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Determining probiotic potential of exopolysaccharide producing lactic acid bacteria isolated from vegetables and traditional Indian fermented food products
Food Bioscience 5 (2014) 27 –33
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/fbio
Determining probiotic potential of exopolysaccharide producing lactic acid bacteria isolated from vegetables and traditional Indian fermented food products Ami Patela,b,n, J.B. Prajapatia, Olle Holstb, Asa Ljunghc a
Department of Dairy Microbiology, SMC College of Dairy Science, Anand Agricultural University, Anand, Gujarat, India b Department of Biotechnology, Lund University, Lund, Sweden c Department of Medical Microbiology, Lund University, Lund, Sweden
art i cle i nfo
ab st rac t
Article history:
Exopolysaccharide producing lactic acid bacterial (LAB) were tested in vitro to select a
Received 21 February 2013
candidate probiotic strain by testing their tolerance to low pH and bile, bile salt hydrolase
Received in revised form
(BSH) activity, antibiotics susceptibility pattern and antimicrobial activity. Results indi-
30 September 2013
cated that LAB isolates were highly sensitive against oxbile (0.3%) but could grow in the
Accepted 12 October 2013
presence of sodium taurocholate (0.3%). Seven out of 9 isolates were found to be BSH positive. Two of the isolates, Lactobacillus plantarum 86 and Weissella cibaria 92 showed
Keywords:
considerable antimicrobial activity against Gram-positive and Gram-negative pathogens.
Exopolysaccharides
The study reveals that traditional fermented products of India could be an alternative and
BSH activity
readily available resource for LAB starter cultures with interesting functional character-
Weissella
istics.
Dhokla
& 2013 Elsevier Ltd. All rights reserved.
Idli batter Antimicrobial activity
1.
Introduction
Lactic acid bacteria (LAB) are associated with fermented foods where they flair to produce technologically important substances such as exopolysaccharides (EPS). EPS producing LAB are especially relevant in yoghurt, cheese, sour cream and other cultured dairy products by conferring beneficial rheological and functional properties as natural thickening agents, giving the product a suitable viscosity and reducing syneresis (Ruas-Madiedo & de los Reyes-Gavilán, 2005; De Vuyst & Degeest, 1999). EPS have been found to enhance
gastrointestinal (GI) colonization of probiotic bacteria in the GI tract and thus, also play a significant role as prebiotics (Welman & Maddox, 2003). Many of the LAB strains considered as probiotic are found to be effective against diverse GI exertions either by adhering to the gut mucosa, combating pathogens by virtue of producing antimicrobial compounds and/or exerting beneficial effects on human health (Servin, 2004). Before using bacterial strains as probiotics, they should be screened for certain imperative characteristics such as resistance to bile and low pH, antibiotic susceptibilities and antimicrobial activity
n Corresponding author at: Department of Dairy Microbiology, SMC College of Dairy Science, Anand Agricultural University, Anand, Gujarat, India. Tel.: þ91 2692 640170. E-mail address: [email protected] (A. Patel).
2212-4292/$ - see front matter & 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fbio.2013.10.002
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Food Bioscience 5 (2014) 27 –33
(Lee & Salminen, 2009; Kruszewska et al., 2002). Several probiotics bacteria are found to produce bile salt hydrolase (BSH) that helps to reduce serum cholesterol (Reynier et al., 1981) and hence BSH activity is also considered as an additional criterion for the selection of probiotics. Traditional fermented products could be an alternative and readily available resource for LAB starter cultures with interesting functional characteristics and improved technological and probiotic properties. In this context, based on EPS producing trait, total 17 LAB strains were isolated from different vegetables such as carrot, cabbage, turmeric, cucumber and tomato and traditional fermented foods including dhokla batter, idli batter, dahi, and cabbage (Patel, Shah, & Prajapati, 2012). The present study is aiming at determining potential probiotic characteristics of these EPS producing LAB isolates that were, tested in vitro for tolerance to low pH and bile, bile salt hydrolase (BSH) activity, antibiotics susceptibility and antimicrobial activity.
2.
Materials and methods
2.1. Bacterial strains—isolation, strain identification and growth conditions All 17 EPS producing isolates were characterized biochemically and genotypically using 16S rDNA sequencing. The GenBank and EMBL accession numbers for reference 16S rRNA gene sequences are represented in Table 1. All 17 LAB isolates that were preserved at 20 1C on De Man, Rogosa and Sharpe (MRS, MERCK) broth containing 10% glycerol (v/v) and in freeze-dried form and were propagated twice prior to begin any experiment. The incubation temperature was
maintained at 37 1C for lactobacillus spp. and Weissella isolates and 30 1C for Pediococcus spp. during the experiments whenever their normal growth was required. All isolates are available at the host institution and processed for submission in Microbial type culture collection-MTCC, Chandigarh, India. To perform antimicrobial assay, the clinical isolates of Escherichia coli ESBL and methicillin-resistant Staphylococcus aureus (MRSA) were obtained from the department of Medical Microbiology, Lund University, Lund (Sweden). They were propagated in brain heart infusion (BHI, Oxoid) broth and maintained as frozen stocks at 20 1C in BHI broth containing 10% (v/v) glycerol. Before use, frozen cultures were grown on blood agar plate followed by two successive transfers into BHI broth.
2.2.
Tolerance to low pH
Tolerance to low pH was determined using the method of Chou and Weimer (1999) with some modifications. The active strains grown in MRS broth were inoculated (1.5%) in 10 ml of fresh MRS broth adjusted to pH 3.0 with hydrochloric acid (1.0 M) for 2.5 h at 37 1C. Samples were withdrawn at 0 h and at the end of 2.5 h of incubation to measure the initial bacterial population and residual cell population by plating suitable dilutions on MRS agar plates. The plates were incubated at 37 1C/30 1C for 48 h and the number of colonies grown was counted.
2.3.
