured butter milk
The starting ingredient for buttermilk is skim or low-fat milk. The milk is pasteurized at 82° to 88°C for 10 - 30 minutes. This heating process is done to destroy all naturally occurring bacteria and to denature the protein in order to minimize wheying off (separation of liquid from solids). The milk is then cooled to 22°C and starter cultures of desirable bacteria, such as Lactococcus lactis, L. cremoris, L. citrovorum and L. dextranicum are added to develop buttermilk’s acidity and unique flavour. These organisms are used in proper combination to obtain the desired flavour. The ripening process takes about 12 to 14 hours (overnight). At the correct stage of acid and flavour, the product is gently stirred to break the curd, and it is cooled to 7.2°C (45°F) in order to stop fermentation. It is then packaged and stored under refrigeration. 2.5.5.5 Cheese A dairy product prepared from cow, buffalo, goat or sheep’s milk that is set aside to thicken until it separates into liquid, called whey, and semisolids, called curd. The whey is drained off and the curd is formed into the shape as per specification of cheese. It is packaged immediately; making it a fresh cheese like Ricotta cheese or cottage cheese, or it is aged using various curing methods. Types of cheeses There are 400 varieties of cheeses, of which 18 are distinct. Important varieties of cheeses are given below: •
Cheddar
Cheddar is a hard variety with about 40% moisture and has a diverse selection of tastes that range from mild to sharp. This is dependent upon the age of the cheese. Mild Cheddar is perfect for sandwiches because it has a mellow balance of flavors. Sharp Cheddar is good for cooking because its flavour is released when heated and it shreds well with other cheeses. •
Mozzarella
Mozzarella has a mild, milky taste and is more of a cooking cheese due to its good binding properties, moist texture and ability to melt. It is a “stretched-cured” cheese meaning that during the manufacturing process the curd is pulled, kneaded and shaped while it is still pliable. Therefore, it absorbs the flavors and juices of the ingredients surrounding it and is perfectly designed for cooking. Mozzarella is also low in fat; therefore, it is ideal to use even when dieting. Mozzarella is an ideal cheese for Pizza making. •
Swiss
Swiss cheese, which is also known as Emmental or Schweizer, is a firm cheese with a sweet, mildly nutty flavour. This cheese is known for the holes or eye formation that develops as it ripens. These holes or eyes range in diameter from ½ inch to 1 inch and begin forming when the cheese is about 3 weeks old. •
Camembert
Camembert has a soft texture with a buttery taste and mushroom smell. It tastes best when it is at room temperature and the center becomes soft and it is a mold-ripened cheese. •
Processed Cheeses
It is prepared by melting one or more pressed cooked or uncooked cheeses, and adding milk, cream, butter and sometimes flavouring agents. One or several ripened cheeses are heated and mixed, then pasteurized at high temperature (130-140°C) after other dairy products, such as liquid or powdered milk, cream, butter, casein, whey, and seasoning have been added.
Chapter 3 Material & Methods
3.1
CHEMICAL AGENTS, NUTRIENTS AND CULTURES. All chemicals used for chemical reaction were of analytical grades and media
ingredients like yeast extract, casein enzyme hydrolysate, casein peptone, soya peptone, skim milk powder, beef extract, tryptone, tryptose, protease peptone were of extra pure grade purchased from Himedia, India. Other chemicals like sodium acetate, triammonium citrate, di-potassium hydrogen sulphate, magnesium sulphate, manganous sulphate, di-sodium glycerol phosphate, ascorbic acid, sucrose, lactose, maltose, dextrose, arabinose, malto dextrin, sodium chloride, potassium chloride, sodium hydroxide, hydrochloric acid, sulphuric acid were obtained from Fisher Scientific, India; Himedia, India; Rankem, India; Fluka, UK and Merck, Germany. Some pre-prepared agar medium were also obtained from Himedia, India like Streptococcus thermophilus isolation agar (M948), M-16 (M600), Lactobacillus selection agar (M1180), Lactobacillus bulgaricus agar (M927), Lactobacillus MRS agar (M369), M-17 agar (M929), Brain Heart infusion agar (M211A), Tomato Juice agar (M048) and L.S differential media (M582). Reference bacterial strains were purchased from American Type Culture Collection (ATCC), three lactic acid bacterial strains for reference were purchased from Microbial Type Culture Collection and Gene Bank (MTCC) Chandigarh, India and five reference strains were obtained from National Collection of Industrial Microrganism, Pune, India.
Lactococcus lactis (ATCC 19435)
Lactobacillus acidophilus (ATCC 4356)
Streptococcus lactis (MTCC 460)
Lactobacillus acidophilus (MTCC 10307)
Lactococcus lactis (MTCC 440; ATCC 11454)
Streptococcus thermophilus (NCIM 2904)
Streptococcus thermophilus (NCIM 2412)
Lactobacillus acidophilus (NCIM 2903)
3.2
Lactobacillus bulgaricus (NCIM 2057)
Lactobacillus lactis (NCIM 2368)
SCREENING AND IDENTIFICATION OF SELECTED ISOLATES OF LACTIC ACID BACTERIA 3.2.1 Sample Collection
Lactic acid bacteria (LAB) in this study were isolated from different sources which includes cow milk, buffalo milk, goat milk, sheep milk, camel milk, Indian traditional curd and grapes collected from the surrounding area of Gwalior district of Madhya Pradesh, India. The sources of cow, buffalo and goat milk were urban dairy farms while as sheep and camel milk samples were obtained from local milk suppliers. Curd samples were collected from both dairy farms and local curd suppliers. Grape samples were randomly picked from different fruit markets and grape farms in the city. Grape sample was taken aseptically and packaged into clean bag, then stored at 4°C during their transport. These fruit samples were analyzed within 24 hours of acquisition at the grape farms. On the other hand raw milk samples were collected in sterile tubes and maintained in chilled condition (8-10o C) during their transport to Microbiology Biotechnology Laboratory, Tropilite Foods Pvt. Ltd. Gwalior, for further analysis and research. 3.2.2 Selective Enumeration and isolation methods
Serial dilutions of all raw milk samples in 0.1% peptone saline were used for microbial isolation separately. All the milk samples were serially diluted one by one and were pour plated on MRS (DE MAN, ROGOSA and SHARPE), M17 agar, Streptococcus thermophilus isolation agar and Lactobacillus selection agar. Plates were incubated for 24-48 hours at 15, 32, 37 and 45°C in both anaerobic and aerobic conditions. Same protocol was followed every time for different raw milk samples of cow, buffalo, goat, sheep and camel (Sharma et al., 2013). In case of grapes, 10 g of grape sample was homogenized with 90 mL of peptone water (mother solution), 1 mL of
mother solution was transferred into 9 mL of slain solution (8.5 g NaCl, 1000 mL distilled water, pH 7.0) and serial dilutions up to 10 were made. Then, 1 mL form each dilution was cultivated in the following selective media: M17 (Himedia, India) and Streptococcus thermophilus isolation agar to count Streptococcus, incubation at 45°C/48h (Terzaghi and Sandine, 1975), MRS (Himedia, India) to count Lactobacillus and Pediococcus, incubation at 30°C/48h (De Man et al., 1960) And Elliker (Himedia, India) to count Lactococcus, incubation at 30°C/48h (Elliker et al., 1956). Randomly picked colonies were transferred to suitable media and purification of colonies was made by repeated streaking on suitable media. Randomly picked colonies were transferred to suitable media and purification of colonies was made by repeated streaking on suitable media (Patil et al., 2010). The colonies were randomly (different colony types) picked from plates with 30-300 colonies. Several representative strains displaying the general characteristics of lactic acid bacteria were chosen from each plate for further studies. Each of the isolates were repeatedly streaked in order to purify the isolates, which were maintained on MRS agar slants for immediate use and in 15% glycerol for storage at -20 °C. Strains of LAB were identified according to their microscopical, morphological, physiological and biochemical properties (Samelis et al., 1994; Bisen 2014). 3.2.3 Morphological Characterization
Cell morphology was observed by Gram’s staining under bright field microscopy (CX 21i, Olympus). Selected isolate was heat fixed on a glass slide followed by flooding the sample with crystal violet for 30 seconds. Crystal violet dissociates in aqueous solution CV+ and chloride (Cl–) ions. These ions penetrate through the cell wall and cell membrane of both gram-positive and gram-negative cells. The CV+ ion interacts with negatively charged components of bacterial cells to stain the cells purple. Gentle rinsing was required to wash the violet layer followed by flooding with iodine. Iodine, in the form of negatively charged ions, interacts with CV+ to form large complexes of crystal violet and iodine (CV–I complexes) within the inner and outer layers of the cell. Iodine acts as a trapping agent to retain the purple crystal violet color in the cell. This step was followed by decolourization in which the decolorizing agent will remove the crystal violet stain from both gram-positive and negative cells. Counter stain is
applied last to stain the decolorised gram-negative bacteria a pink or red shade. Basic fuchsin, which stains anaerobic bacteria more intensely, may be substituted for safranin, but it is less commonly used. Gram-positive bacteria were stained dark blue or violet by crystal violet and safranin. Gram positive bacteria stain violet due to the presence of a thick layer of peptidoglycan in their cell walls, which retains the crystal violet. Catalase activity was initially tested by placing a drop of 3% hydrogen peroxide solution on the cells (Guyot et al., 1998). Immediate formation of bubbles indicated the presence of catalase in the cells. Catalase test differentiates between two or more similar looking unknown coccus bacteria. Streptococcus bacteria can be differentiated from staphylococcus, planococcus and micrococcus as streptococcus are always catalase negative while as the other three mentioned are catalase positive. A catalase positive will produce bubbles of oxygen within one minute after addition of hydrogen peroxide, while as no bubble release will determines catalase negative. For gas production determination, bacterial cells were added in MRS test tubes containing the Duram tube (Nomura et al., 2006) to determine whether the isolate strains produces carbon dioxide during fermentation (CO2 from glucose or gluconate). The identification procedures were determined according to the criteria established in Bergey’s Manual of Determinative Bacteriology. An isolate was deemed to be a homo-fermentative lactic acid producer if no gas was produced (no CO2 from glucose or gluconate). Plates were incubated at 30 °C for 24-48 h (Holt et al., 1994). All tests were performed in duplicate. 3.2.4 Biochemical Characterization
The previous procedure was repeated in order to purify the isolates, and to be able to select the high ability in term of growth and good characterization. According to these tests, the isolates were selected as potential lactic acid bacteria. Biochemical characterization of the selected strains was carried out with following biochemical tests: 3.2.4.1 MR-VP tests (Methyl red and Voges-Proskauer test) MR-VP test differentiates bacteria upon the fermentative end product they produce. Some bacterial strains produce large amount of acids and some produce a neutral acetoin as the end product. MR-VP are basically two different tests but the reason
behind performing the two tests together is the results they produce as if methyl red tests gives positive results then Voges-Proskauer must be negative of the same and vice-versa. Material requirements: • • • •
MRVP broth tubes (Peptone 7g, Dextrose 5g, and Potassium phosphate 5g) Methyl Red pH indicator Napthol solution 40% potassium hydroxide
MR-VP broth was prepared and selected strains were tested against the standard. Eight tubes were prepared for each bacterial strain separately. Six were inoculated and incubated at 35°C for 48 hours while as two were kept un-inoculated. On completion of incubation, five drops of methyl red were added to any three tubes with clear turbidity, while as 12 drops of napthol solution and 2-3 drops of 40% potassium hydroxide were added to the remaining three. No color change in the tubes added with methyl red indicates MR positive and color changing to yellow indicates MR negative. Ruby pink color on the tubes with napthol indicates VP positive and no color change shows VP negative. If the MR test is positive it means that the organism produces a large amount of organic acids like lactic acid, formic acid, succinic acid by fermenting glucose but a negative MR test shows that the organic acids produced by the organism faced some enzymatic conversion and get converted into non acidic products like ethanol or acetoin. On the other hand VP test also determines the presence of acetoin in the under testing broth. The organisms capable of converting pyruvate into acetoin shows VP positive (showing red colour) while as no acetoin in broth shows negative test (no colour change). 3.2.4.2 Nitrate reduction test Bacterial species can also be differentiated on the basis of their ability to reduce nitrate to nitrite or nitrogenous gases. The reduction of nitrate may be coupled to anaerobic respiration in some species. Selected isolated strains were also tested against their nitrate reducing capacity. Material requirements: • • •
Nutrient broth and KNO3 (Peptone: 5g, Beef Extract: 3g, Nacl: 5g, KNO3: 5g) Solution A (Sulfanilic acid 8 g + acetic acid 5N 1000 mL) Solution B (Alpha-naphthylamine 5 g + acetic acid 5N 1000 mL)
Inoculate nitrate broth with an isolate and incubate for 48 hours. Add 10-15 drops each of sulfanilic acid and followed by addition of Alpha-naphthylamine. If the bacterium produces nitrate reductase, the broth will turn a deep red within 5 minutes at this step. If no color change is observed, then the result is inconclusive. Add a small amount of zinc to the broth. If the solution remains colorless, then both nitrate reductase and nitrite reductase are present. If the solution turns red, nitrate reductase is not present. 3.2.4.3 Casein Hydrolysis Caseinase is an exoenzyme that is produced by some bacteria in order to degrade casein. Casein is a large protein that is responsible for the white color of milk. This test is conducted on milk agar which is a complex media containing casien, peptone and beef extract. If an organism can produce caseinase, then there will be a zone of clearing around the bacterial growth. Material requirements are: •
SMP 100 g, peptone 5 g, agar 15 g pH 7.2
•
Actively growing culture
Inoculate the organism on the plate either in a straight line or a zig-zag and incubate at 25 or 37 0C. Hold the plate up to the light to see the zones. Positive reactions may be recorded as strong + or weak + reactions. There is no reagent or indicator in the agar. A zone of clearing around the growth area identifies the presence of the enzyme caseinase
3.2.4.4 Carbohydrate Fermentation Carbohydrate fermentation tests detect the ability of microorganisms to ferment a specific carbohydrate. Fermentation patterns can be used to differentiate different bacterial groups or species (Forbes et al., 2007). During the fermentation process, an organic substrate serves as the final electron acceptor. The end-product of carbohydrate fermentation is an acid or acid with gas production (Mahon et al., 2011). The end-product depends on the organisms involved in the fermentation reaction, the substrate being fermented, the enzymes involved, and environmental factors such as pH and temperature. Common end-products of bacterial fermentation include lactic acid, formic acid, acetic acid, butyric acid, butyl alcohol, acetone, ethyl alcohol, carbon dioxide, and hydrogen (MacFaddin, 2000). Fermentation reactions are detected by the
color change of a pH indicator when acid products are formed. This is accomplished by adding a single carbohydrate to a basal medium containing a pH indicator. Because bacteria can also utilize peptones in the medium resulting in alkaline by-products, the pH changes only when excess acid is produced as a result of carbohydrate fermentation. Phenol red is commonly used as a pH indicator in carbohydrate fermentation tests because most of the end-products of carbohydrate utilization are organic acids. However, other pH indicators such as bromocresol/bromcresol purple, bromothymol/bromthymol blue, and Andrade’s can be used (Table 3.1). Table: 3.1 pH indicators for carbohydrate fermentation media pH indicator Andrade’s Bromocresol purple Bromothymol blue Phenol red
Uninoculated Acid media (fermentation) pH Color pH Color 7.1- Light pink 5.0 Pink-red 7.2 7.4 Deep purple 5.2 Yellow
pH 12.014.0 6.8
Alkaline (negative) Color Yellow, colorless Purple
7.0
Green
6.0
Yellow
7.6
Deep Prussian blue
7.4
Reddishorange
6.8
Yellow
8.4
Pink-red
When using phenol red as the pH indicator, a yellow color indicates that enough acid products have been produced by fermentation of the sugar to lower the pH to 6.8 or less. A delayed fermentation reaction may produce an orange color. In such cases, it is best to re-incubate the tube. Bubbles trapped within the Durham tube indicate the production of gas. Even a single bubble is significant and denotes evidence of gas production. No bubbles within the Durham tube indicate a non-gas-producing or anaerogenic organism. A reddish or pink color indicates a negative reaction. In negative tubes, the presence of turbidity serves as control for growth. A reddish or pink color in a clear tube could indicate a false negative.
3.2.5
Molecular Characterization Genomic DNA from nearly all prokaryotic and eukaryotic organisms is also
complexed with protein and termed chromosomal DNA. Each gene is located at a particular position along the chromosome, termed the locus, whilst the particular form of the gene is termed the allele. In mammalian DNA each gene is present in two allelic forms which may be identical (homozygous) or which may vary (heterozygous). The use of 16S rRNA gene sequences to study bacterial phylogeny and taxonomy has been by far the most common house keeping genetic marker used for a number of reasons. 3.2.5.1
16s rDNA gene sequencing 16S rDNA sequencing has played a pivotal role in the accurate identification of
bacterial isolates and the discovery of novel bacteria in clinical microbiology laboratories. For bacterial identification, 16S rDNA sequencing is particularly important in the case of bacteria with unusual phenotypic profiles, rare bacteria, slow-growing bacteria, uncultivable bacteria and culture-negative infections. The hyper variable regions of 16S rRNA gene sequences provide species-specific signature sequences useful for bacterial identification. In medical microbiology, 16S rRNA sequencing serves as a rapid and cheap alternative to phenotypic methods of bacterial identification.
The genetic interrelationships of members of the lactic acid bacteria have been studied extensively in 16S rDNA sequence, and DNA-DNA hybridization experiments. Total genomic DNA of all isolated strains was prepared by using the following procedure (Cardinal et al., 1997). DNA (214 ng/µl) was subjected to PCR utilizing the primer 1 (AGA GTT TGA TCC TGG CTC AG) and primer 2 BOX A1R (CTA CGG CAA GGC GAC GCT GAC G) as Versalovic et al described. Each 27 µl PCR reaction contained 5 µl 5× Gitschier buffer (1 M (NH4)2SO4, 1 M Tris-HCl (pH 8.8), 1 M MgCl2, 0.5 M EDTA (pH 8.8) and 14.4 M β -mercapto-ethanol add double distilled water till 200 mL), 0.6 mg/ mL BSA (Sigma, A-7906), 100% DMSO (Sigma, D-8418), 0.2 mM dNTP (Sigma, D7295), 0.5 µM oligonucleotide primer, 1 units of Taq DNA polymerase (Sigma, D1806) and distilled water. PCR amplifications were performed in a DNA thermal cycler with an initial denaturation step (95°C, 7 min), followed by 30 cycles of
denaturation (94°C, 1 min), annealing (53°C, 1 min) and extension (65°C, 8 min), and a single final extension step (65°C, 16 min). The amplified fragments were fractionated on a 1.5% w/v agarose gel during 200 min at a constant voltage of 40 V in 0.5×TAE (TrisAcetat EDTA) at 4°C. A 10-kb reference marker (Sigma, D7058) was used to allow standardization, followed by staining with ethidium bromide and visualization. 3.2.5.2
Whole Genome Sequencing De-novo sequencing and assembly of novel strain of ST-500 (Streptococcus
thermophilus) on Ion torrent platform. 1.6 ug of Qubit quantified DNA was sonicated for 300 sec on Covaris. 950 ng of DNA was taken for library preparation (Table 3.2). Endrepair and adapter ligation was done according to the protocol. The sample was bar coded at this step. The sample was cleaned using Ampure XP beads. Sample was size selected using 2% Low melting agarose gel. The gel purified sample was amplified as per the protocol. The amplified product was cleaned up using Ampure XP beads.
The sample
was run on Bioanalyzer HS chip to check the distribution and was quantified using Qubit Fluorometer. Table: 3.2 Details of the analytical tools and reference genome used
[1]
Sequencing Platform Ion torrent
[2]
Data Analysis Tools
[3]
Reference genome (1)
[4]
Reference genome (2)
SeqQC, MIRA-3.4.0, MUMmer, T-MAP, BLAST, RAST Streptococcus thermophilus MTCC_5461.Only scaffolds are available. (gi|444751733|ALIL01000001ALIL01000144) a. Link: http://www.ncbi.nlm.nih.gov/nuccore/NC_003888.3 b. Genome size: 1619896 bp (1.61MB) c. GC content: 39.33 % Streptococcus thermophilus MN-ZLW-002 (gi|387908808|ref|NC_017927.1|) a. Link: http://www.ncbi.nlm.nih.gov/nuccore/NC_017927.1 b. Genome size: 1848520 bp (1.84MB) c. GC content: 39.01 %
The experimental process of whole genome sequencing was conducted at Genotypic Technology Pvt. Ltd, Bangalore, India, on paid basis as analysis of the whole genome sequences and genome mapping requires a number of latest bioinformatics tools and techniques.
