Sephadex G-100 Column Chromatography Research Articles

Structural characterization of Astragalus polysaccharide-D1 and its improvement of low-dose metformin effect by enriching Staphylococcus lentus

To explore the adjuvant therapy drugs of low-dose metformin, one homogeneous polysaccharide named APS-D1 was purified from Astragalus membranaceus by DEAE-52 cellulose and Sephadex G-100 column chromatography. Its chemical structure was characterized by molecular weight distribution, monosaccharide composition, infrared spectrum, methylation analysis, and NMR. The results revealed that APS-D1 (7.36 kDa) consisted of glucose, galactose, and arabinose (97.51 %:1.56 %:0.93 %). It consisted of →4)-α-D-Glcp-(1→ residue backbone with →3)-β-D-Galp-(1→ residue and terminal-α/β-D-Glcp-(1→ side chains. APS-D1 could significantly improve inflammation (TNF-α, LPS, and IL-10) in vivo. Moreover, APS-D1 improved the curative effect of low-dose metformin without adverse events. APS-D1 combined with low-dose metformin regulated several gut bacteria, in which APS-D1 enriched Staphylococcus lentus to produce l-carnitine (one of 136 metabolites of S. lentus). S. lentus and l-carnitine could improve diabetes, and reduction of S. lentusl-carnitine production impaired diabetes improvement. The combination, S. lentus, and l-carnitine could promote fatty acid oxidation (CPT1) and inhibit gluconeogenesis (PCK and G6Pase). The results indicated that APS-D1 enhanced the curative effect of low-dose metformin to improve diabetes by enriching S. lentus, in which the effect of S. lentus was mediated by l-carnitine. Collectively, these findings support that low-dose metformin supplemented with APS-D1 may be a favorable therapeutic strategy for type 2 diabetes.

Purple sweet potato polysaccharide ameliorates concanavalin A-induced hepatic injury by inhibiting inflammation and oxidative stress

BackgroundAutoimmune hepatitis (AIH) is a prevalent liver disease that can potentially lead to hepatic fibrosis and cirrhosis. The prolonged administration of immunosuppressive medications carries significant risks for patients. Purple sweet potato polysaccharide (PSPP), a macromolecule stored in root tubers, exhibits anti-inflammatory, antioxidant, immune-enhancing, and intestinal flora-regulating properties. Nevertheless, investigation into the role and potential mechanisms of PSPP in AIH remains notably scarce. PurposeOur aim was to explore the possible protective impacts of PSPP against concanavalin A (Con A)-induced liver injury in mice. MethodsPolysaccharide was isolated from purple sweet potato tubers using water extraction and alcohol precipitation, followed by purification through DEAE-52 cellulose column chromatography and Sephadex G-100 column chromatography. A highly purified component was obtained, and its monosaccharide composition was characterized by high performance liquid chromatography (HPLC). Mouse and cellular models induced by Con A were set up to investigate the impacts of PSPP on hepatic histopathology, apoptosis, as well as inflammation- and oxidative stress-related proteins in response to PSPP treatment. ResultsThe administration of PSPP significantly reduced hepatic pathological damage, suppressed elevation of ALT and AST levels, and attenuated hepatic apoptosis in Con A-exposed mice. PSPP was found to mitigate Con A-induced inflammation by suppressing the TLR4-P2X7R/NLRP3 signaling pathway in mice. Furthermore, PSPP alleviated Con A-induced oxidative stress by activating the PI3K/AKT/mTOR signaling pathway in mice. Additionally, PSPP demonstrated the ability to reduce inflammation and oxidative stress in RAW264.7 cells induced by Con A in vitro. ConclusionPSPP has the potential to ameliorate hepatic inflammation via the TLR4-P2X7R/NLRP3 pathway and inhibit hepatic oxidative stress through the PI3K/AKT/mTOR pathway during the progression of Con A-induced hepatic injury. The results of this study have unveiled the potential hepatoprotective properties of purple sweet potato and its medicinal value for humans. Moreover, this study serves as a valuable reference, highlighting the potential of PSPP-1 as a drug candidate for the treatment of immune liver injury.

Inhibition of Proliferation and Induction of Apoptosis in Prostatic Carcinoma DU145 Cells by Polysaccharides from Yunnan Rosa roxburghii Tratt.

