DEAE Sephadex Research Articles

The structural features and anti-inflammatory properties of a glucogalactan from Holotrichia diomphalia Bates (Qi Cao)

Ethnopharmacological relevanceThe dried larvae of Holotrichia diomphalia Bates, named Qi Cao, is a traditional Chinese medicine treat for liver diseases and arthritis. Polysaccharides is a principal component in Qi Cao, which exhibiting antioxidant and anti-inflammatory effects. However, the structural characteristics and underlying mechanisms of the polysaccharides remain inadequately elucidated. Aim of the studyTo analyze the primary structure and elucidate the molecular anti-inflammatory mechanisms of the active polysaccharide in Qi Cao. Materials and methodsThe total polysaccharide was extracted by water extraction and alcohol precipitation, and further isolated and purified by DEAE Sephadex A-25 column and Sephadex G-100 column. The anti-inflammatory properties of four major fractions (HDPS-1, HDPS-2, HDPS-3, HDPS-4) and the pure homogeneous polysaccharides (HDPS-1I and HDPS-1II) were assessed using a RAW 264.7 cell model induced by lipopolysaccharide (LPS), and HDPS-1II was identified as the polysaccharide exhibiting significant anti-inflammatory activity in Qi Cao. The structural characteristics of HDPS-1II were subsequently analyzed using high-performance size-exclusion chromatography (HPSEC), fourier-transform infrared spectroscopy (FT-IR), gas chromatography-mass spectrometry (GC-MS), and nuclear magnetic resonance (NMR) spectroscopy. The TLR4, NF-κB, COX-2 and iNOS expressions were determined by Western blot analysis to investigate the anti-inflammatory mechanism of HDPS-1II in vitro. Finally, the in vivo anti-inflammatory activity of HDPS-1II were evaluated by measuring the serum levels of pro-inflammatory factors, inflammatory cell infiltration and organelle damage in the lung tissues of sepsis model mice. ResultsA homogeneous polysaccharide (HDPS-1II) with molecular weight of 1.7 × 104 Da was isolated from Holotrichia diomphalia Bates. HDPS-1II contains a backbone of α-T-Glcp-(1 → 6)-α-Glcp-(1 → 4)-α-Galp-(1 → 4)-α-Galp-(1 → 6)-α-Galp-(1 → 3)-α-Galp-(1 → . It inhibited activation of the TLR4/NF-κB signaling and reduced pro-inflammatory factors and NO in LPS-stimulated macrophage. Moreover, HDPS-1II increased the survival rate, inhibited inflammatory cells infiltration, and ameliorated the lung tissue damage in septic mice. ConclusionsHDPS-1II exhibits anti-inflammatory effects in vitro and in vivo, which is the active polysaccharide components of the anti-inflammatory activity of Qi Cao.

Screening of sweet potatoes varieties, physicochemical and biological activity studies of glycoproteins of selected varieties

In order to meet the nutritional and health needs of different consumers, the main nutrients of eight new sweet potatoes (SPs) varieties were to characterize by principal component analysis. The watermelon red (WR) variety is proving to be the preferred SPs variety because of its high nutritional value and unique sensory features. Three homogeneous fractions SPFG-1, SPFG -2 and SPFG-3 with yield of 53.02% ± 3.20%, 10.50% ± 0.61% and 5.20% ± 0.35% were obtained from the WR by multifrequency ultrasonic extraction (MUE), isolated and purified with DEAE Sepharose CL-6B and Sephadex G-100 gel chromatography. Physiochemical and structural characteristics of three fractions were studied with chemical components analysis, HPLC, HPGPC, and β-elimination, UV, FT-IR and CD spectrum analysis. SPFG-1, SPFG-2 and SPFG-3 were all conjugates with O-glycosylation with the molecular weight of 30.42 ± 2.12, 391.15 ± 25.12 and 6.85 ± 0.78 kDa, including seven monosaccharides and 17 amino acids with quite different mole percentages, that they were predominantly α-helix, β-folding and irregularly curled with a relatively small β-turning content. Meanwhile, the three conjugates exhibited excellent antioxidant and hypoglycemic activities by radical scavenging, α-glycosidase and α-amylase inhibition assays. These findings are helpful to promote the breeding, cultivation and promotion of new SPs varieties with higher nutritional value, as well as the development and application of glycoproteins.

