PROBIOTIC POTENTIAL OF SOME LACTIC ACID BACTERIA ISOLATED FROM ROMANIAN FERMENTED VEGETABLES Silvia Simona Grosu-Tudor, Medana Zamfir

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1 PROBIOTIC POTENTIAL OF SOME LACTIC ACID BACTERIA ISOLATED FROM ROMANIAN FERMENTED VEGETABLES Silvia Simona Grosu-Tudor, Medana Zamfir INSTITUTE OF BIOLOGY BUCHAREST OF THE ROMANIAN ACADEMY 296 SPLAIUL INDEPENDENTEI, BUCHAREST, P.O. BOX 56-53, ROMANIA Summary Probiotics are live microbial food supplements which beneficially affect the host by improving the intestinal microbial balance. To be used as a probiotic, a bacterial strain must have a good tolerance to the acidity of the stomach and to the bile salts present in the upper small intestine. The purpose of the present work was to evaluate the probiotic potential of six LAB strains isolated from Romanian fermented vegetables. The strains were investigated for tolerance to acidity (ph values of 2.0, 3.0, and 4.0, respectively) and to bile salts (concentrations of 0.3% and 0.5%, w/v, respectively). All six strains tested were resistant to ph 3.0 and ph 4.0, after 24 h of incubation showing viability rates of 10 9 CFU/ml and CFU/ml, respectively. After 3 h of exposure to ph 2.0, four strains (Leuc. citreum 344, Lb. brevis 183, Leuc. mesenteroides 348 and Lb. plantarum 327) reached viability rates of at least 10 8 CFU/ml. Moreover, Lb. brevis 183 survived even after 24 h of exposure to ph 2.0, reaching a viability of 10 4 CFU/ml. Concerning the bile salt resistance, all six strains showed a high resistance to a concentration of 0.3% (w/v), reaching a cell viability of CFU/ml after 24 h of treatment. After 2 h of exposure to 0.5% (w/v) of bile salts, the viability of strains Lb. brevis 183 and Leuc. mesenteroides 348, reached about 10 6 CFU/ml, a value still adequate for the use of these strains as probiotics. The high resistance to low ph values and in the presence of bile salts enables these strains to survive in the stomach and intestine, or even to compete with other bacterial groups in this environment and to colonize the GIT of the host, having a promising probiotic potential. Key words: lactic acid bacteria, probiotics, acid and bile tolerance, fermented vegetables Introduction Lactic acid bacteria (LAB) constitute a heterogeneous group of industrially important bacteria that are used to produce fermented foods and beverages, using various substrates, such as milk, vegetables, cereals, meat, cocoa beans etc. (Doyle and Beuchat, 2007; Wood and Holzapfel, 1995; Wood, 1997). During the last few decades, development of the functional food concept and, more specifically, the application of certain LAB strains as life vaccines, pro- and prebiotics, nutraceuticals, have created new perspectives for LAB research and human consumption, attracting the attention of 234 silvia.grosu@ibiol.ro both food scientists and health professionals (Mozzi et al., 2010; De Vuyst et al., 2004; Hardy et al., 2002). Actually, an increasing demand exists for new strains that show desirable effects on the product characteristics or strains promoting well-being and health (the so-called probiotics ). Traditionally fermented foods are the best places to look for such new microorganisms. On the other hand, the use of a probiotic strain in fermented foods of plant origin, would be of great importance, since in recent years, consumers who are lactose intolerant, as well as vegetarians, increasingly demand for non-dairy-based probiotics.

