Jun-Qiang Jia

Production of ACE inhibitory peptides from sweet sorghum grain protein using alcalase: Hydrolysis kinetic, purification and molecular docking study.

In this study, sweet sorghum grain protein (SSGP) was hydrolyzed using alcalase yielding ACE inhibitory peptides. A kinetic model was proposed to describe the enzymolysis process of SSGP. The kinetic parameters, a and b, were determined according to experimental data. It was found that the model was reliable to describe the kinetic behaviour for SSGP hydrolysis by alcalase. After hydrolysis, the SSGP hydrolysate with DH of 19% exhibited the strongest ACE inhibitory activity and the hydrolysate was then used to isolate ACE inhibitory peptides. A novel ACE inhibitory peptide was successfully purified from this hydrolysate by ultrafiltration, ion exchange chromatography, gel filtration chromatography, and reversed-phased high performance liquid chromatography (RP-HPLC). The amino acid sequence of the purified peptide was identified as Thr-Leu-Ser (IC50=102.1 μM). The molecular docking studies revealed that the ACE inhibition of the tripeptide was mainly attributed to its C-terminal Ser, which can effectively interact with the S1 and S2 pockets of ACE. Our studies suggest that the tripeptide from the SSGP hydrolysate can be utilized to develop functional food ingredients or pharmaceuticals for prevention of hypertension.

Characterization, antioxidant and antitumor activities of polysaccharides from purple sweet potato.

Three polysaccharides, PSPP1-1, PSPP2-1 and PSPP3-1, were isolated from purple sweet potato. The three polysaccharides belonged to β-type polysaccharides and contained low proportions of proteins and uronic acids. PSPP1-1 and PSPP3-1 with molecular weights of 33.3 and 75.3 kDa, respectively, were composed of rhamnose, xylose, glucose and galactose, whereas PSPP2-1 with molecular weight of 17.8 kDa was composed of rhamnose and galactose. The three polysaccharides possessed in vitro antioxidant (scavenging DPPH radicals, chelating ferrous ions and reducing power) and antitumor activities (against SGC7901 and SW620 cells) in a dose-dependent manner. Among the three polysaccharides, PSPP2-1 exhibited the strongest reducing power, scavenging activity on DPPH radicals and chelating capability on ferrous ions. PSPP1-1 showed the strongest inhibitory activities on the growth of SGC7901 and SW620 cells. In addition, flow cytometry results showed that PSPP1-1 could induce apoptosis in SGC7901 and SW620 cells. These results suggest that polysaccharides from purple sweet potato are potential natural antioxidant and antitumor agents that can be used as drugs or functional food ingredients.

A novel angiotensin-І converting enzyme (ACE) inhibitory peptide from gastrointestinal protease hydrolysate of silkworm pupa (Bombyx mori) protein: Biochemical characterization and molecular docking study

Silkworm pupa (Bombyx mori) protein was hydrolyzed using gastrointestinal endopeptidases (pepsin, trypsin and α-chymotrypsin). Then, the hydrolysate was purified sequentially by ultrafiltration, gel filtration chromatography and RP-HPLC. A novel ACE inhibitory peptide, Ala-Ser-Leu, with the IC50 value of 102.15μM, was identified by IT-MS/MS. This is the first report of Ala-Ser-Leu from natural protein. Lineweaver-Burk plots suggest that the peptide is a competitive inhibitor against ACE. The molecular docking studies revealed that the ACE inhibition of Ala-Ser-Leu is mainly attributed to forming very strong hydrogen bonds with the S1 pocket (Ala354) and the S2 pocket (Gln281 and His353). The results indicate that silkworm pupa (B. mori) protein or its gastrointestinal protease hydrolysate could be used as a functional ingredient in auxiliary therapeutic foods against hypertension.

Comparison of the structural characterization and biological activity of acidic polysaccharides from Cordyceps militaris cultured with different media.

