Investigation of Acid Tolerance Mechanism of Acetobacter pasteurianus under Different Concentrations of Substrate Acetic Acid Based on 4D Label-Free Proteomic Analysis.

Acetobacter pasteurianus is always used to brew vinegar because of its ability of producing and tolerating a high concentration of acetic acid. During vinegar fermentation, initial acetic acid contributes to acetic acid accumulation, which varies with initial concentrations. In this study, to investigate the mechanisms of tolerating and producing acetic acid of Acetobacter pasteurianus under different concentrations of substrate acetic acid, four-dimensional label-free proteomic technology has been used to analyze the protein profiles of Acetobacter pasteurianus at different growth stages (the lag and exponential phases) and different substrate acetic acid concentrations (0%, 3%, and 6%). A total of 2093 proteins were quantified in this study. The differentially expressed proteins were majorly involved in gene ontology terms of metabolic processes, cellular metabolic processes, and substance binding. Under acetic acid stress, strains might attenuate the toxicity of acetic acid by intensifying fatty acid metabolism, weakening the tricarboxylic acid cycle, glycerophospholipid and energy metabolism during the lag phase, while strains might promote the assimilation of acetic acid and inter-conversion of substances during the exponential phase by enhancing the tricarboxylic acid cycle, glycolysis, pyruvate, and energy metabolism to produce and tolerate acid. Besides, cell cycle regulation and protein translation might be potential acid tolerance pathways under high acid stress. The result contributes to the exploration of new potential acid tolerance mechanisms in Acetobacter pasteurianus from four-dimensional label-free relative quantitative proteomics analysis.

Effects of 2,2'-Azobis(2-methylpropionamidine) Dihydrochloride Stress on the Gel Properties of Duck Myofibrillar Protein Isolate.

The aim of this study was to investigate the biochemical properties and gel-forming capacity of duck myofibrillar proteins under the effects of 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH)-mediated oxidation. Duck myofibrillar proteins were extracted and treated with different concentrations of AAPH solutions (0, 1, 3, 5, 10 mmol/L) and then analysed for carbonyl content, dynamic rheology, protein profiles and gel-forming properties (colour, water holding capacity, gel strength and microstructure). The results showed that with increasing AAPH concentration, the carbonyl content of the proteins exhibited an increasing trend (p < 0.05); SDS-PAGE pattern changes indicated that moderate oxidation (3 mmol/L AAPH) induced myosin aggregation via covalent bonds including disulfide, enhanced protein-protein interactions, and thus affected the gel strength of the DMPs' heat-induced gels. However, high oxidation (5 and 10 mmol/L AAPH) led to the partial degradation of the myosin heavy chain (MHC) isoforms, as evidenced by lower storage modulus and irregular microstructures, which significantly reduced gelation ability. These results suggest that the internal relationship between alkylperoxyl radical-induced oxidation should be taken into account in the processing of duck meat, as mild protein oxidation is conducive to improving gel quality.

Flavor Characterization of Native Xinjiang Flat Peaches Based on Constructing Aroma Fingerprinting and Stoichiometry Analysis.

The flat peach is a high economic value table fruit possessing excellent quality and a unique aroma. This article investigated the quality characteristics and aroma fingerprinting of flat peaches (Qingpan, QP; Ruipan 2, R2; Ruipan 4, R4; Wanpan, WP) from Xinjiang in terms of taste, antioxidant capacity, and volatile aroma compounds using high-performance liquid chromatography (HPLC) and HS-SPME-GC-MS. The results showed that the flat peaches had a good taste and high antioxidant capacity, mainly due to the high sugar-low acid property and high levels of phenolic compounds. This study found that sucrose (63.86~73.86%) was the main sugar, and malic acid (5.93~14.96%) and quinic acid (5.25~15.01%) were the main organic acids. Furthermore, chlorogenic acid (main phenolic compound), epicatechin, rutin, catechin, proanthocyanidin B1, and neochlorogenic acid were positively related to the antioxidant activity of flat peaches. All flat peaches had similar aroma characteristics and were rich in aromatic content. Aldehydes (especially benzaldehyde and 2-hexenal) and esters were the main volatile compounds. The aroma fingerprinting of flat peaches consisted of hexanal, 2-hexenal, nonanal, decanal, benzaldehyde, 2,4-decadienal, dihydro-ß-ionone, 6-pentylpyran-2-one, 2-hexenyl acetate, ethyl caprylate, γ-decalactone, and theaspirane, with a "peach-like", "fruit", and "coconut-like" aroma. Among them, 2,4-decadienal, 2-hexenyl acetate, and theaspirane were the characteristic aroma compounds of flat peaches. The results provide a theoretical basis for the industrial application of the special aroma of flat peaches.

