Broad-spectrum cytotoxicity to cancer cells of Brevilaterin C from Brevibacillus laterosporus and its specific mechanism on human epidermal cancer cells.

Antimicrobial peptides (AMP) from Brevibacillus laterosporus have good prospects as clinical treatments for cancer. Nevertheless, details about their anticancer spectrum and mode of cytotoxicity remain poorly understood. A newly found AMP (named Brevilaterin C) secreted by B. laterosporus S62-9 exhibited strong inhibition on almost cancer cell lines examined at a concentration of 8 µg/ml but was relatively safe for normal cells. We further systematically examined its cytotoxicity and mechanism toward human epidermal cancer cell A431. A dosage of 3 µg/ml of Brevilaterin C could significantly increase lactate dehydrogenase release of tumor cells. Moreover, it could remarkably increase the ratio of apoptosis and reactive oxygen species generation of A431, indicating effective induction of apoptosis. Moreover, the formation of JC-1 aggregates was effectively prevented by a low concentration of Brevilaterin C, indicating its effective induction of A431's apoptosis. Brevilaterin C exhibited broad-spectrum cytotoxicity to cancer cells, indicating a good potential prospect in the medical field.

An overview on antioxidant peptides from rice bran proteins: extraction, identification, and applications.

Rice by-products, generated through the milling processes, have recently been recognized as a potential source of bioactive compounds, such as proteins, essential amino acids, and phenolics. Owing to their antioxidant capacity (which improve the storage stability of foods), these compounds have gained much attention because of their beneficial impacts on human health. It has to be noted that large quantities of rice by-products are not efficiently utilized, which may result in industrial wastes and environmental consequences. Thence, the aim of this review is to provide a comprehensive insight on the antioxidant capabilities, extraction, identification, functional attributes, and applications of bioactive hydrolysates and peptides derived from rice bran protein. This overview would provide an insight on rice bran proteins, which are abundant in bioactive peptides, and could be used as value-added products in food and pharmaceutical applications. Inclusion of bioactive peptides to prevent food spoilage while maintaining food safety has also been highlighted.

An accurate strategy for pointing the key biocatalytic sites of bre2691A protein for modification of the brevilaterin from Brevibacillus laterosporus.

BACKGROUND: Brevilaterin A-E, a novel class of multi-component cationic antimicrobial lipopeptides, were biosynthesized by a non-ribosomal peptides synthetase (NRPS) in Brevibacillus laterosporus. However, the antimicrobial abilities of different brevilaterin components varied greatly, and this multi-component form was impeding the scale production of the excellent component, and a little information about the brevilaterin biosynthesis mechanism was available to apply in brevilaterin design modification. In this study, we used an accurate strategy that revealed the reason for producing multi-component was the substrate selectivity of bre2691A protein being not enough specific and pinpointed the key design sites to make the specificity of bre2691A enhanced. RESULTS: Bioinformatic analysis revealed that the biocatalytic site of bre2691A, which was an adenylation domain catalyzed and recognized methionine, leucine, valine and isoleucine and thus introduced them into brevilaterins and caused different components (brevilaterin A-E), was consisted of A1 ~ A10 residues named specificity-conferring code. Coupling molecular docking simulations with mutation studies identified A2 and A7 as critical residues, where determined substrate-specificity and impacted activity. The in virto activity assay showed that the A2 mutant (G193A) would lose activity against methionine and have no effect on the other three amino acids, the A7 mutant (G285C) would enhance the catalytic activity against four substrates, especially against leucine at almost a double activity. When the A2 and A7 residues were synchronously mutated, this mutant would be more focused on recognizing leucine. CONCLUSIONS: An accurate strategy that combined with bioinformatics and site-directed mutation techniques revealed the pivotal site A2 and A7 positions of bre2691A protein that could be used to design and modify brevilaterins, thus further providing a reasonable direction of genetic engineering for Brevibacillus laterosporus. A deeper understanding of the function of crucial residues in the adenylation domain would make it get more accurate and highly efficient design and more fully utilized. Furthermore, it would contribute to biotechnological applications, namely for the large centralized synthesis of antimicrobial peptides, or for the optimization of their production.