This study aimed to investigate methodologies for the extraction and purification of polysaccharides from Rosa roxburghii Tratt fruits and their impact on various cellular processes in prostate cancer DU145 cells, including survival rate, migration, invasion, cell cycle, and apoptosis. Compared to the control group, the polysaccharide exhibited a significant reduction in the viability, migration, and invasion rates of DU145 cells in a time- and dose-dependent manner within the polysaccharide-treated groups. Additionally, it effectively arrested the cell cycle of DU145 cells at the G0/G1 phase by downregulating the expressions of CDK-4, CDK-6, and Cyclin D1. Furthermore, it induced apoptosis by upregulating the expressions of Caspase 3, Caspase 8, Caspase 9, and BAX. Polysaccharides were extracted from Rosa roxburghii Tratt sourced from Yunnan, China. Extraction and decolorization methods were optimized using response surface methodology, based on a single-factor experiment. Polysaccharide purification was carried out using DEAE-52 cellulose and Sephadex G-100 column chromatography. The optimal dosage of R. roxburghii Tratt polysaccharide affecting DU145 cells was determined using the CCK-8 assay. Cell migration and invasion were assessed using transwell and scratch assays. Flow cytometry was employed to analyze the effects on the cell cycle and apoptosis. Western blotting and Quantitative real-time PCR were utilized to examine protein and mRNA expressions in DU145 cells, respectively. Rosa roxburghii Tratt polysaccharides, consisting of D-mannose, L-rhamnose, N-acetyl-D-glucosamine, D-galacturonic acid, D-glucose, D-galactcose, D-xylose, L-arabinose, and L-fucose, possess the ability to hinder DU145 cell proliferation, migration, and invasion while inducing apoptosis through the modulation of relevant protein and gene expressions.

Purification and Structural Characterization of Polysaccharides from Polygonum multiflorum Thunb. and Their Immunostimulatory Activity in RAW264.7 Cells.

Polygonum multiflorum Thunb. (PM) and derived products are broadly utilized in Chinese traditional medicine. According to our previous research, PM mostly contains polysaccharides, which display a wide range of biological activities. Two water-soluble polysaccharides (PMPs-1 and PMPs-2) were obtained from PM by DEAE-Cellulose and Sephadex G-100 column chromatography. Colorimetry, HPGPC-MALLS-RID, HPLC-PDA, methylation, FT-IR, NMR, and SEM were used to characterize these polysaccharides. PMPs-1 and PMPs-2 had average molecular weights of 255.5 and 55.7 kDa, respectively. PMPs-1 consisted of Man, Glc, Gal, and Ara at 0.9:78.6:1.0:1.6 and was a glucan with → 4)-Glcp-(1 → as a backbone. Meanwhile, PMPs-2, an acidic polysaccharide, comprised Rha, GalA, Glc, Gal, and Ara at 3.2:20.3:2.7:1.0:8.3. PMPs-1 and PMPs-2 significantly improved the proliferation of RAW 264.7 cells and induced NO, TNF-α, and IL-6 release. This study reveals that these two polysaccharides can be explored as novel immunomodulators and provide a basis for further development of PM in food and pharmaceutical industries.

The structural characteristic of acidic-degraded polysaccharides from seeds of Plantago ovata Forssk and its biological activity

In this study, a response surface methodology (RSM) was used to determine the best combination for acid degradation parameters to reduce the viscosity of Plantago ovata Forssk seed polysaccharide (POFP). Then, the two major homogeneous polysaccharides (AH-POFP1 and AH-POFP3) were obtained by DEAE-650 M and Sephadex G-100 column chromatography. The apparent structure of the main fraction AH-POFP1 was characterized by SEM, TG and XRD, and the linkage of AH-POFP1 was determined by a combination of partial acidolysis, Smith's degradation, methylation analysis and 2D NMR analysis. Structural analysis showed that AH-POFP1 was mainly composed of xylose, with a molecular weight of 618.1 kDa, and had a backbone of 1 → 4-linked Xylp, as well as branches of T-linked Xylp, 1 → 4-linked Xylp attached to the O-2 position. The antioxidant activity assays showed that the both AH-POFP1 and AH-POFP3 possess strong scavenging radical ability. Moreover, AH-POFP1 inhibits the secretion of pro-inflammatory factors, and promotes the secretion of anti-inflammatory factors, thereby exerting anti-inflammatory effects. These findings may help to guide future applications of Plantago ovata Forssk in the fields of food, health care, and pharmacy.

