The antibacterial mechanism of (-)-epigallocatechin-3-gallate (EGCG) against Campylobacter jejuni through transcriptome profiling.

(-)-Epigallocatechin-3-gallate (EGCG) has been shown antibacterial activity against Campylobacter jejuni; however, the relevant antibacterial mechanism is unknown. In this study, phenotypic experiments and RNA sequencing were used to explore the antibacterial mechanism. The minimum inhibitory concentration of EGCG on C. jejuni was 32 µg/mL. EGCG-treated was able to increase intracellular reactive oxygen species levels and decline bacterial motility. The morphology and cell membrane of C. jejuni after EGCG treatment were observed collapsed, broken, and agglomerated by field emission scanning electron microscopy and fluorescent microscopy. The RNA-seq analysis presents that there are 36 and 72 differential expressed genes after C. jejuni was treated by EGCG with the concentration of 16 and 32 µg/mL, respectively. EGCG-treated increased the thioredoxin expression, which was a critical protein to resist oxidative stress. Moreover, downregulation of the flgH and flgM gene in flagellin biosynthesis of C. jejuni was able to impair the flagella, reducing cell motility and virulence. The primary antibacterial mechanism revealed by RNA-seq is that EGCG with iron-chelating activity competes with C. jejuni for iron, causing iron deficiency in C. jejuni, which potentially impacts the survival and virulence of C. jejuni. The results suggested a new direction for exploring the activity of EGCG against C. jejuni in the food industry. PRACTICAL APPLICATION: A deeper understanding of the antibacterial mechanism of EGCG against C. jejuni was more beneficial in improving the food safety, eliminating concerns about human health caused by C. jejuni in future food, and promoting the natural antibacterial agent EGCG application in the food industry.

Transcriptomic analyses of Vibrio parahaemolyticus under the phenyllactic acid stress.

Phenyllactic acid (PLA) generally recognized as a natural organic acid shows against Vibrio parahaemolyticus activity. In this study, V. parahaemolyticus ATCC17802 (Vp17802) was cultured under the stress of 1/2MIC PLA, and then the antibacterial mechanisms were explored via transcriptomics. The minimum inhibitory concentration (MIC) of PLA against Vp17802 was 3.2 mg/mL, and the time-kill analysis resulted that Vp17802 was inhibited. PLA was able to destroy the bacterial membrane, leading to the leakage of intracellular substances and decline of ATP levels. The RNA-sequencing analysis results indicated that 1616 significantly differentially expressed genes were identified, among which 190 were up-regulated and 1426 were down-regulated. Down-regulation of the icd2 gene in the TCA cycle mediates blockage of tyrosine metabolic, arginine biosynthesis, and oxidative phosphorylation, causing insufficient energy supply of Vp17802. Moreover, PLA could cause amino acids, metal ions, and phosphate transporters to be blocked, affecting the acquisition of nutrients. The treatment by PLA altered the expression of genes encoding functions involved in quorum sensing, flagellar assembly, and cell chemotaxis pathway, which may be interfering with the biofilm formation in Vp17802, reducing cell motility. Overall, 1.6 mg/mL PLA inhibited the growth of Vp17802 by disrupting to uptake of nutrients, cell metabolism, and the formation of biofilms. The results suggested a new direction for exploring the activity of PLA against Vp17802 and provided a theoretical basis for bacterial pathogen control in the food industry. KEY POINTS: •RNA sequencing was carried out to indicate the antibacterial mechanism of Vp17802. •The icd2 gene in the TCA cycle mediates blockage of metabolic of Vp17802. •The biofilm formation has interfered with 1.6 mg/mL PLA, which could reduce cell motility and virulence.

Effect of allyl isothiocyanate on the growth and virulence of Clostridium perfringens and its application on cooked pork.

