Probiotic Growth Pattern and Physicochemical Evaluation of Water Kefir Fermentation


  • Phin Yin Sin Faculty of Industrial Sciences and Technology, Universiti Malaysia Pahang, Lebuhraya Persiaran Tun Khalil Yaakob, 26300 Gambang, Kuantan, Pahang Darul Makmur, Malaysia
  • Suat Hian Tan Faculty of Industrial Sciences and Technology, Universiti Malaysia Pahang, Lebuhraya Persiaran Tun Khalil Yaakob, 26300 Gambang, Kuantan, Pahang Darul Makmur, Malaysia
  • Mohd Fazli Farida Asras Faculty of Industrial Sciences and Technology, Universiti Malaysia Pahang, Lebuhraya Persiaran Tun Khalil Yaakob, 26300 Gambang, Kuantan, Pahang Darul Makmur, Malaysia
  • Chin Mei Lee Faculty of Industrial Sciences and Technology, Universiti Malaysia Pahang, Lebuhraya Persiaran Tun Khalil Yaakob, 26300 Gambang, Kuantan, Pahang Darul Makmur, Malaysia
  • Thong Chuan Lee Faculty of Industrial Sciences and Technology, Universiti Malaysia Pahang, Lebuhraya Persiaran Tun Khalil Yaakob, 26300 Gambang, Kuantan, Pahang Darul Makmur, Malaysia


brown sugar, fermentation, probiotics, palm sugar, water kefir


Probiotics are live-friendly microorganisms that can confer a health benefit on the host if it is consumed in sufficient amounts. Water kefir is a probiotic-rich fermented beverage that contains multi-species of live cultures. Brown sugar and palm sugar were used for water kefir fermentation due to their high sucrose and mineral contents. The objective of this study was to determine the probiotic growth pattern of water kefir and to evaluate the physicochemical parameters, including the pH changes, lactic acid content, reducing sugar content, and total soluble solids. The fermented water kefir was collected at every 6-hour interval, until the end of 72 hours of fermentation. The growth curve was determined by enumerated probiotics on De Man, Rogosa, and Sharpe (MRS) agar, Yeast Extract-Peptone-Dextrose (YPD) agar, and Gluconobacter (GM) agar plates, respectively. MRS, YPD, and GM agar plates were used to enumerate lactic acid bacteria, yeast, and acetic acid bacteria, respectively. The result showed increased probiotic growth as fermentation time increased with different phases observed from the growth curve. The stationary phase of probiotics was recorded at 30-42 h and was recommended as the optimal harvesting point. Besides, longer fermentation time produced lower pH values and lower total soluble solids while higher lactic acid and higher reducing sugars. At the end of fermentation, the concentration of lactic acid and reducing sugars were 2.16 ± 0.09 g/L and 13.66 ± 0.14 mg/mL, respectively. In conclusion, probiotics from water kefir fermentation are suggested to be best harvested between 30-42 hours and can be used for self-consume or downstream processing.


Download data is not yet available.


Metrics Loading ...


Abedi, E. & Hashemi, S.M.B. 2020. Lactic acid production – producing microorganisms and substrates sources-state of art. Heliyon, 6(10): e04974. DOI:

AOAC. 2012. Official Methods of Analysis of the Association of Analytical Chemists International. 20th ed. AOAC international, Gaithersburg, USA.

Azizi, N.F., Kumar, M.R., Yeap, S.K., Abdullah, J.O., Khalid, M., Omar, A.R., Osman, M.A., Mortadza, S.A.S. & Alitheen, N.B. 2021. Kefir and its biological activities. Foods, 10(6): 1210. DOI:

Broeckx, G., Kiekens, S., Jokicevic, K., Byl, E., Henkens, T., Vandenheuvel, D., Lebeer, S. & Kiekens, F. 2020. Effects of initial cell concentration, growth phase, and process parameters on the viability of Lactobacillus rhamnosus GG after spray drying. Drying Technology, 38(11): 1−19. DOI:

Calatayud, M., Börner, R.A., Ghyselinck, J., Verstrepen, L., De Medts, J., Van den Abbeele, P., Boulangé, C.L., Priour, S., Marzorati, M. & Damak, S. 2021. Water kefir and derived pasteurized beverages modulate gut microbiota, intestinal permeability and cytokine production in vitro. Nutrients, 13(11): 3897. DOI:

Çevik, T., Aydoğdu, N. S., Özdemir, N. & Kök Taş, T. 2019. The effect of different sugars on water kefir grains. Turkish Journal of Agriculture - Food Science and Technology, 7(sp1): 40−45. DOI:

Dahiya, D. & Nigam, P.S. 2023. Therapeutic and dietary support for gastrointestinal tract using kefir as a nutraceutical beverage: Dairy-milk-based or plant-sourced kefir probiotic products for vegan and lactose-intolerant populations. Fermentation, 9(4). DOI:

Destro, T.M., Prates, D.da F., Watanabe, L.S., Garcia, S., Biz, G. & Spinosa, W.A. 2019. Organic brown sugar and jaboticaba pulp influence on water kefir fermentation. Ciencia e Agrotecnologia, 43(6). DOI:

Dwiloka, B., Rizqiati, H. & Setiani, B.E. 2020. Physicochemical and sensory characteristics of green coconut (Cocos nucifera L.) water kefir. International Journal of Food Studies, 9(2): 346–359. DOI:

Egea, M.B., Santos, D.C. dos, Oliveira Filho, J.G. de, Ores, J.da C., Takeuchi, K.P. & Lemes, A.C. 2022. A review of nondairy kefir products: their characteristics and potential human health benefits. Critical Reviews in Food Science and Nutrition, 62(02): 1536–1552. DOI:

FAO/WHO. Food and Agriculture Organization and World Health Organization (FAO/WHO) 2001. Probiotics in Food: Health and Nutritional Properties of Probiotics in Food Including Powder Milk With Live Lactic Acid Bacteria. World Health Organization, Food and Agriculture Organization of the United Nations, Rome.

Gamba, R.R., Yamamoto, S., Sasaki, T., Michihata, T., Mahmoud, A.H., Koyanagi, T. & Enomoto, T. 2019. Microbiological and functional characterization of kefir grown in different sugar solutions. Food Science and Technology Research, 25(2): 303–312. DOI:

Gökırmaklı, Ç., Yüceer, Y.K. & Guzel-Seydim, Z.B. 2023. Chemical, microbial, and volatile changes of water kefir during fermentation with economic substrates. European Food Research and Technology, 249(7): 1717–1728. DOI:

Kareena, A., Siripongvutikorn, S., Usawakesmanee, W. & Wichienchot, S. 2022. In vitro evaluation of probiotic bacteria and yeast growth, pH changes and metabolites produced in a pure culture system using protein base products with various added carbon sources. Food Science and Technology, 42: 1–10. DOI:

Koirala, S. & Anal, A.K. 2021. Probiotics-based foods and beverages as future foods and their overall safety and regulatory claims. Future Foods, 3: 100013. DOI:

Kongkaew, S., Chaijan, M. & Riebroy, S. 2014. Some characteristics and antioxidant activity of commercial sugars produced in Thailand. Kmitl Science and Technology Journal, 14(1): 1–9.

Laureys, D. & De Vuyst, L. 2014. Microbial species diversity, community dynamics, and metabolite kinetics of water Kefir fermentation. Applied and Environmental Microbiology, 80(8): 2564–2572. DOI:

Laureys, D., Aerts, M., Vandamme, P. & De Vuyst, L. 2019. The buffer capacity and calcium concentration of water influence the microbial species diversity, grain growth, and metabolite production during water kefir fermentation. Frontiers in Microbiology, 10: 02876. DOI:

Laureys, D., Leroy, F., Hauffman, T., Raes, M., Aerts, M., Vandamme, P. & De Vuyst, L. 2021. The type and concentration of inoculum and substrate as well as the presence of oxygen impact the water kefir fermentation process. Frontiers in Microbiology, 12: 628599. DOI:

Laureys, D., Leroy, F., Vandamme, P. & De Vuyst, L. 2022. Backslopping time, rinsing of the grains during backslopping, and incubation temperature influence the water kefir fermentation process. Frontiers in Microbiology, 13: 8571550. DOI:

Laureys, D., Van Jean, A., Dumont, J. & De Vuyst, L. 2017. Investigation of the instability and low water kefir grain growth during an industrial water kefir fermentation process. Applied Microbiology and Biotechnology, 101(7): 2811–2819. DOI:

Lengkey, H.A.W. & Balia, R.L. 2014. The effect of starter dosage and fermentation time on pH and lactic acid production. Biotechnology in Animal Husbandry, 30(2): 339–347. DOI:

Limbad, M., Gutierrez-Maddox, N., Hamid, N., Kantono, K., Liu, T. & Young, T. 2023. Microbial and chemical changes during fermentation of coconut water kefir beverage. Applied Sciences, 13(12): 7257. DOI:

Łopusiewicz, Ł., Drozłowska, E., Trocer, P., Kwiatkowski, P., Bartkowiak, A., Gefrom, A. & Sienkiewicz, M. 2020. The effect of fermentation with kefir grains on the physicochemical and antioxidant properties of beverages from blue lupin (Lupinus angustifolius L.) seeds. Molecules, 25:5791. DOI:

Lynch, K.M., Wilkinson, S., Daenen, L. & Arendt, E.K. 2021. An update on water kefir: Microbiology, composition and production. International Journal of Food Microbiology, 345: 109128. DOI:

Miller, G.L. 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry, 31(3): 426–428. DOI:

Pendón, M.D., Bengoa, A.A., Iraporda, C., Medrano, M., Garrote, G.L. & Abraham, A.G. 2021. Water kefir: Factors affecting grain growth and health-promoting properties of the fermented beverage. Journal of Applied Microbiology, 133(1): 162−180. DOI:

Ram, Y., Dellus-Gur, E., Bibi, M., Karkare, K., Obolski, U., Feldman, M.W., Cooper, T.F., Berman, J. & Hadany, L. 2019. Predicting microbial growth in a mixed culture from growth curve data. Proceedings of the National Academy of Sciences of the United States of America, 116(29):14698–14707. DOI:

Rickett, L.M., Pullen, N., Hartley, M., Zipfel, C., Kamoun, S., Baranyi, J. & Morris, R.J. 2015. Incorporating prior knowledge improves detection of differences in bacterial growth rate. BMC Systems Biology, 9(1): 60. DOI:

Sharifi, M., Moridnia, A., Mortazavi, D., Salehi, M., Bagheri, M. & Sheikhi, A. 2017. Kefir: A powerful probiotics with anticancer properties. Medical Oncology, 34(11):183. DOI:

Srikaeo, K., Sangkhiaw, J. & Likittrakulwong, W. 2019. Productions and functional properties of palm sugars. Walailak Journal of Science and Technology, 16(11):897–907. DOI:

Stadie, J., Gulitz, A., Ehrmann, M.A. & Vogel, R.F. 2013. Metabolic activity and symbiotic interactions of lactic acid bacteria and yeasts isolated from water kefir. Food Microbiology, 35(2): 92–98. DOI:

Staniszewski, A. & Kordowska-Wiater, M. 2021. Probiotic and potentially probiotic yeasts—characteristics and food application. Foods, 10(6): 1–13. DOI:

Tzavaras, D., Papadelli, M. & Ntaikou, I. 2022. From milk kefir to water kefir: Assessment of fermentation processes, microbial changes and evaluation of the produced beverages. Fermentation, 8(3): 135. DOI:

Verce, M., De Vuyst, L. & Weckx, S. 2019. Shotgun metagenomics of a water kefir fermentation ecosystem reveals a novel Oenococcus species. Frontiers in Microbiology, 10:479. DOI:

Veselovsky, V.A., Dyachkova, M.S., Bespiatykh, D.A., Yunes, R.A., Shitikov, E.A., Polyaeva, P.S., Danilenko, V.N., Olekhnovich, E.I. & Klimina, K.M. 2022. The gene expression profile differs in growth phases of the Bifidobacterium longum culture. Microorganisms, 10(8): 1683. DOI:

Winarni, S., Arifan, F., Wisnu Broto, R.T.D., Fuadi, A. & Alviche, L. 2018. Nira acidity and antioxidant activity of palm sugar in Sumowono Village. Journal of Physics: Conference Series, 1025(1): 012052. DOI:

Yang, X., Hong, J., Wang, L., Cai, C., Mo, H., Wang, J., Fang, X. & Liao, Z. 2024. Effect of lactic acid bacteria fermentation on plant-based products. Fermentation, 10(1): 48. DOI:



How to Cite

Sin, P. Y., Tan, S. H., Asras, M. F. F., Lee, C. M., & Lee, T. C. (2024). Probiotic Growth Pattern and Physicochemical Evaluation of Water Kefir Fermentation. Malaysian Applied Biology, 53(2), 21–30.



Research Articles

Funding data