Isolation and genomic characterization of Vmp-1 using Vibrio mimicus as the host: A novel virulent bacteriophage capable of cross-species lysis against three Vibrio spp.

Vibrio mimicus is a zoonotic pathogen that is widely distributed in aquatic habitats/environments (marine coastal water, estuaries, etc). The development of biocontrol agents for V. mimicus is imperative for the prevention and control of aquatic animal diseases and human food-borne infections. In this study, a broad-spectrum bacteriophage Vmp-1 was isolated from dealt aquatic product in a local market by double-layer agar plate method using V. mimicus CICC21613 as the host bacteria. Results indicated that Vmp-1, which belongs to the family Podoviridae, showed good pH tolerance (pH 3.0-12.0) and thermal stability (30-50 °C). The optimal multiplicity of infection (MOI) of Vmp-1 was 0.001 for a 20-min incubation and 100-min lysis period. Vmp-1 effectively controlled V. mimicus CICC21613 in LBS model (MOI = 0.0001, 0.001, 0.01, 0.1, 1) within 8 h. The full length of the Vmp-1 genome was 43,312 bp, with average GC content of 49.5%, and a total of 44 protein-coding regions. This study provides a novel phage strain that has the highest homology with vB_VpP_HA5 (GenBank: OK585159.1, 95.96%) for the development of biocontrol agents for V. mimicus.

Active secretion of a thermostable transglutaminase variant in Escherichia coli.

BACKGROUND: Streptomyces mobaraenesis transglutaminase (smTG) is widely used to generate protein crosslinking or attachment of small molecules. However, the low thermostability is a main obstacle for smTG application. In addition, it is still hard to achieve the secretory expression of active smTG in E. coli, which benefits the enzyme evolution. In this study, a combined strategy was conducted to improve the thermostability and secretory expression of active smTG in E. coli. RESULTS: First, the thermostable S. mobaraenesis transglutaminase variant S2P-S23V-Y24N-S199A-K294L (TGm1) was intracellularly expressed in pro-enzyme form in E. coli. Fusing the pro-region of Streptomyces hygroscopicus transglutaminase (proH) and TrxA achieved a 9.78 U/mL of intracellular smTG activity, 1.37-fold higher than the TGm1 fused with its native pro-region. After in vitro activation by dispase, the TGm1 with proH yielded FRAPD-TGm1, exhibiting 0.95 â„ƒ and 94.25% increases in melting temperature and half-life at 60 â„ƒ compared to FRAP-TGm1 derived from the expression using its native pro-region, respectively. Second, the TGm1 with proH was co-expressed with transglutaminase activating protease and chaperones (DnaK, DnaJ, and GrpE) in E. coli, achieving 9.51 U/mL of intracellular FRAPD-TGm1 without in vitro activation. Third, the pelB signal peptide was used to mediate the secretory expression of active TGm in E. coli, yielding 0.54 U/mL of the extracellular FRAPD-TGm1. A script was developed to shuffle the codon of pelB and calculate the corresponding mRNA folding energy. A 1.8-fold increase in the extracellular expression of FRAPD-TGm1 was achieved by the Top-9 pelB sequence derived from the coding sequences with the lowest mRNA folding energy. Last, deleting the gene of Braun's lipoprotein further increased the extracellular yield of FRAPD-TGm1 by 31.2%, reached 1.99 U/mL. CONCLUSIONS: The stabilized FRAPD-smTG here could benefit the enzyme application in food and non-food sectors, while the E. coli system that enables secretory expression of active smTG will facilitate the directed evolution for further improved catalytic properties. The combined strategy (N-terminal modification, co-expression with chaperones, mRNA folding energy optimization of signal peptide, and lipoprotein deletion) may also improve the secretory expression of other functional proteins in E. coli.

Isolation and genomic characterization of P.A-5, a novel virulent bacteriophage against Enterobacter hormaechei.