The objective of this study is to explore the antibacterial action modes and virulence-inhibitory effects of allyl isothiocyanate (AITC) against Clostridium perfringens (C. perfringens). The minimum inhibitory concentration (MIC) of AITC against vegetative cells of Cp 13124 was 0.1 µL/mL, and the time-kill kinetics analysis revealed that AITC could significantly suppress the growth of Cp 13124. According to the results from scanning electron microscopy (SEM), fluorescence microscopy, and UV absorbance substance detection, the cell membrane of Cp 13124 was damaged upon AITC treatment, causing a loss of integrity and the release of intracellular substances. Meanwhile, the fluorescence quenching experiment indicated the interaction of AIT-C with membrane proteins, which caused changes in the conformation of membrane proteins. Measurement of reactive oxygen species (ROS) and flow cytometry analysis demonstrated that AITC could induce apoptosis through oxidative stress. The formation of Cp 13124 biofilms was inhibited by AITC using the crystalline violet method, which was possibly related to the inhibition of sliding motility. Finally, low concentrations of AITC could be used as an antibacterial agent to inhibit the outgrowth of Cp 13124 in cooked pork, suggesting that AITC is a promising candidate for novel preservatives in the meat business.

The alleviating effect of ellagic acid on DSS-induced colitis via regulating gut microbiomes and gene expression of colonic epithelial cells.

The anti-inflammatory effect of ellagic acid (EA) and its possible underlying mechanism in dextran sulfate sodium (DSS)-induced mouse chronic colonic inflammation were studied. It was observed that EA administration significantly alleviated the colonic inflammation phenotypes, including decreasing the disease activity index (DAI), enhancing the body weight loss, and improving the shortened length of the colon and pathological damage of colon tissue. Additionally, EA reshaped the constitution of the gut microbiota by elevating the ratio of Bacteroidetes along with Bacteroides and Muribaculaceae, while decreasing the proportion of Firmicutes. The Phylogenetic Investigation of Communities by Reconstruction of Unobserved States 2 (PICRUSt2) revealed that the metabolic function of the gut microbiota was also changed. Furthermore, mouse colon transcriptome analysis showed that the tight junction and peroxisome proliferator-activated receptor (PPAR) signaling pathways were activated and the expressions of related genes were upregulated after EA intervention. These results showed that EA could remodel the gut bacterial composition, change the intestinal epithelial cell gene expressions in mice, and consequently improve the colonic inflammatory symptoms.

Dextran-Sulfate-Sodium-Induced Colitis-Ameliorating Effect of Aqueous Phyllanthus emblica L. Extract through Regulating Colonic Cell Gene Expression and Gut Microbiomes.

The anti-inflammation effect of aqueous Phyllanthus emblica L. extract (APE) and its possible underlying mechanism in dextran sulfate sodium (DSS)-induced mice chronic colonic inflammation were studied. APE treatment significantly improved the colitic symptoms, including ameliorating the shortening of the colon, increasing the DSS-induced body weight loss, reducing the disease activity index, and reversing the condition of colon tissue damage of mucus lost and goblet cell reduction. Overproduction of serum pro-inflammatory cytokines were suppressed by the treatment of APE. Gut microbiome analysis showed that APE remodeled the structure of gut bacteria in phylum and genus levels, upregulating the abundance of phylum Bacteroidetes, family Muribaculaceae, and genus Bacteroides and downregulating the abundance of phylum Firmicutes. The reshaped gut microbiome caused metabolic functions and pathway change with enhanced queuosine biosynthesis and reduced polyamine synthesis pathway. Colon tissue transcriptome analysis further elucidated APE-inhibited mitogen-activated protein kinase (MAPK), cytokine-cytokine receptor interaction, and tumor necrosis factor (TNF) signaling pathways and the expressions of the genes that promote the progress of colorectal cancer. It turned out that APE reshaped the gut microbiome and inhibited MAPK, cytokine-cytokine receptor interaction, and TNF signaling pathways as well as the colorectal-cancer-related genes to exert its colitis protective effect.

