Contribution of ultrasound in combination with chlorogenic acid against Salmonella enteritidis under biofilm and planktonic condition.

The antibacterial and antibiofilm mechanisms of ultrasound combined with chlorogenic acid treatment for Salmonella enteritidis under biofilm and planktonic condition were investigated. S. enteritidis under biofilm and planktonic cells were treated with ultrasound, chlorogenic acid, and their combination for 5, 10, 20, 30, and 60 min. Results showed that the combined treatments exhibited synergistic effects that inactivated the S. enteritidis biofilm and planktonic cells. The nucleic acids and ATP leakage and CLSM imagines showed that the combining chlorogenic acid and ultrasound treatment significantly increased the permeability of the S. enteritidis cell membrane. SEM indicated that the combining chlorogenic acid and ultrasound treatment quickly destroyed the integrity of the S. enteritidis cell membrane, and the activity of respiratory chain dehydrogenase sharply decreased. Additionally, the amounts of polysaccharides in the S. enteritidis biofilms significantly decreased after the combined treatments. Hence, the combining chlorogenic acid and ultrasound treatment have potential applications in food preservation.

Novel sample treatment method for the determination of free (E)-4-hydroxy-2-nonenal in meat products by liquid chromatography/tandem mass spectrometry using 4-hydroxy-2-nonenal-d3 as internal standard.

RATIONALE: (E)-4-Hydroxy-2-nonenal (HNE) is a reactive secondary product of lipid oxidation with biological significance. The analysis of HNE is a challenge due to its volatility and high activity. Developing sample preparation and analytical tools for the determination of free HNE is crucial for better understanding the actual level of free HNE in meat products. METHODS: Liquid nitrogen freezing, subzero-temperature extraction and derivatization were employed for meat sample treatment. Liquid chromatography/tandem mass spectrometry with electrospray ionization in negative ion mode was used for the determination of free HNE after isotope-coded derivatization. RESULTS: High repeatability and good recoveries with a limit of quantification as low as 0.25 pmol/g were found. Nineteen out of 24 samples, including chilled/processed meat products and meat-based instant foods, were found to contain free HNE with a range of 0.014-1.160 nmol/g. CONCLUSIONS: The proposed method showed satisfactory reliability, sensitivity and accuracy. We believe that such a sample preparation strategy will provide a powerful tool for better understanding the actual level of free HNE in meat products.

Inhibition of biofilm formation and exopolysaccharide synthesis of Enterococcus faecalis by phenyllactic acid.

This study aimed to evaluate the inhibitory activity of phenyllactic acid (PLA) against the biofilm formation of Enterococcus faecalis and to explore its potential molecular mechanism. The MIC value of PLA that inhibited the growth of E. faecalis R612-Z1 in BHI broth was 5 mg/mL. PLAs at subinhibitory concentrations of 1.25 and 2.50 mg/mL were found to inhibit biofilm formation by a crystal violet staining assay. The cell swimming and swarming motilities of E. faecalis were reduced in the presence of PLA. An apparent decrease in the thickness of PLA-treated biofilms was observed through confocal laser scanning microscopy analysis. The exopolysaccharide production in E. faecalis biofilms was inhibited by EPS quantification assay and scanning electron microscopy (SEM). qRT-PCR analyses showed that PLA down-regulated the transcription of Ebp pili genes (ebpABC) and Epa polysaccharide genes (epaABE). PLA inhibited the biofilm formation by interfering with cell mobility and EPS production of E. faecalis. In addition, PLA at concentrations of 10.0 mg/mL can effectively control the bacterial cells in a three-day-old mature biofilm of E. faecalis grown on 24-well flat-bottom polystyrene plates and stainless-steel surfaces. Thus, PLA is potentially an effective agent to control E. faecalis biofilms.

Antibacterial activity and action mode of chlorogenic acid against Salmonella Enteritidis, a foodborne pathogen in chilled fresh chicken.

