A polysaccharide from Grifola frondosa relieves insulin resistance of HepG2 cell by Akt-GSK-3 pathway

Grifola frondosa is an important fungal research resource. However, there was little report about hyperglycemic activity of Grifola frondosa polysaccharide on insulin resistance in vitro. In this study, the hypoglycemic activity of a polysaccharide obtained from Grifola frondosa (GFP) on HepG2 cell and hpyerglycemic mechanism were investigated. The purity of the isolated polysaccharides was examined by HPLC. In this research, it was found that GFP enhanced the absorption of glucose of HepG2 cells in a dose dependent manner at 24 h of 30 ugmL^?1. GC-MS and FT-IR spectroscopy analysis results showed that glucose and galactose were the dominant monosaccharides in GFP and the major component of GFP was β-pyranoside. Western-blotting results showed that the HepG2 cell model treated with GFP activated the insulin receptor protein (IRS) in the cell membrane and increased phosphorylated-AktSer473 expression, which had an inhibition of glycogen synthase kinase (GSK-3). The down-regulation of GSK-3 stimulated synthesis of intracellular glycogen. The results above suggested that the GFP increased the metabolism of glucose and stimulated synthesis of intracellular glycogen through the Akt/GSK-3 pathway.     

A novel breeding strategy for new strains of Hypsizygus marmoreus and Grifola frondosa based on ligninolytic enzymes

A novel breeding strategy for new strains of Hypsizygus marmoreus and Grifola frondosa using ligninolytic enzymes as markers was evaluated with the detection and analysis of activities and composition of 15 edible fungi. The results showed that the activity and composition of ligninolytic enzyme system varied in response to changes of fungal strains. By analyzing the growth rate of mycelia and their ability to produce ligninolytic enzymes, H. marmoreus and P. geesteranus, G. frondosa and P. sajor-caju were screened for further study. Three colonies of 26 regenerated colonies of H. marmoreus and P. geesteranus protoplast fusion and one colony of 48 regenerated colonies of G. frondosa and P. sajor-caju were selected respectively. At the same time, these four strains were identified using RAPD and ISSR molecular markers. The results showed that the strains HM5G1and PS7F1are new strains and have low similarity to parental strains H. marmoreus and G. frondosa. These results are supported by the results of antagonism tests. These two fusants were significantly higher in their ligninolytic enzyme activity than H. marmoreus and G. frondosa. The growth rates of strains HM5G1and PS7F1 were also noticeably higher than those of H. marmoreus and G. frondosa, by 1.36 and 1.5 times respectively. The biological efficiency of the strain HM5G1 was 11.5 % higher than that of the parental strain H. marmoreus. This work suggests that it is an efficient way of breeding new strains to use the decolorization of ligninolytic enzymes as a preliminary screening marker.     

Evaluation of Nutritive Value of Raw and Fermented De-oiled Physic Nut, Jatropha curcas Seed Meal in the Formulated Diets for Rohu, Labeo rohita (Hamilton) Fingerlings

Rohu (Labeo rohita) fingerlings, were fed de-oiled Jatropha curcas seed meal (DJSM) supplemented diets for 60 days and the effectiveness, if any, on the growth was evaluated. Nine isonitrogenous (35 % crude protein) and isocaloric (4.0 kcal g^?1) diets were formulated of which one was reference diet (RD, fishmeal based control diet) and the other eight were experimental diets prepared by incorporating raw (D1–D4) and fermented (D5–D8) DJSM at 10, 20, 30 and 40 % levels by weight, respectively. Autoclaved DJSM was processed through solid state fermentation (SSF) for 15 days at 37 ± 1 °C by an exo-enzyme producing bacterium, Bacillus cereus Lr.H.23 isolated from the hindgut of rohu, L. rohita. Processing through SSF caused decrease in the contents of crude fibre and anti-nutritional factors, but increase in the levels of free amino acids and free fatty acids. In terms of growth performance, feed utilization efficiency and apparent protein digestibility, fish fed diet D7 containing 30 % fermented DJSM showed the best performance, which differed significantly (P < 0.05) from that of the fish fed diets containing raw DJSM. The results indicated that an inclusion level up to 30 % fermented DJSM replacing 15 % FM in the practical diet for rohu fingerlings can be proposed when compared to the RD. However, further experiments are required to recommend the ingredient for use in industry.     

