Structure and immunomodulatory activity of Lentinus edodes polysaccharides modified by probiotic fermentation

Plant-based fermentations provide an untapped source for novel biotechnological applications.In this study,a probiotic named Lactobacillus fermentum 21828 was introduced to ferment Lentinus edodes.Polysaccharides were extracted from fermented and non-fermented L.edodes and purified via DEAE-52 and Sephadex G-100.The components designated F-LEP-2a and NF-LEP-2a were analyzed by FT-IR,HPGPC,HPAEC,SEM,GC-MS and NMR.The results revealed that probiotic fermentation increased the molecular weight from 1.16×10^(4) Da to 1.87×10^(4) Da and altered the proportions of glucose,galactose and mannose,in which glucose increased from 45.94%to 48.16%.Methylation analysis and NMR spectra indicated that F-LEP-2a and NF-LEP-2a had similar linkage patterns.Furthermore,their immunomodulatory activities were evaluated with immunosuppressive mice.NF-LEP and F-LEP improved immune organ indices,immunoglobulin(Ig G and Ig M)and cytokines concentrations;restored the antioxidation capacity of liver;and maintained the balance of gut microbiota.F-LEP displayed better moderating effects on the spleen index,immunoglobulin,cytokines and the diversity of gut microbiota than NF-LEP(200,400 mg/kg).Our study provides an efficient and environment-friendly way for the structural modification of polysaccharides,which helps to enhance their biological activity and promote their wide application in food,medicine and other fields.

Fermented Lentinus edodes extract containingα-glucan ameliorates concanavalin A-induced autoimmune hepatitis in mice

Autoimmune hepatitis(AIH)is a chronic inflammatory liver disease that threatens human health worldwide.The aim of this study was to detect the protective effect of a fermented Lentinus edodes extract containingα-glucan(FLA),in a concanavalin A(Con A)-induced AIH mouse model and to determine the underlying liver-protective mechanism.The results showed that compared with the model group,the level of proinflammatory cytokines in serum of FLA pretreated mice was significantly decreased,and the degree of inflammatory cell infiltration in liver,thymus and spleen was significantly reduced.Quantitative polymerase chain reaction,immunohistochemistry,and Western blotting showed that FLA pre-treatment inhibited the Con A-induced apoptosis of hepatocytes by down-regulating the expression of BAX and up-regulating the expression of BCL-2.Further research found that FLA may improve liver injury in mice by activating NRF2 signaling pathway and inhibiting TRAF6/NF-κB signaling pathway.Thus,FLA may improve liver injury in mice by shifting gut microbial composition to reduce the release of inflammatory cytokines in the serum and prevent the necrosis of hepatocytes.Up-regulation of NRF2 signaling pathway,down-regulation of TRAF6/NF-κB signaling pathway,and an increase in the relative abundance of Lactobacillus_johnsonii and Ligilactobacillus_murinus play a protective role in liver.

Bioremediation of Textile Azo Dyes Amido Black 10B, Reactive Black 5, Reactive Blue 160 by Lentinus squarrosulus AF5 and Assessment of Toxicity of the Degraded Metabolites

Bioremediation is an eco-compatible and economical approach to counter textile dye menace. The isolated Lentinus squarrosulus AF5 was assessed for decolourization of textile azo dyes, and had shown ~93%, 88% and 70% decolorization of Reactive blue 160 (RB160), Reactive black 5 (RB5) and Amido black 10B (AB10B) respectively. Further analysis using UV-vis, HPLC, and FTIR, 1H NMR had shown the degradation of the dyes. Toxicity analysis of the metabolites was performed using seed germination and plant growth on two agriculturally important plants Guar (Cyamopsis tetragonoloba) and wheat (Triticum aestivum) as well as cytotoxicity analysis using the human keratinocyte cell line (HaCaT). The dye mix appeared inhibitory for seed germination (20% - 40%), whereas metabolites were non-inhibitory for germination. Treatment of HaCaT cells with of dye mix and metabolites led into 45% and ~100% of cell viability of HaCaT cells respectively. Therefore, metabolites following degradation of the dye mix were observed to be non-toxic.

PEG-mediated Transformation of Lentinus edodes

Expression vector p301-bG1 contains a Sw gene and a bialaphos resistance gene both driven by glyceraldehydes-3-phosphate dehydrogenase (GPD) gene promoter isolated from Lentinus edodes ( Berk.) Sing. Using p301-bG1, PEG-mediated transformation of protoplast of L. edodes was studied. Mixed with PEG-purified plasmid DNA, the protoplasts of L. edodes were treated with PEG solution and cultured on CYM regeneration plate containing 40 mug/mL bialaphos. Bialaphos-resistant and GUS-positive transformants were obtained using this transformation system. Although the transformation efficiency was relatively low, the protocols release large expenses on expensive instrument and restriction enzymes, providing a simple and economical method for mushroom breeding at the molecular level.

