A Small RNA, SaaS, Promotes Salmonella Pathogenicity by Regulating Invasion, Intracellular Growth, and Virulence Factors.

Salmonella enterica serovar Enteritidis is a common foodborne pathogen that infects both humans and animals. The S. Enteritidis virulence regulation network remains largely incomplete, and knowledge regarding the specific virulence phenotype of small RNAs (sRNAs) is limited. Here, we investigated the role of a previously identified sRNA, Salmonella adhesive-associated sRNA (SaaS), in the virulence phenotype of S. Enteritidis by constructing mutant (ΔsaaS) and complemented (ΔsaaS/psaaS) strains. SaaS did not affect S. Enteritidis; it was activated in the simulated intestinal environment (SIE), regulating the expression of virulence target genes. We discovered that it directly binds ssaV mRNA. Caco-2 and RAW 264.7 cell assays revealed that SaaS promoted S. Enteritidis invasion and damage to epithelial cells while suppressing macrophage overgrowth and destruction. Furthermore, a BALB/c mouse model demonstrated that the deletion of SaaS significantly reduced mortality and attenuated the deterioration of pathophysiology, bacterial dissemination into systemic circulation, and systemic inflammation. Our findings indicate that SaaS is required for S. Enteritidis virulence and further highlight its biological role in bacterial pathogenesis. IMPORTANCE Salmonella is a zoonotic pathogen with high virulence worldwide, and sRNAs have recently been discovered to play important roles. We explored the biological characteristics of the sRNA SaaS and developed two cell infection models and a mouse infection model. SaaS is an SIE-responsive sRNA that regulates the expression of virulence-targeted genes. Additionally, it differentially mediates invasion and intracellular growth for survival and infection of the epithelium and macrophages. We further found that SaaS enhanced bacterial virulence by promoting lethality, colonization, and inflammatory response. These findings provide a better understanding of the critical role of sRNA in bacterial virulence.

A new picrotoxane-type sesquiterpene from Dendrobium nobile Lindl.

A picrotoxane-type sesquiterpene, dendroterpene E (1), together with five benzene derivatives (2-6), were isolated from the stems of Dendrobium nobile Lindl. Their structures were elucidated by spectroscopic analysis and X-ray diffraction analysis. Compound 1 was a new picrotoxane-type sesquiterpene with a C-9/C-1/O/C-11 oxetane ring, which was first encountered in this type of compounds. Compounds 1-3 exhibited inhibitory activities against α-glycosidase.

Enhanced flavor strength of broth prepared from chicken following short-term frozen storage.

This study investigated the effect of frozen storage periods (0, 2, 4, 6, or 8 weeks) of raw meat and stewing on the flavor of chicken broth. With the increased storage duration of frozen raw material, the contents of the free amino acids, nucleotides and mineral elements in the broth decreased significantly, especially within the first 4 weeks, and then increased significantly. Meanwhile, the volatile compounds showed the reverse trend. The results from the E-nose, E-tongue and sensory evaluation indicated a progressive difference in overall flavor profiles between the samples. The sensory scores of the meaty and fatty traits reached a maximum as raw chicken meat was stored for 4 weeks at -18 °C, which should be related to the increased contents of aldehydes and 2-pentyl furan. Overall, the limited storage duration of frozen raw meat can enhance the flavor of chicken broth.

Structural basis for high-pressure improvement in depolymerization of interfacial protein from RFRS meat batters in relation to their solubility.

High-pressure processing (HPP) can modify the construction of interfacial proteins (IPs) to improve the properties of reduced-fat and reduced-salt (RFRS) meat batters. In this study, the relationship between the construction of IPs and their solubility at fat droplet/water interface in RFRS meat batters with HPP treatments was investigated. When 200 MPa for 2 min was applied, the IPs exhibited the highest solubility due to a high concentration of absorbed myosin with the content of random coil 65.62%, but the particle diameter was in reverse. The microscopy revealed the depolymerization of IPs occurred at low pressure, while macromolecular aggregates were produced as the cross-linking of IPs to some degree at pressure ≥ 200 MPa. This phenomenon was supported by the result of SDS-PAGE and the sulfhydryl of IPs. In conclusion, the HPP induced solubility alteration of IPs was achieved by modifying their construction through adjusting the secondary structures and regulating bond interactions.