3.3 MEDIA FORMULATIONS AND EFFECTS OF DIFFERENT SOURCES OF NUTRITION
ON
MASS
PRODUCTION
OF
STREPTOCOCCUS
THERMOPHILUS Since a potential lactic acid bacterium was isolated, it was essential to test the growth parameters of the strain on the minimal media and other selective media prescribed for the strain to check the final bacterial mass. Selective media were studied for a brief idea on the nutrition requirements of the strain on specific source basis. Effect of carbon, nitrogen, amino acids, buffers, surfactants were studied. 3.3.1
Effect of carbon sources on mass production of S. thermophilus The optimum conditions for growth and fermentation were investigated by using
the selected lactic acid bacterium. To investigate the requirements for complex carbon sources, the experiments were performed either omitting one of the organic carbon sources from the modified MRS medium. Effects of different carbon sources like sucrose, dextrose, lactose, maltose, glucose, arabinose, malodextrine, and fructose were studied. The initial concentrations of all the selected carbon sources varied from 20-100 g/L (210%). For cultivation, a modified MRS and M948 (Himedia, India) was used: carbon source (2- 10%), nitrogen source (5%), sodium acetate (0.5%), tri-ammonuim citrate (0.2%), di-hydrogen potassium phosphate (0.2%). All the compositions were uniformly mixed sterilized under total controlled conditions and later inoculated with 5% inocolum size. The concentrations were in triplicates prepared in 500 mL conical flasks with working volume of 200 mL. Experiments were carried out with incubating temperature of 40°C in orbital shaking incubator (Remi, India) with agitation speed of 150 RPM. Media formulations resulting in high cell mass were selected for further analysis by cultivation on 2L fermenter (BIOSTAT A PLUS, Sartorius Stedim, Germany) with working volume of 1.5 L. Formulations were selected on the basis of their spectra-photometric results
(UV-Visible spectrophotometer 119, Systronics, India) and differentiated on the basis of optical density (OD) taken at 660 nm. Sampling was done every 5 hour interval for first 15 hours and then every 2 hour for total 24 hours. The optimized condition and medium were further used in process development. 3.3.2
Effect of nitrogen sources on mass production of S. thermophilus To investigate the requirements for organic nitrogen sources, the experiments
were performed omitting one of the organic nitrogen sources from the modified MRS medium. The highly effective lactic acid bacterium which possesses desirable characteristics such as homo-fermentative ability, good fermentation performance was selected. Different nitrogen sources were investigated such as casein peptone, meat peptone, tryptone, soya peptone, beef extract, tryptose, whey protein concentrate, corn steep liquor, sweet whey powder, yeast extract, respectively. The initial concentrations of all the selected nitrogen sources varied from 20-80 g/L (2-8%). For cultivation, a modified MRS and M948 (Himedia, India) was used: carbon source (4%), nitrogen source (2-8%), sodium acetate (0.5%), tri-ammonium citrate (0.2%), di-hydrogen potassium phosphate (0.2%). However, it was reported that spent yeast extract lacks growth factors especially iron. Therefore, 2 mg/L of ferrous sulfate (FeSO4) was added. Effect of polysorbate 60 (0.1-0.2%), polysorbate 80 (0.1-0.2%), KH2PO4 (0.2-0.4%), K2HPO4 (0.2-0.4%), MgSO4 (0.05-0.1%), MnSO4 (0.005-0.01%) NaCl (0.2-0.4%), sodium acetate (0.3-0.5%), tri-ammonium citrate (0.2-0.4%), sodium glycerophosphate (1.5-2.5%) ammonium nitrate (0.1-0.2%) was also studied. Formulations were selected on the basis of their spectra-photometric results along with the morphological observations of the cells to check cell rupturing and viability staining for further analysis. Experiments were carried out with the same protocol followed for the standardization of carbon sources.
3.4 EFFECT OF OTHER FACTORS AFFECTING THE GROWTH OF S. THERMOPHILUS Other than nutrition sources, there were several other factors and conditions which needs to be standardized for successful process optimization like incubation temperature and pH standardization. 3.4.1
Temperature Optimization The temperature giving the highest productivity was in some cases lower than the
temperature resulting in highest lactic acid concentration and yield, whereas in others the same temperature gave the best results in all categories (Hofvendahl and Hagerdal, 2000). The effect of temperature between 30 °C, 37 °C, 40 °C, 45 °C, 50 °C, or 55 °C were investigated for lactic acid fermentation (Busairi, 2002). All of determinations were analyzed and the optimum temperature was selected for further study 3.4.2
pH optimization Some enzymes have ionic groups on their active site, and these ionic groups must
be in the correct form (acid or base) to function. Variation in the pH of the medium results in changes in the ionic form of the active site. Therefore, the activity of the enzymes were significantly affected the reaction rate for cell growth and lactic acid production. The effects of initial pH (4.0, 5.0, 6.0, 7.0, or 8.0) were investigated for lactic acid fermentation (Yuwono and Kokugan, 2008). The optimum pH was selected for further study. 3.5
UP-SCALING PROCESS STANDARDIZATION The up-scaling process for bacterial mass production is one of the critical
standardization points in order to obtain maximum cell mass during the fermentation process for which a number of parameters were standardized staring from inocolum preparation to viable cell mass as end product.
3.5.1
Inocolum Preparation
Pure viable colonies of the isolated strain were selected after complete morphological and biochemical observations and were cultured on agar media (M948 Himedia) followed by incubation at 37°C for 24 hours. Sub-cultured colonies were checked again for any traces of other bacterial contaminants and were cultured on slants followed by storage at 8°C. Each experiment was performed using a preserved slant by inoculating loop-full culture in 80 mL of broth medium. After incubation and visible turbid growth, broth to broth sub-culturing was done for four to five bacterial generations and further used as inocolum for different generations (Figure 3.1).
Figure 3.1: Step elabaration of Inocolum preparation from 1st. 2nd and 3rd generation
3.5.2
Process standardization for incubating conditions Different incubation conditions were provided to the isolated strains in order to
check the effect of the same on viability of the cells. Both anaerobic and aerobic conditions were provided and results for oxygen and carbon dioxide demand were concluded. Temperature was controlled with an attached heating jacket on the fermenter along with a chilling rod supplied with cold water, while as amount of dissolved oxygen in the fermenter was calculated with an attached probe. Air supply inside the fermenter vessel was managed externally through a 0.20 µm air filter (Midisart 2000). Extra pressure was managed with the help of an attached exhaust. The acid-base ratio inside the vessel was maintained by an external supply of 1N hydrochloric acid and 5N sodium hydroxide. The effects of pH, air pressure, dissolved oxygen, temperature, working volume were studied. The external supply of sodium hydroxide and hydrochloric acid on the basis of its normality was studied from 1N to 5N and 0.1N to 1N respectively on the basis of the pH drop due to lactic acid secretion and managed according to the working volume of the vessel. 3.5.3
Commercial viability of the standardized process
In order to check the commercial viability of the developed process, it was essential to calculate the final CFU (colony forming units) of the end product of the upstreaming process, as cell mass with low viability is unsuitable for commercial production. Final number of CFU was calculated by taking 1 mL sample of the turbid broth followed by serial dilution of the same. Plates were incubated for 24 hours and colonies were counted on colony counter (Remi, India). The effect of various parameters on CFU was also studied and process with maximum CFU was selected for further experiments. 3.6
VIABILITY AND CFU STANDARDIZATION
Cell viability is defined as the number of living cells present based on a total cell sample. Quantifying bacteria can be a difficult task to achieve using direct methods of enumeration. Cell-counting instruments exist that can be used to count numbers of
organisms in a sample using electrical or light impedance, but these tools are often not found in every lab setting. The viable cell count is an estimate of bacterial population in an original sample being tested. To perform viable cell counts on agar plates, it is often necessary to dilute the original sample to make counting easier. Countable plates are typically considered to hold 30-300 colonies on the surface of the agar. Serial dilution was performed as per following protocol: One milliliter of the original sample was transferred to a tube containing 9 mL of sterile water which makes it a total of 10 mL of solution, 1 part sample and 9 parts water. One milliliter of solution was moved from this tube to another tube with 9 mL of sterile water creating a 1:100 dilution. This process is carried out until desired dilution factors were met. After the proper dilutions the contents of each tube were used to create plates. • • • • • • • • 3.7
Using a sample of milk, transfer 1 mL into the first sterile water blank. Label this tube 1:10. Mix the tube contents using the vortex mixer on the end of each table. Using another transfer pipette, transfer 1 mL of the 1:10 tube to the next sterile blank tube. Label this tube 1:100. Continue making transfers until 1:100 and 1:10000 dilutions have been made. After all dilution have been completed, transfer ½ mL from the 1:10 dilution and place it on a plate labeled 1:20. Spread the liquid across the surface of the plate with a clean spreading rod. Continue making plates in this fashion from each of the dilution tubes until created four plates: 1:20, 1:200, 1:2000, 1:20000. Place the plates in inverted position in an incubator at fixed temperature. DOWN-STREAM PROCESS STANDARDIZATION
Down stream process for production of starter culture includes a lot of bioinstrumentation and a number of processes like centrifugation, ultra micro-filtration, cryoprotectant standardization, freezing and lyophilization. It also includes the application testing of the final product on different parameters. The effects of all the processes were studied in order to obtain a remarkable commercially viable product.
3.7.1
Centrifugation and micro-filtration
Centrifugation of the turbid broth was standardized to overcome the issue of viability loss during the process. Specific number of revolutions per minute (rpm) were standardized by studying the effect of revolutions on cells starting from 4000 to 6000 (4000, 4500, 5000, 5500, 6000 rpm). It was observed during the process that, excessive centrifugal time was leading to cell rupturing, so the effect of centrifugal time was tested from 5 minutes to 20 minutes. On the other hand, the effect of micro-filtration on cells was also studied with both single and double membrane system and it was observed that the process was less viable compared to centrifugation. The cell loss during centrifugation was also studied by taking 0.1 g of the fresh pellet sample followed by serial dilution and colony counting after incubation. 5000 rpm for 15 minutes was standardized with maximum CFU or lowest cell loss and was selected for performing further experiments. The 15 minute centrifugal time was again distributed into two phases with 10 minutes of centrifugation followed by discarding the supernatant and 5 minute washing of pellet at the same 5000 rpm with saline water.
3.7.2
Cryoprotectant preparation and standardization The turbid broth samples were then centrifuged at 5000 RPM for 10 minutes
followed by pellet washing with physiological NaCl (0.9%) solution. The pellet were resuspended in 10% sterile solution of defatted skim milk powder (Himedia, India) and then distributed in sterile vials of 1 mL capacity followed by immediate freezing at -80° C for further use. The following compounds were used to check the viability improvement. Lactose, monohydrate (min 99.5%, milk sugar, Himedia, India), Sucrose, Extra pure (min 99.5%, Himedia, India), D-(-)-Fructose, Extra pure (min 99%, Himedia, India), D-(+)-Glucose monohydrate (min 99.5%, Himedia, India), D-(-)-Mannitol, Extra pure (min 99%, Himedia, India), D-(-)-Sorbitol (min 99%, Himedia, India), D-(+)-Maltose monohydrate (min 95%, Himedia, India), Maltodextrine (Himedia, India), D-(-)-Ribose (Min 99%, Himedia, India), D-(-)-Arabinose (Min 99%, Himedia, India). Polymers,
inorganic compounds and other media: Distilled water, Monosodium-L-glutamate monohydrate (min 98%, Himedia, India), meso-Inositol (min 98%, Himedia, India), glycerol, purified (min 99%, Himedia, India), sodium chloride, extra pure (min 99%, Himedia, India), Skim Milk powder, defatted (Himedia, India), di-Potassium hydrogen phosphate (min 99%, Himedia, India), Potassium dihydrogen orthophosphate, purified (min 99%, Himedia, India), tri-ammonium citrate, extra pure (min 97%, Himedia, India), whey protein concentrate (Fonterra, New Zealand), sweet whey powder (Fonterra, New Zealand), sodium caseinate (Arla Foods, Denmark). Different lyoprotectant combinations were prepared to check the cell viability after lyophilization. A total of 18 lyoprotectants were used to develop 40 combinations for evaluating viability results and escalation. All the lyoprotectants (10% w/w) were prepared and sterilized except monosodium glutamate (1% w/w), meso-inositol (0.5% w/w), KH2PO4 (1% w/w), K2HPO4 (1% w/w), ammonium citrate (1% w/w) which were sterilized with mentioned w/w. These protectants in single and in combinations were mixed with freezed vials in 1:5 ratios (1 mL inocolum vial and 5 mL lyoprotectant). Lyophilization of the samples was done at 0.04mbar vacuum at -50° C (Alpha 1-2, LO plus, Martin Christ). 3.7.3
Freezing temperature and incubation Freezing is a critical step in lyophilization process as randomness of ice
nucleation and growth is not easy to control. Impact of freezing on lyophilization includes inter vial and intra vial uniformity, cake appearance, reconstitution time, cake integrity, container-closure system, in-process stability and long term shelf life stability. Two phase freezing system was tested in current study. Primary freezing temperature range from -20°C to -60°C and secondary freezing from -60°C to -80°C were studied. Freezing cycle time and high degree of intra-vial heterogeneity was also studied. 3.7.4
Lyophilization standardization Freeze drying technique is a dehydrating method in which biological materials are
first frozen followed by sublimation (primary drying) and desorption (secondary drying), generally used for long term preservation of lactic acid bacteria starters, but freeze drying also brings changes in physical state of membrane lipids and structure of sensitive
proteins leading to viability issues (Leslie et al., 1995). However, demand of starter in modern era is continuously expanding the interest to create ready to use lyophilized starter with improved stress and shock tolerance (Broadbent and Lin, 1999). Consequently, some compounds such as polyols, polysaccharides, disaccharides, amino acids, proteins, vitamins, and various salts have been examined for their potential role to improve the survival of LAB throughout freeze drying process (Champagne et al., 1991). Use of S. thermophilus as starter culture for curd, depends on the concentration and preservation technologies employed, which are required to guarantee long-term delivery of stable cultures in terms of viability and functional activity. Lyophilization process was standardized by studying the effect of vacuum pressure from 0.02 mbar to 0.1 mbar and results were compared and finalized with different freezing period and temperature. The results were evaluated on the basis of CFU present on different vacuum pressures analyzed by serial dilution.
3.8
APPLICATION TESTING OF ISOLATED STARTER CULTURE Monitoring and screening starter culture is of pivotal importance in modern dairy
technology to guarantee quality of products like e. g. curd, yoghurt, cheese, or butter milk. During milk fermentation organic acids, mainly lactic acid, are produced by the microorganisms lowering the pH value. This acid production can be used to characterize milk fermentation and is therefore an important method to test the activity of starter cultures. 3.8.1
Curd Preparation Curd is an important part of Indian diet. In most Indian homes curd is prepared
almost every day. Homemade curd is not only very simple to prepare but is also delicious. Moreover, it has no preservatives and is also economical.
Figure 3.2:
Chart for preparation of Indain traditional curd from starter culture
The milk was slowly stirred to distribute the culture organisms uniformly and the inoculated milk was poured into 250 mL capacity beakers which were then incubated at 42°C for about 6 h. After fermentation, the beakers were shifted to refrigerator to cool the curd for about 24 h so that it was cooled to about 5°C. The curd was then analysed for sensory quality, rheological attributes and physico-chemical attributes. Following parameters are checked on curd formation:
Texture of Curd: The texture of curd depends mainly upon the heat treatment given to milk. Cow milk (3.5% fat and 8.5% SNF) was subjected to two separate treatments: (1) heating at 63oC for 30 min and (2) boiling treatment without holding period. The milk was cooled to about 40oC and inoculated with S. thermophilus culture and incubated at 42oC for about 6 hours. The curd formed was chilled to 5oC and evaluated for quality. Firmness, consistency and index of viscosity as measured by Texture Analyser increased with increased heat
treatment and the highest values were observed in curd prepared from boiled milk. Boiling treatment of milk resulted in least syneresis of whey in the curd. Based on the results, it was recommended that milk be subjected to boiling treatment to produce best quality curd.
Sensory: The chilled curd (5°C) was served to a panel of seven judges and its colour and appearance, flavor, body and texture and overall acceptance were evaluated on 9- point Hedonic scale (Amerine et al., 1965). The sensory evaluation was conducted in Sensory Evaluation Laboratory of the Institute under fluorescent lights. The sensory acceptance data were tabulated by taking average of scores awarded by all the judges for different treatments in three replications.
Syneresis: The set curd at 5°C was slowly transferred to 15 mL capacity centrifuge tubes causing minimum disturbance to the coagulum. The centrifuge tubes were balanced by adjusting their weights and centrifuged at 2000 rpm in a Remi centrifuge for 5 min. The quantity of whey separated at the top of the coagulum inside centrifuge tubes was recorded as mL. The higher the volume of whey separated, the higher will be the syneresis and vice versa.
pH: pH was determined by potentiometric method i.e. by potential difference between the sample and electrolyte solution present inside the electrode of pH meter, using digital pH meter (Systronic Co., Bangalore). The electrode of the pH meter was directly dipped in the set curd and the pH was recorded (5°C). pH was recorded to check the lactic acid secreting capability of the culture in specific duration with favorable temperature. Curd was prepared in cups with 100 mL volume and was incubated. pH was recorded after 3, 3.5, 4, 4.5, 5, 5.5, 6 hours on incubation from different cups of same experiment, so that the coagulation will not get disturbed.
Acidity: The acidity of the curd samples was analyzed by BIS method and expressed as per cent lactic acid (Bureau of Indian Standards, 1981).
Rheological: Firmness, consistency and viscosity of curd are important rheological or textural parameters that govern the quality of curd. These attributes can be measured objectively by Texture Analyzer or cone penetrometer. The following method was employed for measuring firmness, consistency and index of viscosity by Texture Analyzer (Stable Microsystems, UK). The working of Texture Analyser is based on the principle that a cylindrical steel probe penetrates into the curd samples and experiences resistance during the penetration. The resistance offered by the curd sample (5ºC) during the penetration of the probe up to a specified distance is recorded as firmness of curd.
3.8.2
Comparative testing between prepared starter and starters available in market Starter cultures are available in Indian market for curd preparation. Starter
culture JAMA and Yoflex (CHR Hansen, Denmark) and FlavoGard (Danisco Dupont, Copenhagen) collected from sources and local distributors for comparative studies on performance for curd preparation. Culture packs were equally diluted in milk and were inoculated in conditions which were maintained similar for both self isolated and market starters. The conditions provided were: Milk FAT 6%, Milk SNF 8.5%, Inoculation temperature 42° C, Incubation temperature 42° C, pH check 3-6 hours. Parameters observed •
Gel formation
•
Cut ability
•
Creaminess
•
Texture
•
Taste
•
Acidity
•
Mouth feel
•
Water separation
•
Shelf life
3.9
COMMERCIAL UP-SCALING TRIALS Commercial up-scaling trials were conducted on 300 L fermenter at a rented
facility with TBI (Technology Based Incubator) Center, Delhi University South campus, New Delhi. The facilities used were – Shaking incubator, seed fermenter (14, 30 L), pilot fermenter (300L), Micro and Ultra filtrate, pilot centrifuge and lyophilizer. At commercial facility inocolum was first prepared in 500 mL flasks (10) with 180 mL each and lyophilized vials were used as inocolum multiplication. 7 L media was prepared for 14 L fermenter and sterilized at 15psi. After 8 hours of incubation the total volume in the 14 L fermenter was transferred to 250 L media (working volume) in a 300 L fermenter as its inocolum.
3.10 PROTOCOL FOLLOWED AT TECHNOLOGY BASED INCUBATOR CENTER (TBI) DELHI UNIVERSITY FOR FERMENTATION TRIALS ON PILOT PLANT MEDIA PREPARATION IN 500 mL FLASKS WITH WORKING VOLUME 180 mL INOCULATION IN FLASKS WITH PREPARED LYOPHILIZED VIAL INOCOLUM INCUBATION IN ORBITAL SHAKER INCUBATOR FOR 12-16 HOURS TILL MORPHOLOGICAL OBSERVATION OF THE STRAIN AFTER MULTIPLICATION ST
PREPARATION OF 10L MEDIA FOR 14L SEED FERMENTER FOR INOCOLUM 1.44 L BROTH WAS TRANSFERRED TO 14L FERMENTER AS SEED INOCOLUM FERMENTATION WAS CARRIED OUT AT 200RPM & 40°C FOR 10-12 HOURS MORPHOLOGICAL OBSERVATION AFTER SEED PREPARATION (2ND gen) 10.5 L BROTH WAS TRANSFERRED TO 250L MEDIA IN 300L FERMENTER FERMENTATION WAS CARRIED OUT AT 200rpm & 40°C FOR 10-12 HOURS 1N HCL AND 5N SODIUN HYDROXIDE WERE PREPARED AS BUFFERS CENTRIFUGATION WAS CARRIED OUT AT 5000rpm FOR 10 MINUTES LYOPHILIZATION MEDIA WAS PREPARED AND MIXED WITH PELLET PELLET AND LYOPHILIZATION MEDIA WERE MIXED IN RATIO OF 1:1 FREEZING WAS DONE AT -60°C FOR 12 HOURS (UNTILL CAKE FORMATION) LYOPHILIZATION WAS CARRIED OUT AT 0.04MBAR VACUUM AT -40°C FOR 12 CREAMISH WHITE POWDER WAS RECOVERED AS END PRODUCT
Chapter 4 Results & Discussion
4.1
SCREENING AND ISOLATION OF LACTIC ACID BACTERIA
4.1.1
Isolation of Lactic acid bacteria Streptococcus thermophilus Isolation (STI) agar, M 17 agar and MRS agar were
employed as selective media for isolation of lactic acid bacteria. 10 colonies were randomly picked from different agar plates containing 20 to 200 lactic acid bacteria in each isolation experiment and more than 500 isolation experiments were conducted by serial dilution of different samples from different region. Plates were examined by eye, and the different colony types were individually picked. They were propagated twice and streaked on the selective media to obtain cultures. Gram positive and catalase negative cultures were maintained on selective media slants for further studies while others were discarded. A total of 128 isolates of LAB were isolated from curd, ripen grapes, cow milk, buffalo milk, camel milk, sheep milk and goat milk. Initially, the isolated strains were named according to their origin. For example; thirty seven isolates were isolated from curd were named as C (n), twenty two from goat milk were named as G (n), twenty eight from buffalo milk were named B (n), Twelve from cow milk were named CW (n), seven from camel milk were named CAM (n), six from grapes were named GP (n), sixteen from sheep milk were named S (n), respectively.
