The Inhibitory Effect of Resveratrol from Reynoutria japonica on MNV-1, a Human Norovirus Surrogate.

As a natural nonflavonoid polyphenol compound, resveratrol is the main functional component of Reynoutria japonica and has anti-inflammatory, antioxidant, antiviral, and other physiological activities. In this study, the effect of resveratrol on the viability of RAW264.7 cells was examined, and murine norovirus (MNV-1) was used as a surrogate for human norovirus to evaluate the inhibitory effect of resveratrol. The concentrations of resveratrol resulting in 50% cytotoxicity (CC50) for RAW264.7 cells were 21.32 and 24.97 µg/mL after 24 and 48 h of incubation, respectively, and resveratrol at a concentration lower than the half-effective inhibitory concentration (EC50) could not damage cell DNA. The EC50 of resveratrol on MNV-1 in infected RAW264.7 cells was determined to equal 5.496 µg/mL. After RAW264.7 cells, virus, and a fresh mixture of virus and RAW264.7 cells were treated with resveratrol solution for 1 h (denoted cell pre-treatment, virus pre-treatment, and mixture coprocessing), the RAW264.7 cells obtained after cell pre-treatment exhibited lower virus infection, and MNV-1 obtained after virus pre-treatment and mixture coprocessing showed a decreased infectious capacity. The inhibition ratio of resveratrol on MNV-1 did not significantly differ between the treatments at 4 and 25 °C or among the various pH values except for the lower acidic condition (pH 2). TEM revealed significant changes in the morphology of MNV-1 after treatment with resveratrol, and molecular docking indicated that resveratrol strongly binds to the viral capsid protein of MNV-1. In addition, resveratrol regulated the expression of cytokine that protects against MNV-1 infection. Therefore, at a lower concentration, resveratrol, a natural component from Reynoutria japonica, exerts an inhibitory effect on MNV-1 growth and could be used as a safe additive in food products to improve the nutritional status and control norovirus.

Carrageenan oligosaccharide alleviates intestinal damage via gut microflora through activating IMD/relish pathway in female Drosophila melanogaster.

Recent evidence suggests the anti-inflammatory effect of carrageenan oligosaccharides (COS). The effects of COS on intestinal injury induced by 0.6% sodium dodecyl sulfate (SDS) and the molecular mechanisms involved were investigated in this study. 0.625, 1.25, and 2.5 mg/mL COS in diet had no toxic effect in flies, and they could all prolong SDS-treated female flies' survival rate. 1.25 mg/mL COS prevented the development of inflammation by improving the intestinal barrier integrity and maintaining the intestinal morphology stability, inhibited the proliferation of intestine stem cells (ISCs), and the production of lysosomes induced by SDS, accompanied by a decrease in the expression of autophagy-related genes. Moreover, COS decreased the active oxygen species (ROS) content in gut and increased the antioxidant activity in SDS-induced female flies, while COS still played a role in increasing survival rate and decreasing intestinal leakage in CncC-RNAi flies. The improvement of anti-inflammation capacity may be associated with the regulation of intestinal microflora with COS supplementation for Drosophila melanogaster. COS changed the gut microbiota composition, and COS had no effect on germ-free (GF) flies. It is highlighted that COS could not work in Relish-RNAi flies, indicating relish is required for COS to perform beneficial effects. These results provide insights into the study of gut microbiota interacting with COS to modulate intestinal inflammation in specific hosts.

Ginseng Extract Attenuates the Injury from Ultraviolet Irradiation for Female Drosophila melanogaster through the Autophagy Signaling Pathway.

Ginseng is an ancient medicinal and edible plant with many health benefits, and can serve as a drug and dietary supplement, but there are few relevant studies on its use to ease ultraviolet (UV) irradiation damage. After 0.8 mg/mL ginseng extract (GE) was added to the medium of female Drosophila melanogaster subjected to UV irradiation, the lifespan, climbing ability, sex ratio, developmental cycle, and antioxidant capacity of flies were examined to evaluate the GE function. In addition, the underlying mechanism by which GE enhances the irradiation tolerance of D. melanogaster was explored. With GE supplementation, female flies subjected to UV irradiation exhibited an extension in their lifespan, enhancement in their climbing ability, improvement in their offspring sex ratio, and restoration of the normal development cycle by increasing their antioxidant activity. Finally, further experiments indicated that GE could enhance the irradiation tolerance of female D. melanogaster by upregulating the gene expressions of SOD, GCL, and components of the autophagy signaling pathway. Finally, the performance of r4-Gal4;UAS-AMPKRNAi flies confirmed the regulatory role of the autophagy signaling pathway in mitigating UV irradiation injury.