Broad-spectrum antifungal activity of lipopeptide brevilaterin B and its inhibition effects against Fusarium oxysporum and Penicillium chrysogenum.

AIMS: Brevilaterin B is a natural antimicrobial lipopeptide produced by Brevibacillus laterosporus S62-9. However, its antifungal spectrum and modes of action are still unclear. Herein, we investigated the detailed antifungal activity of brevilaterin B against 33 pathogenic fungi and the antifungal effects against two sensitive fungi in vitro and in vivo. METHODS AND RESULTS: Brevilaterin B exhibited inhibitory activity against 33 pathogenic fungi involved in plant disease and food spoilage at the minimum inhibitory concentrations (MICs) range of 16-128 µg ml-1 . The antifungal effects were further studied by Fusarium oxysporum and Penicillium chrysogenum. Both spore germination and mycelium growth were inhibited by brevilaterin B at sub-MIC. Transmission electron microscopy and fluorescent dye staining assays indicated brevilaterin B damaged cell integrity and induced apoptosis. In vivo tests, brevilaterin B inhibited the infection of F. oxysporum to Dendrobium officinale and P. chrysogenum to mandarin (Citrus reticulata) at 500 µg ml-1 , respectively. CONCLUSIONS: Brevilaterin B showed broad-spectrum antifungal activity against 33 pathogenic fungi. And its antifungal modes of action were proposed as damaging cell integrity and inducing cell apoptosis. The lipopeptide is promising to control F. oxysporum in the D. officinale and P. chrysogenum in the mandarin. SIGNIFICANCE AND IMPACT OF STUDY: The research provided insights into antifungal modes of action of brevilaterin B. The lipopeptide brevilaterin B is potential to be developed as a broad-spectrum antifungal agent for agricultural biocontrol and postharvest storage.

Brevilaterin B from Brevibacillus laterosporus has selective antitumor activity and induces apoptosis in epidermal cancer.

Brevilaterins as antimicrobial peptides (AMPs) secreted by a newly discovered species Brevibacillus laterosporus, had been demonstrated to display excellent antibacterial and antifungal activities; however, very limited information about their new bioactivity was ever developed. Herein, we discovered Brevilaterin B, an AMP produced by Br. laterosporus S62-9, exhibited a new anticancer activity and investigated its anticancer details. Proliferation, membrane permeability and apoptotic rate of cell lines were studied by methods of CCK-8 Assay, LDH Assay and Annexin V-FITC/PI Kits, respectively. ROS levels and mitochondrial membrane potential of tested cells were further detected through the fluorescent probes DCFH-DA and JC-1. Brevilaterin B exhibited broad-spectrum anticancer activity in a dose-dependent manner. It selectively inhibited the proliferation of epidermal cancer cell A431 but had no effect on its control normal cells in a dose of 2.0 µg/mL. In comparision, typical morphological characteristics of apoptosis and an apoptotic ratio of 71.0% in A431 were observed after treatment by 2.0-3.0 µg/mL of Brevilaterin B. The ROS levels increased by 21.3% and mitochondrial membrane potential reduced by 48.8% from A431 were further occurred, indicating Brevilaterin B's anticancer action was mainly focus on the mitochondrion of cancer cells. In total, Brevilaterin B we reported above maybe believed to be a potential application as an anticancer medicament, increasing its commercial value.

Antibacterial mechanism of brevilaterin B: an amphiphilic lipopeptide targeting the membrane of Listeria monocytogenes.

Antimicrobial peptides (AMPs) are recognized as promising safe alternatives to antibiotics for its low drug-resistance. Brevilaterin B, a newly discovered antimicrobial lipopeptide produced by Brevibacillus laterosporus S62-9, exhibits efficient antibacterial activity on Listeria monocytogenes with a minimum inhibitory concentration of 1 µg mL-1. The present research aimed to investigate the antibacterial mechanism of brevilaterin B against Listeria monocytogenes. Brevilaterin B caused membrane depolarization and the breakup of the cytomembrane as measured by 3,3-dipropylthiadicarbocyanine iodide and transmission electron microscopy, respectively. Using 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and 1,2-dipalmitoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (7:3) as a model membrane, results proved that brevilaterin B could bind to liposomes, integrate into the lipid bilayer, and consequently increase the permeability of liposomes to calcein. The secondary structure of brevilaterin B also changed from an unstructured coil to a mainly ß-sheet conformation as measured by circular dichroism. Brevilaterin B exhibits antibacterial activity by a membrane interaction mechanism, which provides a theoretical basis for using brevilaterin B as a promising natural and effective antimicrobial agent against pathogenic bacteria. KEY POINTS: • Brevilaterin B exhibited antibacterial activity against Listeria monocytogenes. • Brevilaterin B exhibited membrane interaction mechanism. • Brevilaterin B showed conformational change when interacted with liposome.

