Dual bacterial strains TTI for monitoring fish quality in food cold chain.

Most microbial time-temperature indicators (TTIs) considered only one spoilage strain. This research compared single and dual spoilage strains-based microbial TTI for quality changes of chilled grouper fish (Epinephelus fuscoguttatus x E. lanceolatus) fillet products during distribution. The next-generation sequencing (NGS) and traditional plate count approach showed that Pseudomonas fragi and Vibrio parahaemolyticus were specific spoilage bacteria at 7 and 15°C. A dual-strain TTI response provides more accurate results than a single-strain TTI and provides an irreversible color change from yellow to reddish-brown, showing levels of fish freshness. The microbial TTI comprises fish spoilage bacteria strains with 3 log CFU/ml, a nutrient broth supplemented with 2% NaCl as a medium, and phenol red with 0.25 mg/ml as a pH indicator. Overall, this study points to the applicability of a dual-strain microbial TTI as a valuable tool for monitoring fish quality changes during cold chain break condition.

Applications of Nisin and EDTA in Food Packaging for Improving Fabricated Chitosan-Polylactate Plastic Film Performance and Fish Fillet Preservation.

This study aimed to increase the antibacterial activity of chitosan-polylactic acid (PLA) composite film by adding nisin and ethylenediaminetetraacetic acid (EDTA). We evaluated the mechanical, physicochemical, and antibacterial properties of various PLA composite films, as well as the enhancement effect of PLA composite films with EDTA + nisin on the preservation of grouper fillets. Films of PLA alone, PLA plus chitosan (C5), PLA plus nisin + EDTA (EN2), and PLA plus chitosan plus nisin + EDTA (C5EN1 and C5EN2) were prepared. The addition of EDTA + nisin to the chitosan-PLA matrix significantly improved the antibacterial activity of the PLA composite film, with C5EN1 and C5EN2 films showing the highest antibacterial activity among the five films. Compared with the fish samples covered by C5, the counts of several microbial categories (i.e., mesophilic bacteria, psychrotrophic bacteria, coliforms, Aeromonas, Pseudomonas, and Vibrio) and total volatile basic nitrogen content in fish were significantly reduced in the samples covered by C5EN1. In addition, the counts of samples covered by C5EN1 or C5 were significantly lower compared to the uncovered and PLA film-covered samples.

Anti-Allergic Diarrhea Effect of Diosgenin Occurs via Improving Gut Dysbiosis in a Murine Model of Food Allergy.

Although the anti-allergic and prebiotic activities of diosgenin have been reported, the influence of diosgenin on intestinal immune and epithelial cells remains unclear. As the gut microbiota plays an important role in allergic disorders, this study aimed to investigate whether the anti-allergic diarrhea effect of diosgenin occurs via improving gut dysbiosis. In a murine food allergy model, the density of fecal bacterial growth on de Man, Rogossa and Sharpe (MRS) plates was diminished, and growth on reinforced clostridial medium (RCM) and lysogeny broth (LB) agar plates was elevated. However, the oral administration of diosgenin reduced the density of fecal bacteria and ameliorated diarrhea severity. Concordantly, reshaped diversity and an abundance of fecal microbes were observed in some of the diosgenin-treated mice, which showed a milder severity of diarrhea. The relevant fecal strains from the diosgenin-treated mice were defined and cultured with Caco-2 cells and allergen-primed mesenteric lymph node (MLN) cells. These strains exhibited protective effects against the cytokine/chemokine network and allergen-induced T-cell responses to varying degrees. By contrast, diosgenin limitedly regulated cytokine production and even reduced cell viability. Taken together, these findings show that diosgenin per se could not directly modulate the functionality of intestinal epithelial cells and immune cells, and its anti-allergic effect is most likely exerted via improving gut dysbiosis.

Antibacterial Activity of Chitosan-Polylactate Fabricated Plastic Film and Its Application on the Preservation of Fish Fillet.

