Volume 13, Issue 2 (June 2026)                   J. Food Qual. Hazards Control 2026, 13(2): 121-131 | Back to browse issues page

Ethics code: Not applicable.


XML Print


Download citation:
BibTeX | RIS | EndNote | Medlars | ProCite | Reference Manager | RefWorks
Send citation to:

Satiyarti R, Windayani N, Cahyanto T, Juwitaningsih T, Hidayat R. Effect of Refrigerated Fermentation on Lactic Acid Bacteria Growth in Milk Kefir. J. Food Qual. Hazards Control 2026; 13 (2) :121-131
URL: http://jfqhc.ssu.ac.ir/article-1-1401-en.html
Department of Magister Science Education, State Islamic University Sunan Gunung Djati, Bandung, Indonesia , nenengwinda.ftk@uinsgd.ac.id
Abstract:   (160 Views)
Background: Kefir, a probiotic-rich fermented milk product, offers significant health benefits. However, its ethanol content remains a concern for halal compliance. This study assesses the impact of low-temperature fermentation on ethanol content, Lactic Acid Bacteria (LAB) viability, and antioxidant activity of milk kefir as key quality indicators relevant to halal compliance and potential health benefits.
Methods: Milk kefir was fermented at 2–10 °C using varying starter concentrations (5–50%) and analyzed for pH, LAB viability, ethanol content (determined by iodometric titration), and antioxidant activity (using the 2,2-diphenyl-1-picrylhydrazyl [DPPH] method). Sensory properties were assessed using a hedonic quality test with 15 semi-trained panelists. Statistical analysis was conducted using analysis of variance (ANOVA) with Tukey's post hoc test (p<0.05), and LAB growth kinetics were modeled using Gaussian regression.
Results: Fermentation at low temperatures (2–10 °C) for 72 h reduced ethanol levels (<0.5%), ensuring compliance with halal certification standards while maintaining optimal LAB growth (107 Colony Forming Unit [CFU]/ml) and strong antioxidant activity (Half Maximal Inhibitory Concentration [IC50] = 1.7586 parts per million [ppm]). The Gaussian kinetic model best represented LAB growth dynamics (R² = 0.948). The optimized kefir formulation with 50% starter concentration exhibited enhanced sensory properties and favorable probiotic characteristics.
Conclusion: In summary, low-temperature fermentation offers a practical approach to better control ethanol formation while maintaining LAB viability and enhancing antioxidant activity. These improvements support the production of kefir with more favorable quality attributes relevant to halal compliance and product acceptability; however, broader health-related claims should be confirmed in future studies through targeted metabolite profiling and expanded functional assays.

DOI: 10.18502/jfqhc.13.2.22141

 
Full-Text [PDF 632 kb]   (123 Downloads)    
Type of Study: Original article | Subject: Special
Received: 25/08/04 | Accepted: 26/06/27 | Published: 26/07/01