巴西革耳Lentinus striguellus深层发酵的研究

报道了巴西革耳Lentinusstriguellus深层培养的适宜发酵培养基、摇瓶发酵条件。试验结果表明 ,Lentinusstriguellus对单糖、双糖、多糖和糖醇都有一定的利用 ,但葡萄糖等单糖对菌丝生长效果较好 ;酵母粉等蛋白质含量高的氮源对菌丝生长较为有利 ;通过L9(3 4 )正交试验确定了Lentinusstrigullus发酵的最适培养基为 :5 %葡萄糖 ,0 .7%酵母粉 ,0 .2 %KH2 PO4,0 .3 %MgSO4·7H2 O ,0 .0 0 1%VB1 ;一定浓度内 ,Fe2 + 、Ca2 + 对菌丝体生长有促进效果。适宜的摇瓶发酵条件为 :培养基起始 pH5 .0～ 6.0、培养温度 2 6℃、接种量 10 % ,摇床转速为 2 0 0r/min ,2 5 0mL三角瓶最适装液量为 80mL ,发酵周期为 6d。

Mathematical Modeling and Effect of Various Hot-Air Drying on Mushroom(Lentinus edodes)

An experimental study was performed to determine the characteristics and drying process of mushroom （Lentinus edodes） by 6 different hot-air drying methods namely isothermal drying, uniform raise drying, non-uniform raise drying, uniform intermittent drying, non-uniform intermittent drying and combined drying. The chemical composition （dry matter, ash, crude protein, crude fat, total sugars, dietary fiber, and energy）, color parameters （L, a＊, b＊, c＊, and h~） and rehydration capacities were determined. Among all the experiments, non-uniform intermittent drying reached a better comprehensive results due to the higher chemical composition, better color quality associated with high bright （26.381＋5.842）, high color tone （73.670＋2.975）, low chroma （13.349a：3.456） as well as the highest rehydration （453.76% weigh of dried body）. Nine kinds of classical mathematical model were used to obtained moisture data and the Midili-kucuk model can be described by the drying process with the coefficient （R2 ranged from 0.99790 to 0.99967）, chi-square （X2 ranged from 0.00003 to 0.00019） and root mean square error （RMSE ranged from 0.000486 to 0.0012367）.

STRUCTURE,MOLECULAR WEIGHT AND BIOACTIVITIES OF(1→3)-β-D-GLUCANS AND ITS SULFATED DERIVATIVES FROM FOUR KINDS OF LENTINUS EDODES

Lentinan samples,(1→3)-β-D-glucans containing 4.6-15.2 wt% proteins,coded as L-I_1 L-I_2 L-I_3 and L-I_4(L-I)were isolated from four kinds of Lentinus edodes.These glucans were treated with acetone to remove the protein in orderto obtain free protein glucans coded as LNP-I_1,LNP-I_2.LNP-I_3 and LNP-I_4(LNP-I).The free-protein polysaccharideswere sulfated to give derivatives(S-LNP-I)with degree of substitution(DS)from 0.4-0.8.The structural features andweight-average molecular weight(M_w)of the samples were investigated by using infrared spectroscopy,elemental analysis,^(13)C-NMR,size exclusion chromatography combined with laser light scattering(SEC-LLS)and viscometry.The effects ofstructure and conformation of the polysaccharides on antitumor activities were assayed in vivo(Sarcoma 180 solid tumors)and in vitro(Sarcoma 180,HL-60,MCF-7 and Vero tumors).The results indicated that the predominant species of thesamples L-I and LNP-I in 0.2 mol/L NaCl aqueous solution existed as triple-helical chains with high rigidity and in dimethylsulfoxide(DMSO)as single-flexible chains.Interestingly,the antitumor activities of LNP-I are lower than those of the nativeglucans(L-I),whereas their sulfated derivatives have higher inhibition ratio against Sarcoma 180 than LNP-I.The resultsreveal that the binding of protein,sulfated modification and the triple helix conformation are important factors in theenhancement of the antitumor activities of polysaccharides on the whole.

