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BACKGROUND: More than 50% of the world's population is infected with Helicobacter pylori, which is classified as a group I carcinogen by the World Health Organization (WHO). RESULTS: Corn protein dual-functional peptides were identified and functionally analyzed in vitro and in silico. Two novel dual-functional peptides were identified as Cys-Gln-Asp-Val-Pro-Leu-Leu (CQDVPLL, CQ7) and Thr-Ile-Phe-Pro-Gln-Cys (TIFPQC, TI6) using nanoscale liquid chromatography coupled to tandem mass spectrometry (nano-LC-MS/MS). The antiadhesive effects against H. pylori of CQ7 and TI6 were 45.17 ± 2.41% and 48.62 ± 1.84% at 4 mg mL-1 , respectively. In silico prediction showed that CQ7 and TI6 had good physicochemical properties. Molecular docking demonstrated that CQ7 and TI6 could bind to the adhesins BabA and SabA by hydrophobic interactions and hydrogen bonds, preventing H. pylori infection. Moreover, CQ7 showed strong antioxidant activity due to its unique amino acid composition. CONCLUSION: The present study demonstrated that the identified peptides, CQ7 and TI6, possess antioxidant and antiadhesive effects, preventing H. pylori infection and alleviating oxidative injury to the gastric mucosa. © 2023 Society of Chemical Industry.
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Helicobacter pylori , Antioxidantes/farmacologia , Hidrolisados de Proteína/farmacologia , Simulação de Acoplamento Molecular , Zea mays , Sequência de Aminoácidos , Espectrometria de Massas em Tandem , Peptídeos/farmacologia , Peptídeos/químicaRESUMO
In order to classify and set different prices on basis of difference of beet sugar content in the acquisition process and promote the development of beet sugar industry healthily, a fast, nondestructive, accurate method to detect sugar content of beet was determined by applying near infrared spectroscopy technology. Eight hundred twenty samples from 28 representative varieties of beet were collected as calibration set and 70 samples were chosen as prediction set. Then near infrared spectra of calibration set samples were collected by scanning, effective information was extracted from NIR spectroscopy, and the original spectroscopy data was optimized by data preprocessing methods appropriately. Then partial least square(PLS)regression was used to establish beet sugar quantitative prediction mathematical model. The performances of the models were evaluated by the root mean square of cross-validation (RMSECV), the coefficient of determination (R2) of the calibration model and the standard error of prediction (SEP), and the predicted results of these models were compared. Results show that the established mathematical model by using first derivative (FD) and standard normal variate transformation (SNV) coupled with partial least squares has good predictive ability. The R2 of calibration models of sugar content of beet is 0.908 3, and the RMSECV is 0.376 7. Using this model to forecast the prediction set including 70 samples, the correlation coefficient is 0.921 4 between predicted values and measured values, and the standard error of prediction (SEP) is 0.439, without significant difference (p > 0.05) between predicted values and measured values. These results demonstrated that NIRS can take advantage of simple, rapid, nondestructive and environmental detection method and could be applied to predict beet sugar content. This model owned high accuracy and can meet the precision need of determination of beet sugar content. This detection method could be used to classify and set different prices on basis of difference of beet sugar content in the acquisition process.
