Discovery of a temperature-dependent protease spoiling meat from Pseudomonas fragi: Target to myofibrillar and sarcoplasmic proteins rather than collagen.

Chilled meat frequently suffered microbial spoilage because bacteria can secrete various proteases that break down the proteins. In this study, Pseudomonas fragi NMC 206 exhibited a temperature-dependent secretion pattern, with the ability to release the specific protease only below 25 °C. It was identified as alkaline protease AprA by LC-MS/MS, with the molecular weight of 50.4 kDa, belonging to the Serralysin family metalloprotease. Its significant potential for meat spoilage in situ resulted in alterations in meat color and sensory evaluation, as well as elevated pH, total volatile basic nitrogen (TVB-N) and the formation of volatile organic compounds (VOCs). The hydrolysis of meat proteins in vitro showed that AprA possessed a considerable proteolysis activity and degradation preferences on meat proteins, especially its ability to degrade myofibrillar and sarcoplasmic proteins, rather than collagen. These observations demonstrated temperatures regulated the secretion of AprA, which was closely related to chilled chicken spoilage caused by bacteria. These will provide a new basis for the preservation of meat products at low temperatures.

Revealing extracellular protein profile and excavating spoilage-related proteases of Aeromonas salmonicida based on multi-omics investigation.

Aeromonas is a ubiquitous aquatic bacteria, and it is a significant factor contributing to meat spoilage during processing and consumption. The abilities of Aeromonas salmonicida 29 and 57, which exhibit spoilage heterogeneity, to secrete protease, lipase, hemolysin, gelatinase, amylase, and lecithinase were confirmed by plate method. A total of 3948 proteins were identified by ITRAQ in extracellular secretions of A. salmonicida, and 16 proteases were found to be potentially related to spoilage ability. The complete genome sequence of A. salmonicida 57 consists of one circular chromosome and three plasmids, while A. salmonicida 29 consists of one circular chromosome, without a plasmid. Transcriptomic analysis revealed a significant number of DEGs were up-regulated in A. salmonicida 29, which were mainly enriched in metabolic pathways (e.g., amino acid metabolism, carbohydrate metabolism), indicating that A. salmonicida 29 had better potential to decompose and utilize nutrients in meat. Six protease genes (2 pepB, hap, pepA, ftsI, and pepD) were excavated by combined ITRAQ with transcriptome analysis, which potentially contribute to bacterial spoilage ability and exhibit universality among other dominant spoilage bacteria. This investigation provides new insights and evidence for elucidating metabolic and spoilage phenotypic differences and provides candidate genes and strategies for future prevention and control technology development.

The interaction of an effector protein Hap secreted by Aeromonas salmonicida and myofibrillar protein of meat: Possible mechanisms from structural changes to sites of molecular docking.

Microbial spoilage of meat products is a significant problem in the food industry. Aeromonas salmonicida is a significant microorganism responsible for spoilage in chilled meat. Its effector protein, hemagglutinin protease (Hap), has been identified as an effective substance for degrading meat proteins. The ability of Hap to hydrolyze myofibrillar proteins (MPs) in vitro demonstrated that Hap has obvious proteolytic activity, which could alter MPs' tertiary structure, secondary structure, and sulfhydryl groups. Moreover, Hap could significantly degrade MPs, focusing primarily on myosin heavy chain (MHC) and actin. Active site analysis and molecular docking revealed that the active center of Hap was bound to MPs via hydrophobic interaction and hydrogen bonding. It may preferentially cleave peptide bonds between Gly44-Val45 in actin, and Ala825-Phe826 in MHC. These findings suggest that Hap may be involved in the spoilage mechanism of microorganisms and provide crucial insights into the mechanisms of meat spoilage induced by bacteria.

SaaS sRNA promotes Salmonella intestinal invasion via modulating MAPK inflammatory pathway.

Salmonella Enteritidis is a foodborne enteric pathogen that infects humans and animals, utilizing complex survival strategies. Bacterial small RNA (sRNA) plays an important role in these strategies. However, the virulence regulatory network of S. Enteritidis remains largely incomplete and knowledge of gut virulence mechanisms of sRNAs is limited. Here, we characterized the function of a previously identified Salmonella adhesive-associated sRNA (SaaS) in the intestinal pathogenesis of S. Enteritidis. We found that SaaS promoted bacterial colonization in both cecum and colon of a BALB/c mouse model; it was preferentially expressed in colon. Moreover, our results showed that SaaS enhanced damage to mucosal barrier by affecting expressions of antimicrobial products, decreasing the number of goblet cells, suppressing mucin gene expression, and eventually reducing thickness of mucus layer; it further breached below physical barrier by strengthening invasion into epithelial cells in Caco-2 cell model as well as decreasing tight junction expressions. High throughput 16S rRNA gene sequencing revealed that SaaS also altered gut homeostasis by depleting beneficial gut microbiota while increasing harmful ones. Furthermore, by employing ELISA and western blot analysis, we demonstrated that SaaS regulated intestinal inflammation through sequential activation P38-JNK-ERK MAPK signaling pathway, which enabled immune escape at primary infection stage but strengthened pathogenesis at later stage, respectively. These findings suggest that SaaS plays an essential role in the virulence of S. Enteritidis and reveals its biological role in intestinal pathogenesis.