This research prepared chitosan-PLA plastic films by extrusion, analyzed the physical and mechanical properties and antibacterial activity of the fabricated plastic films, and used them to preserve grouper fillet. We added chitosan (220 kDa, 93% DD) in the weight ratio of 0.5-2% into the PLA to prepare the chitosan-PLA films. With the increasing chitosan dosage, both the water vapor transmission rate and moisture content of chitosan-PLA films increased. Among the three doses of chitosan (0.5%, 1%, and 2%) added to PLA, 0.5% chitosan-PLA film had the highest antibacterial activity. This plastic film had an inhibitory efficiency of over 95% against Escherichia coli, Pseudomonas fluorescens, and Staphylococcus aureus. The action of covering the fish fillet with 0.5% chitosan-PLA film significantly reduced several microbes' counting (i.e., mesophiles, psychrophiles, coliforms, Pseudomonas, Aeromonas, and Vibrio) and total volatile basic nitrogen (TVBN) value in the grouper fillets stored at 4 °C. Thus, such action prolongs the fish fillets' shelf life to up to at least nine days, and this 0.5% chitosan-PLA film shows promising potential for preserving refrigerated fish.

Polysaccharide-rich red algae (Gelidium amansii) hot-water extracts ameliorate the altered plasma cholesterol and hepatic lipid homeostasis in high-fat diet-fed rats.

We have demonstrated that red algae Gelidium amansii (GA) hot-water extract (GHE) is a polysaccharide-rich fraction, containing 68.54% water-soluble indigestible carbohydrate polymers; the molecular weight of major polysaccharide is 892. Here, we investigated the mechanisms of GHE on plasma and hepatic lipid metabolisms in high-fat (HF) diet-fed rats. Rats were divided into: normal diet group, HF-diet group, HF-diet+5% GHE group, and HF-diet+1% cholestyramine group. GHE supplementation for 8 weeks significantly decreased plasma cholesterol, LDL-C, and VLDL-C levels and increased the fecal triglyceride and bile acid excretion in HF diet-fed rats. GHE group has lower lipid contents in the liver and adipose tissues. GHE supplementation decreased the activities of acetyl-CoA carboxylase, fatty acid synthase, and HMG-CoA reductase in the livers. The levels of increased phosphorylated AMP-activated protein kinase (AMPK), peroxisome proliferator activated receptor (PPAR)-α, farnesoid-X receptor (FXR), low density lipoprotein receptor (LDLR), and cytochrome P450-7A1 (CYP7A1) protein expression, and the decreased PPAR-γ protein expression in the livers were observed in GHE group. These results suggest that GHE supplementation is capable of interfering in cholesterol metabolism and increasing hepatic LDLR and CYP7A1 expression to decrease blood cholesterol, and activating FXR and AMPK to inhibit lipogenic enzyme activities and reduce the hepatic lipid accumulation.

Extracellular production of a novel endo-ß-agarase AgaA from Pseudomonas vesicularis MA103 that cleaves agarose into neoagarotetraose and neoagarohexaose.

The gene agaA, of the isolated marine bacterium Pseudomonas vesicularis MA103, comprised 2958-bp nucleotides encoding a putative agarase AgaA of 985 amino acids, which was predicted to contain a signal peptide of 29 amino acids in the N-terminus, a catalytic domain of glycoside hydrolase 16 (GH16) family, a bacterial immunoglobulin group 2 (Big 2), and three carbohydrate binding modules 6 (CBM 6). The gene agaA was cloned and overexpressed in Escherichia coli, and the optimum temperatures for AgaA overexpression were 16, 20 and 24 °C. The agaA was cloned without its signal peptide for cytosolic production overexpression, whereas it was cloned with the heterologous signal peptide PelB and its endogenous signal peptide for periplasmic and extracellular productions, respectively. Extracellular and periplasmic rAgaA showed greater activity than that of cytosolic rAgaA, indicating that membrane translocation of AgaA may encourage proper protein folding. Time-course hydrolysis of agarose by rAgaA was accomplished and the products were analyzed using thin layer chromatography and matrix-assisted laser desorption inoization-time of flight mass spectrometry, indicating that AgaA from P. vesicularis was an endo-type ß-1,4 agarase that cleaved agarose into neoagarotetraose and neoagarohexaose as the final products.

Monitoring and preparation of neoagaro- and agaro-oligosaccharide products by high performance anion exchange chromatography systems.

A series of neoagaro-oligosaccharides (NAOS) were prepared by ß-agarase digestion and agaro-oligosaccharides (AOS) by HCl hydrolysis from agarose with defined quantity and degree of polymerization (DP). Chain-length distribution in the crude product mixtures were monitored by two high performance anion exchange chromatography systems coupled with a pulsed amperometric detector. Method 1 utilized two separation columns: a CarboPac(™) PA1 and a CarboPac(™) PA100 connected in series and method 2 used the PA100 alone. Method 1 resolved the product in size ranges consisting of DP 1-46 for NAOS and DP 1-32 for AOS. Method 2 clearly resolved saccharide product sizes within DP 26. The optimized system utilizing a semi-preparative CarboPac(™) PA100 column was connected with a fraction collector to isolate and quantify individually separated products. This study established systems for the preparation and qualitative and quantitative measurements as well as for the isolation of various sizes of oligomers generated from agarose.