Isolation and characterization of a novel mycovirus infecting an edible mushroom,Grifola frondosa

Grifola frondosa (Maitake mushroom) is an important cultivated mushroom due to its medicinal and nutrient values. In this study, we isolated and characterized a novel partitivirus (named Grifola frondosa partitivirus 1, GfPV1) infecting a standard G. frondosa strain Gf-N2. This virus has a two-segmented dsRNA genome (dsRNA1 and dsRNA2) with nucleotide lengths of 2.3 and 2.2 kbp, respectively. The coding strand of dsRNA1 and dsRNA2 segments carries single open reading frame encoding RNA-dependent RNA polymerase (RdRp) and a coat protein (CP), respectively. BLAST searches and phylogenetic analyses showed that GfPV1 is most closely related to a betapartitivirus, Lentinula edodes partitivirus 1 (RdRp <70% and CP <60% amino acid sequence identities), but the sequence divergence suggests that GfPV1 is classifiable as a new member of the genus Betapartitivirus, family Partitiviridae. The presence of GfPV1 does not affect colony morphology and fruiting body development of G. frondosa. This is the first report investigating the effects of a mycovirus infection on the colony morphology and fruiting body development of G. frondosa. Interestingly, GfPV1 accumulations markedly decreased along with the fruiting body maturation stages, suggesting the inhibition of virus multiplication during sexual phase of the G. frondosa life cycle.     

Purification and molecular cloning of a serine protease from the mushroom Hypsizigus marmoreus

A protease with a molecular mass of 28 kDa, designated as hmsp, was isolated from fresh fruiting bodies of the edible mushroom Hypsizigus marmoreus. The purification protocol entailed ion exchange chromatography on DEAE-cellulose, CM-cellulose, and FPLC-gel filtration on Superdex 75. The protease was unadsorbed on DEAE-cellulose but adsorbed on CM-cellulose. hmsp was thermolabile, and exhibited a temperature optimum at 50 °C and a pH optimum at pH 7.5. The activity of the protease was adversely affected by PMSF, EGTA and aprotinin, indicating that it is a serine protease. Based on the N-terminal sequence, the cDNA of hmsp was cloned by using RACE combined with the TAIL-PCR method. The deduced protease sequence contained a signal peptide with 19 amino acids, a pro-region with 82 amino acids, and a mature protease with 285 amino acids and a molecular mass of 28.07 kDa. It possessed the three active sites characteristic of the subtilisin family (S8A). hmsp demonstrated 63%, 57% and 44% identity in amino acid sequence respectively to Absp1, Absp2, and Gf-spr1, which are serine proteases from Agaricus bisporus and Grifola frondosa.     

棒状乳杆菌FZU63发酵对红茶汤风味品质改善的作用

乳酸菌发酵可赋予茶饮料独特的香气与滋味,且可改变其物质组成,产生益生因子等。目前,针对乳酸菌在不同发酵阶段对茶汤中风味物质形成影响的研究较少。本研究以从中国传统泡菜中筛选获得的棒状乳杆菌FZU63为发酵菌株,对不同发酵阶段红茶汤中的挥发性香气成分、还原糖、游离氨基酸、有机酸等含量的变化过程进行分析,并对发酵红茶汤的感官品质进行评价。结果表明,棒状乳杆菌FZU63以红茶汤中的葡萄糖、果糖、甘露糖和木糖作为发酵过程中的主要碳源物质。红茶汤经棒状乳杆菌FZU63发酵作用后,香气成分丰度显著增加,且主要香气组分结构发生改变,发酵红茶汤在花香、坚果香的基础上增添了水果香;此外,部分苦味氨基酸含量下降,甜味和鲜味氨基酸含量增加;并且,乳酸、苹果酸、柠檬酸等有机酸含量在发酵过程中呈现积累。同时,感官评定结果表明棒状乳杆菌FZU63发酵可改善红茶汤的感官品质,且在发酵48h后达到较优。本文系统分析了经棒状乳杆菌发酵不同阶段对红茶汤风味的影响,可为乳酸菌发酵茶饮料的品质控制与产业化应用提供理论参考。     

