Insights into ultrasonic treatment on the mechanism of proteolysis and taste improvement of defective dry-cured ham.

To investigate the effects of ultrasonic treatment on proteolysis and taste development of defective dry-cured ham, sensory attributes, enzyme activities, protein degradation and free amino acids were evaluated after different ultrasonic treatments. The ultrasonic treatment of 1000 W & 50 °C significantly increased the intensities of overall taste, umami, sweetness and richness, and decreased bitterness values compared with other groups. The residual activities of DPP I and cathepsin B + L in 1000 W & 50 °C maintained 48.71% and 24.94% of control group, respectively; the intense degradation of structural proteins was observed by label-free proteomics, accordingly. The contents of total free amino acids from 4522.64 mg/100 g muscles in control group increased to 5838.75 mg/100 g muscles in 1000 W & 50 °C; the largest increase of sweet and umami amino acids observed in 1000 W & 50 °C was responsible for the improvement of taste quality of defective dry-cured ham.

A comprehensive review on molecular mechanism of defective dry-cured ham with excessive pastiness, adhesiveness, and bitterness by proteomics insights.

Excessive bitterness, pastiness, and adhesiveness are the main organoleptic and textural defects of dry-cured ham, which often cause a lot of financial losses to manufacturers and seriously damage the quality of the product. These sensory and textural defects are related to the protein degradation of dry-cured ham. Proteomics shows great potential to improve our understanding of the molecular mechanism of sensory and textural defects and identify biomarkers for monitoring their quality traits. This review presents some of the major achievements and considerations in organoleptic and textural defects of dry-cured ham by proteomics analysis in the recent decades and gives an overview about how to correct sensory and textural defects of dry-cured ham. Proteomics reveals that muscle proteins derived from myofibril and cytoskeleton and involved in metabolic enzymes and oxygen transport have been identified as potential biomarkers in defective dry-cured ham. Relatively high residual activities of cathepsin B and L are responsible for the excessive degradation of these protein biomarkers in defective dry-cured ham. Ultrasound-assisted mild thermal or high-pressure treatment shows a good correction for the organoleptic and textural defects of dry-cured ham by changing microstructure and conformation of muscle proteins by accelerating degradation of proteins and polypeptides into free amino acids.

Penicillin­binding protein 1A mutation­positive Helicobacter pylori promotes epithelial­mesenchymal transition in gastric cancer via the suppression of microRNA­134.

Evidence suggests that Helicobacter pylori (H. pylori) is not only the main cause of gastric cancer (GC), but is also closely associated with its metastasis. One of the major virulence factors in H. pylori is the cytotoxin­associated gene A (CagA). With the growing proportion of amoxicillin­resistant H. pylori strains, the present study aimed to explore the effects of CagA­ and penicillin­binding protein 1A (PBP1A) mutation­positive H. pylori (H. pyloriCagA+/P+) on GC cells, and its clinical significance. The clinical significance of H. pyloriCagA+/P+ infection was analyzed in patients with GC. In vitro, GC cells were infected with H. pyloriCagA+/P+ to investigate whether it was involved in the epithelial­mesenchymal transition (EMT) of SGC­7901 cells using immunofluorescence and western blot analysis. The results of clinical analysis demonstrated that, although CagA­negative H. pylori infection had no significant association with the characteristics of patients with GC, H. pyloriCagA+/P+ infection was significantly associated with various clinicopathological parameters, including invasion depth, lymphatic metastasis and distant metastasis. In vitro, the results indicated that H. pyloriCagA+/P+ promoted proliferation, invasion and EMT of SGC­7901 cells. MicroRNA (miR)­134 was downregulated in H. pyloriCagA+/P+ infected tissues compared with in those with H. pyloriCagA+/P­ infection. miR­134 overexpression significantly reversed H. pyloriCagA+/P+ infection­associated cell proliferation, invasion and EMT. Furthermore, the results revealed that Forkhead box protein M1 (FoxM1) was a direct target of miR­134, and FoxM1 knockdown impeded H. pyloriCagA+/P+­induced EMT. In conclusion, the present study demonstrated that miR­134 may suppress the proliferation, invasion and EMT of SGC­7901 cells by targeting FoxM1, and may serve a protective role in the process of H. pyloriCagA+/P+­induced GC. These findings may lead to an improved understanding of H. pyloriCagA+/P+­associated poor clinical characteristics in patients with GC.

