In vitro evaluation of the probiotic attributes of two pediococci strains producing pediocin PA-1 with selective potency as compared to nisin Anita Kumari Garsa, Rashmi Kumariya, Anil Kumar, Puja Lather, Suman Kapila, S. K. Sood & Meena Kapasiya European Food Research and Technology Zeitschrift für LebensmittelUntersuchung und -Forschung A ISSN 1438-2377 Eur Food Res Technol DOI 10.1007/s00217-014-2243-7
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Author's personal copy Eur Food Res Technol DOI 10.1007/s00217-014-2243-7
Original Paper
In vitro evaluation of the probiotic attributes of two pediococci strains producing pediocin PA‑1 with selective potency as compared to nisin Anita Kumari Garsa · Rashmi Kumariya · Anil Kumar · Puja Lather · Suman Kapila · S. K. Sood · Meena Kapasiya
Received: 7 February 2014 / Revised: 21 April 2014 / Accepted: 28 April 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract Eighteen dairy starter cultures, spoilage and food-borne pathogenic strains were analyzed for susceptibility to antimicrobial peptides pediocin PA-1 (PedPA-1) and nisin, through the individual 50 % inhibitory concentrations (IC50) determination. The IC50 of purified PedPA-1 was found to be more potent than nisin against five spoilage and food-borne pathogenic strains, i.e., Bacillus cereus NCDC 240, Enterococcus faecalis NCDC 114, Enterococcus faecium NCDC 124, Streptococcus agalactiae NCDC 208 and Staphylococcus aureus NCDC 110. The IC50 of PedPA-1 and nisin ranged from 6.58 to 0.29 µM and 18.91 to 0.03 µM, respectively. Further, PedPA-1 producing Pediococcus pentosaceus NCDC 273 and Pediococcus acidilactici NCDC 252 strains were evaluated for potential probiotic attributes by in vitro studies. Both pediococci strains were able to survive at low pH and 2 % bile with a good bile salt hydrolase activity, cell surface hydrophobicity and β-galactosidase activity that makes them potentially good candidates for probiotics. These strains with proven promising probiotic attributes are good candidates for further investigation through in vivo studies to elucidate their potential health benefits. Keywords Probiotic · Pediococcus · Pediocin PA-1 · β-Galactosidase · BSH activity · Cell surface hydrophobicity A. K. Garsa (*) · R. Kumariya · P. Lather · S. Kapila · S. K. Sood · M. Kapasiya Division of Animal Biochemistry, National Dairy Research Institute, Karnal 132001, Haryana, India e-mail:
[email protected] A. Kumar Division of Dairy Cattle Physiology, National Dairy Research Institute, Karnal 132001, Haryana, India
Introduction Consumers’ increased reluctance to use the chemically preserved foods favored the use of natural and fresh foods with no chemical preservatives added. In addition, increasing demand for minimally processed foods with long shelf life has stimulated research interest in finding natural and effective preservatives known as biopreservatives. Bacteriocins from lactic acid bacteria (LAB) are a diverse group of antimicrobial proteins/peptides, offering potential as biopreservatives [7]. Nisin A (NisA), is approved by Food and Drug Administration (FDA), used in more than 48 countries as a natural food preservatives [7, 12]; however, pediocins, a group of class IIa bacteriocins produced by Pediococcus strains, have gained great attention in recent years and are extensively studied as well characterized [10, 20, 21]. Pediocin-like bacteriocins (36–48 residues) are far the most investigated [22] and share considerable amino acid sequence similarity when the corresponding sequences are aligned [18, 45]. Pediocin PA-1/AcH (PedPA-1/PedAcH) was shown to have an extra C-terminal disulfide bond which improves its potency at increased temperatures and widens its antimicrobial spectrum [17]. Probiotics are defined as “live microorganisms that when administered in adequate amounts, confer a health benefit on the host” [35]. One of the desirable properties of a probiotic strain is the ability to produce antimicrobial substances such as bacteriocins [29]. In addition, potential probiotic organisms should have desirable properties like resistance to gastric and intestinal juices and adhesion. Organisms also must be resistant to antibiotics, produce β-galactosidase, exhibit cholesterol-reducing and cancer prevention property which has additional health benefits for the host [24]. The ability of bacteriocin-producing LAB to survive in low pH (gastric juice) and in presence of bile
