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TABLE OF CONTENTS Subject TABLE OF CONTENTS«««««««««««««««««« LIST OF FIGURES«««««««««««««««««««« INTRODUCTION««««««««««««««««««««« CHAPER ONE «««««««««««««««««««««« CONCEPT OF STARTER CULTURE What is the starter culture«««««««««««««««««« Types of starter cultures««««««««««««««««««« Functions of Starter Cultures««««««««««««««««« Characteristics of a good starter culture««««««««««««« Ecology of starter bacteria««««««««««««««««« CHAPTER TWO««««««««««««««««««««« CLASSIFICATION OF STARTERS CULTURES«««««««« Classification /Taxonomic Groups as Per Bergey's Manual««««« Bacteria««««««««««««««««««««««««« Moulds««««««««««««««««««««««««««« Types of Starters«««««««««««««««««««««« CHAPTER THREE««««««««««««««««««««« LACTIC STARTER CULTURE Lactic acid bacteria««««««««««««««««««««« Metabolic activity of lactic acid bacteria CHAPTER FOUR««««««««««««««««««««« FERMENTATIVE CHANGES IN MILK BROUGHT ABOUT BY STARTER CULTURES Lactic acid fermentation««««««««««««««««««« Propionic acid fermentation««««««««««««««««« Microbiology of the propionic acid fermentation««««««««« Alcoholic fermentation««««««««««««««««««« Citric acid fermentation«««««««««««««««««««« Microorganisms used for citric acid production««««««««« CHAPTER FOUR««««««««««««««««««««« FACTORS AFFECTING FERMENTATION CHARACTERISTICS OF STARTER CULTURES CHAPTER SIX«««««««««««««««««««««« PREPARATION OF STARTER CULTURES Principles to maintain active tarter culture«««««««««««« Preparation of mother culture«««««««««««««««««
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Page No. i v 1 4 4 5 6 7 8 9 9 10 13 15 17 17 29 33
32 33 34 35 38 39 42
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Preparation of working cultures«««««««««««««««« Preparation of bulk starters«««««««««««««««««« Continuous starter production««««««««««««««««« Preparation of master culture«««««««««««««««««« CHAPTER SEVEN««««««««««««««««« MAINTENANCE AND PRESERVATION OF STRTER CUTURES Importance of maintenance and preservation««««««««««« Methods of starter cultures preservation««««««««««««« Regular transfer of starter culture«««««««««««««««« Storage at the refrigerator««««««««««««««««««« Paraffin Method««««««««««««««««««««««« Storage in soil«««««««««««««««««««««««« Storage in Silica Gel«««««««««««««««««««««« Storage by freezing«««««««««««««««««««««« Storage by drying methods««««««««««««««««««« i- Vacuum drying««««««««««««««««««««««« ii-Spray drying«««««««««««««««««««««««« Freeze Drying (Lyophilization) ««««««««««««««««« Quality control of the preserved starter culture«««««««««« CHAPTER EIGHT «««««««««««««««««««« FACTORS CAUSING INHIBITION OF STARTER CULTURES Components naturally present in milk««««««««««««« 1. Antibiotics««««««««««««««««««««««« 2- Bacteriophage«« ««««««««««««««««««« 3. Detergents and disinfectants residues«««««««««««« 4. Production of nisin«««««««««««««««««««« 5. Free fatty acids««««««««««««««««««««« CHAPTER NINE««««««««««««««««««««««« YEASTS STARTER CULTURES Principles of yeast growth and fermentation««««««««««« Sources of yeast strains««««««««««««««««««« Yeast strain identity and purity«««««««««««««««« Preservation of yeast starter cultures«««««««««««««« Supply of yeast starter culture«««««««««««««««« Quality assurance of yeast cultures«««««««««««««« CHAPTER TEN««««««««««««««««««««««« APPLICATION OF STARTER CULTURE IN FOOD INDUSTRY Food industry««««««««««««««««««««««« Selection of starter cultures««««««««««««««««« CHAPTER ELEVEN«««««««««««««««««««« UTILIZATION OF STARTERCULTURE IN CHEESE MAKING Lactic acid bacteria in cheese production«««««««««««.
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53 53 53 54 55 55 56 57 58 58 59 59 60 61 61 62 63 64 65 65 68 70 72 73 74 75 75 76 77 78 79 80 81 81 85 87 87
LAB food safety and cheese technology«««««««««««... Role of fungi in cheese making«««««««««««.................. i. Role of yeasts««««««««««««««««««««««. Ii. Role of moulds«««««««««««««««««««««.. Roquefort cheese«««««««««««««««««««««... Camembert cheese««««««««««««««««««««««. Improvement of new starter cultures for cheese preparation«««««. CHAPTER TWELVE«««««««««««««««««««....... FERMENTED MILK PRODUCTS«««««««««««««««. Selecting probiotic bacteria for milk products««««««««««« Beneficial effects of probiotics«««««««««««««««««« Beneficial probiotic strains««««««««««««««««««« Application of probiotic bacteria in dairy foods««««««««««.. Examples of probiotic fermented milk products««««««««««.. Acidophilus milk«««««««««««««««««««««........ Bifidus milk«««««««««««««««««««««««........ Mil-Mil product«««««««««««««««««««««««.. Yakult product«««««««««««««««««««««««« Yogurt«««««««««..«««««««««««««««««« Butter milk robe«««««««««««««««««««««««.. Garris product«««««««««««««««««««««««« Kefir product««««««««««««««««««««««««.. Koumiss-Kumiss product«««««««««««««««««««.. Acidophilin product«««««««««««««««««««««« Nono product««««««««««««««««««««««««.. Maas and Inkomasi««««««««««««««««««««««. Channa product«««««««««««««««««««««««.. CHAPTER THIRTEEN««««««««««««««««««««.. UTILZATION OF STRTER CULTURES IN PRODUCTION OF FERMENED MEAT PRODUCTS Composition of sausages««««««««««««««««««««. Types of sausages«««««««««««««««««««««««. Advantages of meat starter cultures«««««««««««««««« Starter cultures for fermented sausages««««««««««««««.. Classification of sausage starter cultures«««««««««««««« Microorganisms involved in sausage fermentation«««««««««« Advantages of sausage starter cultures««««««««««««««. Flavour of fermented sausages«««««««««««««««««« CHAPTER FOURTEEN««««««««««««««««««««. UTILIZATION OF STARTER CULTURES IN PRODUCTION OF BAKED PRODUCTS The microflora of sourdough bread«««««««««««««««..
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89 92 92 94 95 97 98 100 101 103 104 106 108 108 109 110 110 111 112 113 113 114 115 116 116 117 118
121 122 122 123 124 125 126 127 130
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Production of sourdough bread«««««««««««««««««« Types of sourdoughs«««««««««««««««««««««« Benefits of sourdough bread««««««««««««««««««« CHAPTER FIFTEEN«««««««««««««««««««««.. USE OF STARTER CULTURE FOR VETABLES FERMENTATION Benefits of vegetables fermentations«««««««««««««««. Species of LAB for vegetable fermentations««««««««««««. Rapid and predominant growth«««««««««««««««««« How to ferment vegetables«««««««««««««««««««... CHAPTER SIXTEEN««««««««««««««««««««.
133 136 140 141 143 145 146 148 151
FUTURE OF STARTER CULTRES TECHNOLOGY REFERNCES «««««««««««««««««««««««««.
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LIST OF FIGURES Figure No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
Subject Classification of starter culture«««««««««««««««««. Classification of lactobacillus based on glucose fermentation««««« Leuconostoc cells««««««««««««««««««««««.. Streptococcus thermophiles««««««««««««««««««« Lactobacillus bulgarius««««««««««««««««««««.. Lactobacillus acidophilus«««««««««««««««««««.. Lactobacillus casei«««««««««««««««««««««« Lactobacillus helveticus«««««««««««««««««««« Lactococcus lactis subsp. lactis««««««««««««««««« Generalized scheme for the fermentation of glucose in lactic acid bacteria Propionibacterium shermanii«««««««««««««««««« A propionic acid production plant«««««««««««««««. %UHZHU¶V\HDVW«««««««««««««««««««««««.. Alcoholic fermentation««««««««««««««««««««. Citric acid cycle««««««««««««««««««««««« Spray dryer««««««««««««««««««««««««.. Freeze dryer««««««««««««««««««««««««.. Lactoperoxidase«««««««««««««««««««««««. Penicillin«««««««««««««««««««««««««.. Streptomycin«««««««««««««««««««««««« Bacteriophage«««««««««««««««««««««««« Yeast cells«««««««««««««««««««««««««... Gorgonzola cheese ripening««««««««««««««««««. kaasmisdrijf cheese ripening«««««««««««««««««« Yeast cell««««««««««««««««««««««««««. Penecillium roqueforti fungus«««««««««««««««««.. Roquefort cheese««««««««««««««««««««««« Camembert cheese««««««««««««««««««««««. Acidophilus milk««««««««««««««««««««««« Bifidus milk««««««««««««««««««««««««« Yakult product«««««««««««««««««««««««.. Yoghurt product««««««««««««««««««««««« Kefir product««««««««««««««««««««««««. Koumiss product««««««««««««««««««««««.. Maas product««««««««««««««««««««««««. Channa product«««««««««««««««««««««««. Fermented sausage««««««««««««««««««««««
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Page No. 9 12 20 24 25 26 26 27 29 30 34 35 36 37 40 62 64 66 69 69 71 76 93 93 94 96 96 97 108 109 110 112 114 115 116 117 121
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Fermented dried sausage«««««««««««««««««««.. Sour dough bread««««««««««««««««««««««.. Type I sourdough««««««««««««««««««««««.. Type II sourdough««««««««««««««««««««««. Type III sourdough«««««««««««««««««««««« Vegetables starter cultures««««««««««««««««««« Fermented olive««««««««««««««««««««««« Fermented sauerkraut«««««««««««««««««««««. Fermented cabbage««««««««««««««««««««««..
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124 131 138 139 140 143 146 148 150
INTRODUCTION
Fermentation can be defined as the anaerobic breakdown of an organic substrate by an enzyme system in which the final hydrogen acceptor is an organic compound. It is also a metabolic process in which chemical changes are brought about in an organic substrate through activities secreted by microorganisms. It can be also defined as the anaerobic enzymatic conversion of organic compounds, especially carbohydrates, to simpler compounds, especially to lactic acid or ethyl alcohol, producing energy in the form of ATP used commercially in the preparation of alcoholic beverages and the generation of by-products used as animal feed. Also the basic process in the manufacture of antibiotics Microorganisms of various types are employed in the fermentation processes. These include species or strains of bacteria, yeasts and moulds. The microorganisms of fermentation differ greatly in morphology, size, manner of reproduction , reaction to free oxygen, growth requirements, ability to attack different substrate. The scientific rationale of the function of microorganisms in fermentation started to be built with the discoveries of Louis Pasteur in the nineteenth century. Extensive scientific study continues to characterize microbial food cultures traditionally used in food fermentation taxonomically, physiologically, biochemically and genetical ly. This allows better understanding and improvement of traditional food processing and opens up new fields of applications. Fermented foods originated many thousands of years ago when presumably micro-organism contaminated local foods. Micro-organisms cause changes in the foods which: help to preserve the food, extend shelflife considerably over that of the raw materials from which they are made, improve aroma and flavour characteristics and increase its vitamin
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content or its digestibility compared to the raw materials. Fermented foods comprise about one-third of the world wide consumption of food and 20- 40 % (by weight) of individual diets. A food is regarded fermented when one or more of its components have been acted upon by microorganisms to produce considerably altered final product acceptable for human utilization. Natural fermentations are initiated without the addition of microorganisms and their control is limited to maintenance of external environmental conditions. Fermented milk products are known for their taste, nutritive value and WKHUDSHXWLF SURSHUWLHV )HUPHQWHG PLONV DUH ³SURGXFWV SUHSDUHG IURP milks, entire, mostly or completely skimmed, concentrated or milk substituted from partially or fully skimmed dried milk, somewhat or completely skimmed pasteurized or sterilized and fermented by means of VSHFLILF PLFURRUJDQLVPV´ 0LON IURP HLJKW VSHFLHV RI GRPHVWLFDWHG mammals (cow, buffalo, sheep, goat, horse, camel, yak and zebu) has been used to make traditional fermented milk products throughout the world. The people who had domesticated these milk animals usually acknowledge fermented milks by necessity. These foods that have been subjected to the action of micro-organisms or enzymes, in order to bring about a desirable change. They owe their production and characteristics to the fermentative activities of the microorganisms. A starter cultures is a preparation 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. The type of starter culture used depends on the desired product. Culture supply companies can provide processors with a variety of cultures tailored for their operation that can be purchased frozen or dehydrated, typically as a mixture of several strains. It is very important to follow the
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suppliHU¶V DGYLFH RQ WKH KDQGOLQJ VWRUDJH URWDWLRQ XVH UDWH DQG incubation temperature for their cultures. Microbial starter cultures are considered as food ingredients and are allowed in the production of foodstuffs everywhere throughout the world. Commercially accessible microbial starter cultures are sold as preparations, which are formulations, comprising of concentrates of at least one microbial species and/or potentially strains including unavoidable media components carried over from the fermentation and components, which are fundamental for their survival, storage, standardization and to encourage their application in the food production process. Safety of microbial food cultures, contingent upon their characteristics and use, can be based on genus, species or strain levels.
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CHAPER ONE CONCEPT OF STARTER CULTURE
What is the starter culture: A starter culture has many definitions: it can be defined as one or more strains of one or more species of desirable bacteria used to inoculate a raw or pasteurized product to start a fermentation to produce a fermented food by accelerating and steering its fermentation process. Starter cultures are also those microorganisms (bacteria, yeasts, and molds or their combinations) that initiate and carry out the desired fermentation essential in manufacturing cheese and fermented dairy productsAnother definition: Starter cultures are preparations of live microorganisms or their resting forms, whose metabolic activity has desired effects in the fermentation substrate, the food. The arrangements may contain unavoidable deposits from the culture substrate and added substances that help the essentialness and innovative usefulness of the microorganisms (such as antifreeze or antioxidant compounds). This definition includes a multitude of preparations, which is based on the history of starter cultures. The advancement of fermented foods was controlled by the way that initially an exceptional microbial affiliation created affected by biological variables prevailing in the respective substrate. Such as the fermentation of sauerkraut (pickled cabbage), olives or pickles are still state-of-the-art. In other places, fermenting substrate was utilized to inoculate new fermentation methods. Still standard practice today is for LQVWDQFH µROGQHZ LQRFXODWLRQ¶ ZLWK FKHHVHV µEDFN-VORSSLQJ¶ RU µEDFNVKXIIOLQJ¶ ZLWK sourdough. Vinegar is ,likewise produced in this way. The inoculum obtained in this manner (having been propagated many times) experiences a high level of organism selection and is practically V\QRQ\PRXV ZLWK VWDUWHU FXOWXUHV 6XFK µXQGHILQHG FXOWXUHV¶ are still being used today, for H[DPSOH µ)ORUD 'DQLFD¶ XVHG DV D PLON VWDUWHU
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culture from more than 100 Leuconostoc and Lactococcus strains or µ5HLQ]XFKWVDXHU¶used as a sourdough starter. These cultures are liable to a nonstop change in structure, as strains may vanish or transform, or may change their properties after phage assaults. Types of starter cultures: The utilization RIµGHILQHGFXOWXUHV¶ allows for a greater degree of control over the fermentation process. Mäyrä-Mäkinen and Bigret, (1998) made a distinction between: - Mother starters: are small-volume cultures( usually 1 litre or less) prepared and sometimes stored in the laboratory, and used to inoculate the bulk starter medium. - Bulk starters: are large-volume cultures used to initiate the fermentation of raw or pasteurized product - Single-strain cultures: contain one strain of a species; - Multi-strain cultures: contain more than one strain of a single species; - Multi-strain mixed cultures: contain different strains from different species. These different cultures are used in the fermentation of milk, meat, wine, fruit, vegetables and cereals. To keep up their strength, viability and appropriateness, they are prepared, packaged, bundled, frozen or freezedried.
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Functions of Starter Cultures: The main function of lactic starters is the production of lactic acid from milk sugar (lactose), this acid is important as preservative agents and generating flavour of the products.. In addition to lactic acid production the starter cultures are also useful in different ways as stated below. Table (1): Functions of starter cultures Function
Result
Production of acids: x Gel formation Homofermentative LAB x Expulsion (syneresis) of whey for texturing produce lactic acid, while x Preservation of milk Heterofermentative x Helps in the development of flavour produce lactic acid, acetic acid, ethanol and CO2 Production of flavour Impart the characteristics flavour of fermented products components such as diacetyl, acetaldehyde and acetoin Production of Texture formation exopolysaccharides Preservation of fermented x Lowering of pH and redox potential products x Production of lactic acid x Production of antibiotics x Production of H2O2 x Production of acetate x Eye formation in certain cheeses x Production of open texture Ex. blue veined cheese Stabilizer formation * Development of body and viscosity Ex. Polysaccharide materials Lactose utilization * Reduces the development of gas and off flavours * Suitable for people having lactose intolerant Lowering of redox x Helps in food preservation potential x Helps in development of food flavour Proteolysis and lipolysis Helpful in the ripening/maturation of cheeses Miscellaneous Production of alcohol in some products like kefir, koummis, robe compounds and garris. Gas formation
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Starters are used today to initiate milk fermentations under conditions which allow the selected bacteria to grow and produce lactic acid at as fast rate as possible. This discourages the growth of pathogens which may be present in the raw milk or enter milk after pasteurization. Knowledge about culture organisms is primarily limited to those cultures that are commercially available as there are diverse cultures poorly documented LQ WD[RQRPLFDO WHUPV WKDW DUH SURSDJDWHG µLQ-KRXVH¶ RU DUH produced by biotech firms for practical application. Basically it is known that these may contain bacteria, yeasts and moulds. The most progressive state of application of starter cultures has been accomplished in the dairy industry. The manufacture of fermented dairy products produced using pasteurized milk ± therefore containing very low levels of bacteria - would be practically impossible without cultures. However, high-TXDOLW\ FKHHVH LV DOVR SURGXFHG XVLQJ UDZ PLON RU µLQKRXVH¶FXOWXUHV Characteristics of a good starter culture: The following desirable characteristics must be looked into for exploring them for full fermentation potentials while selecting a particular LAB starter culture, either single or in combination with others for production of fermented milk products. These characteristics include: i- The lactic starter culture must produce sufficient lactic acid at desirable rate to suit the plant schedule and produces high quality product. ii- Good LAB culture must continue producing acid over the appropriate range of temperatures, at which it is likely to be utilized during the processing of milk into production of fermented dairy products. iii-The LAB starters must be resistant to antibiotics and bacteriophages. iv- The LAB starter must be active in the presence in inhibitory substances as well as residual amounts of chemical, sanitizers and detergents in milk.
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v- A good starter culture should not produce bacteriocins and bacteriocin like substances or any other antibiotic type of substance inhibiting other strains in the mixed culture. vi- A good LAB starter culture should produce desirable flavour, aroma, consistency, body and texture in the fermented products. vii- The use of starter cultures producing certain defects like ropy body, malty or any other undesirable flavour and similar other defects should be immediately discontinued. viii- In case of mixed cultures, the individual cultures must be able to synergize maximum aroma and flavour production without inhibition of acid production. ix- The associative action of mixed cultures must be quite stable and contributes towards the development of a good starter culture even after repeated subcultures. Ecology of starter bacteria: Most starters being used to today have begun from lactic acid bacteria initially present as part of the contaminating microflora of milk. These bacteria have probably originated from vegetation in the case of lactococci or the intestinal tract in the case of Bifidobacterium spp., enterococci and Lactobacillus acidophilus. Modern starter cultures have created from the act of holding little amounts of whey or cream from the successful manufacture of a fermented product on a previous day and utilizing this as the inoculum or VWDUWHU IRU WKH SUHYLRXV GD\¶V SURGXFWLRQ 7KLV DFW KDV EHHQ FDOOHG various names but the term 'back-slopping' is used widely particularly in fermented sausage manufacture.
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CHAPTER TWO CLASSIFICATION OF STARTERS CULTURES Classification /Taxonomic Groups as Per Bergey's Manual: Starter cultures are generally classified based on their ability to utilize the lactose as shown in Fig. (2). Starter cultures
Lactic
NON-Lactic
Rods
Cocci
Bacteria
Yeasts
Moulds
Lactobacilus
Lactococcus
Bifidobacteria
Candida kefir
Streptococcus
Brevibacterium linens
Kluyrverumyces
Penecillium camemberti
Acetobacter acetii
Saccharomyces
Leuconostoc Pediococcus
Torulospora
Propionibacteria
Penecillium roqueforti Aspergillus oryze Mucor rasmusen Geotrichum candidum
Fig. 1. Classification of starter cultures
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Bacteria: Genus lactococcus Bergey¶s Manual of Systematic Bacteriology (1986), combined all the mesophilic lactic acid bacteria (LAB) with Lactococcus lactis to form a single species as they possess 1. 2. 3. 4.
Identical isoprenoid TXLQLQHVDQGWKHHQ]\PHȕ-phosphotase Indistinguishable lactic dehydrogenase Identical percentage of guanine and cytosine. High DNA homology
Examples include: Lactococcus lactis subsp. cremoris : Acid producer but non-flavour producer Lactococcus lactis subsp. cremoris : Acid producer but non-flavour producer Lactococcus lactis subsp. lactis biovar. diacetylactis: Both acid and flavour producer
All the these Lactococcus species are mesophilic in nature and their optimum growth temperature is between 25-30°C. All are homofermentative organisms. Genus streptococcus:
The members of the Streptococcus are Gram-positive organisms that usually form pairs or chains. In 1937, Sherman separated the genus according to physiological and growth characteristics, especially with regards to temperature limitations on growth. Four general groups designated by Sherman are (1) pyogenic, (2) viridans, (3) enterococcus,
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and (4) lactic. This categorization has become somewhat obsolete as relationships between species have been shown to overlap. The only species used as starter culture is Streptococcus salivarius subsp thermophilus. This is a yoghurt culture, which is thermophilic in nature with optimum growth temperature of 3842°C. All are homofermentative organisms. Genus leuconostoc All are heterofermentative organisms capable of producing lactic acid, CO2 and aromatic compounds (ethanol and acetic acid) from glucose. These organisms are normally used along with lactic acid bacteria (LAB) in multiple or mixed strain cheese starter cultures, which produces flavour compounds. Leuconostoc cremoris Leuconostoc citrovorum Leuconostoc dextranicum Genus lactobacillus: Lactobacillus delbruekii subsp bulgaricus is used for the preparation of yoghurt along with Streptococcus salivarius subsp thermophilus. These two organisms exhibit a symbiotic relationship. Lactobacillus acidophilus is a probiotic culture, used for preparation of acidophilus milk and other probiotic milk products like Bifighurt, Bioghurt, etc. The members of lactobacillus are classified based on fermentation of glucose into 3 groups as shown in Figure (2).