Two acidic polysaccharide fractions, CM-jd-CPS2 and CM-jd(Y)-CPS2, were isolated from the fruiting bodies of cultured Cordyceps militaris grown on solid rice medium and silkworm pupa, respectively, by hot-water extraction, ethanol precipitation and fractionation using ion-exchange column (DEAE-cellulose-52) and gel-filtration column (Sephadex G-100) chromatography. Their structural characterizations were performed by gas chromatography and fourier-transform infrared spectroscopy. Some differences existed between their structures, which indicated that culture media could influence the structure of polysaccharides of C. militaris. The antioxidant activities of CM-jd-CPS2 and CM-jd(Y)-CPS2 were evaluated by various methods in vitro. They had strong 2,2-diphenyl-1-picrylhydrazyl radical-scavenging activity and ferrous ion-chelating capacity, but moderate reducing power. The antioxidant activities of CM-jd(Y)-CPS2 were slightly higher than those of CM-jd-CPS2. These two acidic fractions were evaluated for proliferation of mouse splenocyte activity in vitro. They both possessed does-dependent mitogenic effects on mouse splenocytes, and could synergistically promote murine T- and B-lymphocytes induced by Con A and LPS. CM-jd(Y)-CPS2 exhibited stronger stimulatory activities upon immunomodulation than CM-jd-CPS2. These results are beneficial for the interpretation of the connection between polysaccharide structures and their biological activities.

Improved 1-Deoxynojirimycin (DNJ) production in mulberry leaves fermented by microorganism

DNJ, an inhibitor of α-glucosidase, is used to suppress the elevation of postprandial hyperglycemia. In this study, we focus on screening an appropriate microorganism for performing fermentation to improve DNJ content in mulberry leaf. Results showed that Ganoderma lucidum was selected from 8 species and shown to be the most effective in improvement of DNJ production from mulberry leaves through fermentation. Based on single factor and three factor influence level tests by following the Plackett-Burman design, the optimum extraction yield was analyzed by response surface methodology (RSM). The extracted DNJ was determined by reverse-phase high performance liquid chromatograph equipped with fluorescence detector (HPLC-FD). The results of RSM showed that the optimal condition for mulberry fermentation was defined as pH 6.97, potassium nitrate content 0.81% and inoculums volume 2 mL. The extraction efficiency reached to 0.548% in maximum which is 2.74 fold of those in mulberry leaf.

Identification of ecdysone response elements (EcREs) in the Bombyx mori cathepsin D promoter.

Bombyx mori Cathepsin D (BmCatD) is specifically expressed in the fat body, and plays a critical role for the programmed cell death of the larval fat body and pupal gut during metamorphosis. To better understand the transcriptional control of BmCatD expression, we conducted this study to identify the ecdysone response elements (EcREs) in the BmCatD promoter and clarify their regulational functions. We inserted EcREs into a recombinant AcMNPV (Autographa californica multiple nucleopolyhedrovirus) vector and performed luciferase assay with a dual-luciferase quantitative assay system. Three putative EcREs were located at positions -109 to -99, -836 to -826 and -856 to -846 relative to the transcription start site. Overlapping deletion studies of this EcRE region showed that the three EcREs could suppress the ectopic expression of the BmCatD promoter. EcRE mutations resulted in the loss of the fat body-specific expression of the BmCatD gene. These results suggest that the EcREs are vital for activation of the promoter by 20-hydroxyecdysone (20E) in the larval fat body and further support the crucial role of ecdysone signaling to control cathepsin D gene transcription. It may suggest that the heterodimeric complex EcR/USP mediates the activation of ecdysone-dependent BmCatD transcription in the larval fat body of B. mori.

Isolation and characterization of an antimicrobial endophytic bacterium ME-2 from mulberry twig in China

An antimicrobial endophytic bacterium strain, ME-2, was isolated from fresh mulberry twig tissues, and was identified. In addition, antimicrobial activities of the isolated strain were evaluated against Bacillus thuringiensis, Escherichia coli, Staphylococcus aureus, Beauveria bassiana and Mycoid bacillus using the antagonism test method. The results showed that, strain ME-2 was gram-positive, aerobic, motile, nitrate-reduction-positive, spore-formation-positive, methyl-red-negative, 0.6-0.7 × 1.5-1.6 μm long and rods. The strain could utilize D-glucose, D-lactose, sucrose, starch, citrate, gelatin and casein, and it was slightly halophilic with growth occurring at 10% (w/v) NaCl. According to the morphological, biochemical and physiological properties and sequence analysis of 16S rRNA gene, the strain was identified as Bacillus subtili (GeneBank accession No. JQ900623). Moreover, the results also showed that strain ME-2 exhibited strong antimicrobial activities against B. thuringiensis (76.0%), E. coli (69.8%), S. aureus (45.8%), B. bassiana (64.2%) and M. bacillus (86.3%). It was concluded that, strain ME-2 and its antimicrobial materials might be considered as a potential antimicrobial agents in the food and pharmaceutical industries.

