Cronobacter sakazakii is a major pathogen that contaminates infant milk powder, which usually attaches to abiotic surfaces in the form of biofilms and causes diseases. However, few in-depth studies have been conducted on the properties of biofilms formed on different abiotic surfaces. Therefore, this study explored the characteristics of biofilm formation by C. sakazakii on commonly used food contact surfaces, including stainless steel (SS), glass (GS), poly(phenylene sulfone) resin (PPSU), and soft silicone (SSI), with a focus on the biofilm formation ability and the biofilms' functional, structural, and compositional properties. Correlation analysis was used to confirm the leading factors affecting C. sakazakii biofilm formation, including surface hardness and stiffness, cell length, and extracellular nucleic acid content. Finally, proteomic analysis revealed menaquinone biosynthesis, amino acid metabolism, SOS response, and transport systems as being crucial for C. sakazakii biofilm formation. This study provides valuable insights for a deeper understanding of the bacterium's biofilm formation.

Blackcurrant is rich in anthocyanins, which are key functional components contributing to its nutritional value and antioxidant capacity. However, anthocyanins are highly susceptible to oxidative degradation during postharvest storage. This study investigated the effects of different concentrations (0, 0.2, 0.4, 0.6, and 0.8 mmol·L− 1) of exogenous nitric oxide (NO), applied via its donor sodium nitroprusside (SNP), which was freshly dissolved in distilled water immediately before use, on the postharvest quality of blackcurrant fruit during cold storage (0 °C, 42 days). Results demonstrated that 0.6 mmol·L− 1 NO treatment exhibited the most pronounced effects in maintaining postharvest quality. NO treatment significantly inhibited respiration intensity and weight loss, while preserving higher levels of nutrient substances. Importantly, NO treatment significantly slowed the degradation of total anthocyanin content, especially at 0.6 mmol·L− 1. NO treatment also enhanced antioxidant enzyme activities, facilitating the scavenging of superoxide anion and hydrogen peroxide, thereby reducing reactive oxygen species (ROS) accumulation and oxidative stress. Moreover, NO treatment strongly suppressed polyphenol oxidase (PPO) activity, delayed the degradation of antioxidant compounds, and inhibited cell wall-degrading enzyme activities, thus preserving high antioxidant capacity and fruit firmness. In conclusion, NO treatment effectively alleviated oxidative damage in blackcurrant fruit during cold storage by activating the endogenous antioxidant enzyme system, maintaining ROS metabolic homeostasis, inhibiting PPO-mediated anthocyanin degradation, and slowing cell wall degradation. This study demonstrates that NO treatment effectively alleviates oxidative damage and preserves anthocyanin content through a dual mechanism involving enhanced antioxidant defense and direct protection of anthocyanin stability. These findings offer a new and practical strategy for extending the shelf life of non-climacteric berries.

The intestinal microbiome is fundamental to host physiological homeostasis, while deviations from its balanced state have been linked to inflammatory bowel diseases (IBD). To address the limitations of conventional antibiotic therapies, this study explored snake gut microbiota as a novel source of anti-inflammatory probiotics. We explored the gut microbiota of five snake species (Deinagkistrodon acutus, Trimerodytes annularis, Trimerodytes percarinatus, Lycodon rufozonatus, and Trimeresurus stejnegeri) through metagenomic sequencing. Community composition analysis revealed that the phylum-level composition was mainly Proteobacteria, Bacteroidetes, Actinomycetota, and Firmicutes. We further detected some potential probiotic species, such as Enterococcus, Lactobacillus, and Limosilactobacillus. From 196 isolated strains, Lactobacillus johnsonii DA0116 and Limosilactobacillus reuteri DA0218 were selected through rigorous safety and functional assessments, including acid/bile tolerance, pathogen inhibition, and adhesion capacity. In a DSS-induced murine colitis model, both strains significantly reduced disease activity index (DAI), pro-inflammatory cytokines (TNF-α, IL-6, and IL-8), and restored gut microbiota diversity. Additionally, whole-genome analysis identified bacteriocin synthesis clusters (gassericin-S/T) and carbohydrate metabolism genes, explaining their antimicrobial and immunomodulatory properties. This study not only emphasizes the untapped latent value of reptilian gut microbiota for probiotic discovery but also provides two candidate strains with therapeutic promise for IBD and functional food applications.