Osmotic stress relief antibiotic tolerance of 1,8-cineole in biofilm persister cells of Escherichia coli O157:H7 and expression of toxin-antitoxin system genes.

The control of E. coli activity from forming biofilm and persister cells is an essential factor in both the health and food industries. The efficacy of antimicrobial treatment is often limited due to their low penetrability as biofilm formation protect cells within from physical or chemical threats. Among other factors, osmotic stress has shown to have a high capacity to enhance the antimicrobial activities against various pathogens. Thus, this study aimed to test the hypothesis that the antimicrobial activity of cineole (CN) could be enhanced under osmotic stress to inhibit biofilm and persister cells. Time-kill analysis revealed that CN under NaCl-induced osmotic stress (CN-S) had better inhibitory effect on E. coli biofilm. 5% CN-S altered the integrity, hydration, motilities and exopolysaccharide production of E. coli cells. Also, the outer membrane permeability, surface roughness and hydrophobicity which determine initial cell adhesion, aggregation and colony assembly were significantly perturbed. Furthermore, the expression levels of virulence genes stx1, stx2, eae, flhD, and the TA system antitoxin genes mazE, hipB were downregulated. When applied to cucumber, the rate of increase in internalized bacterial cells significantly reduced after storage at 4 °C for 48 h. Thus, the results suggested that the application of osmotic stress could minimize the working concentration of antimicrobials in real food systems, which could be helpful in counteracting the growing concern of microbial resistance.

Transcriptomic and proteomic analysis of Staphylococcus aureus response to cuminaldehyde stress.

The previous study indicated that cuminaldehyde (CUM) could be used as an antibacterial agent in sauced beef to reduce the propagation of Staphylococcus aureus (S. aureus). This research took sauced beef treated with 0.4 µL/mL CUM as the research object. Transcriptomic and proteomic methods were used to comprehensively analyze the changes in genes and proteins of S. aureus under CUM stress. A total of 258 differentially expressed genes (DEGs, 178 up-regulated and 80 down-regulated) and 384 differentially expressed proteins (DEPs, 61 up-regulated and 323 down-regulated) were found. It was observed that CUM destroyed the cell wall and cell membrane by inhibiting the synthesis of peptidoglycan and fatty acid. Low energy consumption strategies were formed by reducing glycolysis and ribosome de novo synthesis. The levels of genes and proteins associated with the glycine, serine, threonine, methionine, cysteine, and branched-chain amino acids were dramatically changed, which impaired protein synthesis and reduced bacterial viability. In addition, the up-regulated DEGs and DEFs involved in DNA replication, recombination and single-stranded DNA-binding contributed to DNA repair. Moreover, ATP-binding cassettes (ABC) transporters were also perturbed, such as the uptake of betaine and iron were inhibited. Thus, this study revealed the response mechanism of S. aureus under the stress of CUM, and provided a theoretical basis for the application of CUM in meat products.

Comparative transcriptomics to reveal the mechanism of enhanced catalytic activities of Aspergillus niger whole-cells cultured with different inducers in hydrolysis of citrus flavonoids.

A new Aspergillus niger whole-cell catalyst was cultured for the cascade hydrolysis of hesperidin (HES) to produce high-value hesperetin-7-O-glucoside (HG) and hesperetin with high conversion (above 90%). Moreover, the inducers used were shown to be useful for cell growth and to induce cells to produce specific enzymes. Remarkably, the type of inducers determined whether the cells can hydrolyze HES. The product composition was also controllable by adjusting different inducers. Transcriptome analysis suggested that both naringin-vs-blank group and saccharose-vs-blank group had obviously difference in gene expression. The naringin-vs-blank group was mainly up-regulated differentially expressed genes (DEGs), while saccharose-vs-blank group was mainly down-regulated DEGs. The Gene Ontology (GO) analysis showed that whether naringin or saccharose was added as an inducer would greatly affect the catalytic activity of cells. Furthermore, 3 genes related to rhamnosidase, 14 genes related to glucosidase and 5 genes related to hydrolase activity were found. These genes were not only involved in rhamnosidase and glucosidase activities, but also spliceosome and the sucrose and starch metabolic pathways. The quantitative real-time polymerase chain reaction (qRT-PCR) analysis indicated that the results of transcriptome sequencing were reliable. This study gave a new approach to hydrolyze HES, and new perspectives to understand the mechanisms associated with the hydrolysis of whole-cell catalyst.