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Genus bifid bacterium: Found in the gut of infants, intestines of man, various animals and honeybees. These organisms are generally used in preparation of therapeutic fermented milk products in combination with yoghurt, acidophilus milk or yakult starter cultures. Eg: Bifidbacterium bifidum, Bifidobacterium longum, Bifidobacterium inf antis, Bifidobacterium breve, etc. The optimum growth temperature is 37 - 41°C. Anaerobic conditions are essential for optimum growth. Milk fermented with bifidobacteria has a distinctive vinegar taste due to the production of acetate plus lactate from the metabolism of carbohydrates. Genus propionibacterium Propionibacterium freudenreichii and Propionibacterium shermanii are used in swiss cheese. It has the ability to produce large gas holes in the cheese during ripening / maturation period. P. jensenii, P. thoenii and P. acidipropionici are other organisms present in these genera. Genus brevebacterium: Brevebacterium linens is used as starter culture in preparation of bacterial surface ripened cheeses. It imparts distinctive, reddish orange colour to the rind of (or formation of smear on) Brick and Limburger cheese or Camembert cheese. Moulds: Moulds are used for the manufacture of some semi soft cheese varieties and in some fermented milk products. Moulds enhance the flavour and modify slightly the body and texture of curd.
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White mould: It is used in manufacture of surface mould ripened cheeses like Camembert and Brie cheeses. Eg: Penicillium camemberti, Penicillium caseicolum, Penicillium candidum Blue mould: It is used in manufacture of internal mould ripened cheeses like Roquefort, Blue Stilton, Danish blue, Gorgonzola and mycella cheeses. Eg: Penicillium roquefortii Other moulds: Mucor rasmusen used in Norway for the manufacture of ripened skim milk cheese. Asperigillus oryzae used in Japan for the manufacture of Soya milk cheese. Geotricum candidum used in the manufacture of Villi a cultured product of Finland. The mould grows on the surface of the milk to form the white velvety layer. Yeasts: Yeasts are used in the manufacture of Kefir and Kumiss Kefir grains: Kefir grains consist of a mixture of different microorganisms such as Candida kefir, Kluyeromyces marxianus, Saccharomyces kefir, Torulopsis kefir. Kumiss: The important starter microflora of kumiss include Torulopsis spp. Kluyeromyces marxianus var lactis, Saccharomyces cervisiae
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Types of Starters Starters are grouped under different categories based on composition of microflora, growth temperature, type of products, flavour production and type of fermentation into the following categories: 1- Based on the composition of micro flora/ organisms a. Single: Always used as a single organism in the preparation of dahi or cheese. The only problem is there will be sudden failure of starter due to bacteriophage attack which leads to heavy loss to the industry. b. Paired compatible strain: Two strains of cultures having complementary activities in know proportion are used. This will reduce chances of culture failures. . In case of bacteriophage attack, only one type of organism will be affected and the other organism will carry out the fermentation without any problem. c. Mixed Strain: More than two organisms which may have different characteristics like, acid production, flavour production, slime production etc. in unknown proportion are used. d. Multiple mixedstrain: More than two strains in known proportion are used. The quality and behaviour of these strains is predictable. 2 Based on the growth temperature Based on the growth temperature organisms can be divided into mesophilic and thermophilic. i-
Mesophilic starter cultures: The optimum growth temperature of these cultures is 30°& and they have a growth temperature range of 22- 40°&. The mesophilicstarter cultures generally contain the organisms of Lactococci.
Ex. Dahi cultures : Lactococcus spp. such as
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x x x x
ii-
x x x x x x
Lactococcus lactis subsp. cremoris L. delbrueckii subsp. lactis L. lactis subsp. lactis biovar diacetylactis Leuconostoc mesenteroides subsp. cremoris Thermophilic starter cultures: The optimum growth temperature of these cultures is 40°C and they have a growth temperature range of 32- 45°C. Examples include: Streptococcus salivarius subsp. thermophilus (S.thermophilus) Lactobacillus delbrueckii subsp. bulgaricus L. delbrueckii subsp. lactis L. casei L. helveticus L. plantarum
3- Based on the flavour production The starters are grouped into B, D, BD and N type based on their ability of flavour production B (L) type: Leuconostocs as flavour producer (old name is Betacocccus) D type: L. lactis subsp lactis biovar diacetylactis BD (LD) type: Mixer of both of the above cultures N or O type: Absence of flavour producing organism 4- Based on the type of fermentation The starters are classified as homo or hetero fermenter based on end products resulting from glucose metabolism. Homo fermentative cultures: eg. Lactococcus lactis subsp lactis Hetero fermentative cultures eg. Leuconostoc dextranicum
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CHAPTER THREE LACTIC STARTER CULTURE Lactic acid bacteria: Lactic fermentation has been known for a long time and the expression ³/DFWLFDFLGEDFWHULD´ZDVXWLOL]HGE\WKHHDUO\EDFWHULRlogists to describe those bacteria that immediately soured conventional lactic acid fermented foods. In developed countries, the vast majority the lactic acid fermentation has been amassed in dairy and vegetable products while in the developing countries and in particularly Africa, lactic acid fermentation predominates all the indigenous processing of cereals like maize, sorghum millet and root crops such as cassava. The bacteria utilized in the manufacture of fermented dairy products are generally lactic acid bacteria (LAB); however, Propionibacterium shermanii and Bifidobacterium spp. which are not lactic acid bacteria, although Bifidobacterium species do produce lactic acid, are also used. In addition, other bacteria including Brevibacterium linens, responsible for the flavour of Limburger cheese; and moulds (Penicillium species) are used in the manufacture of Camembert, Roquefort and Stilton cheeses. Lactic acid fermentation processes have survived in Africa throughout the centuries because of the following benefits of this technology: 1- It serves as a household technology for improving food safety. 2- It serves as low-cost method of food preservation in Africa. 3- It contributes to the improvement of the nutritional value and digestibility of food raw materials in Africa. In addition, lactic acid fermentations have survived due to the traditional believes, good taste and appearance of product as well as long shelf life.
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Table 2. Main LAB used in food fermentation Species
subspecies
Lactococcus
Lc.lactis subsp. lactis
Lc.lactis subsp. Lactis biovar. diacetylactis Lactococcus Lc.lactis subsp. cremoris Streptococcus Sc. thermophilus Lactococcus
Lactobacillus
Lb. acidophilus
Lb. delbrueckii subsp. bulgaricus Lb. delbrueckii subsp. lactis Lb. helveticus
Lb.casei
Lb. plantarum
Lb. rhamnosus
Leuconostoc
Ln. mesentroides subsp. cremoris
Type of starter Mesophilic Mesophilic
Mesophilic Thermophilic
Probiotic adjunct culture Thermophilic Thermophilic Thermophilic
Probiotic adjunct culture Probiotic adjunct culture Probiotic adjunct culture Mesophilic
Source: Gemechu (2015).
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Main uses Many cheeses, butter, butter milk Gouda, edam, laxtic butter, sour cream, butter milk Many cheeses, butter, butter milk Yoghurt and many hard and semi-hard high- cook cheeses Cheese and yoghurt
Yoghurt and many hard and semi-hard cheeses Fermented milk, high cook cheese Fermented milk and many hard and semi-hard highcook cheeses Cheese ripening
Cheese ripening
Cheeses
Edam, gouda, fresh cheese, lactic butter, sour cream.
LAB is currently being used to produce diversity in foods by altering flavour, texture and appearance of raw commodities in a desirable way. The sour aromatic flavours imparted by lactic acid fermentation are desirable traits in fermentation products and give a natural image to the products. Lactic acid bacteria are also reported to colonize the human intestinal mucosa leading to beneficial effect. There are currently sixteen genera of the (LAB). Many species of the genera Enterococcus, Lactobacillus,Lactococcus, Oenococcus, Oenococcus, Ped iococcus, Streptococcus and Tetragenococcus are important in food fermentations and have recently been reviewed. These microorganisms differ in their composition from one product to the other. Therefore, a great number of fermented milk types have developed. Variables include heat treatment of the milk, fermentation temperature, inoculum percentage and the concentrating of the milk. According to these conditions, various types of lactic acid bacteria become predominant e.g., producing various flavour components. Species of many genera of LAB are utilized as starter cultures. The genera include: Leuconostocs: The original classification of bacteria placed the genus Leuconostoc close to the Streptococcus, since was largely based on morphology, albeit in a VHSDUDWH JHQXV DV WKH ³ KHWHURIHUPHQWDWLYH FRFFL´ IRUPHUO\ FDOOHG WKH betacocci by Orla- Jensen. They produce D (-) lactate from glucose as opposed to L (+) lactate that is produced by the lactococci and DL-lactate by heterofermentative lactobacilli with whom, they share many characteristics. Leuconostoc is the predominant genus among the LAB on plants, with Leuconostoc mesenteroides subsp. mesenteroides as the principal isolate All Leuconostocs specie are capable of producing lactic acid, CO2 and aromatic compounds (ethanol and acetic acid) from glucose. These organisms are normally used along with lactic acid bacteria (LAB) in
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multiple or mixed strain cheese starter cultures, which produces flavour compounds. Leuconostoc cremoris Leuconostoc citrovorum Leuconostoc dextranicum Leuconostocs species are important flavour producers in some fermented dairy products. There is general agreement that two species, Leuconostoc mesenteroides subsp. cremoris and Lecon. lactis are important in starter cultures. Unlike lactococci, leuconostocs grow on Rogosa agar and are hetrofermentative producing carbon dioxide from glucose and usually fructose. While the carbon dioxide production is undesirable in Cheddar cheese gas production is desirable in some varieties e.g. Emmental.
Fig. (3). Leuconostoc cells
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On microscopic examination, leuconostocs generally appear as Grampositive cocci similar in size and shape (occur in pairs and in usually short chains) to lactococci. However, small rods can often be found and since leuconostocs grow on Rogosa agar, there can be a tendency to assume that these cultures are contaminated, with lactobacilli for example. Unlike lactococci, leuconostocs do not produce ammonia from arginine and produce the D isomer of lactic acid. With some exceptions leuconostocs only grow weakly in milk, and are not capable of reducing litmus before coagulation in litmus milk medium. Isolation and identification of leuconostocs in starters is time consuming and laborious (see Billie et al., 1992) and the author has found that the use of Rogosa agar to obtain initial isolates helpful. Carbohydrate fermentation and identification of the lactic acid isomer are useful elements in an identification protocol. Streptococcus: Streptococci were among the earliest bacteria to be perceived by microbiologists in view of their association in a large number of human and animal diseases. The genus Streptococcus was initially depicted in view of morphologically, serological, physiological and biochemical characteristics and it involved an extensive variety of organisms including the highly pathogenic bacteria, S. preumoniae , S pyogenes and S. agalactiae; the intestinal group D streptococci. S faecalis and S. faecium ; and the economically important group N starter bacteria S.cremoris and S. lactis. The members of the Streptococcus are Gram-positive organisms that usually form pairs or chains. In 1937, Sherman separated the genus according to physiological and growth characteristics, especially with regards to temperature limitations on growth. Four general groups designated by Sherman are (1) pyogenic, (2) viridans, (3) enterococcus,
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and (4) lactic. This categorization has become somewhat obsolete as relationships between species have been shown to overlap. The streptococcus is classified as a thermophile growing at 45°C, and higher, and is widely used in the production of yoghurt and some types of cheese such as Mozzarella cheese. Streptococcus thermophilus is the only species of this genus found in starter cultures. Recently, it has been used widely in the production of Cheddar cheese. It is a component, along with lactococci, in some DVI / DVS cultures in which it produces acid rapidly during scalding. Its incorporation in Cheddar-cultures also has the advantage of reducing the production costs of DVI/DVS cultures and controlling retail prices. Growth stops at around 15°C. In the production of yoghurt; the cocci are cells of Streptococcus thermophilus and the rods are cells of Lactobacillus delbrueckii subsp. bulgaricus. Like lactococci and many leuconostocs, strains of Streptococcus thermophilus are catalase-negative; coccus shaped and occur in pairs and chains. Generally, most strains produce long chains. L-lactic acid only is produced and carbon dioxide is not produced from glucose. Some strains produce urease and have the potential to produce CO2 from urea. Since Streptococcus thermophilus and Streptococcus thermophilus-like organisms can grow in the regeneration section of pasteurisers high levels can every so often happen in cheese. Urease-producing strains have the potential to cause openness in cheese. Moreover the failure of many strains to process galactose can bring about cheddar with noteworthy convergences of a fermentable starch that could be utilized by NSLAB for gas generation. The potential association of Streptococcus thermophilus ought to be considered during examinations of occurrences of open texture or unmistakable gas generation in cheese. It is likely that the intermittent
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issues of excessive early acidification experienced by Mozzarella makers utilizing extended production runs with pasteurised milk are due to NSLAB and in particular Str. thermophilus-like organisms that had grown to high cell densities in the regeneration section of pasteurisers. Strains differ in their ability to utilise galactose. Use of non-galactose fermenting strains will result in high levels of this reducing sugar in products. Since galactose and other reducing sugars react with amino acids in the Maillard reaction it is usual to only select galactose-utilising strains to reduce the probability of undesirable colour changes occurring in heated products. Streptococcus salivarius, usually found in salivation, has been appeared by DNA: DNA hybridisation concentrates to be like Streptococcus thermophilus. Along these lines, for a few years Streptococcus thermophilus was classified as a subspecies of Streptococcus salivarius. However, it is now accepted that Streptococcus thermophiles while similar, adequately unmistakable to to justify species designation. Streptococcus thermophilus is sensitive to low levels of salts and in particular to sodium chloride concentrations of around 2%. This sensitivity is important in using DVI / DVS cultures in Cheddar and similar cheeses. Cheese producers should understand that once the salt in moisture concentration exceeds 2 - 3% lactose utilization and acid production stops. M17 medium broadly utilized as a part of concentrates in studies with lactococci is not an ideal medium for the growth of some strains unless modified to reduce its glycerophosphate concentration. Note that large portions of the typically develop attractively on M17 agar.-like strains isolated from pasteurizers are are significantly less sensitive to salt and typically develop attractively on M17 agar.
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Fig. (4). Streptococcus thermophilus Lactobacillus: Lactobacillus genus is composed of a large group of Gram positive, catalase negative, rod-shaped bacteria. Some species are homofermentative while others are hetrofermentative. While some species produce mainly L-lactate from glucose, others produce D-lactate. Since some strains exhibit significant racemase activity, a racemase is an isomerase enzyme, D/L lactic acid is also produced. Strains may also exhibit coccoid morphology and this can lead to confusion with leuconostocs and perhaps even lactococci. Lactobacilli are used as starters in the production of yoghurt and Mozzarella cheese. They are also used as starter subordinates to promote faster ripening of Cheddar and comparable cheeses, to decrease the rate of intensity of bitterness and as probiotics in yoghurt type products. Lactobacillus delbrueckii subsp. bulgaricus is widely used along with Streptococcus thermophilus as a starter in yoghurt production. This
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subspecies is homofermentative, produces about 2% w/v lactic acid in milk, has an optimum temperature of 42°C and grows at temperatures of 45°C and higher. It will not grow in low concentrations of salt and is sensitive to bile salts.
Fig. (5). Lactobacillus bulgarius Lactobacillus acidophilus, which is typically present in the digestive system, is generally not utilized as a starter; it is widely used as a probiotic. This bacterium, is homofermentative, producing high concentrations of D-lactic acid in milk, has an optimum temperature of 37°C, and is relatively tolerant of oxygen, compared with Bifidobacterium species that are frequently used in conjunction with this organism. Little growth occurs at temperatures less than 20°C and most strains show no growth at 15°C. Because Lactobacillus acidophilus produces D-lactate there have been some concerns about its use in infant nutrition.
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Fig. (6). Lactobacillus acidophilus Lactobacillus casei is also a normal inhabitant of the small intestine and impervious to bile. It is used as a probiotic although it is found in some starter cultures and is commonly one of a number of non-starter lactic acid bacteria (NSLAB) found in Cheddar cheese. Rogosa agar is widely used as a general isolation medium for lactobacilli.
Fig. (7). Lactobacillus casei
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Lactobacillus helveticus is frequently utilized alongside other thermophilic lactic acid bacteria in the production of a range of fermented milk products including Emmental cheese, Mozzarella and yoghurt. One advantage of this species along with Lb. delbrueckii subsp. bulgaricus is that Lactobacillus helveticus utilizes galactose and this can be useful if products free of reducing sugars are required. Since many strains have been appeared to have proline-iminopeptidase-like action, Lactobacillus helveticus has been used to produce modified 'Cheddar-type cheese' with some of the 'sweetness' attributes of Swiss cheeses like Emmental. More recently, assigned strains have been utilized a as starter subordinates to decrease bitterness in a scope of cheeses, to enhance or potentially to quicken maturing.
Fig. (8). Lactobacillus helveticus Bitterness is reduced due to peptidase action on starter-derived hydrophobic peptides. The species is homofermentative and produces high concentrations of D/L lactic acid in milk. Many strains grow at 45°C despite the fact lower temperatures 42-43°C for the most part give higher recuperations when enumerated using selective media such as Rogosa or modified MRS agars. demonstrate no or little development at 15 °C
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(some atypical strains may take several weeks to develop at 15 °C or below). Lactococcus: Basically, the bacteria in this group were classified as members of the genus Streptococcus and were designated as lactic streptococci. They were differentiated from other streptococci, some of which are pathogens, by their specific reaction with Group N antiserum and by their tolerance to temperature, salt and dyes. It is now known that serotyping lactic LAB has limited value in species differentiation; strains of the same species may react with different sera and some strains may exhibit no group antigen. The ovoid shape of lactococci can be hard to interpret in light of the fact that the cells are some cases elongated in the plane of chain formation bringing about misclassification of some lactococci as lactobacilli. The most perceived habitat for the lactococci is dairy products. They are nonmotile, coccus- shaped homofermentative bacteria that grow at 10°Cbut not at 45° Cand produce L ± lactic acid from glucose. Some strains become well adapted to develop in milk because of their efficient uptake and fermentation of lactose. Strains with the capacity to utilize citrate with production of diacetyl were classified as S. diacetylactis and subsequently as Lc. lactis subsp. lactis. The utilization of lactococci is widespread and has the longst convention in industrial starter culture technology. Strains of Lc. lactis produce nisin, which is a relatively wide range bacteriocin that is active against gram- positive bacteria including Clostridium botulinum and its spores Lactococci can be differentiated to the species or biovariant level using the scheme developed for lactic streptococci. The lactococci will not grow on Rogosa agar. Differential, but not selective, media are available and can be useful for quality control and strain isolation purposes. The
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medium, Reddys' Differential Agar, developed by Reddy et al. (1972) is still of value. This medium contains the differential ingredients lactose, calcium citrate, L-arginine and the pH indicator bromocresol purple. This indicator gives yellow and blue/purple colours under acid and alkaline conditions respectively. Lactococcus lactis subsp. cremoris gives yellow colonies due to acid production from lactose. Lactococcus lactis subsp. lactis while producing acid also produces ammonia from arginine. The ammonia neutralizes the acid and eventually produces an alkaline reaction that results in blue/purple coloured colonies. Lactococcus lactis subsp. Lactis biovar. diacetylactis also gives a blue/purple colony. Because some strains of Lactococcus lactis subsp. lactis possess only weak arginase activity streaking techniques on an improved version of this medium may be helpful in identifying these strains.
Fig. (9). Lactococcus lactis subsp. lactis
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Metabolic activity of lactic acid bacteria: Lactic acid bacteria (LAB) are for the most part mesophilic, however can develop at temperatures as low as 5 ºCor as high as 45 ºC So also, while the majority of strains grow at pH 4.0-4.5, some are active at pH 9.6 and others at pH 3.2. Strains are generally weakly proteolytic and Lipolytic and require amino acids, purine and pyrimidine bases and B vitamins for growth. Glucose
Homolactic
Heterolactic
Gluccose-6 phosphate
Glucose 6-p
Fructose-6-p
6 phosphogluconate
Fructose 1,6-DP
Riboulose ± 5-p
Xylulose-5-p
Glyceraldehyde3-p H2O
Dihydroxacetone-p Glyceraldehyde-3p
Acetyl.P
2pyruvate Pyrurate
Acetaldehyde
Lactate
Ethanol
2 lactate
Fig. (10). Generalized scheme for the fermentation of glucose in lactic acid bacteria Source: Cogan and Hill (1993)
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All LAB produce lactic acid from hexoses and since they lack functional electron transport chains and a functional Krebs cycle, they obtain energy via substrate level phosphorylation. The lactic acid produced may be L (+) or less frequently, D(-) or a mixture of both. It should be noted that D(-) lactic acid is not recommended for infants and young children (WHO, 1974). The pathways for hexoses divide lactic acid bacteria into two groups, homofermentative and heterofermentative (Fig. 10). Homofermenters such as Pediococcus, Streptococcus, Lactococcus and some Lactobacillus produce lactic acid as the a sole end product of glucose fermentation. However, under modified development conditions and when the underlying substrate is a pentose this may change. Homofermenters use the Glycolysis pathway to generate two moles of lactate per mole of glucose and derive approximately twice as much energy per mole of glucose as heterofermenters. Heterofermenters such as Weisella and Leuconostoc and some Lactobacillus produce equimolar amounts of lactate, CO2 and ethanol from glucose through hexose monophosate or pentose pathway.
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CHAPTER FOUR FERMENTATIVE CHANGES IN MILK BROUGHT ABOUT BY STARTER CULTURES Starter cultures can bring about at least 4 major fermentation reactions in milk: 1- Lactic acid fermentation 2-Propoinic acid fermentation 3- Alcoholic fermentation 4- Citric acid fermentation 1- Lactic acid fermentation: Lactic acid fermentation is the simplest type of fermentation- a onestep reaction. In this reaction no gas is formed. Since 2 ATP molecules are consumed in formation of hexose diphospate from glucose and 4 molecules are subsequently produced, the net yield is 2 ATPs per hexose molecule metabolized. As 2 hexose molecules are generated from one molecule of lactose, there is a net yield of 4 ATP molecules. This fermentation is identical with glycolytic pathway. There are two types of lactic fermentation:
1-Homolactic:lactate (lactic acid) is the end product, and lactic acid bacteria (LAB involved) include: Lactobacillus casei, Lactococcus lactis subsp. lactis,Lactococcus lactis subsp. cremoris. 2-Heterolactic: only half of each glucose molecule is converted into lactate (lactic acid), the other half being converted into other products. Both types are responsible for acidification of milk and providing desirable flavours.