Cronobacter sakazakii is a foodborne pathogen that mainly infects infants and the elderly, which can form biofilms that are difficult to remove. D-carbohydrates are common quorum sensing inhibitors during bacterial biofilm formation, while the roles of L-carbohydrates and their corresponding efflux pumps in biofilm formation are less studied in C. sakazakii. In this study, the role and mechanism of multidrug resistance efflux pump MdtG in biofilm formation of C. sakazakii was investigated with gene knockout method. The results showed that MdtG, which was validated as a novel L-arabinose efflux pump, could efflux L-arabinose as an active inhibitor of the quorum-sensing signaling molecule AI-2 and inhibited the level of biofilm formation by downregulating the peptidoglycan content, exopolysaccharide secretion, membrane permeability, motility, self-aggregation capacity, and cell length of C. sakazakii. This study revealed the effect and molecular mechanism of the multidrug resistance efflux pump MdtG on biofilm formation in C. sakazakii and identified a potential novel quorum sensing inhibitor, L-arabinose, for the control of its biofilm formation.

The outstanding desiccation tolerance of Cronobacter sakazakii (C. sakazakii) enables long-term persistence in food products with low-water activity to increase the infection risk, especially in low-birth-weight, immuno-compromised neonates, and infants less than 4 weeks of age. In our previous study, the disruption of glutathione transport-related gene gsiD by transposon was found to significantly increase its inactivation rate under drying stress challenges. However, the mechanism underlying the association between glutathione transport and desiccation tolerance of C. sakazakii remains to be clarified. In this study, the mechanism underlying their association was investigated in detail by constructing the gsiD gene deletion mutant. gsiD gene deletion was found to cause the dysfunction of the glutathione transport system GsiABCD and the limitation of glutathione import. The resulting decrease in intracellular glutathione caused the decreased potassium ions uptake and increased potassium ions efflux, inhibited the proline synthesis process, limited extracellular glutathione utilization, increased oxidant stress, reduced biofilm formation, and increased outer membrane permeability, which may be the main reasons for the significant reduction of the desiccation tolerance of C. sakazakii.IMPORTANCEContributing to its superior environmental adaptability, Cronobacter sakazakii can survive under many abiotic stress conditions. The outstanding desiccation tolerance makes this species persist in low-water activity foods, which increases harm to humans. For decades, many studies have focused on the desiccation tolerance of C. sakazakii, but the existing research is still insufficient. Our study found that gsiD gene deletion inhibited glutathione uptake and further decreased intracellular glutathione content, causing a decrease in desiccation tolerance and biofilm formation and an increase in outer membrane permeability. Moreover, the expression level of relative genes verified that gsiD gene deletion made the mutant not conducive to surviving in dry conditions due to restricting potassium ions uptake and efflux, inhibiting the conversion of glutamate to compatible solute proline, and increasing the oxidative stress of C. sakazakii. The above results enrich our knowledge of the desiccation tolerance mechanism of C. sakazakii.

Cronobacter sakazakii (C. sakazakii), a food-borne pathogen, can cause severe intestinal diseases, such as intestinal damage and necrotizing enterocolitis, and even death in neonates and infants through its adhesion to intestinal epithelium cells by protein-protein interaction and translocation across the intestinal barrier. Nevertheless, the mechanisms of adhesion, translocation, and pathogenesis remain unclear. This study aimed to screen and validate the outer membrane proteins of C. sakazakii capable of adhering to the surface of intestinal cells through biotinylated protein adhesion and LC-MS/MS identification. A total of 319 proteins were identified. Gene Ontology and KEGG analysis revealed significant enrichment of ligand proteins in metabolism-related processes and catalytic activity. The identified 37 outer membrane proteins and 41 proteins of unknown subcellular localization or function mainly enriched in five protein-protein interaction clusters. Ten of these outer membrane/unknown proteins were selected to test their roles on C. sakazakii adhesion and invasion of intestinal cells by silencing the encoding gene expression and nine of the ten proteins, Protein_26/30/34/38/42/98/104/194/282, were shown to be critical in this process. This study contributes to the understanding of proteins involved in the host adhesion process by C. sakazakii as well as the mechanisms for its translocation across the intestinal barrier.

