Study of Exopolysaccharide Production by Lactic Acid Bacteria used in the Baking Industry and Comparison of Methods for Their Determination

Introduction: Exopolysaccharides are high molecular weight compounds consisting of monosaccharide residues and their derivatives, which have biological activity and play a protective role in physiological processes. The search for strains of lactic acid bacteria producing exopolysaccharides is a promising area of research due to their proven positive effect on the rheological properties of fermented food products, as well as human health.

Purpose: To study the ability of lactic acid bacteria strains from the collection of FGANU NIIHP to produce exopolysaccharides, as well as to compare different methods of their determination - gravimetric, phenol-sulfuric acid Dubois method, anthrone reagent method and titrimetric Bertrand method.

Materials and Methods: To determine the ability to produce exopolysaccharides, cultures of lactic acid bacteria were cultured on 12% malt mash for 48 hours at the optimum temperature for each strain. After protein removal and dialysis, the amount of exopolysaccharides was determined in the culture fluid by different methods.

Results: It was found that all the collection strains formed exopolysaccharides in different amounts. The strains producing the highest amount of exopolysaccharides were identified as L.amilolyticus-2, L. plantarumA-63(d), L.amilolyticus-1, L.brevis-78(d), L. paracasei-6. The yield of the product containing exopolysaccharides produced by lactic acid bacteria ranged from 7 to 14.4 mg in 1 ml of culture liquid. The results of the study allowed us to conclude the correctness of Dubois method for estimating the content with respect to exopolysaccharides produced by lactic acid bacteria.

Conclusion: The conducted studies confirm the ability of strains of lactic acid bacteria, from the collection of FGANU NIIHP, to produce exopolysaccharides. The correctness of the Dubois method for the determination of exopolysaccharides produced by lactic acid bacteria was established.

1. Абрамова, А.Л. (2009). Методы определения экзополисахаридов (ЭПС). Научное обеспечение молочной промышленности (ВНИМИ - 80 лет) (c. 8-12). Место издания: ВНИМИ.

2. Винокуров, В. А., Грайфер, В. И., Гринберг, Т. А., Пирог, Т. П., Владимиров, А. И., & Исмагилов, А. М. (2004). Способ получения экзополисахаридов (Патент РФ № 2 241 037). Российское патентное ведомство. Опубликовано 27.11.2004, Бюл. № 33.

3. Григорьев, Е. Ф., Болоховская, В. А., Халабузарь, В. Г., Кравец, Л. Ф., Дерябин, В. В., & Бовина, Е. В. (1990). Способ выделения экзополисахаридов (Патент СССР № 1 549 996). Патентное ведомство СССР. Опубликовано 15.03.1990.

4. Еникеев, Р. Р., Бобошко, Д. Н., Руденко, Е. Ю., & Зимичев, А. В. (2011). Способ количественного анализа полисахарида, производимого молочнокислыми бактериями (Патент РФ № 2 437 092). Российское патентное ведомство. Опубликовано 20.12.2011, Бюл. № 35.

5. Кичемазова, Н.В. (2019). Экзополисахариды бактерий родов Xanthobacter и Ancylobacter: характеристика и их биологические свойства [Диссертация на соиск. уч. ст. к. биол. н.]. ФГБОУ ВО Вавиловский Университет.

6. Куис, Л. В., & Маркевич, Р. М. (2009). Выделение, фракционирование и анализ экзополисахаридов Bacillus mucilaginosus. Труды БГТУ. Серия 2: Химические технологии, биотехнология, геоэкология (c. 170-173). Минск: БГТУ.

7. Локачук, М. Н., Савкина, О.А., Павловская, Е. Н., Фролова, Ю. М., Костюченко, М. Н., & Мартиросян, В. В. (2023). Современная таксономия и разнообразие молочнокислых бактерий в заквасках. Хлебопродукты, (6), 28-35. https://doi.org/10.32462/0235-2508-2023-32-6-28-35

8. Фокина, Н.А. (2015). Экзополисахарид Streptococcus Thermophilus: условия выделения и свойства. Актуальная биотехнология, 14(3), 41-42.

