DOI QR코드

DOI QR Code

Modification of Substrate and Fermentation Process to Increase Mass and Customize Physical Properties of Lacticaseibacillus rhamnosus and Limosilactobacillus fermentum Exopolysaccharides in Kefir Grain

  • Dandy Yusuf (Research Centre for Applied Microbiology, National Research and Innovation Agency (BRIN)) ;
  • Raden Haryo Bimo Setiarto (Research Centre for Applied Microbiology, National Research and Innovation Agency (BRIN)) ;
  • Andi Febrisiantosa (Research Center for Food Technology and Processing, National Research and Innovation Agency (BRIN)) ;
  • Angga Maulana Firmansyah (Research Center for Food Technology and Processing, National Research and Innovation Agency (BRIN)) ;
  • Taufik Kurniawan (Research Center for Food Technology and Processing, National Research and Innovation Agency (BRIN)) ;
  • Ahmad Iskandar Setiyawan (Research Center for Food Technology and Processing, National Research and Innovation Agency (BRIN)) ;
  • Rina Wahyuningsih (Research Center for Food Technology and Processing, National Research and Innovation Agency (BRIN)) ;
  • Tri Ujilestari (Research Center for Food Technology and Processing, National Research and Innovation Agency (BRIN)) ;
  • Satyaguna Rakhmatulloh (Department of Animal Products Technology, Faculty of Animal Science, Universitas Gadjah Mada) ;
  • Heni Rizqiati (Department of Agriculture, Faculty of Animal and Agricultural Sciences, Diponegoro University, Tembalang Campus)
  • Received : 2024.01.12
  • Accepted : 2024.03.18
  • Published : 2024.06.28

Abstract

The microbial starter used to produce kefir beverages, kefir grain, contains a microbial exopolysaccharide called kefiran. Kefir grain consisting of water-insoluble polysaccharides, proteins, and fats, which can be applied as a multi-functional biopolymer. The mass of kefir grain can increase in the fermentation process of Kefir, but it is considered very slow. The purpose of this research is to study the impact of ammonium sulfate supplementation and yeast extract on reconstituted skim milk to increase the mass kefir grain and physical properties of kefiran. Results showed that the ammonium sulfate-supplemented substrate increased the mass of kefir grain by 547% in 14 days, with the condition that the substrate must be renewed every 2 days. Refreshing the substrate is considered one of the important factors. Supplementation on substrate did not appear to affect the viability of bacterial and yeast cells. Kefir grain produced from supplemented substrate also yields better thermal stability properties and has more functional groups than without supplementation. Two Lacticaseibacillus rhamnosus (RAL27 and RAL43) and one Limosilactobacillus fermentum (RAL29) were found to produce EPS. The three isolates also showed good skim milk fermentation ability after purification from kefir grain. The kefir grain produced in this study has the potential for wider application. This study also showed that kefir grain can be adjusted in quantity and quality through fermentation substrate engineering.

Keywords

Acknowledgement

All authors had equal contributions as the main contributors to this manuscript paper. The authors would like to thank LPDP Indonesia and BRIN Indonesia for fully supporting this research through the Program Research and Innovation for Advanced Indonesia (RIIM) 2023. Thanks to the facilities, scientific and technical support from the Bandung Advanced Characterization Laboratory, National Research and Innovation Agency.