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In Swiss cheese the late formation of Co2 is responsible for the formation of eyes, while propionic acid contributes to the flavour development. 2-Propionic acid fermentation: Propionic acid and its salts are generally utilized as a part of industry and particularly in the food industry as antifungal agents. A substantial part of this production is by petrochemical routes. Nevertheless, fermentation processes have been depicted since 1923. The expanding customer interest for biological products and the more efficient performance of new fermentation processes have restored scientist and industrial interest for a new investigation of biological propionic acid production. . expansion comes about because of the rise of high density cell bioreactor technology. Generally, pyruvate is carboxylated to yield oxaloacetae which is reduced to succinate and is then decarboxylated to yield propionate (propionic acid). Species of the genus Propionibacterium species are utilized in production of propionic acid. The varying ratios of propionic to acetic acid as a result of the fermentation of glucose are attributed to differences in carbon dioxide tension and in the initial and final pH values of the cultures. Propionic acid and its salts are utilized in a number of processes such as the production of cellulose plastics, herbicides, in the manufacture of ester solvents, fruit flavours (citronellyl propionate and geranyl propionate), perfume bases and butyl rubber to improve process ability. In addition, the association of propionic acid with lactic and acetic acids has been recommended for the preservation of foods.
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Fig. (11). Propionibacterium shermanii
Microbiology of the propionic acid fermentation: The first works on propionic acid fermentation resulted in the formulation of the Fitz equation: 3 lactic acid -> 2 propionic acid + 1 acetic acid + 1 CO2 + 1 H20 or 1.5 glucose -> 2 propionic acid + 1 acetic acid + 1 CO2 + 1 H20 The maximum yields are 54.8% (w/w) as propionic acid and 77% as total acids. Formation of propionic acid is accompanied by the formation of acetic acid. The dicarboxylic acid pathway is the most common pathway for the formation of propionic acid. The acrylic pathway, restricted to a few species of bacteria (Clostridium propionicum, Megasphaera elsdenii, Bacteroides ruminicola), also leads to propionic acid formation.
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Fig. (12). A propionic acid production plant 3-Alcoholic fermentation: Alcohol fermentation, also known as ethanol fermentation, is the anaerobic pathway carried out by yeasts in which simple sugars are changed over to ethanol and carbon dioxide. Yeasts commonly work under vigorous conditions, or within the sight of oxygen, but on the other hand are fit for working under anaerobic conditions, or without oxygen. At the point when no oxygen is promptly accessible, alcohol fermentation occurs in the cytosol of yeast cells. The Process of Alcohol Fermentation: Alcoholic fermentation is a complex process that includes twelve different chemical reactions. Each of the twelve chemical reactions in alcoholic fermentation of glucose requires an enzyme.
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The fundamental condition for alcohol fermentation demonstrates that yeast begins with glucose, a kind of sugar, and completes with carbon dioxide and ethanol. a type of sugar, and finishes with carbon dioxide and ethanol. . Nonetheless, to better comprehend the process, we have to investigate some of the steps that take us from glucose to the final products. The process of alcohol fermentation can be divided into two parts. In the first part, the yeast breaks down glucose to form 2 pyruvate molecules. This part is known as glycolysis. In the second part, the 2 pyruvate molecules are converted into 2 carbon dioxide molecules and 2 molecules of ethanol, otherwise known as alcohol. This second part is called fermentation. The fundamental reason for alcohol fermentation is to produce ATP, the energy currency for cells, under anaerobic conditions. So from the yeast's viewpoint, the carbon dioxide and ethanol are waste products. That's the fundamental overview of alcohol fermentation.
Fig.(13). %UHZHU¶V\HDVW
ϯϲ
Fig. (14). Alcoholic fermentation
Fermentation conditions: The fermentation of sugars to ethanol is promoted by the following conditions: 1. The sugars being in solution (involving mashing of grain or fruit if necessary). Not all sugars are fermentable. Non fermentable sugars in solution will remain after fermentation and will result in a sweeter end product. Malt has non fermentable sugars which can be used to balance the bitterness of the hops. The amount of sugar in the solution can be too much and this can prevent fermentation͘ 2. The presence of yeast (which contains certain enzymes):The ability of yeast cells to convert sugar into Carbon dioxide and alcohol is down to enzymes. Several enzymes are involved each does its step in the process. The final step is Zymase reduction which takes the end product of the other enzymes (acetaldehyde/glycerol), and turns this into ethyl alcohol. In fact, high concentrations alcohol actually
ϯϳ
destroys enzymes and kills the yeast cell. Different strains of yeast can tolerate different concentrations of alcohol. 3. A temperature. The fermentation process has limits such as temperature. Greater than 27C kills the yeast less and than 15C results in yeast activity which is too slow. 4. The exclusion of air, which provides low oxygen concentrations. 4-Citric acid fermentation:
Citric acid is the most vital organic acid produced in tonnage and is widely utilized in food and pharmaceutical industries. It is produced principally by submerged fermentation using Aspergillus niger or Candida sp. from various sources of carbohydrates, such as molasses and starch based media. However, other fermentation techniques, e.g. solid state fermentation and surface fermentation, and alternative sources of carbon such as agroindustrial buildups have been intensively studied indicating extraordinary point of view to its generation. Citric acid (C6H8O7, 2 ± hydroxy ± 1,2,3 ± propane tricarboxylic acid), a characteristic constituent and normal metabolite of plants and animals, is the most is the most flexible and broadly utilized organic acid in the field of food (60%) and pharmaceuticals (10%). Applications of citric acid: Citric acid is basically utilized as a part of food industry industry as a result of its pleasant acid taste and its high dissolvability in water. It is worldwide acknowledged DV ³*5$6´ JHQHUDOO\ UHFRJQL]HG DV VDIH endorsed by the Joint FAO/WHO Expert Committee on Food Additives. The pharmaceutical and cosmetic industries retain 10% of its usage and the rest of utilized for various other purposes.
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Microorganisms used for citric acid production: A large number of micro-organisms including bacteria, fungi and yeasts have been e utilized to produce citric acid. The majority of them, however, are not ready to produce industrially worthy yields. This reality could be clarified by the fact that citric acid is a metabolite of energy metabolism and its accumulation rises in considerable amounts only under conditions of drastic imbalances. Among the micro-organisms used to produce citric acid, only Aspergillus niger and certain yeasts such as Saccharomycopsis sp. are employed for commercial production of citric acid. However, the fungus Aspergillus has remained the organism of choice for commercial production. The main advantages of using this microorganism are: (a) its ease of handling, (b) its ability to ferment a variety of cheap raw materials, and c) high yields. Among lactococci only Lc. lactis subsp. lactis biovar diacetylactis has the ability to metabolise the citrate present in milk. The metabolic end products of citrate metabolism are diacetyl, acetoin, 2,3 butanediol, acetic acid and carbon dioxide which contribute to the flavour development in fermented dairy products.
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acids, has a molecular mass of 3354. belongs to the group of lantibiotics, and has a practical use in food preservation. It is successfully used in the production of cheeses, melted cheeses, milk desserts, fermented drinks and canned vegetables. In 1988 the US Food and Drug Administration (FDA) accepted it as a preservative for prevention of delayed clostridial bloating in cheeses. In addition to Lc. lactis subsp. lactis, the certain strains of Lc. lactis subsp. cremoris and Lc. lactis subsp. lactis biovar diacetylactis possess an ability to produce different bacteriocins with a range of inhibition. However, their possible value in the growth control of microorganisms causing decay, and of pathogenic microorganisms, remains to be investigated.
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CHAPTER FOUR FACTORS AFFECTING FERMENTATION CHARACTERISTICS OF STARTER CULTURES
The fermentation procedure of lactic acid bacteria (LAB) starters can be affected by many factors, for example, temperature, pH, strain capability, growth medium, inhibitors, bacteriophage, incubation period, heat treatment of milk, etc. There is a need to play it safe to accomplish the optimal activity of lactic acid bacteria during the production of fermented milks. The important factors influencing the growth and activity of lactic starter cultures include the followings: 1-Temperature: Temperature is one of the essential elements, which specifically impact the development of microorganisms. Although different types of LAB have different optimal growth temperature, most Lactic starters grow optimally at 27-32 C. including: L.lactis subsp.lactis, L.lactis subsp. cremoris«HWF. On the other hand, S.thermophilus and some lactobacilli grow best at 37-42 C. And Leuconostocs grow best at 20-30 C. The variation in temp affect strain dominance in mixed and multiple starters. 2-pH: Control of pH in milk during propagation of starter culture is important because extreme acidic could be detrimental for viability of LAB. Despite the fact that milk is thought to be about the ideal food for man and bacteria, it still needs some enrichment for use as a bulk starter medium. Lactic starter produce lactic acid at the level of more than 10 percent of their weight per minute after their development in milk and
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along these lines, and thus pH of milk is lowered. The extreme acidic pH could be detrimental for the viability of LAB. Hence, control of pH in milk during propagation of starters is very important . This fact is generally ignored during the commercial production for bulk starters. externally and internally pH controlled media used for the preparation of bulk starters. pH can be controlled via: 1-Preparation of frozen concentrates is done under continuous neutralization. 2- Diffusion culture technique for removal of toxic end products of metabolism of S.C. 3-Genetic manipulation of these organisms by introducing a pH sensitive promoter for regulation of structural genes involved in acid production. 3-Strain compatibility Mixed starters have been utilized or the preparation of several fermented dairy products. In any case, maintenance of mixed strain starters in cheese processing plants is very little honed any more, partially because repeated subculture of mixed strains of lactococci may bring about decrease in number or loss of all but one of the strains that the long run a single strain remains in the mixed starter preparation. The factors lead to strain dominance include: differences in generation times, acid sensitivity, production of antibiotic or bacteriocin by the component strains and differences in optimal temperature. In addition, antibiotic resistance of some strains in the mineral cultures which will not be influenced by the presence of inhibitory substances in milk and the milk fermentation cultures also plays a very important role in bringing about the desirable changes in milk. In mixed cultures if one strain is infected, others will not. The associative action of between individual strain in mixed cultures plays important role in bringing about desirable changes in milk.
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4-Growth medium:
The media used for the cultivation of LAB are quite complex. MRS, M17 and Lactic or Eliker medium are the complex medium used for cultivation of LAB. Lactic medium and M-17 are excellent growth medium and is extensively used for the growth of lactococci. A few plating media have been depicted for separation of various types of lactococci, specifically, L. lactis subsp. lactis, L. lactis subsp. cremoris and L. lactis subsp. Diacetilactis. LAB also grow very well in milk, and at specific seasons of the year more inoculum is needed for milk fermentation. Late lactation milk and the milk produced in winter is usually deficient in certain factors that promote growth of LAB, so some stimulatory factors should be added to this milk. 5-Inhibitors
The growth and activity of the starter cultures in milk is unfavorably influenced because of the presence of residual antibiotics and sanitizers in milk, and additionally the production of antibiotic-like substances (bacteriocins) by certain wild strains of Lactococcus lactis subsp. lactis and other lactic cultures in raw milk. Antibiotics, for example, penicillin or streptomycin may enter milk because of their unpredictable use in the treatment of mastitis or udder sicknesses. Consequently, milk must be altogether checked for the presence of residual antibiotics before addition of starter cultures. The methods based on immunological reactions as well as isotopic tracer dilution procedures (Charm test) are very effective.
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6-Bacteriophage:
%DFWHULRSKDJHVRU³SKDJHV´DUHYLUXVHVWKDWLQIHFWEDFWHULD7KH\DUHQRZ believed to represent the most abundant biological entities. These bacterial viruses are present in ecosystems where bacteria have been found, including man-made ecological niches such as food fermentation vats. These bacteriophage cause slow acid production by LAB. Despite extensive efforts, however, phage infection of starter LAB cultures remains the most common cause of slow or incomplete fermentation in the dairy industry. Phage infection represents the most significant biological factor influencing industries that depend on bacterial growth and metabolic activities. Depending on the process stage in which the infection proceeds, consequences may vary from slow acid production to completely lost batches. High pH values, high residual lactose concentration and insufficient lactic acid content are the result of phage attacks occurring during the early stages of the fermentation͘ The promising solution by replacing the phage sensitive strains with phage resistant ones. The use of defined single strains and their phage resistant mutants is used widely in some parts of the world. 7-Incubation period
The incubation period of is another important factor, which can influence the growth of lactic acid bacteria. The higher the incubation temp. up to certain limits, the faster development of S.C. Normally 16-24 hrs at the optimum temp. is adequate for maximum growth of these organisms. Storage of ripened starters for about 18h at low temp. does not affect their activity , although over-rippened cultures are affected on prolonged storage.
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8-Heat treatment of milk
Heat treatment of milk usually improves its value as a medium for starter organisms and other lactic acid bacteria. Different species of LAB behave differently in heat treated milk. Adequate heat treatment of milk results in many advantages as follows: 1- It drives out the dissolved oxygen, 2- It brings about formation of sulphydryl compounds (acting as growth factors), 3- Destruction of inhibitory substances naturally present in milk and killing of antagonistic bacteria. However, with more severe heating, slight protein breakdown may occur with the formation of peptides and amino acids, which act as nutrients. In addition to these, different species of lactic acid bacteria appears to behave differently in heattreated milks. For example, the growth of S. thermophilus appears to be favored, while L. lactis subsp. cremorisdisfavored by drastically heat treatment of milk.
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9-Degree of aeration The lactic acid bacteria (LAB) prefers a medium with reduced oxygen tension than that of atmosphere, so the acid production is faster at the bottom of the container or under controlled oxygen (reduced oxygen tension). Possibly, a reduced level of oxygen tension is ideal for the initiation of growth as it manages energy for growth by a mechanism somewhat more effective than the lactic fermentation, which releases only a small fraction of the energy available in the lactose. However, agitation is clearly a dubious condition and may sometimes quicken souring. It unmistakably includes two unique factors, namely, oxygenation and movement of the medium, which appear to have opposing effects on the starter cultures. Although excessive aeration might be the reason for slow starter, its effects might be neutralized by heating milk or by adding sulphydryl compounds. 10-Effect of carbon dioxide
A minimum concentration ofcarbon dioxide is essential for the initiation of bacterial growth. Complete removal of carbon dioxide from the medium results in extended the lag phase until the bacteria have slowly produced sufficient carbon dioxide to sustain normal growth. For LAB the optimum concentration of carbon dioxide varies from 0.2-2.3%. Small amount of CO2 Sterilized skim milk may lead for the prolonged lag phase of the given starter culture. However, incorporation of yeast extract in milk at a concentration of 0.5% can get rid of this problem. 11-Storage conditions:
Storage of lactic acid bacteria is yet another imperative factor influencing their performance during the manufacture of fermented milk products. It
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is recommended not to store the mature cultures in presence of acid that they have produced during growth. Storage under these conditions will result in cellular injury and the cultures will become slow and can no longer be useful for the preparation of fermented products. It is important therefore to transfer the cultures to fresh milk and in a refrigerator without incubation, when a new culture is needed, it is removed from the refrigerator, incubated and transferred to fresh medium and placed in the refrigerator again. Mature cultures may also be stored at 2-5 °C in milk with added calcium carbonate. Cultures can also be frozen and stored at -40 °C or below in the freeze dried state. Frozen storage in liquid nitrogen (-196 °C) in the form of starter concentrates.
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CHAPTER SIX PREPARATION OF STARTER CULTURES
The propagation and preparation of starter culture is one of the most important operations in a plant. Since the quality of a starter has a direct bearing on the quality of the fermented product, starter must be in good shape. For the best performance and maintenance milk ought to be cleaned by heating at 90°C for 1 hour. The breakdown products of milk by utilizing heat treatment act as bacterial growth factors. This is most likely one reason why lactic acid bacteria develop more quickly heated than unheated milk. After giving sufficient heat treatment milk is cooled to 22-25°C and inoculated with appropriate inoculum size. After inoculation, the culture is incubated at 22-25°C until clotting takes place, and thereafter it is stored in a refrigerator. Further, a small aliquot of the culture is inoculated into a similar container containing sterilized milk for storage of mother culture. The remaining culture is inoculated into the starter can or vessel containing sterilized milk. The purity of mother culture however is very essential. The inoculums generally is added at the rate of 1 percent from a culture having approximately 0.8 percent acidity. The acidity should not exceed 0.9 percent lactic acid. In case of poor performance including slow activity, any visible abnormality in behaviour, flavor and colour and appearance of the starter, it should be immediately discarded and fresh starters should be used. The purity and activity of the starter culture should be maintained by any means to achieve desirable fermentation efficiently in the manufacture of cultured dairy products. The starter culture must contain maximum number of viable organisms and must be very active under production
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conditions of the plant. For the preparation of fermented dairy products like cheese, dahi, yoghurt, etc. starter cultures are often maintained in milk, heated to 90°C for 1 hour and bulk starters may be grown in milk held at 90oC for 30 min. This heat treatment is adequate to kill phages and all other vegetative bacterial cells. Principles to maintain active tarter culture : 1- By reducing the metabolic activity of the S.C. through refrigeration 2- By separating the organisms from their waste products through concentration and preservation. The success in the preparation of cultures depend on several factors: 1- The choice of milk: Some specific types of milk are unsuitable for growth of starters due to alteration in the chemical composition of milk as a result of disease e.g mastitis. Such milk may have high bacterial counts and may contaminate the starter and produce off-flavours, so it should not be used for starter preparation. The chosen milk must be from a healthy cow that are secreting normal and clean milk, free from inhibitory substances. Also milk with high lipolytic activity and milk low in total solids should not be used. 2- Heat treatment of milk: A minimum of 30 min at 71.1 C is necessary. Sometimes use of sterile milk is recommended, but it has some disadvantages: 1- more drastic heating may inhibit the culture 2-The formed culture is soft and sloppy.
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Steaming or boiling of milk for 30-60 min. After heat treatment, milk must be immediately cooled to inoculation temperature to minimize physicochemical changes in milk. 3- Containers/Utensils: Stainless steel aluminium enameled wares and glasses are the best for starter propagation and among them the stainless steel vessels are the best though expensive. The best form of a starter vessel is a jar or cylindrical container at least twice as high as its diameter and having a smooth surface free from crevices to ease its cleaning. After cleaning, the utensils should be sterilized by steaming at least 30 min. at 100 C. 4- Amount of inoculum: Amount of inoculum may affect performance of a starter, this depends on the time and temp. , individual culture characteristics and condition of culture at time of inoculation. The ideal inoculum size for strater prepartion is 0.5-2.0%. In mixed cultures, some cultures may be inoculated at higher levels than others if desirable characteristics are to be maintained. 5- Aseptic culture transfer: Strict aseptic conditions should be maintained while transferring S.C to avoid aerial contamination. There should be a separate inoculation chamber equipped with a UV lamp and devices for the spray of proper detergents and sanitizers. The sterilization of inoculation needles , pipettes, spoons and other equipments is necessary.
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6- Time of inoculation: After inoculation, most cultures should be incubated at 21.1-30 C , and time of incubation depend upon the inoculum size and the individual culture characteristics. Normally inoculation time is 14-16 hr is ideal when inoculum size is 1.0%. Maintenance of proper temp. is important particularly during summer. 7-Cooling:
After achieving the desirable growth of S.C. it should be immediately cooled to stop further development so that it is in a good condition for reuse. Refrigeration give the appropriate cooling effect, in majority of the cases, the mother cultures are stored in refrigerator until further use. Preparation of mother culture: Preparation of mother culture is a very important step in the production of bulk starter. The mother cultures are maintained in narrow neck poly ethylene bottle which are washed in detergent, sterilized over steam and filled with 0.1% hypochlorite solution, the seals and caps are also autoclaved and held in the hypochlorite solution. This process has advantage that contamination of starter at any one stage does not entail contamination at the next stage. The preparation steps are as follows: 1-The sterilized bottles filled with ¾ th milk is heated to 90 C for 1 hr, cooled to 22-25 C and closed inverted cap. 2-This milk is then inoculated aseptically with starter of guaranteed purity form a reliable resource. 3-After inoculation, the culture is incubated at 22-25C until clotting takes
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place, subsequently stored in a refrigerator. 4-At the appropriate time, a small aliquot of the culture is re-inoculated into a similar container containing sterilized milk for storage of mother culture. The remaining culture is inoculated into the starter can or vessel which is filled with milk. 5- The inoculum should be added at the level of 1.0% from a culture with 0.8% acidity Preparation of working cultures: Working cultures are the same as small mother cultures in bottles. To avoid making big number of these working cultures for a series, few large bottles could be used as working cultures to inoculate the whole series. Preparation of bulk starters: 1-The used vessels are steam sterilized and filled with bulk raw milk. 2- Milk in the container is steam sterilized at 72-73 C for 45 min. and cooled to the incubation temp. 3- Then inoculated in the same way as mother culture and agitated in order to mix the starter into the milk by rotating the vessel. 4- incubated under selected conditions. Continuous starter production: This technique is more economical, convenient. Many cheese plants today may use up to 100.000 gallons of milk for cheese production in one day requiring almost 500-1000 gallons of starters every.Such bulk starters require many items of equipments and the maintenance costs for cleaning and sterilization which are costly.
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A serious disadvantage, is that if the culture is attacked by a phage, the whole bulk and all equipments get contaminated, so control of purity and activity is important. Preparation of master culture: For preparation of master cultures, litmus milk previously sterilized in glass bottles is filled into polyethylene tubes. The method of inoculation is the same as for mother culture, however, ilk is sterilized or pasteurized. Such cultures may be maintained indefinitely with careful checking and testing after 3 months interval.
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CHAPTER SEVEN MAINTENANCE AND PRESERVATION OF STRTER CUTURES
Maintenance and preservation of microbial culture is noteworthy undertaking. The basic principle in preserving the cultures is to keep the morphological and physiological characteristics of the organism place .Large collections of starter cultures are used in a laboratory for various reasons. So it is essential to preserve them properly for further use. Microbial culture protection aims at maintaining a microbial strain alive, uncontaminated, and without variety or transformation, as like unique disengage. Many types of work require promptly accessible microorganisms. The caused in gaining them from other sources or attempting to re-isolate them from their natural habitat can be unacceptable. Sometimes it is impossible to obtain the same isolate again. Sometimes repeated attempts of reisolation of the same organism have been failed. Importance of maintenance and preservation: Considerable work has been devoted to finding methods of maintaining cultures in a vigorous and stable condition. The productive routine with regards to microbiology depends on the utilization of cultures of microorganisms. Authentic reference strains are required for comparison with laboratory isolates, for control cultures in standard methods of analysis, and for use in research and teaching. The great increase in number and size of industrial fermentations has highlighted the benefit of maintaining collections of microorganisms, particularly of production strains, assay organisms and related species. Industrially important microorganisms are also maintained for utilization in various industrial processes. The preservation of bacterial stock cultures to maintain viability and biochemical or virulence characteristics
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is an integral requirement for the continuity of microbiological research. Simple access to effectively growing cultures is a necessity of most microbiological laboratories. Cultures are routinely required generally on a day-to-day basis for quality control, comparative testing, inoculum for bioassays and for various other reasons. Methods of starter cultures preservation: The objective of preservation methods is to maintain the viability and genetic stability of the culture by reducing the organism's metabolic rate thereby extending the period between subcultures. Many preservation techniques have been employed to preserve starter culture microorganisms. The techniques that have been developed and used can be divided into three categories: 1- Continuous growth 2-Dehydration 3- Frozen storage
The factors which increase the time period between subcultures include: manipulation of growth conditions by limiting carbon, nitrogen and energy sources, lowering the temperature, or preventing dehydration. Other than this, dehydration can be used to preserve microorganisms: techniques include air-drying, desiccation in or above a desiccant, or drying in a vacuum either from the liquid or frozen state. Frozen storage is storage at a temperature where the organism is frozen to decrease or completely prevent metabolism and physical change. Success of the preservation depends upon the use of the proper medium and cultivation procedure and on the age of the culture at the time of preservation.