The study evaluated the antibacterial activity of chlorogenic acid (CA) against Salmonella Enteritidis S1, a foodborne pathogen in chilled fresh chicken. Its minimum inhibitory concentration for S. Enteritidis S1 was 2 mM. 1 MIC CA treatment reduced the viable count of S. Enteritidis S1 by 3 log cfu/g in chilled fresh chicken. Scanning electron microscopy examination indicated that CA induced the cell envelope damage of S. Enteritidis S1. Following this, 1-N-Phenylnaphthylamine assay and LPS content analysis indicated that CA induced the permeability of outer membrane (OM). Confocal laser scanning microscopy examination further demonstrated that CA acted on the inner membrane (IM). To support this, the release of intracellular protein and ATP after CA treatment was also observed. CA also suppressed the activities of malate dehydrogenase and succinate dehydrogenase, two main metabolic enzymes in TCA cycle and electron transport chain. Thus, damage of intracelluar and outer membranes as well as disruption of cell metabolism resulted in cell death eventually. The finding suggested that CA has the potential to be developed as a preservative to control S. Enteritidis associated foodborne diseases.

Crystal structures of the kinase domain of PpkA, a key regulatory component of T6SS, reveal a general inhibitory mechanism.

The type VI secretion system (T6SS) is a versatile and widespread export system found in many Gram-negative bacteria that delivers effector proteins into target cells. The functions of T6SSs are tightly regulated by diverse mechanisms at multiple levels, including post-translational modification through threonine phosphorylation via the Ser/Thr protein kinase (STPK) PpkA. Here, we identified that PpkA is essential for T6SS secretion in Serratia marcescens since its deletion eliminated the secretion of haemolysin co-regulated protein, while the periplasmic and transmembrane portion of PpkA was found to be disposable for T6SS secretion. We further determined the crystal structure of the kinase domain of PpkA (PpkA-294). The structure of PpkA-294 was determined in its apo form to a 1.6 Šresolution as well as in complex with ATP to a 1.41 Šresolution and with an ATP analogue AMP-PCP to a 1.45 Šresolution. The residues in the activation loop of PpkA-294 were fully determined, and the N-terminus of the loop was folded into an unprecedented inhibitory helix, revealing that the PpkA kinase domain was in an auto-inhibitory state. The ternary MgATP-PpkA-294 complex was also inactive with nucleotide ribose and phosphates in unexpected and unproductive conformations. The αC-helix in the inactive PpkA-294 adopted a conformation towards the active site but with the conserved glutamate in the helix rotated away, which we suggest to be a general conformation for all STPK kinases in the inactive form. Structural comparison of PpkA with its eukaryotic homologues reinforced the universal regulation mechanism of protein kinases.

The Antibacterial Activity and Mechanism of Chlorogenic Acid Against Foodborne Pathogen Pseudomonas aeruginosa.

Chlorogenic acid (CA), an ester of caffeic acid, is a major phenolic compound in herbs. The antimicrobial activity of CA against Pseudomonas aeruginosa P1, a foodborne pathogen, was investigated in this study. To understand how CA injured target cells, the influence of CA on cell morphology was assessed. A sunken cell surface and detachment of outer membrane components in P. aeruginosa P1 were observed after being treated by CA. Following this, the intracellular membrane permeability and the content of lipopolysaccharide (LPS), a main component of outer membrane, were determined. The release of intracellular protein and ATP from P. aeruginosa P1 indicated that CA increased intracellular membrane permeability and resulted in the leakage of intracellular materials. The uptake of propidium iodide, a compromised cell membrane nucleic acid stain, further demonstrated that CA acted on the intracellular membrane. CA resulted in the decrease of LPS contents of P. aeruginosa P1, which supported the detachment of outer membrane. CA also downregulated the expression of major genes in LPS biosynthesis, suggesting that CA may inhibit intracellular metabolism of P. aeruginosa P1 cells. Thus, CA increased the intracellular membrane permeability, induced the exfoliation of outer membrane, and disturbed the intracellular metabolism. Damage of intracellular and outer membranes as well as disruption of cell metabolism resulted in cell death eventually. The finding suggested that CA has the potential to be developed as a preservative to control P. aeruginosa-associated foodborne diseases.