References
1. Abou Ayana, I. and Saber, W. (2016) 'Optimization of milk type and physical factors for reduction of alcohol content in kefir', Journal of Food and Dairy Sciences, 7(3), pp. 161-166. Available at: [DOI:10.21608/jfds.2016.42959]
2. Abou Ayana, I.A.A., Al-Otibi, F.O., Elgarhy, M.R., Omar, M.M., EL-Abbassy, M.Z., Khalifa, S.A., Helmy, Y.A. and Saber, W.I.A. (2025) 'Chemical, physical, microbial, and sensory properties of innovative sesame milk kefir, focusing on the ultrastructure of kefir grains', ACS Omega, 10(8), pp. 7752-7769. Available at: [DOI:10.1021/acsomega.4c08044] [PMID] [PMCID]
3. Alves, E., Ntungwe, E.N., Gregório, J., Rodrigues, L.M., Pereira-Leite, C., Caleja, C., Pereira, E., Barros, L., Aguilar-Vilas, M.V., Rosado, C. and Rijo, P. (2021) 'Characterization of kefir produced in household conditions: physicochemical and nutritional profile, and storage stability', Foods, 10(5), p. 1057. Available at: [DOI:10.3390/foods10051057] [PMID] [PMCID]
4. AOAC International (2019) 'Method 920.57 - Alcohol by volume in beer', In: Official Methods of Analysis of AOAC International. 21st ed. Gaithersburg, MD: AOAC International
5. Apalowo, O.E., Adegoye, G.A., Mbogori, T., Kandiah, J. and Obuotor, T.M. (2024) 'Nutritional characteristics, health impact, and applications of kefir', Foods, 13(7), p. 1026. Available at: [DOI:10.3390/foods13071026] [PMID] [PMCID]
6. Atma, Y., Taufik, M. and Seftiono, H. (2018) 'Identifikasi resiko titik kritis kehalalan produk pangan: studi produk bioteknologi', Jurnal Teknologi, 10(1), pp. 59-65. Available at: [DOI:10.24853/jurtek.10.1.59-66]
7. Awasti, N. and Anand, S. (2020) 'The Role of yeast and molds in dairy industry: an update', In: Dairy Processing: Advanced Research to Applications. Singapore: Springer Singapore, pp. 243-262. Available at: [DOI:10.1007/978-981-15-2608-4_12]
8. Azizi, N.F., Kumar, M.R., Yeap, S.K., Abdullah, J.O., Khalid, M., Omar, A.R., Osman, M.A., Mortadza, S.A.S. and Alitheen, N.B. (2021) 'Kefir and its biological activities', Foods, 10(6), p. 1210. Available at: [DOI:10.3390/foods10061210] [PMID] [PMCID]
9. Blasche, S., Kim, Y., Mars, R.A.T., Machado, D., Maansson, M., Kafkia, E., Milanese, A., Zeller, G., Teusink, B., Nielsen, J., Benes, V., Neves, R., Sauer, U. and Patil, K.R. (2021) 'Metabolic cooperation and spatiotemporal niche partitioning in a kefir microbial community', Nature Microbiology, 6(2), pp. 196-208. Available at: [DOI:10.1038/s41564-020-00816-5] [PMID] [PMCID]
10. Ciani, M., Morales, P., Comitini, F., Tronchoni, J., Canonico, L., Curiel, J.A., Oro, L., Rodrigues, A.J. and Gonzalez, R. (2016) 'Non-conventional yeast species for lowering ethanol content of wines', Frontiers in Microbiology, 7, p. 642. Available at: [DOI:10.3389/fmicb.2016.00642]
11. De Sainz, I., Redondo-Solano, M., Solano, G. and Ramírez, L. (2025) 'Optimization of process conditions and kinetic microbial growth for milk fermentation using domestic kefir grains from Costa Rica', Italian Journal of Food Safety, 14(3). Available at: [DOI:10.4081/ijfs.2025.12477] [PMID] [PMCID]
12. Dong, J., Bobe, G., Guan, Y., Li, G., Zuo, R., Shu, X., Wang, Y., Sun, X., Chen, X. and Li, X. (2020) 'Mitochondrial membrane protein mitofusin 2 as a potential therapeutic target for treating free fatty acid-induced hepatic inflammation in dairy cows during early lactation', Journal of Dairy Science, 103(6), pp. 5561-5574. Available at: [DOI:10.3168/jds.2019-17652] [PMID]
13. Egea, M.B., Santos, D.C. dos, Oliveira Filho, J.G. de, Ores, J. da C., Takeuchi, K.P. and 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(6), pp. 1536-1552. Available at: [DOI:10.1080/10408398.2020.1844140] [PMID]
14. Fernández-Martínez, L.T., Javelle, A. and Hoskisson, P.A. (2024) 'Microbial primer: bacterial growth kinetics', Microbiology, 170(2). Available at: [DOI:10.1099/mic.0.001428] [PMID] [PMCID]
