SDS-PAGE Research Articles

Isolation and Characterization of Cellulolytic Pseudomonas Aeruginosa from Teak Forest Soil for its Cellulase Production and Cellulolytic Activity

A cellulolytic bacterium was isolated from Tectona forest soil and provisionally identified as Pseudomonas aeruginosa. The bacterium showed the highest level of reducing sugar and extracellular protein production when incubated for 3 days at 40°C, pH 8.5 in Winstead’s broth having 1.5% carboxymethyl cellulose (CMC) and 0.2% Yeast Extract as carbon and nitrogen sources, respectively. The bacterium produced the maximum 462 µg/ml reducing sugar and 450 µg/ml extracellular proteins. Crude cellulase produced by the bacterium showed the highest carboxymethyl cellulase (CMCase) activity rather than filter paperase (FPase), Avicelase and β-Glucosidase activities. The crude cellulase showed highest CMCase activity when incubated for 90 minutes at pH 8.5, 25°C using CMC and Yeast Extract as carbon and nitrogen sources, respectively as well as in presence of Hg2+ ion and SDS (inhibitor and reductant). The highest enzyme activity was found to be 315 U/ml. The molecular weight of the crude cellulase was found as 33 kDa through SDS PAGE analysis. According to Worthington Enzyme Manual it may be Endoglucanase IV. The Chittagong Univ. J. B. Sci., Vol. 10(1 &2):167-179, 2020

Co-crosslinking strategy for dual functionalization of small magnetic nanoparticles with redox probes and biological probes

Surface functionalization strategy is becoming a crucial bridge from magnetic nanoparticles (MNPs) to their broad bio-application. To realize the multiple functions of MNPs such as magnetic manipulation, target capture, and signal amplification in their use of electrochemical biosensing, co-crosslinking strategy was proposed here to construct dual-functionalized MNPs by combining ultra-sensitive redox moieties and specific biological probes. In this work, MNPs with a TEM size of 10 nm were synthesized by co-precipitation for amination and PEGylation to maintain colloid stability once dispersed in high-ionic-strength buffer (such as phosphate-buffered saline). Then, MNPs@IgG were prepared via the bis(sulfosuccinimidyl) suberate (BS3) cross-linker to conjugate these IgG onto the MNP surface, with a binding efficiency of 73%. To construct dual-functionalized MNPs, these redox probes of ferrocene-NHS (Fc) were co-crosslinked onto the MNP surface, together with IgG, by using BS3. The developed MNPs@Redox@IgG were characterized by SDS‒PAGE to identify IgG binding and by square wave voltammetry (SWV) to validate the redox signal. Additionally, the anti-CD63 antibodies were selected for the development of MNPs@anti-CD63 for use in the bio-testing of exosome sample capture. Therefore, co-crosslinking strategy paved a way to develop dual-functionalized MNPs that can be an aid of their potential utilization in diagnostic assay or electrochemical methods.Graphical

Dermatoprotective effect of Moringa oleifera leaf extract on sodium valproate-induced skin damage in rats

Valproic acid is an antiepileptic drug associated with skin-related issues like excessive hair growth, hair loss, and skin rashes. In contrast, Moringa oleifera, rich in nutrients and antioxidants, is gaining popularity worldwide for its medicinal properties. The protective properties of M. oleifera extract against skin-related side effects caused by valproic acid were investigated. Female rats were divided into control groups and experimental groups such as moringa, sodium valproate, and sodium valproate + moringa groups. A 70% ethanolic extract of moringa (0.3 g/kg/day) was given to moringa groups, and a single dose of sodium valproate (0.5 g/kg/day) was given to valproate groups for 15 days. In the skin samples, antioxidant parameters (such as glutathione, glutathione-S-transferase, superoxide dismutase, catalase, and total antioxidant capacity), as well as oxidant parameters representing oxidative stress (i.e. lipid peroxidation, sialic acid, nitric oxide, reactive oxygen species, and total oxidant capacity), were examined. Additionally, boron, hydroxyproline, sodium-potassium ATPase, and tissue factor values were determined. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was also carried out for protein analysis in the skin samples. The results showed that moringa could increase glutathione, total antioxidant capacity, sodium-potassium ATPase, and boron levels, while decreasing lipid peroxidation, sialic acid, nitric oxide, total oxidant capacity, reactive oxygen species, hydroxyproline, and tissue factor levels. These findings imply that moringa possesses the potential to mitigate dermatological side effects.