Chitosan oligosaccharide improves intestinal homeostasis to achieve the protection for the epithelial barrier of female Drosophila melanogaster via regulating intestinal microflora.

Chitosan oligosaccharide (COS) is a new type of marine functional oligosaccharide with biological activities such as regulating intestinal microflora and improving intestinal immunity. In this study, female Drosophila melanogaster was used as a model organism to evaluate the effect of COS on intestinal injury by H2O2 induction, and its mechanism was explored through the analysis of intestinal homeostasis. The results showed that 0.25% of COS could effectively prolong the lifespan of stressed female D. melanogaster by increasing its antioxidant capacity and maintaining intestinal homeostasis, which included protecting the mechanical barrier, promoting the chemical barrier, and regulating the biological barrier by affecting its autophagy and the antioxidant signaling pathway. Additionally, the protective effect of COS on the intestinal barrier and homeostasis of D. melanogaster under oxidative stress status is directly related to its regulation of the intestinal microflora, which could decrease excessive autophagy and activate the antioxidant system to promote health. IMPORTANCE: The epithelial barrier plays an important role in the organism's health. Chitosan oligosaccharide (COS), a new potential prebiotic, exhibits excellent antioxidant capacity and anti-inflammatory effects. Our study elucidated the protective mechanisms of COS on the intestinal barrier of Drosophila melanogaster under oxidative stress, which could provide new insights into COS application in various industries, such as food, agriculture, and medicine.

Protective effect of agar oligosaccharide on male Drosophila melanogaster suffering from oxidative stress via intestinal microflora activating the Keap1-Nrf2 signaling pathway.

Agar oligosaccharide (AOS) is a new kind of marine functional oligosaccharide with generous biological activities. To investigate the antioxidative effects of AOS in vivo, 3 % aqueous hydrogen peroxide (H2O2) was used to induce oxidative stress in male Drosophila melanogaster (D. melanogaster) fed 5 % sucrose (SUC). AOS (0.125 %) in the medium extended the lifespan of D. melanogaster suffering from oxidative stress by improving antioxidant capacity and intestinal function. Electron microscopic observation of epithelial cells showed that AOS alleviated the damage caused by H2O2 challenge in the intestine of D. melanogaster, including a reduction of gut leakage and maintenance of intestinal length and cell ultrastructure. The Keap1-Nrf2 (analogues of CncC gene in D. melanogaster) signaling pathway was significantly activated based on gene expression levels and a reduction in ROS content in the intestine of D. melanogaster suffering from oxidative stress. The improvement of antioxidant capacity may be related to the regulation of intestinal microflora with AOS supplementation for D. melanogaster. Nrf2-RNAi, sterile and gnotobiotic D. melanogaster were used to validate the hypothesis that AOS activated the Keap1-Nrf2 signaling pathway to achieve antioxidant effects by regulating intestinal microflora. The above results contribute to our understanding of the antioxidative mechanism of AOS and promote its application in the food industry.

Structure characteristics of low molecular weight pectic polysaccharide and its anti-aging capability by modulating the intestinal homeostasis.

Pectic polysaccharide has attracted increasing attention for their potential biological properties and applications in health industries. In this study, a low-molecular-weight pectic polysaccharide, POS4, was obtained from citrus peel. The structure of POS4 was preliminarily analyzed by gel-permeation chromatography, monosaccharide analysis, infrared spectroscopy (IR) and nuclear magnetic resonance spectroscopy (NMR). Results showed that the molecular weight of POS4 was 4.76 kDa and it was a galacturonic acid enriched pectic polysaccharide. The anti-aging activity in vivo showed that POS4 could notably prolong the average lifespan of fruit flies by suppressing the generation of reactive oxygen species (ROS). Further studies demonstrated that POS4 could enhance intestinal homeostasis by modulating gut microbiota in a positive way and regulating autophagy associated genes. Taken together, we proposed that galacturonic acid enriched low molecular weight pectic polysaccharide have great potential in the development of healthy foods such as anti-aging health care products.