Enterobacter hormaechei is a foodborne pathogen responsible for neonatal sepsis in humans and respiratory disease in animals. In this work, a new virulent phage (P.A-5) infecting E. hormaechei was isolated from domestic sewage samples and characterized. Transmission electron microscopy revealed that P.A-5 belonged to the family Myoviridae having a head size of 77.53 nm and a tail length of 72.24 nm. The burst size was 262 PFU/cell after a latent period of 20 min. Phage P.A-5 was able to survive in a pH range of 4-9 and resist temperatures up to 55 °C for 60 min. The genome sequence of P.A-5 had homology most similar to that of Shigellae phage MK-13 (GenBank: MK509462.1). Pork artificially contaminated with E. hormaechei was used as a model to evaluate the potential of P.A-5. The results clearly showed that P.A-5 treatment can completely inhibit E. hormaechei growth in pork within 8 h, indicating the potential use of P.A-5 as a biocontrol agent for E. hormaechei.

Isolation of virulent phages infecting dominant mesophilic aerobic bacteria in cucumber pickle fermentation.

Pickle is a type of mildly lactic acid fermented vegetable and is a traditional dish favored in China, Japan, and Korea. Corruption of spoilage bacteria and accumulation of nitrite during vegetable fermentation are common problems that affect the pickle industry and consumer health. In this work, cucumber juice was used as a vegetable model to study the dominant mesophilic aerobic bacteria (MAB) producing nitrite during pickle fermentation. Virulent phages infecting the dominant MABs combined with Lactobacillus plantarum M6 were used to control these bacteria. Enterobacter cloacae and Pseudomonas fluorescens are the dominant MABs in the fermentation of cucumber juice containing 4% or 8% NaCl, with isolation percentages reaching 30.6% and 23.1%, respectively. Virulent phages PspYZU5415 and EcpYZU01 were isolated using P. fluorescens J5415 and E. cloacae J01 as the host bacteria, respectively. These two phages show a broad host range and strong lytic activity, and their genomes contain no toxins and antibiotic resistance genes. PspYZU5415 and EcpYZU01 were combined into a cocktail (designated as Phage MIX) that effectively inhibits the growth of E. cloacae and P. fluorescens in cucumber juice with different salt concentrations. PhageMIX combined with L. plantarum M6 decreased the counts of P. mendocina and E. cloacae to undetectable levels at 48 h during the fermentation of cucumber juice artificially contaminated with P. mendocina and E. cloacae. In addition, nitrite content increased to 11.3 mg/L at 20 h and then degraded completely at 36 h. By contrast, P. mendocina and E. cloacae remained in the groups without PhageMIX during fermentation (0-48 h). Nitrite content rapidly increased to 65.7 mg/L at 12 h and then decreased to 21.6 mg/L at 48 h in the control group. This study suggests that PhageMIX combined with lactic acid bacterial strains can be used as an ecological starter for controlling the dominant MABs P. mendocina and E. cloacae and for reducing nitrate production during the early stage of pickle fermentation.

Three-dimensional porous Cu@Cu2O aerogels for direct voltammetric sensing of glucose.

