Xing Wan

This study aimed to screen lactic acid bacteria (LAB) with enhanced antioxidant activity and evaluate their potential as starter cultures for dry-cured fermented sausages by assessing their effects on oxidative stability and physicochemical quality during processing. The antioxidant activities of different cellular fractions of five LAB strains were first compared, and the results indicated that the antioxidant activity was mainly associated with extracellular metabolites. Based on their good in vitro antioxidant activities and oxidative tolerance, Leuconostoc carnosum AMS1 and Lactiplantibacillus plantarum P4 were subsequently selected as starter cultures for sausage fermentation. Individual inoculation with AMS1 and P4 modulated the bacterial community structure during fermentation and reduced the abundance of opportunistic pathogens. Compared with the uninoculated control, starter inoculation accelerated acidification, reduced water activity, and positively modulated color and texture properties. Moreover, inoculation improved oxidative stability, as indicated by reduced thiobarbituric acid reactive substances (TBARS) levels and lipoxygenase (LOX) activity, and alleviated protein oxidative changes, as evidenced by lower carbonyl accumulation, reduced sulfhydryl group loss, and a lower increase in surface hydrophobicity. In conclusion, AMS1 and P4, particularly AMS1, showed potential as antioxidative starter cultures for dry-cured fermented sausages by improving physicochemical quality and oxidative stability during fermentation and maturation.

Fermented vegetables in Yunnan Province, China, harbor abundant microbial diversity. However, the development of indigenous starter cultures remains under-utilized. Genomic information regarding Leuconostoc (L.) mesenteroides isolates from this region is particularly scarce. To assess the genomic characteristics of eight L. mesenteroides isolates from traditional Yunnan fermented vegetables, we performed whole-genome sequencing and conducted a comparative analysis with 21 publicly available vegetable-derived genomes. Comparative genomic analysis revealed marked variation in genome size and plasmid content, and pangenome analysis indicated an open configuration. Core-genome multilocus sequence typing (cgMLST) of the eight indigenous isolates showed high allelic diversity, indicating a genetically heterogeneous and non-clonal population. Phylogenomic analysis revealed that the evolutionary relationships among the 29 strains were not strictly correlated with their vegetable sources, suggesting an influence from other factors, such as geographic origin and region-specific processing methods. Similar to the profiles of the 21 publicly available genomes, inactive prophages, intrinsic vancomycin resistance genes, and genomic island fragments were detected in eight isolates, whereas no known virulence genes were identified. Bacteriocin gene clusters varied among strains, while stress tolerance and probiotic-related genes were conserved. Overall, these results provide genomic indications relevant to the safety, adaptability, and fermentation potential of indigenous L. mesenteroides from Yunnan. However, because these functional traits are inferred solely from genomic predictions, subsequent experimental validation is essential to confirm their phenotypic properties and technological efficacy.

Background: Laccases can oxidize a broad spectrum of substrates, offering promising applications in various sectors, such as bioremediation, biomass fractionation in future biorefineries, and synthesis of biochemicals and biopolymers. However, laccase discovery and optimization with a desirable pH optimum remains a challenge due to the labor-intensive and time-consuming nature of the traditional laboratory methods. Results: This study presents a machine learning (ML)-integrated approach for predicting pH optima of basidiomycete fungal laccases, utilizing a small, curated dataset against a vast metagenomic data. Comparative computational analyses unveiled the structural and pH-dependent solubility differences between acidic and neutral-alkaline laccases, helping us understand the molecular bases of enzyme pH optimum. The pH profiling of the two ML-predicted alkaline laccase candidates from the basidiomycete fungus Lepista nuda further validated our computational approach, showing the accuracy of this comprehensive method. Conclusions: This study uncovers the efficacy of ML in the prediction of enzyme pH optimum from minimal datasets, marking a significant step towards harnessing computational tools for systematic screening of enzymes for biotechnology applications. Graphical