Leptin-mediated suppression of lipoprotein lipase cleavage enhances lipid uptake and facilitates lymph node metastasis in gastric cancer.

Lymph node metastasis (LNM) is the primary mode of metastasis in gastric cancer (GC). However, the precise mechanisms underlying this process remain elusive. Tumor cells necessitate lipid metabolic reprogramming to facilitate metastasis, yet the role of lipoprotein lipase (LPL), a pivotal enzyme involved in exogenous lipid uptake, remains uncertain in tumor metastasis. Therefore, the aim of this study was to investigate the presence of lipid metabolic reprogramming during LNM of GC as well as the role of LPL in this process. Intracellular lipid levels were quantified using oil red O staining, BODIPY 493/503 staining, and flow cytometry. Lipidomics analysis was employed to identify alterations in intracellular lipid composition following LPL knockdown. Protein expression levels were assessed through immunohistochemistry, Western blotting, and enzyme-linked immunosorbent assays. The mouse popliteal LNM model was utilized to investigate differences in LNM. Immunoprecipitation and mass spectrometry were employed to examine protein associations. In vitro phosphorylation assays and Phos-tag sodium dodecyl-sulfate polyacrylamide gel electrophoresis assays were conducted to detect angiopoietin-like protein 4 (ANGPTL4) phosphorylation. We identified that an elevated intracellular lipid level represents a crucial characteristic of node-positive (N+) GC and further demonstrated that a high-fat diet can expedite LNM. LPL was found to be significantly overexpressed in N+ GC tissues and shown to facilitate LNM by mediating dietary lipid uptake within GC cells. Leptin, an obesity-related hormone, intercepted the effect exerted by ANGPTL4/Furin on LPL cleavage. Circulating leptin binding to the leptin receptor could induce the activation of inositol-requiring enzyme-1 (IRE1) kinase, leading to the phosphorylation of ANGPTL4 at the serine 30 residue and subsequently reducing its binding affinity with LPL. Moreover, our research revealed that LPL disrupted lipid homeostasis by elevating intracellular levels of arachidonic acid, which then triggered the cyclooxygenase-2/prostaglandin E2 (PGE2) pathway, thereby promoting tumor lymphangiogenesis. Leptin-induced phosphorylation of ANGPTL4 facilitates LPL-mediated lipid uptake and consequently stimulates the production of PGE2, ultimately facilitating LNM in GC.

Laccase/caffeic acid-catalyzed crosslinking coupled with galactomannan alters the conformational structure of ovalbumin and alleviates Th2-mediated allergic asthma

Ovalbumin (OVA) is the major allergenic protein that can induce T helper 2 (Th2)-allergic reactions, for which current treatment options are inadequate. In this study, we developed a polymerized hypoallergenic OVA product via laccase/caffeic acid (Lac/CA)-catalyzed crosslinking in conjunction with galactomannan (Man). The formation of high molecular weight crosslinked polymers and the IgG-binding were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. The study indicated that Lac/CA-catalyzed crosslinking plus Man conjugation substantially altered secondary and tertiary structures of OVA along with the variation in surface hydrophobicity. Gastrointestinal digestion stability assay indicated that crosslinked OVA exhibited less resistance in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF). Mouse model study indicated that Lac-Man/OVA ameliorated eosinophilic airway inflammatory response and efficiently downregulated the expression of Th2-related cytokines (interleukin (IL)-4, IL-5, and IL-13), and upregulated IFN-γ and IL-10 expression. Stimulation of bone marrow-derived dendritic cells with Lac-Man/OVA suppressed the expression of phenotypic maturation markers (CD80 and CD86) and MHC class II molecules, and suppressed the expression levels of proinflammatory cytokines. The knowledge obtained in the present study offers an effective way to acquire a hypoallergenic OVA product that can have a therapeutic effect in alleviating OVA-induced allergic asthma.