Purification and characterization of a new xylanase with excellent stability from Aspergillus flavus and its application in hydrolyzing pretreated corncobs.

A new extracellular xylanase was purified from a non-toxic mesophilic fungus Aspergillus flavus, and characterized as the ß-1, 4-endoxylanase (designated as AfXynA) that appeared in a single protein band on SDS-PAGE with a molecular mass of 20.2 kDa, which is different from all other reported xylanases from the same strain. The AfXynA exhibited a specific activity of 838.2 U/mg. Its optimal temperature and pH were determined to be 55 °C and 7.5, respectively. It was stable up to 50 °C and within pH 3.5-10.5. AfXynA also exhibited an excellent tolerance to various proteases. This new xylanase had an endohydrolytic mode of action and could hydrolyze xylotriose to xylobiose through transglycosylation. It could efficiently degrade xylan to mainly yield xylobiose, xylotriose, xylopentose and xylohexaose. In addition, the AfXynA was effective in hydrolyzing pretreated corncobs, and shows a great potential in the production of xylooligosaccharides. These unique enzymatic properties make the AfXynA attractive for more biotechnological applications.

Characterization of a novel xylanase from Aspergillus flavus with the unique properties in production of xylooligosaccharides.

A novel xylanase from the filamentous fungus Aspergillus flavus was purified and characterized as the ß-1, 4-endoxylanase (designed as AfXynB) with a molecular mass (32.2 kDa), which is different from all of the previously reported xylanases from the same strain. AfXynB was optimally active at pH 7.5 and 55 °C, respectively. It was stable up to 50 °C within range of pH 4.0-9.5, and displayed an excellent tolerance to various cations, reagents, and proteases. AfXynB showed specific activity toward beechwood xylan but no detected activity toward CMC and pNP-ß-D-xylopyranoside. The xylanase is a typical endo-xylanase; it could hydrolyze beechwood xylan to only yield xylobiose (X2) and xylopentaose (X5). Actually, this may be the first report for the endo-xylanases that displayed such a unique hydrolytic property. These findings in the present study have great implications for its future applications of the novel xylanase.

Purification and characterization of a novel ß-glucosidase from Aspergillus flavus and its application in saccharification of soybean meal.

Aspergillus flavus has been regarded as a potential candidate for its production of industrial enzymes, but the details of ß-glucosidase from this strain is very limited. In herein, we first reported a novel ß-glucosidase (AfBglA) with the molecular mass of 94.2 kDa from A. flavus. AfBglA was optimally active at pH 4.5 and 60 °C and is stable between pH 3.5 and 9.0 and at a temperature of up to 55 °C for 30 min remaining more than 90% of its initial activity. It showed an excellent tolerance to Trypsin, Pepsin, Compound Protease, and Flavourzyme and its activity was not inhibited by specific certain cations. AfBglA displayed broad substrate specificity, it acted on all tested pNP-glycosides and barley glucan, indicating this novel ß-glucosidase exhibited a ß-1, 3-1, 4-glucanase activity. Moreover, the AfBglA could effectively hydrolyze the soybean meal suspension into glucose and exhibit a strong tolerance to the inhibition of glucose at a concentration of 20.0 g/L during the saccharification. The maximum amount of the glucose obtained by AfBglA corresponded to 67.0 g/kg soybean meal. All of these properties mentioned above indicated that the AfBglA possibly attractive for food and feed industry and saccharification of cellulolytic materials.

Decellularized ovine arteries as biomatrix scaffold support endothelial of mesenchymal stem cells.