A thermo-tolerant cellulase enzyme produced by Bacillus amyloliquefaciens M7, an insight into synthesis, optimization, characterization, and bio-polishing activity

Abstract In the current study, among 36 isolates, the bacterial strain M7 was selected as the highest cellulase producer and underwent traditional and molecular identification as Bacillus amyloliquefaciens M7. The productivity of the cellulase enzyme was optimized using the one-factor-at-a-time method. The optimization analysis showed that the best pH value for cellulase production was 7, in the presence of 1% bacterial inoculum size, 5 g·L−1 of carboxymethyl cellulose, 5 g·L−1 of peptone as nitrogen source, and incubation period of 24 h at a temperature of 35°C. The highest cellulase activity (64.98 U·mL−1) was obtained after optimizing conditions using BOX-Behnken Design. The maximum cellulase yield (75.53%) was obtained after precipitation by 60% ammonium sulfate, followed by purification by dialysis bag and Sephadex G-100 column chromatography. The purified cellulase enzyme was characterized by 6.38-fold enrichment, with specific activity (60.54 U·mg−1), and molecular weight of approximately 439.0 Da. The constituent of purified cellulase was 18 amino acids with high concentrations of 200 and 160 mg·L−1 for glycine and arginine, respectively. The purified cellulase enzyme was more stable and active at pH 8 and an incubation temperature of 50°C. The metal ions CaCl2, NaCl, and ZnO enhanced the activity of purified cellulase enzyme. Finally, the B. amyloliquefaciens M7-cellulase exhibits high bio-polishing activity of cotton fabrics with low weight loss (4.3%) which was attained at a maximum concentration (1%, v/v) for 90 min.

Construction of 131I-RGDyC-PEG-PAMAM-DTX targeted drug delivery system and study of its physicochemical properties and biological activity.

To construct a novel targeted drug delivery nanoprobe: iodine-131-arginine-glycine-aspartate-tyrosine-cysteine peptide-polyethylene glycol-fifth generation polyamide-amine-docetaxel (131I-RGDyC-PEG-PAMAM-DTX) and to investigate its physicochemical properties and biological activity. Docetaxel was wrapped by solvent volatilization method, and the regression curve of DTX was constructed by high-performance liquid chromatography to determine its drug loading. The particle size of RGDyC-PEG-PAMAM-DTX was detected by dynamic light scattering. The 131I labeling was performed by a chloramine-T method and purified by Sephadex-G50 column chromatography, and it is in vitro stability and lipid-water partition coefficient was investigated. The cytotoxicity of RGDyC-PEG-PAMAM-DTX and 131I-RGDyC-PEG-PAMAM-DTX on A549 cells in vitro was detected by Cell Counting Kit-8 assay. Arginine-glycine-aspartate-tyrosine-cysteine peptide-PEG-PAMAM-DTX was successfully prepared by solvent volatilization with a loading capacity of about 44μg/mg. The average particle size of RGDyC-PEG-PAMAM-DTX was 57.8nm; the labeling rate of 131I-RGDyC-PEG-PAMAM-DTX by the chloramine-T method was 74.09%-76.09%, and the radiochemical purity was 88.9%-92.6% after purification. The in vitro stability showed that the radiochemical purity was above 80% after 72h in fetal bovine serum and PBS buffer (25oC and 37oC).CCK-8 assay showed that RGDyC-PEG-PAMAM-DTX and 131I-RGDyC-PEG-PAMAM-DTX had more pronounced cytotoxic effects than free DTX and 131I. Iodine-131-RGDyC-PEG-PAMAM-DTX has good physicochemical properties and apparent cytotoxic effectsandis expected to be used in treating tumors.