Three-dimensional Cu@Cu2O aerogels with excellent electrocatalytic activity were prepared and used as electrode matrix for constructing novel electrochemical glucose sensors. The aerogels were obtained by adding a fresh solution of NaBH4 into a mixture of CuCl2 and NaOH aqueous solutions under stirring at room temperature. The aerogels were assembled with Cu or Cu2O nanoparticles. The materials show superfine spongy-like structures with large surface-to-volume ratio, numerous active sites and good solubility. The Cu@Cu2O aerogels show highly efficient electrochemical activity toward glucose oxidation with a relatively low-onset potential (0.25 V) in 0.1 M NaOH solution. This non-enzymatic glucose sensor offers a low detection limit of 0.6 µM (S/N = 3), a high sensitivity (195 mA M-1 cm-2), and two wide linear ranges (0.001-5.2 mM, 5.2-17.1 mM) at a working voltage of 0.6 V (vs. Ag/AgCl) in alkaline solution. While in neutral pH values, the respective data are a linear analytical range from 0.1 to 10 mM; a detection limit of 54 µM (S/N = 3) and a sensitivity of 12 mA M-1 cm-2 at scan rate of 100 mV s-1. The sensor possesses high selectivity, good reproducibility and long-time stability. It was utilized to determine glucose levels in (spiked) human serum samples, and satisfactory results were obtained. Graphical abstract Schematic presentation of a glassy carbon electrode modified with 3D porous Cu@Cu2O aerogels. The aerogels were obtained by a reduction reaction at room temperature (Scheme 1A). The aerogel networks were used to develop a highly sensitive electrochemical sensing platform for the detection of glucose (Scheme 1B).

Ultrafast and Energy-saving Synthesis of Nitrogen and Chlorine Co-doped Carbon Nanodots via Neutralization Heat for Selective Detection of Cr(VI) in Aqueous Phase.

In this work, it is presented for the first time that nitrogen and chlorine co-doped carbon nanodots (N,Cl-CDs) were synthesized by simply mixing glucose, concentrated hydrochloric acid (HCl), and 1,2-ethylenediamine (EDA). No external heat was employed; the neutralization reaction served as the heat source. The glucose served as the carbon source while EDA and HCl were the N and Cl dopants, respectively. The fluorescence of N,Cl-CDs was adequately quenched by hexavalent chromium Cr(VI) based on a combination of dynamic quenching and inner filter effect (IFE). Accordingly, an efficient N,Cl-CDs-based fluorescence probe was established for sensitive and selective detection of Cr(VI). The proposed fluorescence sensor provides a linear recognition range for Cr(VI) determination from 3 to 40 µM with a limit of detection (LOD) of 0.28 µM (14.6 µg/L). The proposed fluorescence method was successfully utilized to detect Cr(VI) in different water samples with satisfactory results. The spike recoveries vary from 97.01% to 103.89% with relative standard deviations (RSDs) of less than 0.82%. This work highlights the development of a simple, ultrafast, and energy-saving one-step synthetic route to fabricate N,Cl-CDs for highly selective and sensitive detection of Cr(VI) in real water samples. It is anticipated that the proposed fluorescence method could be further explored and widely used for Cr(VI) detection in the environmental industry.

Soluble expression and purification of the recombinant bioactive peptide precursor BPP-1 in Escherichia coli using a cELP-SUMO dual fusion system.

A bioactive peptide precursor (BPP-1, 14.3 kDa/115AA), a newly designed polypeptide that may exert a potential antihypertensive effect in vivo, is composed of many different ACE inhibitory peptides and antioxidant peptides tandemly linked according to the restriction sites of gastrointestinal proteases. In this report, we present a novel method to obtain soluble BPP-1 in Escherichia coli using cationic elastin-like polypeptide and SUMO (cELP-SUMO) tags. The cELP-SUMO-tagged fusion protein was expressed in soluble form at 20 °C for 20 h. After purification based on the inverse transition cycling (ITC) method, the purified cELP-SUMO-CFPP fusion protein was subsequently cleaved by a SUMO protease to release the mature BPP-1. After a subsequent simple salt precipitation process, approximately 167.2 mg of recombinant BPP-1 was obtained from 1 l of bacterial culture with at least 92% purity. The molecular mass (Mr) of the recombinant BPP-1 was confirmed by MALDI-TOF MS to equal 14,347. The purified BPP-1 was subjected to simulated gastrointestinal digestion, and the resulting hydrolysates exhibited notable ACE inhibitory and antioxidant activities in vitro. This report provides the first description of the soluble production of a bioactive peptide multimer with potential ACE inhibitory and antioxidant activities in E. coli using a cELP-SUMO tag.