Aqueous Extract of Phyllanthus emblica L. Alleviates Functional Dyspepsia through Regulating Gastrointestinal Hormones and Gut Microbiome In Vivo.

Phyllanthus emblica L. fruits were extracted by a hot water assistant with ultrasonication to obtain aqueous Phyllanthus emblica L. extract (APE). The ameliorating functional dyspepsia (FD) effect of a low dose (150 mg/kg) and a high dose (300 mg/kg) of APE was exhibited by determining the gastrointestinal motility, gastrointestinal hormones, and gut microbiome shifts in reserpine induced FD male balb/c mice. APE increased the gastrointestinal motility including the gastric emptying (GE) rate and small intestinal transit (SIT) rate. The level of serum gastrointestinal hormones such as motilin (MTL) and gastrin (GAS) increased, and the vasoactive intestinal peptide (VIP) level decreased after the administration of APE. Furthermore, the gut microbiome analysis demonstrated that APE could regulate the microbiome structure and restore homeostasis by elevating useful bacterial abundance, while simultaneously decreasing harmful bacterial abundance. This study demonstrated the ameliorating FD effect of APE and its potential efficacy in curing functional gastrointestinal disorders and maintaining a healthy digestive tract.

Effects of citronellal on growth and enterotoxins production in Staphylococcus aureus ATCC 29213.

Staphylococcus aureus (S. aureus) is known to be one of the most common foodborne pathogens capable of secreting a wide range of exotoxins such as enterotoxin, which severely threatens the health of consumers. Over the past few years, the development of safe and effective strategies in inhibiting the growth and enterotoxins generation of S. aureus in food turns out to be the research focus and emphasis. This research explores citronellal (CIT), a native compound with extensive existence in spices, which could effectively inhibit the growth and enterotoxins generation of S. aureus (ATCC 29213). Results from minimal inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and time-kill curves, showed that CIT could tremendously inhibit the growth of S. aureus. Analysis on hemolysin showed that CIT at sub-MIC could significantly (p < 0.05) inhibit the hemolytic activity of S. aureus. As revealed by the results of ELISA, the production of enterotoxins in the culture supernatant and pork meat decreased significantly (p < 0.05) after exposure to CIT at sub-MIC. Furthermore, a significant (p < 0.05) decrease in dose-dependent was found in the transcription levels of virulence-related genes. In all, CIT proved to be a possible inhibitor of the growth and enterotoxins production of S. aureus with highly promising application in the food industry.

The combination of thymol and cinnamaldehyde reduces the survival and virulence of Listeria monocytogenes on autoclaved chicken breast.