The differentiation rate of adipose-derived mesenchymal stem cells (Ad-MSCs) into endothelial cells is always lower under normal condition, which limits further clinical application of Ad-MSCs for angiogenesis regenerative medicine and needs to be enhanced. In the present study, the tissue-specific-derived decellularized ovine arteries matrix (DCS) was used as scaffold to investigate the pro-endothelial differentiation ability of decellularized ovine arteries matrix as well as the underlying mechanisms. The prepared decellularized ovine arteries matrix by the combination of enzymatic and chemical decellularization approaches preserved macroscopic 3D architecture, native composition and ultrastructure of natural ovine arteries. The RT-PCR, histopathological and immunofluorescence assay results suggested that DCS could increase the proliferation ability of MSC. What's more, the DCS could also induce the endothelial differentiation of MSC, which was further enhanced by adding VEGF. Our results showed that natural 3D matrix from decellularized ovine arteries could induce the endothelial differentiation of AD-MSCs alone or with the combination of VEGF. Our results indicated that the decellularized ovine arteries matrix would serve as an efficient culture system for promoting endothelial differentiation of Ad-MSCs.

[Study on the choice of functional monomer before preparation of myclobutanil molecularly imprinted polymer].

To prepare myclobutanil molecularly imprinted polymer, a method was established for the choice of the appropriate functional monomer and its dosage. UV spectra was applied to study the combination form, the effect intensity, the optimal concentration ratio and the numbers of binding sites between myclobutanil and methyl acrylic acid (MAA) or acrylamide (AM) functional monomer. The results showed that hydrogen-bonding interaction could be formed between myclobutanil and methyl acrylic acid (MAA) or acrylamide (AM) functional monomer. The pi electron of the triazole ring conjugated double bond in my clobutanil could transit to pi* conjugate antibonding orbital when it absorbed energy. The formation of hydrogen bond could make pi-->pi* absorption band transit. Maximum absorption wavelength produced red shift with the increase in the functional monomer concentration in the system. The research revealed that the optimal concentration ratios between myclobutanil and the two monomers were c(M):c(MAA) = 1:4, c(M):c(AM) = 1:2. Myclobutanil and the both the functional monomers had the bonding ability, and strong bonding force. The prepared molecularly imprinted polymer using AM as a functional monomer had better stability and specificity of recognition for myclobutanil.

Inactivation mechanism of phenyllactic acid against Bacillus cereus spores and its application in milk beverage.

Phenyllactic acid (PLA) as a natural phenolic acid exhibits antibacterial activity against non-spore-forming bacteria, while the inhibitory effect against bacterial spore remained unknown. Herein, this study investigated the inactivation effect of PLA against Bacillus cereus spores. The results revealed that the minimum inhibitory concentration of PLA was 1.25 mg/mL. PLA inhibited the outgrowth of germinated spores into vegetative cells rather than germination of spores. PLA disrupted the spore coat, and damaged the permeability and integrity of inner membrane. Moreover, PLA disturbed the establishment of membrane potential due to the inhibition of oxidative metabolism. SEM observations further visualized the morphological changes and structural disruption caused by PLA. Besides, PLA caused the degradation of DNA of germinated spores. Finally, PLA was applied in milk beverage, and showed promising inhibitory effect against B. cereus spores. This finding could provide scientific basis for the application of PLA against spore-forming bacteria in food industry.

Preparation, Isolation and Antioxidant Function of Peptides from a New Resource of Rumexpatientia L. ×Rumextianshanicus A. Los.

Rumexpatientia L. ×Rumextianshanicus A. Los (RRL), known as "protein grass" in China, was recognized as a new food ingredient in 2021. However, the cultivation and product development of RRL are still at an early stage, and no peptide research has been reported. In this study, two novel antioxidant peptides, LKPPF and LPFRP, were purified and identified from RRL and applied to H2O2-induced HepG2 cells to investigate their antioxidant properties. It was shown that 121 peptides were identified by ultrafiltration, gel filtration chromatography, and LC-MS/MS, while computer simulation and molecular docking indicated that LKPPF and LPFRP may have strong antioxidant properties. Both peptides were not cytotoxic to HepG2 cells at low concentrations and promoted cell growth, which effectively reduced the production of intracellular ROS and MDA, and increased cell viability and the enzymatic activities of SOD, GSH-Px, and CAT. Therefore, LKPPF and LPFRP, two peptides, possess strong antioxidant activity, which provides a theoretical basis for their potential as food additives or functional food supplements, but still need to be further investigated through animal models as well as cellular pathways.