Heat shock and cold shock treatments affect the survival of Listeria monocytogenes and Salmonella Typhimurium exposed to disinfectants.

The foodborne pathogens Listeria monocytogenes and Salmonella Typhimurium were subjected to heat shock at 48°C for 10 and 30 min, respectively, and then cold shocked at 15°C for 3 h. The effect of these shocks on the viability of test organisms exposed to chlorine dioxide and quaternary ammonium compounds was then determined. After exposure to the disinfectants, the viable population of each test organism, regardless of heat shock or cold shock treatment, decreased as the exposure period was extended. Both heat shock and cold shock treatments reduced the susceptibility of L. monocytogenes to both disinfectants at 25°C. However, for Salmonella Typhimurium, exposure to the chlorine dioxide disinfectant or quaternary ammonium compounds at 25°C significantly reduced (P < 0.05) survival of heat-shocked cells but significantly increased (P < 0.05) survival of cold-shocked cells compared with control cells. Survival of both L. monocytogenes and Salmonella Typhimurium generally was reduced after exposure to disinfectants at 40°C compared with 25°C.

Evaluation of HPSEC-ELSD method for precise measurement of ß-agarase activity.

ß-agarase activity was monitored by traditional reducing sugar content methods: Somogyi-Nelson's arsenomolybdate, Miller's dinitrosalicylic acid and Kidby and Davidson's ferricyanide methods, as well as by high-performance size exclusion chromatography coupled with a refractive index detector and an evaporative light scattering detector (ELSD). Calibration curves were established separately for each method to measure the amounts of the neoagaro-oligosaccharides (NAOS) in the reaction mixtures, which are the products from 1-10 units (U) of ß-agarase cleavage activity on agarose. Product quantities from each monitoring method were compared with the isolated NAOS products. The graphs plotted by agarase activity unit and product concentration clearly displayed that the ELSD method closely followed the results of the isolated products. The percentage deviation of results measured by the five methods away from those of the isolated NAOS product mixture amounted to -13.1-35.1, -21.1-25.5, -27.1-23.81, 6.1-24.3 and 16.2-22.8%, respectively. When the loss during product isolation, about 15-17%, was taken into account, the high precision of the ELSD method was confirmed. HPSEC-ELSD methods also accurately measured the enzyme kinetics as well as enabling partial identification of oligosaccharides assembled in the NAOS product mixture. This study established the HPSEC-ELSD system as an alternative method for monitoring agarase activity.

Separation and quantification of neoagaro- and agaro-oligosaccharide products generated from agarose digestion by beta-agarase and HCl in liquid chromatography systems.

A series of neoagaro-oligosaccharides (NAOS) were separated and isolated by beta-agarase digestion and agaro-oligosaccharides (AOS) by HCl hydrolysis from agarose with defined quantity and degree of polymerization (DP). Profiles of the oligomer length in the crude product mixtures were monitored by two high-performance liquid chromatography (HPLC) systems: size-exclusion chromatography (SEC) and NH2-column chromatography (NH2-HPLC), coupled with an evaporative light-scattering detector (ELSD). Calibration curves were established separately to identify the DP and quantify the amount of the oligomer products analyzed in the two systems. Each system was optimized to generate a spectrum of saccharide oligomers with various DP, where the reaction yield for NAOS was 52.7% by 4U/mg beta-agarase and for AOS was 45.6% by 0.4M HCl. SEC resolved the product in size ranges consisting of DP 1-22 for NAOS and DP 1-14 for AOS. NH2-HPLC clearly resolved both distinct saccharide product sizes within DP 12. The optimized system was connected with a fraction collector to isolate and quantify these individually separated products. The total product yields of the recovered NAOS of DP 1-22 and AOS of DP 1-14 by the SEC system were 84.7% and 82.9%, respectively. NH2-HPLC recovered NAOS and AOS, both with a DP of 1-10 with total product yields of 48.9% and 90.0%, respectively. Isolated NAOS and AOS product fractions were inspected by (1)H NMR spectroscopy and ESIMS spectrometry to confirm structure, molecular mass, and purity. This study established feasible systems for the preparation and qualitative and quantitative measurements, as well as for the isolation of various sizes of oligomers generated from agarose.