Enhanced extracellular production of alpha-lactalbumin from Bacillus subtilis through signal peptide and promoter screening

Alpha-lactalbumin (α-LA) is a major whey protein found in breast milk and plays a crucial role in the growth and development of infants. In this study, Bacillus subtilis RIK1285 harboring AprE signal peptide (SP) was selected as the original strain for the production of α-LA. It was found that α-LA was identified in the pellet after ultrasonic disruption and centrifugation instead of in the fermentation supernatant. The original strain most likely only produced α-LA intracellular, but not extracellular. To improve the expression and secretion of α-LA in RIK1285, a library of 173 homologous SPs from the B. subtilis 168 genome was fused with target LALBA gene in the pBE-S vector and expressed extracellularly in RIK1285. SP YjcN was determined to be the best signal peptide. Bands in supernatant were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and purified by nickel column to calculate the highest yield signal peptide. In addition, different promoters (PaprE, P43, and Pglv) were compared and applied. The results indicated that the strain RIK1285-pBE-Pglv-YjcN-LALBA had the highest α-LA yield, reaching 122.04 μg/mL. This study demonstrates successful expression and secretion of human α-LA in B. subtilis and establishes a foundation for simulating breast milk for infant formulas and developing bioengineered milk.

Experimental Production and Efficacy Testing of Mono-specific Antibodies against the Venom of Carpet viper (Echis ocellatus) from Savannah Nigeria

Echis ocellatus is one of the commonest snakes responsible for envenomation in Nigeria. Antivenom is the only effective treatment, but the country suffers from a limited supply of effective antivenom. This study therefore aimed to explore the feasibility of effective, mono-specific antibodies production through immunization in rabbits using the venom of Echis ocellatus from Nigeria. The World Health Organization guide on antivenom production was employed in the immunization and the resultant antibodies were purified using protein A agarose column chromatography. Antibody titer reached a high plateau by 2-month immunization, and SDS PAGE of the sera suggests the presence of intact immunoglobulins accompanied with the heavy (50 kDa) and light (25 kDa) chains. The venom has an intravenous LD50 of 0.35 mg/kg in mice, and the venom lethality at a challenge dose of 2 LD50 was effectively neutralized by the antibodies with a potency value of 0.83 mg venom per g antibodies. The antibodies also neutralized the procoagulant activity of the venom with an effective dose (ED) of 13±0.66 ul, supporting its use for hemotoxic envenomation. The study establishes the feasibility of developing effective, mono-specific antibodies against the Nigerian Carpet viper.

Antibacterial Properties of Peptide and Protein Fractions from Cornu aspersum Mucus.

The discovery and investigation of new natural compounds with antimicrobial activity are new potential strategies to reduce the spread of antimicrobial resistance. The presented study reveals, for the first time, the promising antibacterial potential of two fractions from Cornu aspersum mucus with an MW < 20 kDa and an MW > 20 kDa against five bacterial pathogens-Bacillus cereus 1085, Propionibacterium acnes 1897, Salmonella enterica 8691, Enterococcus faecalis 3915, and Enterococcus faecium 8754. Using de novo sequencing, 16 novel peptides with potential antibacterial activity were identified in a fraction with an MW < 20 kDa. Some bioactive compounds in a mucus fraction with an MW > 20 kDa were determined via a proteomic analysis on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and bioinformatics. High homology with proteins and glycoproteins was found, with potential antibacterial activity in mucus proteins named aspernin, hemocyanins, H-lectins, and L-amino acid oxidase-like protein, as well as mucins (mucin-5AC, mucin-5B, mucin-2, and mucin-17). We hypothesize that the synergy between the bioactive components determined in the composition of the fraction > 20 kDa are responsible for the high antibacterial activity against the tested pathogens in concentrations between 32 and 128 µg/mL, which is comparable to vancomycin, but without cytotoxic effects on model eukaryotic cells of Saccharomyces cerevisiae. Additionally, a positive effect, by reducing the levels of intracellular oxidative damage and increasing antioxidant capacity, on S. cerevisiae cells was found for both mucus extract fractions of C. aspersum. These findings may serve as a basis for further studies to develop a new antibacterial agent preventing the development of antibiotic resistance.