CE–MS-based metabolomics reveals the metabolic profile of maitake mushroom (Grifola frondosa) strains with different cultivation characteristics

Maitake mushroom (Grifola frondosa [Dicks.] Gray) is generally cultured using the sawdust of broadleaf trees. The maitake strain Gf433 has high production efficiency, with high-quality of fruiting bodies even when 30% of the birch sawdust on the basal substrate is replaced with conifer sawdust. We performed metabolome analysis to investigate the effect of different cultivation components on the metabolism of Gf433 and Mori52 by performing CE–MS on their fruiting bodies in different cultivation conditions to quantify the levels of amino acids, organic acids, and phosphorylated organic acids. We found that amino acid and organic acid content in Gf433 were not affected by the kind of sawdust. However, Gf433 contained more organic acids and less amino acids than Mori52, and Gf433 also contained more chitin compared with Mori52. We believe that these differences in the metabolome contents of the two strains are related to the high production efficiency of Gf433.     

Isolation and characterization of a lectin from Grifola frondosa fruiting bodies

An N-acetylgalactosamine-specific lectin (GFL) was isolated from Grifola frondosa fruiting bodies by affinity chromatographies on acid-treated Sepharose CL-4B and then GalNAc-Toyopearl. The isolated lectin agglutinated all types of erythrocytes equally. Molecular masses estimated by gel filtration under various buffers and matrices varied from 30 to 52 kDa. On the other hand, SDS-PAGE in the presence or absence of 2-mercaptoethanol showed three major bands of 33, 66 and 100 kDa and a faint band of 65 kDa. This lectin exhibited GalNAc-specificity. The protein was a glycoprotein containing 3.3% total sugar, and the amino acid analysis revealed a high content of acidic and hydroxy amino acids and a low content of methionine and histidine. GFL was cytotoxic against HeLa cells. The toxicity did not appear after preincubating the lectin with the haptenic sugar N-acetylgalactosamine.     

Development of a beverage from red grape juice fermented with the Kombucha consortium

Kombucha is a health-promoting fermented beverage traditionally made by fermenting a sweetened tea with a symbiotic culture of yeast species and acetic acid bacteria. The aim of this work was to develop a beverage using red grape juice as an alternative substrate. Grape juice contains various nutrient elements and phytochemicals, such as polyphenols, which possess a wide range of biological activities. We investigated the chemical characteristics and sensory and antimicrobial activities of the fermented grape juice Kombucha beverage. The pH decreased from 3.95 to 2.9 during the fermentation process and remained fairly constant thereafter, and the acetic acid bacteria and yeast counts in the broth increased up to 6 days of fermentation and subsequently decreased. Phenolic and anthocyanin contents and the antioxidant activity of the fermented beverage were higher after fermentation, with the maximum increase observed on the sixth day of fermentation when values were approximately 2.47- and 1.59-fold higher than pre-fermentation values, respectively, as assessed by 2,2-diphenyl-1-picrylhydrazyl and 2,2′-azino-bis (3-ethylbenzothiazoline-6- sulfonic acid) radical scavenging assays. Fourier transform infrared spectroscopy was used for the qualitative analysis of the grape juice before and after fermentation. Distinct peak variations in the spectral region between 2500 and 1650 cm^?1 were observed, which matched the appearance of organic acids and changes in phenolic compounds. Fermented juice Kombucha showed antibacterial activity toward all tested bacteria, which can be primarily ascribed to the increased production of acetic acid, but also to the biosynthesis of other metabolites, during the fermentation process. The 6-day fermented juice was the most appreciated by the taste panel based on the overall quality evaluation; with prolongation of fermentation the fermented juice acquired a distinct sour flavor.     

