Sucrose contributed to the biofilm formation of Tetragenococcus halophilus and changed the biofilm structure.

Based on the previous research results that the addition of sucrose in the medium improved the biofilm formation of Tetragenococcus halophilus, the influence of sucrose on biofilm formation was explored. Moreover, the influence of exogenous expression of related genes sacA and galE from T. halophilus on the biofilm formation of L. lactis NZ9000 was investigated. The results showed that the addition of sucrose in the medium improved the biofilm formation, the resistance of biofilm cells to freeze-drying stress, and the contents of exopolysaccharides (EPS) and eDNA in the T. halophilus biofilms. Meanwhile, the addition of sucrose in the medium changed the monosaccharide composition of EPS and increased the proportion of glucose and galactose in the monosaccharide composition. Under 2.5% (m/v) salt stress condition, the expression of gene sacA promoted the biofilm formation and the EPS production of L. lactis NZ9000 with the sucrose addition in the medium and changed the EPS monosaccharide composition. The expression of gene galE up-regulated the proportion of rhamnose, galactose, and arabinose in the monosaccharide composition of EPS, and down-regulated the proportion of glucose and mannose. This study will provide a theoretical basis for regulating the biofilm formation of T. halophilus, and provide a reference for the subsequent research on lactic acid bacteria biofilms.

Improvement of the viability of Tetragenococcus halophilus under acidic stress by forming the biofilm cell structure based on RNA-Seq and iTRAQ analyses.

BACKGROUND: Tetragenococcus halophilus is a halophilic lactic acid bacterium (LAB) isolated from soya sauce moromi. During the production of these fermented foods, acid stress is an inevitable environmental stress. In our previous study, T. halophilus could form biofilms and the cells in the biofilms exhibited higher cell viability under multiple environmental stresses, including acid stress. RESULTS: In this study, the effect of preformed T. halophilus biofilms on cell survival, cellular structure, intracellular environment, and the expression of genes and proteins under acid stress was investigated. The result showed that acid stress with pH 4.30 for 1.5 h reduced the live T. halophilus cell count and caused cellular structure damage. However, T. halophilus biofilm cells exhibited greater cell survival under acid stress than the planktonic cells, and biofilm formation reduced the damage of acid stress to the cell membrane and cell wall. The biofilm cells maintained a higher level of H+ -ATPase activity and intracellular ammonia concentration after acid stress. The RNA-Seq and iTRAQ technologies revealed that the genes and proteins associated with ATP production, the uptake of trehalose and N-acetylmuramic acid, the assembly of H+ -ATPase, amino acid biosynthesis and metabolism, ammonia production, fatty acid biosynthesis, CoA biosynthesis, thiamine production, and acetoin biosynthesis might be responsible for the stronger acid tolerance of T. halophilus biofilm cells together. CONCLUSION: These findings further explained the mechanisms that allowed LAB biofilm cells to resist environmental stress. © 2023 Society of Chemical Industry.

Multispecies biofilms in fermentation: Biofilm formation, microbial interactions, and communication.

Food fermentation is driven by microorganisms, which usually coexist as multispecies biofilms. The activities and interactions of functional microorganisms and pathogenic bacteria in biofilms have important implications for the quality and safety of fermented foods. It was verified that the biofilm lifestyle benefited the fitness of microorganisms in harsh environments and intensified the cooperation and competition between biofilm members. This review focuses on multispecies biofilm formation, microbial interactions and communication in biofilms, and the application of multispecies biofilms in food fermentation. Microbial aggregation and adhesion are important steps in the early stage of multispecies biofilm formation. Different biofilm-forming abilities and strategies among microorganisms lead to several types of multispecies biofilm formation. The spatial distribution of multispecies biofilms reflects microbial interactions and biofilm function. Then, we discuss the intrinsic factors and external manifestations of multispecies biofilm system succession. Several typical interspecies cooperation and competition modes and mechanisms of microbial communication were reviewed in this review. The main limitations of the studies included in this review are the relatively small number of studies of biofilms formed by functional microorganisms during fermentation and the lack of direct evidence for the formation process of multispecies biofilms and microbial interactions and communication within biofilms. This review aims to provide the food industry with a sufficient understanding of multispecies biofilms in food fermentation. Practical Application: Meanwhile, it offers a reference value for better controlling and utilizing biofilms during food fermentation process, and the improvement of the yield, quality, and safety of fermented products including Chinese Baijiu, cheeese,kefir, soy sauce, kombucha, and fermented olive.