Assuntos
Beta vulgaris/química , Carboidratos/análise , Espectroscopia de Luz Próxima ao Infravermelho , Calibragem , Análise dos Mínimos QuadradosRESUMO
A novel fibrinolytic enzyme was produced by the liquid fermentation of Coprinus comatus. The enzyme was purified from the culture supernatant by hydrophobic interactions, gel filtration, and ion exchange chromatographies. It was purified by 241.02-fold, with a specific activity of 3619 U/mg and a final yield of 10.02%. SDS-PAGE analysis confirmed the purity of the enzyme, showing a single band with a molecular weight of 19.5 kDa. The first nine amino acids of the N-terminal of the purified enzyme were A-T-Y-T-G-G-S-Q-T. The enzyme exhibited optimal activity at a temperature of 42 °C and pH 7.6. Its activity was significantly improved by Zn2+, K+, Ca2+, Mn2+, and Mg2+ while being inhibited by Fe2+, Fe3+, Al2+, and Ba2+. The activity of the enzyme was completely inhibited by ethylenediamine tetraacetic acid (EDTA), and it was also dose-dependently inhibited by phenylmethylsulfonyl fluoride (PMSF) and soy trypsin inhibitor (SBTI). However, inhibitors such as N-α-tosyl-L-phenylalanine chloromethyl ketone (TPCK), aprotinin, and pepstatin did not significantly affect its activity, suggesting that the enzyme was a serine-like metalloproteinase. The enzyme acted as both a plasmin-like fibrinolytic enzyme and a plasminogen activator, and it also exhibited the capability to hydrolyze fibrinogen and fibrin. In vitro, it demonstrated the ability to dissolve blood clots and exhibit anticoagulant properties. Furthermore, it was found that the enzyme prolonged activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT), and reduced the levels of fibrinogen (FIB) and prothrombin activity (PA). Based on these studies, the enzyme has great potential to be developed as a natural agent for the prevention and treatment of thrombotic diseases.
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Cordycepin production in the submerged culture of Cordyceps militaris was demonstrated using hydrolyzed corn processing protein by-products, known as corn steep liquor hydrolysate (CSLH), as an alternative nitrogen source. The growth, metabolism, and cordycepin production of Cordyceps militaris were evaluated under various concentrations of CSLH induction. The results demonstrated that CSLH addition had positive effects on the growth and cordycepin production with various C. militaris strains. The optimum strain, C. militaris GDMCC5.270, was found to effectively utilize CSLH to promote mycelium growth and cordycepin production. Low concentrations of CSLH (1.5 g/L) in the fermentation broth resulted in 343.03 ± 15.94 mg/L cordycepin production, which was 4.83 times higher than that of the group without CSLH. This also enhanced the metabolism of sugar, amino acids, and nucleotides, leading to improved cordycepin biosynthesis. The increase in key amino acids, such as glutamic acid, alanine, and aspartic acid, in the corn steep liquor hydrolysate significantly enhanced cordycepin yield. The corn steep liquor hydrolysate was confirmed to be a cost-effective accelerator for mycelium growth and cordycepin accumulation in C. militaris, replacing partial peptone as a cheap nitrogen source. It serves as a suitable alternative for efficient cordycepin production at a low cost.
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H. pylori is a highly pathogenic and prevalent pathogen that is a class I carcinogen. More than 50% of the world's population is infected with H. pylori. An anti-adhesive strategy is an effective way to antagonize H. pylori infection, which does not cause H. pylori resistance and is safer compared to antibiotic therapy. In the present study, to obtain rice bran protein-derived anti-adhesive activity peptides against H. pylori, an efficient enzymatic hydrolysis system was established, and it was found that rice bran protein hydrolysate prepared under specific conditions possessed anti-adhesive activity against H. pylori. The anti-adhesive activity of rice bran protein hydrolysate (RPH) was 43.74 ± 1.12% (4 mg mL-1), and gastric digestion (RPHA) had no significant effect on its activity. Hydrophobic amino acids and aromatic amino acids were important for its anti-adhesive activity. Further, 284 peptide sequences with potential anti-adhesive activity were isolated and identified from RPHA. Combined with molecular docking results, four novel anti-adhesive activity peptides were finally screened, namely LS5 (LSFRL), SN8 (SNTPGMVY), VV7 (VVNFGNL) and PV9 (PVLWGVPKG). Among them, PV9 showed the highest anti-adhesive activity of 59.64 ± 2.00% (4 mg mL-1). These four peptides could bind H. pylori adhesins BabA and SabA, occupying the binding sites of cell receptors and acting as anti-adhesion agents. In conclusion, four rice bran protein-derived anti-adhesive activity peptides against H. pylori can be used for the development of novel functional foods antagonizing H. pylori infection.