Bile resistance
The ability of isolated LAB strains to grow in presence of two different biles, namely Oxbile and sodium-taurocholate was
Table 1 – Genetic and phenotypic characterization data for the isolates and EPS production in semi-defined media. Isolate code
Source of isolation
Similarity based on 16S rRNA
Sequence length (no. base pairs)
% Similarity
Gene accession number
EPS production (mg/l)
AD1 AI2 AI3 AV2 AV3 AV4 138 AD29 86 AI10 AV1 85 92 142 145 AI1 AV5
Dahi Dhokla batter Dhokla batter Fermented cabbage Carrot Cabbage Fresh turmeric Dahi Dahi Idli batter Fermented cabbage Dahi Idli batter Cucumber Cabbage Idli batter Tomato
L. fermentuma L. fermentumb L. fermentumc L. fermentuma L. fermentuma L. fermentumb L. fermentuma L. plantarumd L. plantarumd W. confusae W. cibariaf W. cibariaf W. cibariaf W. cibariaf W. cibariaf P. parvulusg P. parvulusg
1114 1017 1357 1397 1041 1031 1395 985 1027 1445 932 1375 1109 1141 938 1100 825
99 99 98 99 98 98 97 99 98 96 99 97 99 98 99 99 100
JN792470 JN792468 JN792457 JN792461 JN792462 JN792463 JN792459 JN792465 JN792454 JN792460 JN792467 JN792458 JN792466 JN792456 JN792455 JN792469 JN792464
360 570 280 250 600 680 350 390 960 610 500 570 570 570 480 470 410
a
% Sequence similarity of 16S rDNA gene with the type strains L. fermentum CIP 102980T. % Sequence similarity of 16S rDNA gene with the type strains L. fermentum OMZ 1117T. c % Sequence similarity of 16S rDNA gene with the type strains L. fermentum IFO 3956T. d % Sequence similarity of 16S rDNA gene with the type strains L. plantarum CIP 103151T. e % Sequence similarity of 16S rDNA gene with the type strains W. confusa JCM 1093T. f % Sequence similarity of 16S rDNA gene with the type strains W. cibaria NRIC 0136T. g % Sequence similarity of 16S rDNA gene with the type strains P. parvulus DSM 20331T. b
Food Bioscience 5 (2014) 27 –33
studied according to method of Vinderola and Reinheimer (2003) with few modifications. Each strain was inoculated (2% v/v) into 10 ml MRS broth containing 0.3 (w/v) of Oxbile or sodium taurocholate (Sigma, USA) along with a control i.e., without bile, all tubes were incubated at 37 1C. After 24 h of incubation the bacterial concentration was checked by viable count determination on MRS agar by plating suitable dilutions.
2.4.
Antibiotic susceptibility assay
The antibiotic susceptibility of LAB isolates were evaluated against ampicillin (10 μg), erythromycin (15 μg), chloramphenicol (30 μg), norfloxacin (10 μg), polymyxin (150 μg), vancomycin (30 μg), gentamycin (10 μg), tetracycline (30 μg), and kanamycin (30 μg) using Rosco Neo-Sensitabs™ antibiotic discs. Fifty microliters of the active bacterial suspension was spread evenly on the surface of the MRS agar plate after dilution of the culture to about 105–106 cfu/ml (Zhou, Pillidge, Gopal, & Gill, 2005). The antibiotics discs were placed on the plates and incubated at 37 1C/30 1C for 24–48 h. Inhibition zone diameters were measured inclusive of the diameter of the discs and the results were expressed as sensitive, S; intermediate, I; and resistant, R; according to the instructions given by the manufacturer.
2.6.
Statistical analysis
All the experiments were carried out in three independent experiments and the results are shown as mean7standard deviation (s.d.).
3.
Results and discussion
Bile salt hydrolase activity
The ability of strains to deconjugate or hydrolyze bile salts was determined according to the method of Dashkevicz and Feighner (1989). Test plates were prepared with 0.5% (w/v) of taurodeoxycholic acid (TDCA) in the MRS agar medium. The strains were streaked on the media and the plates were incubated anaerobically using AnaerocultR A (Merck, Germany) in anaerobic jar at 37 1C/30 1C for 48–72 h. The presence of precipitated bile acid around colonies (opaque halo) was considered as positive result.
2.5.
2.7.
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Antimicrobial activity using microtiter plates
The antimicrobial activity of LAB isolates was determined using method of MCVay and Rolfe (2000) with some modifications. The cell free supernatant (CFS) obtained by harvesting 24 h old cultures from MRS broth by centrifugation (using Eppendorf Centrifuge 5816) at 4000 g, 15 min at 4 1C was sterilized through 0.22 μm filter. Further, CFS was serially diluted in BHI broth to prepare 1:1, 1:10 and 1:100 dilutions; and from each dilution 100 μl CFS was added into the wells of microtiter plate in triplicates and allowed to equilibrate at room temperature for 15 min. Pathogens grown on blood agar plates for 24 h at 37 1C in aerobic (static) or microaerophilic conditions were harvested, washed twice and suspended in sterile phosphate buffer saline (PBS, pH 7.2). From that, 10 μl of each pathogen adjusted to an OD620 ¼0.4 in BHI broth was added to the wells of microtiter plate previously equilibrated with CFS of the LAB strains and incubated at 37 1C in plastic box for overnight. The microtiter plates were subjected to measure OD595 nm using iMark™ Microplate Reader (BIO-RAD) and results were recorded to find out percentage of growth inhibition of each pathogen with reference to growth in respective pathogen control.
Several strains of Lactobacilli, Weissella and Bacteroides found to reside into the GI tract, remarkably in the small intestine where less is known about host-microbiome interactions and are required to resist an extreme environment with low pH, presence of bile salts and other natural growth inhibitors (Walter & Ley, 2011). Regardless of source of origin, their part in the normal gut microflora raises the interest to further investigate their potential as health beneficiary microorganisms. EPS producing Lactobacillus, Weissella and Pediococcus spp. has been frequently isolated from various fermented foods (Galle, Schwab, Arendt, & Ganzle, 2010; Van der Meulen et al., 2007). Several studies indicate that biopolymer produced by LAB appear to be associated in cellular recognition, adhesion and the formation of biofilms (Klein, Duarte, Xiao, Mitra, & Foster, 2009; De Vuyst & Degeest, 1999). In one of the reported studies EPS producing LAB found to increase the efficiency of adhesion to the epithelial layer of the GI tract (De Palencia et al., 2009). In connection to this, the investigated LAB strains found to produce EPS ranged from 250 to 900 mg/L in a semi-defined media in earlier experiments (Patel, Lindström, Patel, Prajapati, & Holst, 2012). Based on their ability to grow at low pH and in the presence of bile salts, out of 17 LAB isolates only eight potential strains were further selected to study their probiotic temperament.
3.1.
Tolerance to low pH
Before reaching the intestinal tract, probiotic bacteria must pass through the stomach where the pH can be as low as 1.5–2 (Dunne et al., 2001). Both the strains of Lactobacillus plantarum showed different performance to low pH (Table 2). The strain 86 remained unaffected in acidic condition while strain AD29 showed reduction in the viable count from 7.28 to 6.98 log cfu ml 1 within 2.5 h. Other two isolates, Weissella cibaria strain 92 and P. parvulus AI1 also showed decrease viability from initial count 6.94 and 6.76, to 6.13 and 6.63 log cfu ml 1, respectively. W. cibaria strain 142 did not show any significant change in the viable count (6.71–6.83 log cfu ml 1) while remaining three isolates did not show significant decrease in the viable count. Results are in accordance with other reported studies (De Palencia et al., 2009; Gu, Yang, Li, Chen, & Luo, 2008). In a recent study, Lee et al. (2012) reported significant degree of resistance against low pH (pH 3.0), 0.3% bile salts and sensitivity towards the common antibiotics of Weissella spp. which is comparable with the current results.