The influence of ultrasound and adenosine 5'-monophosphate marination on tenderness and structure of myofibrillar proteins of beef

Objective: The aim was to investigate the influence of ultrasound and adenosine 5'-monophosphate (AMP) marination (UAMP) on tenderness and structure of myofibrillar proteins of beef. Methods: Five groups, the untreated meat (Control), deionized water marination (DW), ultrasound followed by DW (UDW), AMP marination (AMP), and ultrasound followed by AMP (UAMP) were studied. Myofibrillar fragmentation, cooking loss, shear force, thermograms, histological observation of meats and myofibrillar proteins properties were investigated in these different treatments. Results: The results showed that UAMP significantly increased MFI from 152 (control), 231 (AMP) and 307 (UDW) to 355 (P < 0.05), respectively. The lowest cooking loss, shear force and peak denaturation temperature were observed in UAMP. In histological observation, UDW and UAMP had more fragmented muscular bundles than the others. Furthermore, a drastic increase in α-helix and decrease in ß-sheet of myofibrillar proteins was observed in UAMP, implying the disaggregation of protein samples. The synchronous fluorescence spectra of myofibrillar proteins in UAMP suggested the combination of ultrasound and AMP could accelerate the unfolding molecular structure and destroying hydrophobic interactions. The results of circular dichroism and synchronous fluorescence spectra for myofibrillar proteins coincided with the microstructures of beef. Conclusion: The results indicate that ultrasound combined with AMP improved meat tenderness not only by disruption in muscle integrity, increasing water retention, but also altering their spatial structure of myofibrillar proteins.

Interaction of Hsp90 with phospholipid model membranes.

Heat shock protein 90 (Hsp90) is an essential molecular chaperone with versatile functions in cell homeostatic control under both normal and stress conditions. Hsp90 has been found to be expressed on the cell surface, but the mechanism of Hsp90 association to the membrane remains obscure. In this study, the direct interaction of Hsp90 and phospholipid vesicles was characterized, and the role of Hsp90 on membrane physical state was explored. Using surface plasmon resonance (SPR), we observed a strong interaction between Hsp90 and different compositions of lipid. Hsp90 had a preference to bind with more unsaturated phospholipid species and the affinity was higher with negatively charged lipids than zwitterionic lipids. Increasing the mole fraction of cholesterol in the phospholipid led to a decrease of binding affinity to Hsp90. Circular dichroism (CD) spectroscopy of Hsp90 in PC membranes showed more α-helix structure than in aqueous buffer. The differential scanning calorimeter (DSC) and fluorescence polarization results showed Hsp90 could affect the transition temperature and fluidity of the bilayer. We postulate from these results that the association between Hsp90 and membranes may involve both electrostatic and hydrophobic force, and constitute a possible mechanism that modulates membrane lipid order during thermal fluctuations.

Class III bacteriocin Helveticin-M causes sublethal damage on target cells through impairment of cell wall and membrane.

Helveticin-M, a novel Class III bacteriocin produced by Lactobacillus crispatus exhibited an antimicrobial activity against Staphylococcus aureus, S. saprophyticus, and Enterobacter cloacae. To understand how Helveticin-M injured target cells, Helveticin-M was cloned and heterologously expressed in Escherichia coli. Subsequently, the cell wall organization and cell membrane integrity of target cells were determined. The mechanism of cellular damage differed according to bacterial species. Based on morphology analysis, Helveticin-M disrupted the cell wall of Gram-positive bacteria and disorganized the outer membrane of Gram-negative bacteria, therefore, altering surface structure. Helveticin-M also disrupted the inner membrane, as confirmed by leakage of intracellular ATP from cells and depolarization of membrane potential of target bacteria. Based on cell population analysis, Helveticin-M treatment caused the increase of cell membrane permeability, but the cytosolic enzymes were not influenced, indicating that it was the sublethal injury. Therefore, the mode of Helveticin-M action is bacteriostatic rather than bactericidal.

Antimicrobial Activity of Phenyllactic Acid Against Enterococcus faecalis and Its Effect on Cell Membrane.