Antiaging effect of anthocyanin extracts from bilberry on natural or UV-treated male Drosophila melanogaster.

Anthocyanins from bilberry (Vaccinium vitis-idaea) are one of the most abundant sources of polyphenols and are widely used in the food, medicine and cosmetics industries due to their antioxidation properties, but few studies have investigated their antiaging properties. Based on our previous examination, the effect of anthocyanin extracts from bilberry (BANCs) on several characteristics of natural and UV-treated male Drosophila melanogaster, including their lifespan, fecundity, and antioxidant capacity, was studied, and the related mechanisms were preliminary explored. The results indicated that BANCs can effectively prolong the average and maximum lifespan and improve the reproductive capacity and antioxidant capacity of natural and UV-treated flies. In particular, BANCs significantly changed the growth cycle, sex ratio and content of ROS in the fat bodies of the offspring and decreased the expression levels of antioxidant- and autophagy-related genes in UV-treated flies. Collectively, the results demonstrate that BANC supplementation in the medium effectively alleviated the aging process, and this effect was not directly correlated with the antioxidant and autophagy signaling pathways in the body of D. melanogaster.

Protective Effect of Bilberry Anthocyanin Extracts on Dextran Sulfate Sodium-Induced Intestinal Damage in Drosophila melanogaster.

Inflammatory bowel disease (IBD) is a chronic recurrent disease that can be controlled by various natural extracts. Anthocyanins (ANCs) from bilberry have significant antioxidant capacity and are widely used as food colors and antioxidants. In this study, we investigated the protective effects of bilberry anthocyanin extracts (BANCs) against dextran sulphate sodium (DSS)-induced intestinal inflammation in a Drosophila melanogaster (D. melanogaster) model, and the effects on the lifespan, antioxidant capacity, intestinal characteristics, and microbiome and gene expression profiles were analyzed to elucidate the underlying biological mechanisms. In DSS-induced normal and axenic D. melanogaster, BANCs significantly increased the survival rate, maintained the intestinal morphology and integrity, and reduced the number of dead intestinal epithelial cells and the ROS level of these cells. BANC supplementation had no significant effect on the intestinal microflora of DSS-induced D. melanogaster, as demonstrated by a 16S rDNA analysis, but improved the antioxidant capacity by activating the relative gene expression of NRF2 signaling pathways in the intestine of D. melanogaster with DSS-induced inflammation. Therefore, the results demonstrate that BANCs effectively alleviate intestinal inflammatory injury induced by DSS and improve the antioxidant capacity of D. melanogaster by modulating NRF2 signaling pathways, and could thus promote the application of BANCs as functional foods.

Inhibition of Liver Cancer HepG2 Cell Proliferation by Enzymatically Prepared Low-molecular Citrus Pectin.

BACKGROUND: Low-molecular citrus pectin (LCP) is a pectin polysaccharide with low molec-ular weight, low degree of crux, and no branching. It is obtained by degrading natural citrus pectin (CP) through physical, chemical and enzymatic methods. LCP has received considerable attention in recent years due to its potential applications in the medical and biological fields. METHODS: In our previous study, LCP was prepared from CP by using recombinant Bacillus subtilis pectate lyase B. Monosaccharide comparative analysis revealed that the galacturonic acid content of LCP was higher than that of CP. The cell viability effect of LCP was elucidated by using HepG2 cells and the Cell Counting Kit-8 (CCK-8) assay. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining, Annexin V-FITC/PI staining, and flow cytometer propidium iodide stain-ing were performed to detect the effects of LCP on apoptosis and cell cycle arrest in HepG2 cells. Mi-tochondrial membrane potential (MMP) was observed through 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-imidacarbocyanine assay. RESULTS & DISCUSSION: The Mw of the prepared LCP was 7.6 kDa, which was significantly lower than that of CP (140 kDa). Cell viability decreased with the increase in the concentration of LCP. The half-inhibitory concentration of 1.46 ± 0.02 mg/mL was determined. Treatment with 1.6 mg/mL LCP in-duced the apoptosis of HepG2 cells with the inhibition rate of 83.10% ± 4.72%, and the cell cycle was arrested in the S phase. Furthermore, the MMP of HepG2 cells decreased with the increase in LCP concentration. CONCLUSION: The enzymatically prepared LCP could inhibit the proliferation of HepG2 cells. This study provided a partial experimental basis and reference for LCP to become a potential functional food for anti-liver cancer.