AIMS: To reveal the antibacterial mechanism of the combination of thymol and cinnamaldehyde to Listeria monocytogenes ATCC 19115 on autoclaved chicken breast. METHODS AND RESULTS: In this study, L. monocytogenes ATCC 19115 on autoclaved chicken breast was exposed to the stress of 125 µg/ml thymol and 125 µg/ml cinnamaldehyde, and transcriptome analysis was used to reveal the crucial antibacterial mechanism. According to the results, 1303 significantly differentially expressed genes (DEGs) were identified. Treated by thymol and cinnamaldehyde in combination, pyrimidine and branched-chain amino acid biosynthesis of L. monocytogenes were thwarted which impairs its nucleic acid biosynthesis and intracellular metabolism. The up-regulated DEGs involved in membrane composition and function contributed to membrane repair. Besides, pyruvate catabolism and TCA cycle were restrained which brought about the disturbance of amino acid metabolism. ABC transporters were also perturbed, for instance, the uptake of cysteine, D-methionine, and betaine was activated, while the uptake of vitamin, iron, and carnitine was repressed. Thus, L. monocytogenes tended to activate PTS, glycolysis, glycerol catabolism, and pentose phosphate pathways to obtain energy to adapt to the hostile condition. Noticeably, DEGs involved in virulence factors were totally down-regulated, including genes devoted to encoding flagella, chemotaxis, biofilm formation, internalin as well as virulence gene clusters. CONCLUSIONS: The combination of thymol and cinnamaldehyde is effective to reduce the survival and potential virulence of L. monocytogenes on autoclaved chicken breast. SIGNIFICANCE AND IMPACT OF STUDY: This work contributes to providing theoretical information for the application and optimization of thymol and cinnamaldehyde in ready-to-eat meat products to inhibit L. monocytogenes.

Evaluation of Cronobacter sakazakii biofilm formation after sdiA knockout in different osmotic pressure conditions.

This study characterizes the impact of sdiA on biofilm formation under normal or osmotic stress conditions in Cronobacter sakazakii by constructing a sdiA deletion mutant (ΔsdiA). Here, the downregulation of flagellar assembly-related genes and upregulation of capsular, cellulose and lipopolysaccharide biosynthesis-associated genes in ΔsdiA were observed when compared to the wild type strain (WT) through transcriptomic analysis. Meanwhile, reduced ability of motility, enhanced cell surface hydrophobicity and stronger biofilms with extracellular matrix were observed in WT with deletion of sdiA. Both WT and ΔsdiA formed more biofilm in low osmotic stress medium, while in hyperosmolarity conditions, formation of biofilm was dramatically reduced. Our findings supported that sdiA might suppress biofilm formation of C. sakazakii by regulating biosynthesis of flagellar and extracellular polymeric substances. This study investigates the role of sdiA on biofilm formation in C. sakazakii, and provides the basis for the inhibition of C. sakazakii in food industry and infant-feeding.

Synergistic inactivation of Escherichia coli O157:H7 and Staphylococcus aureus by gallic acid and thymol and its potential application on fresh-cut tomatoes.

Antibacterial activity against Escherichia coli O157:H7 and Staphylococcus aureus of five typical plant-derived compounds [gallic acid (G.A), citral (Cit), thymol (Thy), salicylic acid (S.A), lauric acid (L.A)] were investigated by determining the minimum inhibitory concentration (MIC) and the fractional inhibitory concentration index (FICI). The results showed that only a combination of Thy and G.A (TGA), with a concentration of 0.1 and 1.25 mg/mL, respectively, had a synergistic effect (FICI = 0.5) on both E. coli O157:H7 and S. aureus. The amount of Thy and G.A in mixture were four-fold lower than the MICs of the individuals shown to cause the equivalent antimicrobial activity in trypticase soy broth (TSB). The microbial reduction obtained in TSB with addition of TGA were significantly higher (P < 0.05) than the reduction shown for the broth supplemented with the separated phenolics. TGA caused the changes of morphology and membrane integrity of bacteria. Additionally, the application of TGA on fresh-cut tomatoes are investigated. Fresh-cut tomatoes inoculated with E. coli O157:H7and S. aureus were washed for 2min, 5min, 10min at 4 °C, 25 °C, 40 °C in 0.3% NaOCl, or water containing TGA at various concentrations. Overall, the reduction of TGA achieved against S. aureus is higher than E. coli O157:H7. Same concentrations of combined antimicrobials at a temperature of 40 °C further increased the degree of microbial inactivation, with an additional 0.89-1.51 log CFU/g reduction compared to that at 25 °C. Moreover, 1/2MICThy+1/2MICG.A at 25 °C for 10min or 40 °C for 5min were generally acceptable with sensorial scores higher than 7. Our results showed that TGA could work synergistically on the inactivation of the tested bacteria and may be used as an alternative disinfectant of fresh produce.