Synergistic effects of psyllium husk powder and different levels of methylcellulose on the storage stability of sodium caseinate emulsion.

The study investigated the effects of compound fibers composed of psyllium husk powder (PHP, 0.3%) and methylcellulose (MC, 0, 0.3, 0.6, 0.9, and 1.2%) on the storage stability, rheology, and microstructure of sodium caseinate emulsions. Results showed that the emulsion stability was enhanced with the increased concentrations of MC, especially at the concentration of 1.2%. The oil droplet size in the emulsions was decreased as the concentrations of compound fibers increased, which was further confirmed by the optical microscope analysis. The rheological measurements and cryo-scanning electron microscopy results indicated that compound fibers improved the viscosity of the emulsions, and formed a strong three-dimensional network structure. The results of confocal laser scanning microscope and surface protein concentration measurements showed that compound fibers were evenly distributed into the oil droplet surface. The above results demonstrate that compound fibers are an effective thickener and emulsifier in enhancing the stability properties of oil-in-water (O/W) emulsions stabilized by sodium caseinate.

Physicochemical properties and gel-forming ability changes of duck myofibrillar protein induced by hydroxyl radical oxidizing systems.

This paper focuses on the changes of physicochemical properties and gel-forming ability of duck myofibrillar proteins (DMPs) induced using hydroxyl radical oxidizing systems. DMPs were firstly extracted and then oxidized at various H2O2 concentrations (0, 4, 8, and 12 mmol/L) using Fenton reagent (Fe3+-Vc-H2O2) to generate hydroxyl radicals, and the effects of hydroxyl radical oxidation on the physicochemical changes and heat-induced gel-forming capacity of DMPs were analyzed. We observed obvious increases in the carbonyl content (p < 0.05) and surface hydrophobicity of DMPs with increasing of H2O2 concentrations (0-12 mmol/L). In contrast, the free thiol content (p < 0.05) and water retention ability of DMPs decreased with increasing H2O2 concentrations (0-12 mmol/L). These physicochemical changes suggested that high concentrations of hydroxyl radicals significantly altered the biochemical structure of DMPs, which was not conducive to the formation of a gel mesh structure. Furthermore, the gel properties were reduced based on the significant decrease in the water holding capacity (p < 0.05) and increased transformation of immobilized water of the heat-induced gel to free water (p < 0.05). With the increase of H2O2 concentrations, secondary structure of proteins analysis results indicated α-helix content decreased significantly (p < 0.05), however, random coil content increased (p < 0.05). And more cross-linked myosin heavy chains were detected at higher H2O2 concentrations groups through immunoblot analysis (p < 0.05). Therefore, as H2O2 concentrations increased, the gel mesh structure became loose and porous, and the storage modulus and loss modulus values also decreased during heating. These results demonstrated that excessive oxidation led to explicit cross-linking of DMPs, which negatively affected the gel-forming ability of DMPs. Hence, when processing duck meat products, the oxidation level of meat gel products should be controlled, or suitable antioxidants should be added.

Effects of Cherry (Prunus cerasus L.) Powder Addition on the Physicochemical Properties and Oxidation Stability of Jiangsu-Type Sausage during Refrigerated Storage.

Effects of different levels (1%, 3% and 5%) of cherry powder on the physiochemical properties and antioxidant activity of Jiangsu-type sausages were investigated at 4 °C for 30 days. The results show that the sensory evaluation values and physicochemical properties of the sausages had no significant differences compared to the control group when cherry powder addition was 1%, and the alcohols, aldehydes and esters were increased after the addition of cherry powder improved the flavor of sausages. However, higher concentration of cherry powder (3% and 5%) exerted adverse influences on sensory evaluation values and physicochemical properties of sausages compared with the control. The addition of cherry powder could better inhibit lipid and protein oxidation of sausages, and the cherry powder concentration has a positive correlation with its effect on the inhibition of lipid and protein oxidation. In addition, cherry powder could effectively control TVB-N values of sausages during chilled storage. All these results indicate that 1% cherry powder could not only guarantee the physicochemical properties of sausages, but also inhibited the oxidation of sausages during chilled storage.