The Influence of Microwave Sterilization on the Ultrastructure, Permeability of Cell Membrane and Expression of Proteins of Bacillus Cereus.

Bacillus cereus was isolated from ready-to-serve brine goose, identified by 16S rRNA gene sequencing analysis and treated with a commercial microwave sterilization condition (a power of 1,800 W at 85°C for 5 min). The influence of microwaves on the morphology, the permeability of membrane and the expression of total bacterial proteins was observed. Microwave induced the clean of bacterial nuclear chromatin, increased the permeability and disrupted the integrity of membrane. Twenty-three proteins including 18 expressed down-regulated proteins and 5 expressed up-regulated proteins were identified by HPLC-MS/MS in the samples treated with microwave. The frequencies of proteins changed after microwaves treatment were labeled as 39.13% (synthesis and metabolism of amino acid or proteins), 21.74% (carbohydrate metabolism), 8.70% (anti-oxidant and acetyl Co-A synthesis), and 4.35% (the catalyst of catabolism of bacterial acetoin, ethanol metabolism, glyoxylate pathway, butyrate synthesis and detoxification activity), respectively. This study indicates that microwaves result in the inactivation of Bacillus cereus by cleaning nuclear chromatin, disrupting cell membrane and disordering the expression of proteins.

The effect of cooking temperature on the aggregation and digestion rate of myofibrillar proteins in Jinhua ham.

BACKGROUND: In order to evaluate the effect of cooking temperature on the nutrition quality of dry-cured hams, 60 biceps femoris samples from 16 Jinhua hams were divided into four groups (control, 70, 100 and 120 °C) and cooked for 30 min. Carbonyl content, sulfhydryl groups, surface hydrophobicity, microstructure, protein aggregation and digestibility of myofibrillar proteins were investigated. RESULTS: Cooking promoted carbonylation and decreased sulfhydryl groups in a temperature-dependent way. Scanning electron microscopy and Nile Red revealed that protein aggregation became a main phenomenon at 120 °C; it coincided with surface hydrophobicity. The increased carbonyl content and decreased sulfhydryl groups contributed to the formation of aggregates. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles showed the initial difference in proteolysis rate among four groups. The in vitro digestibility of pepsin and of trypsin and α-chymotrypsin increased from the control to 100 °C and decreased from 100 to 120 °C. CONCLUSION: The increased digestibility could be attributed to the oxidation of proteins and exposing recognition sites of digestive enzymes, while the decreased digestibility was due to the formation of aggregates. Cooking was a main factor that affected the digestibility of Jinhua ham, and cooking at 100 °C could be an ideal way to gain the highest digestibility of Jinhua ham. © 2018 Society of Chemical Industry.

The effect of ATP marination on the depolymerization of actin filament in goose muscles during postmortem conditioning.

In order to study the tenderization mechanism of ATP treatments by depolymerizing actin filaments, breast muscles of Eastern Zhejiang White Geese were randomly divided into 3 groups: control, 10 and 20 mM groups. Shear force (SF), sarcomere length (SL) and myofibrillar fraction index (MFI), the content of F-actin and G-actin, the expression of actin associated proteins (cofilins and tropomodulins) were investigated during conditioning. In 20 mM group, cofilins content increased from 48 to 168 h, while tropomodulins decreased; the content of F-actin decreased from 24 to 168 h, while the increased G-actin was observed upto 48 h. In the control, the degraded tropomodulins were observed at 168 h, and the increased cofilins and G-actin were detected at the same time; the increase of MFI and decrease of F-actin content were shown at 96 and 168 h. Compared to control group, 20 mM group accelerated the transformation of F-actin into G-actin; it showed higher SL and MFI, and lower SF at 48, 96 and 168 h, respectively. We concluded that depolymerization of actin filaments, which was regulated by cofilins and tropomodulins, contributed to myofibrillar fraction and low SF during conditioning.