Antioxidant and Hemolysis Protective Effects of Polyphenol-Rich Extract from Mulberry Fruits

Background: Mulberry fruits are a superior source of polyphenol, especially anthocyanins that contribute potentially to the beneficial effects which include reducing the risk of cardiovascular diseases and cancers with antioxidant, anti-inflammatory, and chemoprotective properties. Objectives: In this study, purification of the polyphenol-rich extract from mulberry fruit (MPE) was purified and assessed the activities of antioxidant and hemolysis protective in vivo and in vitro. Materials and Methods: Antioxidant activities in vitro was measured by quantifying its 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity, reducing power and Fe2+-chelating ability. MPE was purified by high-pressure liquid chromatography (HPLC) and analyzed individual polyphenols using liquid chromatography–mass spectrometry (LC-MS)/MS. Results: The total polyphenol content was 147.69 ± 0.02 mg gallic acid equivalents (GAE)/g dried weight (DW) in the extract and 403.55 ± 0.02 mg GAE/g DW in the purified extract. Further identification by HPLC-ultraviolet-visible and LC-MS/MS analysis indicated in MPE, an anthocyanin compound, cyanidin-3-O-glucoside. With regard to in vitro assays, MPE possessed antioxidant effect, especially in Fe2+ chelating ability with an IC50value of 1.016 mg/mL. The protective effects on mouse red blood cell hemolysis and lipid peroxidation ex vivo were dose and time dependent. Conclusion: It indicates that MPE could be a good candidate for future biomedical applications to promote human health with limited side effects. Abbreviations used: MPE: Purification of the polyphenol-rich extract from mulberry fruit, LC-MS: Liquid chromatography–mass spectrometry, HPLC: High-pressure liquid chromatography, DPPH: 2,2-diphenyl-1-picrylhydrazyl scavenging activity, RBC: Red blood cell, GAE: Gallic acid equivalent, FeCl2: Ferrous chloride, H2O2: Hydrogen peroxide, EDTA-2Na: Ethylenediaminetetraacetic acid disodium salt, PBS: Phosphate-buffered saline, TCA: Trichloroacetic acid, TBA: 2-thiobarbituric acid, FeSO4: Ferrous sulphate, MDA: Malondialdehyde, VC: Vitamin C, DW: Dried weight.

Genotypic analysis of degenerative Cordyceps militaris cultured in the pupa of Bombyx mori

The chemical composition and pharmacological effects of Cordyceps militaris are similar to those of Cordyceps sinensis, with the former undergoing greater development and utilization. Strain degeneration is a common phenomenon that occurs with high frequency during the subculturing of C. militaris, however, and the mechanism underlying strain degeneration remains unclear. In this study, we used touch‐down PCR to compare the ITS1 + 5.8S + ITS2, 18S, 28S and mating‐type (MAT) regions sequence of wild‐type and degenerated strains of C. militaris. We also used quantitative real‐time PCR to analyze expression levels of the CmMAT gene. Sequence analysis showed that the ITS1 + 5.8S + ITS2 and 28S regions of degenerated and wild‐type strains were completely identical, the 18S region of the degenerated strain contained seven single‐base mutations, including six base substitutions and one single‐base insertion. Compared with the wild‐type strain, the degenerated strain contained a deletion of the MAT1–2‐1 region, three base substitutions in the MAT1–1‐1 region, and a base substitution in the MAT1–1‐2 region that causes a glycine‐to‐valine amino acid substitution. Quantitative real‐time PCR analysis detected no CmMAT1–2‐1 gene expression in the degenerated strain, confirming the deletion of the CmMAT1–2‐1 gene. Expression levels of the CmMAT1–1‐1 and CmMAT1–1‐2 genes were significantly down‐regulated to only 7.5 % and 4.4 %, respectively, that of the wild‐type strain. These results indicate that 18S and MAT region mutations, as well as down‐regulated of CmMAT gene expression levels, may play important roles in C. militaris degeneration. This study provides a theoretical basis for further elucidation of the molecular mechanisms of C. militaris degeneration.