To elucidate the structural characteristics and transport mechanism of the signaling molecule auto inducer-2 (AI-2) transporter GXS4 from Limosilactobacillus fermentum, this study utilized multiple bioinformatics methods to systematically analyze its physicochemical properties and structural features. Molecular docking and molecular dynamics simulations were conducted to investigate the binding sites and interaction mechanism between GXS4 and AI-2. The results showed that GXS4 consisted of 366 amino acid residues with a molecular mass of 40.0 kDa. It was a membrane protein lacking a signal peptide and possessed 8 transmembrane helices. The secondary structure consisted of up to 77.32% α-helix, contributing to maintaining the stability of the protein backbone and facilitating transmembrane transport. The three-dimensional structure presented a typical “half-moon” conformation, suggesting that AI-2 transport occurs through an “alternating access” mechanism. Molecular docking revealed that GXS4 preferentially binds to the (R)-2,3,3,4-tetrahydroxytetrahydrofuran borate (R-THMF) configuration of the AI-2 molecule, with key residues Val233 and Ala263 forming a stable complex with AI-2 through hydrogen bonds. Molecular dynamics simulations further verified the structural stability of the complex and found that a mutation at Ala263 significantly reduced the complex’s stability, identifying Ala263 as a critical target site of GXS4. This study provides a theoretical basis and new strategies for targeted regulation of the quorum sensing system in L. fermentum.

S-ribosylhomocysteinase (also referred to as LuxS protein) is an important enzyme involved in the biosynthesis of autoinducer-2 (AI-2) as a signaling molecule. The physicochemical properties, hydrophilicity, hydrophobicity, signaling peptides, transmembrane structure, phosphorylation sites, structural domains and spatial structure of the LuxS protein in Limosilactobacillus fermentum A51 were studied by bioinformatics. In addition, its structural and functional properties were explored by protein-protein interaction (PPI) network analysis. The results showed that the LuxS protein was encoded by 158 amino acids, with a molecular mass of 17718.91 Da, a theoretical isoelectric point of 5.29, an instability coefficient of 32.16, and it was an acidic, stable and hydrophilic protein. The LuxS protein, without signal peptide or transmembrane domain, was presumed to mainly play an intracellular role as an endocrine protein, and it had 16 phosphorylation sites, belonging to the LuxS superfamily, and contained one PRK02260 structural domain. The LuxS protein was a relatively stable protein with a secondary structure consisting mainly of 39.87% random coils and 31.65% α-helices, and its three-dimensional structure was folded into a baseball-like structure. The results of PPI network showed that the LuxS protein mainly interacted with pfs, metE, metC, metC-2, yhcE, mmuM, patB and cysK, and were involved in the metabolism and synthesis of cysteine, methionine, and sulfur-containing amino acids, suggesting that it is involved in the activation of the activated methyl cycle and thus regulates the quorum sensing system and the metabolism and synthesis of functional substances in the strain. This study provides a theoretical basis for an in-depth study of LuxS protein regulation of the AI-2 quorum sensing system in L. fermentum.

Rubing cheese was manufactured by lactic acid bacterial fermentation or traditional direct acidification. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and liquid chromatography-mass spectrometry (LCMS) were used to determine protein degradation and free amino acid contents in rubing cheese, respectively. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to identify peptides in rubing cheese. The biological activity of the hydrolysate of rubing cheese was characterized by in vitro antioxidant activity, α-glucosidase-inhibiting activity and angiotensin-converting enzyme (ACE)-inhibiting activity. According to the results of SDS-PAGE, both lactic acid bacterial fermentation and direct acidification could promote the degradation of milk proteins. Lactic acid bacterial fermentation caused a higher degree of degradation of whey protein, promoting the release of free amino acids. From the fermented rubing cheese and the directly acidified one, 23 and 13 potential ACE inhibitory peptides, six and four antioxidant peptides and five and one antiglycemic peptides were identified, respectively. Furthermore, lactic acid bacterial fermentation significantly improved the α-glucosidase-inhibiting activity and ACE-inhibiting activity of rubing cheese peptides (P < 0.05). The half-maximum inhibitory concentration (IC₅₀) values of the fraction separated by ultrafiltration with a molecular mass of less than 3 kDa for α-glucosidase and ACE inhibitory activity were 1.250 and 0.416 mg/mL, respectively. In conclusion, lactic acid bacterial fermentation promotes protein degradation to release biologically active peptides and produce free amino acids, enhancing the biological value of rubing cheese.

Using third-generation sequencing technology, full-length 16S and internal transcribed spacer (ITS) gene sequencing were performed on bacteria and fungi in Zhuganzha from Heqing, Yunnan, respectively. The diversity of bacteria and fungi was analyzed and annotated at the species level. As a result, the microbial community of Zhuganzha was complex and diverse. The microbial community structure analysis showed that at the phylum level, the dominant bacteria and fungi were Firmicutes and Ascomycota, respectively. At the genus level, the dominant bacterium was Tetragonococcus and the fungal genus was Millerozyma. At the species level, the dominant bacterium was T. halophilus, and the dominant fungus was M. farinosa. More fungal and bacterial species were involved in the fermentation process. The heatmap analysis indicated that the bacterial and fungal community compositions of samples ZGZ201, ZGZ202, and ZGZ302 were the most similar. The microbial community composition of Heqing Zhuganzha was dominated by fungi, followed by bacteria. The annotated T. halophilus and M. farinosa can provide a reference for the screening and development of potential starters.