Biocatalytical Acyl-Modification of Puerarin: Shape Gut Microbiota Profile and Improve Short Chain Fatty Acids Production in Rats.

Gut microbiota balance and metabolites have become a potentially mechanism in maintaining health. The specific aim of this study was to compare the modulation of puerarin and puerarin acid esters on gut microbial composition and metabolites. Male mice were fed a control diet or diets supplemented with puerarin, puerarin propanoate ester, puerarin hexanoate ester, puerarin myristate ester for 24 h, respectively. The result revealed that puerarin acid esters with different chain lengths showed different activities to create more own impacted bacterial. Puerarin propanoate and puerarin hexanoate ester significantly improved the diversity of microbiota and promoted the relative abundance of beneficial gut microbiota such as Lactobacillus, Barnesiella, Clostridium IV, Prevotella. Additionally, the puerarin propanoate ester group showed the capacity to deliver specific propionic acid to the colon. But esters with medium-long chain lengths had more opportunity to alter gut microbiota for enhancing the short chain fatty acids production. As a whole, puerarin acid esters with different chain lengths supplements shaped different gut microbial and short chain fatty acids metabolism, which could improve human health.

Evaluation of the digestion and transport profiles and potential immunocompetence of puerarin and its acylated derivatives.

Acylation has become one of the most widely used methods to improve the lipid solubility and bioavailability of flavonoids. In this study, puerarin acid esters (PAES) with different chain lengths were synthesized via biocatalytic acylation. This was the first study to evaluate the digestion and transport profiles and immunocompetence of PAES. The relationship between the digestion and transport profiles and potential immunocompetence of the acylated derivatives in Caco-2 cell monolayers was also explored. Puerarin and PAES remained stable in gastric phases, whereas different degrees of hydrolysis of PAES were found in the intestine. PAES with less than 12 carbon chains were positively correlated with the degree of hydrolysis, while those with more than 12 carbon chains showed higher resistance to hydrolysis by the artificial human digestive juice. The apparent permeability coefficients of puerarin, puerarin acetate, puerarin propanoate, puerarin butyrate, puerarin hexanoate, puerarin octanate and puerarin laurate were 1.62 ± 0.09, 1.70 ± 0.15, 1.89 ± 0.19, 1.86 ± 0.18, 2.29 ± 0.12, 4.06 ± 1.01 and 2.32 ± 0.88 × 10-6 cm s-1, respectively, in Caco-2 cell monolayers. The results of the immune factor assays indicated that puerarin propanoate, puerarin hexanoate and puerarin myristate could significantly promote the secretion of IL-6, TNF-α and IL-10. These findings suggested that a better absorption could be predicted after oral intake using PAES. Meanwhile, the concentration of esters and their metabolites (puerarin) found in the digestion and transport profiles directly affected their potential immunocompetence.

Bioavailability of quercetin in zein-based colloidal particles-stabilized Pickering emulsions investigated by the in vitro digestion coupled with Caco-2 cell monolayer model.

Protein-based Pickering emulsions have received considerable attention as nutraceutical vehicles. However, the oral bioavailability of nutraceuticals encapsulated in Pickering emulsions was not well established. In this work, a simulated gastrointestinal tract/Caco-2 cell culture model was applied to investigate the oral bioavailability of quercetin encapsulated in zein-based Pickering emulsions with quercetin in zein particles as the control. Pickering emulsions with shell (ZCP-QE) and core quercetin (ZCPE-Q) were constructed, and quercetin bioaccessibility, cell uptake and secretion, and the overall bioavailability were evaluated and compared. The overall oral bioavailability of quercetin was increased from 2.71% (bulk oil) to 38.18% (ZCPs-Q) and 18.97% (ZCPE-Q), particularly reached 41.22% for ZCP-QE. This work took new insights into the contributions of bioaccessibility and absorption (cell uptake plus secretion) to the overall oral bioavailability of quercetin. A schematic representation is proposed to relate the types of colloidal nanostructures in the digesta to the uptake, cell absorption, and overall oral bioavailability of quercetin. This study provided an attractive basis for identifying effective strategies to improve the oral bioavailability of hydrophobic nutraceuticals.