3-Phenyllactic acid (PLA) was reported to have an effective antimicrobial activity. This study evaluated the antimicrobial activity of PLA against foodborne Enterococcus faecalis and its effect on cell membrane. The minimum concentration of PLA to inactivate E. faecalis in brain heart infusion broth was 5 mg/mL. PLA solutions of 5 and 10 mg/mL can inactivate E. faecalis population ≥6 log CFU/mL within 60 and 30 min, respectively. The cell membranes of most E. faecalis cells were damaged after PLA treatment according to the images of scanning electron microscopy and transmission electron microscopy. The differences in the regions of cell membrane protein, fatty acid, and polysaccharide were revealed by Fourier transform infrared spectroscopy, which further indicated cell membrane damages. The cell membrane permeability was increased when the concentration of PLA treatment was increased in the membrane permeability assays. Finally, almost all bacterial cells were damaged after treatment with 10 mg/mL PLA for 30 min, further confirmed by flow cytometry analysis. This study concluded that PLA is effective in inactivating E. faecalis cells through the leakage of intracellular components caused by cell membrane damage.

Construction of Chitosan-Zn-Based Electrochemical Biosensing Platform for Rapid and Accurate Assay of Actin.

This work reports a study on the development of a sensitive immunosensor for the assay of actin, which is fabricated using sensing material chitosan-Zn nanoparticles (NPs) and anti-actin modified on glassy carbon electrode respectively. The prepared materials were characterized using transmission electron microscope (TEM), fourier transform infrared spectra (FTIR), X-ray diffraction (XRD) spectra, and circular dichroism (CD) techniques. Meanwhile, the electrochemical properties were studied by linear sweep voltammetric (LSV), electrochemical impedance spectra (EIS), and differential pulse voltammetry (DPV). According to the experiments, under the optimum conditions, the linear fitting equation was I (µA) = -17.31 + 78.97c (R² = 0.9948). The linear range was from 0.0001 to 0.1 mg/mL and the detection limit (LOD, S/N = 3) was 21.52 ng/mL. The interference studies were also performed for checking the sensors' selectivity to actin. With better properties of the chitosan-Zn NPs, the modified electrode is considered as a better candidate than Western blot or immunohistochemical method for real-time usability. The detection limit reported is the lowest till date and this method provides a new approach for quality evaluation.

Differential expression of heat shock protein 90, 70, 60 in chicken muscles postmortem and its relationship with meat quality.

OBJECTIVE: The aim of this study was to investigate the expression of heat shock protein (HSP) 90, 70, and 60 in chicken muscles and their possible relationship with quality traits of meat. METHODS: The breast muscles from one hundred broiler chickens were analyzed for drip loss and other quality parameters, and the levels of heat shock protein (HSP) 90, 70, and 60 were determined by immunoblots. RESULTS: Based on the data, chicken breast muscles were segregated into low (drip loss≤5%), intermediate (5%0.05). CONCLUSION: Results of this study suggests that higher levels of HSP90 and HSP60 may be advantageous for maintenance of cell function and reduction of water loss, and they could act as potential indicator for better water holding capacity of meat.

The effect of adenosine 5'-monophosphate (AMP) on tenderness, microstructure and chemical-physical index of duck breast meat.

BACKGROUND: Adenosine 5'-monophosphate (AMP) is often used in meat and poultry soups as a flavor enhancer (flavor modifier), or as food additives for specific nutritional purposes. Our previous research as well as evidence from others showed that actomyosin could be dissociated into myosin and actin by AMP in extracted muscle solution. However, there is no report available on the application of AMP to dissociate actomyosin and to improve meat tenderness. The objectives of this study were to evaluate the effect of AMP on duck meat tenderness and other quality traits and to explore the mechanism of the action of AMP on meat tenderness. RESULTS: Duck breast muscle was treated with 0, 10, 20, 30, 40 mmol L(-1) AMP at 5 °C for 10 h and examined for shear force, microstructure, actomyosin dissociation, myofibril fragmentation index (MFI), pH, water content, cooking loss, CIE* color (L*, a*, b*), inosine monophosphate (IMP) and free amino acid (FAA) contents. Results showed that shear force, cooking loss, L* and b* of the muscles significantly decreased after AMP treatment (P < 0.05); actomyosin dissociation, MFI, pH, water content, fiber diameter, sarcomere length, IMP and ammonia significantly increased (P < 0.05); no significant change in a* or other FAA content was observed (P > 0.05), and muscle shrinkage in transverse and longitudinal directions were restrained after AMP treatment. CONCLUSION: The results suggest that AMP could notably improve meat tenderness, and this effect was probably mainly through increasing muscle pH, promoting actomyosin dissociation and disrupting the Z-line; meanwhile, the conversion of AMP to IMP may contribute to the flavor of meat.

