Exopolysaccharides May Increase Gastrointestinal Stress Tolerance of Lactobacillus reuteri

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Abstract

This study investigated a possible relationship between exopolysaccharides (EPS) production and the resistance to bile salts and low pH in intestinal strains of Lactobacillus reuteri. The strains displayed a mucoid phenotype, when grown in the presence of 10 % sucrose. Scanning electron microscopy (SEM) revealed strands of exopolysaccharide linking neighbouring cells. The strains (except L. reuteri B1/1) produced EPS in the range from 15.80 to 650.70 mg.l−1. The strains were tested for tolerance to bile salts (0.15; 0.3 %) and low pH (1.5—2.0—2.5—3.0). The survival rate, after the treatment with artificial gastric and intestinal juices, was determined by flow cytometric analysis. The strains of L. reuteri that produced 121—650 mg.l−1 of EPS showed a significantly higher tolerance (P < 0.001) to the gastric juice at pH 3 and 2.5, throughout the entire exposure time, in comparison to the strains that produced less than 20 mg.l−1 of EPS. L. reuteri L26, with the highest production of EPS, exhibited the highest survival rate (60 %) at pH 2 after the 120 minutes of in-cubation and was able to tolerate pH 1.5 for 30 minutes. Higher production of EPS significantly (P < 0.001) increased the strains’ tolerance against the intestinal juice in the presence of 0.15 and 0.3 % bile salts and was time dependent. L. reuteri L26 showed the highest tolerance (P < 0.001) against 0.3 % bile salts. This investigation revealed a positive correlation between the EPS production and the resistance of intestinal L. reuteri to the stress conditions of the gastrointestinal tract (GIT).

1. Alp, G., Aslim, B., 2010: Relationship between the resistance to bile salts and low pH with exopolysaccharide (EPS) production of Bifidobacterium spp. isolated from infants feces and breast milk. Anaerobe, 16, 101—105.

2. Amund, O. D., Ouoba, L. I. I., Sutherland, J. P., Ghoddusi, H. B., 2014: Assessing the effects of exposure to environmental stress on some functional properties of Bifidobacterium animalis spp. lactis. Benef. Microbes, 5, 461—469.

3. Amund, O. D., 2016: Exploring the relationship between exposure to technological and gastrointestinal stress and pro-biotic functional properties of lactobacilli and bifidobacteria. Can. J. Microbiol., 62, 715—725.

4. Azcarate-Peril, M. A., McAuliffe, O., Altermann, E., Lick, S., Russell, W. M., Klaenhammer, T. R., 2005: Microarray analysis of a two-component regulatory system involved in acid resistance and proteolytic activity in Lactobacillus acidophilus. Appl. Environ. Microbiol., 71, 5794—5804.

5. Badel, S., Bernardi, T., Michaud, P., 2011: New perspectives for Lactobacilli exopolysaccharides. Biotechnol. Adv., 29, 54—66.

6. Ben Amor, K., Breeuwer, P., Verbaarschot, P., Rombouts, F. M., Akkermans, A. D. L., De Vos, W. M., Abee, T., 2002: Multiparametric flow cytometry and cell sorting for the assessment of viable, injured, and dead Bifidobacterium cells during bile salt stress. Appl. Environ. Microbiol., 68, 5209—5216.

7. Bermudez-Brito, M., Plaza-Díaz, J., Muñoz-Quezada, S., Gómez-Llorente, C., Gil, A., 2012: Probiotic mechanisms of action. Ann. Nutr. Metab., 61, 160—174.

8. Bernal, P., Llamas, M. A., 2012: Promising biotechnological applications of antibiofilm exopolysaccharides. Microbiol. Biotechnol., 5, 670—673.

9. Boke, H., Aslim, B., Alp, G., 2010: The role of resistance to bile salts and acid tolerance of exopolysaccharides produced by yogurt starter bacteria. Arch. Bio. Sci. Belgrade, 62, 323—328.

10. Bradford, M. M., 1976: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72, 248—254.

11. Chapot-Chartier, M. P., Monnet, V., De Vuyst, L., 2011: Cell walls and exopolysaccharides of lactic acid bacteria. In Lede-boer, A., Hugenholtz, J., Kok, J., Konings, W., Wouters, J. (Eds.): The 10th LAB Symposium. Thirty Years Research on Lactic Acid Bacteria. Media Labs, Rotterdam, 37—59.

12. Chen, Y., Woodward, A., Zijlstra, R. T., Gänzle, M. G., 2014: Exopolysaccharides synthesized by Lactobacillus reuteri protect against enterotoxigenic Escherichia coli in piglets. Appl. Environ. Microbiol., 80, 5752—5760.