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The method of preservation is mainly of two types: i- short- term preservation: include mainly the serial transfer of organisms to fresh medium storage at low temperature, maintenance of spores of spore formers in dry sterile soil etc. long term. ii- long-term preservation methods: are now widely used and use either freeze drying or ultrafreezing in liquid nitrogen (-196ºC) It is known that there is no universal method of preservation that is successful for all microorganisms. Different group of microorganisms respond differently to different preservation methods. The preservation methods used reflect the distinctive biological properties of the different groups of microorganisms such as bacteria, viruses, fungi, algae. The choice of preservation method depends on the nature of the microorganism, availability of equipment and skilled personnel and on the preservation objective. The most commonly used methods are discussed below. 1- Regular transfer of starter culture: Microbial starter cultures can be maintained by periodically preparation of fresh culture from a previous stock culture. The culture preserved in this way is maintained by alternate cycles of active growth and storage periods acquired by arrangement of subcultures. The frequency of transfer varies with the organism. Many organisms remain viable for several weeks or month . After growth for 24 hours at 37ºC, the slants can be stored at low temperature for 20- 30 days. The frequency of the starter culture can be decreased if developing it on a medium containing minimal nutrition decreases the metabolism of the organism. Several factors are considered while maintaining a microbial culture by using subculture method. The chosen temperature should support slow growth of the microorganisms rather than rapid growth.
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Cheese cultures (S. lactis, S.cremoris, L. cremoris ) can be propagated up to 50 times without any fear of mutation. And the sterilized `medium is inoculated at a rate of 1% and incubated at 22 or 30 C for 18 hr or 6 hr, respectively. While yoghurt S.C. are normally sub-cultured only 15-20 times as safe guard against mutation, and the incubation is carried out at 42C for 3-4 hr or at 30C for 16-18 hr. Starter culture activity is affected by the rate of cooling after incubation, level of acidity at the end of the incubation period and duration of storage. The reserve stock-culture can be maintained in a liquid form, and incubation for inoculated medium for a short time and stored under normal refrigeration. Reactivation is only necessary every 3 months. 2- Storage at the refrigerator: Live starter cultures on a culture medium can be successfully stored in refrigerators or cold rooms (at 4ºC). At this temperature the metabolic activities of microbes slows down greatly. Consequently, bacterial metabolism will be very slow and only less amounts of nutrients will be utilized. The cultures should be prepared using standard technique and contamination should be avoided and then sealed before storage. In this method, vials are filled with I ml agar medium and sterilized. Microorganisms are then brought into the set agar. The starter culture is incubated for overnight and then stored at 4ºC. This method can't be utilized for a very long time because toxic products get accumulated which can execute the microbes. 3- Paraffin Method: Paraffin method is a very simple and most economical method of preserving cultures of starter cultures for longer time at room temperature. In this method, the agar slants are inoculated and incubated
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until good growth appears. They are then covered with sterile mineral oil to a depth of 1 cm above the tip of slant surface The layer of paraffin prevents dehydration of the medium and by ensuring an aerobic condition, the microorganism remain in dormant state. Transfers are made by removing a loop full of the growth, touching the loop to the glass surface to drain off excess oil, inoculating a fresh medium and then preserving the initial stock culture. The most common used oil is paraffin or Vaseline with a layer of 1-2 cm thickness. This method slows the metabolic activity by reduced growth through reduced oxygen tension. Cultures can also be maintained by covering the agar slants with a layer of sterile mineral oil about half inch above the surface of the slant. The cell viability in this technique is high when preserving bacteria from the genera of Azotobacter , Mycobacterium and Bacillus. 4- Storage in soil: Many fungus genera such as Fusarium, Penicillium, Alternaria, Rhizopus, Aspergillus etc. demonstrated fruitful for storage in sterile soil. Soil storage involves inoculation of 1 ml of spore suspension into soil (that has been autoclaved twice) and incubating at room temperature for 5-10days. This initial growth period enables the fungus to utilize the accessible and gradually to become dormant. The bottles are then stored at refrigerator. Spraying few soil particles on an appropriate medium recovers culture.
5- Storage in Silica Gel: The bacteria and yeast can be stored in silica gel powder at low temperature for a period of 1-2 years. In this method, finely powdered, heat sterilized and cooled silica powder is blended with a thick suspension of cells and stored at low temperature. The essential rule in this method is quick drying at low temperature, which enables the cell to remain viable for a long period.
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6- Storage by freezing: Freezing is a typical procedure for storage of bacteria. Accordingly, thick bacterial suspensions can be frozen at a temperature of - 30ºC. In general, the colder the storage temperature, longer the culture will remain viable. The metabolic rates are diminished by bringing down the temperature and in the extreme case of storage in liquid nitrogen at -196ºC, are considered to be reduced to nil. Freezing and thawing is a well-known technique for actually disrupting cells. In addition, as water is expelled during freezing as ice, electrolytes become increasingly concentrated in unfrozen water, and this too may be harmful, since electrolyte concentrations outside cells become very different from inside those cells, leading to osmotic stress. Cultures can be maintained effectively if frozen in the presence of a cry protectant, which reduces damage from ice crystals. Glycerol or dimethylsulphoxide (DMSO) are commonly used as cryoprotectants. The simplest way to preserve a culture is to add 15%(v/v) glycerol to the culture and then to store it at -20ºC or -80ºC in a freezer. Cultures can be preserved for many years in glycerol, at a temperature of -40ºC in a freezer. In this technique, about 2 ml of glycerol solution is added on to the agar slant culture. Shaking can emulsify the culture. Emulsion is then transferred to ampoules, with each ampoule having 5 ml of the culture. These ampoules are placed in a mixture of industrial methylated spirit and carbon dioxide and frozen rapidly to -70ºC. Ampoules are then removed and placed directly in a deep freeze at -40ºC for utilization of the stock cultures. The ampoules are kept in a water bath at 45ºC for about a few seconds and then used for plate cultures. The use of freezing in liquid nitrogen at -196ºC is the best method and the advantages include: Convenience, culture reliability, greater
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flexibility, better control of phages and possible improvements of the quality. However, the liquid nitrogen storage has many disadvantages such as the cost of the apparatus and regular supplies of liquid nitrogen, risk of explosion, loss of large numbers of cultures if careful monitoring of liquid nitrogen levels is not carried out and possible contamination of the liquid nitrogen in the storage container.
7- Storage by drying methods: Some bacterial strains can be preserved by drying from the liquid state rather than the frozen state. A several methods for drying suspensions of bacteria for preservation purposes have been developed which are useful in laboratories that cannot afford the expensive equipment used for storing at very low temperatures or for freeze drying, or in which preservation of cultures is performed infrequently. Some of the following procedures of drying method are preservation by drying technique is an alternative method for culture retention. The development of such processes seeks to overcome the work involved in maintaining liquid stock-cultures. It also facilitates the dispatch of the dried cultures by post without any loss in activity.
i- Vacuum drying :
was the normal practice. The process consists of mixing a liquid culture with lactose and then neutralizing the excess acid with calcium carbonate. The mixture is partially concentrated by separation the whey , so yielding granules which are dried under vacuum. The dried starters contain only 1-2% viable bacteria, and may require several sub-culturing before returning maximum activity.
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ii-Spray drying: The spray-drying of bacteria enables a larger production scale; energy costs are lower and the process is sustainable. This is also a promising way to microencapsulate bacteria within various protective matrices to ensure their improved resistance during storage, technological rocesses and digestive stresses. Higher survival rates can be achieved when using spay drying. This system has not been used commercially.
Fig. (16). Spray dryer
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7- Freeze Drying (Lyophilization): In this method the microbial suspension is placed in small vials. A thin film is frozen over the inside surface of the vial by rotating it in mixture of dry ice (solid carbon dioxide) and alcohol, or acetone at a temperature RIíoC. The vials are instantly connected to a high vacuum line. This dries the organism while still frozen. Finally, the ampules are sealed off in a vacuum with small flame. The freeze drying can damage the bacterial cell-membrane, but the damage can be minimized by addition of certain compounds. Starter cultures preserved by freeze-drying are mainly used as inoculants for the propagation of mother cultures. Large quantities are needed for direct inoculation of the bulk starters, and an extended incubation time may be required.
Preservation of bacteria by lyophilization requires suspension of bacteria in a medium keeps up its feasibility by means of freezing, water removal and subsequent storage.
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Fig. (17). Freeze dryer Quality control of the preserved starter culture: Whichever strategy utilized for the safeguarding and upkeep of mechanically imperative life forms it is fundamental to check the nature of the saved creatures stock. Whichever method utilized for the preservation and maintenance of industrially important starter culture organisms, it is essential to check the quality of the preserved organisms stocks. Each batch of recently saved cultures routinely checked to guarantee their quality. A single colony moved into a shake-flask to ensure growth of particular kind of microorganism; further shake-flask subculture used for the preparation of gigantic amount of vials. For the evaluation of purity, viability, and productivity of cultures, few vials are examined. If samples fail any one of these tests, the whole batch destroyed. Thus, by the use of such a quality-control system stock cultures retain and used with confidence.
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CHAPTER EIGHT FACTORS CAUSING INHIBITION OF STARTER CULTURES
There are many factors that can cause an inhibition and /or reduction in S.C. activity and can lead to poor quality fermented dairy products reaching the consumers and financial loss to the manufacturers, So it is recommended that milk intended for bulk starter produc6tion or manufacture of these dairy products should be free these factors: Components naturally present in milk: There are various antimicrobial systems present in milk and their major role is protection of suckling animal against diseases or infection. Presence of these inhibitory systems can inhibit lactic acid bacteria. The inhibitory compounds known as lactenins, are heat sensitive and can be destroyed by heating milk at 68-74°C. Another antibacterial compound found naturally in milk is lactoperoxidase system, which consists of Lactoperoxidae/thiocyanate/ hydrogen peroxide. Lactoperoxidase is a basic glycoprotein that contains a heme group. It is synthesized in mammary glands and milk may contain sufficient to activate the lactperoxidase system (LPS). Thiocyanate is widely distributed in animal secretions and possibly derived from a rhodanase-catalysed reaction with thiosulphate in the liver and kidney.
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Fig. (18). Lactoperoxidase
The hydrogen peroxide (H2O2) does not present naturally in milk, and its presence in milk is the result of metabolic activity of lactic acid bacteria, or from anaerobic growth of other microorgansims.
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Table 3. Inhibitory compounds produced by lactic acid bacteria and their mechanisms of action Inhibitory compound Lactic acid and other volatile acids Hydrogen Peroxide
Carbon doxide
Diacelyl Bacteriocins metabolites)
Mechanisms of action Disruption of cellular metabolism
Inactivation of essential biomolecules by superoxide anion chain reaction, activation of lactoperoxidase system. Anaerobic environment and/or inhihition of enzyme decarboxylation and/or disruption of the cell membrane. Interference with the arginine (secondary utilization. Little is known disruption of cytoplasmic membrane.
Source: Maloney (1990) In the Lactopreoxidase system (LPS), the inhibitory compound is the result of an oxidation reaction whereby, in the presence of hydrogen peroxide (H2O2), the lactoperoxidase (LP) catalysis the oxidation of thiocyanate to non-inhibitory compound (SO4-2, CO2 and NH3) followed by further oxidation to form intermediate inhibitory substances, such as hypothiocyanate or higher oxyacids. Generally, most starter organisms are resistant to Lactopreoxidase system (LPS), however, some lactic starter cultures can produce mutants. On the other hand, continual propagation of starter cultures in autoclaved milk can affect the susceptibility of organisms to the Lactopreoxidase system (LPS).
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Other inhibitory compound found in milk include: a. Bacterial agglutinin which causes agglutination of the starter organisms, thus affecting their metabolic activity and growth. b. 30% oxygen dissolved in milk stimulated growth of Streptococcus thermophilus 15HA, but decreased but decreased growth of Lactobacillus delbrueckii bulgaricus. c. Certain types of forage. e.g. mouldy silage, turnips or vetech reduces growth, these products may result in a milk containing inhibitory compounds which can reduce acid production of yoghurt starter cultures. 1. Antibiotics: Residues of antibiotics in milk results from mastitis treatment in the dairy cow . Mastitis inflammation of the udder. Although this term includes all inflammatory conditions of the udder, it is defined here as a bacterial infection of the udder. The common causative organisms of mastitis are Streptococcus agalactiae, Streptococcus dysgalactiae, coagulase-negative staphylococci and Staphylococcus aureus.
The antibiotics commonly used belong to six major groups:Aminoglycosides e.g. gentamicin, Penicillins and cephalosporins (ßLactams) e.g. cloxacillin, Macrolides-e.g. erythromycin, Quinolones and fluroquinolones. Sulphonamides e.g. trimethoprim, Tetracyclines e.g. tetracycline The level and duration of antibiotic dissemination into milk depends upon several factors including the specific antibiotic, its concentration and method of preparation (aqueous solution, nature of suspending medium). The method of preparation notably impacts maintenance and can influence attachment of the antibiotic to equipment and pipelines.
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Starter culture are susceptible to very low concentrations of antibiotics. Low levels of penicillin in the bulk starter milk had a more pronounced impact on acid production when the culture grown in that milk was used in cheese manufacture compared with acid production by a normal starter inoculated into cheese milk containing a high level of penicillin. For this reason, it is imperative that milk used for starter production is antibiotic free. This is one of the many reasons why so many companies use commercial frozen or freeze-dried starter cultures for direct vat inoculation rather than manufacture their own bulk starter.
The inhibitory levels of streptomycine, chloramphenicol and tetracycline seem rather high, and this apparent resistance could be attributed to culture strain variation, variation in the commercial preparations of antibiotics used. 2- Bacteriophage: Bacteriophage, also called phage or bacterial virus , any of a group of viruses that infect bacteria. Bacteriophages also infect the single-celled prokaryotic organisms known as archaea. Phages can attack and destroy starter culture organisms, the result is the failure of lactic acid production. ,
Various varieties of phage exist, each of which may infect only one type or a few types of bacteria. Lactic streptococci and lactobacilli are the most vulnerable organisms of the starter culture attacked by phage. The morphology of these phages show a head and a tail made up of nucleic acid core (DNA and RNA). Which is protected with a protein layer. These bacterial viruses are present in ecosystems where bacteria have been found, including man-made ecological niches such as food fermentation vats.
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Despite broad endeavors, however, phage infection of starter LAB cultures remains the most well-known reason of slow or incomplete fermentation in the dairy industry.
Fig. (21). Bacteriophage
It is now acknowledged that the most permanent source of new phages within dairy environments is through raw milk, with their concentration ranging between 10 and 104 phages per ml. Based on sensitivity to phages, starter culture are classified into 3 main groups: 1- Phage insensitive 2- Phage carriers (slight reduction in the rate of lactic acid being produced 3- Phage sensitive ( the bacteria undergo complete lyses)
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The phage host interactions is divided into 3 categories
1- Virulent phages (which can cause lysis of the cells or partial lysis and the survivors become resistant. 2-Temperate phages 3- Carrier phages (phage is present in the host cells and can be removed by phage antiserum Many precaution measures which can be followed to minimize the effect of phages: 1-Employing aseptic technique for propagation of S.C. 2-Heat treatment of bulk starter milk 3-Use of phage resistant strains in the dairy 4- Effective filtration of air in the starter room 5- Properly sanitizing of the equipments e.g. by heat or chemical 6- Location of starter room far away from production room. 7- Plant personnel should not be allowed into starter handling room. 8- Propagation of S.C. in phage inhibitory medium. 9- Development of phage resistant strains 10- Use of mixed strain starters. -Fogging the air space of starter room with hypochlorite solution or use of UV light. 3-Detergents and disinfectants residues: Detergents and disinfectants comprise just a part of chemical contaminants in milk. Other chemical contaminants include antibiotics and sulfonamides, pesticides, herbicides, fungicides, dioxins and mycotoxins. Chemical contaminants possess the potential to cause toxicological harm to consumers. Deposits of chlorine mixes in milk are not dangerous in this regard due to their fast disintegration, however different disinfectants, for example,
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quaternary ammonium mixes are fairly steady in milk. The levels of these mixes (0.00001% - 0.00005%) still show a bacteriostatic action in milk. Some detergents and disinfectants as well as antibiotics and sulfonamides, can cause real risk in manufacturing cheese and cultured milk products due to diminished starter activity. Residues of these compounds (alkaline detergents, iodophore, quaternary ammonium compounds and ampholytes) can affect the starter activity. The minimum concentration required for inhibition varies with the different anti-microbial agents and between different strains of starter bacteria. Residues gain entry to milk at the (a) farm, (b) during transport to the factory and (c) the factory due to careless use of sterilants or detergents, incomplete draining or inadequate rinsing of equipment. 4. Production of nisin: Nisin is an antimicrobial peptide produced by certain Lactococcus species. If favourable conditions are accessible, high number of lactococci bacteria will develop in raw milk. Nisin has been acknowledged as a safe and natural preservative in more than 50 countries. Nisin is wide range antibiotic and if produced it will inhibit some starter cultures. This peptide represses the vegetative growth of a range of gram-positive bacteria. Since, specifically, nisin restrains the food-borne pathogens Listeria monocytogenes,Staphylococcus aureus, and psychrotrophic enterotoxigenic Bacillus cereus, the effectiveness of nisin as a food preservative against these organisms under various preservation conditions has been investigated in detail. Providing there is sufficient control of temperature during the production, storage and distribution of milk, nisin production will not be a problem. Not just the utilization of nisin-producing lactic acid bacteria (LAB) as a fermentation starter culture yet additionally the immediate option of nisin the direct addition of nisin to various kinds of foods, such as cheese,
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margarine, flavored milk, canned foods, and so on, is permitted. The improvement of successful nisin production systems utilizing LAB is a new field of interest. 5. Free fatty acids: Free fatty acids are available at low concentration in freshly drawn milk. Their concentration may increase because of the activity of milk lipase. Pseudomonas spp. if allowed to grow in refrigerated milk will produce lipases and high concentrations of free fatty acids. However, such milk typically contains a total bacterial count in the region of 1 x 107 CFU/ml or higher. Fatty acids are inhibitory to lactococci and in particular to Lactococcus lactis subsp. cremoris. However, relatively high levels of fatty acids are required; 0.1% butyric, decanonic, hexanoic and oleic acid were required for the inhibition of Lc. lactis subsp. cremoris. Such high concentrations of free fatty acids do not normally occur in modern hygienically produced milk that has been held at correct storage temperatures.
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CHAPTER NINE YEASTS STARTER CULTURES
Yeasts are eukaryotic, single-celled microorganisms classified as members of the fungus kingdom. The first yeast originated hundreds of millions of years ago, and 1,500 species are currently identified. Yeasts are unicellular organisms which evolved from multicellular ancestors with some species having the ability to develop multicellular characteristics by forming strings of connected budding cells known as pseudohyphae Yeast sizes change significantly, contingent upon on species and environment, ordinarily measuring 3±4 µm in diameter, although some yeasts can grow to 40 µm in size. Most yeasts reproduce asexually by mitosis, and many do so as such by the unbalanced division process known as budding. Principles of yeast growth and fermentation: Yeast is a facultative anaerobe which can survive and grow in the presence or absence of oxygen. The oxygen determines the metabolic fate of the cell. In terms of the yeast cell, its survival, growth and metabolism is optimal in the presence of oxygen. In this case, yeast will rapidly grow to high densities and will convert glucose to carbon dioxide and water. Under anaerobic conditions, yeast develops considerably more gradually and to bring down densities and glucose is not entirely processed to ethanol and carbon dioxide. The motivation behind a yeast starter culture is not to create a pleasant fermented beverage but to delver an adequate amount of yeast for resulting fermentation. Propagation conditions ought to be with the end goal that a maximal measure of yeast is delivered which gives ideal
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from your currently-used yeast culture was isolated within the laboratories, or acquired from an outsider ± whether that was yesterday or may decades prior. It is usually preferable to select the yeast strain on the basis of its possession of a number of desirable traits, and the absence of undesirable traits. Table 3. Yeast species frequently found in dairy products. Identification according Kreger-van Rij (1984)
to Some equivalent names in earliest Literature
Debaryomyces hansenii Candida famata Kluyveromyces marxianus Candida Kefyr Candida stellata Saccharmyces (Yarrovia) Lipolytica Candida holmii Saccharomyces exigubs Pichia membranaefaciens Pichia fermentans Rhodotorula glutinis Rhodotorula rubra Source: Kreger-van-Rij, (1984).
D.subglobosus: Torulaspora hansenii Torulopsis candida: T. famata Kluy.bulgaricus:Saccharomyces lactis: S.fragilis C.Pseudotropicalis:Torulopsis Kefyr: Torula cremoris. Torulopsis stellata Candida lipolytica Torulopsis holmii Candida krusei Candida krusei
Yeast strain identity and purity: It is essential that strategies are set up to ensure that what is planned has been achieved. Genetic tests of identity and purity can be carried out using PCR-based DNA fingerprinting or by karyotyping. Phenotypic testing ± for example, tests for flocculation, fermentation performance, and the presence of certain traits, such as the ability to grow at different
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temperatures, possession of certain enzymes, or the ability to grow in the presence of specific antimicrobial agents ± are also useful. Tests for the presence of contaminant microorganisms (wild yeasts and bacteria) must also be carried out. All of the laboratory tests have to perform as intended and all methods should be validated at frequent intervals to assure performance. Preservation of yeast starter cultures: The preservation technique utilized is basic. The overlooked details are the main problem. Preservation in liquid nitrogen is the technique for decision. Preserved in straws or cryovials, yeast strains will stay unaltered for a considerable length of time, and most likely hundreds of years under such conditions. accurately this procedure makes an environment in which the yeast cells are placed in a state of suspended animation. Most importantly the process has no negative effects on genetic or physiological properties of the cells. Viability of the cells is additionally kept up under these conditions. For the preservation process there is a requirement for master hardware, authority staff, and access to a reliable source of liquid nitrogen are just three. Staff safety is paramount, and precautions have to be taken to protect anyone using liquid nitrogen from the potential suffocating impacts of oxygen starvation and nitrogen gas. Practically, it's typically best to have two sorts of cultures ± master cultures and working cultures. Given adequate storage room, enough working cultures must be available to produce all the needed yeast a long time ahead.