Optimization, partial purification, and characterization of a novel high molecular weight alkaline protease produced by Halobacillus sp. HAL1 using fish wastes as a substrate

BackgroundHydrolytic enzymes from halophilic microorganisms have a wide range of industrial applications. Herein, we report the isolation of Halobacillus sp. HAL1, a moderately halophilic bacterium that produces a novel high molecular weight extracellular alkaline protease when grown in fish processing wastes as a substrate. ResultsResults showed that the isolated strain belonged to the genus Halobacillus, and it was designated as Halobacillus sp. HAL1 with the GenBank accession number OK001470. The strain secreted an extracellular alkaline protease, and the highest yield was obtained when it was grown in a medium with fish wastes substrate as the sole nutritional source (10 g/L) and incubated at 25 °C under shaking conditions. The enzyme was partially purified by Sephadex G-100 column chromatography. Zymographic analysis showed two casein degrading bands of about 190 and 250 KDa. The optimum enzyme activity was at a temperature of 50 °C at pH 8. The proteolytic activity was enhanced in the presence of metal ions (Ca2+, Mg2+, and Mn2+), surfactants (Tween 80, SDS, and Triton-X100), H2O2, and EDTA. ConclusionOur study indicates that Haobacillus sp. HAL1 is a moderately halophilic strain and secrets a novel high molecular wight alkaline protease that is suitable for detergent formulation.

OPTIMUM CONDITIONS FOR PHENYLALANINE AMMONIA LYASE EXTRACTION AND PURIFICATION FROM LOCALLY CULTIVATED GRAPE SEEDS

In the present study, Phenylalanine ammonia lyase (PAL) activity was detected in different parts of grape plant (leaves, pulp, whole seeds and defatted seeds) extracts. The results revealed that the whole seed possess highest PAL activity (69.44 U), followed by defatted seed (44.22 U), whereas extracts of leaves and pulp gave the lowest activity values (22.77 and 5.11 U, respectively). The optimum conditions for crude PAL extraction achieved using 50mM potassium phosphate buffer (pH 7) at mixing ratios of (1:7) for 1 h. extraction time. PAL was shown to precipitate out at the saturation range between 20-90%. For further purification, gel filtration chromatography using Sephadex G-200 column (1.5×70) and ion exchange chromatography using DEAE- Cellulose (DE 52) column (3.2× 10) were applied. The specific activity, number of folds and yield of purified PAL was 1157 U/mg, 19.94 and 15.32 % respectively.

Structural characterization and immunomodulatory activity of a polysaccharide from finger citron extracted by continuous phase-transition extraction

FCP-2-1, a water-soluble polysaccharide rich in galacturonic acid was isolated by continuous phase-transition extraction and purified with DEAE-52 cellulose and Sephadex G-100 column chromatography from finger citron with essential oil and flavonoids removed. The structural characterization and immunomodulatory activity of FCP-2-1 were further investigated in this work. FCP-2-1 with a Mw and Mn of 1.503 × 104 g/mol and 1.125 × 104 g/mol, respectively, was predominantly composed of galacturonic acid, galactose, and arabinose in a molar ratio of 0.685: 0.032: 0.283. The main linkage types of FCP-2-1 were proved to be →5)-α-L-Araf-(1→ and →4)-α-D-GalpA-(1→ based on methylation and NMR analysis. Moreover, FCP-2-1 was demonstrated to have significant immunomodulatory effects on macrophages in vitro by improving the cell viability, and enhancing phagocytic activity and secretion of NO and cytokines (IL-1β, IL-6, IL-10 and TNF-α), indicating that FCP-2-1 could be used as a natural agent in immunoregulation functional foods.

Purification, structural identification, antioxidant activity, and inhibitory effects on digestive enzymes of α-glucan from Chuanminshen violaceum

Chuanminshen violaceum, as a food and folk medicine, is cultivated and grown only in China. Its polysaccharides play an important role in medicinal benefits. Herein, crude C. violaceum polysaccharides (CVPs) were purified to yield two polysaccharide fractions (CVPs-1-G and CVPs-2-G) using DEAE Cellulose-52 and Sephadex G-100 column chromatography. Structural analyses indicated that both CVPs-1-G and CVPs-2-G were glucans with average molecular weights of 8.1 kDa and 187.5 kDa, respectively. The main chain of the major component (CVPs-1-G) might be composed of (1 → 4)-linked α-Glcp, and the branched-chain might be an α-Glcp → 4)-α-D-Glcp-(1 → 6)-α-D-Glcp-(1 → 4)-α-D-Glcp, which connected to the main chain through the O-6 position of → 3,6)-α-D-Glcp-(1 →. In addition, CVPs-1-G displayed ABTS, DPPH, and hydroxyl radical scavenging activity, as well as competitive inhibitory activities of α-amylase and non-competitive inhibitory activities of lipase and pepsin. According to molecular docking, its inhibitory activity on digestive enzyme might be caused by hydrogen bond and hydrophobic interaction between them. Thus, CVPs exhibit strong potential for use in functional foods and pharmaceuticals.