Enhanced thermal stability of Pseudomonas aeruginosa lipoxygenase through modification of two highly flexible regions.

Lipoxygenase (LOX; EC 1.13.11.12) is an enzyme which is widely used in food industry to improve aroma and rheological or baking properties of foods. A series of studies have proven that the flexible regions negatively relates to the thermal stability of enzymes. In this study, two highly flexible regions, residues(20-49) and residues(201-206), were modified to improve the thermal stability of LOX from Pseudomonas aeruginosa. Deletion of the first 20 and 30 residues of the former region increased the thermal stability of the LOX by 1.3- and 2.1-fold, respectively. Although deletion of the residues(201-206) led to a sharp reduction of both thermal stability and catalytic activity of the enzyme, the residue substitutions with the glycines (G204P, G206P, and G204P/G206P) or even glycine-rich linker (L6/PT) within this region increased the thermal stability of LOX by values ranging from 0.46- to 3.45-fold. To be noted, over 85% of the specific activity was maintained in all thermally stabilized LOX mutants. Circular dichroism and fluorescence analysis showed that the overall secondary and tertiary structures were not significantly changed by these modifications. To the best of our knowledge, this is the first report on increasing the thermal stability of LOX by protein engineering without remarkably affecting the catalytic rate.

Thermal inactivation of a recombinant lipoxygenase from Pseudomonas aeruginosa BBE in the absence and presence of additives.

BACKGROUND: Bacterial lipoxygenase (EC 1.13.11.12, LOX) is an important enzyme used as a brightener and strengthening agent during breadmaking. In this study, thermal inactivation of a recombinant LOX of Pseudomonas aeruginosa BBE was characterized by kinetic and thermodynamic analysis in the absence and presence of additives. RESULTS: As the heating temperature increased from 25 to 55 °C, the thermal inactivation rate (k) values for LOX without the additives ranged from 0.0407 to 0.2627 min(-1), while the half-life (t1/2) values were between 17.08 and 3.25 min. The activation energy (ΔE) values were increased with rise in heating temperatures from 13.26 to 108.9 kJ mol(-1) . Separate tests at 45 °C in the presence of additives (polyols, sugars and ions) at specific concentrations showed that xylitol (1 mol L(-1)) was the most effective stabilizer for recombinant LOX and increased the t1/2 value by 297%. CONCLUSION: Recombinant LOX was sensitive to heat treatment, and addition of polyols, sugars and ions could enhance its thermal stability. Our findings may provide useful information for stabilizing emerging bacterial LOXs.

Acid-Heat-Induced Fabrication of Nisin-Loaded Egg White Protein Nanoparticles: Enhanced Structural and Antibacterial Stability.

As a natural cationic peptide, Nisin is capable of widely inhibiting the growth of Gram-positive bacteria. However, it also has drawbacks such as its antimicrobial activity being susceptible to environmental factors. Nano-encapsulation can improve the defects of nisin in food applications. In this study, nisin-loaded egg white protein nanoparticles (AH-NEn) were prepared in fixed ultrasound-mediated under pH 3.0 and 90 °C. Compared with the controls, AH-NEn exhibited smaller particle size (112.5 ± 2.85 nm), smaller PDI (0.25 ± 0.01), larger Zeta potential (24 ± 1.18 mV), and higher encapsulation efficiency (91.82%) and loading capacity (45.91%). The turbidity and Fourier transform infrared spectroscopy (FTIR) results indicated that there are other non-covalent bonding interactions between the molecules of AH-NEn besides the electrostatic forces, which accounts for the fact that it is structurally more stable than the controls. In addition, by the results of fluorescence intensity, differential scanning calorimetry (DSC), and X-ray diffraction (XRD), it was shown that thermal induction could improve the solubility, heat resistance, and encapsulation of nisin in the samples. In terms of antimicrobial function, acid-heat induction did not recede the antimicrobial activity of nisin encapsulated in egg white protein (EWP). Compared with free nisin, the loss rate of bactericidal activity of AH-NEn was reduced by 75.0% and 14.0% following treatment with trypsin or a thermal treatment at 90 °C for 30 min, respectively.

