Small Molecular Peptides and Their Potential Antifungal Activities During the Pile-Fermentation of Post-Fermented Tea

This study systematically investigated the dynamic diversity, potential sources, and antifungal activities of small molecular peptides during the pile-fermentation process of post-fermented tea. By analyzing the damaging effects of small molecular peptide extracts from tea samples at different pile-fermentation stages on the spore cell membranes of Aspergillus carbonarius (A. carbonarius) and the inhibitory activity against β-1,3-glucan synthase (β-1,3-GS), it was confirmed that some small molecular peptides exhibit significant antifungal effects. The main findings are as follows: (1) The number of identified small molecular peptides showed a trend of first increasing and then decreasing with the progress of pile-fermentation, peaking at 4453 species on the 35th day of pile-fermentation, and were dominated by hexapeptides and heptapeptides with molecular weights ranging from 600 to 800 Da. (2) Based on orthogonal partial least squares discriminant analysis (OPLS-DA), the samples were divided into three characteristic stages according to the differences in small molecular peptide composition at different stages, and 156 characteristic peptides with a relative abundance higher than 0.1% were screened out. Their precursor proteins were derived from 148 proteins belonging to 16 genera, including Camellia, Aspergillus, Saccharomyces, Penicillium, and Bacillus. (3) BLAST alignment results showed that five out of the 156 characteristic peptides were degradation fragments of known antifungal peptides originating from Aspergillus and Bacillus. (4) Combining molecular docking screening and in vitro verification of synthetic peptides, a total of 27 small molecular peptides with antifungal activity were obtained, and their mechanism of action was the inhibition of β-1,3-GS activity. (5) The small molecular peptides related to antifungal activity could be classified into two categories: enzymatic hydrolysates of known antifungal peptides, and the enzymatic hydrolysates of tea-derived proteins or macromolecular peptides. Both categories were mainly distributed in the three stages of pile-fermentation, and there was a significant positive correlation among the population size of dominant microorganisms, microbial peptidase activity, and the abundance of small molecular peptides. This study reveals the dynamic generation pattern and antifungal potential of small molecular peptides during the pile-fermentation of post-fermented tea, providing a new scientific basis for evaluating the dynamic changes in microbial communities in tea and effectively controlling the contamination of harmful fungi during the pile-fermentation process.