Here, we examined the functionality of Lactobacillus fermentum strain JDFM216, a newly isolated probiotic bacterium, using a Caenorhabditis elegans model. We determined bacterial colonization in the intestinal tract of C. elegans by plate counting and transmission electron microscopy and examined the survival of C. elegans using a solid killing assay. In addition, we employed DNA microarray analysis, quantitative real time-polymerase chain reaction, and immunoblotting assays to explore health-promoting pathways induced by probiotic bacteria in C. elegans. Initially, we found that the probiotic bacterium L. fermentum strain JDFM216 was not harmful to the C. elegans host. Conditioning with JDFM216 led to its colonization in the nematode intestine and enhanced resistance in nematodes exposed to food-borne pathogens, including Staphylococcus aureus and Escherichia coli O157:H7. Interestingly, this probiotic strain significantly prolonged the life span of C. elegans. Whole-transcriptome analysis and transgenic worm assays revealed that the health-promoting effects of JDFM216 were mediated by a nuclear hormone receptor (NHR) family and PMK-1 signaling. Taken together, we described a new C. elegans-based system to screen novel probiotic activity and demonstrated that preconditioning with the probiotic L. fermentum strain JDFM216 may positively stimulate the longevity of the C. elegans host via specific pathway.

Osteoporosis is a systemic skeletal disease characterized by low bone mass and micro-architectural deterioration of bone tissue, with a consequent increase in bone fragility and fracture susceptibility. In an aged society with increased life expectancy, the incidence rate of osteoporosis is also rapidly increasing. Inadequate nutrition may negatively influence bone metabolism. Recently, many studies have investigated the functionality of milk-derived exosomes, which play important roles in cell-to-cell communication. However, there are few reports of how milk-derived exosomes influence osteoblast proliferation and differentiation. Here, we determined whether bovine colostrum-derived exosomes promote anti-osteoporosis in vitro and in vivo. Tartrate-resistant acid phosphatase-stained cells were significantly inhibited in Raw264.7 cells treated with exosomes, indicating reduced osteoclast differentiation. We induced osteoporosis in mice using glucocorticoid pellets after orally administering exosomes for 2 mo. Interestingly, the bone mineral density of exosome-fed mouse groups was significantly improved compared with the glucocorticoid-induced osteoporosis group without exosome treatment. In addition, Lactobacillus were decreased in the gut microbiota community of osteoporosis-induced mice, but the gut microbiota community composition was effectively restored by exosome intake. Taken together, we propose that exosomes isolated from bovine colostrum could be a potential candidate for osteoporosis prevention, bone remodeling improvement, and inhibition of bone resorption. To our knowledge, this is the first time that a protective effect of milk exosomes against osteoporosis has been demonstrated in vivo. Our results strongly suggest that bovine colostrum exosomes might be used as a prophylaxis to prevent the onset of osteoporosis. Indeed, our results offer promising alternative strategies in the nutritional management of age-related bone complications.

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.

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.

This study aimed to develop an in vivo screening platform using Caenorhabditis elegans to identify a novel bacteriocin for controlling the mastitis-causing pathogen Staphylococcus aureus strain RF122 in dairy cows. Using Bacillus spp. isolated from traditional Korean foods, we developed a direct in vivo screening platform that uses 96-well plates and fluorescence image analysis. We identified a novel bacteriocin produced by Bacillus licheniformis strain 146 (lichenicin 146) with a high in vivo antimicrobial activity using our liquid C. elegans-Staph. aureus assay. We also determined the characteristics of lichenicin 146 using liquid chromatography-mass spectrometry and confirmed that it shared homologous sequences with bacteriocin family proteins. In addition, RNA-sequencing analysis revealed genes encoding cell surface or membrane proteins (SAB0993c, SAB0150, SAB0994c, and SAB2375c) that are involved in the bactericidal activity of lichenicin 146 against Staph. aureus strain RF122 infection as well as those encoding transcriptional regulators (SAB0844c and SAB0133). Thus, our direct in vivo screening platform facilitates simple, convenient, cost-effective, and reliable screening of potential antimicrobial compounds with applications in the dairy field.