Co-culture with Tetragenococcus halophilus improved the ethanol tolerance of Zygosaccharomyces rouxii by maintaining cell surface properties.

The accumulation of ethanol has a negative effect on the viability and fermentation performance of microorganisms during the production of fermented foods because of its toxicity. In this study, we investigated the effect of co-culture with Tetragenococcus halophilus on ethanol stress resistance of Zygosaccharomyces rouxii. The result showed that co-culture with T. halophilus promoted cell survival of Z. rouxii under ethanol stress, and the tolerance improved with increasing co-culture time when ethanol content was 8%. Physiological analysis showed that the co-cultured Z. rouxii cells maintained higher intracellular content of trehalose and amino acids including tyrosine, tryptophan, arginine and proline after 8% ethanol stress for 90 min. The membrane integrity analysis and biophysical analysis of the cell surface indicated that the presence of ethanol resulted in cell membrane damage and changes of Young's modulus value and roughness of cell surface. While the co-cultured Z. rouxii cells exhibited better membrane integrity, stiffer and smoother cell surface than single-cultured cells under ethanol stress. As for transcriptomic analyses, the genes involved in unsaturated fatty acid biosynthesis, trehalose biosynthesis, various types of N-glycan biosynthesis, inositol phosphate metabolism, MAPK signaling pathway and tight junction had higher expression in co-cultured Z. rouxii cells with down-regulation of majority of gene expression after stress. And these genes may function in the improvement of ethanol tolerance of Z. rouxii in co-culture.

Formation of biofilm changed the responses of Tetragenococcus halophilus to ethanol stress revealed by transcriptomic and proteomic analyses.

Biofilms were found to promote the survival of Tetragenococcus halophilus, a functional halophilic lactic acid bacterium in the production of high-salt fermented foods under various environmental stresses including ethanol stress. Here, a comprehensive exploration of the response of T.halophilus biofilms and planktonic cells to ethanol stress was performed. Biofilms showed an ability to reduce death and damage of cell membrane and wall under 12% ethanol stress The formation of biofilm changed the characteristic of Fourier transformed infrared spectroscopy (FT-IR). RNA-seq technology and iTRAQ technology revealed the differential expression of genes and proteins in biofilm and planktonic cells with or without ethanol treatment. The differentially expressed genes and proteins played positive roles in the biosynthesis of polysaccharides, proteins, and DNA, benefitting biofilm matrix production. The shelter provided by biofilms and the differential expression of genes and proteins involved in citrate formation, malate utilization, and the biosynthesis of tryptophan, fatty acid, lipoteichoic acid, and peptidoglycan might contribute to the stress tolerance of biofilm cells together. Results presented in this study may contribute to our understanding of biofilm formation by T. halophilus and the roles of bacterial biofilm in stress tolerance.

Effects of salt concentration on the quality and microbial diversity of spontaneously fermented radish paocai.

Paocai is a traditional Chinese fermented vegetable product popular in Asian countries. As an important additive, salt concentration is closely related to the quality of paocai. The aim of this study was to investigate the effect of salt concentration on the physicochemical characteristics, microbial diversity, and flavor profiles of spontaneously fermented radish, and the cross-correlation between microorganisms and flavor compounds was also revealed. Analysis of the microbial diversity of paocai showed that Firmicutes, Proteobacteria, and Ascomycota were detected as the main phyla with different salt concentrations, Weissella and Lactobacillus were the predominant bacterial genera, and Yarrowia dominated the fungal genera. Based on LEfSe analysis, Lactobacillus, Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium, Microbacterium, Lactococcus, Staphylococcus, and Weissella were regarded as differential genera caused by differences in salinity. Analysis of the flavor compounds showed that 17 free amino acids, 5 isothiocyanates, 3 terpenes, 15 sulphur-containing compounds, 16 esters, 8 organic acids, 9 aldehydes, 8 ketones, 25 alcohols, 7 nitriles, 2 lactones, and 10 hydrocarbons were detected. Then, the correlation between the microbial community and flavor compounds was revealed, and the results indicated that several bacterial genera significantly correlated with flavors, including Lactobacillus, Kosakonia, Weissella, Leuconostoc, and Staphylococcus, while fungi had weak correlations with flavors. In addition, Metacyc pathway analysis was carried out to elucidate the effect of salt content on the metabolic pathways, showing that most flavor-related pathways were up-regulated with the increase in salt content. Results presented in this study may contribute to further understanding the role of salt in the fermentation of paocai and provide effective references for quality control of traditional fermented vegetables.