Purification of a chemotherapeutic agent, L-Arginase, from Pseudomonas aeruginosa isolated from soil.

L. arginase refers to the enzyme arginase found in the genus Lactobacillus, it plays a crucial role in the urea cycle, and has implications in various biological applications. This study aimed to purify arginase from Pseudomonas aeruginosa, isolated from soil, and apply it as an anticancer. 28 soil samples of P. aeruginosa were collected from different places of Baghdad, and rice lands in Najaf and Diwaniyah governorates. Different standard laboratory and biochemical assays, and Vitik system were used in diagnosis and growth of arginase enzyme under certain pH, temperature, incubation period. The purified enzyme was precipitated by ammonium sulfite (60-80%), dialyses bag 8000-1000KD, ion exchange by DEAE cellulose and sephadex G100 in gel filtration. Cytotoxicity of arginase against breast t cancer AJM-13 and rat embryo fibroblast REF normal cell line was evaluated for (48 and 72 hours). The inhibition rate increased in the low concentration of abnormal cell (AMJ-13) while decreased in the normal cell (REF), this study takes different concentration (0.392-12.5mg/mL), and low concentration (1562-0.048 mg/mL), the result in high concentration was IR 54.7% during 72 hours for AJM-13 and 14.3% for REF in the same time, while the low concentration was IR 91% in the 1562 mg/mL in the AMJ-13, and 51% in ERF, LD50 of arginase enzyme was 0.781 mg/mL that 41% during 72 hours for ERF, its save to normal cells. Arginase enzyme, at low concentrations, may have an inhibitory effect on cancer cells, and simultaneously, protect normal cell lines.

Enzyme-Assisted Ultrasonic Extraction and Antioxidant Activities of Polysaccharides from Schizochytrium limacinum Meal.

Enzyme-assisted ultrasonic extraction (EAUE) was utilized and optimized for extracting polysaccharides from Schizochytrium limacinum meal (SLMPs) via the response surface methodology. The optimal EAUE conditions were determined as follows: enzyme concentration at 5.18%, ultrasonic temperature at 53 °C, ultrasonic duration of 40 min, ultrasonic power at 60 W, and a liquid-to-material ratio of 34 mL/g, achieving a polysaccharide extraction yield of 11.86 ± 0.61%. The purified polysaccharide component, SLMP1-1, isolated using DEAE Sepharose Fast Flow and Sephadex G-100 columns, exhibited potent antioxidant activity. SLMP1-1, with a molecular weight of 25.5 kDa, comprises glucose, mannose, arabinose, and galactose in a molar ratio of 16.39:14.75:1:693.03. 1H NMR analysis revealed the α configuration of SLMP1-1. Antioxidant assessments, including DPPH, ABTS, and ferric ion reduction assays, were detected with inhibitory values at 21.82-82.98%, 38.21-98.46%, and 3.30-20.30% at 0.2-1.0 mg/mL. This confirmed the effective antioxidant capacity of SLMP1-1, which is notably enhanced post oral and gastric digestion. The findings suggest that polysaccharides extracted from Schizochytrium limacinum meal hold significant promise as natural antioxidants.

Synovial Fluid Proteomics and Serum Metabolomics Reveal Molecular and Metabolic Changes in Osteoarthritis

Background: Osteoarthritis (OA) is a common joint disorder with a complex and multifactorial pathogenesis. Proteomics analysis using two-dimensional gel electrophoresis (2DE) and mass spectrometry (MS) enables high-throughput identification of differentially expressed proteins related to OA. However, the etiology, pathophysiology, and early diagnostic markers of OA are still poorly understood. Methods: Synovial fluid protein biomarkers were compared between OA patients and healthy controls. It was fractionated using DEAE cellulose and Sephadex G-200 columns, followed by SDS‒PAGE and 2D-PAGE for visualization and identification. Mass spectrometry and Mascot were used for protein analysis, and serum metabolite profiles were also investigated using 1D 1H CPMG NMR spectra. Multivariate data analysis, including PCA and PLS-DA, was performed to detect metabolic differences between groups. Results: Proteomics analysis revealed differential expression of synovial fluid proteins, such as serine protease inhibitors, complement components, and apolipoproteins, which may be involved in inflammation and cartilage breakdown. Additionally, serum metabolite profiles differed significantly between OA patients and controls, involving amino acid, lipid, glucose, and energy metabolism. The pathway analysis indicated disruption of the metabolic pathways associated with these metabolites. Conclusions: This study provides insights into the molecular and metabolic changes in OA. Protein biomarkers and serum metabolite alterations enhance the understanding of OA pathogenesis and offer potential opportunities for early diagnosis and disease management. Further validation and translation of these findings into clinical applications are needed for improved OA detection and intervention strategies.