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salts and to remain viable in the gastrointestinal tract prove them a good probiotic strains, as these characteristics allow them to carry out antimicrobiosis and immunomodulation in host which are the important criteria in defining probiotic strains [27]. Once colonized, the probiotics get an opportunity to exhibit their antimicrobial and immunomodulatory properties in favor of the host organism [39]. The antimicrobial activity exhibited by the pediococci strains may be due to the production of bacteriocins like pediocin and other metabolites such as lactic acid, H2O2 and organic acids. Comparisons of data obtained in culture media with those obtained in food systems such as meat reveal that the efficacy of bacteriocins is often much lower in the latter [40]. Sometimes, at least tenfold higher bacteriocin concentrations must be added to foods in order to achieve an equivalent inhibitory effect. Variations in strain sensitivities for pediocin and nisin show that pediocin can be added to suppress the growth of the undesirable bacteria without affecting the growth of desirable bacteria at the added concentration. Recently, a commercial preparation of PedPA-1 by P. acidilactici known as Alta™ 2341 was formulated. This formulation is known to extend shelf life of a variety of foods, particularly by inhibiting the growth of Listeria monocytogenes in ready-to-eat products. This indicates that pediocin-like bacteriocins are next or even best in line after successful use of nisin for preservation of dairy foods from contamination by undesirable microorganisms. In previous research, we demonstrated that pediocin 273 produced by Pediococcus pentosaceus NCDC 273 (GenBank accession no. FJ825757.1) and pediocin 252 produced by Pediococcus acidilactici NCDC 252 (GenBank accession no. FJ827481.1) are identical to PedPA-1 at nucleotide sequence as well as protein level [45, 52]. In the present study, we purified pediocin 273 [52] to determine the IC50 against dairy starter cultures, spoilage and food-borne pathogenic strains and compared with nisin (supplied by Sigma-Aldrich). Further, for use as potential probiotics, PedPA-1 producing P. pentosaceus NCDC 273 and P. acidilactici NCDC 252 strains were evaluated for potential probiotic attributes in terms of acid and bile tolerance, cell surface hydrophobicity, bile salt hydrolase activity, β-galactosidase activity and antibacterial activity by in vitro studies.
Materials and methods Bacterial cultures, media and growth conditions Bacterial cultures used as indicator were procured from National Collection of Dairy Cultures (NCDC, NDRI, Karnal, India) in freeze-dried form (Table 1). Experiments
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Eur Food Res Technol Table 1 Bacterial cultures used in the study Bacterial cultures
Culture conditions
Pediococcus pentosaceus NCDC 273
MRS broth, 37 °C
Pediococcus acidilactici NCDC 252
MRS broth, 37 °C
Pediococcus acidilactici Lb42
MRS broth, 37 °C
Lactobacillus bulgaricus NCDC 09
MRS broth, 37 °C
Lactobacillus acidophilus NCDC 195
MRS broth, 37 °C
Lactobacillus brevis NCDC 01
MRS broth, 30 °C
Lactobacillus helveticus NCDC 05
MRS broth, 37 °C
Lactobacillus casei NCDC 17
MRS broth, 37 °C
Lactobacillus plantarum NCDC 21
MRS broth, 37 °C
Lactobacillus rhamnosus NCDC 19
MRS broth, 37 °C
Lactobacillus delbrueckii NCDC 10
MRS broth, 37 °C
Lactobacillus acidophilus NCDC 11
MRS broth, 37 °C
Lactobacillus rhamnosus GG
MRS broth, 37 °C
Lactococcus lactis NCDC 60
M17 broth, 30 °C
Enterococcus faecium NCDC 124
BHI broth, 37 °C
Enterococcus faecalis NCDC 114
NB, 37 °C
Streptococcus thermophilus NCDC 75
M17 broth, 37 °C
Streptococcus agalactiae NCDC 208
M17 broth, 37 °C
Bacillus cereus NCDC 240
NB, 30 °C
Staphylococcus aureus NCDC 110
NB, 37 °C
Micrococcus luteus NCDC 131
NB, 37 °C
Escherichia coli NCDC 178
BHI broth, 37 °C
were conducted with P. pentosaceus NCDC 273 and P. acidilactici NCDC 252 previously characterized as the pediocin producer strains [45]. MRS (deMan Rogasa Sharpe) media, NB (nutrient broth), BHI (brain–heart infusion) broth, agar and other reagents were purchased from HiMedia. Mueller–Hinton broth No. 2 and all the antibiotics used for broth microdilution test were purchased from HiMedia and Sigma-Aldrich, respectively. All the fine chemicals used in the experiments were of analytical grade. For shortterm preservation, streaks of the culture were made on agar slants, incubated at 37 °C for 12 h and stored at 2–8 °C. For long-term preservation of cultures, the glycerol stocks (30 %, v/v) were prepared of both the producers as well as the indicator cultures and stored at −20 °C in a deep freezer. Antibacterial activity The antibacterial activity of pediococci was determined by the agar spot test as described by Schillinger and Lucke [42] with minor modifications. Briefly, 5 µL each of selected pediococci strains was spotted on the surface of an MRS agar plate and incubated at 37 °C for 24 h. A 100µL sample of each of the test strains grown in BHI broth/ NB at 37 °C for 12 h was vigorously mixed with 10 mL of BHI/NB soft agar (0.6 % agar, w/v) and poured over the