15. Fiorda, F.A., de Melo Pereira, G.V., Thomaz-Soccol, V., Rakshit, S.K., Pagnoncelli, M.G.B., Vandenberghe, L.P. de S. and Soccol, C.R. (2017) 'Microbiological, biochemical, and functional aspects of sugary kefir fermentation - a review', Food Microbiology, 66, pp. 86-95. Available at: [DOI:10.1016/j.fm.2017.04.004] [PMID]
16. Fitrianingsih, F., Hafid, H., Kimestri, A.B., Sulfitrana, A. and Toba, R.D.S. (2022) 'The effect of different kefir grain starter concentration on pH, alcohol percentage, and organoleptic properties of ultra high temperature (UHT) milk kefir', in Proceedings of the International Conference on Improving Tropical Animal Production for Food Security (ITAPS 2021), pp. 399-403. Available at: [DOI:10.2991/absr.k.220309.078]
17. Ganatsios, V., Nigam, P., Plessas, S. and Terpou, A. (2021) 'Kefir as a functional beverage gaining momentum towards its health promoting attributes', Beverages, 7(3), p. 48. Available at: [DOI:10.3390/beverages7030048]
18. Garofalo, C., Osimani, A., Milanović, V., Aquilanti, L., De Filippis, F., Stellato, G., Di Mauro, S., Turchetti, B., Buzzini, P., Ercolini, D. and Clementi, F. (2015) 'Bacteria and yeast microbiota in milk kefir grains from different Italian regions', Food Microbiology, 49, pp. 123-133. Available at: [DOI:10.1016/j.fm.2015.01.017] [PMID]
19. Gökırmaklı, Ç., Yüceer, Y.K. and 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), pp. 1717-1728. Available at: [DOI:10.1007/s00217-023-04242-9]
20. Gul, O., Mortas, M., Atalar, I., Dervisoglu, M. and Kahyaoglu, T. (2015) 'Manufacture and characterization of kefir made from cow and buffalo milk, using kefir grain and starter culture', Journal of Dairy Science, 98(3), pp. 1517-1525. Available at: [DOI:10.3168/jds.2014-8755] [PMID]
21. Güzel-Seydim, Z.B., Ece Cagdas, M.D. and Seydim, A.C. (2016) 'Effect of kefir on fusobacterium nucleatum potentially causing intestinal cancer', Functional Foods in Health and Disease, 6(7), p. 469. Available at: [DOI:10.31989/ffhd.v6i7.272]
22. Guzel-Seydim, Z.B., Kok-Tas, T., Greene, A.K. and Seydim, A.C. (2011) 'Review: functional properties of kefir', Critical Reviews in Food Science and Nutrition, 51(3), pp. 261-268. Available at: [DOI:10.1080/10408390903579029] [PMID]
23. Halim, Y., Sukmawardani, Y. and Windayani, N. (2023) 'Determination of bioethanol in fermented products with redox titration', in Proceedings of the Symposium on Advance of Sustainable Engineering 2021 (SIMASE 2021), p. 030019. Available at: [DOI:10.1063/5.0114198]
24. Hsieh, K.-C., Lin, T.-C. and Kuo, M.-I. (2022) 'Effect of whole chia seed flour on gelling properties, microstructure and texture modification of tofu', LWT, 154, p. 112676. Available at: [DOI:10.1016/j.lwt.2021.112676]
25. Ilyin, V.K., Komissarova, D. V., Afonin, B. V., Usanova, N.A., Morozova, Y.A., Muravieva, V. V., Bairamova, G.R. and Priputnevich, T. V. (2023) 'Effect of probiotic consumption with a fermented drink on intestinal microbiota, mucosae, and gastrointestinal functions in humans', Human Physiology, 49(7), pp. 845-850. Available at: [DOI:10.1134/S0362119723070083]
26. Jiang, X., Ren, S., Geng, Y., Jiang, C., Liu, G., Wang, H., Yu, T. and Liang, Y. (2021) 'Role of the VirSR-VirAB system in biofilm formation of Listeria monocytogenes EGD-e', Food Research International, 145, p. 110394. Available at: [DOI:10.1016/j.foodres.2021.110394] [PMID]
27. John, S.M. and Deeseenthum, S. (2015) 'Properties and benefits of kefir - A review', Songklanakarin Journal of Science and Technology, 37(3), pp. 275-282. Available at: https://www.cabdirect.org/cabdirect/abstract/20153258255
28. Joni, L.S., Erina, E. and Abrar, M. (2018) 'The total of lactic acid bacteria (lab) on feces of Sambar Deer (Cervus unicolor) in Taman Rusa Aceh Besar', Jurnal Ilmiah Mahasiswa Veteriner, 2(2), pp. 77-85. Available at: [DOI:10.21157/jim20vet..v2i2.6782]
29. Juan, J., Wang, Q., Gao, Z., Xiao, T., Chen, H., Zhang, J., Song, X. and Huang, J. (2022) 'Analysis of the composition of substrate for industrial fermentation of agaricus bisporus based on secondary and tertiary fermentation mode composition analysis of industrial fermentation substrates of A. bisporus', Fermentation, 8(5), p. 222. Available at: [DOI:10.3390/fermentation8050222]