Screening and characterization of thermostable protease-producing bacteria isolated from slaughter site soil samples from Southern India

The current study was isolated and identified Bacillus cereus from an animal slaughter site in the South Indian state of Kerala, as a potent additive to detergents for enhancing stain removal efficacy. Identifying the isolates as B. cereus was confirmed through microbial biomass production, biochemical tests, protease-specific activity, and sequencing of the 16S rRNA gene. Following the growth condition optimization, the protease enzyme was extracted from the biomass, exhibiting a protease activity of 72.212 ± 0.235 U/mL. Calcium, magnesium, and sodium positively influenced protease activity under the tested conditions and were negatively impacted by zinc, copper, and iron. The polar solvent dimethyl sulfoxide (DMSO) supported enzyme activity, whereas the non-polar solvents hexane and chloroform completely inhibited it. The highest protease-specific activity of the crude extract following dialysis was 62.997 U/mg. The purified fractions of Sephadex G-100 and DEAE-Sephadex A50 anion exchange columns showed the highest enzyme-specific activity of 193.652 U/mg and 324.004 U/mg, respectively. The addition of crude protease to a generic detergent significantly enhanced its efficacy in removing fabric stains from cotton, polyester, and silk materials. Therefore, the isolated serine protease demonstrates significant potential for industrial applications.

Nutritional intervention with acidic Stigma Maydis polysaccharide (ASMP-1) in membranous nephropathy: Targeting the PI3K/AKT/mTOR pathway

Membranous nephropathy (MN) is a glomerulonephritis characterized by C5b-9-mediated podocyte injury. Stigma Maydis is a medicinal, polysaccharide-based product derived from corn silks, traditionally used by traditional Chinese medicine physicians to treat chronic kidney disease. However, the mechanism through which Stigma Maydis polysaccharides (SMP) alleviates MN remains unclear. This study aimed to verify the potential protective effects of refined acidic SMP (ASMP-1) in vitro and in vivo. An ASMP-1 was successfully isolated by DEAE Sepharose anion exchange and Sephadex G-100 gel permeation chromatography. Our results showed that ASMP-1 suppressed oxidative stress, promoted autophagy, and repressed apoptosis. This mechanism is believed to be associated with the inhibition of the PI3K/Akt/mTOR signaling pathway. Consequently, owing to its enhanced autophagic activity and resources, ASMP-1 has potential as a functional food or nutritional supplement for kidney health preservation.

Extraction, purification and characterization of Lipoxygenase from African oil bean (Pentaclethra macrophylla Benth.) seed

Lipoxygenase (linoleate:oxygen oxidoreductase, LOX) was extracted from African oil bean seed and purified in three purification steps using ammonium sulphate precipitation, ion exchange chromatography on DEAE-Sephadex A50 column and gel filtration chromatography on Sephadex G 200. The optimum pH and temperature were investigated while the pH and temperature stability were also examined. The effect of metal ions on LOX was also investigated. The molecular weight and kinetic parameters were determined. The single protein band on SDS-PAGE suggested that the enzyme was homogenous. The molecular weight was found to be 77.54 kDa. A purification fold of 32.7 and 10.7% yield of the enzyme was achieved. The optimum temperature of the purified lipoxygenase was 40 °C and the enzyme was thermally stable at 40 °C and rapidly lost activity at 50 °C and above. The purified enzyme had an optimum pH of 6.0 and was stable at pH 5.0 and 6.0. The kinetic parameters Km and Vmax of the purified lipoxygenase were 0.1 mM and 94.40 μmol/min, respectively. The activity of the enzyme was enhanced in the presence of Ca2+ and Mg2+ while Zn2+, Cu2+ and Fe2+ significantly reduced the enzyme activity. Total inactivation of the lipoxygenase activity was observed at alkaline environment. This study confirmed lipoxygenase activity in African oil bean seed and showed properties that could be explored in improving its utilization in industrial food processing.