Kenaf Seed Cysteine Protease (KSCP) Inhibits the Intrinsic Pathway of the Blood Coagulation Cascade and Platelet Aggregation.

Thrombosis is the key event that obstructs the flow of blood throughout the circulatory system, leading to stroke, myocardial infarction and severe cardiovascular complications. Currently, available antithrombotic drugs trigger several life-threatening side effects. Antithrombotic agents from natural sources devoid of adverse effects are grabbing high attention. In our previous study, we reported the antioxidant, anticoagulant and antiplatelet properties of kenaf seed protein extract. Therefore, in the current study, purification and characterization of cysteine protease from kenaf seed protein extract responsible for potential antithrombotic activity was undertaken. Purification of KSCP (Kenaf Seed Cysteine Protease) was carried out using gel permeation and ion exchange column chromatography. The purity of the enzyme was evaluated by SDS PAGE (Sodium Dodecyl-Sulfate Polyacrylamide Gel Electrophoresis). RP-HPLC (Reverse Phase High-Performance Liquid Chromatography), MALDI-TOF (Matrix-Assisted Laser Desorption Ionization Time-Of-Flight) and CD (Circular Dichroism techniques) were employed for its characterization. Proteolytic, fibrinolytic and kinetic study was done using spectroscopy. Plasma recalcification time, Prothrombin Time (PT), Thrombin clotting time (TCT), Activated Partial Thromboplastin Time (APTT), bleeding time and platelet aggregation studies were carried out for antithrombotic activity of KSCP. A single sharp band of KSCP was observed under both reduced and non-reduced conditions, having a molecular mass of 24.1667kDa. KSCP was found to contain 30.3% helix turns and 69.7% random coils without a beta-pleated sheet. KSCP digested casein and fibrin, and its activity was inhibited by iodoacetic acid (IAA). KSCP was optimally active at pH 6.0 at the temperature of 40°C. KSCP exhibited anticoagulant properties by interfering in the intrinsic pathway of the blood coagulation cascade. Furthermore, KSCP dissolved both whole blood and plasma clots and platelet aggregation. KSCP purified from kenaf seed extract showed antithrombotic potential. Hence, it could be a better candidate for the management of thrombotic complications.

Molecular Cloning, Expression and Enzymatic Characterization of Tetrahymena thermophila Glutathione-S-Transferase Mu 34.