Structural characterization and bioactivity of novel exopolysaccharides produced by Tetragenococcus halophilus.

In this study, exopolysaccharides produced by Tetragenococcus halophilus were extracted and purified. Two fractions named EPS-1 and EPS-2 were obtained with average molecular weights of 2613.4 and 93.4 kDa, respectively. Analysis of the structures demonstrated that EPS-1 was mainly composed of galactose, glucose, mannose and glucuronic acid, and EPS-2 mostly consisted of glucose and mannose. 800 MHz nuclear magnetic resonance spectroscopy elucidated different and partial primary structures of EPS-1 and EPS-2. The microtopography was observed by atomic force microscope and scanning electron microscope. In addition, the two fractions exhibited antioxidant activities on DPPH, hydroxyl and ABTS•+ radicals. Furthermore, EPS-1 and EPS-2 could protect Lactococcus lactis against cryogenic and lyophilized stress. These results indicated that exopolysaccharides from T. halophilus in this study would have great potential in the application of food and pharmaceutical industry.

Formation of Biofilm by Tetragenococcus halophilus Benefited Stress Tolerance and Anti-biofilm Activity Against S. aureus and S. Typhimurium.

Tetragenococcus halophilus, a halophilic lactic acid bacterium (LAB), plays an important role in the production of high-salt fermented foods. Generally, formation of biofilm benefits the fitness of cells when faced with competitive and increasingly hostile fermented environments. In this work, the biofilm-forming capacity of T. halophilus was investigated. The results showed that the optimal conditions for biofilm formation by T. halophilus were at 3-9% salt content, 0-6% ethanol content, pH 7.0, 30°C, and on the surface of stainless steel. Confocal laser scanning microscopy (CLSM) analysis presented a dense and flat biofilm with a thickness of about 24 µm, and higher amounts of live cells were located near the surface of biofilm and more dead cells located at the bottom. Proteins, polysaccharides, extracellular-DNA (eDNA), and humic-like substances were all proved to take part in biofilm formation. Higher basic surface charge, greater hydrophilicity, and lower intracellular lactate dehydrogenase (LDH) activities were detected in T. halophilus grown in biofilms. Atomic force microscopy (AFM) imaging revealed that biofilm cultures of T. halophilus had stronger surface adhesion forces than planktonic cells. Cells in biofilm exhibited higher cell viability under acid stress, ethanol stress, heat stress, and oxidative stress. In addition, T. halophilus biofilms exhibited aggregation activity and anti-biofilm activity against Staphylococcus aureus and Salmonella Typhimurium. Results presented in the study may contribute to enhancing stress tolerance of T. halophilus and utilize their antagonistic activities against foodborne pathogens during the production of fermented foods.

Heat Adaptation Induced Cross Protection Against Ethanol Stress in Tetragenococcus halophilus: Physiological Characteristics and Proteomic Analysis.

Ethanol is a toxic factor that damages membranes, disturbs metabolism, and may kill the cell. Tetragenococcus halophilus, considered as the cell factory during the manufacture of traditional fermented foods, encounters ethanol stress, which may affect the viability and fermentative performance of cells. In order to improve the ethanol tolerance of T. halophilus, a strategy based on cross protection was proposed in the current study. The results indicated that cross protection induced by heat preadaptation (45°C for 1.5 h) could significantly improve the stress tolerance (7.24-fold increase in survival) of T. halophilus upon exposure to ethanol (10% for 2.5 h). Based on this result, a combined analysis of physiological approaches and TMT-labeled proteomic technology was employed to investigate the protective mechanism of cross protection in T. halophilus. Physiological analysis showed that the heat preadapted cells exhibited a better surface phenotype, higher membrane integrity, and higher amounts of unsaturated fatty acids compared to unadapted cells. Proteomic analysis showed that a total of 163 proteins were differentially expressed in response to heat preadaptation. KEGG enrichment analysis showed that energy metabolism, membrane transport, peptidoglycan biosynthesis, and genetic information processing were the most abundant metabolic pathways after heat preadaptation. Three proteins (GpmA, AtpB, and TpiA) involved in energy metabolism and four proteins (ManM, OpuC, YidC, and HPr) related to membrane transport were up-regulated after heat preadaptation. In all, the results of this study may help understand the protective mechanisms of preadaptation and contribute to the improvement of the stress resistance of T. halophilus during industrial processes.

