food health Food and Health 2602-2834 Özkan ÖZDEN 10.3153/FH25010 Nutritional Science Public Health Nutrition Food Biotechnology Food Sciences (Other) Beslenme Bilimi Halk Sağlığı Beslenmesi Gıda Biyoteknolojisi Gıda Bilimleri (Diğer) Impacts of milk processing and fermentation on microRNA levels in cow’s milk https://orcid.org/0000-0002-0522-9432 Pirim Dilek Bursa Uludağ Üniversitesi https://orcid.org/0000-0001-5846-7648 Gözüdok İrem Nur BURSA ULUDAG UNIVERSITY https://orcid.org/0000-0002-8247-9347 Çobanoğlu Özden BURSA ULUDAG UNIVERSITY, FACULTY OF VETERINARY MEDICINE https://orcid.org/0000-0002-5187-9380 Güldaş Metin BURSA ULUDAĞ ÜNİVERSİTESİ, SAĞLIK BİLİMLERİ FAKÜLTESİ https://orcid.org/0000-0001-7871-1628 Gürbüz Ozan BURSA ULUDAG UNIVERSITY 03 28 2025 11 2 127 138 11 28 2024 12 27 2024 Copyright © 2015, Food and Health 2015 Food and Health

Recent evidence suggests that milk-derived microRNAs (miRNAs) are bioactive components of milk that can influence host cells through cross-kingdom miRNA transfer mechanisms. Therefore, it is essential to assess the content and stability of these miRNAs in drinking milk and milk products to explore their potential roles in human health. Here, we examined the small RNAs and microRNA levels in raw and processed milk samples, including plain and prebiotic-rich kefir. Total RNA was isolated from milk samples processed with different heat treatments and fermentation. The effects of milk processing on specific miRNAs were investigated by RT-qPCR, which evaluated the quantities of four miRNAs related to human diseases. We found that miR-21 and miR-125b could resist harsh conditions applied in milk processing plants. However, no detectable amounts of the tested miRNAs were found in kefir samples by qPCR. Our study highlights the miRNA-specific effects of milk processing methods on milk miRNA content. Future studies focusing on total small RNA content in kefir and other milk products may offer valuable insights into the functional role of milk-derived miRNA. Overall, miRNAs in drinking milk warrant further attention for their potential importance for public health.

 microRNA Milk Milk-derived miRNAs Kefir Biomarker Bursa Uludag University Scientific Research Projects Unit FYL-2022-1233 and FGA-2022-820 Abou el Qassim, L., Alonso, J., Zhao, K., Le Guillou, S., Diez, J., Vicente, F., … Royo, L.J. (2022). Differences in the microRNAs Levels of Raw Milk from Dairy Cattle Raised under Extensive or Intensive Production Systems. Veterinary Sciences, 9(12), 661. https://doi.org/10.3390/vetsci9120661 Abou el Qassim, L., Le Guillou, S., & Royo, L.J. (2022). Variation of miRNA content in cow raw milk depends on the dairy production system. International Journal of Molecular Sciences, 23(19), 11681. https://doi.org/10.3390/ijms231911681 Abou el Qassim, L., Martínez, B., Rodríguez, A., Dávalos, A., López de las Hazas, M.-C., Menéndez Miranda, M., & Royo, L.J. (2023). Effects of cow’s milk processing on microRNA levels. Foods, 12(15), 2950. https://doi.org/10.3390/foods12152950 Baier, S.R., Nguyen, C., Xie, F., Wood, J.R., & Zempleni, J. (2014). MicroRNAs are absorbed in biologically meaningful amounts from nutritionally relevant doses of cow milk and affect gene expression in peripheral blood mononuclear cells, HEK-293 kidney cell cultures, and mouse livers. The Journal of Nutrition, 144(10), 1495–1500. https://doi.org/10.3945/jn.114.196436 Benmoussa, A., Gotti, C., Bourassa, S., Gilbert, C., & Provost, P. (2019). Identification