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MRS agar plates containing developed colonies of pediococci. The plates were then incubated at 37 °C for 24 h, and the zones of inhibition were measured. The test strains used were Staphylococcus aureus NCDC 110, Escherichia coli NCDC 178, Enterococcus faecalis NCDC 114, Enterococcus faecium NCDC 124 and Bacillus cereus NCDC 240. IC50 determination PedPA-1 and nisin are the two bacteriocins used for IC50 determination. Nisin was procured from Sigma-Aldrich, and PedPA-1 was purified using the protocol described by Vijay Simha et al. [52]. The purified fraction was found to contain 57.5 µg/mL PedPA-1, quantified using RPHPLC peak area compared peak area of standard PedPA-1 (Sigma-Aldrich). The PedPA-1 preparations were pooled together and concentrated using Ultracel®-3 kDa cutoff ultrafiltration membrane (Millipore Ireland Ltd) to the concentration of 4 mg/mL. IC50 was determined using broth inhibition assay described by Cabo et al. [4]. Briefly, 190 µL of sterile medium (inoculated to 0.5 % with test cultures listed in Table 3) was taken in sterile Eppendorf tubes. To the first Eppendorf, 10 µL of antimicrobial peptides, i.e., PedPA-1 (4 mg/mL) or nisin (4 mg/mL) was added. To the rest of Eppendorf tubes, 10 µL of antimicrobial peptides was added after serial double dilutions. This gave a final concentration of 40, 20, 10, 5, 2.5, 1.25, 0.625, 0.3125, 0.156 and 0.078 µg/mL of antimicrobial peptides. A positive control was setup with broth and test cultures and a negative control with broth alone. All the tubes were mixed well and incubated at 37 °C for 6 h. Absorbance of the cultures at different concentrations of antimicrobial peptides was taken at 600 nm with negative control as blank using spectrophotometer (Labomed, Inc.). From absorbance data, dose response of inhibition was calculated using the formula:
% I = 1 − (Ac /Ao ) × 100 where Ac is the absorbance of the culture at different concentrations of antimicrobial peptides, i.e., PedPA-1 and nisin, and Ao is the absorbance of the positive control. The IC50 was then determined as the concentration of antimicrobial peptides resulting in 50 % inhibition from the log10 value of the antimicrobial peptides concentration versus percent inhibition. Acid resistance Tolerance of pediococci to acidic conditions was tested as previously described by Clark et al. [6] with some modifications. Briefly, P. pentosaceus NCDC 273 and P. acidilactici NCDC 252 were grown in MRS broth at 37 °C for 12 h. The actively grown cells (8 log10 cfu/mL) were harvested
by centrifugation (7,200g at 4 °C for 10 min) and re-suspended in equal volume of MRS broth. The pH of MRS broth was adjusted to 3.5, 2.5, and 2.0 with 1 M HCl and with pH 6.5 as control. Survival was evaluated by determining the viable counts of the samples serially diluted in saline (0.9 % NaCl) after 2 h and 4 h of incubation at 37 °C in acidic MRS broth. Bile tolerance The ability of pediococci strains to grow in the presence of bile salts was determined in MRS broth, as described by Gilliland et al. [19]. Briefly, P. pentosaceus NCDC 273 and P. acidilactici NCDC 252 were grown in MRS broth at 37 °C for 12 h. The actively grown cells (8 log10 cfu/ mL) were harvested by centrifugation (7,200g at 4 °C for 10 min) and re-suspended in equal volume of MRS broth supplemented with 0.5, 1, 1.5 and 2 % w/v ox bile (HiMedia) and without supplement as a control was inoculated with actively growing bacteria. Survival was evaluated by plate count on MRS agar, after 2, 4 and 6 h of incubation at 37 °C in MRS broth containing bile salts. Cell surface hydrophobicity Ability of the organism to adhere to hydrocarbons is a measure of their adherence to the epithelial cells in the gut, i.e., cell surface hydrophobicity. Cell surface hydrophobicity of pediococci strains was determined according to the method described by Rosenberg et al. [37] with slight modification using n-hexadecane, n-octane and xylene. Cultures of the pediococci strains were grown in MRS broth at 37 °C for 12 h. Active culture was harvested by centrifugation at 12,000g for 5 min at 5 °C, washed twice and re-suspended in 2 mL of phosphate urea magnesium (PUM) buffer (pH 6.5). The initial absorbance (ODinitial) of the cell suspension was adjusted to approximately 0.8–1.0 at 610 nm. To 2 mL of bacterial suspension, 1 mL of n-hexadecane or n-octane or xylene was added slowly. The suspension was preincubated at 37 °C for 10 min followed by vortexing for 2 min. The hydrocarbon layer was allowed to rise completely. After 1 h and 2 h, aqueous phase was removed carefully with a Pasteur pipette, and the final absorbance (ODfinal) was measured at 610 nm using spectrophotometer (Labomed, Inc.). The decrease in the absorbance was taken as a measure of the cell surface hydrophobicity (% Hydrophobicity) calculated using the following formula:
% Hydrophobicity =
ODinitial − ODfinal × 100 ODinitial
where ODinitial and ODfinal are the absorbance at 610 nm before and after extraction, respectively, with the three hydrocarbons.