Gelatin enhances the flavor of chicken broth: A perspective on the ability of emulsions to bind volatile compounds.

The mechanism of flavor retention in chicken broth from the perspective of gelatin affecting the flavor-binding ability of emulsions was investigated. Results showed that fat was the important reservoir for aroma compounds in the emulsion. The particle sizes of emulsions significantly decreased with prolonged stewing time of gelatin, which was consistent with the results from confocal laser scanning microscopy and interfacial tension. The ability of pre-heated gelatin emulsions to bind volatile compounds with higher hydrophobicity was enhanced. When 0.1% gelatin was added into chicken broth, the total amount of the main volatile compounds (OAV > 1) increased from 458.83 ng/g to 1218.42 ng/g. In summary, the pre-heated gelatin increased the interfacial pressure of water/oil interface, resulting in a smaller particle size of oil droplets, and these further increased the binding area between oil droplets and flavor compounds. This appears to be the mechanism that gelatin enhances the flavor intensity of chicken broth.

Insights into the evolution of myosin light chain isoforms and its effect on sensory defects of dry-cured ham.

To better understand the contribution of myosin light chain (MLC) isoforms to sensory defects in Jinhua ham, dipeptidyl peptidase (DPP) activities, peptide fragments, cleavage sites and the potential of DPP to develop sensory defects of dry-cured ham were evaluated and discussed in normal and defective hams. Higher residual activities of DPP I were found in defective ham compared with normal ham; approximate 3-fold peptide fragments were identified in defective ham than in normal ham. These regions of positions 11-35 and 116-141 in MLC 1, 13-53 and 139-156 in MLC 2, and 18-50 in MLC 3 contributed to the intense generation of peptide fragments in defective ham. PLS-DA further revealed DPP I showing intense response to degrade peptides. Cleavage sites including Glu-128, Tyr-132 and Glu-133 were responsible for the intense release of dipeptides in defective ham. These cleavages could play key role in discriminating taste attributes between defective and normal hams.

Protein degradation and peptide formation with antioxidant activity in pork protein extracts inoculated with Lactobacillus plantarum and Staphylococcus simulans.

This study focused on sarcoplasmic and myofibrillar protein degradation and the formation of peptides with antioxidant activity by mixed starters (Lactobacillus plantarum CD101 and Staphylococcus simulans NJ201). Gel electrophoresis indicated that the mixed starters can hydrolyze both sarcoplasmic and myofibrillar proteins, and the concentration of peptides increased (P < .05). Compared with the control group, using mixed starters led to a significant increase (P < .05) in the DPPH radical scavenging activity, Fe2+ chelating activity, and ABTS radical scavenging activity of sarcoplasmic proteins, but demonstrated no significant difference in myofibrillar proteins. Two hydrophobic fractions (C2, C5) separated by RP-HPLC in the inoculation groups with sarcoplasmic proteins showed high DPPH radical scavenging activity (66.60%, 60.50%). Eighteen peptides were identified by LC-MS/MS, which mainly arose from triosephosphate isomerase, creatine kinase M-type, and glyceraldehyde-3-phosphate dehydrogenase. Hydrophobic amino acids accounted for a large proportion. Our results indicate that mixed starters affect proteolytic characterization and contribute to the formation of peptides with antioxidant capacity in sarcoplasmic proteins.

Evaluating the effect of protein modifications and water distribution on bitterness and adhesiveness of Jinhua ham.

Defects with textures and flavors are a common problem, causing many economic losses in the dry-cured ham industry. To obtain a better understanding of the defects of dry-cured ham, texture, protein denaturation, protein structure, and water distribution of normal and defective hams were investigated. Compared with normal ham, more than 1.5-fold values in adhesiveness and bitterness, and less than 0.8-fold values in hardness were found in defective ham. The intense denaturation of sarcoplasmic proteins and actin, and the dramatic transformation of α-helix to ß-sheet were the key modification of proteins; a high proportion (92.39%) of immobile water contributed to the excessive softness and adhesiveness of defective hams. Furthermore, high denaturation of proteins could accelerate the degradation of proteins, which further developed the bitterness and adhesiveness of defective hams. Partial least squares regression demonstrated that the discrepancies in protein denaturation, protein structure and water distribution were related with bitterness and adhesiveness of Jinhua ham.

Label-free proteomics reveals the mechanism of bitterness and adhesiveness in Jinhua ham.