4.1.2
Selection of LAB for curd trials and lactic acid production All the isolated LAB strains were subjected to study their ability to coagulate milk
to produce curd. Isolated 128 isolates were maintained in MRS, M17 and STI broth depending upon their original selective media of isolation at 32, 37 and 45°C temperature in orbital shaker incubator at 150 rpm for 24 hours. Pellets of the isolates were collected by centrifugation at 4°C in cooling centrifuge at 5000 rpm for 10 minutes followed by washing with saline water and supernatant was discarded. 300 mg of fresh cell mass was inoculated in 300 mL of pasteurized milk (6% FAT, 8.5% SNF) which was allowed to
cool up to 42°C before inoculation. The experiment was carried out in triplicates. After six hours of incubation, pH was recorded (Table 4.1) for each isolate and potent strains were selected for further experiments. The concentrations of lactic acid were determined from the inoculated broth medium after 24 hours of incubation. A 10 mL sample was collected during fermentation aseptically produced from 100 mg of sugar. Centrifugation of fermented broth samples was carried out at several intervals at 12000 RPM for 10 minutes and supernatant was analyzed by HPLC. The cultured medium was filtered through 0.45 µm membrane filter. Organic acids were analyzed by HPLC (Merck– Hitachi). Five microliters of the sample was injected into the HPLC system equipped with an Aminex HPX-87 H column and RI detector. The column temperature was maintained at 65ºC. The mobile phase was 10 mM H2SO4 at flow rate of 0.6 mL/min (Sharma et al., 2013; Thang and Novalin, 2008). Isolates with tendency to ferment sugar into lactic acid efficiently were further allowed to multiply individually and concentrations of lactic acid productivity was determined (Table 4.1) by using HPLC and the yield was calculated by folowing equation.
Percentage yield =
x 100
It was observed that 32 isolates could produce high yields of lactic acid (more than 75%) with pH drop to 4.22 - 4.47 after six hours of incubation (Table 4.1). These 32 isolates were also identified for their better milk coagulating properties, fine texture and taste of the final curd, whereas other 96 isolates may be classified as hetero-fermentative LAB because of their low yield of lactic acid production. As a result, 32 isolates showing high product yield were selected for subsequent characterization. However, they could be readily distinguished by microscopic examination. Identification of strains was further investigated according to their morphological, cultural, physiological, and biochemical characteristics by the procedures described in the Bergey’s Manual of Systematic Bacteriology (Holt et al., 1994).
Table: 4.1 Percentage lactic acid yield and pH drop results of the 128 isolates Isolate Code
C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 C15 C16 C17 C18 C19 C20 C21 C22 C23 C24 C25 C26 C27 C28 C29 B2 B3 B4
Percentage yield of lactic acid production 31.45 27.66 25.31 22.96 33.12 15.56 22.13 44.21 39.32 77.88 47.96 55.17 22.17 40.44 71.64 77.85 19.23 81.88 21.23 29.36 79.11 80.23 46.57 69.77 48.14 22.75 66.53 56.11 23.27 51.23 56.45 61.23
pH after six hours incubation
Isolate Code
5.65 5.74 5.84 6.21 5.55 6.21 5.97 5.09 5.27 4.24 4.97 4.78 5.94 5.21 4.44 4.29 6.08 4.21 5.98 5.64 4.25 4.21 4.97 4.33 4.82 5.92 4.51 4.71 5.94 4.82 4.72 4.63
C30 C31 C32 C33 C34 C35 G1 G2 G3 G4 G5 G6 G7 G8 G9 G10 G11 G12 G13 G14 G15 G16 G17 G18 G19 G20 G21 G22 B1 CW7 CW8 CW9
Percentage pH after yield of lactic six hours acid incubation production 21.56 5.94 22.85 5.91 83.88 4.19 54.55 4.76 71.32 4.39 43.12 5.12 62.66 4.51 77.45 4.24 53.21 4.75 47.39 4.96 56.89 4.69 77.97 4.27 51.64 4.81 55.67 4.71 59.96 4.64 52.56 4.81 52.31 4.81 60.14 4.62 80.56 4.22 78.19 4.31 54.36 4.78 74.66 4.37 59.24 4.66 81.23 4.28 46.57 5.01 79.77 4.33 53.21 4.75 47.39 5.12 55.98 4.71 12.66 6.52 37.89 5.32 31.67 5.62
B5 B6 B7 B8 B9 B10 B11 B12 B13 B14 B15 B16 B17 B18 B19 B20 B21 B22 B23 B24 B25 B26 B27 B28 CW1 CW2 CW3 CW4 CW5 CW6
36.45 59.24 58.36 57.29 59.27 58.36 54.22 68.21 61.23 56.22 45.69 48.32 49.02 48.33 55.89 77.22 58.69 43.22 62.59 57.97 51.64 59.24 58.3 48.22 18.66 19.87 17.33 18.65 22.58 25.69
5.48 4.68 4.65 4.68 4.6 4.68 4.77 4.49 4.59 4.69 5.06 4.98 4.92 4.95 4.77 4.25 4.67 5.09 4.58 4.71 4.82 4.62 4.68 4.92 6.08 6.02 6.11 6.09 5.99 5.85
CW10 CW11 CW12 CAM1 CAM2 CAM3 CAM4 CAM5 CAM6 CAM7 GP1 GP2 GP3 GP4 GP5 GP6 S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13 S14
32.22 27.51 26.36 31.22 37.97 35.98 30.11 39.54 33.78 77.92 21.61 17.26 21.39 14.2 19.58 29.09 15.56 22.13 18.66 19.85 17.32 39.54 21.27 29.39 22.51 25.62 12.67 21.66 17.25 17.31
5.55 5.74 5.79 5.62 5.33 5.42 5.62 5.31 5.58 4.29 5.97 6.18 6.02 6.35 6.08 5.68 6.22 5.94 6.15 6.18 6.15 5.31 6.03 5.61 5.92 5.88 6.48 5.98 6.16 6.15
4.1.3
Morphological and Phenotypic characterization of Lactic acid bacteria The single colonies of the sample isolates were grown on selective media.
Microscopic observation showed them to appear as short rods, long, thin and cocci shape (Figure 4.1). Table 4.2 shows the morphological and physiological diversity of the 32 isolates strains giving details on cell morphology (rod, cocci, and ovoid shape) that were subdivided into two groups: cocci (75%), and rods (25%) and on the basis of growth, and gas production (homo- or hetero-fermentative) (Table 4.2).
Isolates were further
subdivided on the basis of profuse growth and poor growth with 50 % (+++), 40 % (++), and 10 % (+), respectively. Bacterial growth depends on culture medium with its ability to produce a high concentration of biomass. The variation of different nutrients in the medium are needed for lactic acid bacterial strains. The group of complex nutrients, i.e., skimmed milk, yeast extract, whey proteins, tryptone and peptone were used to satisfy the complex demands of the bacteria. A complex medium, especially, M17 and MRS are usually employed and supported the growth.
There were some strains showing good performance of homo-lactic acid fermentative which were selected for further investigation as starter culture in the subsequent fermentation processes.
Table: 4.2 Phenotypic characterization, colony observation and gas production results of the selected 32 isolates. STRAIN CELL TURBID GAS COLOR FORM EVEVATION CODE SHAPE GROWTH PRODUCTION C10 Cocci White Circular Flat +++ C15 Cocci White Circular Raised ++ + C16 Cocci White Circular Raised +++ C18 Rods White Punciform Raised +++ C21 Cocci White Circular Raised +++ C22 Cocci White Circular Raised +++ C24 Cocci Creamish Punciform Flat +++ + C26 Cocci White Circular Flat +++ + C27 Rods White Circular Raised ++ + C32 Cocci Creamish Punciform Flat +++ G1 Cocci White Circular Flat ++ G2 Cocci White Circular Flat ++ G6 Cocci White Circular Raised +++ G8 Rods White Irregular Flat + + G10 Cocci White Circular Raised ++ G13 Cocci White Circular Raised + ++ G16 Cocci White Irregular Raised ++ G18 Cocci White Irregular Flat +++ G20 Cocci White Circular Flat + ++ B4 Cocci White Circular Raised + ++ B6 Cocci White Irregular Flat ++ B11 Rods Creamish Punciform Raised + ++ B14 Rods Creamish Punciform Flat + + B17 Cocci White Circular Flat ++ B20 Cocci White Circular Flat +++ B23 Rods White Irregular Raised ++ B27 Cocci White Circular Raised ++ CW11 Cocci White Circular Raised ++ S8 Cocci White Circular Raised + ++ S15 Cocci White Circular Raised ++ CAM3 Rods Creamish Punciform Raised + ++ CAM7 Rods Creamish Punciform Flat +++ Legend: profuse growth (+++), good growth (++), moderate to poor growth or a positive reaction (+), no growth or no reaction (-).
After continuous sub-culturing of the selected strains, few strains showed poor cell viability (40% viability on sub-culturing were selected for further studies and designated according to their morphology. Isolates from curd C10, C16, C21, C22 and C32 with morphology of cocci in chains (Streptococcus) were re-designated as ST-100, ST-200, ST-300, ST-400 and ST-500 respectively while as isolates from goat milk G6 and G18, from buffalo milk B20 were re-designated as ST600, ST-700 and ST-800 respectively. Isolates from curd C18 and from camel milk CAM7 with rod shaped morphology were considered Lactobacillus and re-designated as LB-100 and LB-200 respectively (Figure 4.2, 4.3).
4.1.3.1 Morphology Table 4.3 showed general characteristics of the 10 isolated strains selected for further studies. Most strains possessed cocci or short rods shape with their cell size ranging from 0.2-1.0 μm. Morphological configuration showed white, creamy, and circular colonies with entire margin. Surface was observed smooth with raised elevation. All the isolates were subjected to Gram staining and they were examined under light microscope. They stained blue- purple hence they all were Gram positive bacteria and none of the isolate showed catalase activity. For physiological characterization most of the strains grew well between 35-37°C except LB-100 growing well at 32° C only. ST500 showed a wide range of temperature compatibility from 32° C to 50° C. The ability to grow at high temperature is a desirable trait as this could translate to increased rate of growth and lactic acid production beneficial for fast curdling of milk and a high fermentation temperature could reduce the chances of contamination by other microorganisms (Mohd Adnan and Tan, 2007). Further, ST-400 and ST-500 were found to tolerat to low pH range from 4.2-5.2. Growth at low pH for a starter is a bit dual impact property as low pH may resist the growth of other pathogenic bacteria but can also effect the shelf life and taste of the product due to continuous production of lactic acid.
ST-500 was the only isolate found to tolerate 3% NaCl. Ability to grow at 3% and 5% NaCl concentrations were tested for all isolates and only three of them were found to be resistant to 3% NaCl concentration. Bacteria adapt to hyper-osmolarity by accumulation, synthesis and transport of compatible solutes to restore turgor. It was well documented that osmo-protectants (thermo-protection) could play additional positive role and beneficial effects have been demonstrated on membrane integrity, protein folding and stability, (Baliarda et al., 2003). Accumulation of osmo-protectants or compatible solutes in the present studies had been considered to be the potential lactic acid bacteria. Similarly, a higher tolerance to lactic acid was a desirable trait for an industrial strain of LAB as it could produce more lactic acid in the fermentation broth without adversely affecting itself.
The strain ST-500 grew at higher NaCl concentration compared with the other isolates (Table 4.3). During fermentation process, lactic acid was produced by the cells whereas alkali was pumped into the broth to prevent excessive reduction in pH. The free acid would be converted to its salt lactate which would increase the osmotic pressure on the cell. Therefore, a LAB strain with high osmo-tolerance would be desirable for industrial application. These rapid screening processes resulted in domination of ST-500 in a wide range of tested conditions. Homo-fermentative lactic acid bacteria grew substantially faster than other bacteria present in the same ecological niche. The higher growth rate of lactic acid bacteria is a result of their simple primary metabolism, and their ability in adaptation to rich environments
Table: 4.3 Complete morphological and phenotypical analysis of 10 selected strains
Code of Isolates
Characteristics
ST-100
ST-200
ST-300
ST-400
ST-500
COLONY MORPHOLOGY Configuration Margin Elevation Surface Pigment Opacity
Circular Entire Raised Smooth Cream Opaque
Circular Entire Raised Smooth Cream Opaque
Circular Entire Raised Smooth White Opaque
Circular Entire Raised Smooth White Opaque
Circular Entire Raised Smooth White Opaque
Cocci in chains 0.20.4μm
Cocci in chains 0.4-0.6μm
Cocci
Cocci in chains 0.4-0.6μm
Cocci in chains 0.40.6μm
+ Homo + + + + + + +
+ Homo + + + + + + + + + + + +
CELL MORPHOLOGY Cell diameter Size PHENOTYPIC CHARACTERSTICS Gram staining Spore Formation Motility Catalase Activity Fermentation Type Glucose Fermentation Nitrate Reduction Casein Hydrolysis Growth at 10o C Growth at 32o C Growth at 37°C Growth at 45o C Growth at 50 o C Growth with 1.5% Nacl Growth with 3% Nacl Growth with 5% Nacl At pH 4.2 At pH 5.2 At pH 6.2
+ Homo + + + + + + +
0.6-0.8μm
+ + Homo Homo + + + + + + + + + + + Code of Isolates
Characteristics
ST-600
ST-700
ST-800
LB-100
LB-200
COLONY MORPHOLOGY Configuration Margin Elevation Surface Pigment Opacity
Circular Entire Raised Smooth Cream Opaque
Circular Entire Raised Smooth Cream Opaque
Circular Entire Raised Smooth White Opaque
Circular Entire Raised Smooth White Opaque
Circular Entire Raised Smooth Cream Opaque
Cocci in chains 0.40.6μm
Cocci in chains 0.5-0.6μm
Cocci
Rods
Rods
0.4-0.5μm
0.6-0.9μm
0.51μm
+ Homo + + + + + + +
+ Homo + + + + +
+ Homo + + + + + +
+ Homo + + + + + + +
+ Homo + + + + + + +
CELL MORPHOLOGY Cell diameter Size
PHENOTYPIC CHARACTERSTICS Gram staining Spore Formation Motility Catalase Activity Fermentation Type Glucose Fermentation Nitrate Reduction Casein Hydrolysis Growth at 10o C Growth at 32o C Growth at 37°C Growth at 45o C Growth at 50 o C Growth with 1.5% Nacl Growth with 3% Nacl Growth with 5% Nacl At pH 4.2 At pH 5.2 At pH 6.2
Figure 4.3:
Microscopic views of ST-200 (A), ST-300(B), ST-400(C), ST-500(D), LB-100(E) and LB-200(F)
4.2)
BIOCHEMICAL CHARACTERIZATION OF ISOLATES The type of carbohydrate and microorganism present in are the important criteria
in the resulting product. They contribute to the aroma, flavor development, and food preservation (pH drop). The capabilities of LAB to grow at the expense of carbohydrates and to produce fast acidification (lactic acid) are two criteria for the selection of suitable candidates for starter strains. From a technological standpoint these criteria are important for the inhibition of contaminating microflora, consistency of the final product, and reduction in production time. Detailed physiological characterization of the sugar fermentation pattern is an important requisite to classify new strains and select those appropriate for maximal acidification or biomass production in a given carbon source. Conventional techniques for studying sugar metabolism are usually very time-consuming, needing long incubation periods and precise standard conditions. For identification of the lactic acid bacteria, API 50 CH test kit (bioMerieuxΤΜ) was used. The API 50 CHL test kit is a standard system associated with the study of the assimilation-fermentation of 49 different compounds (plus one control) as per manufacturer’s guideline. Lactic acid bacteria could live in the absence as well as in the presence of the atmospheric oxygen indicating that they are facultative anaerobes. All the isolates were tested against 42 sugars for their fermentation property (Table 4.4) and results obtained were used for other purpose, for example, epidemiological grouping into types of the lactic acid bacteria, taxonomical analysis of a group of lactic acid bacteria, and classification of an unknown bacterial population into homogeneous groups. Among all the 42 sugars, almost all the selected strains were observed positive fermentation against sucrose, lactose, dextrose, maltose and fructose and negative against sorbitol, trehalose, raffinose, salicin, glycogen, starch etc (Table 4.4)
Table 4.4 Fermentation results of selected isolates from different sugars Code of Isolates Sugars
ST-100
ST-200
ST-300
ST-400
ST-500
D-arabinose
-
-
-
-
-
L-arabinose
-
-
-
-
-
D-xylose
-
-
-
-
-
L-xylose
-
-
-
-
-
D-galactose
+
+
+
+
+
D-glucose
+
+
+
+
+
D-fructose
+
+
+
+
+
D-mannose
-
-
-
-
-
D-mannitol
+
+
+
+
+
D-sorbitol
-
-
-
-
-
D-cellobiose
+
+
+
+
+
D-sucrose
+
-
-
+
+
D-trehalose
-
-
-
-
-
D-melibiose
+
-
+
-
-
D-lactose
+
+
+
+
+
D-maltose
-
-
-
-
-
D-raffinose
-
-
-
+
+
D-melezitose
+
+
+
+
+
D-turanose
+
+
+
+
+
D-fucose
-
-
-
-
-
D-arabitol
-
-
-
-
-
L-arabitol
-
-
-
-
-
Code of Isolates Sugars
ST-100
ST-200
ST-300
ST-400
ST-500
Dulcitol
-
-
-
-
-
Erythritol
-
-
-
-
-
Inositol
+
+
-
-
-
Amygdalin
-
-
-
-
-
Arbutin
-
-
-
-
-
Glycerol
-
-
-
-
-
Esculin
+
+
+
+
+
Salicin
-
-
-
-
-
Inulin
+
+
+
+
+
Glycogen
-
-
-
-
-
Xylitol
+
+
+
+
+
Gentiobiose
+
-
+
-
-
L-rhamnose
-
-
-
-
-
N-acetylglucosamine
+
-
+
-
-
Amidon (starch)
-
-
-
-
-
Potassium gluconate
-
-
-
-
-
potassium 2ketogluconate potassium 5ketogluconate
-
+
-
-
-
-
-
-
-
-
Methyl-αDMannopyranoside
-
-
-
-
-
Methyl-αDGlucopyranoside
-
-
-
-
Continued.
Sugars
ST-600
ST-700
ST-800
LB-100
LB-200
D-arabinose
-
-
-
-
-
L-arabinose
-
-
-
-
-
D-xylose
-
-
-
-
-
L-xylose
-
-
-
-
-
D-galactose
+
+
+
+
+
D-glucose
+
+
+
+
+
D-fructose
+
+
+
+
+
D-mannose
-
-
-
-
-
D-mannitol
+
+
+
+
+
D-sorbitol
-
-
-
-
-
D-cellobiose
+
+
+
+
+
D-sucrose
+
-
-
+
+
D-trehalose
-
-
-
-
-
D-melibiose
+
-
+
-
-
D-lactose
+
+
+
+
+
D-maltose
-
-
-
-
-
D-raffinose
-
-
-
+
+
D-melezitose
+
+
+
+
+
D-turanose
+
+
+
+
+
D-fucose
-
-
-
-
-
D-arabitol
-
-
-
-
-
L-arabitol
-
-
-
-
Continued .
` Code of Isolates
Sugars
ST-600
ST-700
ST-800
LB-100
LB-200
Dulcitol
-
-
-
-
-
Erythritol
-
-
-
-
-
Inositol
-
-
-
+
+
Amygdalin
-
-
-
-
-
Arbutin
-
-
-
-
-
Glycerol
-
-
-
-
-
Esculin
+
+
+
+
+
Salicin
-
-
-
-
-
Inulin
+
+
+
+
+
Glycogen
-
-
-
-
-
Xylitol
+
+
+
+
+
Gentiobiose
+
-
+
-
-
L-rhamnose
-
-
-
-
-
N-acetylglucosamine
+
-
+
-
-
Amidon (starch)
-
-
-
-
-
Potassium gluconate
-
-
-
-
-
potassium 2ketogluconate potassium 5ketogluconate
-
+
-
-
-
-
-
-
-
-
Methyl-αDMannopyranoside
-
-
-
-
-
Methyl-αDGlucopyranoside
-
-
-
-
-
Legend: positive (+), negative (-).
Enzymatic activities of all the selected isolates were studied as there are few enzymes from lactic acid bacteria having many dairy applications including lactase responsible for the treatment of lactose intolerance. Lactase activity was observed in five out of ten
strains and all the lactase positive strains were streptococci. Phosphatase and glycosidase activities were observed negative for all the strains (Table 4.5) as was also observed by Tamang et al., 2007. They were all esculin hydrolase and arylamidase positive.
Table 4.5 Results of enzyme assays conducted on selected isolates CODE OF ISOLATES ENZYME ASSAYS
ST-100
ST-200
ST-300
ST-400
ST500
PHOS (PHOSPHATASE)
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
+
+
-
-
+
-
-
-
-
-
+
+
-
-
+
+
-
-
-
-
+
+
+
+
+
+
+
+
+
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
BGAL (BETAGALACTOSIDASE) BMAN (BETAMANNOSIDASE) LACTASE ProA (L-Proline ARYLAMIDASE) LeuA (Leucine ARYLAMIDASE) TyrA (Tyrosine ARYLAMIDASE) ESC (ESCULIN Hydrolase) APPA (Ala-Phe-ProARYLAMIDASE) NAG (N-ACETYL-DGLUCOSAMINE) BMAN (BETAMANNOSIDASE) ProA (L-Proline ARYLAMIDASE) URE (UREASE)
CODE OF ISOLATES ENZYME ASSAYS
ST-600
ST-700
ST-800
LB-100
LB-200
PHOS (PHOSPHATASE)
-
-
-
-
-
BGAL (BETAGALACTOSIDASE)
-
-
-
-
-
BMAN (BETAMANNOSIDASE)
-
-
-
-
-
LACTASE
+
+
-
-
-
ProA (L-Proline ARYLAMIDASE)
-
-
-
-
-
LeuA (Leucine ARYLAMIDASE)
+
+
-
-
+
TyrA (Tyrosine ARYLAMIDASE)
+
-
-
-
-
ESC (ESCULIN Hydrolase)
+
+
+
+
+
APPA (Ala-Phe-ProARYLAMIDASE)
+
+
+
+
+
NAG (N-ACETYL-DGLUCOSAMINE)
-
-
-
-
-
BMAN (BETAMANNOSIDASE)
-
-
-
-
-
ProA (L-Proline ARYLAMIDASE)
-
-
-
-
-
URE (UREASE)
-
-
-
-
-
S.N.