Purification and characterization of a psychrophilic lipase from Serratia marcescens VT 1 and its application in methyl ester synthesis

Lipase from Serratia marcescens VT 1 with a molecular weight of 62 kDa was purified by 9.42 fold with 16.89 % recovery by DEAE Cellulose and Sephadex G-75 columns. The characterization studies showed the optimum temperature and pH were 30 °C and 7, with good stability from 10 to 30 °C and pH 7. The lipase displayed good stability in the presence of metal ions Ca2+ and Na+, surfactants Gum arabic and Triton X-100, inhibitor EDTA, and organic solvents hexane and methanol. The serine inhibitor PMSF reduced lipase activity by 66 %. Vmax and Km kinetic parameters were 35.21 μmol/ml/min and 0.823 mM with p-nitrophenyl palmitate as substrate. The induction of methyl palmitate, methyl stearate, methyl oleate, and methyl linoleate from palm oil was confirmed by fatty acid methyl ester (FAME) analysis. The present study showed that the Serratia marcescens VT 1 lipase is a psychrophilic, neutral, and serine lipase efficacious of methyl esters genesis.

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.

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.

Purification and characterization of thermostable alpha‐amylase from Geobacillus sp. DS3 from Sikidang Crater, Central Java, Indonesia

Amylases are considered the most essential enzymes in biotechnology since they are widely utilized in the textile, food processing, and detergent industries. It is necessary to explore extracellular enzymatic activity in several microorganisms to discover a new potential application from amylases. In a previous study, thermophilic bacteria Geobacillus sp. DS3 isolated from Sikidang Crater, Dieng Plateau, Central Java, Indonesia showed amylase activity in starch medium at 70 °C. This study aimed to purify and characterize the thermostable alpha‐amylase from Geobacillus sp. DS3. The alpha‐amylase was produced and purified using ammonium sulfate and DEAE Sephadex A‐25 column. The enzyme activity was determined using the 3,5‐dinitrosalicylic acid (DNS) method. Geobacillus sp. DS3 optimally produced the alpha‐amylase at 60 °C for 15 h. The alpha‐amylase exhibited high enzymatic activity in 40–60% saturated ammonium sulfate extract. The molecular weight of the enzyme was estimated to be 58 kDa. The thermostable alpha‐amylase showed activity at the optimum temperature of 50 °C in 200 mM sodium phosphate buffer pH 7.0. The enzyme was inhibited by EDTA, PMSF, 2‐ME, and mostly by HgCl2. The Km and Vmax of the pure enzyme were 235.43 mM and 1428.57 U/mL, respectively. The result suggested that the purified thermostable alpha‐amylase from Geobacillus sp. DS3 offers potential application in areas of the food industry, such as the bakery industry.

Purification, structural characterization and immunological activity of Sibiraea laexigata (L.) Maxim polysaccharide.

Sibiraea laexigata (L.) Maxim (SLM) has been used as an herbal tea for treating stomach discomfort and indigestion for a long time in china. Polysaccharides have been identified as one of the major bioactive compounds in the SLM. In the present paper, ultrasonic-assisted enzymatic extraction (UAEE) method was employed in polysaccharides extraction derived from SLM using polyethylene glycol (PEG) as extraction solvent, two SLM polysaccharides (SLMPs) fractions (SLMPs-1-1 and SLMPs-2-1) were purified by DEAE Cellulose-52 and Sephadex G-100 chromatography in sequence. Then, the preliminarily structure of the two factions were characterized by chemical composition analysis, molecular weight measurement, UVS, HPLC-PMP, FT-IR, nuclear magnetic resonance (NMR) spectra analysis and SEM. The results showed that SLMPs-1-1 and SLMPs-2-1 with different molecular weights of 1.03 and 1.02 kDa, mainly composed of glucose (46.76 and 46.79%), respectively. The results of structural characterization from FT-IR, 1H NMR, and SEM revealed that SLMPs-1-1 and SLMPs-2-1 contained the typical pyranoid polysaccharide with α-glycosidic bond and β-glycosidic bond. Furthermore, it was found that SLMPs-1-1 could increase the levels of tumor necrosis factor-α (TNF-α) and interleukin-2 (IL-2), and alleviated the immune organs tissue damage of cyclophosphamide (Cy)-treated mice. RT-qPCR and Western-Blot analysis showed that SLMPs-1-1 could significantly up-regulated the levels of NF-κB, TLR4, which revealed that SLMPs-1-1 could participate in immunosuppressive protection of Cy-treated mice. These findings suggested that the potential of SLMPs-1-1 as an alternative immunostimulator could be used in food and pharmaceutical industries.