The effect of oxidation on the structure of G-actin and its binding ability with aroma compounds in carp grass skeletal muscle.

To investigate the influence of oxidative modifications of G-actin on its binding ability with aroma compounds, the influence of H2O2 treatments on G-actin structure and the absorption for alcohols and aldehydes was investigated. Raman spectroscopy and scanning electron microscopy were used to evaluate structural changes of G-actin; GC-MS was used to analyze the binding with alcohols and aldehydes. Results showed that 0-5mM H2O2 enhanced the absorption of G-actin toward alcohols involved in the formation of hydrogen bonds by increasing α-helix and carbonyl values. 0-1mM H2O2 caused the release of aldehydes with decreased sulfhydryl sites. 1-20mM H2O2 increased the retention of aldehydes, due to the increased hydrophobic sites by G-actin rebuilding and aggregating. The aggregated G-actin favoured the hydrophobic interactions with aroma compounds, forming the protein-aroma compound complex, thus enhancing the resultant binding ability, as evidenced by scanning electron microscopy and GC/MS analysis.

The changes in the proteolysis activity and the accumulation of free amino acids during chinese traditional dry-cured loins processing.

Twelve pieces of longissimus dorsi were processed into Chinese traditional dry-cured loins. The changes in the proteolylic enzymes activities, myofibrillar proteins degradation, and free amino acids content were investigated during processing. Compared with fresh piece (0 day), the cathepsin B + L and calpains activities decreased after dry-curing and maintained potential activities values of 23.25 and 15.04% in the final products, respectively. The myosin heavy chain (MHC) and C protein were intensely degraded at the dry-ripened stage; the 50 kDa desmin increased at day 2 and then disappeared at day 11. The total free amino acids content increased from 333.18 mg/100 g in the raw to 1096.54 mg/100 g at the end of the dry-ripening. This work provided a mechanism for the accumulation of free amino acids and predicted the proteolysis extent of myofibrillar proteins by monitoring the changes of three marker proteins (MHC, C protein and 50 kDa desmin) during Chinese traditional dry-cured loins processing.

Effect of black pepper essential oil on the quality of fresh pork during storage.

The effect of different concentrations (0, 0.1 and 0.5%, v/v) of black pepper essential oil (BPEO) on thiobarbituric acid reactive substances (TBARS), meat color, the percentage of metmyoglobin (MetMb%), microbiological parameters and total volatile basic nitrogen (TVB-N) of pork loins stored at 4°C for 9days was evaluated. BPEO treatments showed lower TBARS, MetMb%, yellowness (b*) values, Pseudomonas spp. and Enterobacteriaceae count and TVB-N values and higher lightness (L*) and redness (a*) values than the control during storage; the effectiveness of BPEO was dose-dependent. The retardation of the formation of MetMb by adding BPEO ensured higher L* and a* values and lower b* values than the control at 6 and 9days; the MetMb content has a similar trend to the lipid oxidation. The lower TVB-N value of BPEO treatments than the control could be attributed to the inhibition of Pseudomonas spp. and Enterobacteriaceae. Gram-negative bacteria were more sensitive than Gram-positive bacteria to BPEO.

Antibacterial Activity and Mechanism of Action of Black Pepper Essential Oil on Meat-Borne Escherichia coli.

The aim of this study was to investigate the antibacterial activity of black pepper essential oil (BPEO) on Escherichia coli, further evaluate the potential mechanism of action. Results showed that the minimum inhibition concentration (MIC) of BPEO was 1.0 µL/mL. The diameter of inhibition zone values were with range from 17.12 to 26.13 mm. 2 × MIC treatments had lower membrane potential and shorter kill-time than 1 × MIC, while control had the highest values. E. coli treated with BPEO became deformed, pitted, shriveled, adhesive, and broken. 2 × MIC exhibited the greatest electric conductivity at 1, 3, 5, 7, 9, 11, and 13 h, leaked DNA materials at 4, 8, 12, 16, 20, 24, and 28 h, proteins at 4, 6, 8, 10, 12, 14, and 16 h, potassium ion at 0, 0.5, 1, 1.5, and 2 h, phosphate ion at 0.5, 1, 1.5, and 2 h and ATP (P < 0.05); 1 × MIC had higher values than control. BPEO led to the leakage, disorder and death by breaking cell membrane. This study suggested that the BPEO has potential as the natural antibacterial agent in meat industry.