3.2.
Bile resistance
Resistance to bile salts is a prerequisite for colonization and metabolic activity of bacteria in the intestinal tract and hence it is an important characteristic of probiotic microorganisms
30
Food Bioscience 5 (2014) 27 –33
Table 2 – Survival of the LAB strains following exposure to low pH (HCl, pH 3.0, 2.5 h) and with different bile types (0.3% w/ v, 24 h) and their BSH activity. LAB isolates
Viable counts (log cfu ml 1) Bile salt tolerance
L. plantarum strain 86 L. plantarum strain AD29 L. fermentum strain AI2 L. fermentum strain AI3 W. cibaria strain 92 W. cibaria strain 142 W. confusa strain AI10 P. parvulus strain AI1
Tolerance to low pH
Control
Sodium taurocholate
Oxgall
0h
3h
7.8270.03 7.8470.03 7.8970.02 7.5470.08 7.7770.07 7.9470.03 7.8470.02 7.8470.02
8.6270.06 7.6770.03 8.6370.06 8.4170.12 8.5070.09 8.8970.04 8.7270.02 8.6870.05
7.3270.09 6.9770.04 7.2370.11 7.0970.16 7.2970.15 7.6770.01 7.7070.03 7.6270.05
7.4370.01 7.2870.01 7.5770.02 7.4570.01 6.9470.04 6.7170.04 7.3570.05 6.7670.06
7.4370.01 6.9870.02 7.4570.03 7.3770.01 6.1370.12 6.8370.03 7.3070.06 6.6370.06
BSH activity
þ þ þ þ ? þ
Each value in the table is the mean7standard deviation of three experiments each with duplicate: ‘þ’, positive; ‘ ’, negative.
(Lee et al., 2012; Dunne et al., 2001). It was found that none of the strains showed growth in the presence of 0.3% oxgall, however as indicated in Table 2 most of the isolates studied were able to survive the presence of oxgall. Only one of the L. plantarum isolate (strain AD29) showed decrease in viable count (from 7.84 to 6.97 log cfu ml 1). On the other hand, most of the isolates were able to grow in 0.3% sodium taurocholate as indicated from the increase viable log count (Table 2). All bile acids, depending upon their concentration can inhibit the growth of bacteria. However, probiotic bacteria vary considerably in their levels of bile tolerance (Gotcheva et al., 2002; Kruszewska et al., 2002). In our study, isolates could not tolerated presence of oxbile (0.3%) in the growth medium while few isolates showed modest growth in the same medium with another bile sodium taurocholate (0.3%). Additionally, it is documented that bile resistance of some strains vary considerably among the LAB species and between strains themselves (Gu et al., 2008). Two isolates belonging to same species viz. L. plantarum 86 and AD29 showed different resistance towards the bile acids. The strain 86 was able to grow in the presence of sodium taurocholate but the later was unable to survive. The similar pattern was observed with tolerance to low pH. Likewise, out of two W. cibaria isolates, strain 92 was unable to resist low pH but at the same time another strain 142 showed stable behavior as indicated in Table 3.
3.3.
Bile salt hydrolase activity
As reported by Tanaka, Doesburg, Iwasaki, and Mierau (1999), when BSH-producing bacteria streaked out on MRS agar plates containing TDCA (0.5%), taurine-conjugated bile acid is deconjugated to deoxycholic acid and thus, BSH-active strains produced opaque white colonies with or without precipitate halos. In the present study, most of the LAB isolates viz. L. fermentum AI2; P. parvulus AI1; W. cibaria 85 and 92 were found to be BSH-positive. Three of the isolates (AI3, AD29 and AI10) showed poor BSH activity while one of the isolate L. plantarum 86 showed BSH negative character. Strains that were found to be BSH inactive produced similar colony types on plates without TDCA. These findings are comparable with the other studies (Tanaka et al., 1999; Reynier et al., 1981). Some strains of L. acidophilus found to
possess BSH activity and cholesterol co-precipitation ability (Liong & Shah, 2005). In case of genus Weissella, up to authors acquaintance this is the first study reporting presence of BSH activity. Bile resistance of certain bacterial strains is related to specific BSH enzyme activity which helps to hydrolyze conjugated bile and reduce its toxic effect (DuToit et al., 1998). BSH has been found to be present in several bacterial species of the GI tract including Lactobacillus spp., Bifidobacterium longum, Clostridium perfringens and in some bacteroides (Corzo & Gilliland, 1999). BSH activity is found in strains isolated from intestines or from feces of mammals -an environment rich in conjugated and unconjugated bile acids and also in fermented food products. Thus, BSH gene may be acquired horizontally as horizontal gene transfer among microbes is very common. Conversely, in current analysis even though the LAB strains have been isolated from fermented foods and vegetables they are found to possess BSH activity which is quite fascinating.
3.4.
Antibiotic susceptibility assay
LAB strains were assayed for their susceptibility to nine different antibiotics and the results are presented in Table 3. All the Lactobacillus and Weissella isolates found to be susceptible towards chloramphenicol, erythromycin, ampicillin and tetracycline, antibiotics that interrupt either protein biosynthesis or cell wall biosynthesis in the bacteria. At the same time most of them were found to be resistant against vancomycin, norfloxacin, kanamycin and polymyxin. Both the pediococci isolates (strain AV5 and AI1) also showed similar trend and they were susceptible against polymyxin as well. The isolates AD1, AI3, AD29 and AI10 showed moderate susceptibility against polymyxin while L. fermentum isolates AV2 and 138 showed modest susceptibility against vancomycin. Transmission of antibiotic resistance genes to potentially pathogenic bacteria in the gut is of major health concern; hence it is desirable that probiotics should sensitive to commonly prescribed antibiotics at the low concentration (Lee et al., 2012). Recent studies reported presence of antibiotic resistance in probiotic strains and LAB (Drago, Mattina, De Vecchi, & Toscano, 2013; Patel, Lindström, Patel, Prajapati,
Kanamycin (30 μg)
R R R R R R R R S S S S S S S S R R R R R R I R Values are the mean of three experiments each with duplicate: S—susceptible; I—moderate susceptibility; R—resistant.