This chapter presents a comprehensive methodology for the identification, characterization, and functional analyses of potentially toxic hypothetical proteins of unknown function (toxHPUFs) in phages. The methods begin with in vivo toxicity verification of toxHPUFs in bacterial hosts, utilizing conventional drop tests and following growth curves. Computational methods for structural and functional predictions of toxHPUFs are outlined, incorporating the use of tools such as Phyre2, HHpred, and AlphaFold2. To ascertain potential targets, a comparative genomic approach is described using bioinformatics toolkits for sequence alignment and functional annotation. Moreover, steps are provided to predict protein–protein interactions and visualizing these using PyMOL. The culmination of these methods equips researchers with an effective pipeline to identify and analyze toxHPUFs and their potential targets, laying the groundwork for future experimental confirmations. Key words Bacteriophages Hypothetical proteins of unknown function (HPUFs) Antimicrobial resistance Next-generation sequencing Functional prediction

Leuconostoc carnosum is a bacterial species commonly associated with meat spoilage. However, some strains exhibit preservative effects due to bacteriocin production. Here, we report the complete genome sequences for two strains, L. carnosum 4010 and AMS1. Bacteriocin-related gene clusters were found on the plasmids of both strains.

In the escalating battle against antimicrobial resistance, there is an urgent need to discover and investigate new antibiotic strategies. Bacteriophages are untapped reservoirs of such potential antimicrobials. This study focused on Hypothetical Proteins of Unknown Function (HPUFs) from a Staphylococcus phage Stab21. We examined its HPUFs for bactericidal activity against E. coli using a Next Generation Sequencing (NGS)-based approach. Among the 96 HPUFs examined, 5 demonstrated cross-species toxicity towards E. coli, suggesting the presence of shared molecular targets between E. coli and S. aureus. One toxic antibacterial HPUF (toxHPUF) was found to share homology with a homing endonuclease. The implications of these findings are profound, particularly given the potential broad applicability of these bactericidal agents. This study confirms the efficacy of NGS in streamlining the screening process of toxHPUFs, contributes significantly to the ongoing exploration of phage biology, and offers promises in the search for potent antimicrobial agents.

Weissella strains are often detected in spontaneously fermented foods. Because of their abilities to produce lactic acid and functional exopolysaccharides as well as their probiotic traits, Weissella spp. improve not only the sensorial properties but also nutritional values of the fermented food products. However, some Weissella species have been associated with human and animal diseases. In the era of vast genomic sequencing, new genomic/genome data are becoming available to the public on daily pace. Detailed genomic analyses are due to provide a full understanding of individual Weissella species. In this study, the genomes of six Weissella paramesenteroides strains were de novo sequenced. The genomes of 42 W. paramesenteroides strains were compared to discover their metabolic and functional potentials in food fermentation. Comparative genomics and metabolic pathway reconstructions revealed that W. paramesenteroides is a compact group of heterofermentative bacteria with good capacity of producing secondary metabolites and vitamin Bs. Since the strains rarely harbored plasmid DNA, they did not commonly possess the genes associated with bacteriocin production. All 42 strains were shown to bear vanT gene from the glycopeptide resistance gene cluster vanG. Yet none of the strains carried virulence genes.

Lactococcus lactis is a commonly used fermenting bacteria in cheese, beverages and meat products. Due to the lack of simplified chassis strains, it has not been widely used in the fields of synthetic biology. Thus, the construction of lactic acid bacteria chassis strains becomes more and more important. In this study, we performed whole genome sequencing, annotation and analysis of L. lactis N8. Based on the genome analysis, we found that L. lactis N8 contains two large plasmids, and the function prediction of the plasmids shows that some regions are related to carbohydrate transport/metabolism, multi-stress resistance and amino acid uptake. L. lactis N8 contains a total of seven prophage-related fragments and twelve genomic islands. A gene cluster encoding a hybrid NRPS–PKS system that was found in L. lactis N8 reveals that the strain has the potential to synthesize novel secondary metabolites. Furthermore, we have constructed a simplified genome chassis of L. lactis N8 and achieved the largest amount of deletion of L. lactis so far. Taken together, the present study offers further insights into the function and potential role of L. lactis N8 as a model strain of lactic acid bacteria and lays the foundation for its application in the field of synthetic biology.