Assuntos
Aderência Bacteriana , Helicobacter pylori , Oryza , Peptídeos , Helicobacter pylori/efeitos dos fármacos , Helicobacter pylori/fisiologia , Oryza/microbiologia , Oryza/química , Peptídeos/farmacologia , Peptídeos/química , Aderência Bacteriana/efeitos dos fármacos , Simulação de Acoplamento Molecular , Proteínas de Plantas/farmacologia , Proteínas de Plantas/química , Infecções por Helicobacter/microbiologia , Infecções por Helicobacter/tratamento farmacológico , Antibacterianos/farmacologia , Antibacterianos/química , Humanos , Hidrolisados de Proteína/farmacologia , Hidrolisados de Proteína/químicaRESUMO
A comprehensive investigation aimed to access the impacts of ultrasonic, microwave, and ultrasonic-microwave synergistic modification on the physicochemical properties, microstructure, and functional properties of corn bran insoluble dietary fiber (CBIDF). Our findings revealed that CBIDF presented a porous structure with loose folds, and the particle size and relative crystallinity were slightly decreased after modification. The CBIDF, which was modified by ultrasound-microwave synergistic treatment, exhibited remarkable benefits in terms of its adsorption capacity, and cholate adsorption capacity. Furthermore, the modification improved the in vitro hypoglycemic activity of the CBIDF by enhancing glucose absorption, retarding the starch hydrolysis, and facilitating the diffusion of glucose solution. The findings from the in vitro probiotic activity indicate that ultrasound-microwave synergistic modification also enhances the growth-promoting ability and adsorbability of Lactobacillus acidophilus and Bifidobacterium longum. Additionally, the level of soluble dietary fiber was found to be positively correlated with CBIDF adsorbability, while the crystallinity of CBIDF showed a negative correlation with α-glucosidase and α-amylase inhibition activity, as well as water-holding capacity, and oil-holding capacity.
Assuntos
Micro-Ondas , Zea mays , Ultrassom , Fibras na Dieta , Glucose/químicaRESUMO
China faces a persistent deficiency in feed protein resources. Enhancing the utilization efficiency of indigenous feed protein resources emerges as a viable strategy to alleviate the current deficit in protein feed supply. Corn gluten meal (CGM), characterized by a high proportion of crude protein and glutamine, is predominantly employed in animal feed. Nonetheless, the water-insolubility of CGM protein hampers its protein bioavailability when utilized as feed material. The aim of this study was to augment protein bioavailability, liberate glutamine peptides from CGM, and produce glutamine-enriched CGM fermented feed. We executed a co-fermentation protocol using Bacillus subtilis A5, Lactobacillus 02002, and acid protease to generate the CGM fermented feed. Subsequent in vivo experiments with broilers were conducted to assess the efficacy of the fermented product. The findings revealed that the soluble protein, glutamine, small peptides, and lactic acid contents in the fermented feed increased by 69.1%, 700%, 47.6%, and 125.9%, respectively. Incorporating 15% and 30% CGM fermented feed into the diet markedly enhanced the growth performance and intestinal health of broilers, positively modulated the cecal microbiota structure, and augmented the population of beneficial bacteria, specifically Lactobacillus. These results furnish both experimental and theoretical foundations for deploying CGM fermented feed as an alternative protein feed resource.
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More than 50% of the world population is infected with Helicobacter pylori (H. pylori), which is classified as group I carcinogen by the WHO. H. pylori surface adhesins specifically recognize gastric mucosal epithelial cells' (GES-1 cells) receptor to complete the adhesion. Blocking the adhesion with an anti-adhesion compound is an effective way to prevent H. pylori infection. The present study found that corn protein hydrolysate, hydrolyzed by Neutral, effectively alleviated gastric injury induced by H. pylori infection through anti-adhesive and anti-inflammatory effects in vitro and in vivo. The hydrolysate inhibited H. pylori adhesion to GES-1 cells significantly, and its anti-adhesive activity was 50.44 ± 0.27% at 4 mg/mL, which indicated that the hydrolysate possessed a similar structure to the GES-1 cells' receptor, and exhibited anti-adhesive activity in binding to H. pylori. In vivo, compared with the H. pylori infection model group, the medium and high dose of the hydrolysate (400-600 mg/kg·bw) significantly decreased (p < 0.05) the amount of H. pylori colonization, pro-inflammatory cytokines (IL-6, IL-1ß, TNF-α and MPO), chemokines (KC and MCP-1) as well as key metabolites of NF-κB signaling pathway levels (TLR4, MyD88 and NF-κB), and it increased antioxidant enzyme contents (SOD and GSH-Px) and the mitigation of H. pylori-induced pathological changes in the gastric mucosa. Taken together, these results indicated that the hydrolysate intervention can prevent H. pylori-induced gastric injury by anti-adhesive activity and inhibiting the NF-κB signaling pathway's induction of inflammation. Hence, the corn protein hydrolysate might act as a potential anti-adhesive agent to prevent H. pylori infection.