Formation mechanism, environmental sensitivity and functional characteristics of succinylated ovalbumin/ε-polylysine electrostatic complexes: The roles of succinylation modification and ε-polylysine combination.

Biocomplex materials formed by oppositely charged biopolymers (proteins) tend to be sensitive to environmental conditions and may lose part functional properties of original proteins, and one of the approaches to address these weaknesses is protein modification. This study established an electrostatic composite system using succinylated ovalbumin (SOVA) and ε-polylysine (ε-PL) and investigated the impact of varying degrees of succinylation and ε-PL addition on microstructure, environmental responsiveness and functional properties. Molecular docking illustrated that the most favorable binding conformation was that ε-PL binds to OVA groove, which was contributed by the multi­hydrogen bonding and hydrophobic interactions. Transmission electron microscopy observed that SOVA/ε-PL had a compact spherical structure with 100 nm. High-degree succinylation reduced complex sensitivity to heat, ionic strength, and pH changes. ε-PL improved the gel strength and antibacterial properties of SOVA. The study suggests possible uses of SOVA/ε-PL complex as multifunctional protein complex systems in the field of food additives.

Potential Role of Lauric Acid in Milk Fat Synthesis in Chinese Holstein Cows Based on Integrated Analysis of Ruminal Microbiome and Metabolome.

The composition and metabolic profile of the ruminal microbiome have an impact on milk composition. To unravel the ruminal microbiome and metabolome affecting milk fat synthesis in dairy cows, 16S rRNA and internal transcribed spacer (ITS) gene sequencing, as well as ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS/MS) methods were used to investigate the significant differences in ruminal bacterial and fungal communities as well as metabolome among Chinese Holstein cows with contrasting milk fat contents under the same diet (H-MF 5.82 ± 0.41% vs. L-MF 3.60 ± 0.12%). Another objective was to culture bovine mammary epithelial cells (BMECs) to assess the effect of metabolites on lipid metabolism. Results showed that the acetate-to-propionate ratio and xylanase activity in ruminal fluid were both higher in H-MF. Microbiome sequencing identified 10 types of bacteria and four types of fungi differently abundant at the genus level. Metabolomics analysis indicated 11 different ruminal metabolites between the two groups, the majority of which were lipids and organic acids. Among these, lauric acid (LA) was enriched in fatty acid biosynthesis with its concentration in milk fat of H-MF cows being greater (217 vs. 156 mg per 100 g milk), thus, it was selected for an in vitro study with BMECs. Exogenous LA led to a marked increase in intracellular triglyceride (TG) content and lipid droplet formation, and it upregulated the mRNA abundance of fatty acid uptake and activation (CD36 and ACSL1), TG synthesis (DGAT1, DGAT2 and GPAM), and transcriptional regulation (SREBP1) genes. Taken together, the greater relative abundance of xylan-fermenting bacteria and fungi, and lower abundance of bacteria suppressing short-chain fatty acid-producing bacteria or participating in fatty acid hydrogenation altered lipids and organic acids in the rumen of dairy cows. In BMECs, LA altered the expression of genes involved in lipid metabolism in mammary cells, ultimately promoting milk fat synthesis. Thus, it appears that this fatty acid plays a key role in milk fat synthesis.

Potential Application of Ovalbumin Gel Nanoparticles Loaded with Carvacrol in the Preservation of Fresh Pork.