of protein markers for extracellular vesicle (EV) subsets in cow’s milk. Journal of Proteomics, 192, 78–88. https://doi.org/10.1016/j.jprot.2018.08.010 Benmoussa, A., Laugier, J., Beauparlant, C.J., Lambert, M., Droit, A., & Provost, P. (2020). Complexity of the microRNA transcriptome of cow milk and milk-derived extracellular vesicles isolated via differential ultracentrifugation. Journal of Dairy Science, 103(1), 16–29. https://doi.org/10.3168/jds.2019-16880 Benmoussa, A., Lee, C.H.C., Laffont, B., Savard, P., Laugier, J., Boilard, E., … Provost, P. (2016). Commercial dairy cow milk microRNAs resist digestion under simulated gastrointestinal tract conditions. The Journal of Nutrition, 146(11), 2206–2215. https://doi.org/10.3945/jn.116.237651 Cao, X., Yang, Q., & Yu, Q. (2020). Increased expression of mir-487b is associated with poor prognosis and tumor progression of HBV-related hepatocellular carcinoma. Open Forum Infectious Diseases, 7(12), ofaa498. https://doi.org/10.1093/ofid/ofaa498 Chen, Q., Zhang, F., Dong, L., Wu, H., Xu, J., Li, H., … Zhang, C.-Y. (2021). SIDT1-dependent absorption in the stomach mediates host uptake of dietary and orally administered microRNAs. Cell Research, 31(3), 247–258. https://doi.org/10.1038/s41422-020-0389-3 Fabris, L., & Calin, G.A. (2016). Circulating free xeno-microRNAs—The new kids on the block. Molecular Oncology, 10(3), 503–508. https://doi.org/10.1016/j.molonc.2016.01.005 Fox, P.F., Cogan, T.M., & Guinee, T.P. (2017). Chapter 25—Factors That Affect the Quality of Cheese. In P.L.H. McSweeney, P.F. Fox, P.D. Cotter, & D.W. Everett (Eds.), Cheese (Fourth Edition) (pp. 617–641). San Diego: Academic Press. https://doi.org/10.1016/B978-0-12-417012-4.00025-9 Golan-Gerstl, R., Elbaum Shiff, Y., Moshayoff, V., Schecter, D., Leshkowitz, D., & Reif, S. (2017). Characterization and biological function of milk-derived miRNAs. Molecular Nutrition & Food Research, 61(10), 1700009. https://doi.org/10.1002/mnfr.201700009 Herwijnen, M.J.C. van, Driedonks, T.A.P., Snoek, B.L., Kroon, A.M.T., Kleinjan, M., Jorritsma, R., … Wauben, M. H.M. (2018). Abundantly present mirnas in milk-derived extracellular vesicles are conserved between mammals. Frontiers in Nutrition, 5. Howard, K.M., Jati Kusuma, R., Baier, S.R., Friemel, T., Markham, L., Vanamala, J., & Zempleni, J. (2015). Loss of miRNAs during processing and storage of cow’s (Bos taurus) milk. Journal of Agricultural and Food Chemistry, 63(2), 588–592. https://doi.org/10.1021/jf505526w Izumi, H., Kosaka, N., Shimizu, T., Sekine, K., Ochiya, T., & Takase, M. (2012). Bovine milk contains microRNA and messenger RNA that are stable under degradative conditions. Journal of Dairy Science, 95(9), 4831–4841. https://doi.org/10.3168/jds.2012-5489 Izumi, Hirohisa, Tsuda, M., Sato, Y., Kosaka, N., Ochiya, T., Iwamoto, H., … Takeda, Y. (2015). Bovine milk exosomes contain microRNA and mRNA and are taken up by human macrophages. Journal of Dairy Science, 98(5), 2920–2933. https://doi.org/10.3168/jds.2014-9076 Jonas, S., & Izaurralde, E. (2015). Towards a molecular understanding of microRNA-mediated gene silencing. Nature Reviews. Genetics, 16(7), 421–433. https://doi.org/10.1038/nrg3965 Kirchner, B., Pfaffl, M.W., Dumpler, J., von Mutius, E., & Ege, M.J. (2016). microRNA in native and processed cow’s milk and its implication for the farm milk effect on asthma. The Journal of Allergy and Clinical Immunology, 137(6), 1893-1895.e13. https://doi.org/10.1016/j.jaci.2015.10.028 Larrue, R., Fellah, S., Van der Hauwaert, C., Hennino, M.