Glutathione-S-transferase enzymes (GSTs) are essential components of the phase II detoxification system and protect organisms from oxidative stress induced by xenobiotics and harmful toxins such as 1-chloro-2,4-dinitrobenzene (CDNB). In Tetrahymena thermophila, the TtGSTm34 gene was previously reported to be one of the most responsive GST genes to CDNB treatment (LD50 = 0.079mM). This study aimed to determine the kinetic features of recombinantly expressed and purified TtGSTm34 with CDNB and glutathione (GSH). TtGSTm34-8xHis was recombinantly produced in T. thermophila as a 25-kDa protein after the cloning of the 660-bp full-length ORF of TtGSTm34 into the pIGF-1 vector. A three-dimensional model of the TtGSTm34 protein constructed by the AlphaFold and PyMOL programs confirmed that it has structurally conserved and folded GST domains. The recombinant production of TtGSTm34-8xHis was confirmed by SDS‒PAGE and Western blot analysis. A dual-affinity chromatography strategy helped to purify TtGSTm34-8xHis approximately 3166-fold. The purified recombinant TtGSTm34-8xHis exhibited significantly high enzyme activity with CDNB (190µmol/min/mg) as substrate. Enzyme kinetic analysis revealed Km values of 0.68mM with GSH and 0.40mM with CDNB as substrates, confirming its expected high affinity for CDNB. The optimum pH and temperature were determined to be 7.0 and 25°C, respectively. Ethacrynic acid inhibited fully TtGSTm34-8xHis enzyme activity. These results imply that TtGSTm34 of T. thermophila plays a major role in the detoxification of xenobiotics, such as CDNB, as a first line of defense in aquatic protists against oxidative damage.

Bacterial ubiquitin ligases hijack the host deubiquitinase OTUB1 to inhibit MTORC1 signaling and promote autophagy

ABSTRACT Many bacterial pathogens have evolved effective strategies to interfere with the ubiquitination network to evade clearance by the innate immune system. Here, we report that OTUB1, one of the most abundant deubiquitinases (DUBs) in mammalian cells, is subjected to both canonical and noncanonical ubiquitination during Legionella pneumophila infection. The effectors SidC and SdcA catalyze OTUB1 ubiquitination at multiple lysine residues, resulting in its association with a Legionella-containing vacuole. Lysine ubiquitination by SidC and SdcA promotes interactions between OTUB1 and DEPTOR, an inhibitor of the MTORC1 pathway, thus suppressing MTORC1 signaling. The inhibition of MTORC1 leads to suppression of host protein synthesis and promotion of host macroautophagy/autophagy during L. pneumophila infection. In addition, members of the SidE family effectors (SidEs) induce phosphoribosyl (PR)-linked ubiquitination of OTUB1 at Ser16 and Ser18 and block its DUB activity. The levels of the lysine and serine ubiquitination of OTUB1 are further regulated by effectors that function to antagonize the activities of SidC, SdcA and SidEs, including Lem27, DupA, DupB, SidJ and SdjA. Our study reveals an effectors-mediated complicated mechanism in regulating the activity of a host DUB. Abbreviations: BafA1: bafilomycin A1; BMDMs: bone marrow-derived macrophages; DUB: deubiquitinase; Dot/Icm: defective for organelle trafficking/intracellular multiplication; DEPTOR: DEP domain containing MTOR interacting protein; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; L. pneumophila: Legionella pneumophila; LCV: L egionella-containing vacuole; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MOI: multiplicity of infection; MTORC1: mechanistic target of rapamycin kinase complex 1; OTUB1: OTU deubiquitinase, ubiquitin aldehyde binding 1; PR-Ub: phosphoribosyl (PR)-linked ubiquitin; PTM: posttranslational modification; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SidEs: SidE family effectors; Ub: ubiquitin.

Thermo-Alkali-Tolerant Recombinant Laccase from Bacillus swezeyi and Its Degradation Potential against Zearalenone and Aflatoxin B1.

The enzymatic biodegradation of mycotoxins in food and feed has attracted the most interest in recent years. In this paper, the laccase gene from Bacillus swezeyi was cloned and expressed in Escherichia coli BL 21(D3). The sequence analysis indicated that the gene consisted of 1533 bp. The purified B. swezeyi laccase was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis -12% with an estimated molecular weight of 56.7 kDa. The enzyme is thermo-alkali-tolerant, displaying the optimal degradation of zearalenone (ZEN) and aflatoxin B1 (AFB1) at pH 8 and 9, with incubation temperatures of 55 and 50 °C, respectively, within 24 h. The degradation potentials of the 50 μg of the enzyme against ZEN (5.0 μg/mL) and AFB1 (2.5 μg/mL) were 99.60 and 96.73%, respectively, within 24 h. To the best of our knowledge, this is the first study revealing the recombinant production of laccase from B. swezeyi, its biochemical properties, and potential use in ZEN and AFB1 degradation in vitro and in vivo.