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Supply of yeast starter culture: Slant cultures dispatched to breweries works from a single approved inspected production site with a move down production facility access as feature of the risk management plan. It makes sense that your point ought to be to make each and every one of those yeast spreads indistinguishable to each other. That needs to begin with the primary phase of the engendering. These days most lager breweries propagate their yeast every week of the year, or every second week. Commonly that includes inoculating about 10 ml of wort with yeast growth from a slant culture. That yeast can either be taken from the slope utilizing an inoculating loop, or by washing off all of the cells from the slope and using that as the inoculum. Either way, when used once a yeast slope culture should be discarded. Slope cultures should be treated DVµGLVSRVDEOH¶7RXWLOL]HWKHPVHYHUDOWLPHVLVWRLQYLWHLQFRQVLVWHQF\LQ the early stages of propagation which can just become magnified as the culture enters its working life in the brewery. An alternative to yeast slope supply is the utilization of ultra-pure dried yeast cultures. This format is the one of choice when cool transport and storage can't be guaranteed, or when laboratory facilities are not accessible in the brewery. These dried yeast cultures are provided in units of 50 g. Each is adequate for inoculation of a single Carlsberg flask ± enough to start a propagation in a brewery yeast propagator. By using this format of culture you can by-pass the need for laboratory propagation steps. While this can save time, money and equipment, maybe the greatest advantage of this approach is the adaptability it gives you on account of a fizzled yeast propagation. On rare occasions when brewery yeast propagations come up short quality tests (for example due to contamination with foreign microorganisms) production delays caused by having to wait for the
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laboratory to propagate a new yeast culture from slope can be both frustrating and costly. However, by utilizing ultra-pure active dried yeast cultures and thus by-passing the laboratory stages of the propagation, several days can be shaved off the time needed for yeast propagation. Quality assurance of yeast cultures:
Cultures tested preceding dispatch to guarantee, purity, performance and freedom from microbiological contamination ± performance of all test of all test strategies. All yeast cultures utilized as a part of a brewery ought to be checked to guarantee their identity, utilizing either microbiological tests and / or genetic tests such as DNA fingerprinting. Checks on culture purity have to be made, to guarantee that the culture is pure and homogeneous. Giant colony morphology on WLN agar works well for this purpose, supported by DNA fingerprinting. Checks for microbiological contaminants have to be made too ± both bacteria and wild yeasts. Furthermore, as usual, the methods utilized for these tests have to be approved to guarantee sure their performance.
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CHAPTER TEN APPLICATION OF STARTER CULTURE IN FOOD INDUSTRY
Food industry: The food industry is a complex, global collective of diverse businesses that supplies the vast majority of the food consumed by the world population. Only subsistence farmers, those who survive on what they grow, and hunter-gatherers can be considered outside the extent of the current food industry. It is challenging to find an inclusive way to cover all aspects of food production and sale. The utilization of microbial starter cultures not only allows the production of quality foods, but also, in particular, an increase in the reproducibility of their manufacturing process and, thereby, also of food safety. These cultures include both defined single-strain and multi-strain cultures and multi-strain mixed cultures, as well as undefined multiplestrain mixed cultures. Generally, the food industry includes: x x
x
x
x
x
Agriculture: raising of crops and livestock, and seafood Manufacturing: agrichemicals, agricultural construction, farm machinery and supplies, seed, etc. Food processing: preparation of fresh products for market, and manufacture of prepared food products Marketing: promotion of non- specific products (e.g., milk board), new products, advertising, marketing campaigns, packaging, public relations, etc. Wholesale and food distribution: logistics, transportation, warehousing Foodservice (which includes catering)
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x x
x x x
Grocery, farmers' markets, public markets and other retailing Regulation: local, regional, national, and international rules and regulations for food production and sale, including food quality, food security, food safety, marketing/advertising. Education: academic, consultancy, vocational Research and development: food technology. Financial services: credit, insurance
Microbial starter cultures food production include live bacteria, fungi (yeasts and moulds). These microorganisms which carry out the fermentation process in foodstuffs have been utilized by humans since the Neolithic period (around 10 000 years BC). . The fermentation assists in preserving perishable foods and to improve their nutritional and sensory qualities. More than 260 different species of microbial food culture are identified and described for their beneficial utilization in fermented food products globally which indicates these microorganism. The industrial production of microbial food cultures is carried out after watchful selection process and under entirely controlled conditions. First, the microbiology laboratory facility, where the original strains are kept, prepares the inoculation material, which is a small quantity of microbes of a single (pure) strain. At that point At that point, the inoculation material is duplicated and developed either in fermenters (liquid) or on a surface (solid) characterized and monitored conditions. Grown cells of pure culture are harvested, eventually blended with other cultures and, finally, formulated (preserved) for subsequent transportation and storage. They are sold in liquid, frozen or freeze-dried formats. Another and conventional method for starting a food fermentation is regularly alluded to as spontaneous fermentation. Cultures originate
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from raw milk, i.e. milk that has not not experienced any sanitation treatment or from the reuse of a fraction of the previous production (back-slopping). The composition of such cultures is complex and extremely variable. The utilization of such methods is consistently diminishing in developed countries. Some countries even forbid the backslopping technique because of the "potential to magnify pathogen loads to very dangerous levels. Food fermentation has been utilized for a considerable length of time as a technique to safeguard perishable food products. The crude materials traditionally utilized for fermentation are as various as: fruits, cereals, nectars, vegetables, milk, meat and fish. It is conceivable to acquire a vast wide range of different food products by selecting different raw materials, starter cultures and fermentation conditions. Traditional fermented foods include typical products prepared by spontaneous fermentation. However, dependence RQ WKH ³QDWXUH´ RI spontaneous fermentation makes the quality of these products neither predictable nor controllable. Therefore, to optimize and control the food characteristics, most inoculated fermentations are developed by using pure cultures as starter inoculums. For instance, wine fermentation is normally inoculated with chosen indigenous yeasts as starters, and this approach yields homogeneous, high-quality products. The assorted variety covers, yet is not restricted to products as: vinegar, bread, soy sauce, wine, brew, sauerkraut, kimchi, cured olives, fermented milk products as buttermilk and yoghurt, a variety of cheeses and sausages. Lactic acid bacteria are broadly utilized in the production of fermented food, and they constitute most of the volume and the value of the commercial starter cultures. The primary activity of the culture in a food fermentation is to change over sugar to wanted metabolites as alcohol, acetic acid, lactic acid or CO2. Alcohol and organic acids great regular additives, but also appreciated in their own right in the fermented product.
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The CO2 produced by some starter cultures contributes the gas needed to rise the dough, form eyes in the cheese or to make the foam of beer and buttermilk. In the production of wine, a secondary fermentation by lactic acid bacteria is responsible for the reduction of the acidity by changing malic acid to lactic acid. The starter cultures utilized in food fermentations are, likewise contributing E\ µµVHFRQGDU\¶¶ UHDFWLRQV WR WKH IRUPDWLRQ RI IODYRXr and texture. This secondary contribution can regularly be in charge of the contrast between products of different brands, in this way contribute essentially to the value of the product. The manufacturers of fermented foods have the decision of either gaining the starter culture in a ready touse, profoundly thought frame, or to make a propagation of the culture in manufacturing plant. The number of different products produced, degree of automation, presence of expertise in microbiology and finally the economy. The highest level of safety and adaptability is accomplished by utilizing a commercial starter culture for direct inoculation. Such cultures are provided either as frozen or freeze-dried highly concentrated and highly active cultures. All stages of starter cultures production are important for acquiring the wanted character, purity and quality of the culture product. The culture producers are applying the principles of hazard analysis critical control point (HACCP) in the production in order to guarantee stable high quality production procedures (European food and feed cultures association,, http://www.effca.com). In order to make the perfect culture for a specific food application, it is important to comprehend the function we request of the culture, and to have devices to enhance the function of the culture. Both aspects have been progressed significantly through scientific achievements during most recent couple of years. The scan for a starter culture has up to this point been depending on the screening of countless of isolates in small-
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scale food fermentations. The starter culture finally selected would be the one giving an acceptable performance in the process and also giving an acceptable organoleptic evaluation of the food product. Excellent cultures have been isolated this way, and the method will positively likewise later to be utilized to expand the pool of microorganisms to be used as starter cultures͘
Selection of starter cultures: Selection of a starter culture with excellent viability and metabolic activity is important for the success of the fermentation process of food. Starter culture selection is involved in the initial isolation of microorganisms from nature and later in the purification of strains of interest due to the synthesis of desirable metabolic products. In most large-scale screening programs, uncommon, unusual starter cultures are chosen by taxonomists as the probability of new interesting activities is maximized in sensitive specific assays using rare, fastidious microorganisms. The purification of unusual organisms is difficult and involves considerable technique in culture selection. Likewise, pure cultures of microorganisms are inherently variable in development attributes and metabolic activities. Although natural selection and mutation selection are generally successful in improving titers of the above metabolites, culture stability, growth and sporulation characteristics often represent additional critical problems to be resolved. These characteristics are additionally innate hereditary issues and should be settled in a culture change program. The end or abatement in titers of undesirable segments can be settled by culture choice or by changes in the synthetic and physical condition fermentation.
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Table 4. Examples of fermented foods in Europe. Raw material Milk
Product Sour milk products: Sour milk, sour cream, yogurt, kefir, kumis Sour cream butter Cheese
Microorganism LAB, yeasts Acetic acid bacteria
LAB LAB, yeasts, moulds, propionic acid bacteria Meat Fermented sausages LAB, yeasts, moulds, staphylococcii Micrococci, Streptomyces Ham LAB, yeasts, moulds, staphylococci Fish Fish sauce, fermented Staphylococci, Vibrio fish costicola, LAB Dough and pastes from Sourdough, yeast LAB , yeasts cereals dough, Kisra Olives, cabbage, Fermented olives, Lactic acid bacteria cucumbers, tomatoes sauerkraut, pickles (LAB) Malt, Koji, made from Beer, sake, spirits LAB , yeasts, moulds cereals Beer, wines and spirits Vinegar Acetic acid bacteria Grapes and other fruits Wine Yeasts, LAB Soya, Carob Soy sauce, tempeh, LAB, Bacillus spp., natto, Dawadawa moulds, yeasts
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CHAPTER ELEVEN UTILIZATION OF STARTER CULTURES IN CHEESE MAKING
Cheese is a food derived from milk that is produced in an extensive variety of flavors, textures, and forms by coagulation of the milk protein casein. It comprises proteins and fat from milk, usually the milk of cows, buffalo, goats, or sheep. During production, the milk is usually acidified, and adding the enzyme rennet causes coagulation. The solids are separated and pressed into final form. Some cheeses have molds on the rind, the external layer, or all through. Most cheeses liquefy at cooking temperature. Conservation with lactic acid bacteria (LAB) is one of the oldest and profoundly productive types of non-thermal processing method. Cheese production depends on LAB capacity to ferment sugars, especially glucose and galactose, so to produce lactic acid and aroma substances that give common f flavors and tastes to fermented products. LAB also release antimicrobial metabolites so called bacteriocins, which are considered safe and natural preservatives, with great potential to be used on their own, or synergistically with other methods in food preservation. Lactic acid bacteria in cheese production: Fermentation with lactic acid bacteria (LAB) is a modest and successful food preservation technique that can be applied even in more rural/remote places, and prompts change in texture, flavour and nutritional value of many food products. LAB have a long and safe history of application and utilization particular in cheese processing. Cheeses may reach a shelf-life
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up to 5 years (depending on variety) , thus being generally regarded as safe (GRAS). Cheese-making depends on utilization of LAB in the form of defined or undefined starter cultures that relied upon to cause a fast acidification of milk through the production of lactic acid, with the subsequent decrease in pH, in this manner influencing various parts of cheese manufacturing process and eventually cheese composition and quality. The earliest preparations of cheeses depended on the spontaneous fermentation, coming about because of the improvement of the microflora naturally present in the raw milk and its environment. The quality of the end product was a reflex of the microbial load and range of the crude material. Spontaneous fermentation was later enhanced through backslopping, i.e., inoculation of the crude material with a little amount of whey from a previous successful fermentation, and the resulting product characteristics depended on the best-adapted strains dominance. Nowadays, backslopping is still used to produce many rawmilk cheeses, which are regarded as an important source of LAB genetic diversity, as well as being crucial from an economic and even ecologic point of view. The starter-culture applied in this natural fermentation, is generally an ineffectively known microflora blend that in spite of the fact that having a predominance of LAB, may likewise contain non-LAB microorganisms, and its microbial diversity and load is normally variable over time. Studies directed to characterize traditional cheeses demonstrate that those produced using raw milk harbor a decent variety of LAB contingent upon topographical district, however, upon optimization may have industrial applications. For instance, since wild strains need to withstand the competition of other microorganisms to get by in their threatening common habitat, they regularly deliver antimicrobials substances such as bacteriocins, which are natural antibacterial proteins that can be
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incorporated directly into fermented foods or indirectly as starter culture. Moreover, traditional cheeses also obtain their flavor intensity also from the non-starter lactic acid bacteria (NSLAB), which are not part of the normal starter flora but develop in the product, particularly during maturation, as a secondary flora. The isolation and advancement of wildtype strains from traditional products, to be utilized as starter cultures in cheese processing, is for sure exceptionally dynamic field of research in Food Science today. LAB food safety and cheese technology: Cheese is produced world-wide and there are more than 2000 varieties, made from milk of several mammals, processed industrially or by traditional methods. However, despite the large number of varieties, the basic steps required in any cheese processing are basically the same, and slight variations in any of these steps may result in products of various general quality. The major steps include: 1- Milk treatment: In large-scale cheese processing, the milk is heat-treated, e.g. 73 ºC for 15 seconds, to destroy pathogens and reduce microbial numbers, while in most traditional raw-milk cheeses heat treatment is not applied. Also the milk may be standardized (increasing or decreasing of the fat content, or adjusting the casein-to-fat ratio. 2-Addition of starter-culture: The kind of industrially accessible starter preparation to be utilized will be determined by the cheese formula. Large-scale processing depends on utilizing characterized, financially accessible starters, while for traditional cheeses, a natural fermentation (whey from the previous lot) is often.
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3- Coagulation: During coagulation, changes on the milk protein complex happen under characterized states of temperature and by action of a coagulant agent, which changes the physical aspect of milk from fluid to a jelly-like mass. Various coagulants are accessible, e.g. lemon juice, plant rennet or more commonly a proteolytic enzyme such as chymosin (rennin) or ± because of appeal from the cheese industry - proteolytic enzymes from the mould Rhizomucor miehei acquired by means of biotechnology. These enzymes have an acidic nature, meaning they have ideal activity in a marginally acidic environment. Accordingly, the activity of LAB in this stage is crucial as they are required to rapidly release enough lactic acid, to bring down the milk pH from 6.7 to near 6.2, (along these lines creating an appropriate environment for optimum activity of rennin) and later to pH 4.5 as the processing proceeds, continues, making an unwelcoming situation for many undesirable bacteria, thus increasing the end product safety. 4- Cutting the coagulum: The subsequent coagulum might be cut with fitting blades into curd particles of a defined size, e.g. 1±2 cm, or it might be moved into compartments or cheese moulds. The cutting of the coagulum is a very important step in the manufacture of some cheese varieties as it decides the rate of acid development and the body (firmness) and texture of the cheese. 5- Heating or cooking the curds: The curd will be heated to 37±45 ºC, depending on the cheese type). The curds and whey influence the rate at which whey is removed from the
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curd particles and the development of the starter microorganisms. During heating, the curds and whey are frequently mixed to keep up the curd as particular particles. 6- Whey removal: After heating and stirring, and when the curd particles have firmed and the correct acid development have taken place, the whey is expelled enabling the curd particles to tangle together. 7. Milling the curd: In cheeses such as Cheddar, when the curd has achieved the desired texture, it is broken up into little pieces to empower it to be salted uniformly. Milling the curd can be done either by hand or mechanically. Salting is normally done to enhance the taste of the curd and to expand its safety and shelf life. 8- Ripening: Finally, for most cheeses, the subsequent mass is molded and put to ripening for periods that may vary from 15 days to one, two or more years. Ripening is a slow phase, significant for the improvement of aroma and flavor, achieved by the action of the numerous enzymes discharged by LAB. During ripening the protein in cheese is broken down from casein to low molecular weight peptides and amino acids. Proteolysis is the major ± and surely the most complex of biochemical events that take place during ripening of most cheese varieties and LAB play an important role in it. This happens while the cheeses are stored in the curing cupboards and in some cases in caves, usually with temperature and humidity controlled.
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Role of fungi in cheese making: i-Yeasts: Yeasts don't specifically affect the cheese, however, they do help other (secondary) bacteria flourish. There are numerous variants of yeast which are added to cheeses as diverse as Brie and Stilton, Limburger and Tallegio. The finished appearance of the cheese is reliant on the yeast strain being used, it's vital to get the right one VRWKDW\RXGRQ¶WHQGXS with a slimy finish if you were aiming for a velvety texture. A decent cheese making pack provider will have the capacity to help with selection. At the surface of the cheese, they generally work by fermenting the lactose in the cheese, which reduces the surface acidity. In turn, the lower acidity empowers rind-forming bacteria or desirable moulds to flourish, i.e. the yeast makes a situation that is perfect for our desired bacteria or moulds. This brings an extra advantage in light of the fact that expanded growth of desirable mould decreases the amount of undesirables on the surface of our cheese. Yeasts additionally create fragrant mixes, so the desirable (or less than) smell of some cheese is, in part at least due to the addition of yeast as a secondary culture. Most yeasts could be assigned to two groups. One group was characterized by the capacity to ferment glucose, to use lactate and to increase pH values and by the fact that proteolytic activity was not shown. This brought about alcoholic, acidic, fruity or fermented odours (Clavispora lusitaniae, Pichia jadinii and Williopsis californica). The second group was made out of non-fermenting species which used lactate, yset the pH was not influenced (Galactomyces geotrichum, Trichosporon ovoides and Yarrowia lipolytica). These yeasts were proteolytic yielding a cheesy aroma. Debaryomyces hansenii B comprised characteristics of both group.
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Fig. (23) Gorgonzola cheese ripening
Fig. (24). kaasmisdrijf cheese ripening
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Fig. (25). Yeast cell ii- Role of moulds: In spite of the fact that seeing mold in food is an indication that it is contaminated and ought to be disposed of, there are some foods in which the presence of visible fungal mycelium is particularly a part of the product. Among these are some of the cheeses. Two of the most familiar examples are Camembert and Roquefort, also known as blue cheese. These cheeses are among the favorites among gourmets. These cheeses are made from two species of Penicillium, P. camemberti, in Camembert cheese and P. roqueforti in Roquefort cheese. In reality, when looking for a secondary culture, you will run over P. roquerforti and P. camemberti as the two main varieties (P standing for Penicillium) producing the distinctive blue veining and white mould, respectively. In some respects, these are the "feature" strains and there are many varieties accessible, fine tuned to the particular variety of
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cheese being created. They are selected for such properties as how long the mould filaments will grow on the cheese surface, the aroma compounds they encourage, the colour of blue/green vein produced and so on. Just like yeast, these cultures convert lactic acid in and on the cheese into other compounds, which reduces its acidity, and break down proteins (proteolysis), which increase its softness. A great example of the effect proteolysis is often seen in Camembert. The cheese is soft and even runny, whereas at the centre, it is firmer and a lighter colour. This difference is because of the surface mould breaking down the protein near it but not being able to reach the middle of the cheese and so leaving the protein intact. Roquefort cheese In making Roquefort cheese, P. roqueforti is added to the cheese made from sheep's milk. The cheese is salted and openings made all through the cheese for aeration, and left for several days. The cheese is then permitted to age for two to five months. The variation in time is subject to who will eat it. Americans like the cheese to be new and mild, while the French prefer it to be exceptionally old and strongly flavored Roquefort cheeses all contain similar molds, yet are not made with a similar sort of milk. In the United States and Canada, either goat's or cow's milk is utilized, while in Roquefort, sheep's milk is used, exclusively. This is why it is more expensive. In general, the processes are usually referred to as "mold-ripened." This, exhibit how we can change the significance of a procedure to sound more positive when fungi are carrying out a process which benefits us. Normally, when we see fungi growing on our food, we say that it is " spoiled " This latter process is actually what is occurring in mold-ripened cheese
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Fig. (26). Penecillium roqueforti fungus
Fig. (27). Roquefort cheese
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Camembert cheese: Camembert cheese is unique in appearance. The fungus engaged with the manufacture of this cheese is Penicillium camemberti. The mycelial growth of this species happens just on surface of the cheese and does not work its way inside to form veins as in P. roqueforti.
Fig. (28). Camembert cheese Similar to the case with Roquefort cheese, there are a few sorts of cheeses that are made with the aid of P. camemberti. Each one with a marginally extraordinary formula and its own particular taste. Brie, which is made in France, is a very similar cheese. A cheese requiring a fungus and a bacterium is Limburger, a very strong cheese, in both flavor and odor. It is named for its place of origin, Limburg, Belgium and is not an extremely regular cheese that is promptly accessible. It is currently manufactured in Northern Europe and the United States. The cheese requires the action of two microorganism, a
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bacterium, Brevibacterium linens, which is responsible for the strong odor and a yeast. Improvement of new starter cultures for cheese preparation: Traditional raw-milk cheeses are exceedingly esteemed for their flavors, while large-scale products are frequently seen by the customer as ""exhausting"" ± a consequence of theelimination by pasteurization, of the flora that has a key role in flavor development; and this puts the food industry under pressure to look for alternative LAB cultures capable of improving products flavor. Today, the expanded comprehension of the genomics and metabolomics of food microbes opens up new points of view for starter-cultures improvements and through genetic engineering it is now possible to express their attractive properties or suppress undesirable features. Originally, starter cultures for the cheese industry were maintained by daily propagation, and later, they became accessible as frozen concentrates and dried or lyophilized preparations, produced on an industrial scale, some of them allowing direct vat inoculation. Since the first starter cultures were blends of a few undefined microbes, the daily propagation, eventually led to shifts of the ecosystem resulting in the disappearance of certain strains. Because some important metabolic traits in LAB are plasmid-encoded, there was a risk that they would be lost during propagation . Lactococci are by and large utilized as starter cultures in the production of industrial cheeses and cultured milk products. In Traditional cheeses the natural starter cultures may harbor many different species and strains. On the other hand, cheeses manufactured in a standard (large-scale) processing manner, are considered as safer because of the application of pasteurization and following the standard hygienic practices, including the HACCP. Traditional cheeses have their own specific processing
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methods, namely the common use of raw milk, however the hygienic procedures and HACCP approaches adapted to their specificities should be applied as well.