TEST
1
VP
2
Esculin Hydrolysis
3
PYR
4
ONPG
5
Arginine utilization
6
Glucose
7
Lactose
8
Arabinose
9
Sucrose
10
Sorbitol
11
Mannitol
12
Raffinose
Figure: 4.4
PRINCLIPLE Detects acetonin production Detects acetonin production Detects PYR enzyme activity Detects β -galactosidase activity Detects arginine decarboxylation Carbohydrate Ulilization Carbohydrate Ulilization Carbohydrate Ulilization Carbohydrate Ulilization Carbohydrate Ulilization Carbohydrate Ulilization Carbohydrate Ulilization
ORIGINAL COLOR OF THE MEDIUM Colourless/Light yellow
POSITIVE REACTION
NEGATIVE REACTION
Pinkish red/Red
Colourless/Slight copper
Cream
Black
Cream
Cream
Cherry Red
Cream
Colourless
Yellow
Colourless
Olive green to light purple
dark Purple
No change in colour or yellow colour
Pinkish red/Red
Yellow
Red/Pinkish red
Pinkish red/Red
Yellow
Red/Pinkish red
Pinkish red/Red
Yellow
Red/Pinkish red
Pinkish red/Red
Yellow
Red/Pinkish red
Pinkish red/Red
Yellow
Red/Pinkish red
Pinkish red/Red
Yellow
Red/Pinkish red
Pinkish red/Red
Yellow
Red/Pinkish red
Analysis of biochemical results of all selected 10 strains by Kit menthod
MR-VP test differentiates bacteria upon the fermentative end product they produce. Some bacterial strains produce large amount of acids and some produce a neutral acetoin as the end product. MR-VP tests were conducted for all isolates and it was observed that all the isolates were MR -positive and VP-negative (Table 4.6) (Figure 4.5, 4.6). Table: 4.6 Biochemical characterization results on the basis of different biochemical test analysis CODE OF ISOLATES BIOCHEMICAL TEST Methyl Red Test Voges Proskauer Test Casein Hydrolysis Starch Hydrolysis Nitrate Reduction Test Urea Test Catalase Test
ST-100 + + -
ST-200 + + -
ST-300 + + -
ST-400 + + -
ST-500 + + -
LB-100 + + -
LB-200 + + -
CODE OF ISOLATES BIOCHEMICAL TEST Methyl Red Test Voges Proskauer Test Casein Hydrolysis Starch Hydrolysis Nitrate Reduction Test Urea Test Catalase Test
ST-600 + + -
ST-700 + + -
ST-800 + + -
Casein hydrolysis was also conducted in which skim milk powder was used as casein source; all the strains produced a clear zone around the area of bacterial contact indicating the presence of caseinase enzyme. Starch hydrolyzing test was also conducted in which maize and potato starch were used along with nitrogen and carbon sources to check the hydrolysis. All the selected isolates were negative against starch hydrolysis. The strains were urease and catalase negative indicating that the strains belong to LAB origin.
Chapter 5 Molecular Characterization 5.1
MOLECULAR CHARACTERIZATION Despite the wide application of lactic acid bacteria, they are currently
differentiated by the determination of DNA-DNA similarity, their G+C content and, by physiological characterization (Tanasupawat et al., 1993). Biochemical identification is not accurate for determining the genotypic differences of microorganisms. The molecular technique (genotypic characterization) has been applied to characterize the LAB bacteria. Molecular technique is fast, practical and accurate. The use of 16S rRNA gene sequences to study bacterial phylogeny and taxonomy has been by far the most common housekeeping genetic marker used for a number of reasons. These reasons include (i) its presence in almost all bacteria, often existing as a multigene family, or operons; (ii) the function of the 16S rRNA gene over time has not changed, suggesting that random sequence changes are a more accurate measure of time (evolution); and (iii) the 16S rRNA gene (1,500 bp) is large enough for informatics purposes. A preliminary estimation of taxonomic distribution was used as criteria in the selection for further investigations. Identification by using selective PCR amplification and subsequent sequencing of the 16S rDNA genes directly from the isolated LAB was carried out. Ten selected strains were identified on the basis of their 16S rDNA homologies with entries in the GenBankEMBL databases used as an additional taxonomic criterion for distinguishing these bacteria from other members of the family (genotypic characterization). DNA were extracted, and used as template for PCR amplification (Figure 5.1). PCR products were detected for the partial nucleotide sequences. 16S rDNA gene sequences have been performed to infer organismal phylogeny reflecting the organismal evolutionary history. DNA quantity and purity of the sample isolates was estimated using nanodrop spectrophotometer (Table 5.1).
Figure: 5.1
Digest results of the 16s rDNA genes of representative isolates along with the reference. Sample IDs of the different lanes are mentioned on the top of the lane along with the last lane of Ladder (L) Hind III digested lambda ladder.
Table: 5.1 Quality Control reports of all of sample isolates. DNA Concentration and purity of samples estimated using nanodrop spectrophotometer.
S. No .
SAMPLE NAME
ABSORBA NCE VALUE 260/280
ABSORBANC E VALUE 260/230
DNA CONCENTRA TION ng/µl
TOTAL YIELD in ng
QC PURITY
QC YIELD
QC INTEGRIT Y
1
ST-100
2.13
2.21
3677.29
91932.2
Optimal
Optimal
Optimal
2
ST-200
1.94
2.17
4122.24
103057.3
Optimal
Optimal
Optimal
3
ST-300
1.13
1.54
4952.33
123808.7
Optimal
Optimal
Optimal
4
ST-400
0.97
1.12
4958.72
117922.8
Optimal
Optimal
Optimal
5
ST-500
0.99
1.21
3597.68
97823.6
Optimal
Optimal
Optimal
6
ST-600
2.03
2.18
1032.47
71239.8
Optimal
Optimal
Optimal
7
ST-700
1.31
1.43
1071.88
73347.2
Optimal
Optimal
Optimal
8
ST-800
0.92
1.07
897.27
56349.5
Optimal
Optimal
Optimal
9
LB-100
1.02
1.15
2217.23
88763.9
Optimal
Optimal
Optimal
10
LB-200
1.07
1.18
4048.37
103582.1
Optimal
Optimal
Optimal
All the samples were found suitable for Sanger sequencing
5.1 16s rDNA SEQUENCING Molecular characterization of all the ten isolates was evaluated on the basis of 16s rDNA sequencing results. Seven strains with ST designations were identified as Streptococcus thermophilis with percentage similarity from 96.66% to 100% in GenBank-EMBL databases. Two strains with LB designations were L. acidophilus and L. indicus with 99 and 96% similarity in databeses respectively.
5.1.1
16s rDNA sequencing results for ST-100
SANGER TRACE FOR ST-100 PRIMER P1 (AGAGTTTGATCCTGGCTCAG) Best BLAST Hit Results Streptococcus thermophilus ATCC19258 Score
Expect
Identities
Gaps
Strand
2571 bits(1374)
0.0
1391/1398(99%)
5/1398(0%)
Plus/Plus
Query
27
Sbjct 17923
17864
Query
85
Sbjct 17983
17924
Query
145
Sbjct 18043
17984
Query
205
Sbjct 18103
18044
ATA-ATGC-AGTAGAACGCTGAAGAGAGGAGCTTGCTCTTCTTGGATGAGTTGCGAACGG 84 ||| |||| ||||||||||||||||||||||||||||||||||||||||||||||||||| ATACATGCAAGTAGAACGCTGAAGAGAGGAGCTTGCTCTTCTTGGATGAGTTGCGAACGG
GTGAGTAACGCGTAGGTAACCTGCCTTGTAGCGGGGGATAACTATTGGAAACGATAGCTA |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| GTGAGTAACGCGTAGGTAACCTGCCTTGTAGCGGGGGATAACTATTGGAAACGATAGCTA
144
ATACCGCATAACAATGGATGACACATGTCATTTATTTGAAAGGGGCAATTGCTCCACTAC |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ATACCGCATAACAATGGATGACACATGTCATTTATTTGAAAGGGGCAATTGCTCCACTAC
204
AAGATGGACCTGCGTTGTATTAGCTAGTAGGTGAGGTAATGGCTCACCTAGGCGACGATA |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| AAGATGGACCTGCGTTGTATTAGCTAGTAGGTGAGGTAATGGCTCACCTAGGCGACGATA
264
FASTA SEQUENCE FOR ST-100 GGGAAGGGTCAGTACGCTCTGCTGTTATAATGCAGTAGAACGCTGAAGAGAGGAGCTTGCTCTTCTTGGATGAGTTGCG AACGGGAGTAACGCGTAGGTAACCTGCCTTGTAGCGGGGGATAACTATTGGAAACGATAGCTAATACCGCATAACAAT GGATGACACATGTCATTTATTTGAAAGGGGCAATTGCTCCACTACAAGATGGACCTGCGTTGTATTAGCTAGTAGGTGA GGTAATGGCTCACCTAGGCGACGATACATAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCA GACTCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGGGGCAACCCTGACCGAGCAACGCCGCGTGAGTGAAG AAGGTTTTCGGATCGTAAAGCTCTGTTGTAAGTCAAGAACGGGTGTGAGAGTGGAAAGTTCACACTGTGACGGGTAGC TTACCAGAAAGGGACGGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTCCCGAGCGTTGTCCGGATTTATTG GGCGTAAAGCGAGCGCAGGCGGTTTGATAAGTCTGAAGTTAAAGG
5.1.2
16s rDNA sequencing results for ST-200
SANGER TRACE FOR ST-200 PRIMER P1(AGAGTTTGATCCTGGCTCAG) Best BLAST Hit Results Streptococcus thermophilus CNRZ1066 Score
Expect
Identities
Gaps
Strand
2601 bits(1388)
0.0
1423/1437(99%)
13/1437(0%)
Plus/Plus
Query
30
Sbjct 17923
17864
Query
89
Sbjct 17983
17924
Query
149
Sbjct 18043
17984
Query
209
Sbjct 18103
18044
ATA-ATGCAAGTAGAACGCTGAAGAGAGGAGCTTGCTCTTCTTGGATGAGTTGCGAACGG ||| |||||||||||||||||||||||||||||||||||||||||||||||||||||||| ATACATGCAAGTAGAACGCTGAAGAGAGGAGCTTGCTCTTCTTGGATGAGTTGCGAACGG
88
GTGAGTAACGCGTAGGTAACCTGCCTTGTAGCGGGGGATAACTATTGGAAACGATAGCTA |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| GTGAGTAACGCGTAGGTAACCTGCCTTGTAGCGGGGGATAACTATTGGAAACGATAGCTA
148
ATACCGCATAACAATGGATGACACATGTCATTTATTTGAAAGGGGCAATTGCTCCACTAC |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ATACCGCATAACAATGGATGACACATGTCATTTATTTGAAAGGGGCAATTGCTCCACTAC
208
AAGATGGACCTGCGTTGTATTAGCTAGTAGGTGAGGTAATGGCTCACCTAGGCGACGATA |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| AAGATGGACCTGCGTTGTATTAGCTAGTAGGTGAGGTAATGGCTCACCTAGGCGACGATA
268
FASTA SEQUENCE FOR ST-200 TGGTGGCGTTGTTTCTATCATGCGGTGCTATAATGCAAGTAGAACGCTGAAGAGAGGAGCTTGCTCTTCTTGGATGAGT TGCGAACGGGTGAGTAACGCGTAGGTAACCTGCCTTGTAGCGGGGGATAACTATTGGAAACGATAGCTAATACCGCAT AACAATGGATGACACATGTCATTTATTTGAAAGGGGCAATTGCTCCACTACAAGATGGACCTGCGTTGTATTAGCTAGT AGGTGAGGTAATGGCTCACCTAGGCGACGATACATAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACAC GGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGGGGCAACCCTGACCGAGCAACGCCGCGTGA GTGAAGAAGGTTTTCGGATCGTAAAGCTCTTGTTTGTAAGTCAAGGAACGGGGTGGTGAGAGGTGGAAAGTTCACACT GTGGACGGTAGCTTACCAGAAAGGGACGGCTAACTACGTGCCAGCAGCCGCGGTAAAGGCTGTGGCTCAACCATAGTT CGCTTTGGAAACTGTCAAACTTGAGTGCAGAAGGGGAGAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGATATAT GGAGGAACACCGGTGGCGAAAGCGGCTCTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCGTGGGGAGCGAACAGG ATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAGG
5.1.3
16s rDNA sequencing results for ST-300
SANGER TRACE FOR ST-300 PRIMER P1 (AGAGTTTGATCCTGGCTCAG) Best BLAST Hit Results Streptococcus thermophilus MN-ZLW-002 Score
Expect
Identities
Gaps
Strand
2564 bits(717)
0.0
739/748(99%)
7/748(0%)
Plus/Plus
Query
1
Sbjct 18272
18215
Query
61
Sbjct 18328
18273
Query
121
Sbjct 18388
18329
Query
181
Sbjct 18448
18389
AACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAGCTCTGTTGTAAGTCCAAGAACC |||||||||||||||||||||||||||||||||||||||||||||||||||| ||||| | AACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAGCTCTGTTGTAAGTC-AAGAA-C
60
GGGGTGTGAGGAGGTGGAAAGTTCACACTGTGACGGTAGCTTACCAGAAAAGGGACGGCT |||||||| || |||||||||||||||||||||||||||||||||| |||||||||||| -GGGTGTGA-GA-GTGGAAAGTTCACACTGTGACGGTAGCTTACCAG-AAAGGGACGGCT
120
AACTACGTGCCAGCAGCCGCGGTAATACGTAGGTCCCGAGCGTTGTCCGGATTTATTGGG |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| AACTACGTGCCAGCAGCCGCGGTAATACGTAGGTCCCGAGCGTTGTCCGGATTTATTGGG
180
CGTAAAGCGAGCGCAGGCGGTTTGATAAGTCTGAAGTTAAAGGCTGTGGCTCAACCATAG |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| CGTAAAGCGAGCGCAGGCGGTTTGATAAGTCTGAAGTTAAAGGCTGTGGCTCAACCATAG
240
FASTA SEQUENCE FOR ST-300 GGAGGGGGGGCTTTGTTGTCTACTGCACTCTGCACTTTACGCTGAAGAGAGGAGCTTGCTCTTCTTGGATGAGTTGCGA ACGGGTGAGTAACGCGTAGGTAACCTGCCTTGTAGCGGGGGATAACTATTGGAAACGATAGCTAATACCGCATAACAA TGGATGACACATGTCATTTATTTGAAAGGGGCAATTGCTCCACTACAAGATGGACCTGCGTTGTATTAGCTAGTAGGTG AGGTAATGGCTCACCTAGGCGACGATACATAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCC AGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGGGGCAACCCTGACCGAGCAACGCCGCGTGAGTGAA GAAGGTTTTCGGATCGTAAAGCTCTGTTGTAAGTCCAAGAACCGGGGTGTGAGGAGGTGGAAAGTTCACACTGTGACG GTAGCTTACCAGAAAAGGGACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTCCCGAGCGTTGTCCGGATT TATTGGGCGTAAAGCGAGCGCAGGCGGTTTGATAAGTCTGAAGTTAAAGGCTGTGGCTCAACCATAGTTCGCTTTGGAA ACTGTCAAACTTGAGTGCAGAAGGGGAGAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAGGAACAC CGGTGGCGAAAGCGGCTCTCTGGTCTGTAACTGACGCTGAGGC
Figure: 5.2
Results of phylogenetic analysis of ST-100, ST-200 and ST-300 in the form of Phylogenetic tree
5.1.4
16s rDNA sequencing results for ST-400
SANGER TRACE FOR ST-400 PRIMER P1 (AGAGTTTGATCCTGGCTCAG) Best BLAST Hit Results Streptococcus thermophilus ATCC19258 Score
Expect
Identities
Gaps
Strand
1646 bits(891)
0.0
989/1029(96%)
36/1029(0%)
Plus/Plus
Query
8
Sbjct
372
Query
60
Sbjct
432
Query
108
Sbjct
492
Query
157
Sbjct
552
TGTAAGTC-AG-ACCGGTGTGAGAGTGGAA-GTTCAC-TTG-GACGGTAGC-TA-CAG-A |||||||| || || ||||||||||||||| |||||| || ||||||||| || ||| | TGTAAGTCAAGAACGGGTGTGAGAGTGGAAAGTTCACACTGTGACGGTAGCTTACCAGAA
59
A-GGAC-GCT-ACTACGTG-CAGCAG-CGC-GT-ATACGTA-GT-CCGAGCG-TGT-C-G | |||| ||| |||||||| |||||| ||| || ||||||| || ||||||| ||| | | AGGGACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTCCCGAGCGTTGTCCGG
107
A-TTA-T-GGCGT-AAGCGAGCGCA-GCCG-TTGAT-AGTCTG-AG-TAAA-GCTGT-GC | ||| | ||||| ||||||||||| || | ||||| |||||| || |||| ||||| || ATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTGATAAGTCTGAAGTTAAAGGCTGTGGC
156
TC-ACCATAG-TCGCTTT-GAAACTGTCAAAC-TGAGTGCAGAAGGGGAGAGTGGAATTC || ||||||| ||||||| ||||||||||||| ||||||||||||||||||||||||||| TCAACCATAGTTCGCTTTGGAAACTGTCAAACTTGAGTGCAGAAGGGGAGAGTGGAATTC
212
431
491
551
611
FASTA SEQUENCE FOR ST-400 ACGCTCGTGTAAGTCAGACCGGTGTGAGAGTGGAAGTTCACTTGGACGGTAGCTACAGAAGGACGCTACTACGTGCAG CAGCGCGTATACGTAGTCCGAGCGTGTCGATTATGGCGTAAGCGAGCGCAGCCGTTGATAGTCTGAGTAAAGCTGTGCT CACCATAGTCGCTTTGAAACTGTCAAACTGAGTGCAGAAGGGGAGAGTGGAATTCCATGTGTAGCGGTGAAATGCGTA GATATATGGAGGAACACCCGGTGGCGAAAGCGGCTCTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCGTGGGGAGC GAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAGGTGTTGGATCCTTTCCGGGATTCAGTGCC GAAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGC ACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCCGATGCTATTTCTA GAGATAGAAAGTTACTTCGGTACATCGGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTA AGTCCCGCAACGAGCGCAACCCCTATTGTTAGTTGCCATCATTCAGTTGGG
EZTAXON RESULTS Rank
Name/Title
1
Streptococcus salivarius subsp. thermophilus
2
Streptococcus vestibularis
3
Streptococcus salivarius subsp. salivarius
4 5
Authors
Strain
Accession
(Orla-Jensen ATCC 1919) Farrow AY188354 19258(T) and Collins 1984
Pairwise Diff/Total Similarity nt
megaBLAST score
BLASTN score
96.660
34/1018
1679
1592
Whiley and Hardie 1988
ATCC GL831116 49124(T)
96.464
36/1018
1671
1584
Andrewes and Horder 1906
ATCC AY188352 7073(T)
96.464
36/1018
1671
1584
Streptococcus peroris
ATCC Kawamura et al. 700780( GL732464 1998 T)
94.401
57/1018
1514
1461
Streptococcus porcorum
Vela et al. 2011
94.401
57/1018
1493
1429
68203(T)
FN643224
5.1.5
16s rDNA sequencing results for ST-500
SANGER TRACE FOR ST-500 PRIMER P1 (AGAGTTTGATCCTGGCTCAG) Best BLAST Hit Results Streptococcus thermophilus MN-ZLW-002 Score
Expect
Identities
Gaps
Strand
2564 bits(1421)
0.0
1424/1425(99%)
3/1425(0%)
Plus/Plus
Query
1
Sbjct
44
Query
60
Sbjct
104
Query
120
Sbjct
164
Query
180
Sbjct
224
ATACATGC-AGTAGAACGCTGAAGAGAGGAGCTTGCTCTTCTTGGATGAGTTGCGAACGG |||||||| ||||||||||||||||||||||||||||||||||||||||||||||||||| ATACATGCAAGTAGAACGCTGAAGAGAGGAGCTTGCTCTTCTTGGATGAGTTGCGAACGG
59
GTGAGTAACGCGTAGGTAACCTGCCTTGTAGCGGGGGATAACTATTGGAAACGATAGCTA |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| GTGAGTAACGCGTAGGTAACCTGCCTTGTAGCGGGGGATAACTATTGGAAACGATAGCTA
119
ATACCGCATAACAATGGATGACACATGTCATTTATTTGAAAGGGGCAATTGCTCCACTAC |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ATACCGCATAACAATGGATGACACATGTCATTTATTTGAAAGGGGCAATTGCTCCACTAC
179
AAGATGGACCTGCGTTGTATTAGCTAGTAGGTGAGGTAATGGCTCACCTAGGCGACGATA |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| AAGATGGACCTGCGTTGTATTAGCTAGTAGGTGAGGTAATGGCTCACCTAGGCGACGATA
239
103
163
223
283