Cronobacter sakazakii (C. sakazakii) is a foodborne pathogen whose initial process involves intestinal cell adhesion mediated by numerous virulence factors encoded in various genes. The key metabolic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH, also known as GapA), is encoded by gapA in the glycolysis pathway and acts as an adhesin in some bacteria. In C. sakazakii, there is also a key enzyme in the gluconeogenesis pathway, Gap, with type I GAPDH and erythrose-4-phosphate dehydrogenase activities and encoded by gap. This study aimed to investigate the virulence properties of GapA and Gap of C. sakazakii in adhesion to and invasion of HCT-8 and HIEC-6 cells and damage to the colon and brain of neonatal rats, by gene silencing. In addition, the role of both recombinant proteins in intestinal cell adhesion and invasion was investigated, and their role in inducting inflammatory cytokine expression was assessed by ELISA and Western blot. Silencing gapA or gap decreased the viability and swimming motility of bacterial cells and reduced bacterial adhesion to and invasion of both types of intestinal cells. Both recombinant proteins contributed to C. sakazakii adhesion in both cells, enhanced protein phosphorylation of NF-κB, and induced inflammatory cytokine expression. Finally, silenced expression of GapA and Gap also weakened bacterial damage to the brain and colon of neonatal rats. In conclusion, we demonstrated for the first time the virulence properties of GapA and Gap in C. sakazakii adhesion to and invasion of intestinal cells and neonatal rats and induction of inflammatory responses.

Lycium ruthenicum Murray (LR) is a medicine and edible plant in Northwest China, and L. ruthenicum Murray anthocyanins (LRA) are green antioxidants with various pharmacological activities, such as antioxidant and anti-inflammatory activities. However, the protective effect and mechanism of LRA against retinal damage induced by blue light exposure are poorly understood. This study explored the protective effects and potential mechanisms of LRA on retinal damage induced by blue light exposure in vitro and in vivo. The results showed that LRA could ameliorate oxidative stress injury by activating the antioxidant stress nuclear factor-related factor 2 pathway, promoting the expression of phase II detoxification enzymes (HO-1, NQO1) and endogenous antioxidant enzymes (catalase, superoxide dismutase, glutathione peroxidase), and reducing reactive oxygen species and malondialdehyde levels. Additionally, LRA could inhibit inflammatory response by decreasing the expression of blue light exposure-induced nuclear factor-κB (NF-κB) pathway-related proteins (NF-κB and p-IκBα), as well as interleukin (IL)-6, tumor necrosis factor-α, IL-1β pro-inflammatory factors and pro-inflammatory chemokine VEGF, and increasing the expression of anti-inflammatory factor IL-10. Furthermore, LRA could ameliorate oxidative stress-induced apoptosis by upregulating Bcl-2 and downregulating Bax and Caspase-3 protein expression. All these results indicate that LRA can be used as an antioxidant dietary supplement for the treatment or prevention of retinal diseases.

