Effect of Ball-Milling Treatment Combined with Glycosylation on the Structure and Functional Properties of Litopenaeus vannamei Protein.

Litopenaeus vannamei protein (LVP) is a high-quality protein. However, its functional properties do not fully meet the needs of food processing. In this study, LVP-xylose conjugates were prepared by conventional wet heat method (GLVP) and ball-milling-assisted wet heat method (GBLVP), respectively. The changes in structure and functional properties of the glycosylated LVP were explored. The findings revealed that ball-milling pretreatment increased the grafting degree to 35.21%. GBLVP had a sparser surface structure and lower particle size than GLVP. FTIR spectra showed that xylose was grafted onto LVP successfully and GBLVP had the lowest α-helix content. Compared with GLVP, GBLVP had a decrease in intrinsic fluorescence intensity and surface hydrophobicity, and an increase in UV absorption intensity. Moreover, GBLVP had higher foaming capacity, solubility and water-holding capacity, and lower allergenicity than GLVP. However, ball-milling pretreatment had a negative impact on the vitro digestibility and oil-holding capacity of GBLVP. In conclusion, ball-milling-assisted treatment of glycosylation could effectively improve the functional properties of LVP, benefiting the broader application of LVP in the food industry.

Antibacterial Regularity Mining Beneath the Systematic Activity Database of Lipopeptides Brevilaterins: An Instructive Activity Handbook for Its Food Application.

Bacterial contamination is a primary threat to food safety. Therefore, the persistent development of natural antibacterial agents has become essential work. The present essay attempts to establish a systematic antibacterial activity database to instruct the food application of brevilaterins, promising antibacterial lipopeptides from Brevibacillus laterosporus S62-9. Minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) were systematically collected from 43 species of standard bacteria and 140 strains of isolated bacteria (food spoilage bacteria and antibiotic-resistant bacteria) using a broth dilution method. The results showed that brevilaterins performed a broad-spectrum inhibitory (0.5~128 µg/mL) and bactericidal activity (1~256 µg/mL), especially efficient against Gram-positive bacteria and spoilage bacteria from grain products. Moreover, brevilaterins not only inhibit and kill multiple antibiotic-resistant bacteria but do not readily develop resistance, with a small specific value of MBC/MIC (1~8). Furthermore, brevilaterins would interact with negatively charged sodium dodecyl sulfate and bind amphipathic soybean phospholipid with an affinity constant of KD = 4.70 × 10-4 M. No significant activity difference was found between brevilaterin B and brevilaterin C. Collectively, this work contributed rich antibacterial data of brevilaterins and revealed the antibacterial regularity beneath these data, which can be used as an activity handbook to instruct their application in food safety.

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.

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.

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.

Structural Organization of Brevilaterin Biosynthesis in Brevibacillus laterosporus S62-9: A Novel MbtH-Independent Cationic Antimicrobial Peptide Synthetase System.

Cationic antimicrobial peptides, produced by nonribosomal peptide synthetases (NRPSs), have received great attention in different applications, including as biocontrol and antimicrobial agents against foodborne pathogenic bacteria. Also, Brevibacillus spp. is a competent microorganism to produce cationic antimicrobial peptides yet has received little attention. Herein, Brevibacillus laterosporus S62-9 genome mining revealed an integrated cationic antimicrobial peptide synthetase system that synthesized brevilaterin. Combining biochemical analysis with bioinformatics elucidated that the A domain from this system was the MbtH-independent enzyme and showed activity against the same amino acid in the structure of brevilaterin. Moreover, the creations of the first three and position 12 residues in the sequence were targeted to bre261, bre270, bre2691A, and bre2662, respectively. Further analysis of the specificity-conferring code of the A domain suggested that a tiny difference would make the activity of the A domain very diverse and the range of substrate selection would be enlarged or narrowed by changing some residues in the code. The dissection of this biosynthesis mechanism would contribute to the successful realization of reasonable artificial design and the modification of bioactive peptides, and this capable organism also would be more fully utilized.

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.

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 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.

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 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.