The Protective Effects of Carrageenan Oligosaccharides on Intestinal Oxidative Stress Damage of Female Drosophila melanogaster.

Carrageenan oligosaccharides (COS) have been reported to possess excellent antioxidant activities, but the underlying mechanism remains poorly understood. In this study, H2O2 was applied to trigger oxidative stress. The results showed that the addition of COS could effectively extend the lifespan of female Drosophila, which was associated with improvements by COS on the antioxidant defense system, including a decrease in MDA, the enhanced activities of SOD and CAT, the reduction of ROS in intestinal epithelial cells, and the up-regulation of antioxidant-relevant genes (GCL, GSTs, Nrf2, SOD). Meanwhile, the axenic female Drosophila fed with COS showed almost no improvement in the above measurements after H2O2 treatment, which highlighted the antioxidant mechanism of COS was closely related to intestinal microorganisms. Then, 16S rDNA high-throughput sequencing was applied and the result showed that the addition of COS in diets contributed to the diversity and abundance of intestinal flora in H2O2 induced female Drosophila. Moreover, COS significantly inhibited the expression of gene mTOR, elevated its downstream gene 4E-BP, and further inhibited autophagy-relevant genes (AMPKα, Atg1, Atg5, Atg8a) in H2O2 induced female Drosophila. The inhibition of the mTOR pathway and the activation of autophagy was probably mediated by the antioxidant effects of COS. These results provide potential evidence for further understanding of COS as an intestinal antioxidant.

Simultaneous Detection of Seven Alternaria Toxins in Mixed Fruit Puree by Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry Coupled with a Modified QuEChERS.

The presence of Alternaria toxins (ATs) in fruit purees may cause potential harm to the life and health of consumers. As time passes, ATs have become the key detection objects in this kind of food. Based on this, a novel and rapid method was established in this paper for the simultaneous detection of seven ATS (tenuazonic acid, alternariol, alternariol monomethyl ether, altenuene, tentoxin, altenusin, and altertoxin I) in mixed fruit purees using ultra-high performance liquid chromatography-tandem mass spectrometry. The sample was prepared using the modified QuEChERS (quick, easy, cheap, effective, rugged, and safe) method to complete the extraction and clean-up steps in one procedure. In this QuEChERS method, sample was extracted with water and acetonitrile (1.5% formic acid), then salted out with NaCl, separated on an ACQUITY UPLC BEH C18 with gradient elution by using acetonitrile and 0.1% formic acid aqueous as eluent, and detected by UPLC-MS/MS under positive (ESI+) and negative (ESI-) electrospray ionization and MRM models. Results showed that the seven ATs exhibited a good linearity in the concentration range of 0.5-200 ng/mL with R2 > 0.9925, and the limits of detection (LODs) of the instrument were in the range of 0.18-0.53 µg/kg. The average recoveries ranged from 79.5% to 106.7%, with the relative standard deviations (RSDs) no more than 9.78% at spiked levels of 5, 10, and 20 µg/kg for seven ATs. The established method was applied to the determination and analysis of the seven ATs in 80 mixed fruit puree samples. The results showed that ATs were detected in 31 of the 80 samples, and the content of ATs ranged from 1.32 µg/kg to 54.89 µg/kg. Moreover, the content of TeA was the highest in the detected samples (23.32-54.89 µg/kg), while the detection rate of Ten (24/31 samples) was higher than the other ATs. Furthermore, the other five ATs had similar and lower levels of contamination. The method established in this paper is accurate, rapid, simple, sensitive, repeatable, and stable, and can be used for the practical determination of seven ATs in fruit puree or other similar samples. Moreover, this method could provide theory foundation for the establishment of limit standard of ATs and provide a reference for the development of similar detection standard methods in the future.