Structural characterization and anti-inflammatory activity of polysaccharides from Astragalus membranaceus

In this study, two homogeneous polysaccharides (APS-A1 and APS-B1) were isolated from Astragalus membranaceus by DEAE-52 cellulose and Sephadex G-100 column chromatography. Their chemical structures were characterized by molecular weight distribution, monosaccharide composition, infrared spectrum, methylation analysis, and NMR. The results revealed that APS-A1 (2.62 × 106 Da) was a 1,4-α-D-Glcp backbone with a 1,4,6-α-D-Glcp branch every ten residues. APS-B1 (4.95 × 106 Da) was a heteropolysaccharide composed of glucose, galactose, and arabinose (75.24:17.27:19.35). Its backbone consisted of 1,4-α-D-Glcp, 1,4,6-α-D-Glcp, 1,5-α-L-Araf and the sidechains composed of 1,6-α-D-Galp and T-α/β-Glcp. Bioactivity assays showed that APS-A1 and APS-B1 had potential anti-inflammatory activity. They could inhibit the production of inflammatory factors (TNF-α, IL-6, and MCP-1) in LPS-stimulated RAW264.7 macrophages via NF-κB and MAPK (ERK, JNK) pathways. These results suggested that the two polysaccharides could be potential anti-inflammatory supplements.

Isolation, purification, and identification of antifungal protein produced by Bacillus subtilis SL-44 and anti-fungal resistance in apple

Apple anthracnose is a fruit fungal disease that is currently recognized as one of the most severe threats to apples worldwide. In this study, antifungal protein from Bacillus subtilis SL-44 was isolated, purified, identified, and applied for Colletotrichum gloeosporioides control. The antagonistic experiment showed that SL-44 had an excellent broad spectrum against plant pathogenic fungi. The optimal fermentation conditions were as follows: initial pH was 7, inoculum volume was 2%, and rotational speed was 180 r/min. The optimized yield of antifungal protein increased by 45.83% compared with that before. The crude protein was isolated and purified by (NH4)2SO4 precipitation, DEAE-Sepharose Fast Flow, and Sephadex G-100 column chromatography. LC-MS analyzed that antifungal protein was likely to be a novel protein with a molecular weight of 42kDa. The mechanism revealed that the antifungal protein may disrupt the cell wall structure of C. gloeosporioides and function as its antifungal action. Additionally, antifungal protein significantly alleviated the size of the lesion to more than 70% in the apple infection protection test. In conclusion, antifungal protein has remarkable potential in developing fungicides for the biological control of apple anthracnose. HIGHLIGHTS: 1. B. subtilis SL-44 had broad-spectrum antagonism against plant pathogenic fungi. 2. The optimal fermentation conditions for extracting antifungal protein were optimized. 3. The antifungal protein is a novel protein with a molecular weight of 42kDa. 4. The mechanism of antifungal protein may disrupt the cell wall structure of C. gloeosporioides.

Purification and characterization of moderately thermostable raw-starch digesting α-amylase from endophytic Streptomyces mobaraensis DB13 associated with Costus speciosus.

Endophytic actinobacteria are known to produce various enzymes with potential industrial applications. Alpha-amylase is an important class of industrial enzyme with a multi-dimensional utility. The present experiment was designed to characterize a moderately thermostable α-amylase producing endophytic Streptomyces mobaraensis DB13 isolated from Costus speciosus (J. Koenig) Sm. The enzyme was purified using 60% ammonium sulphate precipitation, dialysis, and Sephadex G-100 column chromatography. Based on 12% SDS-PAGE, the molecular weight of the purified α-amylase was estimated to be 55 kDa. The maximum α-amylase activity was achieved at pH 7.0, 50°C and it retained 80% of its activity at both pH 7.0 and 8.0 after incubation for 2 h. The α-mylase activity is strongly enhanced by Ca2+, Mg2+, and inhibited by Ba2+. The activity remains stable in the presence of Tween-80, SDS, PMSF, and Triton X-100; however, β-mercaptoethanol, EDTA, and H2O2 reduced the activity. The kinetic parameters Km and Vmax values for this α-amylase were calculated as 2.53 mM and 29.42 U/mL respectively. The α-amylase had the ability to digest various raw starches at a concentration of 10 mg/mL at pH 7.0, 50°C, where maize and rice are the preferred substrates. The digestion starts after 4 h of incubation, which reaches maximum after 48 h of incubation. These results suggest that S. mobaraensis DB13 is a potential source of moderately thermostable α-amylase enzyme, that effciently hydrolyzes raw starch. It suggesting that this α-amylase is a promising candidate to be use for industrial purposes.