Cronobacter sakazakii, an opportunistic foodborne pathogen, has a strong resistance to osmotic stress and desiccation stress, but the current studies cannot elucidate this resistance mechanism absolutely. A mechanosensitive channel MscM was suspected of involving to desiccation resistance mechanism of C. sakazakii. To investigate the specific molecular mechanism, the mscM mutant strain (ΔmscM) was constructed using the homologous recombination method, and the cpmscM complementary strain was obtained by gene complementation, followed by the analysis of the difference between the wild-type (WT), mutant, and complementary strains. Compared to the wild-type bacteria (WT), the inactivation rate of the ΔmscM strain decreased by 15.83% (p < 0.01) after desiccation stress. The absence of the mscM gene led to an increase in the membrane permeability of mutant strains. Through turbidity assay, it was found that the intracellular content of potassium ion (K+) of the ΔmscM strain increased by 2.2-fold (p < 0.05) compared to the WT strain, while other metal ion contents, including sodium ion (Na+), calcium ion (Ca2+), and magnesium ion (Mg2+), decreased by 48.45% (p < 0.001), 24.29% (p < 0.001), and 26.11% (p < 0.0001), respectively. These findings indicate that the MscM channel primarily regulates cell membrane permeability by controlling K+ efflux to maintain the homeostasis of intracellular osmotic pressure and affect the desiccation tolerance of bacteria. Additionally, the deletion of the mscM gene did not affect bacterial growth and motility but impaired surface hydrophobicity (reduced 20.52% compared to the WT strain, p < 0.001), adhesion/invasion capability (reduced 26.03% compared to the WT strain, p < 0.001), and biofilm formation ability (reduced 30.19% compared to the WT strain, p < 0.05) of the bacteria. This study provides a reference for the role of the mscM gene in the desiccation resistance and biofilm formation of C. sakazakii.

The molecular chaperone GroEL of C. sakazakii, a highly conserved protein encoded by the gene grol, has the basic function of responding to heat shock, thus enhancing the bacterium's adaptation to dry and high-temperature environments, which poses a threat to food safety and human health. Our previous study demonstrated that GroEL was found in the bacterial membrane fraction and caused a strong immune response in C. sakazakii. In this study, we tried to elucidate the subcellular location and virulent effects of GroEL. In live C. sakazakii cells, GroEL existed in both the soluble and insoluble fractions. To study the secretory mechanism of GroEL protein, a non-reduced Western immunoblot was used to analyze the form of the protein, and the result showed that the exported GroEL protein was mainly in monomeric form. The exported GroEL could also be located on bacterial surface. To further research the virulent effect of C. sakazakii GroEL, an indirect immunofluorescence assay was used to detect the adhesion of recombinant GroEL protein to HCT-8 cells. The results indicated that the recombinant GroEL protein could adhere to HCT-8 cells in a short period of time. The recombinant GroEL protein could activate the NF-κB signaling pathway to release more pro-inflammatory cytokines (TNF-α, IL-6 and IL-8), downregulating the expression of tight-junction proteins (claudin-1, occluding, ZO-1 and ZO-2), which collectively resulted in dose-dependent virulent effects on host cells. Inhibition of the grol gene expression resulted in a significant decrease in bacterial adhesion to and invasion of HCT-8 cells. Moreover, the deficient GroEL also caused slow growth, decreased biofilm formation, defective motility and abnormal filamentation of the bacteria. In brief, C. sakazakii GroEL was an important virulence factor. This protein was not only crucial for the physiological activity of C. sakazakii but could also be secreted to enhance the bacterium's adhesion and invasion capabilities.

Cronobacter sakazakii is an opportunistic Gram-negative pathogen that has been identified as a causative agent of severe foodborne infections with a higher risk of mortality in neonates, premature infants, the elderly, and immunocompromised populations. The specific pathogenesis mechanisms of C. sakazakii, such as adhesion and colonization, remain unclear. Previously, we conducted comparative proteomic studies on the two strains with the stronger and weaker infection ability, respectively, and found an interesting protein, ESA_00986, which was more highly expressed in the strain with the stronger ability. This unknown protein, predicted to be a type of invasitin related to invasion, may be a critical factor contributing to its virulence. This study aimed to elucidate the precise roles of the ESA_00986 gene in C. sakazakii by generating gene knockout mutants and complementary strains. The mutant and complementary strains were assessed for their biofilm formation, mobility, cell adhesion and invasion, and virulence in a rat model. Compared with the wild-type strain, the mutant strain exhibited a decrease in motility, whereas the complementary strain showed comparable motility to the wild-type. The biofilm-forming ability of the mutant was weakened, and the mutant also exhibited attenuated adhesion to/invasion of intestinal epithelial cells (HCT-8, HICE-6) and virulence in a rat model. This indicated that ESA_00986 plays a positive role in adhesion/invasion and virulence. This study proves that the ESA_00986 gene encodes a novel virulence factor and advances our understanding of the pathogenic mechanism of C. sakazakii.