Selective and sensitive determination of cypermethrin in fish via enzyme-linked immunosorbent assay-like method based on molecularly imprinted artificial antibody-quantum dot optosensing materials.

Molecularly imprinted silica layers appended to quantum dots (MIP-QDs) with customized selective artificial recognition sites were fabricated in this study by optimizing the ratio of the functional monomer to the template. Scanning electron microscopy, transmission electron microscopy, Brunauer­emmett­teller, Fourier transform infrared spectroscopy, and selectivity assay analyses were also performed. Results demonstrated that the selective fluorescence quenching properties of MIP-QDs toward cypermethrin (CYP) are due to strong interactions between these molecules. An enzyme-linked immunosorbent assay (ELISA)-like method based on the MIP-QDs was established under optimal conditions. The fluorescence quenching observed from this method showed a linear relationship with CYP concentration over the range of 0.05­60.0 mg/kg with a correlation coefficient of 0.9838. Good recovery (82.7­92.4%) and a relative standard deviation of less than 10.1% were obtained from fish samples spiked with three levels of CYP. This method also demonstrated a low detection limit of 1.2 µg/kg. The ELISA-like method based on MIP-QDs can be successfully employed to detect residual of CYP in fish samples.

Structure and anti-inflammatory capacity of peptidoglycan from Lactobacillus acidophilus in RAW-264.7 cells.

Probiotic bacteria, especially the Lactobacillus species, can potentially play a significant role in the antiviral and antimicrobial activity of a host's immune system. Lactobacillus acidophilus, an intestinal bacterium, is involved in the intestinal epithelial cell response when a pathogen adheres to a cell. In this study, the structure of peptidoglycan (PGN) isolated from L. acidophilus was determined with the help of HPLC, NMR, FT-IR and MALDI-TOF/TOF MS. The molecular mass of PGN is 875.260 Da. The anti-inflammatory capacity of PGN was evaluated in model RAW 264.7 cells. Epifluorescence microscopy images and western blot analysis provided compelling evidence of PGN's anti-inflammatory capacity on LPS-induced macrophages. A significant decrease in inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) levels when PGN was added up to 200 µg/ml. These data provide new insight into the cellular and molecular mechanisms by which probiotic bacteria can contribute to maintaining good health.

Characterisation of Lactobacillus fermentum SM-7 isolated from koumiss, a potential probiotic bacterium with cholesterol-lowering effects.

BACKGROUND: Lactic acid bacteria (LAB) have been demonstrated to have cholesterol-reducing effects in many studies. RESULTS: Lactobacillus fermentum SM-7 screened from ten LAB strains isolated from koumiss, a fermented milk drink, reduced cholesterol by 66.8%. It also showed acid and bile tolerance as well as antimicrobial activity against pathogenic Escherchia coli and Staphylococcus aureus. Lactobacillus fermentum SM-7 cells assimilated 61.5% and co-precipitated and absorbed 38.5% of the cholesterol in the media. Co-precipitation of cholesterol with cholic acid increased rapidly at pH levels below 6. In vivo experiments using L. fermentum SM-7 on artificially induced hyperlipidaemial ICR mice significantly decreased serum total cholesterol and total triglyceride levels, low-density lipoprotein cholesterol concentrations and atherogenic index (P < 0.01), while serum high-density lipoprotein cholesterol concentrations did not increase significantly (P > 0.05). The body weight and liver weight/body weight ratio of SM-7 groups were lower than those of mice on a high-cholesterol diet that were not given lactobacilli. There was no bacterial translocation in the liver, spleen or kidney of experimental mice. CONCLUSION: The results suggested that L. fermentum SM-7 is a potential probiotic bacterium with cholesterol-lowering effects.

