J. Microbiol. Biotechnol. J. Microbiol. Biotechnol. (2017), 27(9), 1566–1575 https://doi.org/10.4014/jmb.1705.05006 Research Article jmb Review Quantitative Analysis of Milk-Derived microRNAs and Microbiota during the Manufacturing and Ripening of Soft Cheese Sangnam Oh 1 , Mi-Ri Park 2 , Sangdon Ryu 2,3 , Brighton E. Maburutse 2 , Ji-Uk Kim 4 , and Younghoon Kim 2 * Department of Functional Food and Biotechnology, Jeonju University, Jeonju 55069, Republic of Korea Department of Animal Science and Institute of Agricultural Science & Technology, Chonbuk National University, Jeonju 54896, Republic of Korea National Institute of Agricultural Science, Rural Development Administration, Wanju-gun 55365, Republic of Korea R&D Center, Maeil Dairies, Co. Ltd, Pyeongtaek-si, Gyeonggi-do 17714, Republic of Korea Introduction Bovine milk is widely used as a dairy product, including in infant formula, and it is an important material in the food industry. It contains nutrients required for the growth and development of calves, where it is a source of lipids, proteins, amino acids, vitamins, and minerals. Bovine milk also contains immunoglobulins, hormones, growth factors, cytokines, nucleotides, peptides, polyamines, enzymes, and other bioactive peptides that support human health and immunity [1, 2]. Recent studies have demonstrated that microRNAs (miRNAs) are present in bovine milk and that they can modulate the immune system [3-5]. miRNAs are also abundant in milk derived from other livestock. miRNAs in milk have functional biological roles in infants and in the secretion process of mammary glands. Milk- derived miRNAs are encapsulated in extracellular vesicles such as exosomes, and they are bioavailable; therefore, Received: May 2, 2017 Revised: June 21, 2017 Accepted: June 26, 2017 First published online July 7, 2017 *Corresponding author Phone: +82-63-270-2606; Fax: +82-63-270-2612; E-mail: ykeys2584@jbnu.ac.kr pISSN 1017-7825, eISSN 1738-8872 Copyright © 2017 by The Korean Society for Microbiology and Biotechnology MicroRNAs (miRNAs) are abundant in bovine milk and milk derived from other livestock, and they have functional roles in infants and in the secretion process of mammary glands. However, few studies have evaluated miRNAs in dairy processes, such as during cheese making and ripening. Thus, we investigated the characteristics of milk-derived miRNAs during the manufacturing and ripening of Camembert cheese as well as the microbiota present using the quantitative reverse transcription polymer chain reaction (RT-qPCR) and 16S rRNA pyrosequencing, respectively. Pyrosequencing showed that the cheese microbiota changed dramatically during cheese processing, including during the pasteurization, starter culture, and ripening stages. Our results indicated that the RNA contents per 200 mg/200 μl of the sample increased significantly during cheese-making and ripening. The inner cheese fractions had higher RNA contents than the surfaces after 12 and 22 days of ripening in a time- dependent manner (21.9 and 13.2 times higher in the inner and surface fractions than raw milk, respectively). We performed a comparative analysis of the miRNAs in each fraction by RT-qPCR. Large amounts of miRNAs (miR-93, miR-106a, miR-130, miR-155, miR-181a, and miR- 223) correlated with immune responses and mammary glands were present in aged cheese, with the exception of miR-223, which was not present on the surface. Considerable amounts of miRNAs were also detected in whey, which is usually disposed of during the cheese-making process. Unexpectedly, there were no significant correlations between immune-related miRNAs and the microbial populations during cheese processing. Taken together, these results show that various functional miRNAs are present in cheese during its manufacture and that they are dramatically increased in amount in ripened Camembert cheese, with differences according to depth. Keywords: Camembert cheese, cheese manufacturing, immune-related microRNA, microbiota