FASTA SEQUENCE FOR ST-500 ATACATGCAGTAGAACGCTGAAGAGAGGAGCTTGCTCTTCTTGGATGAGTTGCGAACGGGTGAGTAACGCGTAGGTAA CCTGCCTTGTAGCGGGGGATAACTATTGGAAACGATAGCTAATACCGCATAACAATGGATGACACATGTCATTTATTTG AAAGGGGCAATTGCTCCACTACAAGATGGACCTGCGTTGTATTAGCTAGTAGGTGAGGTAATGGCTCACCTAGGCGAC GATACATAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAG TAGGGAATCTTCGGCAATGGGGGCAACCCTGACCGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAGCT CTGTTGTAAGTCAAGAACGGGTGTGAGAGTGGAAAGTTCACACTGTGACGGTAGCTTACCAGAAAGGGACGGCTAACT ACGTGCCAGCAGCCGCGGTAATACGTAGGTCCCGAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTT TGATAAGTCTGAAGTTAAAGGCTGTGGCTCAACCATAGTTCGCTTTGGAAACTGTCAAACTTGAGTGCAGAAGGGGAG AGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAGGAACACCGG
5.1.6
16s rDNA sequencing results for ST-600
SANGER TRACE FOR ST-600 PRIMER P1 (AGAGTTTGATCCTGGCTCAG) Best BLAST Hit Results Streptococcus infantarius Score
Expect
Identities
Gaps
Strand
2564 bits(1421)
0.0
1424/1425(99%)
3/1425(0%)
Plus/Plus
Query
1
Sbjct
24
Query
61
Sbjct
82
Query
121
Sbjct
141
Query
181
Sbjct
201
ATACATGCAAGTAGAACGCTGAAGACTTTTCGCTTGGCTAAAGTTGGAAGAAGTTGCGAA |||||||||||||||||||||||||||||| |||| |||||||||||||| ||||||||| ATACATGCAAGTAGAACGCTGAAGACTTTTAGCTT-GCTAAAGTTGGAAG-AGTTGCGAA
60
CGGGTGAGTAACGCGTAGGTAACCTAGCCTACTAGCGGGGGATAACTATTGGAAACGATA ||||||||||||||||||||||||| |||||||||||||||||||||||||||||||||| CGGGTGAGTAACGCGTAGGTAACCT-GCCTACTAGCGGGGGATAACTATTGGAAACGATA
120
GCTAATACCGCATAACAGCATTTAACCCATGTTAGATGCTTGAAAGGAGCAATTGCTTCA |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| GCTAATACCGCATAACAGCATTTAACCCATGTTAGATGCTTGAAAGGAGCAATTGCTTCA
180
CTAGTAGATGGACCTGCGTTGTATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGAC |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| CTAGTAGATGGACCTGCGTTGTATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGAC
240
81
140
200
260
FASTA SEQUENCE FOR ST-600 ATACATGCAAGTAGAACGCTGAAGACTTTTCGCTTGGCTAAAGTTGGAAGAAGTTGCGAACGGGTGAGTAACGCGTAG GTAACCTAGCCTACTAGCGGGGGATAACTATTGGAAACGATAGCTAATACCGCATAACAGCATTTAACCCATGTTAGAT GCTTGAAAGGAGCAATTGCTTCACTAGTAGATGGACCTGCGTTGTATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGG CGACGATACATAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCA GCAGTAGGGAATCTTCGGCAATGGGGGCAACCCTGACCGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAA AGCTCTGTTGTAAGAGAAGAACGTGTGTGAGAGTGGAAAGTTCACACAGTGACGGTAACTTACCAGAAAGGGACGGCT AACTACGTGCCAGCAGCCGCGGTAATACGTAGGTCCCGAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGC GGTTTAATAAGTCTGAAGTTAAAGGCAGTGGCTTAACCATTGTTCGCTTTGGAAACTGTTAGACTTGAGTGCAGAAGGG AGAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAGGAAC
Name/Title
Authors
Strain
Accession
Pairwise Similarity
Diff/Total nt
mega BLAST score
BLASTN score
1
UNCULTURE D BD10692
Lin A et al., 2013
BD10692
JQ187473
99.99
1/1417
1679
1214
2
Streptococcus Schlegel et HDP9024 infantarius al. 2003 6(T) subsp. coli
AF429763
99.929
2/1417
2737
1276
3
Streptococcus Poyart et al. CIP lutetiensis 2002 106849(T)
DQ232532
99.859
2/1418
2739
1264
4
Streptococcus infantarius Schlegel et subsp. al. 2003 infantarius
ATCC BAA102(T)
ABJK02000017
99.718
4/1418
2724
1258
5
Andrewes Streptococcus and Horder equinus 1906
ATCC 9812(T)
GL698435
99.647
5/1417
2706
1244
Rank
5.1.7
16s rDNA sequencing results for ST-700
SANGER TRACE FOR ST-700 PRIMER P1 (AGAGTTTGATCCTGGCTCAG) Best BLAST Hit Results Streptococcus thermophilus ATCC19258 Score
Expect
Identities
Gaps
Strand
2643 bits(1421)
0.0
1424/1425(99%)
3/1425(0%)
Plus/Plus
Query
5
Sbjct
7
Query
65
Sbjct
66
Query
125
Sbjct
126
Query
185
Sbjct
186
GGGCGTGCCTATACATGCAGTAGAACGCTGAAGAGAGGAGCTTGCTCTTCTTGGATGAGT ||||||| |||||||||||||||||||||||||||||||||||||||||||||||||||| GGGCGTG-CTATACATGCAGTAGAACGCTGAAGAGAGGAGCTTGCTCTTCTTGGATGAGT
64
TGCGAACGGGTGAGTAACGCGTAGGTAACCTGCCTTGTAGCGGGGGATAACTATTGGAAA |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| TGCGAACGGGTGAGTAACGCGTAGGTAACCTGCCTTGTAGCGGGGGATAACTATTGGAAA
124
CGATAGCTAATACCGCATAACAATGGATGACACATGTCATTTATTTGAAAGGGGCAATTG |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| CGATAGCTAATACCGCATAACAATGGATGACACATGTCATTTATTTGAAAGGGGCAATTG
184
CTCCACTACAAGATGGACCTGCGTTGTATTAGCTAGTAGGTGAGGTAATGGCTCACCTAG |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| CTCCACTACAAGATGGACCTGCGTTGTATTAGCTAGTAGGTGAGGTAATGGCTCACCTAG
244
65
125
185
245
FASTA SEQUENCE FOR ST-700 TTGCGGGCGTGCCTATACATGCAGTAGAACGCTGAAGAGAGGAGCTTGCTCTTCTTGGATGAGTTGCGAACGGGTGAG TAACGCGTAGGTAACCTGCCTTGTAGCGGGGGATAACTATTGGAAACGATAGCTAATACCGCATAACAATGGATGACA CATGTCATTTATTTGAAAGGGGCAATTGCTCCACTACAAGATGGACCTGCGTTGTATTAGCTAGTAGGTGAGGTAATGG CTCACCTAGGCGACGATACATAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTA CGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGGGGCAACCCTGACCGAGCAACGCCGCGTGAGTGAAGAAGGTTTTC GGATCGTAAAGCTCTGTTGTAAGTCAAGAACGGGTGTGAGAGTGGAAAGTTCACACTGTGACGGTAGCTTACCAGAAA GGGACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTCCCGAGCGTTGTCCGGATTTATTGGGCGTAAAGCG AGCGCAGGCGGTTTGATAAGTCTGAAGTTAAAGGCTGTGGCTCAACCATAGTTCGCTTTGGAAACTGTCAAACTTGAGT GCAGAAGGGGAGAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAGGAACACCGGTGGCGAAAGCGG CTCTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCGTGGGG
5.1.8
16s rDNA sequencing results for ST-800
SANGER TRACE FOR ST-800 PRIMER P1 (AGAGTTTGATCCTGGCTCAG) Best BLAST Hit Results Streptococcus thermophilus CGLBL208 Score
Expect
Identities
Gaps
Strand
1454 bits(1421)
0.0
1424/1425(99%)
3/1425(0%)
Plus/Plus
Query
38
Sbjct
30
Query
98
Sbjct
90
Query
158
Sbjct
150
Query
218
Sbjct
210
Query
278
Sbjct
270
Query
338
Sbjct
330
ACGCTGAGAGAGGAGCTTGCTCTTCTTGGATGAGTTGCGAACGGGTGAGTAACGCGTAGG |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ACGCTGAGAGAGGAGCTTGCTCTTCTTGGATGAGTTGCGAACGGGTGAGTAACGCGTAGG
97
TAACCTGCCTTGTAGCGGGGGATAACTATTGGAAACGATAGCTAATACCGCATAACAATG |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| TAACCTGCCTTGTAGCGGGGGATAACTATTGGAAACGATAGCTAATACCGCATAACAATG
157
GATGACACATGTCATTTATTTGAAAGGGGCAATTGCTCCACTACAAGATGGACCTGCGTT |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| GATGACACATGTCATTTATTTGAAAGGGGCAATTGCTCCACTACAAGATGGACCTGCGTT
217
GTATTAGCTAGTAGGTGAGGTAATGGCTCACCTAGGCGACGATACATAGCCGACCTGAGA |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| GTATTAGCTAGTAGGTGAGGTAATGGCTCACCTAGGCGACGATACATAGCCGACCTGAGA
277
GGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAG |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| GGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAG
337
GGAATCTTCGGCAATGGGGGCAACCCTGACCGAGCAACGCCGCGTGAGTGAAGAAGGTTT |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| GGAATCTTCGGCAATGGGGGCAACCCTGACCGAGCAACGCCGCGTGAGTGAAGAAGGTTT
397
89
149
209
269
329
389
FASTA SEQUENCE FOR ST-800 TGGGGAGTTGCTTACATCTTATTATTATTCTCTATTCACGCTGAGAGAGGAGCTTGCTCTTCTTGGATGAGTTGCGAACG GGTGAGTAACGCGTAGGTAACCTGCCTTGTAGCGGGGGATAACTATTGGAAACGATAGCTAATACCGCATAACAATGG ATGACACATGTCATTTATTTGAAAGGGGCAATTGCTCCACTACAAGATGGACCTGCGTTGTATTAGCTAGTAGGTGAGG TAATGGCTCACCTAGGCGACGATACATAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGA CTCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGGGGCAACCCTGACCGAGCAACGCCGCGTGAGTGAAGAA GGTTTTCGGATCGTAAAGCTCTGTTGTAAGTCAAGAACGGGTGTGAGAGTGGAAAGTTCACACTGTGACGGTAGCTTAC CAGAAAGGGACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTCCCGAGCGTTGTCCGGATTTATTGGGCGT AAAGCGAGCGCAGGCGGTTTGATAAGTCTGAAGTTAAAGGCTGTGGCTCAACCATAGTTCGCTTTGGAAACTGTCAAA CTTGAGTGCAGAAGGGGAGAGTGGAATTCCATGTGTAGCGGTGAAATG
Name/Title
Authors
Strain
Accession
Pairwise Similarity
Diff/Total nt
mega BLAST score
BLAST N score
1
Streptococcus thermophilus CGLBL208
Cruciata et al., 2014
CGLBL208
KF286609.1
100
0/1454
1454
1214
2
Streptococcus thermophilus FMA196
Settani et al., 2011
FMA196
HQ721271.1
100
0/1454
2737
1276
Rank
5.1.9
16s rDNA sequencing results for LB-100
SANGER TRACE FOR LB-100 PRIMER P3 (TACGGTTACCTTGTTACGACTT) Best BLAST Hit Results Lactobacillus acidophilus Score
Expect
Identities
Gaps
Strand
1408 bits(762)
0.0
761/762(99%)
1/762(0%)
Plus/Plus
Query
1
Sbjct
1439
Query
61
Sbjct
1379
Query
121
Sbjct
1319
Query
181
Sbjct
1259
CTTTGGGCATTGCAGACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTA |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| CTTTGGGCATTGCAGACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTA
60
TTCACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCAGCTTCGTGCAGTCGAGTTGC |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| TTCACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCAGCTTCGTGCAGTCGAGTTGC
120
AGACTGCAGTCCGAACTGAGAACAGCTTTAAGAGATTCGCTTGCCTTCGCAGGCTTGCTC |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| AGACTGCAGTCCGAACTGAGAACAGCTTTAAGAGATTCGCTTGCCTTCGCAGGCTTGCTC
180
CTCGTTGTACTGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGACTT |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| CTCGTTGTACTGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGACTT
240
1380
1320
1260
1200
FASTA SEQUENCE FOR LB-100 CTTTGGGCATTGCAGACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTGCTGAT CCGCGATTACTAGCGATTCCAGCTTCGTGCAGTCGAGTTGCAGACTGCAGTCCGAACTGAGAACAGCTTTAAGAGATTC GCTTGCCTTCGCAGGCTTGCTCCTCGTTGTACTGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGA CTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCATTAGAGTGCCCAACTTAATGCTGGCAACTAATGA CAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCACCACCTGTCTTAG TGTCCCCGAAGGGAACTCCGTATCTCTACGGATTGCACTAGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAA TTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCG GAGTGCTTAATGCGTTAGCTGCAGCACTGAGAGGCGGAAACCTCCCAACACTTAGCACTCATCGTTTACGGCATGGACT ACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCA GTTGCAGACCAGAGAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTCCACCGCTACACATGGAGTTC
5.1.10 16s rDNA sequencing results for LB-200 SANGER TRACE FOR LB-200 PRIMER P3 (TACGGTTACCTTGTTACGACTT) Best BLAST Hit Results Lactobacillus indicus Score
Expect
Identities
Gaps
Strand
1583 bits(857)
0.0
953/995(96%)
23/995(2%)
Plus/Minus
Query
1
Sbjct
1466
Query
61
Sbjct
1406
Query
121
Sbjct
1346
Query
181
Sbjct
1286
TATCCCACCGACTTTGGGCATTGCAAACTTCCATGGGGGGACGGGCGGGGTGTACAAGGC ||||||||||||||||||||||||| |||||||||| | ||||||||| ||||||||||| TATCCCACCGACTTTGGGCATTGCAGACTTCCATGGTGTGACGGGCGGTGTGTACAAGGC
60
CCGGGAACGTATTCACCGCGGCGGGCTGATCCGCGATTACTAGCGATTCCAGCTTCGGGC ||||||||||||||||||||||| ||||||||||||||||||||||||||||||||| || CCGGGAACGTATTCACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCAGCTTCGTGC
120
AGGCGAGTTGCAGCCTGCAGTCCGAACTGAAAACAGCTTTAAGAGATCCGCTTACCCTCG |||||||||||||||||||||||||||||| ||||||||||||||||||||||||||||| AGGCGAGTTGCAGCCTGCAGTCCGAACTGAGAACAGCTTTAAGAGATCCGCTTACCCTCG
180
GGGGTTCGCTTCTCGTTGTACTGCCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGG ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| CGGGTTCGCTTCTCGTTGTACTGCCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGG
240
1407
1347
1287
1227
FASTA SEQUENCE FOR LB-200 TATCCCACCGACTTTGGGCATTGCAAACTTCCATGGGGGGACGGGCGGGGTGTACAAGGCCCGGGAACGTATTCACCG CGGCGGGCTGATCCGCGATTACTAGCGATTCCAGCTTCGGGCAGGCGAGTTGCAGCCTGCAGTCCGAACTGAAAACAG CTTTAAGAGATCCGCTTACCCTCGGGGGTTCGCTTCTCGTTGTACTGCCCATTGTAGCACGTGTGTAGCCCAGGTCATAA GGGGCATGATGACTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCTCTAGAGGGCCCAACTTAATGA TGGCAACTAAAGACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACAACAGCCATGC ACCACCTGTCTCTGCGTCCCCGAAGGGAACCACCTATCTCTAGGTGTAGCGCAGGATGTCAAGACCTGGTAAGGTTCTT CGCGTTGCTTCAAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTC GTACTCCCCAGGCGGAGCGCTTAATGCGTTTGCTGCGGCACTGAGGACCGGAAAGTCCCCAACACCTAGCGCTCATCGT TTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCAAGCCTCAGCGTCAGTTGCAAACCAAAG AGCCGCCTTCGCCACTGGGGTTCTTCCATATATCTACGCAT
Table: 5.2 16s rDNA sequencing results of selected 10 isolates with best hit similarities at data in NCBI
PERCENTAGE
QUERY
SIMILARITY
COVER
Streptococcus thermophilus strain ATCC 19258
99%
96%
NR 042778.1
Streptococcus thermophilus strain MNZLW-002
99%
96%
NR 074827.1
99%
97%
NR 074827.1
96.66%
95%
AY188354
99%
97%
NR 074827.1
99.92%
97%
AF429763
99%
98%
NR 042778.1
100%
97%
KF286609.1
99%
99%
NR 075045.1
96%
100%
NR 029106.1
S. No.
STRAIN DESIGNATION
STRAIN HOMOLOGY
1
ST-100
2
ST-200
3
ST-300
4
ST-400
5
ST-500
6
ST-600
7
ST-700
8
ST-800
9
LB-100
Streptococcus thermophilus strain MNZLW-002 Streptococcus
salivarius subsp thermophilus strain ATCC 19258(T) Streptococcus thermophilus strain MNZLW-002 Streptococcus infantarius subsp. coli Streptococcus thermophilus strain ATCC 19258 Streptococcus thermophilus CGLBL208 Lactobacillus acidophilus NCFM
ACCESSION
strain 10
LB-200
Lactobacillus delbrueckii subsp. indicus NCC725
Figure 5.3
Results of phylogenetic analysis of ST-400, ST-500 and ST-700 in the form of Phylogenetic tree
Figure 5.4
Results of phylogenetic analysis of LB-100 and LB-200 in the form of Phylogenetic tree
ST-200, ST-300 and ST-500 were found similar to Streptococcus thermophilus strain MN-ZLW-002 (accession NR 074827.1) with percentage similarity of 99% and query cover of 97% (Table 5.2). The phylogenetic trees for all the isolates were prepared (Figure 5.2, 5.3 and 5.4). ST-100 (96%), ST-200 (96%) and ST-700 (97%) were found similar Streptococcus thermophilus strain ATCC 19258 (accession NR 042778.1) with maximum query cover of 98%. ST-600 was identified as Streptococcus infantarius subsp. coli (99.92% similarity) and was excluded form further studies. Isolate LB-100 was identified as Lactobacillus acidophilus and observed 99% similar with Lactobacillus acidophilus NCFM strain (accession NR 075045.1). LB-200 was identified as L. indicus with similarty of 96% with Lactobacillus delbrueckii subsp. indicus NCC725.
ST-500 was selected for whole genome sequencing on the basis of its dairy applications and efficient performance as starter for curd and yogurt. The strain was submitted to NCIM (National Collection of Industrial Microorganisms) facility, Pune, under safe deposit agreement with accession number NCIM 5539.
5.2
WHOLE GENOME SEQUENCING THERMOPHILUS NCIM 5539)
OF
ST-500
(STREPTOCOCCUS
Total genomic DNA of the selected strain was prepared essentially by using Cardinal et al., 1994 procedure. DNA (214 ng/µl) was subjected to PCR utilizing primer 1 (AGAGTTTGATCCTGGCTCAG) and primer 2 BOX A1R (TACGGTTACCTTGTTACGACTT) as per procedure described by Versalovic et al.1994. Each 27 µl PCR reaction contained 5 µl 5× Gitschier buffer (1 M (NH4)2SO4, 1 M Tris-HCl (pH 8.8), 1 M MgCl2, 0.5 M EDTA (pH 8.8) and 14.4 M β -mercapto-ethanol and volume is maintained to 200 mL with double distilled), 0.6 mg/ mL BSA (Sigma, A7906), 100% DMSO (Sigma, D-8418), 0.2 mM dNTP (Sigma, D7295), 0.5 µM oligonucleotide primer, 1 unit of Taq DNA polymerase (Sigma, D1806) and distilled water. PCR amplifications were performed in a DNA thermal cycler with an initial denaturation step (95°C, 7 min), followed by 30 cycles of denaturation (94°C, 1 min), annealing (53°C, 1 min) and extension (65°C, 8 min), and a single final extension step
(65°C, 16 min). The amplified fragments were fractionated on a 1.5% w/v agarose gel during 200 min at a constant voltage of 40 V in 0.5×TAE (Tris-Acetat EDTA) at 4°C. A 10-kb reference marker (Sigma, D7058) was used to allow standardization, followed by staining with ethidium bromide and visualization. The 16s rDNA study of the strain suggests its similarity of 99% with MN-ZLW-002 Streptococcus salivarius subsp. thermophilus and was described with the help of circular genomic map (Figure 5.6). The De-novo sequencing and assembly of novel strain of ST-500 (Streptococcus thermophilus) on Ion torrent platform. Following are the whole genome sequencing details: 1.6 ug of Qubit quantified DNA was sonicated for 300sec on Covaris. 950 ng of DNA was taken for library preparation. End-repair and adapter ligation was done according to the protocol. The sample was bar coded at this step. The sample was cleaned using Ampure XP beads. Sample was size selected using 2% Low melting agarose gel. The gel purified sample was amplified as per the protocol. The amplified product was cleaned up using Ampure XP beads.
