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Application of in vitro methods for selection of Lactobacillus casei strains as potential probiotics

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Abstract

Probiotics have established their efficacy as dietary adjuncts providing benefits to consumers, but the selection of probiotics before incorporation in diet requires close scrutiny in the form of in vitro as well as in vivo tests. The present study was undertaken to check different in vitro characteristics of seven Lactobacillus casei strains. The characteristics studied include acid and bile tolerance, adhesion and cell surface hydrophobicity, antimicrobial effect on common pathogens and cholesterol reduction. All strains were able to resist pH 3 for 3 h, though resistance to pH 2 was exhibited by NCDC 17, C1 and Y strains only. NCDC 63 and VT strains were able to tolerate 1% and 2% bile concentrations for 12 h. There was wide variation in ability of strains to adhere to isolated rat epithelial cells. The index was highest for C1 at 66%. The electron microscopic adhesion studies on the stainless steel chips did not reveal any specific attachment to surfaces by any of strains. The hydrophobic character for octane was highest for strain C1 at 54.06% and lowest for strain C2 at 4.65%. The ability to antagonize common pathogens was observed in all strains but this activity was attributed to production of organic acids and no specific compound caused the inhibitory effect. The cholesterol reducing ability varied not only for strains but also for time of incubation. NCDC 17 showed maximum reduction in cholesterol level after 48 h of incubation with buffalo plasma as the source of cholesterol. Overall there existed variations in different strains with respect to different characters of significance to be a probiotic.

Introduction

The science of probiotics, live microbial cultures which, when ingested in sufficient numbers, provide beneficial effects to the consumer beyond basic nutrition (Schaffsma, 1996), may be traced to Metchinikoff in 1907. The characteristics of a successful probiotic are acid and bile tolerance, antimicrobial activity against intestinal pathogens, and ability to adhere and colonize the intestinal tract. Probiotic organisms include Lactobacillus, lactococci, bifidobacteria and Saccharomyces with several species of the first listed genus the most studied. Beneficial effects include control of diarrhea (Reddy et al., 1998), alleviation of lactose intolerance (Fonden et al., 2000), inhibition of intestinal pathogens (Bhatia et al., 1989). Other effects studied include reduction in cholesterol level (Agarbaek et al., 1995), enhanced immune response (Kimura, 1997) and antimutagenic and anticarcinogenic activity (Fuller and Gibson, 1997). Lactobacillus casei has been used successfully as a probiotic in traditional milk products, as well as cheeses and a number of commercial fermented food products. The present study aimed to apply in vitro methods for selection of suitable strains of L. casei for use as probiotics and incorporation in Dahi, a popular traditional lactococcal fermented milk product of India.

Section snippets

Lactobacillus cultures

L. casei ssp. casei 17, 19 and 63 were procured from National Collection of Dairy Cultures (NCDC), NDRI, Karnal. L. casei ssp. casei VT was a gift from Dr. Hans Heilig, Wagnenigen University and Research Centre, The Netherlands. Other L. casei strains viz. C1, C2 and Y were isolated from milk and milk products in Quality Assurance Laboratory of Dairy Microbiology Division, NDRI, Karnal. The strains were characterized by cell morphology, colony appearance, biochemical methods and substrate

Tolerance to simulated human stomach pH

The survival of L. casei strains at pH 1, 2 and 3 was observed for 0, 1, 2 and 3 h (Table 1). While none of the strains survived at pH 1 for any time period (data not shown), only strains NCDC 17, isolate C1 and Y could survive pH 2 for 1 h. All seven strains except for NCDC 17 showed consistency in terms of tolerance to pH 3. The residual counts were more than 107 cfu/ml even after 3 h of incubation. Survival at pH 3 was promising for all strains but not at pH 2. Survival at pH 3 is

Conclusion

The present investigation was undertaken to identify suitable probiotic L. casei strains for incorporation in Dahi, a lactococcal fermented milk product of India. The strains varied in ability to tolerate acid and bile, adherence properties and cholesterol assimilation which indicate that selection of probiotics has to be carefully undertaken. In the present study L. casei C1 and Y, both isolates from milk products, showed promising results for both acid tolerance and adherence properties.

Acknowledgements

The authors acknowledge Dr. Hans Helig, Wageningen University and Research Centre for providing L. casei VTT E/85225 strain and suggestions made by Dr. William Charteris during the course of study.

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