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Antibiotic sensitivity
Eur Food Res Technol
was stopped by the addition of 0.5 mL 10 % sodium carbonate, and the absorbance was read at 420 nm. One unit was defined as the quantity of enzyme that would liberate 1 mM of o-nitrophenol (ONP) from ONPG per minute under the assay conditions. Units were calculated using the following equation:
Pattern of resistance/susceptibility to antibiotic of pediococci strains was determined by broth microdilution test as recommended by CLSI [8]. MICs of 10 antimicrobial agents were determined in the concentration ranges (mg/L) given in parentheses: ampicillin (0.1–64), amoxycillin (0.1–64), bacitracin (0.1–512), chloramphenicol (0.1–64), gentamycin (0.1–512), penicillin G sodium salt (0.1–64), polymyxin B (0.1–512), streptomycin sulfate (0.1–512), tetracycline (0.1–64) and vancomycin (0.1–4,096). MICs were read as the lowest concentration of an antimicrobial agent at which visible growth was inhibited.
Unit mL−1 = A × dilution factor/(ε × time × enzyme solution)
Bile salt deconjugation
Statistical analysis
The ability of the strains to deconjugate bile salts was determined as follows. Bile salt plates were prepared by adding 0.5 % (w/v) of sodium salts (Sigma-Aldrich) of sodium taurocholic acid (TC), sodium taurodeoxycholate (TDC), sodium tauroglycocholate (TGC) and sodium glycocholate (GC) to MRS agar. The strains were streaked on the media, and the plates were anaerobically incubated (GasPak TM 100 System, BBL Systems, Cockeysville, Maryland, USA) at 37 °C for 72 h. Lactobacillus acidophilus NCDC 11 and P. acidilactici Lb 42 procured from National Culture Collection Centre (NCDC, NDRI, Karnal, Haryana, India) were used as a positive and negative control. The presence of the precipitated bile acids around colonies (white opaque halo) was considered as a positive result.
Calculations of IC50 and the mean ± standard deviations of different parameters, viz., acid tolerance, bile tolerance and percent hydrophobicity, studied were done using GRAPHPAD PRISM 3.0 (GRAPHPAD software, San Diego, California, USA).
Determination of β‑galactosidase activity
Among other probiotic properties, P. pentosaceus NCDC 273 and P. acidilactici NCDC 252 exhibited antibacterial activity against S. aureus NCDC 110, E. coli NCDC 178, E. faecalis NCDC 114, E. faecium NCDC 124 and B. cereus NCDC 240 as shown in Table 2. P. pentosaceus NCDC 273 had strong inhibitory activity against most of the food spoilage/pathogenic bacteria. Eighteen dairy starter cultures, spoilage and food-borne pathogenic strains collected from NCDC were analyzed for susceptibility to the antimicrobial peptides, PedPA-1 and nisin. The individual 50 % inhibitory concentrations (IC50) were determined and expressed in µM. The IC50 of PedPA-1 and nisin ranged from 6.58 to 0.27 µM and 18.91 to 0.04 µM, respectively (Table 3). The purified pediocin was found more potent against most of food spoilage and pathogenic bacteria, viz., S. aureus NCDC 110, E. faecalis NCDC 114, B. cereus NCDC 240, E. faecium NCDC 124, M. luteus NCDC 131 and St. agalactiae NCDC 208 as compared to nisin, and most of the starter cultures was found to be less susceptible to PedPA-1, while only Lb. helveticus NCDC 05 and Lb. plantarum NCDC 21 were found to be susceptible for PedPA-1. Nisin was found to be more
β-Galactosidase activity was determined using the method described by Tari et al. [46]. Pediocin producer cultures were inoculated at 1 % (v/v) level into the 10 mL of the cheese whey medium and incubated at 37 °C for 24 h. The cells were harvested by centrifugation at 3,000g at 4 °C for 15 min. Cells were washed with 10 mL of 0.05 M potassium phosphate buffer (pH 7.0). Cells obtained were re-suspended in 4.5 mL of the same buffer, followed by vigorous mixing. Afterward, 100 mg of lysozyme (Sigma-Aldrich) was added and incubated at 37 °C for 15 min. After this, 0.5 mL of 4 M NaCl solution was added and incubated further at 37 °C for another 50 min followed by centrifugation at 3,000g for 15 min. Following centrifugation, the supernatant was used for the enzyme assay. For enzyme assay, o-nitrophenyl-β-d-galactopyranoside (ONPG) (8.3 × 10−3 M) dissolved in sodium phosphate buffer (50 mM, pH 7.0) was used as substrate. The amount of substrate and enzymes used were 2 and 0.5 mL, respectively. At time zero, 0.5 mL of enzyme solution was added to the ONPG solution and incubated for 15 min. The assay