Antityrosinase Mechanism and Antimelanogenic Effect of Arbutin Esters Synthesis Catalyzed by Whole-Cell Biocatalyst.

Tyrosinase is a key enzyme responsible for enzymatic browning of fruits and vegetables and skin disorders due to overproduction of melanin. Arbutin is an inhibitor of tyrosinase; however, its high polarity and weak transdermal absorption capacity limit its applications. In this paper, a green solvent system was developed to successfully synthesize arbutin esters with improved liposolubilities (Clog P values = 0.27-5.03). Among the obtained esters, arbutin undecenoate (AU) showed the strongest tyrosinase-inhibiting activity (15.6%), which was 9.0 times higher than that of arbutin. An enzyme kinetics study indicated that AU was a competitive inhibitor with reversible inhibition. The esters inhibited tyrosinase by making the secondary structure of tyrosinase looser and less stable; moreover, the interactions between tyrosinase and AU driven by metal interactions and hydrogen bonds also offered a mechanism for inhibition of AU on tyrosinase. In addition, AU (100 µM) reduced the melanin content of B16 mouse melanoma cells to 61.3% of the control group.

The responses of Salmonella enterica serovar Typhimurium to vanillin in apple juice through global transcriptomics.

Salmonella enterica serovar Typhimurium can survive some extreme environment in food processing, and vanillin generally recognized as safe is bactericidal to pathogens. Thus, we need to explore the responses of S. Typhimurium to vanillin in order to apply this antimicrobial agent in food processing. In this study, we exposed S. Typhimurium to commercial apple juice with/without vanillin (3.2 mg/mL) at 45 °C for 75 min to determine the survival rate. Subsequently, the 10-min cultures were selected for transcriptomic analysis. Using high-throughput RNA sequencing, genes related to vanillin resistance and their expression changes of S. Typhimurium were identified. The survival curve showed that S. Typhimurium treated with vanillin were inactivated by 5.5 log after 75 min, while the control group only decreased by 2.3 log. Such a discrepancy showed the significant antibacterial effect of vanillin on S. Typhimurium. As a result, 265 differentially expressed genes (DEGs) were found when coping with vanillin, among which, 225 showed up-regulation and 40 DEGs were down-regulated. Treated with vanillin, S. Typhimurium significantly up-regulated genes involved in cell membrane, acid tolerance response (ATR) and oxidative stress response, cold shock cross-protection, DNA repair, virulence factors and some key regulators. Firstly, membrane-related genes, including outer membrane (bamE, mepS, ygdI, lolB), inner membrane (yaiY, yicS) and other proteins (yciC, yjcH), were significantly up-regulated because of the damaged cell membrane. Then, up-regulated proteins associated with arginine synthesis (ArgABCDIG) and inward transportation (ArtI, ArtJ, ArtP and HisP), participated in ATR to pump out the protons inside the cell in this scenario. Next, superoxide stress response triggered by vanillin was found to have a significant up-regulation as well, which was controlled by SoxRS regulon. Besides, NADH-associated (nuoA, nuoB, nuoK, nadE, fre and STM3021), thioredoxin (trxA, trxC, tpx and bcp) and glutaredoxin (grxC and grxD) DEGs led to the increase of the oxidative stress response. Cold shock proteins such as CspA and CspC showed an up-regulation, suggesting it might play a role in cross-protecting S. Typhimurium from vanillin stress. Furthermore, DEGs in DNA repair and virulence factors, including flagellar assembly, adhesins and type III secretion system were up-regulated. Some regulators like fur, rpoE and csrA played a pivotal role in response to the stress caused by vanillin. Therefore, this study sounds an alarm for the risks caused by stress tolerance of S. Typhimurium in food industry.