-F., Perrais, M., Lionet, A., … Cauffiez, C. (2022). The Versatile role of miR-21 in renal homeostasis and diseases. Cells, 11(21), 3525. https://doi.org/10.3390/cells11213525 Le, T.T., Van de Wiele, T., Do, T.N.H., Debyser, G., Struijs, K., Devreese, B., … Van Camp, J. (2012). Stability of milk fat globule membrane proteins toward human enzymatic gastrointestinal digestion. Journal of Dairy Science, 95(5), 2307–2318. https://doi.org/10.3168/jds.2011-4947 Li, J., Lei, L., Ye, F., Zhou, Y., Chang, H., & Zhao, G. (2019). Nutritive implications of dietary microRNAs: Facts, controversies, and perspectives. Food & Function, 10(6), 3044–3056. https://doi.org/10.1039/c9fo00216b Li, R., Dudemaine, P.-L., Zhao, X., Lei, C., & Ibeagha-Awemu, E.M. (2016). Comparative Analysis of the miRNome of Bovine Milk Fat, Whey and Cells. PLOS ONE, 11(4), e0154129. https://doi.org/10.1371/journal.pone.0154129 Liao, Y., Du, X., Li, J., & Lönnerdal, B. (2017). Human milk exosomes and their microRNAs survive digestion in vitro and are taken up by human intestinal cells. Molecular Nutrition & Food Research, 61(11). https://doi.org/10.1002/mnfr.201700082 López de Las Hazas, M.-C., Del Pozo-Acebo, L., Hansen, M. S., Gil-Zamorano, J., Mantilla-Escalante, D.C., Gómez-Coronado, D., … Dávalos, A. (2022). Dietary bovine milk miRNAs transported in extracellular vesicles are partially stable during GI digestion, are bioavailable and reach target tissues but need a minimum dose to impact on gene expression. European Journal of Nutrition, 61(2), 1043–1056. https://doi.org/10.1007/s00394-021-02720-y Manca, S., Upadhyaya, B., Mutai, E., Desaulniers, A.T., Cederberg, R.A., White, B.R., & Zempleni, J. (2018). Milk exosomes are bioavailable and distinct microRNA cargos have unique tissue distribution patterns. Scientific Reports, 8(1), 11321. https://doi.org/10.1038/s41598-018-29780-1 Melnik, B.C., Kakulas, F., Geddes, D.T., Hartmann, P.E., John, S.M., Carrera-Bastos, P., … Schmitz, G. (2016). Milk miRNAs: Simple nutrients or systemic functional regulators? Nutrition & Metabolism, 13(1), 42. https://doi.org/10.1186/s12986-016-0101-2 Melnik, B.C., & Schmitz, G. (2019). Exosomes of pasteurized milk: Potential pathogens of Western diseases. Journal of Translational Medicine, 17(1), 3. https://doi.org/10.1186/s12967-018-1760-8 Myrzabekova, M., Labeit, S., Niyazova, R., Akimniyazova, A., & Ivashchenko, A. (2021). Identification of Bovine miRNAs with the Potential to Affect Human Gene Expression. Frontiers in Genetics, 12, 705350. https://doi.org/10.3389/fgene.2021.705350 O’Brien, J., Hayder, H., Zayed, Y., & Peng, C. (2018). Overview of MicroRNA Biogenesis, Mechanisms of Actions, and Circulation. Frontiers in Endocrinology, 9. Oh, S., Park, M.R., Son, S.J., & Kim, Y. (2015). Comparison of total RNA isolation methods for analysis of immune-related microRNAs in market Milks. Korean Journal for Food Science of Animal Resources, 35(4), 459–465. https://doi.org/10.5851/kosfa.2015.35.4.459 Shandilya, S., Rani, P., Onteru, S.K., & Singh, D. (2017). Small Interfering RNA in Milk Exosomes Is Resistant to Digestion and Crosses the Intestinal Barrier In Vitro. Journal of Agricultural and Food Chemistry, 65(43), 9506–9513. https://doi.org/10.1021/acs.jafc.7b03123 Shome, S., Jernigan, R.L., Beitz, D.C., Clark, S., & Testroet, E.D. (2021). Non-coding RNA in raw