New insights into microorganism-derived antibiotics based on identification and antimicrobial activity of antibiotic-producing actinomycetes in kusaya gravy that lead to its high preservability.

Kusaya shows a high preservability due to the microorganism-derived antibiotics contained in kusaya gravy, which is important for kusaya manufacturing. However, the antimicrobial compounds and its producing bacteria, as well as the antimicrobial activity of the kusaya gravy itself, have remained unknown. In this study, we isolated antibiotic-producing bacteria of the genus Streptomyces from kusaya gravy from Hachijojima and found that they produced antibacterial substances against various fungi and bacteria. In addition, we demonstrated that kusaya gravy itself shows antimicrobial activity, which was consistent with that of the isolates. This is the first report to directly indicate that kusaya gravy contains microorganism-derived antibiotics, which are assumed to be produced by actinomycetes.

Relationship between microorganisms and volatile components in each fermentation process in the kusaya gravy that plays an important role in the manufacturing of kusaya, a traditional Japanese fermented fish product.

The relationship between the microbiota and volatile components of kusaya gravy involved in the manufacturing of kusaya, a traditional Japanese fermented fish product, in the Izu Islands (Niijima and Hachijojima) and the fermentation processes are not clear. In this study, we aimed to investigate the relationship between the microbiota and volatile compounds involved in the manufacturing and management of kusaya gravy. 16S ribosomal RNA (rRNA) gene-based amplicon sequencing revealed that the microbiota in kusaya gravy was significantly different between the two islands, and the microbiota hardly changed during each fermentation process. Gas chromatography-mass spectrometry analysis also revealed that the volatile components were strongly related to the microbiota in kusaya gravy, with Hachijojima samples containing sulfur-containing compounds and Niijima samples containing short-chain fatty acids. Therefore, our findings suggest that kusaya gravy is a characteristic fermented gravy with a stable microbiota, and the fermented pickling gravy is fermented by microorganisms.

Causality Verification for the Correlation between the Presence of Nonstarter Bacteria and Flavor Characteristics in Soft-Type Ripened Cheeses.

Flavor characteristics of ripened cheese are established by various bacteria, such as lactic acid bacteria, Actinobacteria, and Proteobacteria, which spontaneously develop during the cheese-manufacturing process. We previously revealed the relationship between bacterial microbiota and flavor components in soft-type ripened cheeses by using a multiomics approach that combined metagenomics and metabolomics; however, we could not establish a causal relationship. This study aimed to substantiate the causal nature of the correlations revealed by the multiomics approach by using cheese-ripening tests with single isolate inoculation. The bacterial diversity and composition in surface mold-ripened cheeses from Japan and France varied, depending on the differences between the milks (pasteurized or raw), cheese positions (core or rind), and manufacturers. Although the volatile compounds did not clearly reflect the distinctive characteristics of the cheese samples, nonstarter lactic acid bacteria, Actinobacteria, and Proteobacteria positively correlated with ketones and sulfur compounds, as evidenced by a Spearman's correlation analysis. Cheese-ripening tests conducted after inoculation with single bacterial strains belonging to the above-mentioned taxa confirmed that these bacteria formed volatile compounds, in agreement with the correlations observed. In particular, various flavor compounds, such as acids, esters, ketones, and sulfur compounds, were detected in cheese inoculated with Pseudoalteromonas sp. TS-4-4 strain. These findings provide important insights into the role of nonstarter bacteria in the development of cheese flavor and into the effectiveness of the multiomics approach in screening for bacteria that can improve the quality of cheese products. IMPORTANCE Our previous study revealed that the existence of various bacteria, such as lactic acid bacteria, Actinobacteria, and Proteobacteria, clearly correlated with the abundance of flavor components, such as volatile compounds, in soft-type ripened cheeses via a multiomics approach that used 16S rRNA gene amplicon sequencing and headspace gas chromatography-mass spectrometry. However, this approach only showed correlations derived from statistical analyses rather than causal relationships. Therefore, in the present study, we performed cheese-ripening tests using nonstarter bacteria to substantiate the correlations revealed by the multiomics approach in soft-type ripened cheese. Our results suggest the capability of nonstarter bacteria, such as Proteobacteria, to impart flavor to cheese and the effectiveness of the multiomics approach in screening for microbial isolates that can improve the quality of cheese. Overall, our research provides new insights into the importance of bacteria in cheese production.

Evaluation of the Relationships Between Microbiota and Metabolites in Soft-Type Ripened Cheese Using an Integrated Omics Approach.

Cheese ripening is effected by various microorganisms and results in the characteristic flavors of cheese. Owing to the complexity of the microbiota involved, the relationship between microorganisms and components during ripening remains unclear. In this study, metagenomics and metabolomics were integrated to reveal these relationships in three kinds of surface mold-ripened cheeses and two kinds of bacterial smear-ripened cheeses. The microbiota is broadly divided into two groups to correspond with different cheese types. Furthermore, surface mold-ripened cheese showed similar microbiota regardless of the cheese variety, whereas bacterial smear-ripened cheese showed specific microbiota characterized by marine bacteria (MB) and halophilic and alkaliphilic lactic acid bacteria for each cheese variety. In the metabolite analysis, volatile compounds suggested differences in cheese types, although organic acids and free amino acids could not determine the cheese characteristics. On the other hand, Spearman correlation analysis revealed that the abundance of specific bacteria was related to the formation of specific organic acids, free amino acids, and volatile compounds. In particular, MB was positively correlated with esters and pyrazines, indicating their contribution to cheese quality. These methodologies and results further our understanding of microorganisms and allow us to select useful strains for cheese ripening.