13. De Vuyst, L., Degeest, B., 1999: Heteropolysaccharides from lactic acid bacteria. FEMS Microbiol. Rev., 23, 153—177.

14. Dertli, E., Mayer, M. J., Narbad, A., 2015: Impact of the exopolysaccharide layer on biofilms, adhesion and resistance to stress in Lactobacillus johnsonii FI9785. BMC Microbiol., 15, 8.

15. Donoghue, H. D., Newman, H. N., 1976: Effect of glucose and sucrose on survival in batch culture of Streptococcus mutans C67-1 and a noncariogenic mutant, C67-25. Infect. Immun., 13, 16—21.

16. Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., Smith, F., 1956: Colorimetric method for determination of sugars and related substances. Anal. Chem., 28, 350—356.

17. Durlu-Ozkaya, F., Aslimb, B., Ozkaya, M. T., 2007: Effect of exopolysaccharides (EPSs) produced by Lactobacillus delbrueckii subsp. bulgaricus strains to bacteriophage and nisin sensitivity of the bacteria. LWT-Food Science and Technology, 40, 564—568.

18. Gänzle, M., Schwab, C., 2009: Ecology of exopolysaccharide formation by lactic acid bacteria: sucrose utilization, stress tolerance, and biofilm formation. In Ullrich, M. (Ed.): Bacterial Polysaccharides: Current Innovations and Future Trends. Caister Academic Press, Norfolk, 263—278.

19. Giraffa, G., Chanishvili, N., Widyastuti, Y., 2010: Importance of lactobacilli in food and feed biotechnology. Res. Microbiol., 161, 480—487.

20. Jones, S. E., Versalovic, J., 2009: Probiotic Lactobacillus reuteri biofilms produce antimicrobial and anti-inflammatory factors. BMC Microbiol., 9, 35—43.

21. Kim, Y., Sejong, O. H., Kim, S. H., 2009: Released exopolysaccharide (r-EPS) produced from probiotic bacteria reduce biofilm formation of enterohemorrhagic Escherichia coli O157:H7. Biochem. Biophys. Res. Commun., 379, 324—329.

22. Kimmel, S. A., Roberts, R. F., Ziegler, G. R., 1998: Optimization of exopolysaccharide production by Lactobacillus delbrueckii subsp. bulgaricus RR grown in a semidefined medium. Appl. Environ. Microbiol., 64, 659—664.

23. Kos, B., Šušković, J., Goreta, J., Matošić, S., 2000: Effect of protectors on the viability of Lactobacillus acidophilus M92 in simulated gastrointestinal conditions. Food Technol. Biotech., 38, 121—127.

24. Kšonžeková, P., Bystrický, P., Vlčková, S., Pätoprstý, V., Pulzová, L, Mudroňová, D., et al., 2016: Exopolysaccharides of Lactobacillus reuteri: Their influence on adherence of E. coli to epithelial cells and inflammatory response. Carbohydr. Polym., 141, 10—19.

25. Kubota, H., Senda, S., Nomura, N., Tokuda, H., Uchiyama, H., 2008: Biofilm formation by lactic acid bacteria and resistance to environmental stress. J. Biosci. Bioeng., 106, 381—386.

26. Lambert, J. M., Bongers, R. S., de Vos, W. M., Kleerebezem, M., 2008: Functional analysis of four bile salt hydrolase and penicillin acylase family members in Lactobacillus plantarum WCFS1. Appl. Environ. Microbiol., 74, 4719—4726.

27. London, L. E. E., Price, N. P. J., Ryan, P., Wang, L., Auty, M. A. E., Fitzgerald, G. F., et al., 2014: Characterization of a bovine isolate Lactobacillus mucosae DPC 6426 which produces an exopolysaccharide composed predominantly of mannose residues. J. Appl. Microbiol., 117, 509—517.

28. Mills, S., Stanton, C., Fitzgerald, G. F., Ross, R. P., 2011: Enhancing the stress responses of probiotics for a lifestyle from gut to product and back again. Microb. Cell Fact, 10 (Suppl. 1), 19.

29. Mortazavian, M., Mohammadi, R., Sohrabvandi, S., 2012: Delivery of probiotic microorganisms into gastrointestinal tract by food products. In Brzozowski, T. (Ed.): New Advances in the Basic and Clinical Gastroenterology. InTech, Rijeka, 121—146.

30. Mudroňová, D., 2015: Flow cytometry as an auxiliary tool for the selection of probiotic bacteria. Benef. Microbes, 6, 727—734.