Cysteine, sodium metabisulfite, and glutathione enhance crosslinking between proteins during high moisture meat analog extrusion processing and may improve the fibrousness of the products.

High moisture meat analog (HMMA) products processed using extrusion have become increasingly popular in the last few years. Because the formation of disulfide bonds is believed to play a critical role in the texturization mechanism, this study aimed to understand how chemical compounds capable of reducing disulfide bonds, specifically cysteine, sodium metabisulfite, and glutathione, affect the texture and the chemical interactions between the proteins. Wheat protein blended with cysteine, sodium metabisulfite, or glutathione at levels of 0, 0.5, 1.0, 2.5, 5.0, and 7.5 g kg-1 was extruded at three different temperatures (115, 140, and 165 °C) using a co-rotating twin-screw extruder. The feed rate (85 g min-1), the moisture content (600 g kg-1), and the screw speed (300 rpm) were kept constant. Unextruded and extruded material was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, polymeric protein fractionation, and sulfhydryl group/disulfide bond analysis. Extruded samples were further analyzed for their hardness and their anisotropic index. The inclusion of reductants significantly affected the structure of the obtained extrudates. Although reducing agents had a relatively small impact on the total amount of disulfide bonds, their action significantly enhanced crosslinking between the proteins. At select conditions, samples with high fibrousness were specifically obtained when cysteine or sodium metabisulfite was included at levels of 5.0 g kg-1. In the presence of reducing agents, it is believed that disulfide bonds are split earlier during the process without binding to them, giving the protein strands more time to unravel and align, leading to a better flow behavior and more fibrous products. © 2024 Society of Chemical Industry.

Gelatinization and fermentation synergy: investigating the protein digestibility, mineral bioaccessibility and microstructural transformations of black mash beans through Saccharomyces cerevisiae and Lactobacillus spp

ABSTRACT This study explored the nutritional qualities of black mash bean flour impacted by fermentation, gelatinization and their combination. Protease activity, in vitro protein digestibility (IVPD), and protein solubility were evaluated for both fermented and unfermented black mash bean flour samples. Interestingly, when compared to fermented samples and gelatinized mash bean flour (GMBF), raw mash bean flour (RMBF) showed the highest protein solubility. Due to enzymatic activity and protein hydrolysis, fermentation with Lactobacillus E14 and Saccharomyces cerevisiae MK-157 enhanced protein solubility. Prolonged fermentation timeframes were positively correlated with protease activity and IVPD, suggesting the degradation of complex proteins. The highest levels of IVPD (90.12%) and protease activity (5546.78 U/g) were detected in GMBF fermented by S. cerevisiae MK-157, indicating the impact of fermentation on protein breakdown. The Lactobacillus E14-fermented RMBF exhibited 5521.45 U/g of protease activity and 87.54% IVPD. The mineral content of the fermented samples was substantially greater than that of the unfermented GMBF. The increased calcium, magnesium, potassium, sodium, iron, zinc, and copper levels for the GMBF fermented by S. cerevisiae MK-157 indicated that fermentation facilitated the release of minerals from chelated compounds. Microscopic examination revealed higher protein quantities in the fermented flour as well as hydrolyzed and broken starch granules embedded in the protein matrix. Large mash bean proteins were shown to be proteolyzed during fermentation into smaller peptides by SDS PAGE. Storage proteins were extensively broken down during fermentation with S. cerevisiae MK-157, whereas Lactobacillus E14 displayed a more focused pattern of hydrolysis. The nutritional profile of black mash bean flour was improved by fermentation with Lactobacillus E14 and S. cerevisiae MK-157.

Methanol gel electrophoresis: Separation of human fast and slow myosin light chain 1 and other myofibrillar protein isoforms on a single gel format.