Functional characteristics of Lactobacillus fermentum F1.

In this study, Lactobacillus fermentum (L. fermentum) F1 reduced cholesterol 48.87%. The strain was screened from cattle feces using an API 50 CHL system and the 16S rRNA sequence contrasting method. L. fermentum F1 showed acid and bile tolerance, and antimicrobial activity against pathogenic Escherichia coli and Staphylococcus aureus. L. fermentum F1 deconjugated 0.186 mM of free cholalic acid after it was incubated at 37°C in 0.20% sodium taurocholate (TCA) broth for 24 h. Heat-killed and resting cells of L. fermentum F1 showed small amounts of cholesterol removal (6.85 and 25.19 mg/g, respectively, of dry weight) compared with growing cells (33.21 mg/g of dry weight). The supernatant fluid of the broth contained 50.85% of the total cholesterol, while the washing buffer and cell extracts had 13.53 and 35.39%, respectively. These findings suggest that L. fermentum F1 may remove cholesterol by co-precipitating with deconjugated bile salt, assimilating with cells and by incorporation into cellular membranes. Cholesterol assimilated by cells held 72.0% of the reduced cholesterol, while 21.65% of the reduced cholesterol was coprecipitated with deconjugated bile salt and 5.89% was adsorbed into the surface of the cells.

Protective effect of selenium-enriched Lactobacillus on CCl4-induced liver injury in mice and its possible mechanisms.

AIM: To study the protective effects and mechanisms of Se-enriched lactobacillus on liver injury caused by carbon tetrachloride (CCl4) in mice. METHODS: Seventy-two ICR mice were randomly divided into four groups: normal group, CCl4-induced model group, low Se-enriched lactobacillus treatment group (L-Se group), and high Se-enriched lactobacillus treatment group (H-Se group). During a 3-wk experimental period, the common complete diet was orally provided daily for normal group and model group, and the mice in L-Se and H-Se groups were given a diet with 2 and 4 mg of organoselenium from Se-enriched lactobacillus per kg feed, respectively. From the 2nd wk of experiment, the model group, L-Se group, and H-Se group received abdominal cavity injection of olive oil solution containing 500 mL/L CCl4 (0.07 mL/100 g body mass) to induce liver injury, and the normal group was given olive oil on every other day for over 2 wk. In the first 2 wk post injection with CCl4, mice in each group were killed. The specimens of blood, liver tissue, and macrophages in abdominal cavity fluid were taken. Then the activities of the following liver tissue injury-associated enzymes including glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) as well as malondialdehyde (MDA) content were assayed. Changes of phagocytic rate and phagocytic index in macrophages were observed with Wright-Giemsa stain. Plasma TNF-alpha level was measured by radioimmunoassay. The level of intracellular free Ca2+ ([Ca2+]i) in hepatocytes was detected under a laser scanning confocal microscope. RESULTS: During the entire experimental period, the AST and ALT activities in liver were greatly enhanced by CCl4 and completely blunted by both low and high doses of Se-enriched lactobacillus. The Se-enriched lactobacillus-protected liver homogenate GSH-Px and SOD activities were higher or significantly higher than those in model group and were close to those in normal group. CCl4 significantly increased MDA content in liver homogenates, while administration of Se-enriched lactobacillus prevented MDA elevation. Phagocytic rate and phagocytic index of macro-phages decreased after CCl4 treatment compared to those in normal control, but they were dramatically rescued by Se-enriched lactobacillus, showing a greatly higher phagocytic function compared to model group. CCl4 could significantly elevate plasma TNF-alpha and hepatocyte [Ca2+]i level, which were also obviously prevented by Se-enriched lactobacillus. CONCLUSION: Se-enriched lactobacillus can intervene in CCl4-induced liver injury in mice by enhancing macrophage function activity to keep normal and beneficial effects, elevating antioxidant-enzyme activities and reducing lipid peroxidation reaction, inhibiting excessive release of TNF-alpha, preventing the dramatic elevation of [Ca2+]i in hepatocytes.