A comparative study of pectic polysaccharides from fresh and dried Dendrobium officinale based on their structural properties and hepatoprotection in alcoholic liver damaged mice.

In this study, two pectic polysaccharides from fresh and dried Dendrobium officinale, namely FDP and DDP, were obtained by sour-water extraction, ethanol precipitation and further purification with DEAE cellulose-52 and Sephadex G-100 column chromatography. FDP/DDP had eight similar glycosidic linkages including 1,4-linked-GlcAp, 1,4- and 1,3,4-linked-GalAp, 1,3,4- and T-linked-Glcp, 1,6- and T-linked-Galp, T-linked-Galp and T-linked-Xylp. Besides, FDP was marked by 1,6-, 1,2,6-linked-Manp and 1,2,4-, 1,2-linked-Rhap, and DDP consisted of unique 1,6-linked-GlcAp and 1,3,6-Manp. FDP with a molecular weight of 14.8 kDa generally showed stronger scavenging capacity against DPPH, ABTS and hydroxyl radicals than DDP (p < 0.05). Pretreatment with FDP/DDP alleviated the alcohol-induced liver injury in mice, and their serum aminotransferase and triglyceride levels were 10.3%-57.8% lower than those of the model group (MG). Meanwhile, the FDP/DDP-M and FDP/DDP-H groups (200 and 300 mg kg-1) displayed a remarkable increase in antioxidant enzyme activities and significant reduction in inflammatory cytokine levels in comparison with the MG. Further analysis revealed that FDP-treated mice generally exhibited lower transaminase levels and inflammatory cytokine expression as well as higher antioxidant enzyme activities than DDP-treated ones. The FDP-H group showed significant restoration, which was slightly less than or almost comparable to that of the bifendate-fed positive control. The above results indicate that D. officinale pectin can attenuate oxidative stress and inflammatory cytokine response, and ultimately ameliorate liver injury, and "fresh" pectin with specific structural characteristics is expected to be more promising as hepatoprotective food.

A novel Stauntonia leucantha fruits arabinogalactan: isolation and structural characterization

Polysaccharides were the key ingredients of many herbal medicines, and were responsible for multiple pharmacological activities. In this study, a novel polysaccharide fraction, named SLP-2, was isolated from Stauntonia leucantha fruits, and purified by DEAE-52 and Sephadex G-100 column chromatography. Furthermore, SLP-2 was identified by congo red, methylation, partial acid hydrolysis and NMR. The results indicated that the backbone of SLP-2 was composed of →4)-β-D-Galp-(1 → 4)-β-D-Galp-(1→ substituted at C-6 with 1,5-linked arabinan. SLP-2 had good anti-oxidation ability in vitro. Surprisingly, we found that reduction of carboxyl groups and methylation of hydroxyl groups enhanced the ability to scavenge 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) radicals and inhibit lipid peroxidation, and weakened the activity to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals and reduce ferric iron.

Isolation, Purification, and Antioxidant Activities of Polysaccharides from Choerospondias axillaris Leaves.