and commercially processed milk and putative targets related to growth and immune-response. BMC Genomics, 22(1), 749. https://doi.org/10.1186/s12864-021-07964-w Sohel, M.H. (2016). Extracellular/Circulating MicroRNAs: Release Mechanisms, Functions and Challenges. Achievements in the Life Sciences, 10(2), 175–186. https://doi.org/10.1016/j.als.2016.11.007 Torrez Lamberti, M.F., Parker, L.A., Gonzalez, C.F., & Lorca, G.L. (2023). Pasteurization of human milk affects the miRNA cargo of EVs decreasing its immunomodulatory activity. Scientific Reports, 13(1), 10057. https://doi.org/10.1038/s41598-023-37310-x Wang, F., Ye, B.-G., Liu, J.-Z., & Kong, D.-L. (2020). miR-487b and TRAK2 that form an axis to regulate the aggressiveness of osteosarcoma, are potential therapeutic targets and prognostic biomarkers. Journal of Biochemical and Molecular Toxicology, 34(8), e22511. https://doi.org/10.1002/jbt.22511 Wang, Y., Zeng, G., & Jiang, Y. (2020). The emerging roles of miR-125b in cancers. Cancer Management and Research, 12, 1079–1088. https://doi.org/10.2147/CMAR.S232388 Weber, J.A., Baxter, D.H., Zhang, S., Huang, D.Y., Huang, K.H., Lee, M.J., … Wang, K. (2010). The microRNA spectrum in 12 body fluids. Clinical Chemistry, 56(11), 1733–1741. https://doi.org/10.1373/clinchem.2010.147405 Xu, Y., Wang, G., Hu, W., He, S., Li, D., Chen, P., … Zong, L. (2022). Clinical role of miR-421 as a novel biomarker in diagnosis of gastric cancer patients. Medicine, 101(19), e29242. https://doi.org/10.1097/MD.0000000000029242 Yang, J., Elbaz-Younes, I., Primo, C., Murungi, D., & Hirschi, K.D. (2018). Intestinal permeability, digestive stability and oral bioavailability of dietary small RNAs. Scientific Reports, 8(1), 10253. https://doi.org/10.1038/s41598-018-28207-1 Yu, S., Zhao, Z., Hao, P., Qiu, Y., Zhao, M., Zhou, G., … Li, P. (2022). Biological Functions and Cross-Kingdom Host Gene Regulation of Small RNAs in Lactobacillus plantarum-Derived Extracellular Vesicles. Frontiers in Microbiology, 13. Yu, S., Zhao, Z., Sun, L., & Li, P. (2017). Fermentation Results in Quantitative Changes in Milk-Derived Exosomes and Different Effects on Cell Growth and Survival. Journal of Agricultural and Food Chemistry, 65(6), 1220–1228. https://doi.org/10.1021/acs.jafc.6b05002 Zempleni, J., Baier, S.R., Howard, K.M., & Cui, J. (2015). Gene regulation by dietary microRNAs. Canadian Journal of Physiology and Pharmacology, 93(12), 1097–1102. https://doi.org/10.1139/cjpp-2014-0392 Zhang, L., Chen, T., Yin, Y., Zhang, C.-Y., & Zhang, Y.-L. (2019). Dietary microRNA-a novel functional component of food. Advances in Nutrition (Bethesda, Md.), 10(4), 711–721. https://doi.org/10.1093/advances/nmy127 Zhang, L., Hou, D., Chen, X., Li, D., Zhu, L., Zhang, Y., … Zhang, C.-Y. (2012). Exogenous plant MIR168a specifically targets mammalian LDLRAP1: Evidence of cross-kingdom regulation by microRNA. Cell Research, 22(1), 107–126. https://doi.org/10.1038/cr.2011.158 Zhang, Y., Xu, Q., Hou, J., Huang, G., Zhao, S., Zheng, N., & Wang, J. (2022). Loss of bioactive microRNAs in cow’s milk by ultra-high-temperature treatment but not by pasteurization treatment. Journal of the Science of Food and Agriculture, 102(7), 2676–2685. https://doi.org/10.1002/jsfa.11607 Zhu, K., Liu, M., Fu, Z., Zhou, Z., Kong, Y., Liang, H., … Chen, X. (2017). Plant microRNAs in larval food regulate honeybee caste development. PLoS Genetics, 13(8), e1006946. https://doi.org/10.1371/journal.pgen.1006946