Optimization of medium components using response surface methodology (RSM) for mycelium biomass and exopolysaccharide production by <i>Lentinus squarrosulus</i>

The interaction between sucrose, yeast extract and initial pH was investigated to optimize critical medium components for mycelium biomass and production of exopolysaccharide (EPS) of Lentinus squarrosulus using Response Surface Methodology (RSM). A central composite design (CCD) was applied and a polynomial regression model with quadratic term was used to analyse the experimental data using analysis of variance (ANOVA). ANOVA analysis showed that the model was very significant (p Lentinus squarrosulus are as follows: sucrose concentration 114.61 g/L, yeast extract 1.62 g/L and initial pH of 5.81;sucrose concentration 115.8 g/L, yeast extract of 3.39 g/L and initial pH of 6.44 respectively.

Nutrient Requirements and Fermentation Conditions for Mycelia and Crude Exo-Polysaccharides Production by <i>Lentinus squarrosulus</i>

Lentinus squarrosulus Mont. is an emerging tropical white rot basidiomycete, with nutritional and medicinal benefits. Low levels of commercial cultivation of the mushrooms limit their availability for use as food and medicine. Mycelia from submerged fermentation are a suitable alternative to the mushroom from L. squarrosulus. Three strains, 340, 339 and 218, were studied to determine optimum growth conditions for mycelia mass and crude exo-polysaccharides (CEPS) production. The experiments were conducted in a completely randomized design (CRD) with a factorial structure. Nutrients involving 8 carbon and 8 nitrogen sources were screened, and concentrations of the best sources were optimized. Optimized nutrients, interaction between strains and other parameters such as agitation and medium volume were investigated to obtain optimum fermentation conditions for biomass and CEPS production. Biomass yield varied among strains depending on carbon or nitrogen nutrient sources. Starch and yeast extract at 30 and 25 g/L were identified as the most important nutrients in mycelia and CEPS production. Nutrient optimization resulted in a 3-fold increase in mycelia mass: 12.8, 10.0 and 15.3 g/L in strains 340, 339 and 218 respectively. There was a significant interaction between strain, agitation, and volume (p p = 0.02). Static conditions favored more polysaccharide production. Optimized fermentation conditions resulted in very high increase in biomass: 238.1, 266.9 and 185.0 g/L in strains 340, 339 and 218 respectively. Results obtained could be useful in modeling fermentation systems for large-scale production of mycelia mass, CEPS and other bio-products from L. squarrosulus.

Antioxidant Properties and Glucan Compositions of Various Crude Extract from <i>Lentinus squarrosulus</i>Mycelial Culture

Various crude extracts of Lentinus squarrosulus mushroom mycelium and culture broth were prepared for antioxidant studies, glucan and carbohydrate compound analysis. Crude extract from ethanol, cold and hot water extraction was tested on several complementary test systems namely DPPH free radical scavenging, β-carotene linoleic acid oxidation, total phenolic compound, total flavonoid compound, total carbohydrate content and total glucan compositions. The antioxidant activity of the crude extracts of Lentinus squarrosulus mushroom mycelium and culture broth was determined by 2,2’ diphenyl-1-picrylhydrazyl (DPPH) radical scavenging method and β-carotene linoleic acid assay. Crude extracted from culture broth EPS EE recorded the highest radical scavenging effects at 20 mg/mL (85%) which is closer to the control antioxidants (ascorbic acid 97%;BHT 92.4%;BHA 93.6%). Both the ethanol extract (IPS EE and EPS EE) exhibited the highest β-carotene linoleic acid oxidation by recording IC50 values of 0.6 and 0.65 mg/mL respectively. IPS EE (0.59 mg/g) and EPS WE (0.58 mg/g) showed high flavonoid content whereas total phenol content was high in EPS WE (0.85 mg/g) and IPS EE (0.84 mg/g). Total carbohydrate concentrations were high in IPS HWE and IPS CWE with 19.1 and 16.3 mg/mL respectively and these data correlate with the β-glucan content in both extracts. The highest β-glucan content found in IPS CWE and IPS HWE were 12.2 and 11.4% w/w respectively.