30. Kaur, H., Kaur, G. and Ali, S.A. (2022) 'Dairy-based probiotic-fermented functional foods: an update on their health-promoting properties', Fermentation, 8(9), p. 425. Available at: [DOI:10.3390/fermentation8090425]
31. Kim, H., Oh, S. and Song, S. (2024) 'Lactobacillus Persisters formation and resuscitation', Journal of Microbiology and Biotechnology, 34(4), pp. 854-862. Available at: [DOI:10.4014/jmb.2312.12035] [PMID] [PMCID]
32. Kourkoutas, Y. and Proestos, C. (2020) 'Food preservation: challenges and efforts for the future', Foods, 9(4), p. 391. Available at: [DOI:10.3390/foods9040391] [PMID] [PMCID]
33. Kurniadi, M. and Frediansyah, A. (2017) 'Halal perspective of microbial bioprocess based-food products', REAKTOR, 16(3), p. 147. Available at: [DOI:10.14710/reaktor.16.3.147-160]
34. Lestari, M.W., Bintoro, V.P. and Rizqiati, H. (2018) 'Effect of fermentation time on acidity, viscosity, alcohol concentration, and hedonic quality of coconut (Cocos nucifera) water kefir', Jurnal Teknologi Pangan, 2(1), pp. 8-13. Available at: [DOI:10.14710/jtp.v2i1.20750]
35. Liu, J.-R., Chen, M.-J. and Lin, C.-W. (2005) 'Antimutagenic and antioxidant properties of milk−kefir and soymilk−kefir', Journal of Agricultural and Food Chemistry, 53(7), pp. 2467-2474. Available at: [DOI:10.1021/jf048934k] [PMID]
36. Liu, W., Pang, H., Zhang, H. and Cai, Y. (2014) 'Biodiversity of lactic acid bacteria', in Lactic Acid Bacteria. Dordrecht: Springer Netherlands, pp. 103-203. Available at: [DOI:10.1007/978-94-017-8841-0_2]
37. Magalhães, K.T., Pereira, G.V. de M., Campos, C.R., Dragone, G. and Schwan, R.F. (2011) 'Brazilian kefir: structure, microbial communities and chemical composition', Brazilian Journal of Microbiology, 42(2), pp. 693-702. Available at: [DOI:10.1590/S1517-83822011000200034] [PMID] [PMCID]
38. Molyneux, P. (2004) 'The use of the stable free radical diphenylpicrylhydrazyl (DPPH) for estimating antioxidant activity', Songklanakarin Journal of Science and Technology (SJST), 26(2), pp. 211-219. Available at: https://sjst.psu.ac.th/article/214
39. Nampoothiri, K.M., Beena, D.J., Vasanthakumari, D.S. and Ismail, B. (2017) 'Health benefits of exopolysaccharides in fermented foods', In: Fermented Foods in Health and Disease Prevention. Elsevier, pp. 49-62. Available at: [DOI:10.1016/B978-0-12-802309-9.00003-0]
40. Okoniewski, A., Dobrzyńska, M., Kusyk, P., Dziedzic, K., Przysławski, J. and Drzymała-Czyż, S. (2023) 'The role of fermented dairy products on gut microbiota composition', Fermentation, 9(3), p. 231. Available at: [DOI:10.3390/fermentation9030231]
41. Papadimitriou, K., Alegría, Á., Bron, P.A., de Angelis, M., Gobbetti, M., Kleerebezem, M., Lemos, J.A., Linares, D.M., Ross, P., Stanton, C., Turroni, F., van Sinderen, D., Varmanen, P., Ventura, M., Zúñiga, M., Tsakalidou, E. and Kok, J. (2016) 'Stress physiology of lactic acid bacteria', Microbiology and Molecular Biology Reviews, 80(3), pp. 837-890. Available at: [DOI:10.1128/MMBR.00076-15] [PMID] [PMCID]
42. Plessas, S., Nouska, C., Karapetsas, A., Kazakos, S., Alexopoulos, A., Mantzourani, I., Chondrou, P., Fournomiti, M., Galanis, A. and Bezirtzoglou, E. (2017) 'Isolation, characterization and evaluation of the probiotic potential of a novel Lactobacillus strain isolated from Feta-type cheese', Food Chemistry, 226, pp. 102-108. Available at: [DOI:10.1016/j.foodchem.2017.01.052] [PMID]
43. Pourbaba, H., Anvar, A.A., Pourahmad, R. and Ahari, H. (2022) 'Changes in acidity parameters and probiotic survival of the kefir using Lactobacillus acidophilus and Lactobacillus paracasei complementary probiotics during cold preservation', Iranian Journal of Veterinary Medicine, 16(1), pp. 89-98. Avaialble at: [DOI:10.22059/ijvm.2020.313626.1005141]
44. Priyashantha, H., Johansson, M., Langton, M., Sampels, S., Jayarathna, S., Hetta, M., Saedén, K.H., Höjer, A. and Lundh, Å. (2021) 'Variation in dairy milk composition and properties has little impact on cheese ripening: insights from a traditional swedish long-ripening cheese', Dairy, 2(3), pp. 336-355. Available at: [DOI:10.3390/dairy2030027]