Novel L-lactic acid biosensors based on conducting polypyrrole-block copolymer nanoparticles.

The development of advanced nanomaterials for the highly efficient electrical detection of biological species has attracted great attention. Here, novel polypyrrole-Pluronic F127 nanoparticles (PPy-F127 NPs) with conducting and biocompatibility properties were synthesized and used to construct a L-lactic acid biosensor that could be applied in biochemical assays. The PPy-F127 NPs were characterized by transmission electron microscopy (TEM), elemental analysis and UV-vis spectroscopy. Lactate oxidase (LOx) structure variation on the PPy-F127 NPs was investigated by circular dichroism (CD). The cyclic voltammetric results indicated that LOx immobilized on the PPy-F127 NPs exhibited direct electron transfer reaction with a formal potential value (E(0)') of 0.154 V vs. SCE. Moreover, the biosensor had good electrocatalytic activity toward L-lactic acid with a wide linear range (0.015-37.5 mM) and a low detection limit of 0.0088 mM. The regression equation was I (µA) = 0.02353c (mM) + 1.4135 (R(2) = 0.9939). The L-lactic acid biosensor had a good anti-interference property towards uric acid (UA), ascorbic acid (AA), glucose and cysteine. The idea and method provide a promising platform for the rapid development of biosensors that can be used in the detection of biological species.

Analysis of abietic acid and dehydroabietic acid residues in raw ducks and cooked ducks.

Rosin was once widely used for removal of duck feathers in China and is still being used secretly in some poultry processing enterprises. Abietic acid (AA) and dehydroabietic acid (DHAA) are the major compounds of rosin. In the present study, 90 duck samples were collected for investigation of AA and DHAA residues. Abietic acid and DHAA were simultaneously detected in 13 out 40 raw ducks, 8 out of 26 water-boiled salted ducks, and 7 out of 24 roasted ducks, respectively. In positive samples, averages of AA were significantly higher than those of DHAA in positive samples of the 3 types of ducks (P < 0.05). Averages of AA and DHAA in positive raw ducks were significantly higher than those in positive roasted ducks (P < 0.05). The results indicated that almost one-third of raw ducks were defeathered by means of rosin-containing defeathering agent, and cooking processes could reduce the AA and DHAA residues to some extent, but could not eliminate them completely.

An innovative Pickering W/O/W nanoemulsion co-encapsulating hydrophilic lysozyme and hydrophobic Perilla leaf oil for extending shelf life of fish products.

A Pickering water-in-oil-in-water nanoemulsion co-encapsulating lysozyme (LYS) and Perilla leaf oil (PO) was prepared using whey protein isolate-tannin acid conjugated nanoparticles (WPI-TA NPs) as emulsifiers, called LYS-PO-NE, and subsequently analyzed. The nano size and multiple phases was confirmed based on the results of confocal laser scanning microscope, scanning electron microscope, and droplet size analysis. LYS-PO-NE had high encapsulation efficiencies of 89.36 % (PO) and 43.91 % (LYS) and both could be released at a slow and continuous rate. The PO addition increased the droplet size, and the LYS addition delayed the release of PO. LYS-PO-NE also showed good storage, pH, thermal, and salt stability, and an effective combined bactericidal activity of LYS and PO against spoilage bacteria. Furthermore, the results of chilled salmon storage experiments indicated that LYS-PO-NE could extend the shelf life of chilled salmon to at least 6 days, demonstrating the potential in the shelf life for fish products.

Mushroom alcohol(1-octen-3-ol)and other 7 aroma compounds selected from Chinese dry-cured hams can enhance saltiness perception.