Pharmacokinetics, Pharmacodynamics, Safety, and Tolerability of Stapokibart in Healthy Volunteers and Adult Subjects with Atopic Dermatitis.

INTRODUCTION: Stapokibart, a novel humanized anti-interleukin (IL)-4 receptor alpha monoclonal antibody, inhibits the signaling of IL-4 and IL-13, which are key drivers of type 2 inflammation in atopic dermatitis (AD). This study aimed to assess the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of stapokibart in a randomized, double-blind, placebo-controlled single ascending dose (SAD) study and a multiple ascending dose (MAD) study. METHODS: The SAD study enrolled 33 healthy male adults aged 18-65 years at a single center. The MAD study enrolled 39 patients with moderate-to-severe AD aged 18-70 years at seven centers. Enrolled subjects were randomized to subcutaneous (SC) doses of stapokibart (75-600 mg) or placebo. Serum thymus and activation-regulated chemokine (TARC) and total immunoglobulin E (IgE) were measured as PD biomarkers for stapokibart. RESULTS: Similar PK characteristics were observed in healthy volunteers and subjects with AD after the initial administration. Stapokibart exhibited non-linear pharmacokinetics in both types of subjects. Following single doses, the mean maximum serum concentration (Cmax) ranged from 5.3 to 63.0 µg/mL, median Tmax ranged from 3.0 to 7.0 days, mean terminal half-life (t1/2z) ranged from 2.39 to 7.43 days, and mean apparent volume (Vz/F) ranged from 3.64 to 6.73 L in healthy subjects. The mean AUC accumulation ratio was 2.29 in subjects with AD after three doses of stapokibart 300 mg administered every 2 weeks. The median serum total IgE and TARC levels on day 43 decreased from baseline by 14.9-25.2% and 48.6-77.0%, respectively, among subjects with AD receiving three doses of stapokibart. No subjects developed grade ≥ 3 adverse events (AEs) or serious AEs or discontinued the study because of AEs. The incidence of AEs was similar between stapokibart and placebo groups. CONCLUSION: Stapokibart showed favorable pharmacokinetics, pharmacodynamics, safety, and tolerability in the SAD and MAD studies. Based on these results, phase II and phase III trials of stapokibart have been performed in subjects with moderate-to-severe AD. TRIAL REGISTRATION: ClinicalTrials.gov Identifier NCT06161090 (29 November, 2023), NCT04893941 (15 May, 2021).

Efficacy and safety of CM310 in moderate-to-severe atopic dermatitis: A multicenter, randomized, double-blind, placebo-controlled phase 2b trial.

BACKGROUND: Atopic dermatitis (AD) affects approximately 10% of adults worldwide. CM310 is a humanized monoclonal antibody targeting interleukin-4 receptor alpha that blocks interleukin-4 and interleukin-13 signaling. This trial aimed to evaluate the efficacy and safety of CM310 in Chinese adults with moderate-to-severe AD. METHODS: This multicenter, randomized, double-blind, placebo-controlled, phase 2b trial was conducted in 21 medical institutions in China from February to November 2021. Totally 120 eligible patients were enrolled and randomized (1:1:1) to receive subcutaneous injections of 300 mg CM310, 150 mg CM310, or placebo every 2 weeks for 16 weeks, followed by an 8-week follow-up period. The primary endpoint was the proportion of patients achieving ≥75% improvement in the Eczema Area and Severity Index (EASI-75) score from baseline at week 16. Safety and pharmacodynamics were also studied. RESULTS: At week 16, the proportion of EASI-75 responders from baseline was significantly higher in the CM310 groups (70% [28/40] for high-dose and 65% [26/40] for low-dose) than that in the placebo group (20%[8/40]). The differences in EASI-75 response rate were 50% (high vs . placebo, 95% CI 31%-69%) and 45% (low vs . placebo, 95% CI 26%-64%), with both P values <0.0001. CM310 at both doses also significantly improved the EASI score, Investigator's Global Assessment score, daily peak pruritus Numerical Rating Scale, AD-affected body surface area, and Dermatology Life Quality Index compared with placebo. CM310 treatment reduced levels of thymus and activation-regulated chemokine, total immunoglobulin E, lactate dehydrogenase, and blood eosinophils. The incidence of treatment-emergent adverse events (TEAEs) was similar among all three groups, with the most common TEAEs reported being upper respiratory tract infection, atopic dermatitis, hyperlipidemia, and hyperuricemia. No severe adverse events were deemed to be attributed to CM310. CONCLUSION: CM310 at 150 mg and 300 mg every 2 weeks demonstrated significant efficacy and was well-tolerated in adults with moderate-to-severe AD.