Plant essential oil has attracted much attention in delaying pork spoilage due to its safety, but its low antibacterial efficiency needs to be solved by encapsulation. Our previous research had fabricated a type of ovalbumin gel nanoparticles loaded with carvacrol (OCGn-2) using the gel-embedding method, which had a high encapsulation rate and antibacterial activity. The main purpose of this study was to further evaluate the stability and slow-release characteristics of OCGn-2 and potential quality effects of the nanoparticles on the preservation of fresh pork pieces during 4 °C storage. The particle test showed that the nanoparticles had better heat stability below 85 °C and salt stability below 90 mM. The in vitro release study indicated that the carvacrol in OCGn-2 followed a Fickian release mechanism. The pork preservation test suggested that the OCGn-2 coating treatments could remarkably restrict the quality decay of pork slices compared to free carvacrol or a physical mixture of ovalbumin and carvacrol treatment. Nano-encapsulation of ovalbumin is beneficial to the sustained release, enhanced oxidation resistance, and improved antibacterial activity of carvacrol. The study suggested that ovalbumin gel nanoparticles embedded with carvacrol could be applied as an efficient bacterial active packaging to extend the storage life of pork.

Effects of dual succinylation and ultrasonication modification on the structural and functional properties of ovalbumin.

In this study, the functional properties of ovalbumin (OVA) were improved through dual modification with succinylation (succinylation degrees of 32.1 % [S1], 74.2 % [S2], and 95.2 % [S3]) and ultrasonication (ultrasonication durations of 5 min [U1], 15 min [U2], and 25 min [U3]), and the changes in protein structures were explored. Results showed that as the succinylation degree was increased, the particle size and surface hydrophobicity of S-OVA decreased by the maximum values of 2.2 and 2.4 times, respectively, causing emulsibility and emulsifying stability to increase by 2.7 and 7.3 times, respectively. After ultrasonic treatment, the particle size of succinylated-ultrasonicated OVA (SU-OVA) had decreased by 3.0-5.1 times relative to that of S-OVA. Moreover, the net negative charge of S3U3-OVA had increased to the maximum value of - 35.6 mV. These changes contributed to the further enhancement in functional indicators. The unfolding of the protein structure and the conformational flexibility of SU-OVA were illustrated and compared with those of S-OVA via protein electrophoresis, circular dichroism spectroscopy, intrinsic fluorescence spectroscopy, and scanning electron microscopy. The dually modified OVA emulsion (S3U3-E) presented small droplets (243.33 nm), reduced viscosity, and weakened gelation behavior that were indicative of even distribution, which was visually proven by confocal laser scanning microscopy images. Furthermore, S3U3-E exhibited favorable stability, a particle size that was almost unchanged, and a low polydispersity index (<0.1) over 21 days of storage at 4 °C. The above results demonstrated that succinylation combined with ultrasonic treatment could be an effective dual modification method for enhancing the functional performance of OVA.

Autoinduction AND Gate Inhibits Cell Lysis to Enhance Protein Production in Bacillus subtilis Controlled by Population Density and Cell Physiological State.

The extracellular protease-deficient strain Bacillus subtilis WB600 is commonly used as a chassis cell for the production of industrial proteins. However, B. subtilis WB600 exhibits an increased susceptibility to cell lysis and a reduction in biomass. Inhibition of cell lysis by knocking out lytic genes will impair physiological function. Here, we dynamically inhibited cell lysis in B. subtilis WB600 to balance the impairment of physiological function with the accumulation of biomass. First, the inducible protein degradation systems (IPDSs) were constructed and used to investigate the effects of inhibiting cell lysis on biomass, cell morphology, and protein production at different times (using pullulanase as a test). The highest pullulanase activity was obtained at 20 h of inhibiting cell lysis, 184.8 U/mL, which was 44% higher than the activity of B. subtilis WB600. Then, to avoid addition of inducers, we introduced orthogonal quorum sensing and constructed autoinduction protein degradation systems (AIPDSs). The optimized AIPDS showed similar pullulanase activity to the optimal IPDS (20 h), 181.3 U/mL. Next, we constructed dual-signal input autoinduction protein degradation systems (DSI-AIPDSs) via AND gate to further address two deficiencies of AIPDS, one-time activation and damage to new cells. These DSI-AIPDSs were controlled by quorum sensing and stationary phase promoters that respond to population density and single-cell physiological state, respectively. Finally, the OD600 and pullulanase activity of the strain with optimal DSI-AIPDS were 51% and 115% higher than those of B. subtilis WB600 in pullulanase production, respectively. We provided a B. subtilis chassis strain with considerable potential for biomass accumulation and enhanced protein production.