Cronobacter sakazakii, an important Gram-negative foodborne pathogen, can cause neonatal meningitis and sepsis with high rates of infection and death. Gene ESA_RS15745 encodes a sugar transporter protein, which is not only essential for osmotic pressure maintenance during bacterial growth and reproduction but also associated with their desiccation tolerance, motility, and biofilm formation. Here, a mutant strain of ESA_RS15745 (ΔESA_RS15745) and the complementation strain (cpESA_RS15745) were constructed using a suicide vector knockout and gene complementation. ΔESA_RS15745 was found to have a decrease in its ability to transport maltose and trehalose and resist desiccation, whereas an increase in the ability of motility and biofilm formation, implying that ESA_RS15745 may positively regulate sugar transport and desiccation tolerance and negatively regulate motility and biofilm formation. To further investigate the molecular mechanisms underlying the function of related genes, RNA-seq was performed to explore the differentially expressed genes in the mutants. RNA-seq results showed the upregulation of 114 genes (mainly including those regulating chemotaxis and flagellar motility) and the downregulation of 22 genes (mainly including those regulating sugar transport). qRT-PCR analysis supported the RNA-seq results and showed that ESA_RS15745 may influence the dehydration tolerance though decreasing the intracellular trehalose content and negatively regulate the motility though the chemotactic signaling pathway. In addition, the biofilm formation of C. sakazakii should also be speculated to negatively regulate by ESA_RS15745 by consuming the extracellular carbohydrates concentration and then downregulating the intracellular cyclic diguanosine monophosphate. This study offers a reference for comprehending the molecular mechanism of gene ESA_RS15745 in C. sakazakii.

Cronobacter sakazakii (C. sakazakii), a food-borne pathogen, can infect neonates, elderly and immunocompromised populations with a high infection and mortality rate. However, the specific molecular mechanism of its motility, biofilm formation, cell adhesion, and desiccation resistance remains unclear, and flagellum hook associated protein (FlgK), a main component of the flagellar complex, may be an important determinant of its virulence and desiccation resistance. In this study, the flgK mutant strain (ΔflgK) was constructed using the homologous recombination method, and the cpflgK complementary strain was obtained by gene complementation, followed by analysis of the difference between the wild type (WT), mutant, and complementary strains in mobility, biofilm formation, cell adhesion, and desiccation resistance. Results indicated that flgK gene played a positive role in motility and invasion, with no significant effect on biofilm formation. Interestingly, flagellar assembly gene deletion showed increased resistance of C. sakazakii to dehydration. The mechanism underlying the negative correlation of flgK gene with dehydration resistance was further investigated by using the high-throughput sequencing technology to compare the gene expression between WT and ΔflgK strains after drying. The results revealed up-regulation in the expression of 54 genes, including genes involved in osmosis and formate dehydrogenase, while down-regulation in the expression of 50 genes, including genes involved in flagellum hook and nitrate reductase. qRT-PCR analysis of the RNA-seq data further indicated that the flgK gene played an important role in the environmental stress resistance of C. sakazakii by up-regulating the formate dehydrogenase, betaine synthesis, and arginine deiminase pathways, due to dynamic proton imbalance caused by lack of flagella. This study facilitates our understanding of the roles of flgK in motion-related functions and the molecular mechanism of desiccation resistance in C. sakazakii.