This report describes a novel sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) resolving gel format that consistently yields the electrophoretic separation of the fast and slow isoforms of human sarcomeric myosin light chain 1 (MLC1). The inclusion of methanol as a constituent of the resolving gel impacted the electrophoretic mobility of proteins across a broad range of molecular masses. There was greater separation of the fast and slow isoforms of human MLC1, as well as separation and high resolution of fast and slow isoforms of the three myosin heavy chain isoforms that are expressed in human skeletal muscle on the same gel format. Furthermore, the same resolving gel format substantially altered the electrophoretic mobility of at least one isoform of tropomyosin in human striated muscle. It is possible that the inclusion of methanol in SDS-PAGE resolving gels could improve the separation of other proteins that are expressed in muscle and in other tissues and cell types.

Characterization of tryptanthrin as an antibacterial reagent inhibiting Vibrio splendidus

Isolates of Vibrio splendidus are ubiquitously presented in various marine environments, and they can infect diverse marine culture animals, leading to high mortality and economic loss. Therefore, a control strategy of the infection caused by V. splendidus is urgently recommended. Tryptanthrin is a naturally extracted bioactive chemical with antimicrobial activity to other bacteria. In this study, the effects of tryptanthrin on the bacterial growth and virulence-related factors of one pathogenic strain V. splendidus AJ01 were determined. Tryptanthrin (10 μg/mL) could completely inhibit the growth of V. splendidus AJ01. The virulence-related factors of V. splendidus AJ01 were affected in the presence of tryptanthrin. Tryptanthrin resulted an increase in biofilm formation, but lead to reduction in the motility and hemolytic activity of V. splendidus cells. In the cells treated with tryptanthrin, two distinctly differentially expressed extracellular proteins, proteases and flagellum, were identified using SDS–PAGE combined with LC–MS. Real-time reverse transcriptase PCR confirmed that the genes involved in the flagellar formation and hemolysin decreased, whereas specific extracellular proteases and the genes involved in the biofilm formation were upregulated. Two previously annotated luxOVs genes were cloned, and their expression levels were analyzed at different cell densities. Molecular docking was performed to predict the interaction between LuxOVs and ATP/tryptanthrin. The two sigma-54-dependent transcriptional regulators showed similar ATP or tryptanthrin binding capacity but with different sites, and the direct competitive binding between ATP and tryptanthrin was present only in their binding to LuxO1. These results indicated that tryptanthrin can be used as a bactericide of V. splendidus by inhibiting the growth, bacterial flagella, and extracellular proteases, but increasing the biofilm. Sigma-54-dependent transcriptional regulator, especially the quorum sensing regulatory protein LuxO1, was determined to be the potential target of tryptanthrin.Key points• Tryptanthrin inhibited the growth of V. splendidus in a dose-dependent manner.• The effect of tryptanthrin on the virulence factors of V. splendidus was characterized.• LuxO was the potential target for tryptanthrin based on molecular docking.

Molecular Characterization of Methicillin-resistant Staphylococcus aureus (MRSA) Isolates from Ruminants

Background: Methicillin-resistant Staphylococcus aureus (MRSA) infection is one of the leading causes of infections in animals as well as human beings and is associated with significant morbidity, mortality, length of stay and cost burden. Methods: In this study, 725 samples of nasal swabs and milk were collected randomly from cattle, buffalo, sheep and goats and these samples were inoculated on Mannitol salt agar mixed with Oxacillin Resistance Selective Supplement for the molecular characterization of MRSA isolates through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and polymerase chain reaction (PCR) test. Result: Out of 725 samples, 171 were found positive for Oxacillin resistant S. aureus. The SDS-PAGE showed different bands of molecular weight 13, 17, 20, 24, 26, 28, 33, 36, 39, 43, 47, 59, 64, 72, 86, 97 and 121 kDa. An amplified mec A DNA fragment (137) of 533 base pairs (bp) and PCR product of fem A (133 isolates) of 510 bp were detected in isolates.