The sample was run on Bioanalyzer HS chip to check the
distribution and was quantified using Qubit Fluorometer. A final yield of 1567.2 pg was obtained (Table 5.3). WGS ePCR results showed the highest peak range of 10380-12152 bp (Figure 5.5). Table: 5.3 Qubit values of prepared library Sample
Qubit reading (pg/ul)
Yield (pg)
Ion Xpress Barcode
ST-500
78.36
1567.2
8
Figure 5.5:
Figure presents the library preparation report with peak indicating the ST-500 library optimal of sequencing
Figure 5.6:
Circular genomic map of ST-500 (Streptococcus thermophilus) in comparison with Streptococcus thermophilus MN-ZLW-002
Table: 5.4 Summary of the platforms and alignments of WGS of ST-500 Sample Sequence Data Information: Mean Read length
~231 bp
Type of sequencing: Single/Paired end sequencing
Single end
Sequenced using
Ion Torrent
Reference Sequence Data Information: Organism Name
Streptococcus
Type of Sequence: Genomic/Transcriptomic
Genomic
No. of Chromosomes/Transcripts
NA
Analysis Steps: QC and Raw data processing QC Program used
SeqQC-V2.0
High Quality Reads filtering: Yes/No
NO
Vector (Adapter/Primer) contaminated reads filtering: Yes/No
NO
B block trimming
NO
Alignment Information Alignment Tool used
tmap- v3
Assembly tool used
Mira_3.9.15 and CAP3
Table: 5.5 Details of alignments and Raw data sequences obtained from WGS Raw Data SeqQC Fastq file name
IonXpress_001_R_2013_07_10_13_14_04_user_JAG77_Auto_user_JAG-77_82.fastq
Fastq file size
2.49 GB
Time taken for Analysis
1.55 Minutes
Maximum Read Length
462
Minimum Read Length
8
Mean Read Length
231
Total Number of Reads
5505428 (5.51 millions)
Total Number of HQ Reads 1*
5487966 (5.49 millions)
Percentage of HQ Reads
99.683%
Total Number of Bases
1273865252 bases
Total Number of Bases in Mb Total Number of HQ Bases 2*
1273.86525 Mb 1268504974 bases
Total Number of HQ Bases in Mb
1268.50497 Mb
Percentage of HQ Bases
99.579%
Table: 5.6 Alignment statics of ST-500 in comparison with S. thermophilus MNZLW-002 and S. thermophilus MTCC_5461 Alignment Statistics : Sample ST500 Sample: ST500
S. thermophilus MN-ZLW002
S. thermophilus MTCC_5461
Total number of reads
837186
836570
Total number of reads aligned
779518
724777
93.11
86.64
Reference sequence length (bp)
1848520
1619896
Reference sequence covered (bp)
1848467
1598418
Percentage 1X coverage
100.00
98.67
Percentage 5X coverage
99.76
97.52
Percentage 10X coverage
98.71
96.41
Percentage 15X coverage
96.58
94.49
Percentage 20X coverage
93.38
91.55
Average Read Depth
66.26
67.52
%Reads aligned to reference
GOAL:To identify lactic acid genes (at protein level) in the sequenced sample ST500 METHODS:1 Predicted proteins of Sample ST500 were subjected to homology search (BLAST) against Uniprot bacterial proteins (reviewed) involved in lactic acid metabolism. 2 BLAST output were filtered to pass 30% Identity and 30% Query*/Subject** coverage *Query: Predicted proteins **Subject: Bacterial Lactic Acid proteins (Uniprot reviewed) Results: Total Predicted Protein
2912
Total Predicted proteins passing the filters
907
Chapter 6 Media Standardization 6.1
NUTRITIONAL REQUIREMENTS OF BACTERIA The ultimate expression of microbial organization is the self replication. The
chemicals from the environment that are utilized for bacterial growth are called nutrients. During anabolism, nutrients are taken up and are changed into cell constituents in an energy depending process. Different nutrient groups exist and are broadly classified into those providing energy such as carbohydrates, and those used as components in cellular structures such as peptides. Most of the microorganisms require an organic compound as their carbon source including carbohydrates, peptides or amino acids, fatty acids, organic acids, nitrogen bases and aromatic compounds (Ganzle et al., 2007; Brant et al., 2006 ). LAB has numerous nutritional requirements for growth, especially nitrogen sources. The general assumption is that biomass synthesis in lactic acid bacteria is predominantly from building blocks present in the culture medium. The most important application of LAB is their use as starter strains in the manufacture of various fermented products. LAB are fastidious microorganisms that require an exogenous source of amino acids or peptides, which are provided by the proteolysis of the proteins present in raw material. Nitrogen is the second most abundant element in the cell after carbon. A typical bacterial cell is about 12% nitrogen (by dry weight) and it is a main constituent of protein, nucleic acids, and several other constituents in the cell.
Medium optimization generally involves determining the appropriate nutrients and establishing the concentrations of these nutrients that result in maximum levels of biomass or targeted microbial products. Due to a lack of various biosynthetic pathways, lactic acid bacteria generally require media rich in nutrients. A rich nutrient environment can be provided by complex media comprised mostly of complex components (such as yeast extract, peptone, or tryptone), by semi-defined media formulated mostly with defined chemicals (except for one or two complex nutrients), and by CDMs (synthetic
media) containing no complex components (Zhang and Greasham, 1999). Although in most cases complex and semi-defined media provide greater biomass yields than CDMs, using these types of media in physiological studies focusing on metabolism and regulation makes data more difficult to interpret (Cocaign et al., 1995). For this reason, a CDM that supports reasonable cell growth can be very useful in studies of gene regulation, protein expression, and metabolic fluxes. By systematically adding or removing components from a CDM formulation, the specific nutritional and regulatory requirements for growth and targeted metabolic pathways can be determined. The cheaper sources of media are more useful for commercial production or mass production of bacteria. Among the ten isolated strains, ST-500 was observed with potential for commercialization on the basis of its performance in dairy applications and was selected for experiments on media standardization. Lactic acid production have been studied under the control of various operating factors and media components with the objective to achieve high product concentration and low fermentation cost 6.2
EFFECT OF DIFFERENT SOURCES OF NUTRITION ON ST-500 (STREPTOCOCCUS THERMOPHILUS NCIM 5539)
6.2.1
Carbon Sources The lactic acid bacteria for industrial process have to metabolize the
carbohydrates into optically pure lactic acid through homo-fermentative pathway and required a simple medium composition. Moreover, S. thermophilus could also produce lactic acid with a high cell mass yield if the appropriate carbohydrates are used as carbon sources. The homo-fermentative characteristics of carbohydrate metabolism of S. thermophilus were identified. Lactic acid fermentation by using various carbohydrates as the main carbon source were investigated in modified MRS broth containing 10, 20, 30, 40 g/L of the different carbon sources: lactose, sucrose, maltose, xylose, dextrose and fructose, respectively (Yun et al.,2003). Several carbon sources were tested in order to get the suitable carbon source for cell mass production. First set of experiment was started with 10 g/L of dextrose, sucrose, maltose, xylose, lactose and fructose individually with modified MRS broth contents (Figure 6.1A).
Figure 6.1:
(A) Influence of carbon sources on lactic acid fermentation for mass cell production supplied with modified MRS broth and 1% of the selected carbon sources. All the carbon sources were tested indiviually with their 10g/L content in media.
Figure 6.1:
(B) Influence of carbon sources on lactic acid fermentation for mass cell production supplied with modified MRS broth and 2% of the selected carbon sources. All the carbon sources were tested indiviually with their 20g/L content in media.
Figure 6.1: (C) Influence of carbon sources on lactic acid fermentation for mass cell production supplied with modified MRS broth and 3% of the selected carbon sources. All the carbon sources were tested indiviually with their 30g/L content in media
Figure 6.1:
(D) Influence of carbon sources on lactic acid fermentation for mass cell production supplied with modified MRS broth and 4% of the selected carbon sources. All the carbon sources were tested indiviually with their 40g/L content in media
Prepared media was inoculated and incubated for 24 hours at 42 °C. The experiment was conducted in duplicates and media samples were tested after 24 hours, fresh cell mass calculation, optical density and percentage yield of lactic acid was recorded. The maximum cell growth and the highest lactic acid concentration were obtained after 24 h of the fermentation time. These experiments were operated with non controlled pH: therefore; pH of fermentation broth rapidly decreased due to accumulation of lactic acid. Interestingly, sucrose showed better results than lactose and other carbon sources with more than 2% fresh cell mass and 34% lactic acid yield (Figure 6.1C). Sucrose was followed by lactose with 1.6% cell mass yield and 26% lactic acid yield. Other carbon sources including dextrose, xylose, maltose and fructose showed 1.4%, 0.8%, 1.1% and 1.2% cell mass yield, respectively.
All the carbon sources were increased in quantity with modified MRS broth. The experiments were conducted in duplicates and observed that sucrose showed much better result compared to other carbon sources. Both fresh cell mass yield and lactic acid yield was increased (Figure 6.1D). There was a slight fall in growth in trials when there was more than 4% sucrose was added in media. A fresh cell mass yield of 3.4% was achieved with 2% sucrose content which was increased to 4.5% with 3% sucrose and 5.2% with 4% sucrose content in media. Percentage concentration of lactic acid was also recorded and a symmetrical increase was observed with the increase in cell mass viz. 44%, 55% and 81% lactic acid with 2, 3 and 4% sucrose content, respectively. Lactose also played a crucial role in maintaining viable cell mass with 2.3%, 3.1%, and 3.8% fresh cell mass yield with 2, 3 and 4% lactose in media. Other carbon sources were found to be unsuitable for fast multiplication and fermentation of S. thermophilus. The percentage cell mass yield for xylose, dextrose, fructose and maltose was 1.8%, 2.9%, 2.8% and 2.7%, respectively. As a result, the suitable carbon source for (ST-500) S. thermophilus was in the order of sucrose > lactose > dextrose > fructose > maltose > xylose respectively (Figure 6.1D).
6.2.2
Effect of Nitrogen Sources
Inexpensive and effective nitrogen source is also necessary to reduce the fermentation cost. Supplementation of nitrogen source into the fermentation medium is important in order to maintain quality high yield. Peptone was used as the sole nitrogen source to improve the nutritional quality of the fermentation medium. Peptone contains growth promoting compounds in addition to organic nitrogenand along with other carbon compounds.
Effect of various nitrogen sources supplemented in modified MRS broth containing 10, 20, 30, 40 g/L of tryptone, proteose peptone, beef extract, meat extract, soya peptone and casein peptone were used and the result obtained are presented in Figure 6.2 A. Higher supplementation of peptide nitrogen greatly influenced the cell multiplication and fermentation. Considering this fact, special attention was provided to the maximum and minimum limits of the sources to make an efficient media for optimal growth. First set of experiment was started with 10 g/L of tryptone, proteose peptone, beef extract, meat extract, soya peptone and casein peptone individually with modified MRS broth contents. Prepared media was inoculated and incubated for 24 hours at 42 °C. The experiment was conducted in duplicates and media samples were observed after 24 hours by their optical density, fresh cell mass calculation and percentage yield of lactic acid.
The LAB are able to respond to changes in nitrogen availability by regulating the activity of the proteolytic system to ensure proper nitrogen balance in the cell. It was found that the synthesis of many exoproteins influenced, in part at least, by the level of individual nutrients in the extracellular environment (Rollan et al., 1998). Cell multiplication rate and fresh weight was observed higher in tryptone (casein enzyme hydrolysate) and casein peptone compared to other nitrogen sources. A cell mass yield of 4.1% with 1% tryptone was observed which was increased to 5.1% with 2%, 6% with 3% and 7.6% with 4% tryptone in media, (Figure 6.2 D) while casein peptone produced 3.4%, 4.4%, 5.3% and 6.6% with 1, 2, 3 and 4% concentration, respectively.
Figure 6.2:
(A) Effect of nitrogen sources on lactic acid fermentation for mass cell production supplied with modified MRS broth and 1% of the selected nitrogen sources. All the nitrogen sources were tested indiviually with their 10g/L content in media
Figure 6.2:
(B) Effect of nitrogen sources on lactic acid fermentation for mass cell production of supplied with modified MRS broth and 2% of the selected nitrogen sources. All the nitrogen sources were tested indiviually with their 20g/L content in media
Figure 6.2:
(C) Effect of nitrogen sources on lactic acid fermentation for mass cell production supplied with modified MRS broth and 3% of the selected nitrogen sources. All the nitrogen sources were tested indiviually with their 30g/L content in media
Figure 6.2:
(D) Effect of nitrogen sources on lactic acid fermentation for mass cell production supplied with modified MRS broth and 4% of the selected nitrogen sources. All the nitrogen sources were tested indiviually with their 40 g/L content in media
This reflected the complex nutritional demand of this fastidious lactic acid bacterium due to its limited biosynthesis capacity. The percentage cell mass yield
observed for proteose peptone, meat extract, beef extract and soya peptone was 4%, 4.1%, 4.6% and 4.3%, respectively. The suitable carbon source for (ST-500) S. thermophilus was in the order of tryptone > casein peptone > beef extract > meat extract > soya peptone > proteose peptone respectively (Figure 62 D).
6.2.3
Effect of Vegetable protein and Whey protein sources
Whey, a biological by-product of the cheese industry is usually disposed as waste. It is a source of functional valuable proteins. Addition of 1 or 2% of a whey protein concentrate (WPC) to a whey-based medium used for fermentation with Lactobacillus delbrueckii subsp. bulgaricus 11842 or Streptococcus thermophilus ST 20 has produced significantly higher bacterial count and much faster acidity development than the control whey or whey UF permeate media. Industrial whey fractions of • -lactalbumin or β lactoglobulin used as nutrient supplements in the same concentrations as the WPC resulted in smaller but still noticeable effects (Bury et al., 1998). Vegetable protein sources are also useful in the medium to provide the content of amino acids required by bacterial cells for multiplication. Common vegetable protein sources used in bacterial mass production medium are soy protein and wheat peptones. Milk protein sources include a number of proteins like whey proteins concentrate, sweet whey powder, skim milk powder, casein proteins etc. The protein concentrations were autoclaved separately and mixed at the time of inoculation with the other ingredients of modified MRS. Selected protein sources were: whey proteins concentrate, sweet whey powder, soy protein, wheat peptones, casein proteins and vegetable casein protein. 2% and 4% quantity was selected for all the protein sources and results of the study revealed that whey protein concentrate is a much better option to carry forward followed by casein protein showing good results with 2% content in media (Figure 6.3A). Whey protein concentrate showed 4.5% cell mass yield and 30% lactic acid yield with 2% concentration and 5.7% cell mass yield and 40% lactic acid yield with 4% concentration in medium. Casein protein showed 4.8% cell mass yield and 34% lactic acid yield with 2% concentration and 5.6% cell mass yield and 39% lactic acid yield with 4% concentration in medium.
Figure 6.3:
(A) Effect of vegetable protein and milk protein sources on lactic acid fermentation for mass cell production supplied with modified MRS broth and 2% of the selected sources. All the sources were tested indiviually with their 20g/L content in media
Figure 6.3:
(B) Effect of vegetable protein and milk protein sources on lactic acid fermentation for mass cell production supplied with modified MRS broth and 4% of the selected sources. All the sources were tested indiviually with their 40g/L content in media
The percentage cell mass yield observed for sweet whey powder, soy protein, wheat peptone, veg casein peptone was 4.8%, 4%, 2.2% and 3.7% respectively. As a
result, the suitable source for (ST-500) S. thermophilus was in the order of whey protein concentrate > casein protein > sweet whey powder > soy protein > veg casein peptone > wheat peptone respectively (Figure 6.3 B).
6.2.4
Effect of Yeast Extract, Buffers and other salts used
Normally, commercial yeast extract possesses 18 essential elements such as Al, Ba, Cd, Co, Cr, Cu, Fe, Ga, Mg, Mn, Mo, Ni, Pb, Sn, Sr, Ti, V, and Zn, respectively and is found to be essential for growth.
Figure 6.4: Influence of sodium, potassium, ammonium and magnesium salts on lactic acid fermentation for mass cell production supplied with modified MRS broth and were tested indiviually with their mentioned content in media. Moreover, it also contains nutritious substances such as amino acids, peptides, vitamins, and several organic acids including pyruvic and glyceric acid (Jiang et al., 2010). Initially, the influence of yeast extract concentrations on lactic acid fermentation and cell mass production from modified MRS medium were investigated by supplementing with the concentration between 1-6 g/L, lactic acid fermentations were performed using 250 mL shake flasks at 30 °C with pH 6.0. At 1 g/L of yeast extract, lactic acid fermentation was not completed, and the cell growth was almost stopped after
12 h because the nutrient was depleted. On the other hand, the maximum lactic acid productivity and cell mass was obtained at 5 g/L whereas fresh cell mass weight (28 g/L) was obtained at 6 g/L of yeast extract showing a decrease in cell mass production compared to 5g/L yeast extract which was 33g/L (Figure 6.5).
Figure 6.5:
Influence of yeast extract (amino acid source) on lactic acid fermentation for mass cell production supplied with modified MRS broth and were tested indiviually with 1g/L to 5g/L content in media.
The increase of yeast extract concentration between 4 and 6 g/L had very little effect on increasing the lactic acid production. Bacterial Cells were also provided with sodium, potassium, ammonium and magnesium for their survival. Di potassium hydrogen sulphate, hydrogen potassium suplhate, sodium acetate, tri-ammonium citrate, magnesium sulphate, manganous sulphate were added to modified MRS broth in different concentrations. Cell mass of 4.6% along with lactic acid percentage of 60% was obtained from 5 g/L sodium acetate followed by 2g/L K2HPO4 with 4.5% fresh cell mass yield along with 55% of lactic acid yield (Figure 6.4). It was observed that all the buffers had its own role in media with different conditions provided to the cells and media. Based on the experimental research conducted on different ingredients in different concentrations,
media formulations were designed and tested for combined action of ingredients with standardized concentrations.
Table: 6.1 Results of different media compositions concluded after individual studies of media ingredients for mass cell production of Streptococcus thermophilus NCIM 5539
Media Designation 1 2
3
4
5
6
Nutrient Supplementation (g/L) Yeast Extract 5g/L, Tryptone 20g/L, Sucrose 30g/L, K2HPO4 2g/L Yeast Extract 5g/L, Tryptone 20g/L, Sucrose 30g/L, Lactose 10g/L, K2HPO4 2g/L Yeast Extract 5g/L, Tryptone 20g/L, Sucrose 30g/L, Lactose 10g/L, Sodium acetate 5g/L, K2HPO4 2g/L Yeast Extract 5g/L, Tryptone 20g/L, Sucrose 30g/L, Lactose 10g/L, WPC 10g/L, Sodium acetate 5g/L, K2HPO4 2g/L Yeast Extract 5g/L, Tryptone 20g/L, Sucrose 40g/L, Lactose 15g/L, Sodium acetate 5g/L, K2HPO4 2g/L, MgSO4 0.1g/L. Yeast Extract 5g/L, Tryptone 20g/L, Sucrose 40g/L, Lactose 20g/L, Sodium acetate 5g/L, K2HPO4 2g/L, MnSO4 0.05g/L
Fresh Cell Mass yield g/L
Lactic acid concentration g/L
61.22 ± 0.15
78.62 ± 0.16
63.67 ± 0.22
79.99 ± 0.19
64.29 ± 0.13
81.22 ± 0.23
53.78 ± 0.11
67.47 ± 0.15
73.37 ± 0.26
88.34 ± 0.07
68.26 ± 0.11
84.32 ± 0.17
Media 5 was selected for further studies as the output observed from this media was far better compared to other minimal media and readymade media available in market. Media 5 was also observed better compared to MRS and M17 broth media for the identified isolated strain.
6.3
INFLUENCE OF FACTORS ON LACTIC ACID BACTERIA FERMENTATION
6.3.1
Effect of various temperatures on cell mass production
Temperature adaptation of bacterial growth under different temperature regimes was studied. Understanding of the optimal temperature required on lactic acid fermentation will facilitate improvement of the production process. The temperature were monitored and controlled during the fermentation process at 30, 37, 42, 45 and 50°C with fermentation time 30 h as shown in Figure 6.6. In industrial fermentation processes, the operating temperature of the fermenter is often raised to optimum level to increase microbial activity depending on the characteristics of the microorganism used along with the other environmental conditions. The cell count showed a similar trend. The results indicated that temperature had a pronounced influence on the growth of these microorganisms during lactic acid fermentation. Moreover, the temperature affects the rate of biochemical reactions, the activity of extracellular enzymes, the generation time, and the activity of the microorganisms involved. .
Figure 6.6:
The influence of temperature on cell mass yield on self designed Media 5 by Streptococcus thermophilus NCIM 5539
The optimum temperature and time for high cell mass yield was 42°C for 24 hours on the basis of cell mass obtained by centrifugation and absorbance of the samples. Temperature is the most important factor on nutrient utilization and cell viability. Higher temperature stimulated the rapid growth of lactic acid bacteria resulting in a rapid decline
in pH, and consequently suppressed the growth of S. thermophilus. Although, S. thermophilus could grow at 50 °C, the optimum temperatures for its growth were observed between 40-45 °C. Some literatures reported that the rate of reaction for microorganism increases with increasing temperature until a limiting maximum temperature is reached (55°C), after which the growth rate decreases very rapidly (Peleg, 1996).
However, when the temperature of the medium is above or below that required for optimum growth, the microbial activity is substantially reduced and the organisms may eventually die. The pH value of the fermentation broth decreased from 6.0 to values less than 4.5 as fermentation proceeded reaching conditions unfavorable for cell growth. Cell population was a measure of the increase in biomass over time and it was determined from the exponential phase. Growth rate was one important way of expressing the relative ecological success of a species or strain in adapting to its natural environment. The duration of exponential phase in cultures depended upon the size of the inoculums, the growth rate and the capacity of the medium and culturing conditions to support cell growth. At 37 °C and 42 °C, the yield and productivity of lactic acid were also similar. The production yield and volumetric productivity were calculated with the time at the exponential phase of the cell growth. It was, therefore, important that the fermentation temperature must be maintained as stable as possible since bacteria grow optimally within a narrow temperature range. In general, subsequent experiments were conducted at 42 °C in order to reduce the cost of electricity of the production process. For this reason, the control of temperatures were necessary during lactic acid fermentation for lactic acid production.
6.3.2
Effect of pH on cell mass production The effect of different pH
on cell growth, and substrate metabolism were
performed in batch fermentation. The initial pH of the medium was maintained at 4.0, 5.0, 6.0, 7.0, or 8.0, respectively, with 5M NH4OH and 1N Hydrochloric acid after the pH dropped to these points due to the production of lactic acid. Fermentation was completed
in 18 h with complete utilization of glucose. In addition, different fermentation parameters viz. pH in relation to lactic acid production were studied (Figure 6.7)
Figure 6.7:
The influence of initial set point of pH on cell mass yield on self designed Media 5 by Streptococcus thermophilus NCIM 5539
Figure 6.8:
Reducing sugar concentrations (Sucrose) during batch fermentation of Streptococcus thermophilus NCIM 5539
This was in accordance since the μmax obtained early in the fermentation where the lactic acid concentration was very low. There are some reportes where low level of biomass and lactic acid concentration were synthesized at pH values lower to 5.5 (Bai et al., 2004) which could be due to partial loss of the enzymatic activity involved in the biosynthesis of biomass and lactic acid. Our results showed the high level of biomass and lactic acid when pH was maintained in the pH range 5.0-7.0 with the optimum at pH 6.0. Further, at each pH value, the reciprocal of the productivity of lactic acid was linearly correlated to the specific growth rate. Maximum fresh cell mass was obtained from initial pH range of 6-7 with a maximum cell yield of 70-75 g/L. The cell mass obtained for the different pH values did not present significant variations (Goncalves et al., 1997). The optimum pH for cell growth of S. thermophilus appeared to be 6.0; lactic acid fermentation at pH 6.0 was completed faster than other pH. The microorganism, especially, lactic acid bacteria, did not alter its metabolic pathway in the pH range of 5.07.0. Increase in initial pH beyond 6.5 did not improve the lactic acid production. The higher initial pH brings excessive stress on the microorganism metabolic abilities (Vijayakumar et al., 2008). The lactic acid productivity has been considered as one of the critical factors for economical lactic acid fermentation (pH 6.0). S. thermophilus appears to be suitable LAB for further study on lactic acid fermentation at pH 6.0 and temperature 42 °C.