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where A was the absorbance at 420 nm, dilution factor was the fold dilution of the enzyme solution, enzyme solution was the amount of enzyme (mL) undergoing the reaction, ε was the extinction coefficient (determined from the ONP standard curve), and time was the incubation time (15 min).
Results and discussion PedPA-1 producing P. pentosaceus NCDC 273 and P. acidilactici NCDC 252 strains were evaluated for potential probiotic attributes by in vitro studies. Antibacterial efficacy of PedPA‑1 and nisin
Author's personal copy Eur Food Res Technol Table 2 Spectrum of antimicrobial activity exhibited by P. pentosaceus NCDC 273 and P. acidilactici NCDC 252 Antimicrobial activitya
Indicator strains
Staphylococcus aureus NCDC 110 Escherichia coli NCDC 178
P. pentosaceus NCDC 273
P. acidilactici NCDC 252
+++
+++
+++
++
++
Enterococcus faecalis NCDC 114 Enterococcus faecium NCDC 124
+
+++
Bacillus cereus NCDC 240
++
+++
+++
a
The inhibition zones 1, 2, 2–5 mm and more than 5 mm were classified as strains of no (±), mild (+), strong (++) and very strong (+++) inhibition, respectively
Table 3 Efficacy of pediocin and nisin against dairy starter cultures and spoilage and food-borne pathogenic strains Dairy starter cultures and spoilage and food-borne pathogenic strains
IC50 (µM) PedPA-1 Nisin
Dairy starter cultures Lb. bulgaricus NCDC 09
1.40
2.35
Lb. acidophilus NCDC 195
5.01
0.04
Lb. brevis NCDC 01
3.11
1.54
Lb. helveticus NCDC 05
0.27
1.35
Lb. casei NCDC 17
2.42
1.43
Lb. plantarum NCDC 21
0.39
0.08
Lb. rhamnosus NCDC 19
2.04
0.22
Lb. delbrueckii NCDC 10
5.33
1.12
Lb. rhamnosus GG
2.12
0.16
L. lactis NCDC 60
6.58
4.97
1.56
2.35
E. faecium NCDC 124
0.89
2.80
E. faecalis NCDC 114
1.01
1.25
St. agalactiae NCDC 208
0.29
8.81
B. cereus NCDC 240
1.09
18.91
S. aureus NCDC 110
1.32
10.16
M. luteus NCDC 131
1.03
12.85
E. coli NCDC 178
1.64
2.53
St. thermophilus NCDC 75 Spoilage and food-borne pathogenic strains
effective against most of the dairy starter cultures as compared to the spoilage and pathogenic bacteria (Table 3). For comparison of the potencies of the different antimicrobial peptides, quantitative minimum inhibitory concentrations (MICs) or IC50 are needed [5, 13]. Therefore, the antibacterial efficacy of PedPA-1 was evaluated against food spoilage and pathogenic as well as dairy starter cultures by their IC50 determination. The pediocin-like bacteriocins have attracted most attention since they kill a broad spectrum of gram-positive bacteria including important pathogens. However, only few bacteriocins have been found to possess activities against gram-negative bacteria, e.g., plantaricin 35 days produced by Lb. plantarum [30],
bacteriocin ST34BR produced by L. lactis subsp. lactis [48], bacteriocins ST26MS and ST28MS produced by Lb. plantarum [49], bacteriocin AMA-K produced by Lb. plantarum AMA-K [50] and BacTN635 produced by Lb. plantarum sp. TN635 strain [38]. In our finding, PedPA-1 was also found to be effective against E. coli NCDC 178, which is a gram-negative bacteria. In addition, use of natural antimicrobial biopreservatives such as pediocins has been pursued to reduce the use of chemical preservatives in food. Our results of IC50 determination (Table 3) indicated that PedPA-1 can be used as biopreservative to control both food spoilage such as B. cereus and pathogenic bacteria such as S. aureus and E. coli in probiotic-based functional foods, viz., yoghurt, chesses, acidophilus