Cold plasma for enhancing covalent conjugation of ovalbumin-gallic acid and its functional properties

The utilization of cold plasma (CP) treatment to promote covalent conjugation of ovalbumin (OVA) and gallic acid (GA), as well as its functionality, were investigated. Results demonstrated that CP significantly enhanced the covalent grafting of OVA and GA. The maximum conjugation of GA, 24.33 ± 2.24 mg/g, was achieved following 45 s of CP treatment. Covalent conjugation between GA and OVA were confirmed through analyses of total sulfhydryl (-SH) group, Fourier transform infrared (FTIR) spectroscopy, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Unfolding of the OVA molecule occurred upon conjugation with GA, as evidenced by multiple spectroscopy analyses. Additionally, conjugation with GA resulted in significant improvements in the antioxidant activity and emulsifying properties of OVA. This study demonstrated that CP is a robust and sustainable technique for promoting the covalent conjugate of polyphenols and proteins, offering a novel approach to enhance the functional properties of proteins.

High internal phase emulsions stabilized by physically modified mung bean protein isolates under different pHs

This research explored the impact of modified mung bean protein isolates (MBPIs) on the physicochemical, rheological, and structural properties of 70% oil-containing high internal phase emulsions (HIPEs). MBPIs (4% protein concentration) were subjected to heating (90 °C for 2 h) or sequential heating/ultrasonication (20 kHz, 120 W for 10 min) at different pHs (2, 7, and 10). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of modified MBPIs confirmed heat treatment at pH 2 hydrolyzed MBPI, unlike MBPIs heated at pH 7 and 10. Sequential heating/ultrasonication significantly reduced particle size, improved solubility and surface hydrophobicity of MBPIs, resulting in smaller droplet size and enhanced gel-like properties of HIPEs. Transmission electron microscopy revealed that sequentially heated/ultrasonicated MBPIs in the oil–water interface existed in a fibrillar or randomly aggregated structure at pH 2, a randomly aggregated structure at pH 7, and a particulate aggregated structure at pH 10. Overall, these structural modifications at varied pH conditions influenced MBPI adsorption at the HIPE droplet interface, impacting rheological features and promoting the potential use of protein-stabilized HIPEs across diverse applications.

Development of Drying Methodology for Intact Whole Buffalo Liver, Its Characterization, Shelf Life, and Evaluation of Palatability as Pet Treat

Globally, large quantity of animal byproducts is generated from the slaughter of food animals, but there is lack of research articles related to drying of these byproducts and its use as pet food. Therefore, this study was conducted with the aim of utilization of intact whole buffalo liver by drying for pet treat, evaluating its shelf life and palatability. The intact liver surface was superficially sliced, and the surface was pierced. Thereafter, the livers were pretreated in 3% sugar and 4% salt solution (1 : 3 w/v) for 3 h followed by microwaving for 4 min and hot air drying at 60°C for 40 h (designated as T2L). The livers which were dried the same as T2L except surface piercing were referred as T1L, whereas the livers dried only using hot air oven were referred as control (CL). The moisture and protein contents of the dried CL and T2L were found to be 28.46% and 14.29% and 43.85% and 52.76%, respectively. Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) image of T2L revealed the presence of few low as well as high molecular weight protein bands which were absent in CL and T1L indicating a comparatively lower level of protein degradation in T2L. The shelf life of T1L and T2L samples based on microbiological and lipid oxidation analyses was found to be more than 60 days at 25 and 4°C. Palatability studies using dogs showed that all dried samples were highly palatable. Thus, it could be concluded that intact buffalo liver could be dried using surface slicing, with/without piercing followed by salt and sugar pretreatment, 4 min microwaving, and hot air drying at 60°C for 40 h. Future study should focus on the sensory properties such as aroma, texture, and flavor and sensory analysis of the dried liver by human.