Agar oligosaccharides ameliorate the intestinal inflammation of male Drosophila melanogaster via modulating the microbiota, and immune and cell autophagy.

Agar oligosaccharide (AOS) is a marine prebiotic with apparent improving health and longevity effects. In this study, the protective effect of AOS on the intestine was evaluated in the sodium dodecyl sulfate (SDS)-induced inflammatory model of male Drosophila. The results showed that AOS used as a nutritional additive in basal food could lengthen the life of SDS-stimulated male Drosophila. Additionally, AOS could alleviate the injuries of SDS to microvilli and mitochondria in male Drosophila midgut epithelial cells. AOS could regulate the relative gene expressions in the antibacterial peptides (AMPs), mTOR pathway and autophagy process, and significantly improved the α-diversity of midgut microbiota and decreased the abundance of Klebsiella aerogenes, a kind of bacteria easily causing infections. Collectively, AOS could ameliorate the intestinal inflammation by modulating the microbiota, and the gene expression of immune and cell autophagy.

Carrageenan Oligosaccharides Extend Life Span and Health Span in Male Drosophila Melanogaster by Modulating Antioxidant Activity, Immunity, and Gut Microbiota.

Carrageenan oligosaccharide (CAO), the hydrolysate of carrageenan from marine red algae, is used as a prebiotic additive or medical material. In this study, male Drosophila melanogaster was used as an animal model to explore the possibility that CAO can extend the life span through its relationship with antioxidation, immunity, and gut microbiota in vivo. The results show that a certain amount of CAO effectively prolonged the average life span and improved the climbing vitality and fecundity of male Drosophila. In addition, 0.125% CAO in the diet significantly increased the activity of Cu,Zn-superoxide dismutase (Cu,Zn-SOD) and catalase (CAT), reduced the content of malondialdehyde (MDA), and significantly repressed the expression of nuclear factor kappa B (NF-κB) gene in old male Drosophila tissues. In the intestinal microbiota analysis, 0.125% CAO in the diet increased the diversity of gut microbiota and improved the abundance of Commensalibacter at the genus level in Drosophila on the 40th day. The above results indicated that CAO supplementation could extend the life span of male Drosophila by improving antioxidant activity, immunity, and by regulating intestinal microflora.

Influences of acid and ethanol stresses on Oenococcus oeni SD-2a and its proteomic and transcriptional responses.

BACKGROUND: During winemaking, malolactic fermentation (MLF) is usually induced by Oenococcus oeni owing to its high resistance to wine stress factors. To ensure a controlled and efficient MLF process, starter cultures are inoculated in wine. In previous studies, O. oeni strains with sub-lethal acid or ethanol stresses showed higher freeze-drying vitality and better MLF performance. To explore the mechanisms involved, influences of acid and ethanol stresses on O. oeni SD-2a were investigated in this study to gain a better understanding of the cross-protection responses. RESULTS: The results showed that acid and ethanol stresses both caused damage to cell membranes and decreased cellular adenosine triphosphate concentration. At the same time, acid stress increased the uptake of glutathione, while ethanol stress led to cell depolarization. The results of comparative proteomic analysis highlighted that heat shock protein was induced with almost all acid and ethanol stresses. In addition, the expression of stress-relevant genes (hsp20, clpP, trxA, ctsR, recO, usp) increased greatly with ethanol and acid stress treatments. Finally, the viability of O. oeni was improved with acid and ethanol pretreatments after freeze-drying. CONCLUSIONS: This study demonstrated that acid and ethanol stresses had mixed influences on O. oeni SD-2a. Some physiological and molecular changes would contribute to a more stress-tolerant state of O. oeni, thereby improving the viability of lyophilized cells. © 2020 Society of Chemical Industry.

Anti-aging Effect of Agar Oligosaccharide on Male Drosophila melanogaster and its Preliminary Mechanism.