References

  1. Moradi Z, Kalanpour N. 2019. Kefiran, a branched polysaccharide: Preparation, properties and applications: A review. Carbohydr. Polym. 223: 115100.
  2. Xu Y, Cui Y, Yue F, Liu L, Shan Y, Liu B, et al. 2019. Exopolysaccharides produced by lactic acid bacteria and bifidobacteria: Structures, physiochemical functions and applications in the food industry. Food Hydrocoll. 94: 475-499.
  3. Fujisawa T, Adachi S, Toba T, Arihara K, Mitsuoka T. 1988. Lactobacillus kefiranofaciens sp. nov. isolated from kefir grains. Int. J. Syst. Bacteriol. 38: 12-14.
  4. You X, Li Z, Ma K, Zhang C, Chen X, Wang G, et al. 2020. Structural characterization and immunomodulatory activity of an exopolysaccharide produced by Lactobacillus helveticus LZ-R-5. Carbohydr. Polym. 235: 115977.
  5. You X, Yang L, Zhao X, Ma K, Chen X, Zhang C, et al. 2020. Isolation, purification, characterization and immunostimulatory activity of an exopolysaccharide produced by Lactobacillus pentosus LZ-R-17 isolated from Tibetan kefir. Int. J. Biol. Macromol. 158: 408-419.
  6. Chen Z, Shi J, Yang X, Nan B, Liu Y, Wang Z. 2015. Chemical and physical characteristics and antioxidant activities of the exopolysaccharide produced by Tibetan kefir grains during milk fermentation. Int. Dairy J. 43: 15-21.
  7. Rahbar Saadat Y, Yari Khosroushahi A, Pourghassem Gargari B. 2019. A comprehensive review of anticancer, immunomodulatory and health beneficial effects of the lactic acid bacteria exopolysaccharides. Carbohydr. Polym. 217: 79-89.
  8. Exarhopoulos S, Goulas A, Dimitreli G. 2022. Biodegradable films from kefiran-based cryogel systems. Macromol. 2: 324-345.
  9. Linares-Bravo P, Cabo-Araoz SD, Luna-Solano G, Urrea-Garcia GR, Cantu-Luzano D. 2022. Obtention of new edible biofilms from water kefir grains in comparison with conventional biofilms from taro (Colocasia esculenta) and Cassava (Manihot esculenta) starch. Processes 10: 1804.
  10. Radhouani H, Bicho D, Goncalves C, Maia FR, Reis RL, Oliveira JM. 2019. Kefiran cryogels as potential scaffolds for drug delivery and tissue engineering applications. Mater. Today Commun. 20: 100554.
  11. Cottet C, Ramirez-Tapias YA, Delgado JF, Osa O, Salvay AG, Peltzer MA. 2020. Biobased materials from microbial biomass and its derivatives. Materials 13: 1263.
  12. Lucena MA, Ramos IFS, Geronco MS, Araujo R, Filho FLS, Silva LMLR, et al. 2022. Biopolymer from water kefir as a potential clean-label ingredient for health applications: Evaluation of new properties. Molecules 27: 3895.
  13. Plessas S, Mantzourani I, Bekatorou A. 2020. Evaluation of Pediococcus pentosaceus SP2 as starter culture on sourdough bread making. Foods 9: 77.
  14. Spizzirri UG, Abduvakhidov A, Caputo P, Crupi P, Muraglia M, Rossi CO, et al. 2022. Kefir enriched with carob (Ceratonia siliqua L.) leaves extract as a new ingredient during a gluten-free breadmaking process. Fermentation 8: 305.
  15. Affane ALN. 2012. Impact of environmental factors on the metabolic profiles of kefir produced using different kefir grains and subsequent enrichment of kefir prepared with mass cultured grains - Stellenbosch University. http://scholar.sun.ac.za. Accessed Jan. 20, 2017.
  16. Schoevers A, Britz TJ. 2003. Influence of different culturing conditions on kefir grain increase. Int. J. Dairy Technol. 56: 183-187.
  17. Harta O, Iconomopoulou M, Bekatorou A, Nigam P, Kontominas M, Koutinas AA. 2004. Effect of various carbohydrate substrates on the production of kefir grains for use as a novel baking starter. Food Chem. 88: 237-242.
  18. 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 Chem. 139: 970-977.