Assuntos
Infecções por Helicobacter , Helicobacter pylori , NF-kappa B/metabolismo , Zea mays/metabolismo , Infecções por Helicobacter/complicações , Infecções por Helicobacter/tratamento farmacológico , Infecções por Helicobacter/metabolismo , Hidrolisados de Proteína/farmacologia , Citocinas/metabolismo , Mucosa Gástrica , Células Epiteliais , Interleucina-8/metabolismoRESUMO
Ulcerative colitis is a typical chronic inflammatory disease of the gastrointestinal tract, which has become a serious hazard to human health. The purpose of the present study was to evaluate the antagonistic effect of corn protein hydrolysate with glutamine-rich peptides on ulcerative colitis. The sequential hydrolysis of corn gluten meal by Alcalase and Protamex was conducted to prepare the hydrolysate, and then the mouse ulcerative colitis model induced by dextran sulfate sodium was applied to evaluate its biological activities. The results indicated that the hydrolysate significantly improved weight loss (p < 0.05), reduced the colonic shortening and the disease activity index, diminished the infiltration of inflammatory cells in the colonic tissue, and reduced the permeability of the colonic mucosa in mice. In addition, the hydrolysate decreased the contents of pro-inflammatory factors IL-1ß, IL-6, and TNF-α, increased the anti-inflammatory factor IL-10 and oxidative stress markers GSH-Px and SOD in the animal tests. Moreover, the hydrolysate also regulated the abundance and diversity of the intestinal microbiota, improved the microbiota structure, and increased the content of beneficial bacteria including Lactobacillus and Pediococcus. These results indicated that the hydrolysate might be used as an alternative natural product for the prevention of ulcerative colitis and could be further developed into a functional food.
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Lactic acid bacteria (LAB) can produce a vast spectrum of antifungal metabolites to inhibit fungal growth. The purpose of this study was to elucidate the antifungal effect of isolated Weissella cibaria BYL4.2 on Penicillium chrysogenum, the antifungal activity of W. cibaria BYL4.2 against P. chrysogenum was evaluated by the superposition method, results showed that it had obviously antifungal activity against P. chrysogenum. Studying the probiotic properties of BYL4.2 and determining it as beneficial bacteria. Furtherly, different treatments were carried out to characterize the antifungal activity of cell-free supernatant (CFS) produced by W. cibaria BYL4.2, and it was shown that the CFS was pH-dependent, partly heat-sensitive, and was not influenced by proteinaceous treatment. The CFS of W. cibaria BYL4.2 was analyzed by high-performance liquid chromatography (HPLC) and found the highest content of lactic acid. Screening of metabolic markers by a non-targeted metabolomics approach based liquid chromatography-mass spectrometry (LC-MS). The results speculated that organic acid especially detected D-tartaric acid was the main antifungal substance of CFS, which could cause the down-regulation of metabolites in the ABC transporters pathway, thereby inhibiting the growth of P. chrysogenum. Therefore, this study may provide important information for the inhibitory mechanism of W. cibaria BYL4.2 on P. chrysogenum, and provide a basis for further research on the antifungal effect of Weissella.