Agar oligosaccharide (AOS) is a marine prebiotic with apparent anti-inflammatory, antioxidant and anti-tumor effects. During this study, different doses of AOS are added to a basal diet to evaluate its effects on the lifespan, motor vigor and reproduction of male Drosophila melanogaster. Additionally, the activities of Cu,Zn-superoxide dismutase (Cu,Zn-SOD) and catalase (CAT) and the malondialdehyde (MDA) content in male Drosophila are examined on the 10th, 25th and 40th days. The fly midguts are removed on the 10th and 40th days for analyses of the intestinal microbial community by 16S rDNA sequencing and the expression level of intestinal immunity genes by quantitative real-time PCR (RT-PCR). The results show that AOS significantly prolonged the average and maximum lifespan and increased the antioxidant capacity of male Drosophila. Additionally, AOS significantly regulated the structure of the intestinal flora of "old" flies (40 days) and upregulated the expression of immune deficiency (IMD) genes to improve the intestinal immunity, which could be beneficial for delaying aging in old flies. The above-described results provide a theoretical basis for the application of AOS, a type of marine oligosaccharide, as a nutritional supplement or immunomodulator.

A Lactic Acid Bacteria (LAB)-Based Vaccine Candidate for Human Norovirus.

Human noroviruses (HuNoVs) are responsible for more than 95% of the non-bacterial acute gastroenteritis epidemics in the world. The CDC estimates that every year 21 million individuals suffer from HuNoV-induced gastroenteritis in the United States. Currently, there is no FDA-approved vaccine for HuNoVs. Development of an effective vaccine has been hampered by the lack of an efficient cell culture system for HuNoVs and a suitable small animal model for pathogenesis study. In this study, we developed lactic acid bacteria (LAB) as a vector to deliver HuNoV antigen. A LAB strain (Lactococcus lactis) carrying VP1 gene of a HuNoV GII.4 virus (LAB-VP1) was constructed. It was found that HuNoV VP1 protein was highly expressed by LAB vector and was secreted into media supernatants. To test whether LAB-based HuNoV vaccine candidate is immunogenic, 4-day-old gnotobiotic piglets were orally inoculated with various doses of LAB-VP1. It was found that LABs were persistent in the small intestine of piglets and shed in pig feces for at least 25 days post inoculation. LAB DNA and VP1 were detected in mesenteric lymph nodes and spleen tissue in LAB-VP1 inoculated groups. HuNoV-specific IgG and IgA were detectable in serum and feces respectively at day 13 post-inoculation, and further increased at later time points. After being challenged with HuNoV GII.4 strain, a large amount of HuNoV antigens were observed in the duodenum, jejunum, and ileum sections of the intestine in the LAB control group. In contrast, significantly less, or no, HuNoV antigens were detected in the LAB-VP1 immunized groups. Collectively, these results demonstrate that a LAB-based HuNoV vaccine induces protective immunity in gnotobiotic piglets.

Preparation of alginate oligosaccharide nanoliposomes and an analysis of their inhibitory effects on Caco-2 cells.

The conditions were optimised for preparing Alginate oligosaccharide (AOS) nanoliposomes, and Caco-2 cell experiments were carried out to examine their antitumour effects. The optimal formulation of AOS nanoliposomes was as follows: a phosphatidylcholine-to-cholesterol ratio of 5.12, AOS concentration of 8.44 mg/mL, Tween 80 concentration of 1.11%, and organic phase to aqueous phase ratio of 5.25. Under the above conditions, the experimental encapsulation efficiency was 65.84%, and the AOS nanoliposomes exhibited a small particle size of 323 nm. After Caco-2 cells were treated with AOS liposomes and AOS for 24 h, AOS nanoliposomes inhibited the growth of Caco-2 cells to a greater extent than AOS at concentrations of 0.0625, 0.125, 0.25, 0.5 and 1 mg/mL (P < 0.01). LDH leakage exhibited a concentration-dependent increase following treatment with 0.5-1 mg/mL AOS nanoliposomes, and the inhibitory effect of AOS nanoliposomes exhibited a more significant difference than AOS (P < 0.01). Cells treated with 0.5 mg/mL and 1 mg/mL AOS nanoliposomes displayed a substantial and significant increase in activity compared with AOS (P < 0.01). Based on these results, AOS nanoliposomes exerted a more significant effect on inhibiting Caco-2 cell proliferation than AOS.

