Search

Accurate and highly sensitive detection of viable but nonculturable (VBNC) Vibrio parahaemolyticus is crucial for preventing seafood-related outbreaks. The VBNC state may occur due to bacterial susceptibility to cold shock during food storage. Propidium monoazide (PMA) has been widely applied to detect VBNC foodborne pathogens. In this study, we developed and compared real-time PCR (qPCR) and real-time LAMP (qLAMP) methods combined with an improved propidium monoazide (PMAxx) to detect pathogenic VBNC V. parahaemolyticus. The designed primers and probes were determined to be highly selective for V. parahaemolyticus strains. The combined PMAxx-qPCR method, requiring 100 min, demonstrated a quantification limit of 10.5 colony-forming units (CFU)/mL in pure culture and 28 CFU/g in raw shrimp respectively, which were 10-fold lower than the PMAxx-qLAMP method (45min). When testing mixtures containing different ratios of VBNC to dead V. parahaemolyticus, PMAxx-based methods were notably superior at distinguishing between VBNC and dead bacteria when VBNC cell concentrations were low. Therefore, PMAxx is an effective means for improving the detection and quantification of VBNC V. parahaemolyticus by qPCR and qLAMP. Pretreatment with PMAxx was shown to be suitable for detection of VBNC V. parahaemolyticus in complex food samples, including raw shrimp.

Nutritional interventions play a critical role in modifying the intestinal microbiome of host animals. This study was conducted to interrogate the physiological effects on fecal microflora of weaned piglets via the dietary supplemented with two types of products of Citrus reticulata "Chachi", respectively. For this purpose, A total of 72 piglets with uniform sizes were randomly assigned to four dietary treatment groups consisted of a negative control group (NCG), a fermented citrus reticulata "Chachi" pulp group (FCRPG), a Citri Reticulatae Pericarpium group (CRPG) and a positive control group (PCG) in a 21-day feeding trial. After the raising experiment, fresh feces of piglets were analyzed systematically using multi-omics technologies. Metagenomics method with high-throughput compositional characterization indicated that the architecture and diversity of fecal microbiome were both influenced by these two additives and compound antibiotics. Metabolite analysis showed that FCRPG have an significant effects on fecal short-chain fatty acids (SCFAs) among four treatment groups. Results of functional proteomics approaches found that FCRPG presented the highest butyrate metabolic level, and CRPG showed the highest flavone and flavonol biosynthesis level in feces. In addition, NCP produced an effective effect on adjusting fecal microbiome proflie. Consequently, our findings demonstrate that dietary supplementation with FCRP or CRP modulates the microbial taxa, metabolic and proteomic alterations in fecal microbiota of weaned piglets for health maintaining.

Adulteration of the high-value silver pomfret (Pampus argenteus) is a serious problem worldwide, necessitating accurate identification and quantification of the species. In this study, optimisation of the digital droplet PCR (ddPCR) assay for the identification and quantification of the silver pomfret was carried out. The primer and probe concentrations, melting temperature, and PCR cycle number were optimised by combining single-factor experiments with an orthogonal experimental design. The absolute limits of detection and quantification of the ddPCR were 2copies/μl and 21 copies/μl, respectively. Its sensitivity was 0.1% for meat mixtures and 0.5% for DNA mixtures. The ddPCR was 156 times more sensitive than the real-time PCR, although both methods had similar specificities. However, the overall time needed to complete the ddPCR method was twice that of the real-time PCR. Notwithstanding, the ddPCR methodology established in this study can be a valuable tool for addressing species adulteration issues.

Oenothein B (OeB) is a dimeric macrocyclic ellagitannin isolated from Herbs and fruits that have a variety of biological activities. In order to better understand the effect of OeB on the activity of the digestive enzyme pepsin, interactions between OeB and pepsin were investigated in vitro under simulated physiological conditions based on enzyme inhibition studies, fluorescence, isothermal titration calorimetry, CD, and molecular docking. It was found OeB is an effective inhibitor of pepsin, likely acting in a reversible manner through both competitive and noncompetitive inhibition. Fluorescence quenching of pepsin by OeB was a static quenching. CD spectra showed the addition of OeB causes the main chain of pepsin to loosen and expand and the partial β-sheet structure to be converted to a disordered structure. Isothermal titration calorimetry and docking studies revealed the main binding mechanism of OeB and pepsin was through noncovalent interactions, hydrophobic interactions with OeB and the internal hydrophobic group of pepsin, and then hydrogen bonding between OeB and the Val243 and Asp77 residues of pepsin. Noncovalent bonds between OeB and pepsin change the polarity and structure of enzymes, decreasing enzymatic activity. Compared with small molecular polyphenols, OeB has a weaker hydrophobic interaction with pepsin and less effect on the secondary structure of pepsin. These findings are the first direct elucidation of the interactions between the oligomer ellagitannin OeB and pepsin, further contributing to understanding binding between oligomer ellagitannins and digestive enzymes. PRACTICAL APPLICATION: The results of this study indicate that the interaction between OeB and pepsin has a certain inhibitory effect on pepsin. In order to reduce the impact of OeB on human digestion and its own activities, nano-encapsulation technology can be used in the future to protect oligomeric ellagitannin such as OeB.

Hepatitis diseases are remaining in the list of significant threats to human health. Human hepatitis viruses are basically classified into six major hepatotropic pathogens—hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), hepatitis E virus (HEV), and hepatitis G virus (HGV). Among these different forms of hepatotropic viruses, HAV as the leading cause of acute viral hepatitis is characterized as a kind of tiny ribonucleic acid virus that is linked to atopic disease. As we know, animal models have been instrumental in promoting understanding of complex host-virus interactions and boosting the advancement of immune therapies. So far, animal models such as nonhuman primates (NHPs) have enabled scientists to mimic and study the pathogenicities and host immune responses for hepatitis A infection. With the exception of chimpanzees and marmosets, animals like mice, pigs, guinea pigs, and tree shrews can also be selected as alternative animal models infected with HAV under laboratory conditions. In order to gain a better insight into hepatitis A pathogenesis and relevant contents, this chapter is mainly focused on the research progress in animal models of hepatitis A, and discusses the merits and demerits of these alternative models.