S S S S S S S S R I R I R R I S S S S S S S S S R R R R R R R R R R R R R R R R S S S S S S S S L. plantarum strain 86 L. plantarum strain AD29 L. fermentum strain AI2 L. fermentum strain AI3 W. cibaria strain 92 W. cibaria strain 142 W. confusa strain AI10 P. parvulus strain AI1
Vancomycin (30 μg) Erythromycin (15 μg)
31
& Holst, 2012). All the Lactobacillus and Weissella isolates were found to sensitive towards the common antibiotics plus tetracycline regardless of their source of origin, suggesting their suitability as probiotics. Similar results were also obtained by Zhou et al. (2005) and Danielsen and Wind (2003). Pediococci isolates (strain AV5 and AI1) showed identical susceptibility against above mentioned antibiotics. In addition to that, they were found to be sensitive towards polymyxin as well and these results were comparable with that of Danielsen, Simpson, O’Connor, Ross, and Stanton (2007).
3.5.
LAB isolates
Table 3 – Antibiotic susceptibility of LAB isolates.
Norfloxacin (10 μg)
Chloramphenicol (30 μg)
Polymyxin (150 μg)
Ampicillin (10 μg)
Gentamycin (10 μg)
Tetracycline (30 μg)
Food Bioscience 5 (2014) 27 –33
Antimicrobial activity using microtiter plates
Based on the results of simple well diffusion assay (Vinderola et al., 2008), the LAB isolates that showed inhibitory activity against non pathogenic E. coli K12 (data not shown) were further confirmed by microdilution technique using 96 well microtiter plates. During antimicrobial assay in microtiter plates, the growth of each pathogenic bacterium in serially diluted CFS (1:1, 1:10 and 1:100) was compared with their growth to control BHI broth (without CFS) medium and the antimicrobial activity was measured as % growth inhibition. As it can be seen in Fig. 1a and b, in the 1:1 diluted CFS, all LAB strains exhibited on an average 80%–90% growth reduction compared to their growth in normal BHI broth except the strain P. parvulus AI1. Previous studies (Schneider et al., 2006; Bennik, Smid, & Gorris, 1997) also indicate the inefficiency of Pediococcus spp. towards most of the clinical pathogens. With respect to further dilutions (1:10 and 1:100), the growth of each pathogenic strain successively increased possibly due to the decrease in the amount of CFS added in each well. Results indicate that the inhibitory activity of each LAB strain towards both the clinical isolates were very distinct. Among all LAB isolates, all Weissella spp. (including strain 92, 142 and AI10) and L. plantarum 86 showed significant inhibition of both Gram- positive and Gram- negative pathogen in higher dilutions (1:10 and 1:100) as well. Antimicrobial activities attributable with the production of bacteriocins by Lactobacillus and Pediococcus, spp. have been frequently reported (Klaenhammer, 1988; Yanagida, Chen, & Shinohara, 2006; Schneider et al., 2006), however very little information is available for Weissella spp. In one of the study bacteriocin produced by W. cibaria N23 was found to have a narrow antibacterial spectrum, being able to inhibit only W. confusa N31 (Pringsulaka et al., 2012). In the present work, Weissella isolates showed wide spectrum of inhibition towards both the clinical pathogens (Fig. 1a and b). To the best of our knowledge, this is the first study reporting such broad spectrum of antimicrobial activity by W. cibaria strains. The inhibitory effect in higher dilutions indicates that the antimicrobial activity could be associated with the production of some antimicrobial peptide or bacteriocin like compound rather than from only accumulated organic acids, carbon dioxide and/or H2O2 (Pringsulaka et al., 2012; Servin, 2004). Assimilation of prebiotic oligosaccharides is one of the useful attributes that may lead to formation of short chain fatty acids (SCFA) such as acetate, propionate and butyrate which provide metabolic energy for the host and help in the acidification of the bowel (Swennen, Courtin, & Delcour, 2006).
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Window 15 (EMECW-15) for Doctoral research at the Department of Biotechnology Lund University, Sweden. The study was financially supported by the Lund University Antidiabetic Food Centre, which is a VINNOVA VINN Excellence Centre. Dr. Padma Ambalam is thanked for his kind help during the performance of antimicrobial activity at the department of Medical Microbiology, Lund University, Lund, Sweden.
r e f e r e n c e s
Fig. 1 – (a) Antimicrobial activity of LAB isolates against clinical isolate of E. coli through microtiter plate. (b) Antimicrobial activity of LAB isolates against clinical isolate of S. aureus through microtiter plate.
In connection to this, some of the isolate belonging to genus Weissella, Pediococcus and L. fermentum have been found to utilize prebiotic xylooligosaccharides (XOS) obtained from hydrolysis of birchwood xylan (data under publication).
4.
Conclusion
This study demonstrates the diversity of prospective probiotic LAB in dairy and non-dairy food products from the Western part of India. Few isolates, L. plantarum 86, W. cibaria 142 & 92 and P. parvulus AI1 showed considerable probiotic potential such as considerable tolerance to low pH and bile salts as well as antimicrobial activity against clinical bacterial strains along with EPS production feature. The antibacterial activity exhibited by these isolates may be useful to control the undesirable microbiota either in food system or during GI tract applications. Nevertheless, in future it is essential to prove their in vivo performance for better documentation as a desirable probiotic strain as many other physiological conditions could affect the survival of the strains.