Polysaccharides from Hericium erinaceus Fruiting Bodies: Structural Characterization, Immunomodulatory Activity and Mechanism.

Five fractions from crude Hericium erinaceus polysaccharides (HEPs), including HEP-1, HEP-2, HEP-3, HEP-4 and HEP-5, were obtained through column chromatography with a DEAE Cellulose-52 column and Sephadex G-100 column. The contents of total carbohydrates and uronic acid in HEPs were 53.36% and 32.56%, respectively. HEPs were mainly composed of Fuc, Gal and Glu in a molar ratio of 7.9:68.4:23.7. Its chemical structure was characterized by sugar and methylation analysis, along with 1H and 13C NMR spectroscopy. HEP-1 contains the backbone composed of (1→6)-linked-galactose with branches attached to O-2 of some glucose. The immunological activity assay indicated that HEP-1 significantly promoted the production of nitric oxide, interleukin-6, interleukin-10, interferon-γ and tumor necrosis factor-α and the phosphorylation of signaling molecules. Collectively, these results suggested that HEP-1 could improve immunity via NF-κB, MAPK and PI3K/Akt pathways. Hericium erinaceus polysaccharides might be explored as an immunomodulatory agent for use in dietary supplements.

The antifungal activity of a serine protease and the enzyme production of characteristics of Bacillus licheniformis TG116

The growth of Phytophthora capsica, Rhizoctonia solani, Fusarium graminearum, Fusarium oxysporum and Botrytis cinerea were all inhibited by the fermentation supernatant of Bacillus licheniformis TG116, a biocontrol strain isolated from Typhonium giganteum Engl. previously with broad-spectrum resistance to plant pathogens. The fermentation supernatant of the TG116 has a great stability on temperature and UV, and shows the biological activity of protease and cellulase. The antifungal protease produced by B. licheniformis TG116 was purified to homogeneity by ammonium sulfate precipitation, DEAE Sepharose Fast Flow column chromatography and Sephadex G-50 column chromatography. The inhibition of protease by the three surfactants increased with increasing concentration inhibition. Among these surfactants, EDTA showed the strongest inhibition, with only 25% protein activity at a concentration of 1.1mmol·L-1. Gene amplification verified the presence of a gene fragment of serine protease in the strain TG116. The antimicrobial substance isolated from the fermentation broth of TG116 is a serine protease component.

Anti-glycation level of pectic oligosaccharide in orange peel and its stability in accelerated storage temperature

In this study, pectic oligosaccharides of orange peel (OPOs) were isolated and their structure characterized, and then screened according to anti-glycation level compared with aminoguanidine (AG). The results indicated that OPOs mainly included two components, and the main component has more than 50 % inhibition level for the seven glycation products at 60 °C for 40 h. At the accelerated storage temperature, OPOs demonstrated a better anti-glycation level than AG, and this inhibition was concentration-dependent. In addition, the main component in OPOs was separated into 10 fractions by DEAE Sephadex A-25 gel chromatography, the group of 5–7 monosaccharide polymerization showed the best anti-glycation effect, average anti-glycation capability on six products was over 70 % in the 40th hour. The anti-glycation level of this group was closely related to its high content of GalA and molar ratio of GalA: Rha, and positively correlated with time at moderate temperature.

Anti-tumor and immunomodulation activity of polysaccharides from Dendrobium officinale in S180 tumor-bearing mice

• DOP consisted of D-mannose, D-glucose, and D-galactose in a mole ratio of 3:2:1. • DOP possesses antitumor and immunomodulation activity. • DOP enhanced NK cell and CTL activity and increased the non-specific and tumor antigen-specific antibody titers. • DOP could inhibit the growth of S180 tumors by improving the tumor immune microenvironment. This study aims to explore the structural features and anti-tumor and immunomodulation activity of polysaccharides from Dendrobium officinale that were prepared using DEAE Sephadex A-50 and Sephadex G100. DOP structure was characterized using colorimetry, HPGPC, GC–MS, FT-IR, and NMR. The molecular weight of DOP was 1.89 × 10 5 Da. And the DOP consisted of D-mannose, D-glucose, and D-galactose in a mole ratio of 3:2:1. DOP could inhibit the growth of S180 tumors by promoting splenocytes proliferation, enhancing NK cell and CTL activity, and increasing the non-specific and tumor antigen-specific antibody levels. These findings suggested that DOP might be a potential antitumor agent with immunomodulatory activity.