45. Ribeiro, J.G.T. and Cunha, A. (2025) 'Advanced creep modelling for polymers: a variable-order fractional calculus approach', Proceedings of the Royal Society A: Mathematical, Physical and Engineering Sciences, 481(2322). Available at: [DOI:10.1098/rspa.2024.0861]
46. Sadewi, S.M., Nurhasanah, N., Sudibyo, S., Windayani, N., Kiswandono, A.A. and Satria, H. (2023) 'Antioxidant and antibacterial activities of curd and whey kefir produced from Etawa goat milk', Journal of Multidisciplinary Applied Natural Science, 4(1), pp. 139-145. Available at: [DOI:10.47352/jmans.2774-3047.200]
47. Setiawati, A.E., Sari, I.N.I. and Hasyati, N. (2021) 'Effect of temperature and time storage towards alcohol level in cow milk kefir', IOP Conference Series: Earth and Environmental Science, 733(1), p. 012108. Available at: [DOI:10.1088/1755-1315/733/1/012108]
48. Sharma, R., Garg, P., Kumar, P., Bhatia, S.K. and Kulshrestha, S. (2020) 'Microbial fermentation and its role in quality improvement of fermented foods', Fermentation, 6(4), p. 106. Available at: [DOI:10.3390/fermentation6040106]
49. Simova, E., Beshkova, D., Angelov, A., Hristozova, T., Frengova, G. and Spasov, Z. (2002) 'Lactic acid bacteria and yeasts in kefir grains and kefir made from them', Journal of Industrial Microbiology and Biotechnology, 28(1), pp. 1-6. Available at: [DOI:10.1038/sj.jim.7000186] [PMID]
50. Soutelino, M.E.M., da Silva Rocha, R., Teixeira Mársico, E., Almeida Esmerino, E. and Cristina de Oliveira Silva, A. (2025) 'Innovative approaches to kefir production, challenges, and current remarks', Current Opinion in Food Science, 61, p. 101252. Available at: [DOI:10.1016/j.cofs.2024.101252]
51. Tingirikari, J.M.R., Sharma, A. and Lee, H.-J. (2024) 'Kefir: a fermented plethora of symbiotic microbiome and health', Journal of Ethnic Foods, 11(1), p. 35. Available at: [DOI:10.1186/s42779-024-00252-4]
52. Vitali, M., Gandía, M., Garcia-Llatas, G., González-Sarrías, A., Vallejo, F., Cilla, A. and Gamero, A. (2025) 'Modulation of antioxidant capacity, nutritional composition, probiotic viability after digestion and sensory attributes of plant-based beverages through lactic acid fermentation', Foods, 14(9), p. 1447. Available at: [DOI:10.3390/foods14091447] [PMID] [PMCID]
53. Wang, Yaqi, Wu, J., Lv, M., Shao, Z., Hungwe, M., Wang, J., Bai, X., Xie, J., Wang, Yanping and Geng, W. (2021) 'Metabolism characteristics of lactic acid bacteria and the expanding applications in food industry', Frontiers in Bioengineering and Biotechnology, 9, p. 612285. Available at: [DOI:10.3389/fbioe.2021.612285] [PMID] [PMCID]
54. Widyastuti, Y., Febrisiantosa, A. and Tidona, F. (2021) 'Health-promoting properties of lactobacilli in fermented dairy products', Frontiers in Microbiology, 12. Available at: [DOI:10.3389/fmicb.2021.673890] [PMID] [PMCID]
55. Windayani, N., Kurniati, T. and Listiawati, M. (2019) 'Psychochemical and organoleptic characteristics of colostrum kefir as antibacterial', In: Journal of Physics: Conference Series, p. 012016. Available at: [DOI:10.1088/1742-6596/1175/1/012016]
56. Windayani, N., Kurniati, T. and Rukayadi, Y. (2020) 'Antibacterial activity of colostrum kefir against foodborne pathogen bacteria', In: IOP Conference Series: Earth and Environmental Science, p. 012019. Available at: [DOI:10.1088/1755-1315/472/1/012019]
57. Yilmaz, B., Sharma, H., Melekoglu, E. and Ozogul, F. (2022) 'Recent developments in dairy kefir-derived lactic acid bacteria and their health benefits', Food Bioscience, 46, p. 101592. Available at: [DOI:10.1016/j.fbio.2022.101592]

Add your comments about this article : Your username or Email:
CAPTCHA

Rights and permissions
Creative Commons License This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

© 2026 CC BY-NC 4.0 | Journal of food quality and hazards control

Designed & Developed by : Yektaweb