Inhibition of Camellia sinensis (L.) O. Kuntze on Microcystis aeruginosa and isolation of the inhibition factors

Low concentration of tea (Camellia sinensis (L.) O. Kuntze) was shown to inhibit the growth of the toxic cyanobacterium Microcystis aeruginosa. The inhibition efficiency was 40 % at 0.1 g dry tea/L and 90 % at 0.2 g/L after a 12-day culture. All varieties of tea used in the test could inhibit Microcystis growth, in which the inhibitory effect of green tea was greater than that of black tea. Antialgal allelochemicals were isolated from tea by solvent extraction, gel-chromatography and high performance liquid chromatography. Two algal-inhibition compounds were identified by liquid chromatography/mass spectrometry as epigallocatechin-3-gallate, epicatechin-3-gallate respectively. These are the main polyphenols in tea that have inhibitory effects on the growth of cyanobacteria. The combined effect of these polyphenols makes tea a promising source of algicide to inhibit the growth of algal blooms.     

三株散囊菌属真菌的茶叶液态发酵与其成分分析

散囊菌属真菌(Eurotium spp.)又称“金花菌”,能在六堡茶表面生长出金黄色的子囊果而得名,是一类对发酵茶叶的品质有积极影响的真菌。本研究前期从广西梧州六堡茶中筛选到的三株散囊菌属真菌Aspergillus chevalieri E2(简称E2),Aspergillus chevalieri E3(简称E3)与Aspergillus cristatus E6(简称E6),将菌株接种于自制液体茶粉培养基中培养,检测发酵液中产生的色素物质;测定发酵液中纤维素酶、果胶酶、淀粉酶的酶活力、总糖和还原糖糖含量、氨基酸含量、总抗氧化能力、自由基清除能力和茶多酚含量。结果表明:三株真菌在液体茶粉培养基中生长良好,E3生长尤其迅速。在8 d的培养期限内,E2、E3和E6的最高果胶酶活力分别为2.52、3.44和5.15 U/mL;最高纤维素酶活力分别为0.57、0.82和1.10 U/mL;最高淀粉酶活力分别为0.31、0.35和0.49 U/mL。三株真菌的发酵后培养液中还原糖含量大幅增加,增幅分别达到131.70%、627.02%和459.12%;总糖含量同样呈上升趋势,增幅分别达到6.84%、35.25%和2.07%;E2与E3的氨基酸含量分别下降了17.43%与32.68%,E6则上升了0.52%;E3的总抗氧化活性提升最大,达65.87%;DPPH·自由基清除能力提升最大的是E2,达128.33%;茶多酚含量都显著提升,增幅分别为50.85%、51.58%和53.06%。本研究对改善六堡茶的品质及茶叶的发酵的研究有一定意义和参考价值。     

Physiological and Biochemical Responses Reveal the Drought Tolerance Efficacy of the Halophyte Salicornia brachiata

The drought tolerance of Salicornia brachiata seedlings was assessed by monitoring growth, nutrient uptake, electrolyte leakage, lipid peroxidation, and biochemical responses under drought conditions simulated with 0, 10, 20, and 30 % polyethylene glycol (PEG 6000). After 7 days of drought induction, plants were harvested for measurement of various parameters. The biomass decreased and the plant height remained unchanged with PEG treatment. The total plant water content (TWC%) decreased by 11 % at the highest concentration of PEG (30 %). The electrolyte leakage and lipid peroxidation of shoots increased by 17 and 5 %, respectively, in 30 % PEG-treated plants. K⁺ and Ca²⁺ contents of shoots increased in a dose-dependent manner. However, in roots K⁺ content decreased and Ca²⁺ content remained unaffected by PEG treatment. Mg²⁺ content increased at high concentrations of PEG (20–30 %) in shoots and decreased at the highest concentration of PEG (30 %) in roots. Total free amino acids, proline, and polyphenol contents increased progressively with increase in severity of the drought stress. Total sugar content and reducing sugar content increased in 10 and 20 % PEG-treated plants and decreased in 30 % PEG-treated plants. Our results suggest that proline and other free amino acids, sugars, and polyphenols are the main compatible solutes in S. brachiata for maintenance of osmotic balance, protection of cellular macromolecules, detoxification of the cells, and scavenging of free radicals under drought stress. A greater accumulation of compatible solutes also facilitates the maintenance of nutrient uptake and adequate tissue water status and protection of membranes under drought conditions in S. brachiata. The results from the present study suggest that S. brachiata can be used for restoration of arid and semiarid lands of coastal ecosystems.     

