The proteomic differences between Pichia kudriavzevii under heat stress and that under heat-salt stress were analyzed by tandem mass tag (TMT)-based quantitative proteomics to identify the key proteins related to improved thermotolerance. The expression of heat shock protein (HSP) 12 and the enzymes related to ergosterol biosynthesis, including ergosterol biosynthetic protein (ERG) 28 and ERG25 was significantly improved by salt stress, thereby contributing to the structural and functional stability of intracellular proteins and the cell membrane under heat stress. Salt stress significantly increased the expression of glutathione S-transferase (GST) Y-2, which played an important role in inhibiting heat-induced oxidative damage of lipids and proteins. Meanwhile, salt stress significantly increased the expression of enzymes related to carbohydrate metabolism and energy metabolism under heat stress, including hexokinase (HK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate mutase 1 (PGAM1), PGAM2, phosphoglycerate kinase (PGK), alcohol dehydrogenase (ADHP), cytochrome c oxidase subunit 6A (COX6A), and V-type proton ATPase subunit c’ (ATP6L), thus contributing to the synthesis of intracellular ATP and improvement of its thermotolerance. The results of this study can provide important technical support for the genetic engineering of thermotolerant yeasts and the improvement of their ethanol production at high temperature.

In this study, the effect of fermentation with Bacillus subtilis B-2, a salt-tolerant strain capable of producing high yield of protease, on the microbial community and the contents of biogenic amines and amino acid nitrogen (AAN) during the fermentation of low-salt fish sauce. The results of high-throughput 16S rRNA gene sequencing showed that fermentation with B-2 reduced the richness and evenness of the microbial community, Bacillus was dominant throughout the fermentation process and the abundance of spoilage microorganisms significantly decreased. B-2 significantly inhibited the formation of histamine, putrescine, cadaverine, and tyramine, and their contents at the end of fermentation decreased by 25.9%, 35.6%, 23.6% and 9.3%, respectively, compared with those in naturally fermented fish sauce. The content of AAN in low-salt fish sauce was significantly improved by inoculated fermentation, reaching 1.23 g/100 mL after 15 days of fermentation, which was significantly higher than that of naturally fermented fish sauce (0.79 g/100 mL). The correlation network map showed that the decrease in the abundance of Brevibacterium, Dietzia, Paracoccus, Aequorivita, and Brachybacterium was the major reason for the decrease in microbial diversity at the late stage of fermentation of naturally fermented fish sauce. The abundance of Stenotrophomonas showed a significantly positive correlation with the contents of many biogenic amines in naturally fermented and B-2 fermented fish sauce, suggesting its important role in the formation of biogenic amines in low-salt fish sauce. Comparative analysis of the microbial community and quality attributes at the end of fermentation showed that the metabolism of B-2 was the main reason for the decrease in the species and abundance of spoilage microorganisms, the increase in AAN content, and the decrease in the contents of key biogenic amines. B. subtilis B-2 is expected to be developed as a special fermentation starter for fish sauce to improve the quality and safety of rapidly fermented low-salt fish sauce.

Fermented foods are a potential source to produce novel dipeptidyl peptidase-IV inhibitory peptides (D4IPs). In this study, the fermented mandarin fish (Chouguiyu) was used to screen D4IPs and their formation mechanism was studied by metagenomics and peptidomics. A total of 400 D4IPs with DPP-IV inhibition structure and high hydrophobicity were identified. The correlation network map showed that Lactococcus, Bacillus, Lysobacter, Pelagivirga, Kocuria, Escherichia, Streptococcus, and Peptostreptococcus were significantly correlated with the most D4IPs. Four stable D4IPs, including KAGARALTDAETAT, GEKVDFDDIQK, VVDADEMYLKGK, and GQKDSYVGDEAQ were respectively from the precursor proteins parvalbumin, troponin, myosin, and actin, and were mainly formed by the hydrolysis of subtilisin (EC 3.4.21.62), aspartic proteinase (EC 3.4.23.1), thermolysin (EC 3.4.24.27), oligopeptidase B (EC 3.4.21.83), and proteinase P1 (EC 3.4.21.96) from Bacillus, Kocuria, Lysobacter, Lactococcus, and Peptostreptococcus. The inhibition mainly resulted from the hydrogen bond and salt bridge between D4IPs and DPP-IV enzyme. This study provides important information on the proteases and related microbial strains to directionally prepare D4IPs in Chouguiyu.