2 Acording to Guarner and Shaafsma (1998) probiotics are living microorganisms which upon ingestion in certain numbers, exert health benefits beyond inherent basic nutrition. In order to exert its beneficial effect on the host, a probiotic strain must be able to survive the passage through the host s digestive tract and to reach its sites of action in a viable state and in a sufficient concentration (of about 10 7 cells/ml). In order to survive in and colonize the (gastro-intestinal tract) GIT, probiotic bacteria should express high tolerance to acid and bile (Lee and Salminen, 1995; Kirjavainen et al., 1998). Therefore, in our study, six LAB strains isolated from Romanian traditionally fermented vegetables, strains with antibacterial activity against some Gramnegative pathogenic bacteria, were examined for their tolerance to low ph and to bile salts. Material and methods Bacterial strains, media and culture conditions. Six LAB strains isolated from Romanian fermented vegetales were selected: Leuconostoc citreum 344 and Lactobacillus brevis 183 (showing an antibacterial activity against Listeria monocytogenes ATCC 1911), Leuconostoc mesenteroides 348 and Lactobacillus brevis 312 (with an antibacterial activity against Eschrichia coli ATCC 25922) and Lactobacillus plantarum 327 and Lactobacillus brevis 380 (with an antibacterial activity against Salmonella enterica ATCC 14028) (unpublished results). The cultures were preserved in MRS broth (de Man et al. 1960) and stored at -75 C in the presence of 25% (v/v) of glycerol as cryoprotectant. Prior to use, the strains were subcultured twice in MRS broth and incubated overnight at 37 C. Acid tolerance. Two ml of overnight cultures of the tested strains were centrifuged (10 min at rpm) and the cellular sediment was resuspended in two ml of MRS broth previously adjusted with 235 HCl to ph values of 2.0, 3.0, and 4.0, respectively. The initial bacterial concentration after inoculation was about CFU/ml and this was checked by viable count determination on MRS agar (see below). The cultures were incubated at 37 C for 24 h. Samples were taken at 0, 1, 2, 3, and 24 h after the start, to determine the viable cell counts. Simultaneously, a control at optimal growth ph was used. Bile salt tolerance. In order to determine the bile resistance, two ml of the overnight cultures were centrifuged (10 min at rpm) and the sediment was resuspended in two ml of MRS broth containing 0.3% and 0.5% of bile salts (Sigma-Aldrich Chemie GmbH, Germany), respectively and incubated at 37 C for 24 h. The initial bacterial concentration after inoculation was about CFU/ml and this was checked by viable count determination on MRS agar. Samples were taken at 0, 15, 30, 60, 120 min, and at 24 h after the start, to determine the viable cell counts. The controls were incubated in MRS broth without bile salts. Viable cells enumeration. Viable cells were enumerated by plating 10-fold serial dilutions on MRS agar medium. Plates were incubated at 37ºC for 48 h and the LAB counts were expressed in colony forming units per milliliter (CFU/ml). Results and discussions Six LAB strains previously isolated from Romanian traditional fermented vegetables have been shown to have an antibacterial activity against potential pathogenic or spoilage bacteria (results not published). The evaluation of the probiotic potential of these six LAB strains was carried out by in vitro simulating the environment of the GIT, namely by subjecting the strains to low ph values and by exposure to bile salts. The typical transit time of the food in the stomach is between 20 minutes to 3 hours (Hyronimus et al., 2000, Goldin et al., 1992) and therefore the treatment was done for at least 3 h.

3 Acid tolerance. Before reaching the intestinal tract, probiotic bacteria must first survive the acidity of the stomach. Three ph values of the growth medium were used, ranging from 2.0 to 4.0, taking into account that stomach acidity varies from person to person, either naturally or due to several factors (Dunne et al., 2001; Goldin et al., 1992). The results showed that resistance to low ph is strain dependent. All six strains tested were resistant to ph 3.0 and ph 4.0, showing viability rates of about 10 9 CFU/ml and about CFU/ml after 24 h of incubation at ph 3.0 and 4.0, respectively (data not shown). When exposed to ph 2.0, four of the tested strains (Leuc. citreum 344, Lb. brevis 183, Leuc. mesenteroides 348 and Lb. plantarum 327) showed a good survival afer 3 h of treatment showing viability rates of at least 10 8 CFU/ml (Figure 1). Moreover, the strain Lb. brevis 183 survived even after 24 h of exposure to ph 2.0, reaching a viability rate of 10 4 CFU/ml. Figure 1. Acid tolerance of lactic acid bacteria strains at ph 2.0 In a similar study reported by Mishera et al. (2005), three of their seven isolates of Lb. casei tolerated ph 2.0 and/or 3.0. Although acid tolerance in lactobacilli is highly strain specific, members of the genus Lactobacillus are, in general, acidophilic, albe to grow at ph 4.0 in foods containing a fermentable carbohydrate (Alakomi et al., 2000). The probiotic effect of LAB may partly be based on the production of relevant concentrations of lactic acid in the microenvironment, which, in combination with detergents like bile salts, inhibits the growth of Gram-negative pathogenic bacteria (Stiles and Holzapfel, 1997). The high resistance to low ph values of our six tested strains might be explained by the source of their isolation, namely fermented vegetables, in which the 236 ph decreases after only one day of fermentation to about 4.0. The fact these strains became dominant over other species might be the result of an adaptation to the specific environment. Bile salt tolerance. Bile salts belong to the factors that may significantly affect the viability of LAB in the GIT, influencing the health of the host. Tolerance to bile salts is a prerequisite for colonisation and metabolic activity of bacteria in the small intestine of the host (Havenaar et al., 1992). This will help LAB to reach the small intestine and colon and contribute in balancing the intestinal microflora (Tambekar and Bhutada, 2010). After 2 h of incubation in MRS medium supplemented with 0.3% (w/v) bile salts, all six tested strains showed a viability rate of at least 10 6 CFU/ml (Figure

