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Evaluation and Characterization of Milkderived Microvescicle Isolated from Bovine Colostrum
Brighton E. Maburutse,Mi-Ri Park,Sangnam Oh,Younghoon Kim 한국축산식품학회 2017 한국축산식품학회지 Vol.37 No.5
Extracellular microvesicles are membranous nano-sized cellular organelles secreted by a variety of cells under normal and pathological conditions and heterogeneous in size ranging from 30 nm to 1 μm. They carry functional microRNAs that can influence immunity and development. For a particular application of microvesicles, choice of isolation method is particularly important; however, their isolation methods from colostrum in particular have not been described clearly. In this work, differential ultracentrifugation as a conventional method, ultracentrifugation with some modification such as additional precipitations, ultrafiltration, sucrose gradient separation and ExoQuick™ as a commercial reagent were compared. The goal was to compare mainly microvesicular total microRNA yield, distribution and purity among the methods then select the best isolation method for bovine colostrum microvesicles based largely on microRNA yield with the view of applying the vesicles in work where vesicular micro- RNA cargo is the target bioactive component. Highest yields for vesicular microRNA were obtained using conventional methods and among them, subsequent ultracentrifugation with 100,000 g and 135,000 g conventional method 2 was selected as it had the highest RNA to protein ratio indicating that it pelleted the least protein in relation to RNA an important factor for in vivo applications to assess microvesicle functionalities without risk of contaminating non-vesicular biomaterial. Microvesicles isolated using conventional method 2 were successfully internalized by cells in vitro showing their potential to deliver their cargo into cells in vitro and in vivo in case of functional studies.
Diversity and Characteristics of the Meat Microbiological Community on Dry Aged Beef
( Sangdon Ryu ),( Mi Ri Park ),( Brighton E. Maburutse ),( Woong Ji Lee ),( Dong-jun Park ),( Soohyun Cho ),( Inho Hwang ),( Sangnam Oh ),( Younghoon Kim ) 한국미생물생명공학회(구 한국산업미생물학회) 2018 Journal of microbiology and biotechnology Vol.28 No.1
Beef was dry aged for 40-60 days under controlled environmental conditions in a refrigerated room with a relative humidity of 75%-80% and air-flow. To date, there is little information on the microbial diversity and characteristics of dry aged beef. In this study, we explored the effect of change in meat microorganisms on dry aged beef. Initially, the total bacteria and LAB were significantly increased for 50 days during all dry aging periods. There was an absence of representative foodborne pathogens as well as coliforms. Interestingly, fungi including yeast and mold that possess specific features were observed during the dry aging period. The 5.8S rRNA sequencing results showed that potentially harmful yeasts/molds (Candida sp., Cladosporium sp., Rhodotorula sp.) were present at the initial point of dry aging and they disappeared with increasing dry aging time. Interestingly, Penicillium camemberti and Debaryomyces hansenii used for cheese manufacturing were observed with an increase in the dry aging period. Taken together, our results showed that the change in microorganisms exerts an influence on the quality and safety of dry aged beef, and our study identified that fungi may play an important role in the palatability and flavor development of dry aged beef.
( Sangnam Oh ),( Mi-ri Park ),( Sangdon Ryu ),( Brighton E. Maburutse ),( Ji-uk Kim ),( Younghoon Kim ) 한국미생물생명공학회(구 한국산업미생물학회) 2017 Journal of microbiology and biotechnology Vol.27 No.9
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 timedependent 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.