6.3.3
Effect of various agitation speeds on cell mass production Agitation is important for adequate mixing, mass transfer and heat transfer,
respectively. It assists not only the mass transfer between the different phases presented in the culture, but also maintains homogeneous chemical and physical conditions in the culture by means of mixing, eliminating gradients of concentration. However, at high agitation speed the biomass concentration may decrease because of cell damage, cell loss by the impeller and shear force (Senthuran et al., 1999), while at low agitation speed, the liquid or fermentation broth could not homogeneous mix well and the substrate could not be utilized completely.
The effect of agitation speed on cell mass production were studied at three different agitation speeds (100, 200, 300 rpm). The influence of cultivation factors on the lactic acid fermentations was observed for cell growth, substrate and product concentrations, respectively. 100 rpm agitation speed significantly affected the fermentation performance.
(A)
(B)
(C)
Figure 6.9:
6.4
Effect of agitation speed on the cell growth, reducing sugar concentration and mass production by Streptococcus thermophilus NCIM 5539: 100 rpm (A), 200 rpm (B), and 300 rpm (C).
MEDIA COSTING
Interest in the physiology of lactic acid bacteria has been stimulated by the industrial importance of these bacteria and the potential use of genetic engineering in strain optimization. For metabolic investigations, a defined growth medium for these bacteria is desirable which (i) supports growth at a reasonably high rate and (ii) allows for exponential growth over a wide range of cell concentrations. Fermentation medium can represent almost 30% of the cost for a microbial fermentation (Hofvendahl and Hahn-Hagerdal, 2000). Complex media commonly employed for growth of lactic acid bacteria are not economically attractive due to their high amount of expensive nutrients such as yeast extract, peptone and salts (Jensen and Hammer, 1993).
Cost of media ingredients is one of the major hurdles which are continuously creating a barrier for dairy industries to launch their starter cultures in market. In current study, it was observed that the isolated strains were not performing well on economical ingredients of media. The cost of ingredients based on the composition of MRS or M17 was also too high.
Himedia, India was selected for price comparison of media
ingredients because of its consistent product quality and easy availability. Few other media ingredient vendors were also tested for their availability and price like •
Qualigens Fisher Scientific, India
•
Rankem Synergy Scientific, India
•
Sigma-Aldrich, United States
•
Central Drug House (CDH) Chemicals, India
•
Merck, Germany
There is a minor difference in the prices of media ingredients locally available in India, but chemicals from Sigma-Aldrich and Merck were observed to be on higher side for cost with low ratio of availability in stock. MRS and M17 are well known medium for cultivation and fermentation of lactic acid bacteria (Khedid et al., 2009). The costing of the media was compared between MRS and M17.
Table: 6.2 Media per litre cost comparison between MRS and M17 on individual ingredient basis from Himedia, India catalogue. MEDIA COMPONENTS
MRS
M-17
Price/ Kg for MRS
Price/ Kg for M17
2.5g/L
Catalogue Price for Himedia 2013-14 3000/ Kg
Yeast Extract
5g/L
15/-
7.5/-
Protease Peptone
10g/L
Nil
3580/ Kg
35.8/-
Nil
Dextrose
20g/L
Nil
450/ Kg
9/-
Nil
Lactose
Nil
Nil
650/ Kg
Nil
Nil
Sucrose
Nil
Nil
250/ Kg
Nil
Nil
Beef Extract
10g/L
5g/L
3300/ Kg
33/-
16.5/-
Sodium Acetate
5g/L
Nil
418/ Kg
2.09/-
Nil
Ammonium Citrate Di Potassium Phosphate Magnesium Sulphate Manganese Sulphate Ascorbic Acid
2g/L
Nil
1892/ Kg
3.78/-
Nil
2g/L
Nil
830/ Kg
1.66/-
Nil
0.1g/L
0.25g/L
360/ Kg
0.036/-
0.054/-
0.05g/L
Nil
780/ Kg
0.39/-
Nil
Nil
0.5g/L
5700/ Kg
Nil
2.85/-
Agar
12g/L
15g/L
4676/ Kg
55.75/-
69.68/-
Disodium Glycero Phosphate Casein Peptone
Nil
19g/L
13130/ Kg
Nil
249.47/-
Nil
2.5g/L
3870/ Kg
Nil
9.67/-
Soya Peptone
Nil
5g/L
4064/ Kg
Nil
20.32/-
Meat Peptone
Nil
5g/L
3550/ Kg
Nil
17.75
TOTAL PER LITRE COST OF MRS & M17 AGAR
156.50/-
393.79
TOTAL PER LITRE COST OF MRS & M17 BROTH
100.75/-
324.11/-
Both the media are not economically viable for commercial production of starter culture. In order to reduce the production cost, the media was modified to increase cell mass. Several experiments were conducted for optimization as per variation in methods described in Chapter 5. We developed a medium, self optimized media MEDIA 5 (M5),
and the cost was calculated on the basis of prices/Kg of different ingradients from Himedia (Table 6.3). The cost per ingredient provided in Himedia catalogue was for laboratory scale experimental use only.
Table: 6.3 Costing of the designed media on the basis of ingredient prices available with Himedia S. No.
Media Ingredient
Amount
Catalogue Price from
g/L
Himedia 2013-14
Price / L
1
Yeast Extract
5g/L
3000/ Kg
15/-
2
Sucrose
40g/L
250/ Kg
10/-
3
Lactose
15g/L
650/ Kg
9.75/-
4
Sodium Acetate
5g/L
418/ Kg
2.09/-
5
Tryptone
20g/L
4728/Kg
94.56/-
6
K2HPO4
2g/L
830/ Kg
1.66/-
7
MgSO4
0.1g/L
360/ Kg
0.036/-
TOTAL PER LITRE COST OF MEDIA 5
133.09/-
Himedia was later contacted for their commercial prices. Surprisingly, the total cost per litre for media 5 was reduced to almost 50% by new prices of ingredients from Himedia (Table 6.4). Still other Indian suppliers of media ingredients were contacted for their samples in order to reduce the cost of the media. Suppliers contacted were: •
Chaitanya Chemicals, Maharashtra, India
•
Titan Biotech, Delhi, India
•
Warkem Biotech, Mumbai, India
Samples from these companies were tested on the designed parameters for their efficiency to produce cell mass along with the price evaluation and found that Warkem Biotech, Mumbai, India has low commercial price with better quality of raw material. Per Litre media cost from Warkem was around INR 55±2 with 60g/L fresh mass yield.
Table: 6.4 Costing of the designed media on the basis of commercial prices provided by Himedia S. No.
Media Ingredient
Amount g/L
Commercial Price from Himedia 2013-14
Price / L
1
Yeast Extract
5g/L
1550/ Kg
7.75/-
2
Sucrose
40g/L
125/ Kg
5/-
3
Lactose
15g/L
450/ Kg
6.75/-
4
Sodium Acetate
5g/L
336/ Kg
1.68/-
5
Tryptone
20g/L
2410/Kg
48.2/-
6
K2HPO4
2g/L
680/ Kg
1.36/-
7
MgSO4
0.1g/L
274/ Kg
0.027/-
TOTAL PER LITRE COST OF MEDIA 5
70.76/-
The prices of media ingredients from Warkem Biotech, India were also evaluated and implemented on the compositions of MRS and M17 media. Some difference was observed between the cost/L of MRS and cost/L of media 5 (Figure 6.10) but huge difference in overall fresh weight was observed in both media. The morphology and characterization of ST-500 was also critically studied to notice the impact of new ingredients and performance of the same strain in its dairy applications.
Figure 6.10:
Price & Fresh cell mass weight g/L comparison of media from different vendors.
Figure 6.11: Price & Fresh cell mass weight g/L comparison of media M17, MRS and Media 5 from same vendor. The overall performance of the strain ST-500 was studied with new nutritional ingredients and it was observed that highest cell mass of 65g/L was obtained from ingredients of pilot scale (Laboratory grade) due to their content purity. Price variation will depend on the size and quality of packaging. Himedia, India are very economical (Figure 6.11) as compared to lab grade ingredients. But the issue with price was still there. The commercial prices of Chaitanya Chemicals, Maharashtra, India, Titan Biotech, Delhi, India and Warkem Biotech, Mumbai, India were tested. The individual ingredient price issue was not mentioned due to some confidential issues with the dealer. But the final price was calculated to be INR 55 and the yield of fresh cell mass obtained from the same was 58 g/L.
6.5
BATCH FERMENTATION OF ST-500 AND PROCESS STANDARDIZATION In a bioprocess development, two main streams involved are upstream and
downstream processes. The work focused on multiplication and mass production of cell mass from fermentation broth. ST-500 (Streptococcus thermophilus) was successfully isolated and characterized in the laboratory. The time course of pH, cell concentration, reducing sugar concentration, and lactic acid concentration during fermentation were studied. Batch fermentations was first performed in 2 and 5 L fermenters (Biostat A plus, Sartorius stedim) with working volume 1.5 and 4 L, respectively. Up-scaling was standardized in 1.5 L and 4 L media in lab scale. M5 was prepared by mixing all the ingredients uniformly and autoclaved within the fermenter vessel (Figure 6.12, 6.13, 6.14 and 6.15). 5N sodium hydroxide and 1N Hydrochloric acid was prepared for maintaining the pH balance during fermentation. 10% (v/v) inoculum grown in M5 broth for 24 h was aseptically inoculated into the fermenter. Turbidity in batch was observed after 8 hours (Figure 6.16) and regularly monitored up to 24 hours followed by batch closing and cell mass collection in sterile flasks (Figure 6.17, 6.18 and 6.19).
The resulted standardized conditions were provided during the batch. Standard conditions provided were:
Set temperature
:
42º C
Set RPM
:
200
Set pH
:
6.5
Inocolum
:
10%
Base for pH
:
5N Naoh
Acid for pH
:
1N Hcl
Antifoam
:
Not Required
Sterility
:
Fermenter was autoclaved at 15 psi
Sucrose concentration rapidly decreased during the first 16 h followed by a much slower decreasing rate. Utilization of sucrose nearly ceased after 24 h of operation with the remaining concentration of less than 1 g/L. It was observed that S. thermophilus possessed a relatively short lag phase followed by exponential phase. The growth entered its stationary phase at 22 h where cell concentration reached plateau of approximately 64 g/L until the end of the fermentation process (Figure 6.22). Amount of acid-base consumption during the batch run was also recorded by Batch Master Software (Sartorius stedim) and a history plot of the batch was created for evaluation (Figure 6.20).
Cell multiplication rate of ST-500 in 5 L fermenter was observed higher compared to 2 L fermenter, by analyzing OD at 660 nm after every four hour interval (Figure 6.21). Controlled fermentation operation offer special advantages over uncontrolled fermentations in shaker incubators where the pH drops continuously inhibiting the cell growth after 12-14 hours of incubation, where as in fermenter the pH was regularly maintained by continuous feed of buffers.
Figure 6.20: History plot of the 5 L batch of Streptococcus thermophilus showing the change in pH and consumption of acid and base for maintaining the set pH balance.
Figure 6.21: Fermentation profile of ST-500 recorded on the basis of OD at 660nm
Figure 6.22: Batch fermentation profile of ST-500 in 5L fermenter showing cell mass concentration, lactic acid percentage and reducing sugar concentration
On the basis of results from 2L and 5L vessel size fermentation processes, the up-scaling was done on 50L and 300L fermenters. 240 L media was prepared in sterile buckets followed by steam sterilization of vessel and media at 121°C (Figure 6.24). The plant steam pressure was maintained at 3.5 kg/ cm2. 50L fermenter was utilized as seed fermenter to prepare inocolum which was directly transferred to 300L fermenter. 1 micron steam filters were used to maintain aseptic conditions during sample collection between the run. The major issue is to maintain sterility of the batch as a minor chance of contamination can lead to batch failure. The optical density was recoreded to a maximum of 1.324 (Figure 6.25) which was comparatively much higher than 0.934 recorded in 5 L batch at Lab scale.
Figure 6.25:
Fermentation profile of ST-500 recorded on 300L fermenter on the basis of OD at 660nm
Upstreaming of ST-500 on different vessel sizes (2L, 5L, 50L and 300L) of fermenter was evaluated on the basis of final cell mass (g/L), OD, lactic acid content and cell viability. Maximum cell mass and maximum OD was obtained from 300L vessel but maximum cell viability was observed in 5L vessel size of fermenter (Table 6.5). Cell viability and OD from 5L vessel was further explored on the basis of incubation hours (Table 6.6).
Table: 6.5 Comparison of Batch results with complete up-stream analysis of ST-500 Particulates
500 mL Flask
2L Fermenter
5L Fermenter
50L Fermenter
300L Fermenter
Media
Media 5
Media 5
Media 5
Media 5
Media 5
Final pH on Batch closing
4.7
6.54
6.58
6.47
6.42
End Sugar Content
0.82%
0.37%
0.28%
0.22%
0.15%
23%
52%
55%
64%
71%
12g
44g
47g
67g
72 g
Optical Density
0.317
0.911
0.946
1.236
1.267
Cell Viability (CFU/mL)
776×105
790×105
825×105
812×105
817×105
Base consumption
N/A
60 mL
136 mL
1032 mL
8026 L
Acid Consumption
N/A
4 mL
9 mL
120 mL
1200 mL
Lactic acid Percentage Fresh Cell Mass g/L
Table: 6.6 Cell growth rate with respect to incubation hours Incubation hours
Maximum OD at 660nm
Colony forming units per mL sample
8
0.634
339×105
12
0.694
371×105
16
0.755
489×105
20
0.841
592×105
24
0.946
825×105
CFU of the inocolum tube recorded by 285×105, 0.2% EDTA was blank for OD
6.6
DOWN STREAM PROCESSING OF ST-500
Cells were sedimented by centrifugation and high speed of centrifugation cause some cells to loose viability. It was, therefore, thought appropriate to standardize centrifugation to minimize the cell loss. It was observed that 5000 rpm is appropriate with CFU of 7.56×107 and viability loss of 2×106. The batch was centrifuged at 5000 rpm for 10 minutes followed by washing of the pellet with normal saline at 5000 rpm for 5 minutes. Washing also cause viability loss of 1.1×106. A total loss of 3.1×106 cell viability during centrifugation was obsereved. 6.6.1
Lyophilization All the compounds used as lyoprotectants in this study were found to be effective
in most of the cases, providing protection to various lactic acid bacteria (Carvalho et al. 2004). Eighteen lyoprotectants were used separately as medium to provide protection from high vacuum and freeze shock during lyophilization along with a blank sample. Skim milk powder was used in sample preparation before freezing because of its property of preventing cellular injury by stabilizing the cell membrane constituents and creating a porous structure in the freeze dried product (Castro et al., 1996; Salmer-Olsen et al., 1999). Skim Milk also contains proteins that provide a coating to the bacterial cells (Abadias et al., 2001). On testing the viability of S. thermophilus with skim milk (10% w/w) shows highest cell viability of 74% when observed in dry form. On the other hand sugar and sugar derivatives were also used for their protective effect during lyophilization and also during after lyophilization storage (Carvalho et al., 2002, 2003c). Sugar alcohol like sorbitol has found to be one of the strong protective agent during lyophilization and storage of L. bulgaricus, L. plantarum, L. rhamnosus, E. faecalis and E. durans (Carvalho et al., 2003c), but in current study, its performance as protective agent for lyophilization of S. thermophilus was dull and discouraging. Infect cheaper sugar sources like sucrose (72%), glucose (51%), lactose (66%) and maltodextrine (67%) showed better results compared to high cost sugars like mannitol (44%), fructose (45%), maltose (32%), ribose (39%) and arabinose (28%) as high cost compounds would likely to restrict their large scale industrial use. The ability of mono sodium glutamate (MSG) to protect microbial cells during lyophilization and cryopreservation is also described by several
workers (Font de Valdez et al., 1983; Martos et al., 1999). Use of 1% (w/w) MSG for S. thermophilus also showed protective effect with 57% viability, while as more than 1% quantity showed viability loss. Lyophilization of ST-500 with all the seven lyophilization media was conducted at 0.004 mbar vacuum pressure individually in plates and vials (Figure 6.27).
Table: 6.7 Combinations of protectants along with the ratio of addition Lyophilization Medium
Constituents in percentage
Proportion of addition (mL of cell inocolum : protective medium)
1
Skim Milk 10%, Sucrose 5%, Sodium Caseinate 5%
1:5
2
Skim Milk 10%, Lactose 5%, Whey Protein Concentrate 5%
1:7
3
Skim Milk 10%, Maltodextrine 5%, Sweet whey Powder 5%
1:5
4
Sodium Caseinate 10%, Skim Milk 5%, Maltodextrine 5%, Mono sodium glutamate 1% Sodium Caseinate 5%, Whey Protein concentrate 5% Maltodextrine 10%, Meso-inositol 0.5%, Mono sodium glutamate 1% Sodium Caseinate 5%, Lactose 5%, Mono sodium glutamate 1%, Skim Milk 5% Sodium Caseinate 10%, Skim Milk 5%, Sucrose 5% Mono sodium glutamate 1%
5
6
7
1:8
1:8
1:5
1:5
The effect of K2HPO4 and KH2PO4 on cell viability of B. bifidum has been studied by Qin et al. (2013) showing cell survival viability up to 77.80% with KH2PO4 and 79.82% with K2HPO4. But studying the effect of these compounds on S. thermophilus reveals opposite results with K2HPO4 (1% w/w) 21% and KH2PO4 (1% w/w) 17%. Milk proteins present in skim milk leads to stabilization of protein structures
via reactions between the amino group of the microbial cell proteins and with protectant. Other mixtures with casein protein and whey proteins were also tested revealing interesting results by providing high cell viability to S. thermophilus. Sodium caseinate resulted in highest viability of 81% while as whey protein concentrate (WPC) with 65% and sweet whey powder (SWP) 58% (Figure 6.26).
\
Figure 6.26: Effect of different lyoprotectants on the viability after lyophilization. If not other indicated 10% (w/w) solution were used. The error bars show the standard deviation A total of 40 combinations were prepared based on the results obtained on their role as individual protectant in providing the viability to S. thermophilus on lyophilization.
After initial studies combinations producing more than 50% viability were considered for further studies in which only 7 combinations were observed significant with high viability (Table 6.7). The ratio of addition was also studied and implemented on the basis of thickness and viscosity of the prepared lyophilization medium. Lyophilization medium (LM) 2, 4, 6 and 7 were found efficient in maintaining the cell viability both during freezing and drying state of lyophilization. LM 7 was observed as unique viability escalator providing better results compared to other lyophilization media (Figure 6.29). The reason behind the success of LM 7 is the role of protective milk proteins which are in abundance in this particular formulation playing a role in stabilizing the protein structures of this microbe and sugar source playing a crucial part in maintaining the physical state of the membrane lipids and enzyme level. All the protective compounds in lyophilization medium 7 have their unique role in maintaining the cell viability, also the medium is cost efficient and can easily be implemented on large scale industrial production of S. thermophilus for its role in starter culture. After final lab scale evaluation, lyophilization media 7 was used for lyophilization of 300 L batch at TBI (Technology Based Incubator), Delhi University with output of 82% viability (Figure 6.28).
Figure 6.29:
Effect of different Lyophilization media on the viability after lyophilization. The error bars show the standard deviation
Studies on lyophilization of lactic acid bacteria suggest that the stability of lyophilized cells decreases during storage at -8° C. Up to 5% viability loss was observed
on re-examining the stored vials of S. thermophilus after a period of 180 days. On the other hand lyophilized vials of the same microbe stored at -60° C showed higher survival rate with less than 1% viability loss. An organism which survives the various steps of freezing, drying and storage may, nevertheless, lose its viability during rehydration. Therefore, rehydration is a critical step in the recovery of freeze-dried microorganisms, because cells that were subjected to sub lethal injury may not be able to repair said damage if they are rehydrated under inappropriate conditions.
Table: 6.8 Viability loss during up-downstream bio-processing Process Measured with parameters
Colony forming units per mL sample
Maximum OD at 660nm
Viability Loss
Complete fermentation at optimized conditions
7.76×107
0.924
Nil
Centrifugation at 5000 (rpm) for 10 minutes
7.56×107
0.915
2×106
Centrifugal washing of pellet with saline water at 5000 (rpm) for 5 minutes
7.45×107
0.904
1.1×106
Pre-Freezing for Lyophilization at -60°C
7.39×107
0.899
6×105
Freeze drying at 0.04 mbar vacuum pressure
7.15×107
0.884
2.4×106
0.040
6.1×106
Total Viability Loss
Process viability loss during the up-stream and down-stream processing was also recorded by taking samples after regular intervals from fermentation to freeze drying. Inprocess, samples were diluted four times to equalize the proportion of fresh liquid culture and pellet. Optical density was recorded at 660 nm and CFU (colony forming units) were calculated by serially diluting the samples followed by pour-plate method and colony counting. An average viability loss of 1.5×106±2 was observed after each standard step process towards down-streaming with a total viability loss of 6.1×106 (Table 6.8). Cell
multiplication ratio in the final product was also recorded. It was observed that lyophilized culture with viability of CFU size of 5×109 was sufficient enough to work as an inocolum in 1L milk for curd preparation. On inoculation of 4 hours at 42°C the initial CFU of 5×109 was increased to a prebiotic level of 2×1014 with per curd CFU content in curd of 2×1011.