Acknowledgments We are grateful to European Union for granting a scholarship within the program Erasmus Mundus External Cooperation
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Schneider, R., Fernández, F. J., Aguilar, M. B., Guerrero-Legarreta, I., Alpuche-Solís, A., & Ponce-Alquicira, E. (2006). Partial characterization of a class IIa pediocin produced by Pediococcus parvulus 133 strain isolated from meat (Mexican “chorizo”). Food Control, 17, 909–915. Servin, A. L. (2004). Antagonistic activities of lactobacilli and bifidobacteria against microbial pathogens. FEMS Microbiology Review, 28, 405–440. Swennen, K., Courtin, C. M., & Delcour, J. A. (2006). Non-digestible oligosaccharides with prebiotic properties. Critical Reviews in Food Science and Nutrition, 46, 459–471. Tanaka, H., Doesburg, K., Iwasaki, T., & Mierau, I. (1999). Screening of lactic acid bacteria for bile salt hydrolase activity. Journal of Dairy Science, 82, 2530–2535. Van der Meulen, R., Grosu-Tudor, S., Mozzi, F., Vaningelgem, F., Zamfir, M., Font de Valdez, G., et al., (2007). Screening of lactic acid bacteria isolates from dairy and cereal products for exopolysaccharide production and genes involved. International Journal of Food Microbiology, 118, 250–258. Vinderola, C. G., & Reinheimer, J. A. (2003). Lactic acid starter and probiotic bacteria: a comparative “in vitro”study of probiotic characteristics and biological barrier resistance. Food Research International, 36, 895–904. Vinderola, G., Capellini, B., Villarreal, F., Suarez, V., Quiberoni, A., & Reinheimer, J. (2008). Usefulness of a set of simple in vitro tests for the screening and identification of probiotic candidate strains for dairy use. LWT—Food Science and Technology, 41, 1678–1688. Walter, J., & Ley, R. (2011). The human gut microbiome: Ecology and recent evolutionary changes. Annual Review of Microbiology, 65, 411–429. Welman, A. D., & Maddox, I. S. (2003). Exopolysaccharides from lactic acid bacteria: Perspectives and challenges. Trends in Biotechnology, 21, 269–274. Yanagida, F., Chen, Y. S., & Shinohara, T. (2006). Searching for bacteriocin producing lactic acid bacteria in soil. Journal of General and Applied Microbiology, 52, 21–28. Zhou, J. S., Pillidge, C. J., Gopal, P. K., & Gill, H. S. (2005). Antibiotic susceptibility profiles of new probiotic Lactobacillus and Bifidobacterium strains. International Journal of Food Microbiology, 98, 211–217.
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/fbio
Determining probiotic potential of exopolysaccharide producing lactic acid bacteria isolated from vegetables and traditional Indian fermented food products Ami Patela,b,n, J.B. Prajapatia, Olle Holstb, Asa Ljunghc a
Department of Dairy Microbiology, SMC College of Dairy Science, Anand Agricultural University, Anand, Gujarat, India b Department of Biotechnology, Lund University, Lund, Sweden c Department of Medical Microbiology, Lund University, Lund, Sweden
art i cle i nfo
ab st rac t
Article history:
Exopolysaccharide producing lactic acid bacterial (LAB) were tested in vitro to select a
Received 21 February 2013
candidate probiotic strain by testing their tolerance to low pH and bile, bile salt hydrolase
Received in revised form
(BSH) activity, antibiotics susceptibility pattern and antimicrobial activity. Results indi-
30 September 2013
cated that LAB isolates were highly sensitive against oxbile (0.3%) but could grow in the
Accepted 12 October 2013
presence of sodium taurocholate (0.3%). Seven out of 9 isolates were found to be BSH positive. Two of the isolates, Lactobacillus plantarum 86 and Weissella cibaria 92 showed
Keywords:
considerable antimicrobial activity against Gram-positive and Gram-negative pathogens.
Exopolysaccharides
The study reveals that traditional fermented products of India could be an alternative and
BSH activity
readily available resource for LAB starter cultures with interesting functional character-
Weissella
istics.
Dhokla
& 2013 Elsevier Ltd. All rights reserved.
Idli batter Antimicrobial activity
1.
Introduction
Lactic acid bacteria (LAB) are associated with fermented foods where they flair to produce technologically important substances such as exopolysaccharides (EPS). EPS producing LAB are especially relevant in yoghurt, cheese, sour cream and other cultured dairy products by conferring beneficial rheological and functional properties as natural thickening agents, giving the product a suitable viscosity and reducing syneresis (Ruas-Madiedo & de los Reyes-Gavilán, 2005; De Vuyst & Degeest, 1999). EPS have been found to enhance
gastrointestinal (GI) colonization of probiotic bacteria in the GI tract and thus, also play a significant role as prebiotics (Welman & Maddox, 2003). Many of the LAB strains considered as probiotic are found to be effective against diverse GI exertions either by adhering to the gut mucosa, combating pathogens by virtue of producing antimicrobial compounds and/or exerting beneficial effects on human health (Servin, 2004). Before using bacterial strains as probiotics, they should be screened for certain imperative characteristics such as resistance to bile and low pH, antibiotic susceptibilities and antimicrobial activity
n Corresponding author at: Department of Dairy Microbiology, SMC College of Dairy Science, Anand Agricultural University, Anand, Gujarat, India. Tel.: þ91 2692 640170. E-mail address: [email protected] (A. Patel).
2212-4292/$ - see front matter & 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fbio.2013.10.002
28
Food Bioscience 5 (2014) 27 –33
(Lee & Salminen, 2009; Kruszewska et al., 2002). Several probiotics bacteria are found to produce bile salt hydrolase (BSH) that helps to reduce serum cholesterol (Reynier et al., 1981) and hence BSH activity is also considered as an additional criterion for the selection of probiotics. Traditional fermented products could be an alternative and readily available resource for LAB starter cultures with interesting functional characteristics and improved technological and probiotic properties. In this context, based on EPS producing trait, total 17 LAB strains were isolated from different vegetables such as carrot, cabbage, turmeric, cucumber and tomato and traditional fermented foods including dhokla batter, idli batter, dahi, and cabbage (Patel, Shah, & Prajapati, 2012). The present study is aiming at determining potential probiotic characteristics of these EPS producing LAB isolates that were, tested in vitro for tolerance to low pH and bile, bile salt hydrolase (BSH) activity, antibiotics susceptibility and antimicrobial activity.
2.
Materials and methods
2.1. Bacterial strains—isolation, strain identification and growth conditions All 17 EPS producing isolates were characterized biochemically and genotypically using 16S rDNA sequencing. The GenBank and EMBL accession numbers for reference 16S rRNA gene sequences are represented in Table 1. All 17 LAB isolates that were preserved at 20 1C on De Man, Rogosa and Sharpe (MRS, MERCK) broth containing 10% glycerol (v/v) and in freeze-dried form and were propagated twice prior to begin any experiment. The incubation temperature was
maintained at 37 1C for lactobacillus spp. and Weissella isolates and 30 1C for Pediococcus spp. during the experiments whenever their normal growth was required. All isolates are available at the host institution and processed for submission in Microbial type culture collection-MTCC, Chandigarh, India. To perform antimicrobial assay, the clinical isolates of Escherichia coli ESBL and methicillin-resistant Staphylococcus aureus (MRSA) were obtained from the department of Medical Microbiology, Lund University, Lund (Sweden). They were propagated in brain heart infusion (BHI, Oxoid) broth and maintained as frozen stocks at 20 1C in BHI broth containing 10% (v/v) glycerol. Before use, frozen cultures were grown on blood agar plate followed by two successive transfers into BHI broth.
2.2.