To obtain better understanding of the formation mechanisms of bitterness and adhesiveness, protease activities, proteolysis index and protein degradation were investigated among raw, normal and defective hams. Normal and defective hams both showed a decrease in cathepsin B and B + L activities compared with raw ham, while higher residual activities were observed in defective ham. Approximate 1.2-fold values of proteolysis index were observed in defective ham than in normal ham, indicating that cathepsin B and B + L activities were key contributors in degrading muscle proteins of dry-cured ham. 322 proteins were identified by label-free proteomics, and 49 down-regulated proteins were found in the comparison between normal and defective hams. Creatine kinase, myosin, α-actinin and troponin-T showed the most intense response to bitterness and adhesiveness of dry-cured ham, confirmed by partial least squares regression analysis. Myosin could be a suitable biomarker to monitor bitterness and adhesiveness of dry-cured ham.

Morphophysiological responses of detached and adhered biofilms of Pseudomonas fluorescens to acidic electrolyzed water.

Pseudomonas spp. have emerged as the main spoilage bacteria, with many strains easily forming biofilms on food-contact surfaces and causing cross-contamination. The efficacy of disinfectants against bacteria is usually tested with planktonic cells; however, the disinfection tolerance of biofilms, especially detached biofilms, remains unknown. Here, we investigated the tolerance responses of detached and adhered biofilms of Pseudomonas fluorescens to acidic electrolyzed water (AEW) by determining tolerance responses by plate counting, comparing them using a Weibull model, and verifying changes in bacterial morphology by scanning electron microscopy. The experimental data and the responses calculated using Weibull a (scale) and b (shape) parameters agreed well (R2 values: 0.974-0.999), and we found that AEW exhibited effective antimicrobial activity against P. fluorescens, with adhered biofilms were more resistant than detached biofilms and planktonic cells. Additionally, AEW increased the bacterial membrane permeability and decreased the membrane potential, intracellular ATP concentrations, and intracellular pH while also triggering the disruption of extracellular polymeric substances. These results demonstrated that the morphophysiological responses of detached and adhered biofilms differed significantly and provided information on disinfectant-resistance strategies potentially beneficial to the development of novel disinfection approaches.

The effects of insoluble dietary fiber on myofibrillar protein gelation: Microstructure and molecular conformations.

The effects of insoluble dietary fiber (IDF) on heat-induced gelation properties, such as microstructural changes and the molecular conformation of myofibrillar proteins (MP), were analyzed by image analyses and Raman spectroscopy. Scanning electron microscopy revealed that pure MP gelation contained a loose and dispersed network with interconnected water channels. With IDF addition, the lacunarity and the particle size of water pores both significantly decreased (P < 0.05) and fractal dimension significantly increased (P < 0.05), which indicated the formation of a homogenous and compact three-dimensional network. Through Raman spectra, IDF addition resulted in modification of amide I and III regions, by a significant decrease in α-helix content, accompanied by an increase of ß-sheets, ß-turns, and random coil content (P < 0.05); the intensity of CU+005CH stretching vibrations bending vibrations bands were significantly decreased (P < 0.05). Principal component analyses showed significant correlations between textual property, microstructure and molecular conformations. Cluster analyses indicated IDF improved the functionality of MP gel by 1): IDF stable the moisture phase 2) "concentrated" MP promoted structural integrity and compact gelation.

Identification and characterization of the proteins in broth of stewed traditional Chinese yellow-feathered chickens.

Soups and broths are popular in the world due to their nutrition and flavor, and flavor compounds tend to be bound by the proteins in the soups and broth, influencing the flavor perception. Thus, identification of the major proteins in meat-based broth may present a basis for understanding protein adsorption of flavor compounds. The present study aimed to identify the major proteins in traditional Chinese chicken broth and to describe the structural changes of proteins during stewing (1, 2, or 3 h). As stewing time increased, protein content in the broth significantly increased. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) indicated that the macro-molecule proteins (>10 kDa) in the broth were mainly gelatin and actin and that the micro-molecule proteins fractions (<10 kDa) increased substantially. The gelatin had an ordered structure even after 3 h of stewing, as seen by circular dichroism (CD) spectroscopy. The presence of reactive sulfhydryl groups increased remarkably with stewing time. The surface hydrophobicity of the proteins significantly increased within 2 h then deceased slightly after 3 h. The intermolecular crosslinks, as indicated by dispersion index, increased remarkably, consistent with the result of atomic force microscopy (AFM), which together suggested that protein aggregation increased during stewing. These findings suggested that gelatin was the structural protein in the broth system and that intermolecular crosslinks functioned to maintain the broth system.