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The nutritional value and bioactivity of black beans are enhanced when fermented as tempeh, but their bioaccessibility and bioactivity after ingestion remain unclear. In this study, black bean tempeh and unfermented black beans were digested in vitro and changes in protein degradation, phenolic compound release, angiotensin I-converting enzyme (ACE)-inhibitory activity, and antioxidant activity between the two groups were compared. We observed that the soluble protein content of digested black bean tempeh was generally significantly higher than that of digested unfermented black beans at the same digestion stage (P < 0.05). The degree of protein hydrolysis and the content of <10 kDa peptides were also significantly higher in the digested black bean tempeh than in digested unfermented black beans (P < 0.05). SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and reversed-phase high-performance liquid chromatography (RP-HPLC) analysis showed that most macromolecular proteins in tempeh had been degraded during fermentation and more of the small peptides were released from black bean tempeh during digestion, respectively. Compared to that of the unfermented black beans, the level of ACE inhibition of black bean tempeh was lower, but this significantly increased to 82.51% following digestion, closing the gap with unfermented black beans. In addition, the total respective levels of phenolics, flavonoids, and proanthocyanidins released from black bean tempeh were 1.21, 1.40, and 1.55 times those of unfermented black beans following in vitro digestion, respectively. Antioxidant activity was also significantly higher in digested black bean tempeh than in digested unfermented black beans and showed a positive correlation with phenolic compound contents (P < 0.05). The results of this study proved that, compared to unfermented black beans, black bean tempeh retained protein and phenolic compound bioaccessibility and antioxidant activity and showed an improved ACE-inhibitory activity even after consumption.
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This study was designed for determining the effect of particle size on the functional properties of corn bran insoluble dietary fiber (IDF). Results showed that some physicochemical properties were improved with the decrease in particle size. The structure of the IDF was observed by the scanning electron microscope (SEM), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FT-IR). The surface was found wrinkled and sparse, the particle size was smaller, the crystallinity of IDF had increased slightly, and more -OH and C-O groups were exposed. Moreover, the corn bran IDF with a smaller particle size had a better hypoglycemic effect in vitro, and the inhibitory activity of α-glucosidase and α-amylase were also increased significantly with the decrease in particle size (p < 0.05). When the IDF was 300 mesh, the inhibitory rate of α-glucosidase was 61.34 ± 1.12%, and the inhibitory rate of α-amylase was 17.58 ± 0.33%. It had increased by 25.54 and 106.83%, respectively compared to the control treatment (CK) group. In addition, correlation analysis found that the particle size was highly negatively correlated with some functional properties of IDF (p < 0.05), and the content of cellulose was positively correlated with the functional properties of IDF except WHC (p < 0.05). To sum up, reducing particle size was suitable for the development of high value-added IDF products. This study also revealed the potential value of corn bran IDF and provided a new idea for the diversified application of IDF.
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A novel mixture of glycopeptides was prepared from corn glutelin and glucosamine (GlcN). The functional properties and antioxidative activities of this mixture were investigated. Corn glutelin was limited hydrolyzed by Alcalase, and then its hydrolysates were glycosylated with GlcN by transglutaminase (TGase) to modify its main and side chain, respectively. Under the optimized conditions, the content of GlcN conjugated to peptides was 81.98 ± 1.98 mg/g glutelin peptides. According to electrospray ionization mass spectrometry (ESI-MS) analysis, there are two types of glycopeptides in the mixture, TGase and non-enzymatic glycated counterparts. Compared with original glutelin, the glycosylated glutelin hydrolysates exhibited better solubility in the pH range of 2-11 and other functional properties except foaming stability. Meanwhile, it is more easily digested by pepsin and trypsin, and possessed excellent antioxidative activities. It also exhibited cytoprotective effects and intracellular ROS scavenging activities in LO2 cells subjected to oxidative stress by oxidation with ethanol solution.