Inflammation and oxidative stress play key roles in the aging process, while red yeast rice (RYR), a traditional Chinese fermented food, has anti-oxidant and anti-inflammatory effects. To understand the anti-aging function of RYR in vivo, this study established a D-galactose-induced aging mouse model to verify the positive effects of RYR dietary intervention on aging and explore the related underlying mechanism. Eight weeks of RYR dietary intervention was shown to have a significant inhibitory effect on cognitive decline and hippocampal damage. The molecular mechanistic studies showed that the anti-aging effects of RYR were achieved by (i) improving the oxidative stress-related damage (increasing SOD, CAT, and GSH, and reducing MDA), (ii) regulating the NF-κB inflammation pathway induced by oxidative stress (decreasing the pro-inflammatory cytokines IL-6, TNF-α, IFN-γ, iNOs, and IL-1β, increasing the anti-inflammatory cytokine IL-10, and decreasing the expression of the NF-κB protein), (iii) slowing down apoptosis caused by oxidative stress (reducing the expression of P21 and P53), (iv) restoring the abundance of Lactobacillus, Lachnospiraceae and Rikenellaceae downregulated by D-galactose, and (v) reducing the abundance of Akkermansia and Helicobacter enriched by D-galactose. Mass spectrometry revealed orange pigments (rubropunctatin and monascorubrin) as the main antioxidant components in RYR, which might play key roles in aging inhibition. This study provides theoretical support for the wide application of orange pigments as an antioxidant dietary supplement.

为更好地阐释阪崎克罗诺杆菌的耐干燥机制，该研究通过同源重组的方法敲除阪崎克罗诺杆菌ATCC BAA-894的ESA-00281基因，来探究该基因的功能及其在耐干燥中的作用。结果表明，该基因的缺失降低了耐干燥能力，与野生株相比，突变株干燥死亡率提高8.76%，表明此基因在阪崎克罗诺杆菌的耐干燥中发挥重要的正向调节作用。此外，该基因对阪崎克罗诺杆菌的生物膜形成及表面疏水性具有正向调节作用，但对膜透过性和运动性没有影响，表明该基因可能通过改变表面疏水性来调节生物膜的形成和对介质的黏附，从而正向调节菌株的耐干燥能力。

The development of natural antimicrobial agents has attracted long-term attention due to the increasing demand for food preservation. Papaya, a widely cultivated nutritious tropical fruit, has benzyl isothiocyanate (BITC) as one of the most important secondary metabolites in its seeds. And the antibacterial activity of BITC toward different strains and the main antibacterial pathway remain unclear. The current study focused on characterizing the antibacterial effect and exploring the major bacteriostatic pathway of BITC. BITC was shown to have a broad-spectrum antibacterial effect, with a minimum inhibitory concentration of 1 µL/mL for Escherichia coli, Bacillus subtilis, and Aspergillus niger, and 0.5 µL/mL for Salmonella enterica, Staphylococcus aureus, and Penicillium citrinum. Additionally, BITC was identified to affect the integrity of the biological oxidation system rather than the permeability or morphology of cell membranes. Furthermore, BITC was found not only to affect ATP production but also to hinder a series of important chemical reactions of the coenzymes involved in the transfer of hydrogen ions in the respiratory chain. The bacteriostatic pathway of BITC was shown to be implicated in an incomplete respiratory chain and the deregulation of the metabolism system. These results indicate the potential of BITC as a natural preservative in the food industry. PRACTICAL APPLICATION: BITC is present in papaya seeds and can be extracted and purified. Exploring its antibacterial activity and main action pathway may facilitate its application as a new bacteriostatic agent in food industry.

Cronobacter sakazakii, an emerging opportunistic pathogen, is implicated in severe foodborne outbreak infections in premature and full-term infants. Generally, acid tolerance is vital for the pathogenesis of foodborne pathogens; however, its role in C. sakazakii virulence remains largely unknown. To screen out acid-tolerance determinants from transposon mutants, anovel counterselection method using gentamicin and acid was developed. Using the counterselection method and growth assay, we screened several acid-sensitive mutants and found that nlpD encodes an acid-resistance factor in C. sakazakii. Compared to the wild-type strain, the nlpD mutant exhibited attenuated virulence in a rat model. Using macrophage THP-1 cells and a pH probe, we verified that nlpD enables bacteria to resist macrophages by resisting acidification. Finally, we confirmed that nlpD maintains C. sakazakii membrane integrity in acid using propidium iodide permeabilization assays via flow cytometry. Our results confirm that nlpD is a novel virulence factor that permits C. sakazakii to survive under acid stress conditions. Considering that NlpD is a conserved lipoprotein located in the bacterial outer membrane, NlpD could be used as a target for drug development.

