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.

In order to prolong the shelf life of milk fan, casein-chitosan edible films incorporated with antimicrobial peptide BCp12 were prepared. One-factor-at-a-time method combined with orthogonal array design was used to determine the optimum preparation process based on elongation at break and tensile strength. The antimicrobial activity of the composite film was evaluated, and its formation mechanism was explored by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR) and molecular docking. Finally, the composite film was applied to the preservation of milk fan. The results showed that the optimum preparation conditions were casein/chitosan mass ratio 1:1.5, BCp12 concentration 1 mg/mL, glycerin concentration 1.5% (m/m), and drying temperature 55 ℃. SEM showed good compatibility of the composite film and FT-IR analysis revealed that the film exhibited an absorption peak at 1545.67 cm^–1 attributed to the stretching vibration of C–O. Molecular docking showed that the hydrogen bond interaction between BCp12 and casein and chitosan occurred through the active sites of residues Y4 and–NH₂. Compared with the control group, the change of peroxide value of milk fan treated with the antimicrobial film was slower and the shelf life was prolonged by 60 days during storage at 4 ℃, indicating the film could inhibit the growth of spoilage bacteria. The results of this study provide a technical reference for the development and application of chitosan-casein composite films incorporated with BCp12.

This study was undertaken in order to improve the stability, water solubility and gastrointestinal release of phytosterols (PS). Zein-PS nanoparticles were prepared by the anti-solvent method. Single factor experiments and orthogonal array design methods were adopted to optimize the preparation conditions based on the stability and encapsulation efficiency of zein-PS nanoparticles. The structure and functional properties of the prepared samples were characterized by using scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR) and simulated gastrointestinal digestion. The results showed that the optimal preparation conditions were determined as follows: zein/PS ratio of 15:1 (m/m), hydration time of 2 h, hydration temperature of 55 ℃, and ultrasonic treatment time of 20 min. The encapsulation efficiency of the nanoparticles prepared using the optimal conditions was 84.97%, particle size (479.76 ± 0.38) nm, and zeta potential (-22.79 ± 0.015) mV, suggesting small particle size and high stability. The nanoparticles with a zein/PS ratio of 15:1 had better re-solubility and water solubility. SEM revealed the formation of a compact network structure on the surface of the nanoparticles and FTIR spectra showed that it changed at 3313.16 and 1523.51 cm^-1, indicating the presence of hydrogen bonding and electrostatic interaction between zein and PS. Compared with PS, the release rates of the nanoparticles in simulated gastrointestinal fluids were reduced by 49.03% and 28.11%, respectively, showing a good sustained-release effect. In addition, after storage for 30 days, the change in the particle size of the nanoparticles was smaller at 4 ℃ than at 25 ℃, and the encapsulation efficiency remained above 70%. Therefore, zein-PS composite nanoparticles have good stability and sustained release property, which has potential application prospects in the food industry.

The enzymatic hydrolysate of Zhongdian yak milk casein was fractionated by ultrafiltration into two fractions: molecular mass > 3 kDa and < 3 kDa. The cell proliferation activity and antioxidant activity of the two fractions were determined. The bioactive peptides derived from yak milk casein were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), bioinformatics and molecular docking. The results showed that the proliferation rate of RAW264.7 cells was above 80% in the presence of either the molecular mass > 3 kDa or < 3 kDa fraction. Both fractions had the ability to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and 2,2-azino-bis(3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS) cation radical. The cell proliferation and free radical scavenging activity of the molecular mass < 3 kDa fraction were better than those of the > 3 kDa fraction. A total of 895 peptides were identified from the molecular mass < 3 kDa fraction, of which 96 were known bioactive peptides, mainly antioxidant peptides (39.6%) and angiotensinconverting enzyme inhibitory peptides (30.2%), indicating that Zhongdian yak casein is an important source of bioactive peptides. Based on their activity and hydrophobicity, antioxidant peptides GYF and RPW were selected from the predicted 20 potential antioxidant peptides. The results of molecular docking showed that the binding energies of GYF and RPW with ABTS cation and DPPH radicals were all negative, indicating that they had binding potential and exerted antioxidant activity mainly by forming hydrogen bonds and hydrophobic interactions.