Tolerance to low pH
Tolerance to low pH was determined using the method of Chou and Weimer (1999) with some modifications. The active strains grown in MRS broth were inoculated (1.5%) in 10 ml of fresh MRS broth adjusted to pH 3.0 with hydrochloric acid (1.0 M) for 2.5 h at 37 1C. Samples were withdrawn at 0 h and at the end of 2.5 h of incubation to measure the initial bacterial population and residual cell population by plating suitable dilutions on MRS agar plates. The plates were incubated at 37 1C/30 1C for 48 h and the number of colonies grown was counted.
2.3.
Bile resistance
The ability of isolated LAB strains to grow in presence of two different biles, namely Oxbile and sodium-taurocholate was
Table 1 – Genetic and phenotypic characterization data for the isolates and EPS production in semi-defined media. Isolate code
Source of isolation
Similarity based on 16S rRNA
Sequence length (no. base pairs)
% Similarity
Gene accession number
EPS production (mg/l)
AD1 AI2 AI3 AV2 AV3 AV4 138 AD29 86 AI10 AV1 85 92 142 145 AI1 AV5
Dahi Dhokla batter Dhokla batter Fermented cabbage Carrot Cabbage Fresh turmeric Dahi Dahi Idli batter Fermented cabbage Dahi Idli batter Cucumber Cabbage Idli batter Tomato
L. fermentuma L. fermentumb L. fermentumc L. fermentuma L. fermentuma L. fermentumb L. fermentuma L. plantarumd L. plantarumd W. confusae W. cibariaf W. cibariaf W. cibariaf W. cibariaf W. cibariaf P. parvulusg P. parvulusg
1114 1017 1357 1397 1041 1031 1395 985 1027 1445 932 1375 1109 1141 938 1100 825
99 99 98 99 98 98 97 99 98 96 99 97 99 98 99 99 100
JN792470 JN792468 JN792457 JN792461 JN792462 JN792463 JN792459 JN792465 JN792454 JN792460 JN792467 JN792458 JN792466 JN792456 JN792455 JN792469 JN792464
360 570 280 250 600 680 350 390 960 610 500 570 570 570 480 470 410
a
% Sequence similarity of 16S rDNA gene with the type strains L. fermentum CIP 102980T. % Sequence similarity of 16S rDNA gene with the type strains L. fermentum OMZ 1117T. c % Sequence similarity of 16S rDNA gene with the type strains L. fermentum IFO 3956T. d % Sequence similarity of 16S rDNA gene with the type strains L. plantarum CIP 103151T. e % Sequence similarity of 16S rDNA gene with the type strains W. confusa JCM 1093T. f % Sequence similarity of 16S rDNA gene with the type strains W. cibaria NRIC 0136T. g % Sequence similarity of 16S rDNA gene with the type strains P. parvulus DSM 20331T. b
Food Bioscience 5 (2014) 27 –33
studied according to method of Vinderola and Reinheimer (2003) with few modifications. Each strain was inoculated (2% v/v) into 10 ml MRS broth containing 0.3 (w/v) of Oxbile or sodium taurocholate (Sigma, USA) along with a control i.e., without bile, all tubes were incubated at 37 1C. After 24 h of incubation the bacterial concentration was checked by viable count determination on MRS agar by plating suitable dilutions.
2.4.
Antibiotic susceptibility assay
The antibiotic susceptibility of LAB isolates were evaluated against ampicillin (10 μg), erythromycin (15 μg), chloramphenicol (30 μg), norfloxacin (10 μg), polymyxin (150 μg), vancomycin (30 μg), gentamycin (10 μg), tetracycline (30 μg), and kanamycin (30 μg) using Rosco Neo-Sensitabs™ antibiotic discs. Fifty microliters of the active bacterial suspension was spread evenly on the surface of the MRS agar plate after dilution of the culture to about 105–106 cfu/ml (Zhou, Pillidge, Gopal, & Gill, 2005). The antibiotics discs were placed on the plates and incubated at 37 1C/30 1C for 24–48 h. Inhibition zone diameters were measured inclusive of the diameter of the discs and the results were expressed as sensitive, S; intermediate, I; and resistant, R; according to the instructions given by the manufacturer.
2.6.
Statistical analysis
All the experiments were carried out in three independent experiments and the results are shown as mean7standard deviation (s.d.).
3.
Results and discussion
Bile salt hydrolase activity
The ability of strains to deconjugate or hydrolyze bile salts was determined according to the method of Dashkevicz and Feighner (1989). Test plates were prepared with 0.5% (w/v) of taurodeoxycholic acid (TDCA) in the MRS agar medium. The strains were streaked on the media and the plates were incubated anaerobically using AnaerocultR A (Merck, Germany) in anaerobic jar at 37 1C/30 1C for 48–72 h. The presence of precipitated bile acid around colonies (opaque halo) was considered as positive result.
2.5.
2.7.
29
Antimicrobial activity using microtiter plates
The antimicrobial activity of LAB isolates was determined using method of MCVay and Rolfe (2000) with some modifications. The cell free supernatant (CFS) obtained by harvesting 24 h old cultures from MRS broth by centrifugation (using Eppendorf Centrifuge 5816) at 4000 g, 15 min at 4 1C was sterilized through 0.22 μm filter. Further, CFS was serially diluted in BHI broth to prepare 1:1, 1:10 and 1:100 dilutions; and from each dilution 100 μl CFS was added into the wells of microtiter plate in triplicates and allowed to equilibrate at room temperature for 15 min. Pathogens grown on blood agar plates for 24 h at 37 1C in aerobic (static) or microaerophilic conditions were harvested, washed twice and suspended in sterile phosphate buffer saline (PBS, pH 7.2). From that, 10 μl of each pathogen adjusted to an OD620 ¼0.4 in BHI broth was added to the wells of microtiter plate previously equilibrated with CFS of the LAB strains and incubated at 37 1C in plastic box for overnight. The microtiter plates were subjected to measure OD595 nm using iMark™ Microplate Reader (BIO-RAD) and results were recorded to find out percentage of growth inhibition of each pathogen with reference to growth in respective pathogen control.
Several strains of Lactobacilli, Weissella and Bacteroides found to reside into the GI tract, remarkably in the small intestine where less is known about host-microbiome interactions and are required to resist an extreme environment with low pH, presence of bile salts and other natural growth inhibitors (Walter & Ley, 2011). Regardless of source of origin, their part in the normal gut microflora raises the interest to further investigate their potential as health beneficiary microorganisms. EPS producing Lactobacillus, Weissella and Pediococcus spp. has been frequently isolated from various fermented foods (Galle, Schwab, Arendt, & Ganzle, 2010; Van der Meulen et al., 2007). Several studies indicate that biopolymer produced by LAB appear to be associated in cellular recognition, adhesion and the formation of biofilms (Klein, Duarte, Xiao, Mitra, & Foster, 2009; De Vuyst & Degeest, 1999). In one of the reported studies EPS producing LAB found to increase the efficiency of adhesion to the epithelial layer of the GI tract (De Palencia et al., 2009). In connection to this, the investigated LAB strains found to produce EPS ranged from 250 to 900 mg/L in a semi-defined media in earlier experiments (Patel, Lindström, Patel, Prajapati, & Holst, 2012). Based on their ability to grow at low pH and in the presence of bile salts, out of 17 LAB isolates only eight potential strains were further selected to study their probiotic temperament.