The extraction, characterization and antioxidant activity of polysaccharides from Choerospondias axillaris leaves were investigated in the present study. Two purified polysaccharide fractions, CALP-1 and CALP-2, were isolated from crude Choerospondias axillaris leaf polysaccharides (CALP) by DEAE-52 cellulose chromatography and Sephadex G-100 column chromatography. The characteristics of CAL-1 and CALP-2 were determined by using High-performance Gel Permeation Chromatography (HPGPC), High-Performance Anion-Exchange Chromatography, HPAEC (HPAEC-PAD) and Fourier transform infrared spectroscopy (FTIR). CALP-1 with molecular weight of 11.20 KDa was comprised of Rhamnose, Arabinose, Galactose, Glucose, Xylose, Mannose and galacturonic acid in a molar ratio of 5.16:2.31:5.50:27.18:1.00:0.76:1.07. CAL-2 with molecular weight of 8.03 KDa consisted of Rhamnose, Arabinose, Galactose, Glucose, and galacturonic acid at a ratio of 1.38:3.63:18.84:8.28:1.45. FTIR revealed that CALP-1 and CALP-2 were acidic polysaccharides. The antioxidant activity of crude CALP, CALP-1 and CALP-2 was evaluated in vitro. The fraction CALP-2 was demonstrated to be of polysaccharide nature containing a large percentage of Galactose but no Xylose and Mannose. The antioxidant activity assays showed that CALP-1 and CALP-2 exhibited antioxidant and scavenging activities on hydroxyl and DPPH radicals in vitro. Compared with pure polysaccharide, crude CALP exhibited stronger anti-oxidant activities. These results will provide a better understanding of Choerospondias axillaris leaf polysaccharide and promote the potential applications of Choerospondias axillaris leaf polysaccharide in the pharmacological field and as a natural antioxidant.

Co‐production of nattokinase and α ‐amylase from Bacillus natto fermentation using okara

Effective use of okara (soybean residue) requires more application aspects to increase its value. The production of enzyme preparations by okara fermented may be an effective approach. In this paper, Bacillus natto was used in okara fermentation to investigate the production of certain enzymes including nattokinase. The results showed that the fermented okara by B. natto produced a large amount of nattokinase and α-amylase. After the response surface optimization, the nattokinase activity(189.3 IU/mg) and the α-amylase activity (7.8 U/mg) were obtained at the 36 h fermentation time, the rotation speed 170 rpm, the inoculation amount 9%. After simple two-step purification, ammonium sulfate precipitation and Sephadex G-200 column chromatography, the activities of these two enzymes reached 1663.9 IU/mg and 39.93 U/mg, respectively. In short, this research provides a theoretical basis for expanding the application of okara as a low-cost enzyme preparation. Novelty impact statement The co-production of nattokinase and α-amylase from fermented okara by Bacillus natto can effectively increase the added value of okara. Meanwhile this research provides a theoretical basis for expanding the application of okara as a low-cost enzyme preparation.

Structural characterization and immune-enhancing activity of a novel acid proteoglycan from Black soybean

Black soybean ( Glycine max (L.) merr), as an edible seed of the leguminous plant, possesses a high amount of proteins and carbohydrates. However, few studies have been conducted on the protein-polysaccharide polymer (proteoglycan) in Black soybean. Here, we aim to characterize the proteoglycan in Black soybean. Firstly, a crude extract from Black soybean was obtained by water extraction and alcohol precipitation. Then, DEAE-52 cellulose and Sephadex G-100 column chromatography were used to isolate proteoglycans in the crude extract, and a novel proteoglycan named BBWPS2 was obtained. Moreover, the chemical analysis showed that BBWPS2 was a proteoglycan of 2.02 × 10 5 Da, and composed of 7 kinds of monosaccharides and 16 kinds of amino acids. β -elimination reaction identified the carbohydrate chain was connected to peptide chain with O- glycosidic bonds. Finally, the immunomodulatory tests indicated that BBWPS2 can significantly promote the primary splenocyte proliferation and activate mouse peritoneal macrophages to produce TNF-α. Subsequent pathway studies revealed that BBWPS2 activated macrophages through the mannose receptor-mediated NF-κB/MAPK signaling pathway. Our work indicated that BBWPS2 from Black soybean could be explored as a promising natural immunopotentiator in food and pharmaceutical industries.

Characterization of a novel keratinase from Chrysosporium tropicum

A keratinophilic fungus Chrysosporium tropicum was isolated from poultry farm soil and screened for extracellular keratinase activity. The fungus was cultured in basal salts medium and keratinase production was assessed. The novel keratinase was purified by Sephadex G-100 column chromatography and characterized. The molecular weight of the enzyme was estimated to be 14.5 KDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH and temperature of the keratinase were found to be 7.5 and 40oC respectively. The Km for keratin powder from human hair was 6.67 mg and the Vmax of novel keratinase was determined to be 0.33 mg ml-1 by the Lineweaver-Burk plot. The enzyme activity was almost completely inhibited by phenylmethylsulphonyl fluoride (PMSF) suggesting that the keratinase belongs to the serine protease family.