Assuntos
Antioxidantes , Glutens , Glucosamina , Hidrólise , Hidrolisados de Proteína , TransglutaminasesRESUMO
A novel fibrinolytic enzyme, ACase was isolated from fruiting bodies of a mushroom, Agrocybe aegerita. ACase was purified by using ammonium sulfate precipitation, gel filtration, ion exchange and hydrophobic chromatographies to 237.12 fold with a specific activity of 1716.77 U/mg. ACase was found to be a heterodimer with molecular mass of 31.4 and 21.2 kDa by SDS-PAGE and appeared as a single band on Native-PAGE and fibrin-zymogram. The N-terminal sequence of the two subunits of ACase was AIVTQTNAPWGL (subunit 1) and SNADGNGHGTHV (subunit 2). ACase had maximal activity at 47 °C and pH 7.6. It's activity was improved by Cu2+, Na+, Fe3+, Zn2+, Ba2+, K+ and Mn2+, but inhibited by Fe2+, Mg2+ and Ca2+. PMSF, SBTI, aprotinine and Lys inhibited the enzyme activity, which suggested that ACase was a serine protease. ACase could degrade all three chains (α, ß and γ) of fibrinogen. Moreover, the enzyme acted as both, a plasmin-like fibrinolytic enzyme and a plasminogen activator. It could hydrolyze human thrombin slightly, which indicated that the ACase could inhibit the activity of thrombin and acted as an anticoagulant to prevent thrombosis. Based on these results, ACase might act as a therapeutic agent for treating thrombosis, or as a functional food. Further investigation of the enzyme is underway.
Assuntos
Agrocybe/enzimologia , Anticoagulantes/farmacologia , Fibrinolíticos/farmacologia , Serina Proteases/farmacologia , Sequência de Aminoácidos , Anticoagulantes/química , Anticoagulantes/isolamento & purificação , Fenômenos Químicos , Cromatografia por Troca Iônica , Fibrinogênio/metabolismo , Fibrinolíticos/química , Fibrinolíticos/isolamento & purificação , Carpóforos/enzimologia , Proteínas Fúngicas/química , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/farmacologia , Humanos , Concentração de Íons de Hidrogênio , Imunoglobulina G/metabolismo , Peso Molecular , Multimerização Proteica , Serina Proteases/química , Serina Proteases/isolamento & purificação , Albumina Sérica Humana/metabolismo , Trombina/metabolismoRESUMO
Fibrinolytic enzymes are important thrombolytic agents for blood-clotting disorders like cardiovascular diseases. Availability of novel recombinant fibrinolytic enzymes can overcome the shortcomings of current thrombolytic drugs. With the objective of facilitating their cost-effective production for therapeutic applications and for gaining deeper insight into their structure-function, we have cloned and expressed the first fibrinolytic protease gene from Cordyceps militaris. Cordyceps militaris fibrinolytic enzyme (CmFE) has one open reading frame of 759 bp encoding "pre-pro-protein" of 252 amino acids. Recombinant CmFE was expressed as 28 kDa extracellular enzyme in Pichia pastoris which was capable of degrading fibrin clot. A structure homology model of CmFE was developed using urokinase-type plasminogen activator. The active site contains catalytic triad His41, Asp83, Ser177 and consensus sequence of GDSGG. The substrate binding residues are Asp (171), Gly (194) and Ser (192). Its trypsin-like specificity is determined by the critical Asp171 in S1 subsite. The "oxyanion hole" is formed by backbone amide hydrogen atoms of Gly-175 and Ser-177. CmFE contains six conserved cysteines forming three disulfide linkages. This is the first study describing cloning, expression and prediction of structure-function relationship of a mushroom fibrinolytic protease. Hence it has great relevance in application of fibrinolytic enzymes as thrombolytic agents.
Assuntos
Cordyceps/enzimologia , Fibrinólise , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Expressão Gênica , Modelos Moleculares , Homologia Estrutural de Proteína , Sequência de Aminoácidos , Clonagem Molecular , Relação Estrutura-AtividadeRESUMO
New glycopeptides were generated by proteolysis from corn gluten meal (CGM) followed by transglutaminase (TGase)-induced glycosylation with glucosamine (GlcN). The glycopeptides exhibited desirable antioxidant and intracellular ROS-scavenging properties. The amount of conjugated GlcN quantified by high-performance liquid chromatography (HPLC) was 23.0 g/kg protein. The formed glycopeptides contained both glycosylated and glycation types, as demonstrated by the electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS/MS). The glycopeptides exhibited scavenging capabilities against free radical diphenylpicrylhydrazyl (DPPH) and hydroxyl radicals by reducing their power. The potential protection of glycopeptides against ethanol-induced injury in LO2 cells was assessed In Vitro based on methyl thiazole tetrazolium (MTT) testing and intracellular reactive oxygen species (ROS) scavenging capacity, respectively. Glycopeptide cytoprotection was expressed in a dose-dependent manner, with the glycopeptides exhibiting good solubility ranging from 74.8% to 83.2% throughout a pH range of 2-10. Correspondingly, the glycopeptides showed good emulsifying activity (36.0 m2/g protein), emulsion stability (74.9%), and low surface hydrophobicity (16.3). These results indicate that glycosylation of CGM significantly improved its biological and functional properties. Glycopeptides from CGM could be used as potential antioxidants as well as comprising a functional food ingredient.