3.1.
Tolerance to low pH
Before reaching the intestinal tract, probiotic bacteria must pass through the stomach where the pH can be as low as 1.5–2 (Dunne et al., 2001). Both the strains of Lactobacillus plantarum showed different performance to low pH (Table 2). The strain 86 remained unaffected in acidic condition while strain AD29 showed reduction in the viable count from 7.28 to 6.98 log cfu ml 1 within 2.5 h. Other two isolates, Weissella cibaria strain 92 and P. parvulus AI1 also showed decrease viability from initial count 6.94 and 6.76, to 6.13 and 6.63 log cfu ml 1, respectively. W. cibaria strain 142 did not show any significant change in the viable count (6.71–6.83 log cfu ml 1) while remaining three isolates did not show significant decrease in the viable count. Results are in accordance with other reported studies (De Palencia et al., 2009; Gu, Yang, Li, Chen, & Luo, 2008). In a recent study, Lee et al. (2012) reported significant degree of resistance against low pH (pH 3.0), 0.3% bile salts and sensitivity towards the common antibiotics of Weissella spp. which is comparable with the current results.
3.2.
Bile resistance
Resistance to bile salts is a prerequisite for colonization and metabolic activity of bacteria in the intestinal tract and hence it is an important characteristic of probiotic microorganisms
30
Food Bioscience 5 (2014) 27 –33
Table 2 – Survival of the LAB strains following exposure to low pH (HCl, pH 3.0, 2.5 h) and with different bile types (0.3% w/ v, 24 h) and their BSH activity. LAB isolates
Viable counts (log cfu ml 1) Bile salt tolerance
L. plantarum strain 86 L. plantarum strain AD29 L. fermentum strain AI2 L. fermentum strain AI3 W. cibaria strain 92 W. cibaria strain 142 W. confusa strain AI10 P. parvulus strain AI1
Tolerance to low pH
Control
Sodium taurocholate
Oxgall
0h
3h
7.8270.03 7.8470.03 7.8970.02 7.5470.08 7.7770.07 7.9470.03 7.8470.02 7.8470.02
8.6270.06 7.6770.03 8.6370.06 8.4170.12 8.5070.09 8.8970.04 8.7270.02 8.6870.05
7.3270.09 6.9770.04 7.2370.11 7.0970.16 7.2970.15 7.6770.01 7.7070.03 7.6270.05
7.4370.01 7.2870.01 7.5770.02 7.4570.01 6.9470.04 6.7170.04 7.3570.05 6.7670.06
7.4370.01 6.9870.02 7.4570.03 7.3770.01 6.1370.12 6.8370.03 7.3070.06 6.6370.06
BSH activity
þ þ þ þ ? þ
Each value in the table is the mean7standard deviation of three experiments each with duplicate: ‘þ’, positive; ‘ ’, negative.
(Lee et al., 2012; Dunne et al., 2001). It was found that none of the strains showed growth in the presence of 0.3% oxgall, however as indicated in Table 2 most of the isolates studied were able to survive the presence of oxgall. Only one of the L. plantarum isolate (strain AD29) showed decrease in viable count (from 7.84 to 6.97 log cfu ml 1). On the other hand, most of the isolates were able to grow in 0.3% sodium taurocholate as indicated from the increase viable log count (Table 2). All bile acids, depending upon their concentration can inhibit the growth of bacteria. However, probiotic bacteria vary considerably in their levels of bile tolerance (Gotcheva et al., 2002; Kruszewska et al., 2002). In our study, isolates could not tolerated presence of oxbile (0.3%) in the growth medium while few isolates showed modest growth in the same medium with another bile sodium taurocholate (0.3%). Additionally, it is documented that bile resistance of some strains vary considerably among the LAB species and between strains themselves (Gu et al., 2008). Two isolates belonging to same species viz. L. plantarum 86 and AD29 showed different resistance towards the bile acids. The strain 86 was able to grow in the presence of sodium taurocholate but the later was unable to survive. The similar pattern was observed with tolerance to low pH. Likewise, out of two W. cibaria isolates, strain 92 was unable to resist low pH but at the same time another strain 142 showed stable behavior as indicated in Table 3.
3.3.
Bile salt hydrolase activity
As reported by Tanaka, Doesburg, Iwasaki, and Mierau (1999), when BSH-producing bacteria streaked out on MRS agar plates containing TDCA (0.5%), taurine-conjugated bile acid is deconjugated to deoxycholic acid and thus, BSH-active strains produced opaque white colonies with or without precipitate halos. In the present study, most of the LAB isolates viz. L. fermentum AI2; P. parvulus AI1; W. cibaria 85 and 92 were found to be BSH-positive. Three of the isolates (AI3, AD29 and AI10) showed poor BSH activity while one of the isolate L. plantarum 86 showed BSH negative character. Strains that were found to be BSH inactive produced similar colony types on plates without TDCA. These findings are comparable with the other studies (Tanaka et al., 1999; Reynier et al., 1981). Some strains of L. acidophilus found to
possess BSH activity and cholesterol co-precipitation ability (Liong & Shah, 2005). In case of genus Weissella, up to authors acquaintance this is the first study reporting presence of BSH activity. Bile resistance of certain bacterial strains is related to specific BSH enzyme activity which helps to hydrolyze conjugated bile and reduce its toxic effect (DuToit et al., 1998). BSH has been found to be present in several bacterial species of the GI tract including Lactobacillus spp., Bifidobacterium longum, Clostridium perfringens and in some bacteroides (Corzo & Gilliland, 1999). BSH activity is found in strains isolated from intestines or from feces of mammals -an environment rich in conjugated and unconjugated bile acids and also in fermented food products. Thus, BSH gene may be acquired horizontally as horizontal gene transfer among microbes is very common. Conversely, in current analysis even though the LAB strains have been isolated from fermented foods and vegetables they are found to possess BSH activity which is quite fascinating.
3.4.