milk, bulgaricus milk and Yakult™. As pediocin does not pose any harm to most of the tested dairy starter cultures when used at the concentration required for the inhibition of the food spoilage and pathogenic bacteria, it can be used efficiently for the preservation of milk and milk-based products at the concentration of greater than 1.64 µM to suppress the growth of the undesirable bacteria without affecting the growth of desirable bacteria. Therefore, in this context, pediocin is effective and potent antimicrobial peptide (AMP) for use as biopreservative in the dairy food products against the selected target bacteria. Efficacy of PedPA-1 to increase the shelf life of raw milk is being validated presently in our laboratory to explore the potential of PedPA-1 as raw milk biopreservative. Effect of low pH and bile on viability The effect of different pH on the viability of the two pediococci strains is presented in Table 4. P. pentosaceus NCDC 273 showed lower viability in MRS broth at pH 2.0 than at pH 2.5 and pH 3.5 after 2 h. Viability further reduced at 4 h in case of pH 2.0 and pH 2.5. While P. acidilactici NCDC 252 showed lower viability at pH 2.0 and pH 2.5 than at pH 3.5. There was a progressive reduction in viability at pH 2.0 in P. pentosaceus NCDC 273. In the present study, survival up to a log count of 7.4 at pH as low
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Eur Food Res Technol Table 6 Percent hydrophobicity of P. pentosaceus NCDC 273 and P. acidilactici NCDC 252
as 2.0 indicated acid tolerance capability of both strains of pediococci (Table 4). In the present study, at 0.5, 1, 1.5 and 2 % bile salt concentration, both strains showed no log reduction even after 2 h (Table 5). The results indicated that at concentration 0.5 and 1 %, bile salts did not affect the growth of P. pentosaceus NCDC 273 up to 6 h. In case of P. acidilactici NCDC 252, growth remained unaffected even at 2 % bile salts concentration up to 6 h. Our results indicate that both P. pentosaceus NCDC 273 and P. acidilactici NCDC 252 can survive bile conditions pertaining in the small intestine on account of their bile resistance ability. Most of the organisms are known to exhibit the trait of resistance at acidic
n-Hexadecane 14.13 ± 1.05 Xylene 13.02 ± 1.00
17.71 ± 0.80
8.27 ± 0.21
15.96 ± 0.45
Table 4 Acid tolerance of P. pentosaceus NCDC 273 and P. acidilactici NCDC 252 expressed in log (cfu/ml)
Cell surface hydrophobicity
Log (cfu/mL)a
pH
0 hb
2 hb
4 hb
P. pentosaceus NCDC 273 2.0 8.46 ± 0.08 2.5 8.13 ± 0.04 3.5 8.48 ± 0.31 6.5 7.93 ± 0.47
7.53 ± 7.53 7.86 ± 0.14 8.42 ± 0.59 9.23 ± 0.47
6.89 ± 0.89 6.50 ± 0.89 8.67 ± 1.26 9.62 ± 0.20
P. acidilactici NCDC 252 2.0 8.42 ± 0.33 2.5 8.74 ± 0.48 3.5 8.56 ± 0.11
7.13 ± 0.25 7.02 ± 0.28 8.67 ± 0.16
7.31 ± 0.21 7.19 ± 0.34 8.84 ± 0.27
9.03 ± 0.23
10.36 ± 0.61
6.5
8.78 ± 0.28
a
Mean ± SD, n = 3
b
Incubation in hours
Table 5 Bile tolerance of P. pentosaceus NCDC 273 and P. acidilactici NCDC 252 expressed in log (cfu/mL) Bile (%)
Log (cfu/mL)a 0 hb
2 hb
4 hb
6 hb
P. pentosaceus NCDC 273 0 7.93 ± 0.47 9.23 ± 0.47 0.5 8.17 ± 0.13 8.04 ± 0.00 1.0 7.92 ± 0.09 8.06 ± 0.08 1.5 8.00 ± 0.00 7.90 ± 0.00 2.0 8.06 ± 0.08 8.15 ± 0.21
9.62 ± 0.20 7.80 ± 0.28 8.13 ± 0.25 7.90 ± 0.00 8.00 ± 0.00
10.12 ± 0.08 8.28 ± 0.28 8.23 ± 0.11 7.00 ± 0.00 8.00 ± 0.00
P. acidilactici NCDC 252 0 8.78 ± 0.28 9.03 ± 0.23 0.5 8.51 ± 0.30 8.62 ± 0.40 1.0 8.36 ± 0.08 8.58 ± 0.18 1.5 8.36 ± 0.00 8.58 ± 0.18
10.36 ± 0.61 9.04 ± 0.21 8.13 ± 0.25 8.88 ± 0.45
11.24 ± 0.77 9.40 ± 0.51 8.23 ± 0.11 8.94 ± 0.25
8.72 ± 0.26
8.40 ± 0.28
2.0
8.40 ± 0.04
a
Mean ± SD, n = 3
b
Incubation in hours
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8.61 ± 0.01
Hydrocarbons % Hydrophobicitya P. pentosaceus NCDC 273 P. acidilactici NCDC 252
n-Octane
8.87 ± 1.94
a
Mean ± SD, n = 3
and alkaline conditions [8, 9, 16, 23, 28, 31, 51]. Thus, these strains are potentially poised to show better survival through gastrointestinal tract.