Nuciferine Alleviates Renal Injury by Inhibiting Inflammatory Responses in Fructose-Fed Rats.

Nuciferine is a major active component from the lotus leaf. This study examines the effects of nuciferine on fructose-induced renal injury and explores its possible mechanism. Rats consumed drinking water or 10% fructose for 12 weeks. Fructose-fed rats were orally treated with water or 7, 14, or 28 mg/kg of nuciferine for the last 6 weeks. HK-2 cells were exposed to 5 mM fructose alone or in combination with nuciferine (2.5-40 µM) for 24 h. Nuciferine significantly attenuated fructose-induced hyperuricemia, dyslipidemia, and systemic inflammation in rats. More importantly, it alleviated renal pathological injury with proteinuria at 20 and 40 mg/kg (2.58 ± 0.97 and 2.48 ± 1.04 mg/mg·creatinine, P < 0.05) compared with fructose-vehicle group (4.10 ± 1.18 mg/mg·creatinine). Furthermore, nuciferine reduced TLR4, MyD88, PI3K, ILK, p-AKT, p-P65, and NLRP3 inflammasome protein levels (P < 0.05 for all) in the renal cortex of fructose-fed rats (14 and 28 mg/kg) and fructose-exposed HK-2 cells (5-40 µM), which is consistent with its reduction of inflammatory cytokines IL-1ß, IL-6, TNF-α, and MCP-1 (P < 0.05 for all) in vivo and in vitro. These findings suggest that nuciferine alleviated fructose-induced inflammation by inhibiting TLR4/PI3K/NF-κB signaling and NLRP3 inflammasome activation in rat renal cortex and HK-2 cells, which may contribute to the improvement of renal injury.

Nuciferine restores potassium oxonate-induced hyperuricemia and kidney inflammation in mice.

Nuciferine, a major aporphine alkaloid of the leaves of Nelumbo nucifera, was found to decrease serum urate levels and improved kidney function, as well as inhibited system and renal interleukin-1ß (IL-1ß) secretion in potassium oxonate-induced hyperuricemic mice. Furthermore, nuciferine reversed expression alteration of renal urate transporter 1 (URAT1), glucose transporter 9 (GLUT9), ATP-binding cassette, subfamily G, membrane 2 (ABCG2), organic anion transporter 1 (OAT1), organic cation transporter 1 (OCT1), and organic cation/carnitine transporters 1/2 (OCTN1/2) in hyperuricemic mice. More importantly, nuciferine suppressed renal activation of Toll-like receptor 4/myeloid differentiation factor 88/NF-kappaB (TLR4/MyD88/NF-κB) signaling and NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome to reduce serum and renal IL-1ß levels in hyperuricemic mice with renal inflammation reduction. The anti-inflammatroy effect of nuciferine was also confirmed in human proximal renal tubular epithelial cells (HK-2 cells) incubated with 4mg/dl uric acid for 24h. This study firstly reported the anti-hyperuricemic and anti-inflammatory effects of nuciferine by regulating renal organic ion transporters and inflammatory signaling in hyperuricemia. These results suggest that a dietary supplement of nuciferine rich in lotus leaf may be potential for the prevention and treatment of hyperuricemia with kidney inflammation.

Betaine reduces serum uric acid levels and improves kidney function in hyperuricemic mice.

Betaine as a dietary alkaloid has attracted the attention of patients with kidney diseases. This study aimed to investigate the effects of betaine on serum uric acid levels and kidney function, and explore their underlying mechanisms in potassium oxonate-induced hyperuricemic mice. Betaine at 5, 10, 20, and 40 mg/kg was orally administered to hyperuricemic mice for 7 days and found to significantly reduce serum uric acid levels and increase fractional excretion of uric acid in hyperuricemic mice in a dose-dependent manner. It effectively restored renal protein level alterations of urate transport-related molecular proteins urate transporter 1, glucose transporter 9, organic anion transporter 1, and ATP-binding cassette subfamily G member 2 in this model, possibly resulting in the enhancement of kidney urate excretion. Moreover, betaine reduced serum creatinine and blood urea nitrogen levels and affected urinary levels of beta-2-microglobulin and N-acetyl-beta-D-glucosaminidase as well as upregulated renal protein levels of organic cation/carnitine transporters OCT1, OCTN1, and OCTN2, resulting in kidney function improvement in hyperuricemic mice. The findings from this study provide evidence that betaine has anti-hyperuricemic and nephroprotective actions by regulating protein levels of these renal organic ion transporters in hyperuricemic mice.