Pork longissimus dorsi marinated with edible mushroom powders: Evaluation of quality traits, microstructure, and protein degradation.

In this study, we examined the effects of marination with five edible mushroom powders (3%, m/m) on the quality attributes and protein changes of pork longissimus dorsi compared to meat marinated with 0.2% papain and distilled water (control group). The results indicated that pork samples marinated with edible mushrooms displayed better water retention and lower shear force, especially in the 3% Hypsizygus marmoreus (HM) group (12.50 ± 0.73 N). Ultrastructural observation results showed that the Z-disk and M-line of the pork samples marinated with edible mushrooms were fractured and weakened. Furthermore, the degradation extent of desmin in the HM-marinated pork samples was obviously increased (43%) in comparison to the papain group (only 15%), which was further confirmed by the results of the myofibril fragmentation index. Overall, the pork samples marinated with 3% HM exhibited better quality traits among all groups and might be a potential strategy to improve tenderness.

Effects of Casein, Chicken, and Pork Proteins on the Regulation of Body Fat and Blood Inflammatory Factors and Metabolite Patterns Are Largely Dependent on the Protein Level and Less Attributable to the Protein Source.

The impact of meat protein on metabolic regulation is still disputed and may be influenced by protein level. This study aimed to explore the effects of casein, pork, and chicken proteins at different protein levels (40% E vs 20% E) on body weight regulation, body fat accumulation, serum hormone levels, and inflammatory factors/metabolites in rats maintained on high-fat (45% E fat) diets for 84 d. Increased protein levels resulted in a significant reduction in body fat mass and an increase in the serum levels of the anti-inflammatory cytokine IL-10, independent of protein source. Analysis of blood via untargeted metabolomics analysis identified eight, four, and four metabolites significantly altered by protein level, protein source, and a protein level-source interaction, respectively. Together, the effects of casein, chicken, and pork protein on the regulation of body fat accumulation and blood metabolite profile are largely dependent on protein level and less attributable to the protein source.

In Vitro Susceptibility of Oxidized Myosin by µ-Calpain or Caspase-3 and the Determination of the Oxidation Sites of Myosin Heavy Chains.

The effect of susceptibility to in vitro oxidation on the degradation of myosin isolated from beef muscles via µ-calpain or caspase-3 was examined, and the measurement of the oxidation sites of myosin heavy chains was performed. Myosin was incubated with hydroxyl free radical-generating systems, which were composed of 0.01 M FeCl3, 0.1 M ascorbic acid, and 0, 25, 50, and 100 µM H2O2 at 37 °C for 20 min. The oxidized myosin then reacted with µ-calpain or caspase-3 at 37 °C for 30 min, respectively. The results showed that protein oxidation systems in vitro resulted in different levels of myosin oxidation, leading to significant changes in the secondary structure of myosin (P < 0.05). The sodium dodecyl dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting results showed that in vitro oxidation promoted myosin degradation via µ-calpain or caspase-3. Proteomics research suggested that the number of myosin oxidation sites increased constantly with the increase of oxidation levels. Oxidation sites of myosin were mainly cysteine, methionine, arginine, histidine, tyrosine, lysine, and asparagine. These results indicated that oxidation using H2O2 in the range of 0-100 µM could increase the degradation of myosin via µ-calpain and caspase-3 due to increased exposure of the oxidation sites of myosin.

Effects of Oxidation in Vitro on Structures and Functions of Myofibrillar Protein from Beef Muscles.

The main purpose of this study was to investigate the effects of oxidation in vitro on the biochemical properties of myofibrillar protein isolates (MPIs) from beef muscles. MPIs were incubated at 4 °C for 24 h with hydroxyl-radical-generating systems consisting of 0.01 mM FeCl3 and 0.1 mM ascorbic acid plus 0, 0.2, 1, 5, 10, and 20 mM hydrogen peroxide. The results showed that oxidation caused drastically structural changes in bovine MPIs. The carbonyl content, the surface hydrophobicity, and the particle diameter of MPIs were significantly increased, while the free sulfhydryl group content was dramatically decreased with increasing hydrogen peroxide concentrations. Oxidation caused the protein aggregations through cross-linking between proteins and amino acids. Proteomics study identified protein sites in which they were easy to be oxidized. Oxidized catalytic activities and binding sites of enzymes that were susceptible to oxidation were also identified.