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A cell-bound exopolysaccharide (c-EPS) from Lactobacillus fermentum S1 was isolated and purified to near homogeneity by anion exchange and gel filtration chromatography. The c-EPS is a homogeneous heteropolysaccharide with an average molecular weight of 7.19â¯×â¯105â¯Da and comprises mainly mannose, rhamnose, glucose, and galactose. Fourier transform infrared spectroscopy spectrum of the c-EPS exhibited typical characteristic absorption peaks of polysaccharides. Methylation and NMR analyses showed that the c-EPS had a backbone of α-D-Galp-(1â¯ââ¯3), α-L-Rhap-(1â¯ââ¯2), α-D-Glcp-(1â¯ââ¯3), ß-D-Galp-(1â¯ââ¯3), ß-D-Glclp-(1â¯ââ¯2), and ß-L-Rhap-(1â¯ââ¯3,4) residues, terminated with α-D-Manp-(1â¯ââ¯residue. The advanced structure study indicated the c-EPS not to have a triple-helical conformation, while the microstructural study revealed a hollow porous structure for c-EPS. Further, the thermal analysis showed that the degradation temperature for the c-EPS was 288.0⯰C; its peak temperature was 89.4⯰C with an enthalpy value of 273.1â¯J/g. Moreover, the c-EPS exhibited potent DPPH, hydroxyl, and ABTS+ radicals scavenging activities, as well as FRAP in a dose-dependent manner, which could significantly enhance the T-AOC and SOD activity and reduce MDA level in Caenorhabditis elegans. Therefore, this c-EPS could be utilized as a promising natural antioxidant for application in functional foods.
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Antioxidantes/química , Antioxidantes/farmacologia , Limosilactobacillus fermentum/química , Polissacarídeos Bacterianos/química , Polissacarídeos Bacterianos/farmacologia , Antioxidantes/isolamento & purificação , Cápsulas Bacterianas/química , Fenômenos Químicos , Espectroscopia de Ressonância Magnética , Metilação , Peso Molecular , Polissacarídeos Bacterianos/isolamento & purificação , Análise Espectral , TermogravimetriaRESUMO
In this study, we have isolated and characterized a fibrinolytic enzyme from the GRAS (Generally Recognized as Safe) fungus, Neurospora sitophila. The enzyme was purified by fractional ammonium sulfate precipitation, hydrophobic interaction, ion exchange and gel filtration chromatography to 45.2 fold with a specific activity of 415.6U/mg protein. The native molecular mass of the enzyme was 49kDa, while the denatured molecular mass was 30kDa and 17.5kDa, indicating that the enzyme was a hetero-dimer. It was optimally active at 50°C and pH 7.4 and stable at human physiological temperature and pH. It was found to be a chymotrypsin-like serine protease which cleaved the synthetic chromogenic substrate, N-Succinyl-Ala-Ala-Pro-Phe-pNA for which the apparent Km and Vmax values were 0.24mM and 4.17×10-5mM/s, respectively. The enzyme hydrolyzed all the chains of fibrinogen by cleaving α chain first, followed by ß chain and then γ chain. Moreover, the enzyme possessed dual function of direct fibrinolysis as well as plasminogen activation. Due to its attractive biochemical and fibrinolytic properties and being from a GRAS fungus, the fibrinolytic enzyme has application as a safe and efficient thrombolytic drug.