Pneumonia Detection Using an Improved Algorithm Based on Faster R-CNN.

Pneumonia remains a threat to human health; the coronavirus disease 2019 (COVID-19) that began at the end of 2019 had a major impact on the world. It is still raging in many countries and has caused great losses to people's lives and property. In this paper, we present a method based on DeepConv-DilatedNet of identifying and localizing pneumonia in chest X-ray (CXR) images. Two-stage detector Faster R-CNN is adopted as the structure of a network. Feature Pyramid Network (FPN) is integrated into the residual neural network of a dilated bottleneck so that the deep features are expanded to preserve the deep feature and position information of the object. In the case of DeepConv-DilatedNet, the deconvolution network is used to restore high-level feature maps into its original size, and the target information is further retained. On the other hand, DeepConv-DilatedNet uses a popular fully convolution architecture with computation shared on the entire image. Then, Soft-NMS is used to screen boxes and ensure sample quality. Also, K-Means++ is used to generate anchor boxes to improve the localization accuracy. The algorithm obtained 39.23% Mean Average Precision (mAP) on the X-ray image dataset from the Radiological Society of North America (RSNA) and got 38.02% Mean Average Precision (mAP) on the ChestX-ray14 dataset, surpassing other detection algorithms. So, in this paper, an improved algorithm that can provide doctors with location information of pneumonia lesions is proposed.

Incorporation of Exogenous Fatty Acids Enhances the Salt Tolerance of Food Yeast Zygosaccharomyces rouxii.

Fatty acids have great effects on the maintenance of the cell membrane structure, cell viability, and cell metabolisms. In this study, we sought to elucidate the effects of exogenous fatty acids on the salt tolerance of food yeast Zygosaccharomyces rouxii. Results showed that Z. rouxii can grow by using exogenous fatty acids (C12:0, C14:0, C16:0, C16:1, C18:0, C18:1, and C18:2) as the sole carbon source. Four fatty acids (C12:0, C16:0, C16:1, and C18:1) can improve the salt tolerance of cells, enhance the formation of the cell biofilm, regulate the chemical compositions, restore growth in the presence of cerulenin, regulate the contents of membrane fatty acids, and control the expression of key genes in the fatty acid metabolism. Our results reveal that Z. rouxii can synthesize membrane fatty acids from exogenous fatty acids and the supplementation of these fatty acids can override the need for de novo fatty acid biosynthesis.

Effect of co-culture with Tetragenococcus halophilus on the physiological characterization and transcription profiling of Zygosaccharomyces rouxii.

Zygosaccharomyces rouxii and Tetragenococcus halophilus are widely existed and play vital roles during the manufacture of fermented foods such as soy sauce. The aim of this study was to elucidate the effect of T. halophilus CGMCC 3792 on the physiological characterizations and transcription profiling of Z. rouxii CGMCC 3791. Salt tolerance analysis revealed that co-culture with T. halophilus enhanced the salt tolerance of Z. rouxii during salt stress. Analysis of the volatile compounds revealed that co-culture reduced the level of 1-butanol, improved the level of octanoic acid which all were produced by T. halophilus and reduced the level of phenylethyl alcohol produced by Z. rouxii. The presence of Z. rouxii decreased the contents of 3,4-dimethylbenzaldehyde and acetic acid produced by T. halophilus. In addition, co-culture improved the content of benzyl alcohol significantly. Analysis of membrane fatty acid showed that co-culture improved the content of palmitic (C16:0) and stearic (C18:0) acids in cells of Z. rouxii, and reduced the contents of myristic (C14:0), palmitoleic acid (C16:1) and oleic acid (C18:1). In order to further explore the interactions between the two strains, RNA-seq technology was used to investigate the effect of co-culture with T. halophilus on the transcription profiling of Z. rouxii. By comparing cells incubated in co-culture group with cells incubated in single-culture group, a total of 967 genes were considered as differentially expressed genes (DEGs). Among the DEGs, 72 genes were up-regulated, while 895 genes were down-regulated. These DEGs took party in various activities in cells of Z. rouxii, and the result showed co-culture with T. halophilus had a positive effect on proteolysis, the attachment of a cell to another cell, extracellular protein accumulation, energy metabolism, and a negative effect on oxidative phosphorylation, small molecular substances metabolism, DNA replication and repair, and transcription in cells of Z. rouxii. Results presented in this study may contribute to further understand the interactions between Zygosaccharomyces rouxii and Tetragenococcus halophilus.