Comparison of different drying technologies for brocade orange (Citrus sinensis) peels: Changes in color, phytochemical profile, volatile, and biological availability and activity of bioactive compounds.

This study evaluated the effects of freeze drying (FD), heat pump drying (HPD), microwave drying (MD), and far-infrared drying (FID) on the quality of brocade orange peels (BOPs). Although the most attractive appearance, maximum levels of ascorbic acid (0.46 mg/g dry weight (DW)), carotenoids (total 16.34 µg/g DW), synephrine (15.58 mg/g DW), limonoids (total 4.60 mg/g DW), phenols (total 9142.80 µg/g DW), and antioxidant activity were observed in FD-BOPs, many aroma components in FD-BOPs were in the minimum levels. HPD-, and MD-BOPs depicted similar trends to FD-BOPs, but they contained the highest concentrations of limonene and ß-myrcene. Phenols and ascorbic acid in MD-BOPs generally featured the highest levels of bioavailability, being to 15.99% and 63.94%, respectively. In comparison, FID was not beneficial for the preservation of bioactive compounds and volatile. Therefore, considering time and energy costs, HPD and particularly MD are more appropriate for the commercial production of dried BOPs.

Enhanced Thermostability and Catalytic Activity of Streptomyces mobaraenesis Transglutaminase by Rationally Engineering Its Flexible Regions.

Streptomyces mobaraenesis transglutaminase can catalyze the cross-linking of proteins, which has been widely used in food processing. In this study, we rationally modified flexible regions to further improve the thermostability of FRAPD-TGm2 (S2P-S23V-Y24N-E28T-S199A-A265P-A287P-K294L), a stable mutant of the transglutaminase constructed in our previous study. First, five flexible regions of FRAPD-TGm2 were identified by molecular dynamics simulations at 330 and 360 K. Second, a script based on Rosetta Cartesian_ddg was developed for virtual saturation mutagenesis within the flexible regions far from the substrate binding pocket, generating the top 18 mutants with remarkable decreases in folding free energy. Third, from the top 18 mutants, we identified two mutants (S116A and S179L) with increased thermostability and activity. Finally, the above favorable mutations were combined to obtain FRAPD-TGm2-S116A-S179L (FRAPD-TGm2A), exhibiting a half-life of 132.38 min at 60 °C (t1/2(60 °C)) and a specific activity of 79.15 U/mg, 84 and 21% higher than those of FRAPD-TGm2, respectively. Therefore, the current result may benefit the application of S. mobaraenesis transglutaminase at high temperatures.

Enhanced Antibacterial Efficiency and Anti-Hygroscopicity of Gum Arabic-ε-Polylysine Electrostatic Complexes: Effects of Thermal Induction.

The aim of this investigation was to scrutinize the effects of a thermal treatment on the electrostatic complex formed between gum arabic (GA) and ε-polylysine (ε-PL), with the goal of improving the antibacterial properties and reducing the hygroscopicity of ε-PL. The heated complex with a ratio of 1:4 exhibited an encapsulation efficiency of 93.3%. Additionally, it had an average particle size of 350.3 nm, a polydispersity index of 0.255, and a zeta potential of 18.9 mV. The formation of the electrostatic complex between GA and ε-PL was confirmed through multispectral analysis, which demonstrated the participation of hydrogen bonding and hydrophobic and electrostatic interactions, as well as the enhanced effect of heat treatment on these forces within the complex. The complex displayed a core-shell structure, with a regular distribution and a shape that was approximately spherical, as observed in the transmission electron microscopy images. Additionally, the heated GA-ε-PL electrostatic composite exhibited favorable antibacterial effects on Salmonella enterica and Listeria monocytogenes, with reduced minimum inhibitory concentrations (15.6 µg/mL and 62.5 µg/mL, respectively) and minimum bactericidal concentrations (31.3 µg/mL and 156.3 µg/mL, respectively) compared to free ε-PL or the unheated electrostatic composite. Moreover, the moisture absorption of ε-PL reduced from 92.6% to 15.0% in just 48 h after being incorporated with GA and subsequently subjected to heat. This research showed a way to improve the antibacterial efficiency and antihygroscopicity of ε-PL, reducing its application limitations as an antimicrobial substance to some extent.