  19. Pop C, Apostu S, Salanta L, Rotar AM, Sindic M, Mabon N, et al. 2014. Influence of different growth conditions on the Kefir grains production, used in the Kefiran synthesis. Bull. UASVM Food Sci. Technol. 71: 2.
  20. Enikeev R. 2012. Development of a new method for determination of exopolysaccharide quantity in fermented milk products and its application in technology of kefir production. Food Chem. 134: 2437-2441.
  21. Koc EO, Inal M. 2021. Physicochemical properties of polysaccharide kefiran isolated from kefir grains biomass. Int. J. Biotechnol. Biomater. Eng. 3: 1-7.
  22. Radhouani H, Goncalves C, Maia FR, Oliveira JM, Reis RL. 2018. Kefiran biopolymer: Evaluation of its physicochemical and biological properties. J. Bioact. Compat. Polym. 33: 461-478.
  23. Bradford 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.
  24. Martins EF, Moura NK, Moura TK, Araujo TV, Machado JPB, Passador FR, et al. 2022. Determination and standardization of the kefiran extraction protocol for possible pharmacological applications. Carbohydr. Polym. Technol. Appl. 3: 100198.
  25. Yusuf D, Nuraida L, Hariyadi RD, Hunaefi D. 2020. Lactic acid bacteria and yeasts from Indonesian kefir grains and their growth interaction. Asian J. Microbiol. Biotechnol. Environ. Sci. 22: 44-49.
  26. Oleksy-Sobczak M, Klewicka E. 2020. Optimization of media composition to maximize the yield of exopolysaccharides production by Lactobacillus rhamnosus strains. Probiotics Antimicrob. Proteins 12: 774-783.
  27. Yusuf D, Nuraida L, Dewanti-Hariyadi R, Hunaefi D. 2020. In vitro characterization of lactic acid bacteria from Indonesian kefir grains as probiotics with cholesterol-lowering effect. J. Microbiol. Biotechnol. 30: 726-732.
  28. Wang M, Bi J. 2008. Modification of characteristics of kefiran by changing the carbon source of Lactobacillus kefiranofaciens. J. Sci. Food Agric. 88: 763-769.
  29. Gientka I, Bzducha-Wrobel A, Stasiak-Rozanska L, Bednarska AA, Blazejak S. 2016. The exopolysaccharides biosynthesis by Candida yeast depends on carbon sources. Electron. J. Biotechnol. 22: 31-37.
  30. Saadat YR, Khosroushahi AY, Movassaghpour AA, Talebi M, Gargari BP. 2020. Modulatory role of exopolysaccharides of Kluyveromyces marxianus and Pichia kudriavzevii as probiotic yeasts from dairy products in human colon cancer cells. J. Funct. Foods 64: 103675.
  31. Pop CR, Salanta L, Rotar AM, Semeniuc CA, Socaciu C, Sindic M. 2016. Influence of extraction conditions on characteristics of microbial polysaccharide kefiran isolated from kefir grains biomass. J. Food Nutr. Res. 55: 121-130.
  32. Lucena-Aguilar G, Sanchez-Lopez AM, Barberan-Aceituno C, Carrillo-Avila JA, Lopez-Guerrero JA, Aguilar-Quesada R. 2016. DNA source selection for downstream applications based on dna quality indicators analysis. Biopreserv. Biobank. 14: 264-270.
  33. Pearson WR. 2013. An introduction to sequence similarity ("Homology") searching. Curr. Protoc. Bioinforma 42: 1286-1292.
  34. Pfeiffer F, Losensky G, Marchfelder A, Habermann B, Dyall-Smith M. 2020. Whole-genome comparison between the type strain of Halobacterium salinarum (DSM 3754T) and the laboratory strains R1 and NRC-1. MicrobiologyOpen 9: 1-44.
  35. Wang Y, Wu J, Lv M, Shao Z, Hungwe M, Wang J, et al. 2021. Metabolism characteristics of lactic acid bacteria and the expanding applications in food industry. Front. Bioeng. Biotech. 9: 612285.
  36. Ohba T, Uemura K, Nabetani H. 2016. Moderate pulsed electric field treatment enhances exopolysaccharide production by Lactococcus lactis subspecies cremoris. Process Biochem. 51: 1120-1128.