Chapter 7 Dairy Applications 7.1
DAIRY APPLICATIONS The present study relates to the dairy applications of bacterium Streptococcus
thermophilus. S. thermophilus is essentially a lactic acid bacterium widely used as a starter culture for the production of dairy products viz. production of hard cheese of Swiss type, soft cheese, and yogurt (Falentin et al., 2010; Hao et al., 2011). S. thermophilus is classified as a nonpathogenic, single streptococcus species to possess a generally recognized as safe status (Burton et al., 2006). It is also considered as “the second most important industrial dairy starter after Lactococcus lactis” (Mayo et al., 2008). Along with the use in manufacturing fermented food, streptococcus is reported to posses probiotic properties in adequate amount conferring a health benefit to the host (Labeer et al., 2008; Khalil 2009). As noted above, a healthy population of beneficial, mutualistic, and commensal microorganisms in the digestive tract plays a substantial role in maintaining the health and welfare of the host organism. Such microorganisms create benefits to their hosts in many ways through competition with pathogenic microorganisms, aiding in the digestion and absorption of food, helping with vitamin synthesis, and regulating immune responses. Therefore, healthy individuals often display a robust collection of beneficial microorganisms in their digestive systems, which aid them in maintaining a disease free state, and further contribute to the overall well being of the individual. Bacterial isolate ST-500 was tested for various dairy applications like curd, fermented milk, cheese, mishthi dahi and frozen fruit dessert. During milk fermentation, organic acids, mainly lactic acid, are produced by the microorganisms lowering the pH value. This acid production can be used to characterize milk fermentation and is therefore an important method to test the activity of starter cultures. Curd is an important part of Indian diet. In most Indian homes curd is prepared almost every day. Homemade curd is not only very simple to prepare but is also delicious.
The isolate was tested for following applications: •
Curd
•
Yogurt
•
Flavored yogurt
•
Mishti Dahi (Sweet Curd)
•
Butter Milk
•
Fruit dessert
•
Yogurt Softy
•
Cheese
7.1.1
Application testing for curd, yogurt, butter milk, mishti dahi
Pasteurized milk was allowed to cool up to 42°C followed by stirring the milk slowly to distribute the culture organism (ST-500) uniformly throughout the milk and the inoculated milk was poured into 100 mL capacity sterile plastic cups, which were then incubated at 42°C for about 6 hours for curd. (a) Incubation period of 12 hours for butter milk, (b) 7 hours for misthti dahi (10% sugar), (c) Incubation period of 6 hours for yogurt, (d) Incubation period of 7 hours for flavored yogurt. For preparation of yogurt LB-200 and ST-500 were used in combination with ratio of 1:1 mixed culture. Flavored yogurt (set) and flavored yogurt (stirred) were also prepared in 100 mL volume cups with same combination of St-500 and LB-200. Mango, orange, strawberry, pineapple, blueberry and lichi flavors were prepared using both fruit pulp and natural flavors available in market. After fermentation, the cups were shifted to refrigerator for overnight. All the fermented products were then analyzed for sensory quality, rheological attributes and physico-chemical attributes (Table 7.1). 7.1.2
Selected Parameters for application testing
7.1.2.1
Texture of Curd The texture of curd depends mainly upon the heat treatment given to milk. Cow
milk (3.5% fat and 8.5% SNF) was subjected to two separate treatments: (1) heating at 63oC for 30 min and (2) boiling treatment without holding period. The texture of the
prepared curd was smooth with a thick gel (Figure 7.1), while as texture of yogurt was little loose and flow able. A rough and grainy texture was observed in mishti dahi (Table 7.1). 7.1.2.2
Sensory Evaluation The chilled curd, yogurt, butter milk and mishti dahi was served to a panel of
seven judges and its colour and appearance, flavor, body and texture and overall acceptance were evaluated on 9- point Hedonic scale (Amerine et al., 1965). The scores awarded by the judges were better compared to standard curd available in market. The apperence of yogurt, flavored yogurt (Figure 7.2) and mishti dahi were very much liked and appreciated by tasting panel. 7.1.2.3
Syneresis The set curd at 5°C was slowly transferred to 15 mL capacity centrifuge tubes
causing minimum disturbance to the coagulum. The centrifuge tubes were balanced by adjusting their weights and centrifuged at 2000 rpm in a Remi centrifuge for 5 min. The quantity of whey separated at the top of the coagulum inside centrifuge tubes was recorded as milliliters. The higher the volume of whey separated, the higher was the syneresis and vice versa. Syneresis in case of curd and mishti dahi prepared from ST-500 was observed up to mark. Syneresis was not checked for buttermilk as the product is a noncoagulum type fermented drink. 7.1.2.4
Acidity The acidity of the curd samples was analyzed by BIS method and expressed as
per cent lactic acid (Bureau of Indian Standards, 1981). 7.1.2.5 pH pH was determined by potentiometric method i.e. by potential difference between the sample and electrolyte solution present inside the electrode of pH meter, using digital pH meter (Systronic Co., Bangalore). The electrode of the pH meter was directly dipped in the set curd and the pH was recorded (5°C). pH was recorded to check the lactic acid secreting capability of the culture in specific duration with favorable temperature. Curd was prepared in cups with 100 mL volume and was incubated. pH was recorded after 3, 3.5, 4, 4.5, 5, 5.5, 6 hours on incubation from different cups of same experiment, so that
the coagulation will not get disturbed (Figure 7.3). Curd prepared with different fat concentrations was also checked for coagulation and pH drop during incubation (Figure 7.5)
Figure 7.3 Graphical presentation of pH drop rate after inoculation of ST-500 with different fruit and sugar percentages on incubation at 42°C
Figure 7.5:
Graphical presentation of pH drop rate after inoculation of ST-500 with different percentage of milk fat on incubation at 42°
7.2)
APPLICATION TESTING FOR CHEESE PRODUCTION FROM ST-500 Firstly S. thermophilus used for the manufacture of fermented milks such as
yoghurt, it is now increasingly used in cheese production, for example, in production of cheeses that was formerly made with Lactococci bacteria, such as Lactococcus lactis or Lactococcus cremoris. This bacterium converts lactose in milk into lactic acid by acidifing the milk. In the case of cheeses, this acidification not only encourages the action of the coagulant and the synaeresis of the curds, but also inhibits the growth of many undesirable bacteria, certain of which are pathogenic bacteria, and allows their elimination at a greater or lesser speed. On an industrial scale, the hydrolysis of urea by Streptococcus thermophilus poses a number of problems. This is because, in cheese manufacturing for example, the technological operations (cutting of the curds, stirring, etc.) must take place at given values of pH, but in practice these operations are generally carried out at predetermined times. Therefore, the variations in acidifying activity due to urea hydrolysis may lead to defects and variability in the texture, moisture level, and ripening properties of the resulting cheeses. Moreover, because ammonia is basic, the production of ammonia increases the time necessary to reach a given pH. This results in the cheese-making equipment being tied up for longer and in an increase of the risk of contamination by undesirable micro-organisms. It is desirable that the cheese-making whey does not contain an excessive amount of ammonia, because this whey is often used as an ingredient in human food and animal feed. The production of ammonia from urea is difficult to control, in part because the urea content of milk is variable (for example, from 2 to 8 mM) and depends in part on the diet of the livestock that produce the milk. In certain aspects, methods of producing reduced open-texture cheese comprising: a) contacting milk with: i) urease positive Streptococcus thermophilus bacteria and a urease inhibitor, and/or ii) urease negative Streptococcus thermophilus bacteria, which are not able to release ammonia from urea at same level as wild-type bacteria; and b) fermenting the milk under conditions such that initial cheese is produced; and c) aging the initial cheese for a period of time such that reduced-texture cheese is produced which has a reduced amount of open-texture compared to control cheese, wherein the control cheese is produced in the same manner as the reduced open-texture cheese but employs the
urease positive Streptococcus thermophilus bacteria without the urease inhibitor are provided.
Samples of fresh 1% fat milk were treated with various combinations of ST-500 (S. thermophilus NCIM 5539) bacteria. In each testing, an acidification curve was determined by measuring the pH of the milk from the time of addition until 250 minutes after addition. Milk from one source was used as the starting material for each experiment. The temperature of the milk was held at 35° C. for the duration of each experiment. The activity of the S. thermophilus bacteria was correlated to the amount of time that it takes for the pH of the milk to reach a particular level. S. thermophilus, is anaerobic and has little or no activity in the presence of oxygen. When it is active, pyruvate formate lyase is believed to produce formate. When oxygen is present, S. thermophilus activity is believed to decrease because the amount of formate produced by pyruvate formate lyase is reduced. When an external formate source, such as sodium formate, is added, the activity of S. thermophilus is increased. Formate sources other than sodium formate, such as formic acid, potassium formate, magnesium formate, calcium formate, or any other acceptable formate, may also be used for this purpose. In current study, ST-500 took a much longer time to coagulate which was different according to fat concentrations of the milk. The acidity on coagulation was on much higher level and observed between 4-5 final pH. The texture, taste and pH of the cheese were observed on aging period of 10, 20 and 30 days. The taste of the cheese was found increasingly better with aging. The final product obtained after 30 days was with a little loose texture as compared to other lactic cheese available in the market.
Table: 7.1 Results of application testing done on the basis of certain parameters
Parameters tested S.N .
1
2
3
4
5
6
7
Applicatio n Tested Quality Testing of ST-500 for Curd Quality Testing of ST-500 for yogurt Quality Testing of ST-500 for Mishti Dahi Quality Testing of ST-500 for Cheese Quality Testing of ST-500 for Fruit/Flavo r Yogurt Quality Testing of ST-500 for Butter Milk Quality Testing of ST-500 for yogurt softy
Fina l pH
4.45
Post Acidificatio n
Textur e
4.45 ± 0.1
Smoot h
Gel Firmnes s
Taste Flavor/Arom a
Water/Whe y Separation
High
Creamy Mild Curd flavor
Nil
Low
4.92
4.92 ± 0.1
Loose
Low
Cream yogurt flavor and aroma
4.53
4.53 ± 0.2
Smoot h
High
Sweet creamy Dahi aroma
Low
4.87
4.87 ± 0.4
Grainy
N/A
Sour and fatty Cheese aroma
Nil
Low
5.02
5.02 ± 0.2
Loose
Low
Creamy Fruit flavor and aroma
4.44
4.44 ± 0.1
Very Loose
Very Low
Salty butter Curd flavor
N/A
4.88
Smoot h and Soft
N/A
Rich Creamy yogurt flavor and aroma
N/A
4.88
7.3.1
Chemical & Physiological Parameters for Fermented Milk (set curd)
Acidity: Acid production by Starter Culture is assessed by pH measurement of inoculated milk after incubation. Acidity in terms of pH measurement of fermented milk should be between 4.5 to 5. Post Acidification: Post acidification should be very low. Flavor: Like Indian Traditional Curd (Mild acidic). Rheology: Firmness, Texture and viscosity are important rheological or textural parameters that govern the quality of Fermented milk (Set Curd). Gel Firmness: Firmness is the quality of steadiness or immovability (fixed). Texture: Texture is the appearance, or consistency of surface of a Substance. Fermented milk (Set Curd) should be Smooth & Soft in appearance & have the proper cutting ability. Taste: Sour (Mild Acidic) & slightly buttery in taste (Creamy). Water/Whey Separation: Another Parameter which governs the quality of set curd is the separation of water/whey from set curd and it should be very Low after curdling (4 hour). Heat treatment: Milk is heated before the culture inoculation. If pasteurized milk is used, it is heated up to 42-43°c.
7.4.1
TESTING OF ST-500 (STREPTOCOCCUS THERMOPHILUS NCIM 5539) STARTER CULTURE AGAINST AVAILABLE CULTURES IN THE MARKET FOR CURD PREPARATION Market samples of starter culture were brought to laboratory for comparative
testing against ST-500. All the samples were maintained at -20° C storage conditions before use. 100 mg starter culture sample of JAMA
(CHR. Hansen, Denmark),
FlavoGard (Danisco Dupont, Copenhagen) and ST-500 was collected separately and added in 10 mL of milk. All the three 10 mL milk stock samples were mixed uniformly. A 0.3 mL sample was dispensed from all the three stocks and inoculated in three beakers respectively containing 200 mL milk at 40° C with standard parameters (Table 7.2).
Table: 7.2 Selected Parameters and standards for comparative analysis S.N.
PARAMETER
STANDARDS USED
1
Percentage of Fat in Milk
4%
2
Percentage of SNF* in Milk
9%
3
Inoculation temperature
40
4
Incubation temperature
42
5
Incubation Period
4-5 hours
6
Additional Fortification of milk
None
7
Heat Treatment
70 for 10 minutes
8
Starter culture used
JAMA, ST-500, Flavogard
9
Amount of Culture used
Equal
Table: 7.3 Results of comparative testing done on selected parameters Parameters tested S.N.
Starter Testing
Final Post Gel Taste Texture pH Acidification Firmness Flavor/Aroma
Water/ Whey Separat ion
1
ST-500
4.45
4.45 ± 0.1
Smooth
High
Creamy Mild Curd flavor
Nil
2
JAMA
4.48
4.48 ± 0.1
Smooth
High
Creamy Mild Curd flavor
Nil
3
Flavogard
4.63
4.63 ± 0.1
Smooth
High
Creamy yogurt flavor
Nil
Final product ST-500 starter culture was observed effective against all the major applications of dairy industries. Most importantly, the application of its use as starter culture for Indian traditional curd is of great commercial and industrial value. As described in Table 7.3 the curd prepared from ST-500 was observed almost equivalent to the curds prepared by the available starter cultures in the market. The other dairy applications were also observed significant in terms of its commercial use. The culture showed tremendous potential in yogurt production also when analyzed with LB-200. Both the bacteria worked in synchronization to produce both flavored and plain yogurt with rich and creamy taste and texture. However, this synchronization of ST500 and LB-100 is a matter of more experimental study which can be the future prospect.
Chapter 8 Summary & Conclusion
The requirement for making a good meal is to have ingredients of a good quality available, and to have sufficient variety to generate interesting taste and flavor. This is, however, not as simple as it sounds, as a large number of our basic food items are perishable, and quality is also not always easy to recognize. In the industrialized world, the fraction of our food prepared outside the private kitchen is exceeding 50% and this fraction is still rising. With the knowledge about the genetics and the physiology of lactic acid bacteria we now possess, it is possible to engineer starters for a variety of purposes where a suitable starter culture cannot easily be found in nature.
Lactic acid bacteria obtained by biotechnological process is preferred for industrial applications, especially, dairy industry. Lactic acid bacteria are good organisms for lactic acid fermentation. Streptococcus thermophilus (NCIM 5539) is a homofermentative LAB isolated from curd is extensively explored in this study. It exhibited more than 84 % lactic acid production yield along with cell mass yield of more than 6.5% was achieved which is desirable for industrial application. The intention of the present investigation was to isolate, characterise and study various parameters of different environmental conditions to optimize the production of starter culture of Streptococcus thermophilus (NCIM 5539). The isolated strain was further identified from MTCC (Microbial Type Culture Collection) CSIR Labs, Chandigarh, India for genuine confirmation of our identification results. The strain was later deposited to NCIM (National Collection of Industrial Microorganisms), Pune, India, under safe deposit with accession number NCIM 5539. This deposition was followed by Whole Genome Sequencing of the strain conducted by Genotypic, Bangalore, India.
In order to characerise the isolated strain of Streptococcus thermophilus (NCIM 5539), phenotypic (morphology, physiological and biochemical tests) and genotypic
methods (16s rDNA sequencing identification and WGS) were performed. One hundred and twenty eight isolates were identified as homo-fermentative and all were studied for their fermentation, multiplication and lactic acid production capabilities. On the basis of result obtained, 32 were selected for complete phenotypic, morphological and fermentation analysis. Ten isolates with very strong potential as starter culture and other dairy industrial uses were further selected for complete analysis and characterization along with dairy applications of their final product. This selection was made on the basis of biochemical analysis including sugar fermentation, casein hydrolysis, catalase test, starch hydrolysis, urease activity, MRVP test etc. Yeast extract and peptone were used in the fermentative media as nitrogen source for production of lactic acid bacteria though expensive for commercial production. The efficiency of this process could be improved within limits by varying culture conditions and medium composition. Commercial grade sucrose as carbon source and tryptone as nitrogen source were found to reduce the nutrients cost by 60%. This study suggests that sucrose, tryptone and yeast extract could be used as relatively cheaper nutritional source in the fermentation medium for production of biomass of S. thermophilus.
Ten selected strains were, further, identified at molecular level (16s rDNA sequencing) and found that seven strains are of cocci type (chain) in morphology belonging to Streptococcus thermophilus , one strain belong to Streptococcus infantarius subsp. coli and two strains with short and long rod morphology belong to Lactobacillus acidophilus and Lactobacillus delbrueckii subsp indicus, respectively. Phylogenetic tree were prepared for all 10 strains with the help of BLAST at NCBI for best hit results. Whole genome sequencing of one selected strain of S. thermophilus was studied to observe the overall genetic expression of the strain. The selected strain is the new discovery in the world as the ninth strain of S. thermophilus for which the complete genomic profile has been investigated (data will be submitted to NCBI after patenting).
Batch fermentation was conducted with maximum cell mass achieved (7%). More than 100 lab scale fermenter batches (2L & 5L) and 25 pilot scale batches (50L to 300L) were prepared and performed for detailed analysis of cell multiplication,
fermentation, freezing and lyophilization. The final starter culture produced after complete up and down stream processing was tested for its efficiency in dairy industrial application of fermented food products. Curd prepared from S. thermophilus starter was observed completely fine on parameter testing. Other fermented foods like buttermilk, flavored/ plain yogurt, mishti dahi, frozen dessert, yogurt softy and cheese were prepared with fine taste, texture, creaminess, flavor, aroma and low post acidification. Finally, the culture market can also be increased by expanding the application of cultures and by increasing the value of the products. In both cases, the cultures developed will contribute a larger part of the value of the final product compared to the current applications.
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TiTle of Publication Rohit Sharma, Bhagwan S Sanodiya, Gulab S Thakur, Pallavi Jaiswal, Sangeeta Pal, Anjana Sharma and Prakash S Bisen (2013) Characterization of lactic acid bacteria from raw milk samples of cow, goat, sheep, camel and buffalo with special elucidation to lactic acid production. British Microbiology Research Journal 3(4): 743-752. Rohit Sharma, Bhagwan S Sanodiya, Deepika Bagrodia, Mukeshwar Pandey, Anjana Sharma and Prakash S Bisen (2012) Efficacy and Potential of Lactic acid bacteria modulating human health. International Journal of Pharma and Bio Sciences. 3(4): (B) 935-948. Rohit Sharma, Bhuvan Bhaskar, Bhagwan S Sanodiya, Gulab S Thakur, Pallavi Jaiswal, Nitin Yadav, Anjana Sharma, Prakash S Bisen (2014) Probiotic Efficacy and Potential of Streptococcus thermophilus modulating human health: A synoptic review. IOSR Journal of Pharmacy and Biological Sciences. 9(3): 52-58 Rohit Sharma, Bhuvan Bhaskar, Bhagwan S Sanodiya, Gulab S Thakur, Pallavi Jaiswal, Anjana Sharma, Prakash S Bisen. Standardization of Lyophilization medium for Streptococcus thermophilus subjected to viability escalation on freeze drying. (accepted) Mousumi Debnath, Mukeshwar Pandey, Rohit Sharma, Gulab S Thakur and Pushpa Lal (2010). Biotechnological intervention of Agave sisalana: A unique fiber yielding plant with medicinal property. Journal of Medicinal Plants and Research. 4(3): 177-187. Rohit Sharma, Gulab S Thakur, Bhagwan S Sanodiya, Ashish Savita, Anjana Sharma, Prakash S Bisen (2012) Therapeutic potential of Calotropis procera: A giant milkweed. IOSR Journal of Pharmacy and Biological Sciences. 4(2): 45-57. Rohit Sharma, Gulab S Thakur, Bhagwan S Sanodiya, Mukeshwar Pandey and Prakash S Bisen (2012). Saponin: A wonder drug from Chlorophytum species. Global Journal of Research on Medicinal Plants and Indigenous Medicine. 1(10): 503-515 Rohit Sharma, Saxena Nidhi, Thakur Gulab S, Sanodiya Bhagwan, Jaiswal Pallavi, Conventional method for
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saponin extraction from Chlorophytum borivilianum (2014) Global Journal of research on medicinal plant and indigenous medicine, 3(2): 33-39. Gulab S Thakur, Rohit Sharma, Bhagwan S Sanodiya et al. (2013) In vitro induction of tuber formation for the synthesis of secondary metabolites in C.borivilianum sant ed. Fernand. African journal of Biotechnology 12(20)2900-2907
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Rohit Sharma (2014) Micropropagation: An essential tool to flourish endangered medicinal plants. Global J Res. Med. Plants & Indigen. Med. 3 (6), 252-262
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Gulab S Thakur, Rohit Sharma, Bhagwan S Sanodiya, Mukeshwar Pandey, GBKS Prasad and Prakash S Bisen (2011). Factors effecting in-vitro propagation of Momordica balsamina:a medicinal and nutritional climber. Physiology and molecular biology of plants. 17(2): 193-197
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Gulab S Thakur, Rohit Sharma, Bhagwan S Sanodiya, Mukeshwar Pandey, GBKS Prasad and Prakash S Bisen (2012) Gymnema sylvestre: An alternative therapeutic agent for management of diabetes. Journal of Applied and Pharmaceutical Science. 2(12): 001-006. Mukeshwar Pandey, Surendra K Chikara, Manoj K Vyas, Rohit Sharma, Gulab S Thakur, Prakash S Bisen (2012) Tinospora cardifolia: A climbing shrub in Health care management. International Journal of Pharma and Bio Sciences. 3(4): (P) 612-628. Gulab S Thakur, Rohit Sharma, Bhagwan S Sanodiya, Mukeshwar Pandey, Rakesh Baghel, Astha Gupta, GBKS Prasad and Prakash S Bisen (2011). High Frequency invitro shoots regeneration of Momordica balsamina, an important medicinal and nutritional plant. African Journal of Biotechnology. 10(70): 15808-15812.
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