Multi-omics reveals the increased biofilm formation of Salmonella Typhimurium M3 by the induction of tetracycline at sub-inhibitory concentrations.

Exposure to sub-inhibitory concentrations (sub-MICs) of antibiotics could induce the biofilm formation of microorganisms, but its underlying mechanisms still remain elusive. In the present work, biofilm formation by Salmonella Typhimurium M3 was increased when in the presence of tetracycline at sub-MIC, and the highest induction was observed with tetracycline at 1/8 MIC. The integration of RNA-sequencing and untargeted metabolomics was applied in order to further decipher the potential mechanisms for this observation. In total, 439 genes and 144 metabolites of S. Typhimurium M3 were significantly expressed after its exposure to 1/8 MIC of tetracycline. In addition, the co-expression analysis revealed that 6 genes and 8 metabolites play a key role in response to 1/8 MIC of tetracycline. The differential genes and metabolites were represented in 12 KEGG pathways, including five pathways of amino acid metabolism (beta-alanine metabolism, tryptophan metabolism, arginine and proline metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, and glutathione metabolism), three lipid metabolism pathways (biosynthesis of unsaturated fatty acids, fatty acid degradation, and fatty acid biosynthesis), two nucleotide metabolism pathways (purine metabolism, and pyrimidine metabolism), pantothenate and CoA biosynthesis, and ABC transporters. Metabolites (anthranilate, indole, and putrescine) from amino acid metabolism may act as signaling molecules to promote the biofilm formation of S. Typhimurium M3. The results of this work highlight the importance of low antimicrobial concentrations on foodborne pathogens of environmental origin.

Acquisition, loss, and replication of functional modules promote the genetic diversity of Salmonella bacteriophages.

Owing to the threats that Salmonella poses to public health and the abuse of antimicrobials, bacteriophage therapy against Salmonella is experiencing a resurgence. Although several phages have been reported as safe and efficient for controlling Salmonella, the genetic diversity and relatedness among Salmonella phages remain poorly understood. In this study, whole-genome sequences of 91 Salmonella bacteriophages were obtained from the National Center for Biological Information genome database. Phylogenetic analysis, mosaic structure comparisons, gene content analysis, and orthologue group clustering were performed. Phylogenetic analysis revealed four singletons and two major lineages (I-II), including five subdividing clades, of which Salmonella phages belonging to morphologically distinct families were clustered in the same clade. Chimeric structures (n = 31), holin genes (n = 18), lysin genes (n = 66), DNA packaging genes (n = 55), and DNA metabolism genes (n = 24) were present in these phages. Moreover, phages from different subdivided clusters harboured distinct genes associated with host cell lysis, DNA packaging, and DNA metabolism. Notably, phages belonging to morphologically distinct families shared common orthologue groups. Although several functional modules of phages SS1 and SE16 shared > 99% nucleotide sequence identity with phages SI2 and SI23, the major differences between these phages were the absence and replication of functional modules. The data obtained herein revealed the genetic diversity of Salmonella phages at genomic, structural, and gene content levels. The genetic diversity of Salmonella phages is likely owing to the acquisition, loss, and replication of functional modules.