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CHAPTER TWELVE FERMENTED MILK PRODUCTS
Introduction: Milk products that contain probiotic bacteria are those that are produced with various fermentation methods, particularly lactic acid fermentation, by utilizing starter cultures and those that have different textures and smells. Fermented dairy products are popular due to their differences in taste and their favourable physiological impacts. Today, fermented dairy beverages in general are produced locally by utilizing traditional methods. As of late, due to the expanded interest for natural nutrients and probiotic products, fermented dairy beverages have have come to a distinctive position and are considered to importantly affect human health and nutrition. A trend in the production of fermented milk products is the use of probiotic cultures which have some connotation with the promotion of good health. Probiotic bacteria such Lactobacillus acidophillus and Bifidobacterium led to production of a fermented yoghurt-like product which was sold as a health food. The first acidophilus milks were not popular, because they were unpalatable due to their low pH. Sweet acidophillus milk, a low-fat pasteurized milk inoculated with a frozen concentrate of Lactobacillus acidophillus , was much more popular. This was first made in 1970. Probiotic Lactobacillus rhamnosus strain GG is proved to reduce the incidence of antibiotic associated diarrhea. Fermented dairy products are important part of human diet. They are produced with different fermentation types . Probiotics are defined as living microorganisms, which when ingested in adequate amounts, advantageously impact the strength of the host by enhancing the composition of intestinal microflora. In addition to improving gut health, probiotics may play a beneficial role in several
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medical conditions, including lactose intolerance, cancer, allergies, hepatic ailment, Helicobacter pylori infections, urinary tract infections, hyperlipidemia and absorption of cholesterol. A numerous types of dairy products contain probiotic bacteria. The probiotics are known to be beneficial to human health and can be ingested through fermented dairy products, enrichment of various foods with these microorganisms and utilization of pharmaceutical products that are gotten by utilizing feasible cells. Probiotics can be consumed independently or with foods, and they can assist dietary and microbial balance by regulating the mucosal and systemical immunity and independently the consumer's health. Today view of refrigeration and unsafe rural practices like soaking foods with chlorine, the foods contain little or no probiotics and most foods today actually contain antibiotics that kill off the good bacteria in our bodies. By adding more probiotic foods into the diet, all of the following health benefits can be acquired: 1- Stronger immune system 2- Improved digestion 3- Increased energy from production of vitamin B12 4- Better breath since probiotics destroy candida 5- Healthier skin, since probiotics enhance eczema and psoriasis 6- Reduced cold and influenza 7- Healing from broken gut and inflammatory bowel disease 8- Weight loss Therefore in order to acquire all of these benefits, then people must to start consuming these probiotic foods for better health. In fact, a variety of types of probiotics must be eaten as each one offers a different type of beneficial bacteria to help the body in a variety of ways.
Fermented milks are generally produced in many countries. This kind of process is one of the most established used to extend the shelf-life of
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milk, and has been practiced by human beings for thousands of years. The exact origin(s) of the manufacture of fermented milks is hard to build up, but it is safe to accept that it could date to more than 10 000 y ago as the lifestyle of people changed from food gathering to food producing. Selecting probiotic bacteria for milk products:
The human intestinal tract constitutes a complex ecosystem of microorganisms. The bacterial population in the large intestine is high and can achieve maximum counts of 1012 CFU g-1. In the small intestine, the bacterial content is impressively lower at just 104±108 CFU g-1. In the stomach just 10-102 CFU g-1 are found because of the low pH of the environment. It is realized that microbiota in the human digestive system changes during human development. The intestine of infants is fully sterile, however promptly after birth, colonization of many species of bacteria starts. On the first and second days after birth, coliforms, enterococci, clostridia and lactobacilli have been appeared to be available in LQIDQWV¶ IHFHV :LWKLQ WKUHH WR IRXU GD\V ELILGREDFWHULD EHJLQV colonization and becomes predominant around the fifth day. At the same time, coliform counts diminish. Breast-fed babies show 1 log-count more of bifidobacteria in feces than bottle-fed babies. Enterobacteriaceae, streptococci, and other putrefactive bacteria counts are higher in bottlefed babies, recommending that breast-fed babies are more impervious to gastrointestinal infections than the bottle-fed infants͘
In addition to the microbiota changes that happen during human aging, the microbiota in the gastrointestinal system can likewise change due to the food and health conditions of a person. For instance, utilization of antibiotics can harm the balance of intestinal microbiota, lessening counts of bifidobacteria and lactobacilli and increasing clostridia. The ensuing imbalance can cause diarrhea in elderly and immunocompromised people. To help improve the balance of intestinal
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microbiota, probiotic microorganisms can be added to the human diet a specific end goal to stimulate, the development of favored microorganisms, swarm out possibly harmful bacteria, and reinforce the ERG\¶V QDWXUDO GHIHQVH PHFKDQLVPV 7KH VHOHFWLRQ RI SURELRWLF microorganisms is based on safety, functional and technological aspects, as reported by (Saarela et al., 2000). These are summarized in Figure 2. Certain probiotic bacteria have been extensively studied and are already on the market, as shown in Table 1. Before probiotic strains can be delivered to consumers, they must first be able to be manufactured under industrial conditions. They must then survive and retain their functionality during storage as frozen or freeze-dried cultures, as well as in the food products into which they are finally formulated. Moreover, they must be able to be incorporated into foods without producing offflavors or textures. Functional food requirements must take into consideration the following aspects in relation to the probiotics: The preparation should remain viable for large-scale production; it should remain stable and viable during storage and use; it should be able to survive in the intestinal ecosystem͘ Beneficial effects of probiotics: Probiotics hold the key not only for better health and a stronger immune system, yet additionally to treat stomach related problems, mental health illness and neurological disorders. The digestive tracts of humans are critical to the health due to the fact that 80 percent of the entire immune system is located in the digestive tract, that is a great percentage. In addition to the effect of probiotics on the immune system, the digestive system is the second largest part of the neurological system and is located in the gut. Probiotics promote a healthy balance of gut bacteria and have been linked to a wide range of health benefits. More and more studies show that the balance or imbalance of bacteria in your digestive system is
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linked to overall health and disease. The key health benefits linked to probiotics include: 1- Probiotics are live microorganisms. At the point when taken in adequate amounts, they can help reestablish the common adjust of gut microorganisms. Subsequently, health benefits may take after. 2- Probiotics can lessen the hazard and seriousness of diarrhea from from various diverse causes 3-Research demonstrate taking probiotics may help enhance indications of psychological disorders such as depression, anxiety, stress and memory, among others. 4- Probiotics may help secure the heart by diminishing "bad" LDL cholesterol levels and modestly lowering blood pressure. 5- Probiotics may lessen the hazard and seriousness of specific allergies, for example, skin inflammation in infants. 6- Probiotics may help may help decrease the side effects of entrail issue like ulcerative colitis, IBS and necrotizing enterocolitis. 7- Probiotics may help support the immune system and protect against infections. 8- Certain probiotics may enable to lose weight and stomach fat. However, other strains have been connected to weight gain. Beneficial probiotic strains: 1- Bifidobacterium bifidum : The most predominant probiotic in newborn children and in the large intestine, supports production of vitamins in gut, restrains harmful bacteria, supports immune system response and counteracts diarrhea . 2-Bifidobacterium longum: Supports liver function, diminishes inflammation, evacuates lead and heavy metals. 3-Bifidobacterium breve : Colonizes healthy gut community and crowd out bad bacteria. 4-Bifidobacterium infantis: Alleviates IBS manifestations, diarrhea and
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constipation. 5-Lactobacillus casei: Supports immunity, restrains h. pylori and helps fight infections. 6- Lactobacillus acidophilus: Relieves gas, swelling, enhances lactose intolerance, bring down cholesterol levels and formation of vitamin K. In addition, important in GALT immune strength. 7- Lactobacillus bulgaricus: A powerful probiotic strain that has been appeared to light unsafe bacteria that attacks the digestive system and is steady enough to withstand the acidic digestive juices of the stomach. It also neutralizes toxins and naturally produces its own antibiotics. 8- Lactobacillus brevis : shown to survive the GI tract, help cellular immunity, enhanced natural T-killer cells and kill h. pylori bacteria. likewise kills poisons and normally creates its own particular antiinfection agents. 9- Lactobacillus rhamnosus : Supports bacterial balance and supports healthy skin, helps light urinary tract infections, respiratory infections, and reduce anxiety diminishing anxiety hormones and GABA neurotransmitter receptors. Likewise,, survives GI tract. 10-Bacillus subtilis $Q HQGRVSRUH SURELRWLF WKDW¶V KHDW-resistant. Evokes a potent immune response and supports suppresses growth of bad bacteria like salmonella and other pathogens. 11- Bacillus coagulans $QHQGRVSRUHSURELRWLFWKDW¶VKHDW-resistant and enhances nutrient absorption. enhances inflammation and side effects of joint inflamm 12- Saccharomyces boulardii : A yeast probiotic strain that reestablishes natural flora in the large and small intestine and enhances intestinal cell JURZWK ,W¶V GHPRQVWUDWHG HIIHFWLYH LQ WUHDWLQJ LQflammatory bowel GLVHDVHOLNH&URKQ¶VGLVHDVH ,W¶VEHHQDSSHDUHGWRKDYHDQWL-toxin effects, be antimicrobial and reduce inflammation.
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12-Saccharomyces boulardii : a yeast probiotic strain that reestablishes characteristic greenery in the vast and small digestive tract and enhances intestinal cell development. It's demonstrated viable in treating provocative gut sickness like Crohn's illness Application of probiotic bacteria in dairy foods: There is prove that food frameworks assume an imperative part in the valuable health impacts of probiotics on the host. Fermented foods, , especially dairy foods, are are generally utilized as probiotic transporters. Fermented beverages refreshments give an essential commitment to the human diet in in numerous countries since fermentation is an inexpensive technology which preserves food, enhances its nutritional value and improves its sensory properties. However, interest for new probiotic products has encouraged the advancement of different networks to convey probiotics, such as ice cream, infant milk power and fruit juice. Starter cultures containing Streptococcus salivarius ssp. thermophilus and Lactobacillus delbrueckii ssp. bulgaricus, Bifidobacterium longum and Lactobacillus acidophilus have been utilized, in addition, and the fact that culture bacteria did not decrease in the yogurt during frozen storage was verified͘ In addition, the presence of probiotic bacteria did not change the sensory characteristics of the ice cream. The ice cream may offer a decent a good vehicle for probiotic cultures because of its composition, which includes milk proteins, fat and lactose, as well as other compounds. Additionally, its frozen state adds to its proficiency. However, a probiotic ice cream product ought to have generally high pH values ±5.5 to 6.5, so as to support an expanded survival of lactic cultures during storage. The lower acidity additionally brings about expanded consumer acceptance, particularly among consumers who prefer milder products. (Cruz et al., 2009b). Growth of a probiotic yeast, Saccharomyces boulardii, in association with the bio-yogurt microflora, which is done by incorporating the yeast into commercial bio-yogurt, has
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been recommended as an approach to stimulate growth of probiotic organisms and to guarantee t their survival during storage. The probiotic yeast species, S. boulardii, had the capacity to develop in bio-yogurt and achieve maximum counts exceeding 107 CFU g-1. The number of yeast populations was significantly higher in the fruit-based yogurt, principally because the presence of sucrose and fructose got from the fruit. Regardless the inability of S. boulardii to use lactose, the yeast species used accessible organic acids, galactose and glucose got from bacterial metabolism of the milk sugar lactose present in the dairy products. The viability of strains of L. acidophilus and Bifidobacterium animalis ssp. lactis in stirred yoghurts with fruit preparations of mango, mixed berry, passion fruit and strawberry was evaluated during shelf-life. Fermented milks supplemented with lemon and orange fibers expanded the counts of L. acidophilus and L. casei during cold storage compared to the control set. This was not the situation for B. bifidum, conceivably owing to the well-known sensitivity of bifidobacteria species to an acidic environment Domain.
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Examples of probiotic fermented milk products: Acidophilus milk: In this type of fermented milk, Lactobacillus acidophilus is used as a starter culture. For the production, milk is heat processed at 95°C and goes through homogenisation. Then cooled to 37°C and inoculated with 2-5% commercial L.acidophilus pure culture and left for incubation for 12-24 hours. After, milk is cooled to 5°C and kept under cold conditions. However, in some Acidophilus milk productions, milk is subjected to a high temperature (above 120°C) in order to get rid of competition and better L.acidophilus development, then cooled and inoculated with 2-5% commercial L.acidophilus pure culture. Bacteria populate in intestinal system and prevent the activity of harmful gas forming microorganisms. It is beneficial to people with diarrhea and intestinal gas problems.
Fig. (29). Acidophilus milk
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Bifidus milk: Bifidus milk is the first infant product produced with Bifidobacteria and was first produced by Mayer in 1948 at Germany. In the process, milk is standardised and homogenised and kept at 80-120 °C for 5-10 minutes. Then, 10% bifidobacteria starter culture (Bifidobacterium bifidum and Bifidobacterium longum) is inoculated to milk and the milk is left for incubation at 37°C until coagulation. Once it reaches to a pH value of 4.3-4.7, incubation is ended. It is packed and kept in cold conditions. The final product contains 108 -109 cfu/ml bacteria. The product has a 4.3-4.7 pH value and contains 10-100 million bifidobacteria in 1 gram, and has an acidic and spicy aroma which differs it in taste from other products. Bifidus milk is easy to digest and is still used for the treatment of gastrointestinal and liver diseases and for constipation.
Fig. (30). Bifidus milk
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Mil-Mil product: Mil-Mil is a Japanese fermented milk product. A blend of Bifidobacterium bifidum , Bifidobacterium breve and Lb. acidophilus starter cultures is used in the production. It was developed by Yakult Honsha company in Japan. The product is enriched with small amounts of glucose, fructose and carrot juice. Hence, it is rich in provitamin A. The product can also be consumed as soup. Yakult product: Yakult is a probiotic milk product that is produced using L. casei Shirota strain. This strain is resistant to gastric and duedonal acid, can populate and form antimicrobial substances in small intestine and has the ability to enhance the activity and quantity of macrophages. L. casei Shirota strain was discovered by Japanese researcher Dr. Shirota in 1935. It is demonstrated to be beneficial for health and is on the market in 15 different countries including European countries.
Fig. (31).Yakult product
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Yogurt: Yoghurt is a food produced by bacterialfermentation of milk. using a culture consisting of Lactobacillus delbrueckii bulgaricus and Streptococcus thermophiles. In addation, other lactobacilli and bifidobacteria are also sometimes added during or after culturing yogurt. Fermentation of lactose by these bacteria produces lactic acid, which acts on milk protein to give yogurt its texture and characteristic tart flavor. Cow's milk is the milk most commonly used to make yogurt beside milk from buffalo, goats, ewes, mares, camels, and yaks . Milk used may be homogenized or not. Possibly it is the most popular probiotic food if it comes from raw, grass-fed animals. To produce yogurt, milk is first heated, usually to about 85 °C (185 °F), to denature the milk proteins so that they do not form curds. After heating, the milk is allowed to cool to about 45 °C (113 °F).[3] The bacterial culture is mixed in, and a temperature of 45 °C (113 °F) is maintained for four to twelve hours to allow fermentation. The issue is a VXEVWDQWLDO YDULDWLRQ RQ WKH TXDOLW\ RI \RJXUWV RQ WKH PDUNHW WRGD\ ,W¶V recommend when purchasing yogurt to search for three things: First, that LW FRPHV IURP JRDW¶V VKHHS PLON VHFRQG WKDW LW¶V JUDVV-fed; and third, WKDW LW¶V RUJDQLF 7KH SURELRWLF \RJKXUW KDV PDQ\ EHQHILWV LQFOXGLQJ Supports healthy digestion, lowers the Risk of Type 2 Diabetes, lowers the risk of Colorectal cancer, increases bone density and may help prevent osteoporosis, supports weight loss and increases fat loss, boosts the immune system, reduces high blood pressure, reduces bad cholesterol, regulates moods and may help treat chronic pain and brainrelated illnesses.
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Fig. (32). Yoghurt product Butter milk robe: Robe, a traditionally fermented milk product is the most widely produced product. Nomadic tribes produce the entire robe during the rainy season (2-4Months), particularly in central and western Sudan. The bulk of Robe is made from cow's milk, while a smaller proportion is prepared from either goat's or sheep's milk or a mixture of these two milks. Robe is fermented naturally by the bacteria Lactobacillus curvatus, Lactobacillus fermentum and the yeast Pichia membranefaciens. Many uses have been attributed to Robe. Freshly prepared Robe which is available early in the morning is a pleasant sour product with a characteristic buttery flavour. As the day wears on, the product losses its original pleasant flavour and turns more sour till then the whey separates from the curd which floats on top being fully impregnated with gas. Such a Robe is put to a number of uses. In hot climatic conditions, Robe is diluted with 2 or 3 volumes of water to give Gubasha, a thirst quencher. Market samples of Robe was found to contain 7.2% total solids, 3.3% protein, 2.0% lactose, 0.16% fat,
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1.9% total acidity (as lactic acid) and a pH value of 3.5. It seems that little work was accomplished regarding the nutritional quality of Robe. Garris product: Gariss is an uncommon sort of fermented milk prepared from camel milk in the Sudan. The product is made by a semi-continuous or fedbatch fermentation process in huge skin sacks or siin, which contains extensive amount of previously soured product. The product is prepared DQG FRQVXPHG EDVLFDOO\ E\ FDPHO¶V ER\V PHDQGHULQJ SDVWXUHODQGV It is not generally accessible for the family as camels are frequently pushed far away looking for pasture. Fermentation of Gariss takes place while WKHFDPHOVDUHSURJUHVVLQJDQGEHFDXVHRIWKHLQKHUHQWMHUNLQWKHFDPHO¶V walk; the milk in the bags is gently shaken during fermentation. The most predominant Lactobacillus species were identified as Lactobacillus paracasei ssp. paracasei , L. fermentum, L. plantarum. as Lactococcus lactis 7KH IHUPHQWHG FDPHO¶V PLON *DULVV KDV D KLJK nutritive values, which is important for the desert peoples, since they depend only on it. However, the chemical composition and the microbial contents were affected by the management systems and the preparation conditions. Kefir product: Like yogurt, this fermented dairy product is an interesting of milk and fermented kefir grains. Kefir has been consumed for well over 3,000 years, and the term Kefir was started in Russia and Turkey and means ³IHHOLQJ JRRG´ .HILU LV FUHDWHG E\ WKH IHUPHQWDWLRQ RI PLON E\ WKH EDFWHULDDQG\HDVWVLQ.HILUVWDUWHUEUHDNGRZQODFWRVHLQWKHPLON7KDW¶V why Kefir r is suitable for those who are otherwise lactose intolerant. That is the reason Kefir r is suitable for the people who are generally lactose narrow minded. It has a marginally acidic and tart flavor and
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contains anywhere from 10 to 34 strains of probiotics. Kefir r is similar to yogurt, but since iW¶V IHUPHQWHG ZLWK \HDVW DQG PRUH EDFWHULD WKH ILQDO product is higher in probiotics.
Fig. (33). Kefir product Koumiss-Kumiss product: Koumiss is defined as a national beverage produced form mare milk. Different names of koumiss in other languages are: koumis (French), *HUPDQ NXP\VVNXP\Vɤɭɦɵɫ 5XVVVLDQ .RXPLVVLVSURGXFHGZLWK traditional methods in houses and small scaled productions while it is produced with industrial methods. It contains 2% alcohol, 0.5-1.5% lactic acid, 2-4% milk sugar and 2% fat and it tastes like sour ayran. Koumiss is recommended for the treatment of tuberculosis, asthma, pneumonitis, cardiovascular diseases and gynaecological diseases. It is likewise reasonable for weight pick up and expanding vigor and energy .
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Fig. (34). Koumiss product Acidophilin product: Acidophilin is a product, produced from cow milk with a process procedure includes the utilization a high quality starter culture and it tastes rather acidic. The culture principally contains Lb. acidophilus. In addition to Lb. acidophilus, it contains Lc. lactis ssp. lactis or Lb. delbrueckii ssp. bulgaricus. In some cases, kefir culture might be inoculated to the culture with a 1:1:1: ratio. Preceding the inoculation at 18-25°C, the homogenised milk is heated up to 90-92°C for no less than 3 minutes. Milk is left for incubation until it reaches 0.67-0.72% titrable acidity. It is cooled, packed and kept in cold chain. Acidity of Acidofilin is around 0.67-1.08% and the final product contains 97% L. lactis ssp. lactis, 2% L. acidophilus and 1% yeast. It has a lower antimicrobial acitivity than acidophilus milk .
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Nono product: Nono is a naturally fermented milk product produced by the Hausa community, in Northen Nigeria. In the production of Nono, unpasteurized milk is allowed to ferment naturally. Excess whey is allowed to drain, and the product is stirred to obtain a uniform consistency. Nono is a popular product rich in protein. Maas and Inkomasi: Maas and Inkomasi are fermented milk products originated in South Africa. These two products are traditionally produced in clay pots and calabash. Bacteria present on the inner surface of the container are presumed to be responsible for the fermentation of the milk. Mixed fermentation of homo and heterofermentative lactobacilli, streptococci, leuconostocs and yeasts have been reported to be dominant. A portion of the whey might be drained to acquire a product with higher thickness. The preparations of Maas and Inkomasi have also been commercialized.
Fig. (35). Maas product
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Channa product: Channa is Indian product acquired by heat-acid coagulation of milk. The coagulum is granular, a whitish cream in colour, delicate and spongy with a characteristic greasy flavour. Chhana is prepared by boiling milk and then curdling it with a small amount of whey. The resulting coagulated component is collected and wrapped in cheesecloth, strained and beaten thoroughly, until it becomes quite firm. This mixture is kneaded well before use, so that it acquires a very soft and smooth consistency. It is used in the preparation of channa-based sweets such as Rasagolla, champakali, chum-chum, and rasmali. The product is prepared by heating the milk to boiling point for 10 minutes that cooling. Citric acid is added with steady and moderate blending. The coagulated milk is hung in a muslin cloth sack for around two hours to drain the whey. At that point the coagulum is wrapped in aluminum foil and stored in a cool place.