Antibiotic susceptibility assay
LAB strains were assayed for their susceptibility to nine different antibiotics and the results are presented in Table 3. All the Lactobacillus and Weissella isolates found to be susceptible towards chloramphenicol, erythromycin, ampicillin and tetracycline, antibiotics that interrupt either protein biosynthesis or cell wall biosynthesis in the bacteria. At the same time most of them were found to be resistant against vancomycin, norfloxacin, kanamycin and polymyxin. Both the pediococci isolates (strain AV5 and AI1) also showed similar trend and they were susceptible against polymyxin as well. The isolates AD1, AI3, AD29 and AI10 showed moderate susceptibility against polymyxin while L. fermentum isolates AV2 and 138 showed modest susceptibility against vancomycin. Transmission of antibiotic resistance genes to potentially pathogenic bacteria in the gut is of major health concern; hence it is desirable that probiotics should sensitive to commonly prescribed antibiotics at the low concentration (Lee et al., 2012). Recent studies reported presence of antibiotic resistance in probiotic strains and LAB (Drago, Mattina, De Vecchi, & Toscano, 2013; Patel, Lindström, Patel, Prajapati,
Kanamycin (30 μg)
R R R R R R R R S S S S S S S S R R R R R R I R Values are the mean of three experiments each with duplicate: S—susceptible; I—moderate susceptibility; R—resistant.
S S S S S S S S R I R I R R I S S S S S S S S S R R R R R R R R R R R R R R R R S S S S S S S S L. plantarum strain 86 L. plantarum strain AD29 L. fermentum strain AI2 L. fermentum strain AI3 W. cibaria strain 92 W. cibaria strain 142 W. confusa strain AI10 P. parvulus strain AI1
Vancomycin (30 μg) Erythromycin (15 μg)
31
& Holst, 2012). All the Lactobacillus and Weissella isolates were found to sensitive towards the common antibiotics plus tetracycline regardless of their source of origin, suggesting their suitability as probiotics. Similar results were also obtained by Zhou et al. (2005) and Danielsen and Wind (2003). Pediococci isolates (strain AV5 and AI1) showed identical susceptibility against above mentioned antibiotics. In addition to that, they were found to be sensitive towards polymyxin as well and these results were comparable with that of Danielsen, Simpson, O’Connor, Ross, and Stanton (2007).
3.5.
LAB isolates
Table 3 – Antibiotic susceptibility of LAB isolates.
Norfloxacin (10 μg)
Chloramphenicol (30 μg)
Polymyxin (150 μg)
Ampicillin (10 μg)
Gentamycin (10 μg)
Tetracycline (30 μg)
Food Bioscience 5 (2014) 27 –33
Antimicrobial activity using microtiter plates
Based on the results of simple well diffusion assay (Vinderola et al., 2008), the LAB isolates that showed inhibitory activity against non pathogenic E. coli K12 (data not shown) were further confirmed by microdilution technique using 96 well microtiter plates. During antimicrobial assay in microtiter plates, the growth of each pathogenic bacterium in serially diluted CFS (1:1, 1:10 and 1:100) was compared with their growth to control BHI broth (without CFS) medium and the antimicrobial activity was measured as % growth inhibition. As it can be seen in Fig. 1a and b, in the 1:1 diluted CFS, all LAB strains exhibited on an average 80%–90% growth reduction compared to their growth in normal BHI broth except the strain P. parvulus AI1. Previous studies (Schneider et al., 2006; Bennik, Smid, & Gorris, 1997) also indicate the inefficiency of Pediococcus spp. towards most of the clinical pathogens. With respect to further dilutions (1:10 and 1:100), the growth of each pathogenic strain successively increased possibly due to the decrease in the amount of CFS added in each well. Results indicate that the inhibitory activity of each LAB strain towards both the clinical isolates were very distinct. Among all LAB isolates, all Weissella spp. (including strain 92, 142 and AI10) and L. plantarum 86 showed significant inhibition of both Gram- positive and Gram- negative pathogen in higher dilutions (1:10 and 1:100) as well. Antimicrobial activities attributable with the production of bacteriocins by Lactobacillus and Pediococcus, spp. have been frequently reported (Klaenhammer, 1988; Yanagida, Chen, & Shinohara, 2006; Schneider et al., 2006), however very little information is available for Weissella spp. In one of the study bacteriocin produced by W. cibaria N23 was found to have a narrow antibacterial spectrum, being able to inhibit only W. confusa N31 (Pringsulaka et al., 2012). In the present work, Weissella isolates showed wide spectrum of inhibition towards both the clinical pathogens (Fig. 1a and b). To the best of our knowledge, this is the first study reporting such broad spectrum of antimicrobial activity by W. cibaria strains. The inhibitory effect in higher dilutions indicates that the antimicrobial activity could be associated with the production of some antimicrobial peptide or bacteriocin like compound rather than from only accumulated organic acids, carbon dioxide and/or H2O2 (Pringsulaka et al., 2012; Servin, 2004). Assimilation of prebiotic oligosaccharides is one of the useful attributes that may lead to formation of short chain fatty acids (SCFA) such as acetate, propionate and butyrate which provide metabolic energy for the host and help in the acidification of the bowel (Swennen, Courtin, & Delcour, 2006).
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Window 15 (EMECW-15) for Doctoral research at the Department of Biotechnology Lund University, Sweden. The study was financially supported by the Lund University Antidiabetic Food Centre, which is a VINNOVA VINN Excellence Centre. Dr. Padma Ambalam is thanked for his kind help during the performance of antimicrobial activity at the department of Medical Microbiology, Lund University, Lund, Sweden.
r e f e r e n c e s
Fig. 1 – (a) Antimicrobial activity of LAB isolates against clinical isolate of E. coli through microtiter plate. (b) Antimicrobial activity of LAB isolates against clinical isolate of S. aureus through microtiter plate.
In connection to this, some of the isolate belonging to genus Weissella, Pediococcus and L. fermentum have been found to utilize prebiotic xylooligosaccharides (XOS) obtained from hydrolysis of birchwood xylan (data under publication).
4.
Conclusion
This study demonstrates the diversity of prospective probiotic LAB in dairy and non-dairy food products from the Western part of India. Few isolates, L. plantarum 86, W. cibaria 142 & 92 and P. parvulus AI1 showed considerable probiotic potential such as considerable tolerance to low pH and bile salts as well as antimicrobial activity against clinical bacterial strains along with EPS production feature. The antibacterial activity exhibited by these isolates may be useful to control the undesirable microbiota either in food system or during GI tract applications. Nevertheless, in future it is essential to prove their in vivo performance for better documentation as a desirable probiotic strain as many other physiological conditions could affect the survival of the strains.
Acknowledgments We are grateful to European Union for granting a scholarship within the program Erasmus Mundus External Cooperation
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