Although coagulase-negative Staphylococcus (CNS) with technological activities plays a key role in fermented sausage flavour and nutrient production, the molecular mechanism of these activities remains elusive. In this study, 18 CNS strains with high proteolytic activity were isolated from Chinese Dong fermented pork (Nanx Wudl), and their technological and transcriptomic properties were investigated. After biochemical identification and genetic analysis, their technological properties, including nitrate reductase, catalase, antioxidant, and lipolytic activities, as well as their growth under varying temperatures, salt concentrations, and pH levels, were evaluated. Their aroma-producing potential was also determined in a model medium resembling fermented sausages. Transcriptomic analysis was performed using the most promising isolates. Biochemical identification and 16S rDNA sequencing revealed that the 18 Staphylococcus strains belonged to S. xylosus, S. saprophyticus, S. carnosus, S. sciuri, and S. equorum. In terms of technological properties, 16 strains showed a nitrate-reducing ability, while 11 strains had a lipolytic activity. All strains exhibited superoxide dismutase (SOD) and catalase activities; four strains displayed an SOD activity of > 50%. They also tolerated 10% NaCl and 150 mg/kg of nitrite. They showed significant differences in ketone and acid production. The transcriptomic analysis of S. xylosus strains Sx3 and Sx6, which were selected because of their excellent enzymatic activities and aroma-producing ability, revealed the remarkable effect of genes related to pyruvate catabolism and amino acid metabolism on aroma generation. Therefore, this study provided valuable insights into the metabolic mechanisms underlying the technological properties of CNS and identified promising candidates as starter cultures in fermented sausage manufacturing.

The increase in human population has led to imminent pressures to develop new edible proteins with decreased environmental footprints. The most promising approach involves the production of single cell protein (SCP) from yeasts, which have been utilized in a wide variety of foods for thousands of years. In this study, 102 yeast strains isolated from traditional fermented pork (Nanx wudl) were investigated for their potential as SCP producer for the first time. Based on preliminary screening, Saccharomyces cerevisiae Y70 and Candida parapsilosis H5Y13, both showing high protein content and excellent growth capability, were selected for further analysis via 4D-DIA Proteomics technology. Proteomic analysis indicated that the oxidative metabolism pathways, including TCA cycle, oxidative phosphorylation and pentose phosphate pathway, may have a significant impact on global protein synthesis and production. This study provides useful information for selecting SCP-producing yeast from Chinese fermented meat products and contribute to a deeper understanding of the underlying metabolic mechanisms behind global protein synthesis in yeast. Furthermore, these findings also provide potential molecular targets for genetic engineering modifications in yeast, aimed at constructing highly efficient cell factories for protein production.

Yeasts are one of the predominant microbial groups in fermented meats. In this study, yeast communities of Chinese Dong fermented pork (Nanx Wudl) were investigated and the technological properties of 73 yeast isolates were evaluated. Through culture-dependent and high-throughput sequencing methods, the main yeast species identified included Pichia membranifaciens, Kazachstania bulderi, Millerozyma farinosa, Candida zeylanoides, Kazachstania exigua, Candida parapsilosis and Saccharomyces cerevisiae. Among these yeasts, P. membranifaciens, M. farinosa, K. exigua and K. bulderi were detected in fermented meats for the first time. A total of 73 yeast isolates was investigated for their lipolytic and proteolytic activities. All yeast species showed lipolytic activity, while proteolytic activity against meat protein was only detected in S. cerevisiae. Assay of aroma-producing potential was performed in a model simulating fermented sausage condition. Inoculation of yeast strains increased volatiles production, especially esters and alcohols. The highest ester production was observed in S. cerevisiae Y70 strain, followed by K. exigua Y12 and K. bulderi Y19. C. zeylanoides Y10 and S. cerevisiae Y70 were the highest producers of benzeneethanol and 3-methyl-1-butanol. S. cerevisiae Y70 with its highest production of branched alcohols and esters could be a promising candidate as aroma enhancer in the manufacture of fermented sausages.

The effect of a proteolytic starter culture isolated from Nanx Wudl, on microbiological, biochemical and organoleptic attributes of dry fermented sausages was investigated during processing. Based on preliminary screening, the combination of Staphylococcus xylosus SX16 and Lactobacillus plantarum CMRC6, showing excellent proteolytic activity in vitro, was selected as the multi-strain starter (starter LS). For comparison, the single-strain starter culture of L. plantarum CMRC6 (starter LB) and non-inoculated control were also tested. During fermentation, lactic acid bacteria and staphylococci dominated the microbiota and suppressed the Enterobacteriaceae growth in LS-inoculated sausages. The addition of LS starter accelerated acidification and proteolysis during ripening, improving the contents of total free amino acids and several essential free amino acids (Phe, Ile and Leu). Volatile compounds analysis revealed that LS-fermented sausage showed the highest abundance of 3-methyl-1-butanol, probably due to the inoculated S. xylosus. The inoculation of LS starter improved the sensory properties of sausages, especially the flavor attribute. Therefore, S. xylosus SX16 and L. plantarum CMRC6 are promising candidates for inclusion as multi-strain starters in the manufacture of gourmet fermented dry sausage.