Microbial adhesion to hydrocarbons, also known as MATH test, is used to determine microbial cell surface hydrophobicity (CSH). CSH is considered as measure of adhesiveness and colonizing property of bacteria to the intestinal lumen [34, 35, 38]. It has been reported to vary from 2 to 95 % for different probiotic bacteria [34, 36, 41]. The strains under study were evaluated for their CSH toward different hydrocarbons, i.e., n-hexadecane, n-octane and xylene, which may reflect the colonization potential of the organism to intestinal lumen. Both strains showed variable degree of hydrophobicity toward them. As evident from the percent hydrophobicity values shown in Table 6, P. pentosaceus NCDC 273 has relatively more affinity for n-hexadecane (14.13 ± 1.05 %) followed by xylene (13.02 ± 1.00 %) while it was much less in case of n-octane (8.27 ± 0.21 %). On the other hand, P. acidilactici NCDC 252 has relatively more affinity for xylene (17.71 ± 0.80 %) followed by n-octane (15.96 ± 0.45 %) while it was much less in case of n-hexadecane (8.87 ± 1.94 %). Adhesion to host gut epithelial cells and intestinal mucus is an important property of a probiotic strain for temporary colonization of the GI tract and stimulation of beneficial effects. The cultures exhibiting higher cell surface hydrophobicity could be better performers in terms of adherence to the intestinal epithelial cells, thus enhancing their useful property in competitive exclusion of pathogens [33]. Bacterial cells exhibiting higher hydrophobicity are expected to form strong interaction with mucus or epithelial cells [43, 44]. However, other studies showed that hydrophobicity only assists in adhesion but is not mandatory to exhibit strong adherence [47, 51]. Antibiotic susceptibility It is very important to know the antibiotic resistance pattern in pediococci strains before their deliberate use in food
Author's personal copy Eur Food Res Technol Table 7 MICs of antibiotics for P. pentosaceus NCDC 273 and P. acidilactici NCDC 252 Antibiotics
EFSA breakpoints (mg/L)a
MIC (mg/L) P. pentosaceus NCDC 273
P. acidilactici NCDC 252
Ampicillin Amoxycillin Bacitracin Chloramphenicol
2 4 256 1
2 4 256 1
4 – – 4
Gentamycin Penicillin G sodium salt Polymyxin B Streptomycin sulfate Tetracycline
128 0.5 256 512 8
64 0.5 256 256 16
16 1 – 64 8
Vancomycin
1,024
1,024
nr
–, not specified; nr, not required a
Pediococcus strains with MICs higher than EFSA breakpoints are considered as resistants (European Commission 2005)
Table 8 Bile salt hydrolase activity of P. pentosaceus NCDC 273 and P. acidilactici NCDC 252 Bile salt deconjugation Bile salts
P. pentosaceus NCDC 273
P. acidilactici NCDC 252
Sodium deoxycholate (SD) Sodium taurocholate (ST)
– ++
– ++
Sodium tauroglycocholate (STG)
++
++
–, no precipitation; +, slight precipitation; ++, intense precipitation
chain as probiotics. According to Feed Additives and Products (FEEDAP) panel of the European Food Safety Authority (EFSA), bacterial strains carrying an acquired resistance to antibiotics of human and veterinary importance should not be used as food additives, unless it can be demonstrated that it is a result of chromosomal mutation [14]. The results of antibiotic susceptibility of strains investigated using broth microdilution test for a number of clinically important antibiotics according to EFSA [14] are shown in Table 7. Both P. pentosaceus NCDC 273 and P. acidilactici NCDC 252 have comparable susceptibility to ampicillin, amoxicillin, bacitracin, chloramphenicol, penicillin and polymyxin B, while P. pentosaceus was found to be more susceptible to tetracycline than P. acidilactici, which in turn more susceptible to gentamycin and streptomycin sulfate than P. pentosaceus. Both strains were found to be highly resistant for vancomycin (MIC = 1,024 mg/L), and this intrinsic vancomycin resistance in P. pentosaceus is due to a modified peptidoglycan precursor ending in D-Ala– D-lactate [2]. The same mechanism of resistance is likely to cause the resistance among the other Pediococcus species [11]. All the strains of P. pentosaceus isolated from idly batter have been found to be sensitive to tetracycline,