Finite-Level Quantized Min-Consensus Control Based on Encoding-Decoding.

This article studies the min-consensus control of continuous-time real-valued multiagent systems, with sampled information, quantized communication, and switching topologies. Due to the limited bandwidth of the digital communication network, only finite-bit binary symbolic sequence can be exchanged among the agents. In order to realize the min-consensus control with quantized communication and limited bandwidth, a novel finite-level biased quantizer and a nonstrict decreasing scaling function are designed, and correspondingly a set of switching encoders and decoders are constructed. By means of the proposed encoders and decoders, the according sampled-data min-consensus control inputs are carefully constructed, and the memory variables are introduced into the control inputs and are monotonically decreasing no matter how the communication topology is switched. The proposed encoding-decoding-based control scheme can achieve accurate min-consensus with limited bandwidth, as long as the communication graphs are jointly strongly connected. The numerical simulations show the effectiveness of the proposed control scheme.

Novel Property Cytotoxicity and Mechanism of Food Preservative Brevilaterins against Human Gastric Cancer Cells.

Brevilaterins, antimicrobial peptides produced by Brevibacillus laterosporus, are regarded as excellent food preservatives and are popular as antimicrobial applications. Recent research has uncovered their potent cytotoxic effects against diverse cancer cells, thereby underscoring the pressing need for more extensive and intensive investigations into this use. In this study, we explored their novel function in inducing cytotoxicity to cancer cells and systematically investigated the mechanism of action of Brevilaterin B/C (BB/BC) in vivo. Proliferation, membrane permeability, and apoptotic rate were evaluated using CCK-8 assay, LDH assay, and Annexin V-FITC/PI kits. ROS levels and mitochondrial membrane potential were detected using the fluorescent probe DCFH-DA and JC-1. Our results demonstrated that both BB and BC at concentrations of 4-6 µg/mL significantly inhibited the proliferation and migration of gastric cancer cells BGC-823. Treatment with 4 µg/mL of BB/BC rapidly increased LDH levels in the supernatant of BGC-823 cells, leading to further investigation of the mechanism of apoptosis. We found that the apoptotic rate of BGC-823 cells significantly increased upon treatment with BB/BC, demonstrating their potent induction of apoptosis. BB/BC-induced ROS production in BGC-823 cells impaired their growth and induced apoptosis, indicating a close association between apoptosis and ROS elevation. Additionally, JC-1 aggregates rapidly accumulated after treatment with 4 µg/mL of BB/BC, suggesting changes in mitochondrial membrane potential and early apoptosis. Taken together, our findings revealed that BB and BC exhibit significant anticancer effects against gastric cancer cells, highlighting the promising potential of Brevilaterins as anticancer agents.

Dietary Supplementation of Brevibacillus laterosporus S62-9 Improves Broiler Growth and Immunity by Regulating Cecal Microbiota and Metabolites.