4 2). At this bile salts concentration all strains proved a long-term resistance, reaching viability rates between 10 5 and 10 8 CFU/ml. This high tolerance to bile salts represents an advantage for the survival of these bacteria, once introduced in the GIT. Figure 2. Resistance of lactic acid bacteria in the presence of 0.3% bile salts A variable tolerance to higher concentrations of bile salts (0.5%, w/v) was observed for the six tested strains. The viability after 24 h of exposure was in the range of CFU/ml. However, after only 2 h of treatment, the viability of strains Lb. brevis 183 and Leuc. mesenteroides 348, reached about 10 6 CFU/ml (Figure 3), a value still adequate for the use of these strains as probiotics. Figure 3. Resistance of lactic acid bacteria in the presence of 0.5% bile salts The resistance of our tested strains to bile salts might be attributed to their ability to produce bile salt hydrolases (Savage, 1992). Bile salt hydrolases (BSH) 237

5 protect the producing cells from the toxicity of conjugated bile salts by deconjugating the bile acids (Walker & Gillilard, 1993). Compared with their conjugated counterparts, deconjugated bile acids have decreased solubility and diminished detergent activity and may, therefore, be less toxic to bacteria in the intestine (Fernandes et al., 2003). Although the results obtained in in vitro experiments may not necessarily reflect the case of in vivo situations, the high rate of survival of our tested LAB strains to low ph values was an indication of their possible survival in the human s stomach before the transit to the small intestine. In addition, their resistance in the presence of 0.5% bile salts is an indicative of their possible survival in the small intestine (Papamanoli et al., 2003). The survival ability of these LAB strains in the stress conditions mentioned above might result in a temporary colonization of the human GIT (Alander et al., 1997, Johansson et al., 1998), contributing to the final probiotic efect. Conclusions In conclusion, the LAB strains used in this study showed a high resistance to low ph values and to bile salts. These features may enable them to survive in the stomach and intestine, or even to compete with other bacterial groups in this environment and to colonize the GIT of the host. The results of our present study, together with our previous studies concerning the antibacterial activity against pathogenic bacteria, showed that fermented vegetables (and not only dairy products) might be a rich source of LAB strains with a high potential for their use as probiotic strains. Acknowledgements The authors acknowledge their financiar support of the Postdoctoral Research Project PD_33/2010 of the Romanian National Research Plan (PNII-RU). Part of 238 this work was supported by the project no. RO1567-IBB05/2011 from the Institute of Biology Bucharest of Romanian Academy. References Alakomi, H.L.; Skytta, E.; Saarela, M.; Mattila- Sandholm, T.; Latva-Kala, K.; Helander, I.: Lactic acid permeabilizes Gram-negative bacteria by disturbing the outer membrane. Appl. Environ. Microbiol., 66, Alander, M.; Korpela, R.; Saxelin, M.; Vilpponen-Salmela, T.; Mattila-Sandholm, T.; Wright, A: Recovery of Lactobacillus rhamnosis GG from human colonic biopsies. Letters in Applied Microbiol., 24, , De Man, J.; Rogosa, M.; Sharpe, M.: A medium for the cultivation of lactobacilli. J. Appl. Bact., 23, , De Vuyst, L.; Avonts, L.; Makras, E.: Probiotics, prebiotics, and gut health. In C. Remacle & B. Reusens (Eds.), Functional Foods, Ageing and Degenerative Disease Cambridge, UK: Woodhead Publishing, , Doyle, M.P.; Beuchat, L.R.: Food Microbiology: Fundamentals and Frontiers. (eds.) Washington, DC: ASM Press, Dunne, C.; O Mahony, L.; Murphy, L.; Thornton, G.; Morrissey, D.; O Halloran, S.; Feeney, M.; Flynn, S.; Fitzgerald, G.; Daly, C.; Kiely, B.; O Sullivan, G.C.; Shanahan, F.; Collins, K.C.: In vitro selection criteria for probiotic bacteria of human origin: correlation with in vivo findings. Am. J. Clin. Nutr. 73, 386S-392S, Fernandez, M.F.; Boris, S.; Barbes, C.: Probiotic properties of human lactobacilli strains to be used in the gastrointestinal tract. J. Appl. Microbiol., 94, , Goldin, B.; Gorbach, S.; Saxelin, M.; Barakat, S.; Gualtieri, L.; Salminen, S.: Survival of Lactobacillus species (Strain GG) in human gastrointestinal tract. Digestive Dis. Sci., 37, , Guarner, F.; Schaafsma, G.J.: Probiotics. Int. J. Food Microbiol. 39, , Hardy, G.; Hardy, I.; McElroy, B.: Nutraceuticals: a pharmaceutical viewpoint: I. Current Opinion in Clinical Nutrition & Metabolic Care 5, , Havenaar, R.; Huis in t Velt, J.: Probiotics: A general view. In wood ed. The lactic acid

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