chloramphenicol and erythromycin [51]. Moreover, Klare et al. [25] showed that there is no acquired resistance in P. pentosaceus and P. acidilactici to important antimicrobials, viz., streptomycin, vancomycin, erythromycin, clindamycin, oxytetracycline and fusidic acid, which aids to their probiotic attributes. Bile salt deconjugation Bile salt hydrolase (BSH) is one among the recommended probiotic properties (FAO/WHO) [15]. P. pentosaceus NCDC 273 and P. acidilactici NCDC 252 were evaluated for their bile salt deconjugation ability. BSH activity deconjugates bile salts, which are readily excreted from GI tract and rather can be important for the bacteria to grow in and colonize the intestine. Both the strains were tolerant to sodium taurocholate and sodium tauroglycocholate and were able to deconjugate them while in case of sodium deoxycholate completely both showed a complete sensitivity, i.e., got inhibited, and hence, no growth was observed in presence of these salts (Table 8). BSH-positive organisms are preferred to BSH-negative organisms for the use of probiotic as they are essential for
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cholesterol removal [26, 32]. In this study, both P. pentosaceus NCDC 273 and P. acidilactici NCDC 252 were positive. BSH enzyme protects the organism from the toxic effects of bile acid. The conjugated bile acid enters the cell, gets protonated and gets deconjugated; thus, the process is more effective in BSH-positive organisms [3]. However, microbial BSH is still a debatable issue as it also exhibits some deleterious effects in host such as malabsorption of lipids in intestine [1, 51]. β‑Galactosidase activity Both P. pentosaceus NCDC 273 and P. acidilactici NCDC 252 were found to express β-galactosidase activity using ONPG as substrate at 0.086 ± 0.001 U/mL and 0.120 ± 0.001 U/mL, respectively. In previous research, we found that P. pentosaceous NCDC 273 was able to grow in MRS medium containing lactose in place of glucose [52]. Cheese whey being rich in lactose (approximately 60 %), soluble proteins (10–12 %) and ash content (nearly 5 %) with high biological oxygen demand (BOD) may serve as the basis of food-grade inexpensive fermentation medium, if the producer strains are capable of hydrolyzing lactose by producing β-galactosidase. PedPA-1 production by P. pentosaceus NCDC 273 and P. acidilactici NCDC 252 in cheese whey medium is being optimized currently in our laboratory. Deficiency of β-galactosidase is the cause of lactose intolerance but the probiotic organisms utilize lactose and convert it into short-chain fatty acids (SCFA) which is beneficial for the host [51].
Conclusion The purified PedPA-1 was found more potent against five spoilage and food-borne pathogenic bacteria as compared to nisin, indicating that PedPA-1 can be used as food additives or drugs to extend shelf life of food products. For their use as potential probiotic, pediocin producing pediococci strains were evaluated for their capacity to tolerate upper gastrointestinal tract conditions by in vitro studies. Both pediococci strains were found to possess desirable probiotic properties in terms of acid, bile tolerance, cell surface hydrophobicity, bile salt hydrolase activity and β-galactosidase activity. Therefore, these strains may be regarded as potentially probiotics with proven promising probiotic attributes and can be further investigated through in vivo studies to elucidate their potential health benefits and also could be used as potential probiotic starter cultures. Further to explore PedPA-1 as potent raw milk biopreservative, efficacy of PedPA-1 is being checked in our laboratory to increase the shelf life of raw milk.
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Eur Food Res Technol Acknowledgments The research work presented in this paper was partially supported by National Initiative on Climate Resilient Agriculture (NICRA-ICAR, Grant No. 2049/3033) project. We are grateful to the Director, National Dairy Research Institute (NDRI), Karnal, for providing the necessary facilities for this work. Conflict of interest None. Compliance with Ethics Requirements This article does not contain any studies with human or animal subjects.
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