Quercetin and allopurinol reduce liver thioredoxin-interacting protein to alleviate inflammation and lipid accumulation in diabetic rats.

BACKGROUND AND PURPOSE: Thioredoxin-interacting protein (TXNIP), a regulator of cellular oxidative stress, has been associated with activation of NOD-like receptor 3 (NLRP3) inflammasome, inflammation and lipid metabolism, suggesting it has a role in the pathogenesis of non-alcoholic fatty liver disease (NAFLD) in diabetes. In this study we investigated whether TXNIP is involved in type 1 diabetes-associated NAFLD and whether antioxidants, quercetin and allopurinol, alleviate NAFLD by targeting TXNIP. EXPERIMENTAL APPROACH: Diabetes was induced in male Sprague-Dawley rats by a single i.p. injection of 55 mg · kg⁻¹ streptozotocin. Quercetin and allopurinol were given p.o. to diabetic rats for 7 weeks. Hepatic function, oxidative stress, inflammation and lipid levels were determined. Rat BRL-3A and human HepG2 cells were exposed to high glucose (30 mM) in the presence and absence of antioxidants, TXNIP siRNA transfection or caspase-1 inhibitor, Ac-YVAD-CMK. KEY RESULTS: Quercetin and allopurinol significantly inhibited the TXNIP overexpression, activation of NLRP3 inflammasome, down-regulation of PPARα and up-regulation of sterol regulatory element binding protein-1c (SREBP-1c), SREBP-2, fatty acid synthase and liver X receptor α, as well as elevation of ROS and IL-1ß in diabetic rat liver. These effects were confirmed in hepatocytes in vitro and it was further shown that TXNIP down-regulation contributed to the suppression of NLRP3 inflammasome activation, inflammation and changes in PPARα and SREBPs. CONCLUSIONS AND IMPLICATIONS: Inhibition of hepatic TXNIP by quercetin and allopurinol contributes to the reduction in liver inflammation and lipid accumulation under hyperglycaemic conditions. The targeting of hepatic TXNIP by quercetin and allopurinol may have therapeutic implications for prevention of type 1 diabetes-associated NAFLD.

Antihyperuricemic and nephroprotective effects of resveratrol and its analogues in hyperuricemic mice.

SCOPE: Stilbenes, of which, resveratrol is a representative compound in foods and plants, possess a variety of bioactivities including antioxidation, anti-inflammation, chemoprevention, and cardioprotection. This study was conducted to evaluate the antihyperuricemic and nephroprotective effects of resveratrol and its analogues and explore the possible mechanisms. The structure-activity relationships were analyzed. METHODS AND RESULTS: Potassium oxonate-induced hyperuricemic mice were dosed by gavage with eight stilbenes. Uric acid, creatinine, and blood urea nitrogen (BUN) levels in serum and urine, clearance rate of creatinine and BUN, 24-h urate excretion, and fractional excretion of uric acid, uromodulin levels in urine and kidney were determined to evaluate renal urate handling and function. Renal protein levels of organic ion transporters were detected to elucidate the possible mechanisms. Resveratrol, trans-4-hydroxystilbene, pterostilbene, polydatin, and mulberroside A were found to have antihyperuricemic activities. These compounds together with trans-2-hydroxystilbene provided nephroprotection. Trans-3,4',5-trimethoxystilbene and cis-combretastatin A-4 had no effects. CONCLUSION: The uricosuric and nephroprotective actions of resveratrol and its analogues were mediated by regulating renal organic ion transporters in hyperuricemic mice, supporting their beneficial effects for the prevention of hyperuricemia. The number and position, methoxylation and glycosylation of hydroxyl groups in these trans-stilbenes were required for their effects.