Proteomics study unveils ROS balance in acid-adapted Salmonella Enteritidis.

Salmonella Enteritidis is a major cause of foodborne gastroenteritis and is thus a persistent threat to global public health. The acid adaptation response helps Salmonella survive exposure to gastric environment during ingestion. In a previous study we highlighted the damage caused to cell membrane and the regulation of intracellular reactive oxygen species (ROS) in S. Enteritidis. In this study, we applied both physiologic and iTRAQ analyses to explore the regulatory mechanism of acid resistance in Salmonella. It was found that after S. Enteritidis was subject to a 1 h period of acid adaptation at pH 5.5, an additional 1 h period of acid shock stress at pH 3.0 caused less Salmonella cell death than in non-acid adapted Salmonella cells. Although there were no significant differences between adapted and non-adapted cells in terms of cell membrane damage (e.g., membrane permeability or lipid peroxidation) after 30 min, intracellular ROS level in acid adapted cells was dramatically reduced compared to that in non-acid adapted cells, indicating that acid adaption promoted less ROS generation or increased the ability of ROS scavenging with little reduction in the integrity of the cell membrane. These findings were confirmed via an iTRAQ analysis. The adapted cells were shown to trigger incorporation of exogenous long-chain fatty acids into the cellular membrane, resulting in a different membrane lipid profile and promoting survival rate under acid stress. S. Enteritidis experiences oxidative damage and iron deficiency under acid stress, but after acid adaption S. Enteritidis cells were able to balance their concentrations of intracellular ROS. Specifically, SodAB consumed the free protons responsible for forming reactive oxygen intermediates (ROIs) and KatE protected cells from the toxic effects of ROIs. Additionally, acid-labile proteins released free unbound iron promoting ferroptotic metabolism, and NADH reduced GSSH to G-SH, protecting cells from acid/oxidative stress.

Hofmeister Effect-Assistant Fabrication of All-Natural Protein-based Porous Materials Templated from Pickering Emulsions.

Porous materials derived from natural and biodegradable polymers have received growing interest. We demonstrate here an attractive method for the preparation of protein-based porous materials using emulsions stabilized by gliadin-chitosan hybrid particles (GCHPs) as the template, with the addition of gelatin and kosmotropic ions to improve the mechanical strength. The microstructure, mechanical properties, cytotoxicity, and fluid absorption behavior of porous materials were systematically investigated. This strategy facilitated the formation of porous materials with highly open and interconnected pore structure, which can be manipulated by altering the mass ratio of hexane or gelatin in the matrix. The Hofmeister effect resulted from kosmotropic ions greatly enhanced the Young's modulus and the compressive stress at 40% strain of porous materials from 0.56 to 6.84 MPa and 0.26 to 1.11 MPa, respectively. The developed all-natural porous materials were nontoxic to HaCaT cells; they also had excellent liquid (i.e., simulated body fluid and rabbit blood) absorption performance and advantages in resisting stress and maintaining geometry shape. The effects of different concentration amounts and type of salts in the Hofmeister series on the formation and performance of porous materials were also explored. Mechanical strength of porous materials was gradually enhanced when the (NH4)2SO4 concentration increased from 0 to 35 wt %, and the other four kosmotropic salts, including Na2S2O3, Na2CO3, NaH2PO4, and Na2SO4, also showed positive effects. This work opens a simple and feasible way to produce nontoxic and biodegradable porous materials with favorable mechanical strength and controllable pore structure. These materials have broad potential application in many fields involving biomedical and material science, such as cell culture, (bio)catalysis, and wound or bone defect healing.