Use of an isoelectric solubilization/precipitation process to modify the functional properties of PSE (pale, soft, exudative)-like chicken meat protein: A mechanistic approach.

The functionality of pale, soft, exudative (PSE)-like chicken protein was improved by isoelectric solubilization/precipitation (ISP) treatment. PSE-like chicken proteins were solubilized at an acidic pH 3.5 or an alkaline pH 11.0, followed by precipitating at pH 5.5 and 6.2. PSE-like meat paste was treated as control (CON). Precipitated at pH 6.2 led to a more elastic gel than at pH 5.5. Water distribution of ISP-isolated protein was affected by precipitation pH. More tryptophan residues exposed and -SH was partially oxidized to disulfide bond after ISP treatment, which led to large aggregates formation and higher viscosity of ISP isolated proteins than of CON. Absolute zeta potential of alkali-treated protein was higher than other counterparts, indicating stronger electric repulsion. ISP treatments could convert α-helix structure to relatively irregular structures. Overall, solubilizing at pH 11.0, combined with a precipitation pH 6.2 ISP treatment offers a potential for enhanced functionality of PSE-like chicken protein.

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.

Improved gel functionality of myofibrillar proteins incorporation with sugarcane dietary fiber.

The effects of sugarcane dietary fiber (SDF) on the gelation properties of porcine myofibrillar proteins (MP) were studied to understand its mechanism of action in improving gel functionality. Rheological tests on all composite gels (MP with SDF) showed the visco-elastic nature of MP, but the G' significantly increased with contents of SDF and with particle size (P<0.05). However, the δ exhibited the opposite effect. Light microscopy suggested that SDF affected moisture distribution in the gel by drawing water from MP and homogenously embedded in gelation. It is proposed that during the heating the more concentrated MP promoted the unfolding of MP chains and facilitated the formation of ß-sheet instead of α-helices, leading to a compact and homogenous three-dimensioned network. These results indicated that the SDF changed the water distribution and resulted in the enhanced gelation which reacted to firmly bind SDF and form a synergistic interaction system.

Characterization of Extracellular Polymeric Substances Produced by Pseudomonas fragi Under Air and Modified Atmosphere Packaging.

Extracellular polymeric substances (EPS) play an important role in bacterial biochemical properties. The characteristics of EPS from 2 strains of Pseudomonas fragi cultured in meat aerobically (control) and in modified atmosphere packaging (MAP) were studied. The amount and components of EPS, the surface properties, and the effect on biofilm formation of several spoilage organisms were evaluated. The results showed that MAP inhibited the growth of the P. fragi strains. Compared with the control, more loose and less bound EPS (containing protein and carbohydrate) were produced by P. fragi in MAP samples. MAP also caused increased cell autoaggregation and surface hydrophobicity. After the removal of the EPS, the surface property changes were strain-dependent, suggesting that membrane compositions were also changed. In addition, the EPS displayed significant antibiofilm activity on Pseudomonas fluorescens and Serratia liquefaciens. In conclusion, P. fragi strains not only modified the amount, components, and surface properties of EPS but also changed the cell membrane compositions to adapt to MAP stress. Moreover, EPS may play an important role in microbial community competitions.

Combined Effect of Polyphenol-Chitosan Coating and Irradiation on the Microbial and Sensory Quality of Carp Fillets.

Irradiation can extend the shelf-life of fish, but it may cause unacceptable change on quality. Since rose polyphenols have high antioxidant and antibacterial activities, this study evaluated the combined effect of polyphenol-chitosan coatings and irradiation (3 kGy) on the microbial and sensory qualities of carp fillets during storage at 4 °C. A dose of 3 kGy irradiation reduced the initial total viable counts (TVC) and psychrophiles, and increased the initial b* and thiobarbituric acid reactive substance (TBARS) values. During storage, TBARS, TVC and psychrophiles of nonirradiated samples increased faster and were higher than those irradiated. Regardless of irradiation treatment, samples coated with chitosan containing rose polyphenols had lower TBARS, pH and bacteria than that in no coating or chitosan coating batches. Carp treated with combined treatment could preserve an acceptable sensory quality at the end of storage. The result indicated that polyphenol-chitosan coating combined with irradiation can maintain fish quality by preventing bacterial growth, oxidation, and changes in color and sensory acceptability.