31. Nwodo, U. U., Green, E., Okoh, A. I., 2012: Bacterial exopolysaccharides: functionality and prospects. Int. J. Mol. Sci., 13, 14002—14015.

32. Oh, N. S., Joung, J. Y., Lee, J. Y., Kim, Y., 2018: Probiotic and anti-inflammatory potential of Lactobacillus rhamnosus 4B15 and Lactobacillus gasseri 4M13 isolated from infant faeces. PLoS ONE, 13, e0192021.

33. Qurashi, A. W., Sabri, A. N., 2012: Bacterial exopolysaccharide and biofilm formation stimulate chickpea growth and soil aggregation under salt stress. Braz. J. Microbiol., 43, 1183—1191.

34. Ruas-Madiedo, P., Hugenholtz, J., Zoon, P., 2002: An overview of the functionality of exopolysaccharides produced by lactic acid bacteria. Int. Dairy J., 12, 163—171.

35. Ruas-Madiedo, P, de los Reyes-Gavilan, C. G., 2005: Invited review: methods for the screening, isolation, and characterization of exopolysaccharides produced by lactic acid bacteria. J. Dairy Sci., 88, 843—856.

36. Ruas-Madiedo, P., Gueimonde, M., Arigoni, F., de los Reyes-Gavilan, C. G., Margolles, A., 2009: Bile affects the synthesis of exopolysaccharides by Bifidobacterium animalis. Appl. Environ. Microbiol., 75, 1204—1207.

37. Ruiz, L., Ruas-Madiedo, P., Gueimonde, M., de los Reyes-Gavilan, C. G., Margolles, A., Sanchez, B., 2011: How do bifidobacteria counteract environmental challenges ? Mechanisms involved and physiological consequences. Genes Nutr., 6, 307—318.

38. Ryznerová, D., 2013: The Study of the Properties of the Probiotic Bacteria in Terms of their Biological Effects and Applications. Dissertation thesis, University of Veterinary Medicine and Pharmacy in Košice, SR, 146 pp.

39. Sanchez, B., Ruiz, L., van Sinderen, D., Margolles, A., Zomer, A. L., 2010: Acid and bile resistance and stress response in bifidobacteria. In Mayo, B., van Sinderen, D. (Eds.): Bifidobacteria: Genomics and Molecular Aspects. Caister Academic Press, Norfolk, UK, 71—96.

40. Sims, I. M., Frese, S. A., Walter, J., Loach, D., Wilson, M., Appleyard, K., et al., 2011: Structure and functions of exopolysaccharide produced by gut commensal Lactobacillus reuteri 100-23. ISME J., 5, 1115—1124.

41. Stack, H. M., Kearney, N., Stanton, C., Fitzgerald, G. F., Ross, R. P., 2010: Association of beta-glucan endogenous production with increased stress tolerance of intestinal Lacto-bacilli. Appl. Environ. Microbiol., 76, 500—507.

42. Sugimoto, S., Abdullah, Al. M., Sonomoto, K., 2008: Molecular chaperones in lactic acid bacteria: physiological consequences and biochemical properties. J. Biosci. Bioeng., 106, 324—336.

43. Tieking, M., Kaditzky, S., Valcheva, R., Korakli, M., Vogel, R. F., Ganzle, M. G., 2005: Extracellular homopolysaccha-rides and oligosaccharides from intestinal lactobacilli. J. Appl. Microbiol., 99, 692—702.

44. Van Geel-Schutten, G. H., Flesch, F., ten Brink, B., Smith, M. R., Dijkhuizen, L., 1998: Screening and characterization of Lactobacillus strains producing large amounts of exopolysaccharides. Appl. Microbiol. Biotechnol., 50, 697—703.

45. Wall, T., Bath, K. Britton, R. A., Jonsson, H., Versalovic, J., Roos, S., 2007: The early response to acid shock in Lactobacillus reuteri involves the ClpL chaperone and a putative cell wall-altering esterase. Appl. Environ. Microbiol., 73, 3924—3935.

46. Walter, J., Schwab, C., Loach, D. M., Ganzle, M. G., Tannock, G. W., 2008: Glucosyltransferase A (GtfA) and inulosucrase (Inu) of Lactobacillus reuteri TMW1.106 contribute to cell aggregation, in vitro biofilm formation, and colonization of the mouse gastrointestinal tract. Microbiology, 154, 72—80.

47. Zannini, E., Waters, D. M., Coffey, A., Arendt, E. K., 2016: Production, properties, and industrial food application of lactic acid bacteria-derived exopolysaccharides. Appl. Microbiol. Biotechnol., 100, 1121—1135.

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