Fatty acid and sterol compositions in mushrooms of ten species of polyporaceae

The simple lipids present in ten species of Polyporaceae (Piptororus betulinus, Coriolus pargamenus, C. versicolor, C. heteromorphus, Formitopsis cytisina, F. pinicola, Microporus flabelliformis, Gloephyllum saepiarium, Crytoderma citrinum and Grifola frondosa) were investigated. The fatty acids that these species had in common were C₁₆-saturated acids (except in P. betulinus) and C₁₈-unsaturated acids. Ergosterol and ergosta-7,22-dien-3β-ol were isolated from these mushrooms. Lupeol was obtained from G. saepiarium. Ergost-7-en-3β-ol, lanosterol and 24-methylene-24,25-dihydrolanosterol were tentatively identified.     

Hypoglycemic Activity of <Emphasis Type="Italic">Grifola frondosa</Emphasis> Rich in Vanadium

The hypoglycemic activity of fermented mushroom of Grifola frondosa rich in vanadium (GFRV) was studied in this paper. Alloxan- and adrenalin-induced hyperglycemic mice were used in the study. The blood glucose and the HbA1c of the mice were analyzed respectively. After the mice were administered (ig) with GFRV, the blood glucose and the HbA1c of alloxan-induced hyperglycemic mice decreased (p < 0.05, p < 0.01) and ascension of blood glucose induced by adrenalin was inhibited (p < 0.01). Also, the bodyweight of the alloxan-induced hyperglycemic mice was increased gradually. In the fermented mushroom of G. frondosa, vanadium at lower doses in combination with G. frondosa induced significant decreases of the blood glucose and HbA1c levels in hyperglycemic mice.     

Molecular properties and antioxidant activities of polysaccharide–protein complexes from selected mushrooms by ultrasound-assisted extraction

Ultrasound-assisted extraction (UAE) was evaluated for isolation of polysaccharide–protein (PSP) complexes from three important medicinal mushrooms (Grifola frondosa, Coriolus versicolor and Lentinus edodes). Compared with those of conventional hot-water extraction (HWE), the PSP yield of UAE was similar with G. frondosa, notably higher with L. edodes but lower with C. versicolor, and the extraction rate of UAE was notably higher with G. frondosa and L. edodes but much lower with C. versicolor. The PSPs from all three mushrooms by UAE had higher protein but lower carbohydrate contents than those by HWE, and their molecular weight (MW) profiles exhibited an overall shift to lower MW and a major low-MW peak near 1.0 kDa. All PSPs from UAE but none from HWE exhibited 3–4 distinct protein bands between 10 and 130 kDa. The antioxidant activities of PSPs extracted by UAE were generally higher than those by HWE.     

茶氨酸提取纯化工艺研究

系统研究了从茶多酚工业废液中提取纯化茶氨酸的工艺。采用絮凝、吸附、阳离子树脂交换、重结晶工艺来分离纯化茶氨酸。结果表明,絮凝能有效的去除茶多酚工业废液中的蛋白质等杂质,杂质的去除率为50％;吸附能进一步去除色素、多酚类物质及大分子有机物;阳离子交换树脂能较专-吸附氨基酸。茶多酚工业废液经絮凝→吸附→阳离子树脂交换工艺可得纯度50%的茶氨酸,得率为1．8％;通过重结晶可得到纯度90％的茶氨酸,得率为0．8%。     

The use of additives as the stimulator on mycelial biomass and exopolysaccharide productions in submerged culture of Grifola umbellata

In this study, various additives including organic acids, alcohols, vegetable oils, surfactants and polymers were added in the cultural medium to investigate their stimulatory effects on Grifola umbellate mycelia growth and exopolysaccharide (EPS) production. It was found that the commonly used stimulatory additives, effective in other mushrooms’ cultures, exhibited negative results in Grifola umbellata submerged culture. In contrast, the polymer additive, polyethylene glycol (PEG), displayed an effective stimulatory effect on both biomass and EPS productions. With the addition of PEG8 (molecular weight: 8,000 Da), the mycelial biomass production at day 12 was increased from 4.69 to 6.30 g/L, accounting for a 34% increase. Meanwhile, the EPS production was enhanced from 0.478 to 0.767 g/L, accounting for 60% increase.     