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Quimotripsina/química , Quimotripsina/metabolismo , Neurospora/enzimologia , Plasminogênio/química , Plasminogênio/metabolismo , Quimotripsina/isolamento & purificação , Ativação Enzimática/efeitos dos fármacos , Fibrinólise/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Ponto Isoelétrico , Peso Molecular , Plasminogênio/isolamento & purificação , Inibidores de Proteases/farmacologia , TemperaturaRESUMO
Zein and its derived peptides have been used as nanocarriers for bioactive components. Lutein, as well as other xanthophylls, are characterized by blue light filtering and anti-oxidant properties. However, lutein is unstable and has low water solubility, poor absorption, and low bioavailability. In order to protect lutein from oxidative degradation, and to enhance its solubility and dispersibility, stability and bioactivity, lutein-loaded zein nanoparticles (LLZ-NP) and zein-derived peptide nanoparticles (LLZ-PEP-NP) were prepared by the solvent diffusion method. Compared to LLZ-NP, LLZ-PEP-NP possessed good physicochemical properties, including particle size, polydispersity index, zeta potential, entrapment efficiency and in vitro stability. Specifically, transmission electron microscopy (TEM) images showed that LLZ-PEP-NP had a spherical form with a nanometric size and lutein was efficiently loaded into zein-derived peptides through self-assembly. Dynamic light scattering (DLS) results demonstrated that LLZ-PEP-NP had a narrow size distribution in the range of 200-300 nm and a decreased zeta potential compared to that of LLZ-NP. The lutein entrapment efficiency (EE%) of LLZ-NP and LLZ-PEP-NP was more than 85%, while LUT-PEP-NP showed higher lutein entrapment efficiency because of the better capacity of peptides bound with lutein. Nanoencapsulation of lutein into LLZ-PEP-NP resulted in a significantly higher solubility compared to nanoencapsulation of lutein into LLZ-NP and free lutein. The stabilities of lutein in zein-derived peptide nanoparticles in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) were improved. These results suggest that zein-derived peptides have the potential to be used as nanocarriers to enhance the solubility and stability of lutein, which can further improve its bioavailability.
Assuntos
Sistemas de Liberação de Medicamentos/instrumentação , Luteína/química , Nanopartículas/química , Zeína/química , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos/métodos , Estabilidade de Medicamentos , Tamanho da Partícula , Solubilidade , Água/químicaRESUMO
A fibrinolytic enzyme was produced by the medicinal mushroom, Cordyceps militaris using submerged fermentation. The enzyme was purified from culture supernatant by hydrophobic interaction, ion exchange and gel filtration chromatographies. It was purified by 36 fold, with a specific activity of 1,467.4U/mg protein and the final yield was 5.8%. The molecular weight of the enzyme as determined by SDS-PAGE and gel filtration was 28kDa and 24.5kDa, respectively, and its isoelectric point (pI) was 9.0±0.2. It was found to be a glycoprotein with carbohydrate content of 1.67% (w/v). The enzyme was optimally active at 37°C and pH 7.2. The enzyme activity was strongly inhibited by soybean trypsin inhibitor (SBTI) and aprotinin which indicated it to be a serine protease, while other inhibitors like N-α-tosyl-l-phenylalanine chloromethyl ketone (TPCK), phenyl methane sulfonyl fluoride (PMSF), pepstatin and metal chelator EDTA did not inhibit its activity. Amino acid sequences of the purified enzyme were determined partially by Q-TOF2 and they were IEDFPYQVDLR; ANCGGTVISEK; YVLTAGHCAEGYTGLNIR; TNYASVTPITADMICAGFPEGK; KDSCSGDSGGPLVTGGK; VVGIVSFGTGCAR; ANKPGVYSSVASAEIR. Sequences of the seven peptides completely matched with those of a trypsin-like serine protease from Cordyceps militaris CM01 (accession no. EGX95217.1). The purified enzyme degraded α chains of fibrinogen first and then ß and γ chains and also activated plasminogen into plasmin. It can act as an anticoagulant and prevent clot formation by degrading fibrinogen. Based on these studies, the purified enzyme has great potential to be developed as a natural agent for prevention and treatment of thrombolytic diseases.