Fig. (36). Channa product
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CHAPTER THIRTEEN UTILZATION OF STRTER CULTURES IN PRODUCTION OF FERMENED MEAT PRODUCTS
Introduction: Fermented meats are foods with a long-standing convention due their stability and accommodation, their nutritional value, and their appealing sensory properties. They owe improvement of their normal qualities generally to the enzymatic activities of the microorganisms that partake in the fermentation course. As a major example, their unmistakable colour is normally based on the nitrate reductase activity of catalasepositive cocci, enveloping coagulase-negative staphylococci (CNS) and, sometimes, Kocuria species. Fermentation is a simple and cheap technique for conservation of meat and meat products since antiquity. It has an additional preferred advantage of making of particular products with great aroma. Acid production (bringing down the pH), H2O2 production and bacteriocins produced separately or in mix by starter cultures are in charge of keeping the development of food-borne pathogens and spoilage microorganisms in meat. Enthusiasm on the utilization of fermentation techniques in meat has been resuscitated since there is presently a confinement on the utilization of chemical additives for safeguarding of meat and meat products. Meat fermentation is a complex biological phenomenon quickened by the desirable action of specific microorganisms in the presence of an incredible variety of contending or synergistically acting species principally gained from meat. Lactic acid bacteria play a basic part in the production of fermented meat products, Lactobacillus being the fundamental species utilized as a part in
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the European type of fermented products. Keeping in mind the end goal to guarantee sensory quality and good colour formation, lactic acid bacteria are not adequate and the commitment of Staphylococcus carnoses is required. Some strains of meat lactobacilli display critical properties from a technological point of view, such as the production of antimicrobials. A sausage is a cylindrical meat product typically produced using ground meat, frequently pork, beef, or veal, alongside salt, spices and different flavourings, and breadcrumbs, with a skin around it. Normally, a sausage is framed in a casing traditionally made from intestine, but sometimes from synthetic materials. Sausages that are sold uncooked are normally cooked in many ways, including pan-frying, searing and grilling. Some sausages are cooked during processing and the casing may then be removed. Sausages are one of the oldest meat products in which fresh comminuted meats are modified by numerous processing methods to yield desirable and keeping properties. The degree of comminution varies among various processed products and is o\frequently unique characteristic of particular product ranging from coarse comminuted, to finely comminuted to form an emulsion. The primary economic purpose of these products is to present relatively large proportions of fat in palatable ways. Comminution of the fat is therefore a common feature. Dry-cured sausages are one of the established types of meat conservation and are typical of Mediterranean countries with a dry climate (Spain, Italy, France, Portugal and Turkey). Interestingly, smoke cured sausages, or cooked sausages prevail in countries with a colder climate . In general, the subjective attributes of naturally fermented sausages are known to be are known to be generally reliant on the quality of the ingredients and raw materials, the particular conditions of the processing and ripening, and the composition of the microbial population.
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Although commercially grown starter cultures have been around since 1957, it is , just recently that sausage equipment and supplies companies carry them in catalogs. As the specialist -sausage maker becomes more instructed in better parts of the art of sausage making he will undoubtably begin making more fermented sausages at home. Inoculation of the sausage batter with a starter culture made out of selected lactic acid bacteria (LAB), i.e. homofermentative lactobacilli and/or pediococci, and Gram-positive, catalase-positive cocci (GCC), i.e. nonpathogenic, coagulase-negative staphylococci and/or kocuriae, enhances the quality and safety of the final product and standardizes the production process. Nonetheless, small manufacturers keep on using the conventional method of spontaneous fermentation without added starter culture. In this case, the required microorganisms originate from the meat itself or from the environment and constitute a part of the so-called Fhouse flora_ (Back-slopping is additionally utilized, if material from a successful previous batch is added to facilitate the initiation of a new fermentation process. Such artisan fermented sausages are regularly of better quality looked at than controlled maturations immunized of superior quality compared to controlled fermentations inoculated with industrial starters and possess distinctive qualities, partly due to the properties of the raw material and the characteristics of the technology utilized, but also to the particular composition of the house flora. The flavourgenerating, metabolic activity of GCC in artisan chorizo, for example, has been shown to vary with the manufacturing location .
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Fig.(37). Fermented sausage Composition of sausages: Adults and children consume significant amounts of processed meat products (including sausages), regularly utilizing them as substitutes for meat flesh. Therefore, it is vital to keep up the wholesome profile of these products. To aid this, the standard requires that sausages must contain at least 50% fat-free meat flesh. The standard additionally requires that they have a most extreme of fat permitted. This greatest is 50% of the fat-free meat flesh. For instance, in order to make a sausage with 600 g fat-free meat flesh per kg of final sausage, up to 300 g per kg of fat are permitted to have in the final sausage (i.e. 50% of the 600 g fatfree meat flesh per kg). Note that fat-free meat flesh is measured diagnostically by determining the amount of meat protein present, it does not mean meat flesh without noticeable fat. There is no specific restriction on the use of offal as part of the meat content of sausages because the presence of these ingredients must be announced.
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Types of sausages: There are many types of sausages including: 1- Fresh sausage: A type of uncured and uncooked sausage with a short shelf-life. The products have various degrees of comminution, but most products tend to be coarse. The British type of fresh sausage can have various levels of meat content. 2- Emulsion sausages: Sausages which are made from finely comminuted lean meat and fat. Frequently further processed, e.g. by cooking, addition of curing salts, smoking, drying; may therefore have intermediate shelf-life Some contain quite large inclusions of coarsely cut meat, fat, spices, etc. 3- Fermented dried sausage : This type of sausage has a long shelf-life (e.g. 1-2 years) obtained by lactic acid produced by fermentation at the beginning of processing, and curing with nitrite, usually formed microbiologically from nitrate drying out in the later stages of manufacture. The addition of a can of commercially produced concentrated starters to the prepared meat is proffered method. Lyophilized cultures are blended with the seasonings and additives, such cultures can also be dehydrated and then poured over the mix. The meat mix must contain added carbohydrate in form of a fermentable sugar such as dextrose, lactose or sucrose for bacterial growth which results in reduction of pH. Advantages of meat starter cultures:
1- Product safety: The initial rapid population ensures dominance over spoilage or pathogenic bacteria when the pH has not dropped till inhibiting them. 2-Product uniformity: Reliable S.C. ensures uniform flavour, colour and texture of the product.
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3-Drying time: Acidification results in the quicker migration of water to the surface of the sausage and faster drying, with consequent energy cost savings. 4-Increased plant efficiency: Higher volume of the product will be processed with low cost. 5-Product body: Sausages become firmer when the protein is acidified. 6- Shelf-life: Sausage with a low pH and low moisture enables to be sold unrefrigerated and with a long shelf-life. Starter cultures for fermented sausages: Meat is generally subjected to deterioration by the development of several microorganisms. While these spoiling microorganisms are not acceptable in raw meat and meat products, certain types of fermentative microorganisms, particularly the LAB, either exist in crude meat naturally or added by producers, are utilized for the production of fermented meat products. These desirable microorganisms added to the meat batter are called as starter cultures and they can be single species or the blend of specific microorganisms. Starter cultures ferment sausages, develop color and flavor and provide safety. The addition of any commercial culture to the sausage blend provides a safety obstacle, as those large number of crisply presented microbes begin competing for food (moisture, oxygen, sugar, protein) with a modest number living in meat bacteria, keeping them from developing
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Classification of sausage starter cultures: Starter cultures used in fermented sausage can be classified into the following groups: 1- Lactic acid producing starter cultures (fermentation) 2- Colour fixing and flavor forming starter cultures (coulor and flavor) 3- Surface coverage starter cultures (yeasts and molds) 5-Bio-protective starter cultures (producing bacteriocins).
Fig. (38). Fermented dried sausage Microorganisms involved in sausage fermentation: The microorganisms that are basically associated with sausage fermentation include species of LAB, GCC, moulds, and yeasts. In spontaneously fermented European sausages, facultative homofermentative lactobacilli constitute the predominant flora all through ripening. Lactobacillus sakei and/or Lactobacillus curvatus generally dominate the fermentation process.
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Lactobacillus sakei appears to be the most competitive of both strains, frequently representing half to two thirds of all LAB isolates from spontaneously fermented sausage, though Lactobacillus curvatus is as often as possible found in sums up to one fourth of all LAB isolates. Other lactobacilli that might be found, but by and large at minor levels, include Lactobacillus plantarum, Lactobacillus bavaricus (now reclassified as Lactobacillus sakei or Lactobacillus curvatus), Lactobacillus brevis, Lactobacillus buchneri, and Lactobacillus paracasei. Recently, the new species Lactobacillus versmoldensis has been isolated from German, quick-ripened, salami-style sausages where it was present in numbers of up to 108 cfu /g . Pediococci are less frequently isolated from European fermented sausages but occasionally occur in small percentages. They are more typical in fermented sausages from the United States where they are purposely added as starter cultures to quicken acidification of the meat batter. Enterococci are sometimes associated with fermented meat products, specifically artisan products from Southern Europe, where they increase during early fermentation stages and can be detected in the end-product at levels of 102±105 cfu g_1. They are universal in food processing establishments and their presence in the gastrointestinal tract of animals prompts a high potential for contamination of meat at the time of slaughter. These microorganisms may enhance food flavour but also compromise safety if opportunistic pathogenic strains proliferate or antibiotic resistance is spread . Staphylococci and kocuriae can participate in desirable reactions during ripening of dry fermented sausages, such as colour stabilisation, deterioration of peroxides, proteolysis, and lipolysis. They are ineffectively competitive in the presence of actively growing aciduric bacteria. The nonpathogenic, coagulase-negative staphylococci are dominated by Staphylococcus xylosus, Staphylococcus carnosus, and Staphylococcus saprophyticus, yet different species happen as well. In
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addition to staphylococci, Kocuria varians, formerly known as Micrococcus varians, or other kocuriae are sometimes isolated in little amounts from naturally fermented sausage . Moulds, usually Penicillium nalgiovense and Penicillium chrysogenum, are utilized in mould-ripened sausages, especially in Southern Europe. A yeast population, dominated by Debaryomyces hansenii, may also be found on the sausage surface is sometimes added as starter culture . Advantages of sausage starter cultures: The advantages of starter cultures for fermented sausages include: 1- they are of known number and quality. This dispenses with a ton of speculating in the matter of whether there is sufficient bacteria inside meat to start fermentation or whether a strong curing color will be acquired. 2-cultures are upgraded for various temperature ranges that permit production of slow, medium or fast-fermented products. Traditionally produced sausages required three (or more) months to make, starter cultures make this possible within weeks or even days. 3-production of fermented sausages does not rely upon "privileged insights" and a product of consistent quality can be created year round in any climatic zone as long as appropriate natural conditions or fermenting/drying chambers are accessible. 4-they provide safety by contending for food with undesirable bacteria thus repressing their development. Table (4) presents the most important microorganisms used in starter cultures are:
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Table 5. Important microorganisms used in sausage starter cultures. Microorganism Family Lactic Acid Lactobacillus Bacteria Pediococcus
Species Use L.plantarum L.pentosum L.sakei L.curvatus P.acidilactici P.pentosaceus
Use acid production acid production acid production acid production acid production acid production
Curing Bacteria Micrococcus (color and flavor Staphylococcus forming) Yeasts Debaryomeces Candida Moulds Penicillium
K.varians S.xylosus S.carnosus D.hansenii C.famata P.nalgiovense P.chrysogenum
color and flavor color and flavor color and flavor flavor flavor white mold white mold
In addition to being exceptionally competitors for nutrients against pathogenic and spoilage bacteria, lactic acid bacteria are known to create compounds named "bacteriocins"which can act against different microorganisms. Pediococcus acidilactici and Lactobacillus curvatus are known bacteriocins makers particularly successful against the development of Listeria monocytogenes. Flavour of fermented sausages: The flavour of fermented sausage is affected numerous variables, principally, the origin, amount and type of ingredients (e.g. meat, salt, and spices), the temperature, processing time, smoking, and choice of starter culture. Basic flavour brings about from the interaction of taste (primarily determined by lactic acid production and the pattern of peptides and free amino acids resulting from tissue-generated proteolysis)
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and aroma (mainly determined by volatile components derived from bacterial metabolism and lipid autoxidation). However, acetic acid is additionally present and is actually needed in small amounts for full dry sausage flavour. Too high concentrations of acetic acid, however, produce a thorny,, astringent flavour. In Southern European sausage, prevalent acidity is not looked for and might be rejected. In the last kind of sausage, where acidity is gentle and smoking is uncommon, flavour is principally created by proteolytic and lipolytic activities from tissue enzymes, but the starter culture created flavour too because of its aromagenerating metabolic activities. Usually, Mediterranean sausages are likewise inoculated on the surface with fungi that add to the sensory properties too. Proteolytic enzymes, principally those endogenous to the meat, are of are significant for flavour. Meat proteases, especially cathepsin D-like enzymes, appear to be in charge of proteolysis and peptide formation during fermentation (Hierro et al., 1999; Molly et al., 1997), while microbial enzymes rather rather follow up on the discharged d oligopeptides during the later Phases of ripening (). Microbial proteolytic activity against meat proteins is low under conditions found in fermented sausages, however a specific action, yet to a minor degree and in a strain dependent manner, may incompletely add to initial protein breakdown. All the more imperatively, the peptides created by muscle proteolysis can be taken up by bacteria that further split them intracellularly into amino acids and may change over them to aroma components. Lipolysis plays a central role in aroma development. It prompts the arrival of free fatty acids and is for the most part because of tissue lipases, although bacterial lipolytic activity has been depicted as well, in particular by staphylococci . Short chain fatty acids (C < 6) prompt strong cheesy odours, whereas medium and long chain fatty acids can go about as t as antecedents. Lipolysis is Lipolysis is just the initial phase in the process and is followed by further oxidative degradation of the
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liberated fatty acids into alkanes, alkenes, alcohols, aldehydes, ketones, and furanic cycles. The bacterial flora plays a role in the oxidation of free fatty acids despite the fact that non-enzymatic reactions occur too. Except for after death glycolysis, carbohydrate catabolism is primarily of bacterial origin (both LAB and GCC) and compounds such as lactic acid, acetic acid, ethanol, acetoin, and diacetyl, all may play a role in the complexity of sausage flavour. The utilization of selected strains that produce fascinating aroma components as functional starter cultures could prompt more tasty sausages also to a decrease of the ripening time.
Developing of functional starters for enhanced flavor needs to consider learning about crude materials, technology, and sensory quality, and ought to be focused to particular applications. Moreover, starters ought not have undesirable characteristics, such as the formation of toxic compounds or too high quantities of acetic acid or acetoin. Acetoin production, stimulated by low pH and low sugar availability at the end of the ripening period, may prompt fermented sausages with a dairy product odour.
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CHAPTER FOURTEEN UTILIZATION OF STARTER CULTURES IN PRODUCTION OF BAKED PRODUCTS
Introduction: The quality of bread is characterized by its flavour, nutritional value, texture and time span of usability. In the baking industry the characteristics are enhanced by expansion of supposed "improvers" or enzymes (which normally are incorporated in the improvers). On the other hand the expansion of sourdough impacts all aspects of bread quality and consequently meets the consumer demand for a reduced utilization of these improvers which contain all kind of additives. Sourdough bread is made by the fermentation of dough utilizing naturally occurring lactobacilli and yeast. Sourdough bread has a somewhat harsh taste not present in most breads made with baker's yeast and characteristic inherent keeping qualities than other breads, because othe lactic acid produced by the lactobacilli. Sourdough is made from flour and water, which begins to ferment spontaneously and that is permitted to ferment for a specific time at a specific temperature. To be sure flour contains naturally lactic acid bacteria, which will develop in the blend and which will acidify it. Sometimes the baker adds lactic acid bacteria himself. In French this is called a "levain", in Italian or Spanish "madre", in Dutch "zuurdesem" and the German baker will talk about "Sauerteig". As sourdough is a halfway and not a finished result, effect on the bread must be resolved on the premise of the nature of the bread. Biochemical changes during sourdough fermentation occur in protein and carbohydrate segments of the flour. The rate and degree of these
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progressions incredibly impact the properties of the sourdough and consequently the quality of the bread. The impacts are related with the metabolites created by the lactic acid bacteria and yeast during fermentation, including organic acids, enzymes and CO2.
Fig. (39).Sour dough bread
The microflora of sourdough bread:
Lactic acid bacteria (LAB) and yeasts are regularly associated in sourdough. The LAB:yeast proportion in sourdoughs is mostly 100:1. Though in the greater part of fermented foods homofermentative LAB play an essential role, heterofermentative LAB are dominating in sourdough, particularly when traditionally prepared. In reality, acetic acid, vital end product of heterofermentation, plays a major role in the flavour of sourdough. Further, Lactobacillus strains are more successive than Leuconostoc, Weissella, and Pediococcus species; lactococci, enterococci, and streptococci are rarely found. The strength of (commit) heterofermentative lactobacilli in sourdoughs can be clarified
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predominantly by their competitiveness in and adaptation to this specific condition. The microflora of raw cereals is made out of bacteria and fungi (104 ±107 CFU/g), while flour contains 2x104 ±6x106 CFU/g (Stolz, 1999). The bacteria are mainly mesophilic, and are additionally found in spontaneously fermented sourdoughs. They include Gram-negative aerobes (e.g. Pseudomonas) and facultative anaerobes (Enterobacteriaceae), in addition Gram-positive LAB: homofermentative rods (L. casei, L. coryniformis, L. curvatus, L. plantarum, and L. salivarius), heterofermentative rods (L. brevis and L. fermentum), homofermentative cocci (E. faecalis, L. lactis, P. acidilactici, P. parvulus, and P. pentosaceus), and heterofermentative cocci (Leuconostoc and Weissella). Likewise, undesirable Staphylococcus aureus and Bacillus cereus, , and additionally other bacteria, might be available. Many yeasts have been identified, either in the grains (up to 9X104 CFU/g) or flours (up to 2X103 CFU/g), they include: Candida, Cryptococcus, Pichia, Rhodotorula, Torulaspora, Trichosporon, Saccharomyces, and Sporobolomyces. It ought to be emphasised that S. cerevisiae is not found in the raw materials; its occurrence in sourdough may be explained by the appOLFDWLRQRIEDNHU¶V\HDVWLQPRVWGDLO\EDNHU\SUDFWLFH &RUVHWWL et al., 2001; Galli, Franzetti, & Fortina, 1987). Among fungi (ca. 3!104 CFU/g), Alternaria, Cladosporium, Drechslera, Fusarium, Helminthosporium, and Ulocladium (from the field), and Aspergillus and Penicillium (from the storage), are present.
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Production of sourdough bread:
Preparation of starter culture: Before making a loaf of sourdough bread, a sourdough starter must be made. This is a culture of flour and water for growing wild yeast and lactobacilli bacteria. The motivation behind the starter is to deliver a vigorous leaven and to develop the flavour of the bread. In practice there are several kinds of starters, as the ratio of water to flour in the starter varies. A starter might be a liquid batter or a firm dough. Flour naturally contains a variety of yeasts and bacterial spores. At the point when wheat flour comes into contact with water, the naturally occurring enzyme amylase breaks down the starch into the sugars glucose, sucrose, galactose and raffinose, which sourdough's natural yeast can metabolize. Amylase additionally breaks down starch into maltose, which the yeast can't metabolize.[14] With adequate time, temperature, and refreshments with new or fresh dough, the blend builds up a stable culture. This culture will cause a dough to rise if the gluten has been developed sufficiently. The bacteria ferment maltose that the yeast can't use, and the byproducts are metabolized by the yeast which produces carbon dioxide gas, leavening the dough. Acquiring an attractive ascent from sourdough takes longer than a dough leavened with baker's yeast because the yeast in a sourdough is less vigorous. In the presence of lactic acid bacteria, however, some sourdough yeasts have been observed to produce twice the gas of baker's yeast. The acidic conditions in sourdough, alongside the bacteria likewise delivering enzymes that break down proteins, result in weaker gluten and may deliver a denser finished product. Briefly, the starter culture can be made in about five days. On the first day, the flour and water are mixed into a batter, and let them to sit at room temperature overnight. Wild yeast are and they will quickly start to
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thrive in this culture. Over the next few days, the yeast and bacteria are need to fed by pouring off some of the culture and adding fresh flour and water. It will become ready to use to make bread when the culture becomes very bubbly within just a few hours of feeding, and when it smells sour but fresh. Refreshment of the starter culture:
As it ferments, sometimes for several days, the volume of the starter is expanded by periodic additions of flour and water, called "refreshments".As long as this starter culture is fed flour and water regularly it will remain active. The proportion of fermented starter to new flour and water is basic in the development and maintenance of a starter. This proportion is known as the refreshment ratio. Higher refreshment ratios are related with greater microbial steadiness in the sourdough. A high refreshment ratio keeps acidity of the refreshed dough moderately low. Acidity levels of beneath pH 4.0 restrain lactobacilli and favour acid-tolerant yeasts. A drier and cooler starter has less bacterial activity and more yeast development, which brings about the bacterial production of more acetic acid relative to lactic acid. Alternately, a wetter and warmer starter has more bacterial activity and less yeast growth, with more lactic acid in respect to acetic acid. The yeasts produce mainly CO2 and ethanol. High amounts of lactic acid are wanted in rye and blended -rye fermentations, while relatively higher amounts of acetic acid are desired in wheat fermentations. A dry, cool starter produces a sourer loaf than a wet, warm one. Firm starters (for example, the Flemish Desem starter, which covered in a large container of flour to prevent drying out) have a tendency to be more resource-intensive than wet ones.
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Faster starter processes, requiring fewer refreshments, have been devised, sometimes utilizing commercial sourdough starters as inoculants. These starters in geneneral fall into two sorts. One is produced using customarily kept up and stable starter doughs, regularly dried, in which the proportions of micro-organisms are questionable. Another is produced using micro-organisms carefully isolated from Petri dishes, grown into large, homogeneous populations in fermentors, and processed into combined baker's products with numerically defined ratios and known quantities of microorganisms well suited to particular bread styles Local methods: Bakers have have formulated a few methods for empowering a steady culture of micro-organisms in the starter. Unbleached, unbromated flour contains more micro-organisms than more processed flours. Brancontaining (wholemeal) flour gives the best assortment of organisms and extra minerals, however some cultures utilize an underlying blend of white flour and rye or whole wheat flour or "seed" the culture using unwashed organic grapes (for the wild yeasts on their skins). Grapes and grape must are likewise sources of lactic acid bacteria, as are numerous other edible plants. Basil leaves are soaked in room-temperature water for an hour to seed traditional Greek sourdough. Using water from boiled potatoes is said to increase the activity of the bacteria by giving extra starch. Bakers regularly make loaves with fermented dough from a previous batch (which they call "mother dough", "mother sponge", "chef", or "seed sour") rather than making a new starter each time they bake. The original starter culture might be numerous years old. Because of their pH level and the presence of antibacterial agents, such cultures are ready to anticipate colonization by undesirable yeasts and bacteria. Thus, sourdough products inherently keep fresh for a longer time than other breads, and are good at resisting spoilage and mold without the additives required to retard spoiling of other types of bread.