In some Gram-negative bacteria, ompF encodes outer membrane protein F (OmpF), which is a cation-selective porin and is responsible for the passive transport of small molecules across the outer membrane. However, there are few reports about the functions of this gene in Cronobacter sakazakii. To investigate the role of ompF in detail, an ompF disruption strain (ΔompF) and a complementation strain (cpompF) were successfully obtained. We find that OmpF can affect the ability of biofilm formation in C. sakazakii. In addition, the variations in biofilm composition of C. sakazakii were examined using Raman spectroscopy analyses caused by knocking out ompF, and the result indicated that the levels of certain biofilm components, including lipopolysaccharide (LPS), were significantly decreased in the mutant (ΔompF). Then, SDS-PAGE was used to further analyze the LPS content, and the result showed that the LPS levels were significantly reduced in the absence of ompF. Therefore, we conclude that OmpF affects biofilm formation in C. sakazakii by reducing the amount of LPS. Furthermore, the ΔompF mutant showed decreased (2.7-fold) adhesion to and invasion of HCT-8 cells. In an antibiotic susceptibility analysis, the ΔompF mutant showed significantly smaller inhibition zones than the WT, indicating that OmpF had a positive effect on the influx of antibiotics into the cells. In summary, ompF plays a positive regulatory role in the biofilm formation and adhesion/invasion, which is achieved by regulating the amount of LPS, but is a negative regulator of antibiotic resistance in C. sakazakii.

Red jujube fruits and bamboo shoots are rich in many nutrients and have the advantage of high yield in China. However, the storage of fresh fruits is difficult, and there are no fermented products using both as raw materials. In order to develop the two raw materials into novel products and improve their nutritional value, this study reports the production and characterization of a beverage via fermentation of red jujube fruits and bamboo shoots with Lactiplantibacillus plantarum. L. plantarum TUST-232 was selected as the starter from several different strains by comparing pH value and the number of viable cells, which reached 8.91 log CFU/mL in the beverage fermented for 14 h at 37 °C with 0.3% inoculation. After fermentation, the beverage showed improvement in the contents of several nutrients and antioxidant indices, with a decrease of 44.10% in sucrose content, along with increases of 11.09%, 12.30%, and 59.80% in total phenolic content, total antioxidant capacity, and superoxide anion scavenging ability, respectively. These results indicate that L. plantarum fermentation of red jujube fruits and bamboo shoots could be an effective way to develop a new beverage with high nutritional value, high antioxidant capacity, and high dietary fiber content. This research provided experimental support for the development of new fermentation products with the functions of improving health and body functions.

d- Limonene, a cyclic monoterpene, possesses citrus-like olfactory property and multi-physiological functions. In this study, the d- limonene synthase (t LS ) from Citrus limon was codon-optimized and heterologously expressed in Saccharomyces cerevisiae . The metabolic flux of canonical pathway based on overexpressing endogenous geranyl diphosphate synthase gene ( ERG20 ) and its variant ERG20 F96W−N127W was strengthened for improvement d- limonene production in Chinese Baijiu. To further elevate production, we established an orthogonal pathway by introducing neryl diphosphate synthase 1 (t NDPS1 ) from Solanum lycopersicum . The results showed that expressing ERG20 and ERG20 F96W−N127W could enhance d- limonene synthesis, while expressing heterologous NPP synthase gene significantly increase d- limonene formation. Furthermore, we constructed a t LS –t NDPS1 fusion protein, and the best strain yielded 9.8 mg/L d- limonene after optimizing the amino acid linker and fusion order, a 40% improvement over the free enzymes during Chinese Baijiu fermentation. Finally, under the optimized fermentation conditions, a maximum d- limonene content of 23.7 mg/L in strain AY12α-L9 was achieved, which was the highest reported production in Chinese Baijiu. In addition, we also investigated that the effect of d- limonene concentration on yeast growth and fermentation. This study provided a meaningful insight into the platform for other valuable monoterpenes biosynthesis in Chinese Baijiu fermentation.