9. Хусаинов, И.А. (2014). Современные представления о биосинтезе бактериальных экзополисахаридов. Вестник Казанского технологического университета, 17(5), 167-172.

10. Amiri, S., Rezaei Mokarram, R., Sowti Khiabani, M., Rezazadeh Bari, M., & Alizadeh Khaledabad, M. (2019). Exopolysaccharides production by Lactobacillus acidophilus LA5 and Bifidobacterium animalis subsp. lactis BB12: Optimization of fermentation variables and characterization of structure and bioactivities. International Journal of Biological Macromolecules, 123, 752-765. https://doi.org/10.1016/j.ijbiomac.2018.11.084

11. Cerning, J., Bouillanne, C., & Desmazeaud, M. J. (1988). Exocellular polysaccharide production by Streptococcus thermophiles. Biotechnology Letters, 10, 255–260. https://doi.org/10.1007/BF01024415

12. Daba, G. M., Elnahas, M. O., & Elkhateeb, W. A. (2021). Contributions of exopolysaccharides from lactic acid bacteria as biotechnological tools in food, pharmaceutical, and medical applications. International Journal of Biological Macromolecules, 173, 79-89. https://doi.org/10.1016/j.ijbiomac.2021.01.110

13. Das, L., Raychaudhuri, U., & Chakraborty, R. (2015). Effects of hydrocolloids as texture improver in coriander bread. Journal of Food Science and Technology, 52(6), 3671–3680. https://doi.org/10.1007/s13197-014-1296-8

14. Dilna, S. V., Surya, H., Aswathy, R. G., Varsha, K. K., Sakthikumar, D. N., Pandey, A., & Nampoothiri, K. M. (2015). Characterization of an exopolysaccharide with potential health-benefit properties from a probiotic Lactobacillus plantarum RJF4. LWT - Food Science and Technology, 64(2), 1179-1186. https://doi.org/10.1016/j.lwt.2015.07.040

15. Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., & Smith, F. (1956). Colorimetric method for determination of sugars and related substances. Analytical Chemistry, 28(3), 350–356. https://doi.org/10.1021/ac60111a017

16. Galle, S., Schwab, C., Arendt, E., & Gänzle, M. (2010). Exopolysaccharide-forming Weissella strains as starter cultures for sorghum and wheat sourdoughs. Journal of Agricultural and Food Chemistry, 58(9), 5834–5841. https://doi.org/10.1021/jf1002683

17. Goh, K. K. T., Haisman, D. R., Archer, R. H., & Singh, H. (2005). Evaluation and modification of existing methods for the quantification of exopolysaccharides in milk-based media. Food Research International, 38(6), 605-613. https://doi.org/10.1016/j.foodres.2004.11.014

18. Jurášková, D., Ribeiro, S. C., & Silva, C. C. G. (2022). Exopolysaccharides produced by lactic acid bacteria: From biosynthesis to health-promoting properties. Foods, 11(2), 156. https://doi.org/10.3390/foods11020156

19. Katina, K., Maina, N. H., Juvonen, R., Flander, L., Johansson, L., Virkki, L., Tenkanen, M., & Laitila, A. (2009). In situ production and analysis of Weissella confusa dextran in wheat sourdough. Food Microbiology, 26(7), 734–743. https://doi.org/10.1016/j.fm.2009.07.008

20. Kavitake, D., Devi, P. B., Singh, S. P., & Shetty, P. H. (2016). Characterization of a novel galactan produced by Weissella confusa KR780676 from an acidic fermented food. International Journal of Biological Macromolecules, 86, 681–689. https://doi.org/10.1016/j.ijbiomac.2016.01.099

21. Kim, Y., Oh, S., Yun, H. S., Oh, S., & Kim, S. H. (2010). Cell-bound exopolysaccharide from probiotic bacteria induces autophagic cell death of tumour cells. Letters in Applied Microbiology, 51(2), 123-130. https://doi.org/10.1111/j.1472-765X.2010.02859.x

22. Korcz, E., & Varga, L. (2021). Exopolysaccharides from lactic acid bacteria: Techno-functional application in the food industry. Trends in Food Science & Technology, 110, 375-384. https://doi.org/10.1016/j.tifs.2021.02.014