Analysis and Evaluation of the Flagellin Activity of Bacillus amyloliquefaciens Ba168 Antimicrobial Proteins against Penicillium expansum.

Blue mold caused by Penicillium expansum is one of the most common apple diseases, and it is becoming a serious threat in apple production. The strain Bacillus amyloliquefaciens Ba168 showed high levels of antimicrobial activity in our previous study. To analyze the antimicrobial protein of Ba168, a high-resolution LC-MS/MS proteomic analysis was performed. A total of 1155 proteins were identified from 5233 unique peptides. A total of 16 potential antimicrobial-activity-related proteins were identified; 10 of these proteins have direct antimicrobial effects, while 6 of these proteins are associated with the formation of antimicrobial substances. Then, an antifungal protein of Ba168 was isolated and purified by the sequential chromatography of DEAE Bio-sep FF anion exchange and Sephadex G-75. The single protein, named BP8-2, showed antifungal activity towards Penicillium expansum. The peptide mass fingerprinting of the protein band of BP8-2 had a high similarity with the amino acid sequences of flagellin protein. The results showed that BP8-2 significantly inhibited the growth of P. expansum and slowed the spread of apple blue mold. The results indicated that flagellin is one of the important antimicrobial substances from Ba168.

Purification and characterization of thermostable serine alkaline protease from Geobacillus sp. DS3 isolated from Sikidang crater, Dieng plateau, Central Java, Indonesia

Thermostable proteases that optimally withstand the high‐temperature conditions of thermophilic bacteria could be produced and purified, which would be highly beneficial for use in industry. Geobacillus sp. is a thermophilic bacterium that can be found in various environmental conditions. The goal of this study was to isolate and characterize thermostable serine protease that had been produced by thermophilic Geobacillus sp. strain DS3. The proteolytic index was measured in a solid medium. The expression of protease was optimized by Geobacillus sp. DS3 at 50 °C for 18 h. Targeted protease was purified using ammonium sulfate (40‐60%) and DEAE Sephadex A‐25 resin. Using SDS‐PAGE, the molecular weight of the enzyme was predicted to be around 32 kDa. Purified thermostable protease was highly activated at 70 °C, pH 9.6 stable for 1 h, and inhibited by PMSF. Therefore, this enzyme is classified as a thermostable alkaline serine protease. Its kinetic study revealed specific activity of 0.41 U/mg (Vmax) and 0.25 mg/mL (KM). Overall, a thermostable alkaline serine protease from Geobacillus sp. DS3 showed high activity at high temperatures and alkaline pH, which is vital for application in industries such as leather processing and detergent formulation.

Microwave assisted extraction, characterization of a polysaccharide from Salvia miltiorrhiza Bunge and its antioxidant effects via ferroptosis-mediated activation of the Nrf2/HO-1 pathway

A microwave-assisted extraction procedure for the crude Salvia miltiorrhiza polysaccharides (SMPs) obtained from Salvia miltiorrhiza Bunge was optimized. Four independent variables were studied: microwave power, extraction time, solvent-to-solid ratio, and concentration of ethanol, with optimal settings of 1200 W, 12 min, 38, and 86 %, respectively. The SMPs were successively purified by DEAE Sepharose Fast Flow and Sephadex G-100 chromatography to produce a novel polysaccharide termed SMP1. The SMP1 was composed of glucose, galactose, and fructose in a molar ratio of 1:1.67:1.12 with an average molecular weight of 6087 Da. Pharmacological studies showed that SMP1 protected from OGD/R-induced ferroptosis and lipid peroxidation by activating Nrf2/HO-1 pathway in PC12 cells. Our research systematically indicated that polysaccharide could inhibit ferroptosis to alleviate oxidative stress injury, which laid the foundation for the future clinical application of Salvia miltiorrhiza polysaccharide.