Brevibacillus laterosporus has been added as a direct-fed microbiota to chicken. Yet, few studies have reported the effects of B. laterosporus on broiler growth and gut microbiota. The aim of this study was to evaluate the effects of B. laterosporus S62-9 on growth performance, immunity, cecal microbiota, and metabolites in broilers. A total of 160 1-day-old broilers were randomly divided into S62-9 and control groups, with or without 106 CFU/g B. laterosporus S62-9 supplementation, respectively. During the 42 days feeding, body weight and feed intake were recorded weekly. Serum was collected for immunoglobulin determination, and cecal contents were taken for 16S rDNA analysis and metabolome at Day 42. Results indicated that the broilers in S62-9 group showed an increase in body weight of 7.2% and 5.19% improvement in feed conversion ratio compared to the control group. The B. laterosporus S62-9 supplementation promoted the maturation of immune organs and increased the concentration of serum immunoglobulins. Furthermore, the α-diversity of cecal microbiota was improved in the S62-9 group. B. laterosporus S62-9 supplementation increased the relative abundance of beneficial bacteria including Akkermansia, Bifidobacterium, and Lactobacillus, while decreased the relative abundance of pathogens including Klebsiella and Pseudomonas. Untargeted metabolomics revealed that 53 differential metabolites between the two groups. The differential metabolites were enriched in 4 amino acid metabolic pathways, including arginine biosynthesis and glutathione metabolism. In summary, B. laterosporus S62-9 supplementation could improve the growth performance and immunity through the regulation of gut microbiota and metabolome in broilers.

Expression and Transformation Characteristics of a Novel Glutamic Acid Decarboxylase LcGAD10s and Its Application on Sufu Processing.

Gamma-aminobutyric acid (GABA) is an important non-proteinogenic amino acid and a potent bioactive compound with many anti-hypertensive and anti-depressant activities. The bioconversion of GABA by glutamic acid decarboxylase (GAD) has been eagerly studied. Herein, novel pyridoxal-5-phosphate monohydrates (PLP)-dependent GAD, which is not quite similar to reporting, was cloned from Latilactobacillus curvatus and efficiently expressed in E. coli. The conveniently purified GAD (designated LcGAD10s) appeared as a single protein on SDS-PAGE with a molecular mass of 52.0 kDa. LcGAD10s exhibited a specific activity of 303.7 U/mg after purification by Ni-IDA affinity chromatography, with optimal activity at 55 °C and pH 5. LcGAD10s displayed excellent temperature (50 °C) and pH (4-8) stability which relative activity above 80% and 70%, respectively. The enzymatic activity was, respectively, increased and depressed by 130%, and 24% in the presence of Mn+ and Cu2+. Enzyme activity over 90% can be achieved by adding at least 25 mM of PLP. LcGAD10s was able to efficiently transform 15 g/L GABA with a single-factor optimized reaction of pH (5), temperature (50 °C), time (2 h), LcGAD10s dosage (0.4 U) and monosodium glutamate level (5 g/L). Additionally, LcGAD10s can be applied to a tofu fermentation system to achieve GABA conversion and achieved 14.9 mg/g of GABA conversion when added at 2 U/mL, which is higher than most of the commercial sufu and previous application reports, increasing its functional substances.

Expression and characterization of a protease-resistant ß-d-fructofuranosidase BbFFase9 gene suitable for preparing invert sugars from soybean meal.

A novel gene (BbFFase9), with an ORF of 1557 bp that encodes ß-d-fructofuranosidase from Bifidobacteriaceae bacterium, was cloned and expressed in Escherichia coli. The recombinant protein (BbFFase9) was successfully purified and showed a single band with a molecular mass of 66.2 kDa. This was confirmed as a ß-d-fructofuranosidase and exhibited a high specific activity of 209.2 U/mg. Although BbFFase9 was a soluble protein, it exhibited excellent tolerance to proteases such as pepsin, trypsin, acidic protease, neutral protease and Flavourzyme®, indicating its potential applicability in different fields. BbFFase9 exhibited typical invertase activity, and highly catalyzed the hydrolysis of the α1↔2ß glycosidic linkage in molecules containing fructosyl moieties but with no detectable fructosyltransferase activity. It was optimally active at pH 6.5 and 50 °C and stable between pH 6.0 and 9.0 at a temperature of up to 45 °C for 30 min BbFFase9 could also effectively hydrolyze galacto-oligosaccharides, which are a flatulence factor in soybean meal, thus releasing new types of product such as melibiose and mannotriose, or degrading them into invert sugars, the sweeter fructose and glucose. This study is the first to report the application of this type of ß-d-fructofuranosidase.