Characterization of effector protein Hap determining meat spoilage process from meat-borne Aeromonas salmonicida.

The spoilage roles of effector proteins secreted by dominant spoilage bacteria during food spoilage remained unknown. In this investigation, a hemagglutinin protease (Hap) belonging to the M4 family metallopeptidase was identified from Aeromonas salmonicida 29 isolate. It, has a molecular weight of 33.5 kDa, a Vmax of 17.06 µg/mL/min, and a Km of 2.46 mg/mL, and is conserved in various dominant spoilage bacteria. The stability testing demonstrated that Hap could maintain specific activity in the common environments (pH, temperature, and metal ions) of chilled meat. It exhibited high spoilage ability on meat in situ, increasing TVB-N, pH values, and the production of volatile organic compounds (VOCs), which was consistent with proteolytic activity analysis, completely confirming the determinant role of Hap for meat spoilage. These observations will enrich the spoilage theory and provide new insights into the control of food quality and safety.

Assessment of the di- and tri-chlorinated haloacetic acids during broiler prechilling.

Sodium hypochlorite (NaClO) has been widely used at 100 ppm concentration during poultry slaughter to reduce carcass microorganism loads. However, its use in poultry processing is restricted owing to the potential risks of disinfection by-products (DBPs) that can be produced by the reaction of NaClO with poultry meat components. This study assessed whether dichloroacetic acid (DCAA) and trichloroacetic acid (TCAA), as primary DBP representatives, were produced when NaClO was used as a disinfectant in various methods during broiler prechilling. Headspace gas chromatography-mass spectrometry for the quantitative determination of DCAA and TCAA in 180 prechilling water samples and 30 broiler meat samples, obtained from large standard slaughterhouses equipped with an online monitoring system to control the NaClO concentration between 50 and 100 ppm, showed that neither DCAA nor TCAA were detected. In simulation assays, haloacetic acids (HAAs) were not detected when the concentration of the NaClO solution was less than 200 ppm with low frequency addition; however, more than 0.1 mg/L of DCAA and TCAA were detected on applying 200, 300, 400, 500, and 1000 ppm NaClO at high frequency. These findings indicated that adding high concentrations of NaClO and frequently adding low levels pose a potential risk of DBP formation. This investigation provides a basis for the establishment of food risk and the scientific use of NaClO in poultry processing, and contributes to the evaluation of DBPs in poultry slaughter. PRACTICAL APPLICATION: This study confirmed the occurrences of DCAA and TCAA during broiler chilling processing, indicating that formation of HAAs in simulation systems was correlated with NaClO levels and validated the absence of DCAA and TCAA with less than 200 ppm, providing a basic study for food safety standards and regulations on the disinfectants used in food processing.

Modeling the elimination of mature biofilms formed by Staphylococcus aureus and Salmonella spp. Using combined ultrasound and disinfectants.

Biofilm formation by foodborne pathogens on food processing surfaces has contributed to numerous disease outbreaks and food recalls. We evaluated the following strategies for elimination of mature biofilm formed by Staphylococcus aureus and Salmonella spp. on stainless steel surfaces: acidic electrolyzed water (AEW), ozone water (OW), or ultrasound (40 kHz) alone, and combinations of ultrasound and disinfectants. The dynamics of elimination by combinations were determined using the Weibull and biphasic models. Treatment with AEW alone reduced the number of biofilm cells by approximately 3.0 log cfu/cm2, whereas less than 0.8 log cfu/cm2 of cells reduction was observed in biofilm exposed to OW or ultrasound alone, even with treatment for 20 min. The combination of AEW and ultrasound produced an obvious synergistic effect on biofilm reduction, achieving approximately 4.8 log cfu/cm2 reduction in Salmonella spp. biofilm. Interestingly, the biphasic model was a better fit than the Weibull model for the elimination process of mature biofilm formed by both pathogens and subjected to a combination of ultrasound and AEW, as determined by smaller values of the statistical parameters RMSE and AIC, although both models could evaluate the dynamic processes. Our findings indicated that a combination of ultrasound and AEW could effectively reduce the biofilm formed by pathogens on food contact surfaces, and that the biphasic model could predict the number of residual cells after biofilm exposure to this intervention approach.