Purified Dietary Red and White Meat Proteins Show Beneficial Effects on Growth and Metabolism of Young Rats Compared to Casein and Soy Protein.

This study compared the effects of casein, soy protein (SP), red (RMP), and white meat (WMP) proteins on growth and metabolism of young rats. Compared to casein, the ratio of daily feed intake to daily body weight gain of rats was not changed by meat protein but reduced by SP by 93.3% ( P < 0.05). Feeding RMP and WMP reduced the liver total cholesterol (TC) contents by 24.3% and 17.8%, respectively ( P < 0.05). Only RMP increased plasma HDL-cholesterol concentrations (by 12.7%, P < 0.05), whereas SP increased plasma triacylglycerol, TC, and LDL-cholesterol concentrations by 23.7%, 19.5%, and 61.5%, respectively ( P < 0.05). Plasma essential and total amino acid concentrations were increased by WMP (by 18.8% and 12.4%, P < 0.05) but reduced by SP (by 28.3% and 37.7%, P < 0.05). Twenty-five liver proteins were differentially expressed in response to different protein sources. Therefore, meat proteins were beneficial for growth and metabolism of young rats compared to casein and SP.

Identification of S-nitrosylated proteins in postmortem pork muscle using modified biotin switch method coupled with isobaric tags.

The objective of this study was to identify the S-nitrosylated proteins in aging samples of pork longissimus thoracis muscle (aged 0 and 3 d) and to study the effects of exogenous S-nitrosoglutathione (GSNO, concentration at 10 and 100 µM) treatments of aged 0 d sample. After validating modified biotin switch method, the samples were labeled with tandem mass tags (TMT126-129) for the LC-MS/MS analysis. A total of 366 peptides were identified to be S-nitrosylated corresponding to 339 proteins. Comparison of total intensity and individual S-nitrosylated sites between aging samples revealed that S-nitrosylation did occur in pork muscle during postmortem aging through possible pathways of denitrosylation and transnitrosylation. GSNO treatment groups showed a considerable number of potential cysteines could be modified with high thiol-reactivity. It was deduced that S-nitrosylation could be involved in the postmortem metabolic process possibly through the regulation of activity or function of glycolytic enzymes, calcium release, heat shock proteins, antioxidant enzymes and myofibrillar proteins.

Influence of modified atmosphere packaging on protein oxidation, calpain activation and desmin degradation of beef muscles.

BACKGROUND: Protein oxidation is widespread in biochemical systems. The objective of the study was to investigate the differences in protein oxidation, µ-calpain activity, desmin proteolysis and protein solubility of beef psoas major (PM) and semi-membranosus (SM) muscles under three packaging systems during postmortem ageing. At 24 h postmortem, beef muscles were packaged respectively in air-permeable film overwrap (AP), vacuum pack (VP) or modified atmosphere (MAP, 80% O2 + 20% CO2 ), and then displayed for 10 days at 4 °C. RESULTS: Carbonyl group values and thiol group content were significantly influenced by packaging type and storage time. The SM muscles from AP and MAP showed greater µ-calpain activity compared to VP. Desmin of PM and SM from AP and MAP samples showed decreased proteolysis compared with VP. CONCLUSION: The results suggested that the inhibition of µ-calpain activity of beef samples from AP and MAP could be closely associated with protein oxidation which further lowered the level of desmin degradation compared to VP. © 2017 Society of Chemical Industry.

A Review of Antioxidant Peptides Derived from Meat Muscle and By-Products.

Antioxidant peptides are gradually being accepted as food ingredients, supplemented in functional food and nutraceuticals, to positively regulate oxidative stress in the human body against lipid and protein oxidation. Meat muscle and meat by-products are rich sources of proteins and can be regarded as good materials for the production of bioactive peptides by use of enzymatic hydrolysis or direct solvent extraction. In recent years, there has been a growing number of studies conducted to characterize antioxidant peptides or hydrolysates derived from meat muscle and by-products as well as processed meat products, including dry-cured hams. Antioxidant peptides obtained from animal sources could exert not only nutritional value but also bioavailability to benefit human health. This paper reviews the antioxidant peptides or protein hydrolysates identified in muscle protein and by-products. We focus on the procedure for the generation of peptides with antioxidant capacity including the acquisition of crude peptides, the assessment of antioxidant activity, and the purification and identification of the active fraction. It remains critical to perform validation experiments with a cell model, animal model or clinical trial to eliminate safety concerns before final application in the food system. In addition, some of the common characteristics on structure-activity relationship are also reviewed based on the identified antioxidant peptides.