Reducing NaCl content in food while maintaining acceptability poses a significant challenge. Odor-induced saltiness enhancement (OISE) emerges as a promising solution. This study utilized gas chromatography-olfactory (GC-O) in conjunction with gas chromatography-mass spectrometry (GC-MS) to identify 37 key volatile compounds in three representative Chinese dry-cured hams. These compounds had an odor activity value (OAV) of ≥1 or a modification frequency (MF) of ≥30%. Subsequently, quantitative descriptive analysis (QDA) identified eight odorants associated with saltiness. These included 1-octen-3-ol, nonanal, heptanal, 2-methylbutanal, 3-methyl-butanal, benzaldehyde, octanal, and 2,6-dimethylpyrazine. Remarkably, these odorants significantly intensified saltiness (P < 0.05) when added to a low-concentration NaCl solution (0.3%), compared to zero or high concentrations (0.75% and 0.8%). As a result, traditional Chinese salty meat products offer a promising source of odorants for enhancing saltiness, compensating for reduced NaCl content through OISE.

Engineering Escherichia coli for cost-effective production of medium-chain fatty acids from soy whey using an optimized galactose-based autoinduction system.

Medium-chain fatty acids (MCFAs) are essential chemical feedstocks. Microbial production of MCFAs offers an attractive alternative to conventional methods, but the costly media and external inducers limit its practical application. To address this issue and make MCFA production more cost-effective, an E.coli platform was developed using soy whey as a medium and galactose as an autoinducer. We first designed an efficient, stringent, homogeneous, and robust galactose-based autoinduction system for the expression of pathway enzymes by rationally engineering the promoter of the galactose-proton symporter (GalP). Subsequently, the intracellular acetyl-CoA availability and NADH regeneration were enhanced to improve the reversal of the ß-oxidation cycle. The resulting strain yielded 8.20 g/L and 16.42 g/L MCFA in pH-controlled batch fermentation and fed-batch fermentation with glucose added using soy whey as medium, respectively. This study provided a cost-effective and promising platform for MCFA production, as well as future strain development for other value-added chemicals production.

Three-mode ratiometric biosensor based on integrated DNA-driven magnetic beads for Clostridium perfringens detection.

A three-mode biosensor incorporated ratiometric (electrochemical/colorimetric, electrochemical/photothermal) into its design was constructed using DNA-driven magnetic beads (DMBs) as a bridge to detect C. perfringens. It further enhances the accuracy of detection results while maintaining compatibility with applications in multiple scenarios. Briefly, the G-quadruplex was combined with aptamer and immobilized onto magnetic beads through amide-bond, resulting in the integration of DMBs. The DMBs and supernatant were separated by magnetic separation when the target was present. Subsequently, the DMBs were utilized to construct the electrochemical biosensor, whereas the supernatant was used to construct colorimetric and photothermal biosensors. The limits of detection the ratiometric biosensor were ultimately reduced to 0.26 and 0.27 lg CFU g-1, respectively, in comparison to the single three-mode biosensor. Moreover, this biosensor had been applied in real-sample assays successfully. The establishment of this platform provides a new method for detecting pathogens in the fields of food safety and environmental monitoring.

A study on the catalytic domain of pork phospholipase A2: Enzymatic properties and hydrolysis characteristics of phosphatidylcholine and its hydroperoxide.

Endogenous phospholipase A2 (PLA2) plays an important role in phospholipids degradation during cured meat products manufacturing. The present study was undertaken to reveal more information about the endogenous PLA2 in muscles and its role in degradation of intramuscular phospholipids. With the catalytic domain of pork calcium-independent PLA2 (iPLA2cd), impacts of physic-chemical factors on the activity were investigated and substrate specificity of the enzyme were tested respectively. The optimum temperature and pH of pork iPLA2cd were 40 °C and 7.5, respectively. The iPLA2cd could be stimulated by adequate contents of NaCl and ATP, and inhibited by CaCl2 and NaNO2. For native phospholipids, the iPLA2cd was of a little higher affinity towards phosphatidylcholine (PC) than phosphatidylethanolamine (PE), phosphoserine (PS) and phosphatidylinositol (PI). The iPLA2cd could preferentially hydrolyze peroxidized PC over the native PC. The results would help better understand the degradation of phospholipids and the role played by endogenous enzymes during meat products manufacturing.