23. Masuko, T., Minami, A., Iwasaki, N., Majima, T., Nishimura, S., & Lee, Y. C. (2005). Carbohydrate analysis by a phenol-sulfuric acid method in microplate format. Analytical Biochemistry, 339(1), 69-72. https://doi.org/10.1016/j.ab.2004.12.001

24. Moroni, A. V., Arendt, E. K., & Dal Bello, F. (2011). Biodiversity of lactic acid bacteria and yeasts in spontaneously-fermented buckwheat and teff sourdoughs. Food Microbiology, 28(3), 497–502. https://doi.org/10.1016/j.fm.2010.10.016

25. Nguyen, P. T., Nguyen, T. T., Bui, D. C., Hong, P. T., Hoang, Q. K., & Nguyen, H. T. (2020). Exopolysaccharide production by lactic acid bacteria: The manipulation of environmental stresses for industrial applications. AIMS Microbiology, 6(4), 451-469. https://doi.org/10.3934/microbiol.2020027

26. Oleksy-Sobczak, M., Klewicka, E., & Piekarska-Radzik, L. (2020). Exopolysaccharides production by Lactobacillus rhamnosus strains – Optimization of synthesis and extraction conditions. LWT, 122, 109055. https://doi.org/10.1016/j.lwt.2020.109055

27. Patel, S., Majumder, A., & Goyal, A. (2012). Potentials of exopolysaccharides from lactic acid bacteria. Indian Journal of Microbiology, 52(1), 3-12. https://doi.org/10.1007/s12088-011-0148-8

28. Prete, R., Alam, M. K., Perpetuini, G., Perla, C., Pittia, P., & Corsetti, A. (2021). Lactic acid bacteria exopolysaccharides producers: A sustainable tool for functional foods. Foods, 10, 1653. https://doi.org/10.3390/foods10071653

29. Piermaria, J. A., De La Canal, M. L., & Abraham, A. G. (2008). Gelling properties of kefiran, a food-grade polysaccharide obtained from kefir grain. Food Hydrocolloids, 22, 1520–1527. https://doi.org/10.1016/j.foodhyd.2007.10.005

30. Ruhmkorf, C., Jungkunz, S., Wagner, M., & Vogel, R. F. (2012). Optimization of homoexopolysaccharide formation by lactobacilli in gluten-free sourdoughs. Food Microbiology, 32(2), 286–294. https://doi.org/10.1016/j.fm.2012.07.002

31. Rühmann, B., Schmid, J., & Sieber, V. (2015). Methods to identify the unexplored diversity of microbial exopolysaccharides. Frontiers in Microbiology, 6, 565. https://doi.org/10.3389/fmicb.2015.00565

32. Ruijssenaars, H. J., Stingele, F., & Hartmans, S. (2000). Biodegradability of food-associated extracellular polysaccharides. Current Microbiology, 40, 194-199.

33. Ryan, P. M., Ross, R. P., Fitzgerald, G. F., Caplice, N. M., & Stanton, C. (2014). Sugar-coated: Exopolysaccharide producing lactic acid bacteria for food and human health applications. Food & Function, 6(3), 679-693. https://doi.org/10.1039/C4FO00529E

34. Sanalibaba, P., & Çakmak, G. A. (2016). Exopolysaccharides production by lactic acid bacteria. Applied Micro Open Access, 2, 1000115. https://doi.org/10.4172/2471-9315.1000115

35. Zajsek, K., Gorsek, A., & Kolar, M. (2013). Cultivating conditions effects on kefiran production by the mixed culture of lactic acid bacteria imbedded within kefir grains. Food Chemistry, 139(1-4), 970-977. https://doi.org/10.1016/j.foodchem.2012.11.142

36. Zarour, K., Vieco, N., Pérez-Ramos, A., Nácher-Vázquez, M., Mohedano, M. L., & López, P. (2017). Food ingredients synthesized by lactic acid bacteria. In Microbial Production of Food Ingredients and Additives (pp. 89–124). Elsevier. https://doi.org/10.1016/B978-0-12-811520-6.00004-0