Differences in calpain system, desmin degradation and water holding capacity between commercial Meishan and Duroc × Landrace × Yorkshire crossbred pork.

The objective of this study was to examine the differences in calpain system, desmin degradation, pH values and water holding capacity (WHC) between muscles of commercial Meishan and Duroc × Landrace × Yorkshire crossbred pigs. Meishan pork presented better WHC evidenced by lower purge loss at days 1 and 3 and less centrifugation loss at day 1 post mortem (P < 0.05). pH values at 45 min post mortem in Meishan pork were significantly higher compared to crossbred pork (P < 0.05). Calpain-1 messenger RNA (mRNA) expression was lower in Meishan pork compared to that from crossbred pork (P < 0.05). Additionally, calpain-1 activity, the ratio of calpain-1 to calpastatin activity and desmin degradation were lower in Meishan pork compared to those from crossbred pork samples (P < 0.05). The results indicate that the calpain system including mRNA expression and activity were different between commercial Meishan and crossbred pork resulting in difference in the degree of desmin degradation during post mortem aging. pH values at 45 min and 24 h post mortem rather than calpain activity and desmin degradation could explain the higher water holding capacity in commercial Meishan pork.

Enantioselective Organocatalytic Transfer Hydrogenation of 1,2-Dihydroquinoline through Formation of Aza-o-xylylene.

A new way of forming the aza-o-xylylene with easily accessible 1,2-dihydroquinolines as precursor has been developed. The presence of an electron-donating group at the proper position of 1,2-dihydroquinoline was crucial for protonation of the alkene through dearomatization with a simple Brønsted acid. The in situ forming reactive intermediate was trapped with Hantzsch ester to afford tetrahydroquinolines in excellent yield and enantioselectivity.

Activity and expression of nitric oxide synthase in pork skeletal muscles.

The objective of this study was to investigate the biochemical changes of nitric oxide synthase (NOS) in pork skeletal muscles during postmortem storage. Longissimus thoracis (LT), psoas major (PM) and semimembranosus (SM) muscles of pork were removed immediately after slaughter and stored under vacuum condition at 4°C for 0, 1 and 3d. Results showed that all three muscles exhibited NOS activity until 1d while SM muscle retained NOS activity after 3d of storage. The content of nNOS in SM muscle was stable across 3d of storage while decreased intensity of nNOS was detected at 1 and 3d of aging in PM and LT muscles due to the degradation of calpain. Immunostaining showed that nNOS was located at not only sarcolemma but also cytoplasm at 0 and 1d of storage. Our data suggest that postmortem muscles possess NOS activity and nNOS expression depends on muscle type.

Effect of nitric oxide on µ-calpain activation, protein proteolysis, and protein oxidation of pork during post-mortem aging.

The aim of the current research was to examine the influence of nitric oxide (NO) on calpain activation, protein proteolysis, and oxidation in post-mortem pork. Five longissimus muscles were removed from carcass after slaughter, and samples were incubated with water, nitric oxide synthase (NOS) inhibitor, or NO donor for 24 h at 4 °C. The samples were taken out and then stored under 4 °C for 1, 4, and 7 d. Results showed that autolysis of µ-calpain increased by incubation with NOS inhibitor after storage for 1 d (P<0.05). Degradation of titin and nebulin increased by treatment of NOS inhibitor among three treatments (P<0.05). Higher levels of protein oxidation were observed after samples incubated with NO donor than treatment of NOS inhibitor (P<0.05). These data indicated that NO could participate in regulating calpain activation and its proteolysis activity during post-mortem aging.

Alkyl transfer from C-C cleavage: replacing the nitro group of nitro-olefins.

Alkyl substituted Hantzsch esters are rationally used as alkylation reagents to replace the nitro groups of nitro olefins to give excellent yields of trans-olefins. The reaction mechanism is considered to proceed through a free radical mechanism, which is different from the corresponding transfer alkylation of imines.