Variable High-Pressure-Processing Sensitivities for Genogroup II Human Noroviruses.

UNLABELLED: Human norovirus (HuNoV) is a leading cause of foodborne diseases worldwide. High-pressure processing (HPP) is one of the most promising nonthermal technologies for the decontamination of viral pathogens in foods. However, the survival of HuNoVs after HPP is poorly understood because these viruses cannot be propagated in vitro In this study, we estimated the survival of different HuNoV strains within genogroup II (GII) after HPP treatment using viral receptor-binding ability as an indicator. Four HuNoV strains (one GII genotype 1 [GII.1] strain, two GII.4 strains, and one GII.6 strain) were treated at high pressures ranging from 200 to 600 MPa. After treatment, the intact viral particles were captured by porcine gastric mucin-conjugated magnetic beads (PGM-MBs) that contained histo-blood group antigens, the functional receptors for HuNoVs. The genomic RNA copies of the captured HuNoVs were quantified by real-time reverse transcriptase PCR (RT-PCR). Two GII.4 HuNoVs had similar sensitivities to HPP. The resistance of HuNoV strains against HPP ranked as follows: GII.1 > GII.6 > GII.4, with GII.4 being the most sensitive. Evaluation of temperature and matrix effects on HPP-mediated inactivation of HuNoV GII.4, GII.1, and GII.6 strains showed that HuNoV was more easily inactivated at lower temperatures and at a neutral pH. In addition, phosphate-buffered saline (PBS) and minimal essential medium (MEM) can provide protective effects against HuNoV inactivation compared to H2O. Collectively, this study demonstrated that (i) different HuNoV strains within GII exhibited different sensitivities to high pressure, and (ii) HPP is capable of inactivating HuNoV GII strains by optimizing pressure parameters. IMPORTANCE: Human norovirus (HuNoV) is a leading cause of foodborne disease worldwide. Noroviruses are highly diverse, both antigenically and genetically. Genogroup II (GII) contains the majority of HuNoVs, with GII genotype 4 (GII.4) being the most prevalent. Recently, GII.1 and GII.6 have emerged and caused many outbreaks worldwide. However, the survival of these GII HuNoVs is poorly understood because they are uncultivable in vitro Using a novel receptor-binding assay conjugated with real-time RT-PCR, we found that GII HuNoVs had variable susceptibilities to high-pressure processing (HPP), which is one of the most promising food-processing technologies. The resistance of HuNoV strains to HPP ranked as follows: GII.1 > GII.6 > GII.4. This study highlights the ability of HPP to inactivate HuNoV and the need to optimize processing conditions based on HuNoV strain variability and sample matrix.

Obtaining a mutant of Bacillus amyloliquefaciens xylanase A with improved catalytic activity by directed evolution.

This study aimed to obtain xylanase exhibiting improved enzyme properties to satisfy the requirements for industrial applications. The baxA gene encoding Bacillus amyloliquefaciens xylanase A was mutated by error-prone touchdown PCR. The mutant, pCbaxA50, was screened from the mutant library by using the 96-well plate high-throughput screening method. Sequence alignment revealed the identical mutation point S138T in xylanase (reBaxA50) produced by the pCbaxA50. The specific activity of the purified reBaxA50 was 9.38U/mg, which was 3.5 times higher than that of its parent expressed in Escherichia coli BL21 (DE3), named reBaxA. The optimum temperature of reBaxA and reBaxA50 were 55°C and 50°C, respectively. The optimum pH of reBaxA and reBaxA50 were pH 6 and pH 5, respectively. Moreover, reBaxA50 was more stable than reBaxA under thermal and extreme pH treatment. The half-life at 60°C and apparent melting temperature of reBaxA50 were 9.74min and 89.15°C, respectively. High-performance liquid chromatography showed that reBaxA50 released xylooligosaccharides from oat spelt, birchwood, and beechwood xylans, with xylotriose as the major product; beechwood xylan was also the most thoroughly hydrolyzed. This study demonstrated that the S138T mutation possibly improved the catalytic activity and thermostability of reBaxA50.