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The flavour of sourdough bread varies from place to place according to the method utilized, the hydration of the starter and the final dough, the refreshment proportion, the length of the fermentation periods, ambient temperature, humidity, and elevation, all of which contribute to the microbiology of the sourdough. Baking:
The starter is blended with flour and water to make a final dough of the coveted consistency. The starter weight is generally 13% to 25% of the total flour weight, though recipes may vary. The dough is shaped into loaves, left to rise, and then baked. Since the ascent time of most sourdough starters is longer than that of breads made with baker's yeasts, sourdough starters are generally unsuitable for use in a bread machine. However, sourdough that has been demonstrated over numerous hours, utilizing a sourdough starter or mother dough, would then be transferred to the machine, using just the baking portion of the bread-production program, bypassing timed mechanical kneading by the machine's paddle. This might be convenient for single loaf production, but the complex rankled and slashed crust characteristics of oven-baked sourdough bread cannot be achieved in a bread making machine, as this usually requires the utilization of a heating stone in the oven and clouding of the dough to produce steam. perfect hull crust development requires loaves of shapes not achievable in a machine's loaf tin. Types of sourdoughs: On the basis of applied technology for their production, sourdoughs have been grouped into three types. Each type of sourdough is characterized by a specific sourdough LAB microflora. The types include:
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type I: I sourdoughs or traditional sourdoughs ,sourdough which is restarted using a part of the previous fermentation), type II: accelerated sourdoughs generally used as dough-souring supplements in semi-fluid). type III: dried sourdoughs :are dried preparations). Unlike type I sourdoughs, types II and III doughs require the DGGLWLRQRIEDNHU¶V\HDVW (S. cerevisiae) as leavening agent. Type I sourdough:
Type I sourdoughs are generally firm doughs produced with traditional techniques and are characterized by continuous, day by day refreshments to keep the microorganisms in a dynamic state, as demonstrated by a high metabolic activity most importantly with respect to raising, i.e. gas production. The process is performed at encompassing temperature (20± 30 8C) and the pH is about 4.0. Examples of baked goods so obtained are San Francisco sourdough French bread, Panettone and other brioches, Pugliese, Toscanon and Altamura bread, and three-stage sourdough rye bread. The lactic acid bacterial flora dominating type I sourdough includes: Lactobacillus sanfranciscensis, L. pontis L. fermentum, L. fructivorans, L. brevis, and L. paralimentarius. The yeasts include:Saccharomyces exiguus, Candida milleri, or Candida holmii normally populate sourdough cultures symbiotically with Lactobacillus sanfranciscensis. The perfect yeast S. exiguus is associated with the imperfect yeasts C. milleri and C. holmii. Torulopsis holmii, Torula holmii, and S. rosei are synonyms used prior to 1978. Other yeasts reported found include C. humilis, C. krusei, Pichia anomaola, C. peliculosa, P. membranifaciens, and C. valida.
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Fig. (40). Type I sourdough
Type II sourdough:
In Type II sourdoughs, baker's yeast (Saccharomyces cerevisiae) is added to raise the dough. The flora includes: L. pontis and L. panis .They have a pH less than 3.5, and are fermented within a temperature range of 30 to 50 °C (86 to 122 °F) for a few without feedings, which diminishes the flora's activity. This process was received by some in industry, to a limited extent, because of simplification of the multiple-step build typical of Type I sourdoughs. These bakery pre-products serve for the most part as dough acidifiers. A few altered, quickened sourdough fermentation processes exist. Sourdough processes with continuous propagation and long-term one-step fermentations are common now; they ensure more production quality and adaptability. A current pattern of industrial bakeries exists in the instalment of continuous sourdough fermentation plants.
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Fig. (41). Type II sourdough
The obligate homofermentative L. acidophilus, L. delbrueckii, L. amylovorus (rye), L. farciminis, and L. johnsonii, and obligate heterofermentative L. brevis, L. fermentum, L. frumenti, L. pontis, L. panis, L. reuteri, as well as Weissella (W. confusa) species are found in type II sourdoughs. In addition, yeast growth is slowed or stopped because of the higher fermentation temperatures. These doughs are more fluid and once fermented might be chilled and stored for up to a week. They are pumpable and utilized in continuous bread production systems. Type III sourdough: Type III sourdoughs are Type II sourdoughs subjected to a drying process. It is dried doughs in powder form, usually either spray or drum drying, and are mainly used at an industrial level as flavoring agents and aroma carriers during bread making. The drying process additionally leads to an increased shelf-life of the sourdough and turns it into a stock product until further use. Type III sourdoughs are dominated by "dryingresistant lactic acid bacteria for example, Pediococcus pentosaceus, Lactobacillus plantarum, and L. brevis." The drying
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conditions, time and heat applied, may be varied in order to influence caramelization and produce desired characteristics in the baked product.
Fig. (42). Type III sourdough Benefits of sourdough bread: Sourdough does not require much gluten, in contrast to factory-made bread which frequently has gluten added. Individuals with gluten intolerance may in this manner discover sourdough bread more agreeable to eat. Sourdough bread produces a smaller surge in blood glucose and insulin than other types of bread, and there is limited evidence that it is suitable even for coeliac patients.
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CHAPTER FIFTEEN USE OF STARTER CULTURE FOR VETABLES FERMENTATION
Introduction: When vegetables are put in solution of sodium chloride of suitable concentration, they experience fermentation by the naturally occurring micro-organisms on the vegetables. The concentration of sodium chloride utilized relies on the inclination of the specific vegetable to soften during saline solution storage and may range from around 1 to 8%. %. In turn, the sodium chloride concentration enormously impacts the sorts and counts of micro-organisms active during fermentation. Vegetables are not ordinarily washed in commercial brining operations and contain the indigenous microflora when brined. Microbial growth during natural fermentation of vegetables has been arranged into four consecutive stages. The utilization of pure starter cultures for fermentation of vegetables such as olive, cabbage, cucumbers and other products has been explored since several years with varying degrees of success. Starter cultures have been utilized just to a restricted degree commercially. However, late endeavors to enhance fermentation vessels and to develop controlled fermentation methods for fermented vegetables brought about an expanded enthusiasm for developing cultures suitable for application in such methods. The lack of widespread commercial use of these cultures might be due to several variables such as: 1- olive, cabbage and cucumbers undergo natural fermentation by lactic acid bacteria (LAB) if the product is correctly handled and held in salt concentrations that have been established for each particular product.
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2- Heat is the only effective and acceptable treatment known for ridding vegetables of the natural LAB. Heating is costly, and it changes the general characteristics of the product especially the flavor. 3- Saline solution (Brine) from one natural fermentation can be used to inoculate other containers. The salt can promote the fermentation process by inhibiting the growth of undesirable microorganisms, favoring the growth of desired /$% 4-The fermentation devices and common handling processes till now are not compatible with pure culture fermentations. 5- There is no adequately interesting strains of LAB have been revealed up 'til now that have commanded their utilization as starter cultures. While each of the above variables might be a subject to contention, generally they presumably represent the new plug use of starter cultures for vegetables. There are signs, in any case, that expanded utilization of pure starter cultures of LAB may happen within the foreseeable future. Changes in saline solution bringing innovation, acknowledgment of anaerobic fermentation tanks that are more compatible for pure culture utilization, and choice or alteration of LAB with novel and significant properties linger as conceivable purposes behind utilization of pure cultures in certain vegetable fermentations.
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Table 6. Examples of fermented vegetables. Product
Country
Sauerkraut
Germany
Kimchi
Korea
Dhamuoi
Vietnam
Dakguadong
Thailand
Burong mustasa
Philippines
Main ingredients Cabbage, salt
Korean cabbage, radish, various vegetables, salt Cabbage, various vegetables Mustard leaf, salt Mustard
Microorganisms
Usage
Leuconostoc mesenteroides, Lactobacillus brevis, Lactobacillus plantarum L. mesenteroides, Lb. brevis, Lb. plantarum
Salad, dish
side
Salad, dish
side
L. mesenteroides, Salad, side Lb. plantarum dish Lb. plantarum
Salad, side dish Lb. brevis, Salad, side Pediococcus dish cerevisiae
Source: Breidt et. al., (2013).
Fig. (44). Vegetables starter cultures
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Benefits of vegetables fermentations: Upon fermentation of vegetables have been f using starter culture for fresh vegetables, the finished product may offer the following health benefits: 1- The LAB that ferment the fresh vegetables are so advantageous to health that they have frequently been called "life preserving agents". 2- The LAB contribute to the protection of the body against infections and empower the immune system. 3- Fermented vegetables enhance the digestion process by managing the level of acidity in the digestive tract and by empowering the production of beneficial intestinal flora. 4- Fermented vegetables act as anti-oxidants. 5- Live LAB encourage the synthesis of certain vitamins, such as vitamins C and B12. 6- They assist in the breaking down of proteins 7- They drive out pathogenic bacteria, fungi, viruses. 8- Live lactic bacteria are known to have a supporting effect on the nervous system. 9- In producing lactic acid and enzymes, the lactic bacteria likewise encourage the break-down of proteins and thus their absorption. 10- Fermented vegetables are prescribed for diabetics, since the sugar content of vegetables is changed by LAB into a more assimilable forms. 11-The lactic acid produced during fermentation does not have the harmful acidifying impact on the human system that other organic acids have a tendency to have. Truth be told, it might help counteract joint inflammation. 12- Fermented vegetables can be an essential part of a yeast-free diet (as in the case of candidiasis).
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Species of LAB for vegetable fermentations: Three species of LAB historically have been associated with the natural fermentation of cucumber and olives include: s, Pediococcus cerevisiae, Lactobacillus brevis and Lactobacillu plantarum. However, these species in addition of Leuconostoc mesenteroideare also associated with the fermentation of sauerkraut. Characteristics of these microorganisms DUH ODUJHO\ FRQVLVWHQW ZLWK %HUJH\¶V PDQXDO RI 'HWHUPLQDWLYH Bacteriology. Different endeavors have been made to utilize pure cultures of these species for fermentation of vegetables . Presently, nonetheless, pure cultures are are utilized on just a restricted commercial basis. Characteristics of LAB associated with vegetable fermentations are shown in the following table: Table 7. Characteristics of LAB associated with vegetable fermentations. Property Morphology
Optimum temperature Growth at 45°C Growth in 8% NaCl Glucose metabolism
Microorganism L. plantarum
L. brevis
Pediococcus pentosaceus Short to Short rods, Cocci, singly medium rods, singly or in or in pairs or singly short chains in tetrads 30-35 30 35
Leuconostoc mesenteroides Cocco or bacilli, usually in pairs 20-30
No
No
Yes
No
Yes
No
Yes
No
Homofermenter lactic acid
Heterofermenter lacic acid, acetic acid, ethanol, CO2 DL
Homofermenter lactic acid
Heterofermenter lacic acid, acetic acid, ethanol, CO2 D
Lactic acid DL produced from glucose
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DL
The variables that must be considered when developing LAB cultures for use in controlled fermentation of vegetables include: 1- Rapid and dominant growth. 2- Type and extent of acid production 3- Salt tolerance 4- Temperature range 5- Carbon dioxide production 6- Cell sedimentation, , 7- Bacteriophage resistance 8- Nutritional value 9- Ability to survive as concentrated cultures Rapid and predominant growth: Commercial fermentation of vegetables is by naturally occurring microorganisms, Predominance of growth by a species of LAB is affected by the chemical and physical environment under which it must compete. When heat was used to inhibit the natural microbial flora, pure culture fermentations of sauerkraut, cucumbers and olives resulted. For transcendence, in this manner, the additional culture must be exceedingly competitive under the chemical and physical conditions under which the product is held. Salt concentration and temperature are main variables that impact the course of natural fermentations. Direct, mild acidification has been utilized to eliminate growth by numerous undesirable bacteria during controlled fermentation of cucumbers͘
Acidification does not prevent development of other acid-tolerant LAB and yeasts, however. Lactobacillus plantarum characteristically distinctively prevails in the later time of vegetable fermentation, evidently in light of its more prominent acid tolerance. It has been found to finish cucumber fermentations regardless of the species of LAB included. Due to its acid tolerance and tendency to terminate fermentations, Lactobacillus plantarum appears a reasonable decision for
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adjustment or potentially alteration for utilize when homolactic fermentation is desired. Bacteriocins have been shown in numerous LAB and may have an incentive in accomplishing a pure culture fermentation if the starter culture is a producing strain. Etchells et al. (19641 observed in pure culture fermentation of pasteurized cucumbers repressed the development of the Lactobacillus. In later studies, Fleming et al. (1975) demonstrated bacteriocin-like activity in the strain of Pediococcus that Etchells observed to be adversarial to L. planatrum. This inhibitory property might be helpful if controlled by a strain with better fermentation characteristics in order to achieve dominance over the competing natural flora. This approach has been utilized as a part of the improvement of yeast cultures for purpose of wine. A wild strain of yeast containing the 'executioner factor' was mated with starter strains to get hybrids containing desirable fermentation characteristics also the inhibitory executioner factor that is active against contaminating wild yeasts.
Fig. (44). Fermented olive
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Fig. (45). Fermented sauerkraut
How to ferment vegetables: Many steps are employed when preparing a fermented vegetable products, these steps include: 1- Choice of fermentation equipment Fermenting vegetables don't require a lot of specialized equipment. Vegetables can be fermented in a devoted fermenting container, or in a clean glass bowl or glass mason jar. 2- Preparing vegetables for fermenting: Grate: This works well for hard or crunchy vegetables, such as zucchini. Grated fermented vegetables frequently have the texture of a relish once finished.
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Slice: Firm vegetables are sliced thinly and soft vegetables thickly to preserve their shape during fermentation. Chop: The vegetables are chopped to the suitable size Chopped fermented cauliflower and carrot pieces make an easy and healthy snack. Whole: Small vegetables, for example, brussel sprouts and green beans work best if left whole. Pickling cucumbers are likewise awesome. 3-Use salt, whey or a starter culture Salt and water are needed for the fermentation process, with sea salt being the best choice. Numerous formulas call for crisp whey as a ferment starter, but it isn't essential. Utilizing salt will give a similar outcome. A vegetable starter culture can likewise utilized for a faster IHUPHQWDWLRQEXWLWLVQ¶WHVVHQWLDO 4- Use water to prepare the brine Sufficient saline solution (brine) is needed to be able to submerge the vegetables completely. The best fermentation results are acquired with a 2% brine. The simplest way to think about this is in grams. For every 100 grams of vegetables, 2 grams of salt are needed. Filtered water is basic, specifically, water that is free of chlorine, chloramines and fluoride. Chlorine and fluoride won't bolster a healthy ferment as they kill the microbes. Bottled filtered water could be purchased, however a fired water purifier with fluoride filter is awesome without waste alternative. It will likewise give you beautiful filtered drinking water year round. 5-Weigh the vegetables down under the brine Once the vegetables have been prepared, they are placed into the selected fermentation vessel and then weighed under the brine. Keeping them in an anaerobic environment during the fermentation period is important.
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6-Leave the vegetables to ferment at room temperature : The vegetables must be left to ferment at room temperature before moving them to the refrigerator. The fermentation time rely upon various variables, including temperature, the quantity of salt and the nature of the vegetable. After leaving the vegetables to ferment at room temperature for 3 days, taste it. If they are not as acidic might be wanted, they can be left and tasted after an additional 3 days, and so on. Once you are happy with the taste, move them to the refrigerator. The finished product will keep for a considerable length of time in the refrigerator.
Fig. (46). Fermented cabbage
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CHAPTER SIXTEEN FUTURE OF STARTER CULTRES TECHNOLOGY
The preparation of fermented foods originates before the written history of Man. Early people utilized perception of the clear impacts of microbial modification of food characteristics to develop processes for food fermentation. The resultant fermented products regularly have a different texture and flavor contrasted with the unfermented starting materials, consequently making them more satisfactory and edible. Technical advance was at first moderate, as reflected in the long fermentation periods required; it was incremental to the technical know-how and essential scientific data then accessible. It is likely reasonable to say that in the very early days brew-masters were more a greater number of craftsmans than technologists. With the fast advancement in comprehension of the basic sciences of microbiology and biochemistry, combined with the presentation of new equipment, the developed countries have moved forward in enhancing the safety and efficiency of the used to make traditional fermented foods, such as cheese fermentation. With the fast advance in the biological sciences, both fundamental and applied viewpoints, it has been conceivable to gain a better understanding of the mystery that has encompassed fermentation processes. The types of microorganisms involved has been isolated and identified, and the physiology and metabolism of these organisms have been studied. Subsequently, traditional fermented foods would now be able to be improved, faster, and all the more economically. The application of available knowledge to improve traditional food fermentations in developed countries has far outpaced that in developing countries.
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In spite of the fact that change builds the capacity to choose better strains, there can, obviously, be minimal coordinated adjustment of genetic material. The new biotechnology, for example recombinant DNA techniques, conquers this issue. The new biotechnology obviously, be of huge help in producing superstrains of microorganisms that could empower speeding up of fermentation processes, give more proficient use of crude materials, and produce better-quality products. How best can developing nations apply these biotechnologies to traditional fermented foods. In their eagerness to promote the new biotechnology for traditional fermented food applications, researchers from developed countries ought not overlook the changed environments that exist in developed and developing countries. In developed countries the old biotechnology is already well understood and practiced efficiently in fermented food industries. Developing countries may need to get a superior comprehension of the old biotechnology before proficiently retaining and actualizing the new biotechnology to its fullest.
Improvement of starter cultures by using biotechnology Microorganisms, including bacteria, yeasts, and mold, produce an extensive variety of metabolic end products that function as preservatives, texturizers, stabilizers, and seasoning and coloring agents. A few conventional and nontraditional methods have been utilized to enhance metabolic properties of food fermentation microorganisms. These include mutation and selection techniques; the use of natural gene transfer methods, for example, transduction, conjugation and transformation; and, more recently, genetic engineering.
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Mutation and selection: In nature, mutations (changes in the chromosome of an organism) occur happen immediately at low rates (one mutational event in every 106 to 107 cells per generation. These mutations happen indiscriminately all through the chromosome, and an unconstrained mutation in a metabolic pathway of enthusiasm for food fermentations would be a greatly uncommon occasion. The mutation rate can be significantly expanded by presentation of microorganisms to mutagenic agent, for example, ultraviolet light or various chemicals, which initiate changes in the deoxyribonucleic acid (DNA) of host cells. Mutation rates can be increased to one mutational event in every 101 or 102 cells per generation for auxotrophic mutants, and one in 103 to 105 for the isolation of improved secondary metabolite producers. A method of selection is basic for viable screening of mutants as a few thousand individual isolates may need to be assessed to discover one strain with enhanced action in the property of interest. Mutation and selection techniques have been utilized to enhance the metabolic properties of microbial starter cultures utilized for for food fermentations; however, there are serious impediments with this method. Mutagenic agents cause random mutations, therefore specificity and precision are impractical. Possibly malicious undetected mutations can happen, since selection systems might be intended for only the mutation of interest. Moreover, traditional mutation procedures a great degree exorbitant and tedious and there is no chance to extend the gene pool. Regardless of these confinements, mutation and selection techniques have been utilized widely to enhance industrially important microorganisms and, in some cases, yields of greater than 100-times the normal production level of bacterial secondary metabolites have been accomplished. .
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Natural gene transfer : The revelation of natural gene transfer systems in bacteria has enormously encouraged the comprehension of the genetics of microbial starter cultures and at times has been utilized for strain improvement. Genetic exchange in bacteria can happen naturally by three different mechanisms: transduction, conjugation, and transformation.
Transduction: Transduction is a method of transfer of genetic material or characteristics from one bacterial cell to another by a bacteriophage or plasmid. The bacteriophage acquires a part of the chromosome or plasmid from the host strains and transfers it to a recipient during subsequent viral infection. Generally͕ transduction efficiencies are low and gene transfer is not always possible between unrelated strains, limiting the usefulness of the technique for strain improvement. Moreover, bacteriophage have not been isolated and are not well characterized for most strain. Conjugation: Conjugation, or bacterial mating, is a natural gene transfer system that requires close physical contact amongst donors and beneficiaries and is in charge of the spread of plasmids in nature. Various genera of bacteria harbor plasmid DNA. Much of the time, these plasmids are secretive (the functions encoded are not known), but rather now and again important metabolic traits are encoded by plasmid DNA In the event that these plasmids are additionally self-transmissible or on the other hand mobilizable, they can be transferred to beneficiary strains. Once brought into a new strain, the properties encoded by the plasmid can be expressed in the beneficiary. The lactic acid bacteria naturally contain from one to more than ten particular plasmids, and metabolically imperative traits, including lactose-fermenting ability, bacteriophage resistance, and
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bacteriocin production, have been linked to plasmid DNA. Conjugation has been used to transfer these plasmids into beneficiary strains for the construction of genetically improved commercial dairy starter cultures Transformation: Certain microorganisms can take up stripped DNA present in the encompassing medium. This process is called transformation and this gene transfer process is constrained to strains that are naturally competent. Ability subordinate transformation is constrained to a couple, principally pathogenic, genera, and has not been utilized broadly for genetic improvement of microbial starter cultures. For many species of bacteria, the thick peptidoglycan layer present in gram-positive cell walls is considered a potential barrier to DNA uptake. Methods have been developed for enzymatic evacuation of the cell wall to make protoplasts. In the presence of polyethylene glycol, DNA uptake by protoplasts is encouraged. On the off chance that kept up under osmotically balanced out conditions, transformed protoplasts recover cell walls and express the transformed DNA. Protoplast transformation procedures have been developed for some of the lactic acid bacteria; however, the procedures are are dull and tedious, and regularly parameters must be improved for each strain. Transformation efficiencies are regularly low and profoundly variable, restricting the utilization of the technique for strain improvement. Electroporation The above mentioned gene transfer systems have become less popular since the advent of electroporation, a technique involving use of highvoltage electric pulses of short duration to initiate the development of transient pores in cell walls and membranes. Under proper conditions, DNA present in the encompassing medium may enter through the pores. Electroporation is the method for decision for strains that are refractory to other gene transfer techniques; in spite of the fact optimization of
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several parameters (e.g., cell preparation conditions, voltage and duration of the pulse, regeneration conditions, etc.) is still required. Genetic engineering: Genetic engineering gives an alternative technique for enhancing microbial starter cultures. This rapidly extending zone of technology gives strategies for the isolation and transfer of single genes in a precise, controllable, and convenient way. Genes that code for particular desirable traits can be derived from virtually any living organism (plant, animal, microbe, or virus). Genetic engineering is r reforming the science of strain improvement and is destined to have a major impact on the food fermentation industry Although a significant part of the microbial genetic engineering research since the appearance of recombinant DNA technology in the early 1970s has concentrated on the gram-negative bacterium Escherichia coli, critical advance has been made with the lactic acid bacteria and yeast. . Proper hosts have been recognized, multifunctional cloning vectors have been developed, and dependable, higher efficiency gene transfer procedures have been produced. Further, the structural and functional properties, as well as the expression in host strains, of several important genes have been accounted for. Engineered bacteria, yeast, and molds could also be used for the production of other products, including food additives and ingredients, processing aids such as enzymes, and pharmaceuticals.
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