A Comparison of Hypoglycemic Activity of Three Species of Basidiomycetes Rich in Vanadium

The hypoglycemic activity of fermented mushroom of three fungi of basidiomycetes rich in vanadium was studied in this paper. Alloxan- and adrenalin-induced hyperglycemic mice were used in the study. The blood glucose and the sugar tolerance were determined. After the mice were administered (ig) with Coprinus comatus rich in vanadium, the blood glucose of alloxan-induced hyperglycemic mice decreased (p < 0.05), ascension of blood glucose induced by adrenalin was inhibited (p < 0.01) and the sugar tolerance of the normal mice was improved. However, the same result did not occur in Ganoderma lucidum and Grifola frondosa group. Compared with Ganoderma rich in vanadium and Grifola frondosa rich in vanadium, the hypoglycemic effects of Coprinus comatus rich in vanadium on hyperglycemic animals are significant; it may be used as a hypoglycemic food or medicine for hyperglycemic people.     

三叶虫茶营养成分的分析与评价

通过与老鹰茶虫酿茶和一些品牌绿茶的对比,对三叶虫茶的一般营养成分、茶的生化特征、氨基酸和脂肪酸组成、维生素、矿物质元素进行分析和营养评价,为开发三叶虫茶提供科学依据。方法:用常规方法分析其主要营养素和生化成分;用氨基酸分析仪分析氨基酸组成,并采用WHO推荐的蛋白质模式对蛋白质进行营养评价;维生素和矿物元素含量直接说明其营养功能。三叶虫茶含有36.44%的水浸出物,16.28%的茶多酚、1.39%的氨基酸;氨基酸的种类较为齐全,总含量与传统茶叶相当,9种人体必需氨基酸的总量达到0.722%,是传统茶叶的3~12倍,特别是赖氨酸、蛋氨酸、苏氨酸、色氨酸的含量远远高于常规茶叶,而这4种氨基酸在一般植物食品中都是限制性氨基酸;但其茶氨酸、天门冬氨酸、谷氨酸的含量远远低于常规茶叶,而这3种氨基酸对改善茶汤滋味具有重要作用;三叶虫茶中脂肪酸含量为1.23%,其中多不饱和脂肪酸占35.25%;钙、磷、镁含量较高,铁、铜、锌、锰等的含量丰富;维生素C和维生素E含量与普通茶叶相当。三叶虫茶营养丰富,具备常规茶叶的一些生化特征,可作为茶饮料的代用品;若与普通茶叶混合使用,能在滋味和营养价值上实现互补。     

Rapid determination of nucleosides,nucleobases and free amino acids in brown seaweeds using ultra-performance liquid chromatography coupled with triple quadrupole mass spectrometry

In this study the contents of nucleosides, nucleobases and free amino acids (FAAs) in common brown seaweeds Saccharina japonica, Sargassum pallidum, S. fusiforme, S. thunbergii and S. muticum from major production areas in China were determined by ultra-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UPLC-TQ-MS). The total contents of nucleosides and nucleobases varied from 48.46 to 4,105.00 μg g^?1. Among the five seaweeds, levels of nucleosides and nucleobases in S. japonica were higher than the other species. The contents of ribonucleosides in these brown seaweeds were higher than those of deoxynucleosides. Uridine was the predominant constituent in all samples while 2′-deoxycytidine was the least in most samples. FAAs contents ranged from 0.14 to 22.76 mg g^?1. Among the five seaweeds, levels of FAAs in S. muticum were higher than the other species. Essential amino acids accounted for 3.26–51.49 % of total FAAs contents in different species. Glutamic acid was the major constituent in most samples. Four non-protein amino acids, γ-aminobutyric acid, taurine, hydroxyproline and citrulline, were found in the seaweed samples. Furthermore, principal component analysis (PCA) showed that chemical profiles of nucleosides, nucleobases and FAAs in S. pallidum and S. fusiforme were different from S. muticum, S. thunbergii and S. japonica. Common brown seaweeds could be promising natural sources for future industrial research into nucleosides, nucleobases and FAAs with potential benefits for human health.