Characteristic Flavor of Traditional Soup Made by Stewing Chinese Yellow-Feather Chickens.

The traditional recipe for Chinese chicken soup creates a popular taste of particular umami and aroma. The present study investigated the effects of stewing time (1, 2, and 3 h) on the principal taste-active and volatile compounds and the overall flavor profile of traditional Chinese chicken soup by measuring the contents of free amino acids (FAAs), 5'-nucleotides, minerals and volatile compounds and by evaluating the taste and aroma profiles using an electronic nose, an electronic tongue and a human panel. Results showed that the major umami-related compounds in the chicken soup were inosine 5'-monophosphate (IMP) and chloride, both of which increased significantly (P < 0.05) during stewing. The taste active values (TAVs) of the equivalent umami concentration (EUC) increased from 4.08 to 9.93 (P < 0.05) after stewing for 3 h. Although the FAA and mineral contents increased significantly (P < 0.05), their TAVs were less than 1. The volatile compounds were mainly hexanal, heptanal, octanal, nonanal, (E)-2-nonanal, (E)-2-decenal, (E,E)-2,4-decadienal, 1-hexanol, and 2-pentyl furan. With the prolonged stewing time, the aldehydes first increased and then decreased significantly (P < 0.05), while 1-hexanol and 2-pentyl furan increased steadily (P < 0.05). The aroma scores of the chicken soup reached the maximum after stewing for 3 h. The discrepancy in overall flavor characteristics tended to stabilize after 2 h of stewing. In general, stewing time has a positive effect on improving the flavor profiles of chicken soup, especially within the first 2 h.

Physicochemical properties of Pseudomonas fragi isolates response to modified atmosphere packaging.

Pseudomonas spp., in particular Pseudomonas fragi, are dominant in aerobically stored chilled meats. This work isolated P. fragi isolates from spoiled chicken and investigated the effect of modified atmosphere packaging (MAP, CO2/N2 30%/70%) on physicochemical properties of P. fragi isolates compared to the corresponding controls (air). A total of six P. fragi isolates were isolated from aerobically stored spoiled chicken. MAP inhibited the growth of the isolates primarily in the exponential phase without inducing cell death and weakened the isolate's ability to form biofilms. Isolates 2 and 25, which readily form biofilms, exhibited improved stationary-phase growth in MAP compared to the control packaging. Changes in the surface properties, including reduced motility, increased aggregation and hydrophobicity, were also induced by MAP. The surface property results were then confirmed via scanning electron microscopy. In addition, significant differences were observed in cell phenotypic characterization between individual isolates tested, which indicated that the responses to MAP exposure were strain-dependent. These cell responses allowed P. fragi to adapt to MAP. The study provides practical information regarding microbial responses to MAP stresses, which will help further understanding the bacteriostatic mechanism of MAP.

Evaluation of the spoilage potential of bacteria isolated from chilled chicken in vitro and in situ.

Microorganisms play an important role in the spoilage of chilled chicken. In this study, a total of 53 isolates, belonging to 7 species of 3 genera, were isolated using a selective medium based on the capacity to spoil chicken juice. Four isolates, namely Aeromonas salmonicida 35, Pseudomonas fluorescens H5, Pseudomonas fragi H8 and Serratia liquefaciens 17, were further characterized to assess their proteolytic activities in vitro using meat protein extracts and to evaluate their spoilage potential in situ. The in vitro studies showed that A. salmonicida 35 displayed the strongest proteolytic activity against both sarcoplasmic and myofibrillar proteins. However, the major spoilage isolate in situ was P. fragi H8, which exhibited a fast growth rate, slime formation and increased pH and total volatile basic nitrogen (TVBN) on chicken breast fillets. The relative amounts of volatile organic compounds (VOCs) originating from the microorganisms, including alcohols, aldehydes, ketones and several sulfur compounds, increased during storage. In sum, this study demonstrated the characteristics of 4 potential spoilage bacteria on chilled yellow-feather chicken and provides a simple and convenient method to assess spoilage bacteria during quality management.