CRITICAL CONCENTRATIONS OF α(1→3)-D-GLUCAN FROM LENTINUS EDODES IN NaOH AQUEOUS SOLUTION

Critical concentrations of α-(1→3)-D-glucan L-FV-Ⅱ from Lentinus edodes were studied by viscometry andfluorescence probe techniques. The dependence of the reduced viscosity on concentration of the glucan in 0.5 mol/L NaOHaqueous solutions with or without urea showed two turning points corresponding to the dynamic contact concentration c_s andthe overlap concentration c~* of the polymer. The values of c_s and c~* were found to be 1×10^(-3) g cm^(-3) and 1.1×10^(-2) g cm^(-3),respectively, for L-FV-Ⅱ in 0.5 mol/L NaOH aqueous solutions. The two critical concentrations of L-FV-Ⅱ in 0.5 mol/LNaOH aqueous solutions were also found to be 1.2×10^(-3) g cm^(-3) fbr c_s and 9.2×10^(-3) g cm^(-3) for c~* from the concentrationdependence of phenanthrene fluorescence intensities. The overlap concentration c~* of L-FV-Ⅱ in 0.5 mol/L NaOH aqueoussolutions was lower than that of polystyrene with same molecular weight in benzene, owing to the fact that polysaccharidetends to undergo aggregation caused by intermolecular hydrogen bonding. A normal viscosity behavior of L-FV-Ⅱ in 0.5 mol/L urea/0.5 mol/L NaOH aqueous solutions can still be observed in an extremely low concentration range at 25℃.

Lentinus squarrosulus an edible macro-fungus reported from Pakistan

Lentinus squarrosulus(Mont.)Singer:an edible macro-fungus is reported from the moist temperate Himalayan forests of Pakistan.The sample was studied based on morphological characteristics and nucleotide sequence analyses of the ITS region.Studies based on morpho-anatomical and molecular data are consistent with formerly reported specimens.Lentinus squarrosulus represents the first report for Pakistan.

Study of the Production of Lentinus crinitus(L.)Fr.Lignolytic Enzymes Grown on Agro-Industrial Waste

Exclusively Brazilian, the Caatinga biome has been thus far a place of very few studies on the Basidiomycetes fungi. Due to its semiarid climate, fungi found in the region are likely to carry lignolytic enzymes which hold biotechnological potential to be used in industrial processes of agro-industrial residue bioconversion. This study performed a response surface statistical planning to optimize the secretion of enzymes such as laccase (Lac), lignin peroxidase (LiP) and manganese peroxidase (MnP) by Lentinus crinitus. Three variables were under analysis: different concentrations of barley and cassava residue, pH and temperature. MnP enzyme showed the highest enzymatic activity rate (23.5 IU/L). Additionally, MnP had the best results of enzyme secretion for substrate composition of 50% barley and 50% cassava, at pH 7 and temperature at 28°C for a 28-day incubation period. However, further studies are pivotal to test the efficiency in lignin bioconversion by the enzymes synthesized in this work and also to establish their usage pattern on a large scale.

Flavor changes in Lentinus Edodes enzymatic hydrolysate maillard reaction products with different sugars by gas chromatography‐ion mobility spectrometry

The characteristic flavor of the enzymatic hydrolysate of Lentinus edodes,which was involved in the Maillard reaction by xylose,fructose,glucose,sucrose,mannose,maltose,vitamin C(VC)and L-arabinose,was determined and analyzed by gas chromatography-ion mobility spectroscopy(GC-IMS).The characteristic flavor fingerprints of Maillard reaction products(MRPs)of L.edodes enzymatic hydrolysate were constructed.The differences in the characteristic flavors of Maillard reaction with different reducing sugars and VC were compared.The main characteristic flavor,characteristic peak,characteristic marker substance and content were determined,a principal component analysis(PCA)of volatile organic compounds(VOCs)was carried out.The results showed that there were 42 kinds of monomers and some dimers of volatile compounds in the samples including 17 aldehydes,5 alcohols,7 ketones,2 esters,2 acids,7 other compounds and 2 ethers.The volatile VOCs of the sample with added sucrose,VC and L-arabinose were significantly higher than those of the enzymatic hydrolysate of L.edodes.After the Maillard reaction,the flavor of the hydrolysate was significantly improved,and the main components changed significantly.

Immobilized Lentinus edodes residue as absorbent for the enhancement of cadmium adsorption performance

To investigate the potential use of Lentinus edodes （L. edodes） residue for Cd^2＋ adsorption, poly alcohol Na alginate （PVA） was applied to immobilize it. The parameters including contact time, pH, adsorbent dosages, and coexisting metal ions were studied. The suitable pH for immobilized L. edodes was 4-7 wider than that for raw L. edodes （pH 6-7）. In the presence of Pb^2＋ concentration varying from 0 to 30mg.L^-1, the Cd^2＋ adsorption ratios declined by 6.71% and 47.45% for immobilized and raw L. edodes, respectively. While, with the coexisting ion Cu2＋ concentration varied from 0 to 30 mg.L^-1, the Cd^2＋ adsorption ratios declined by 12.97% and 50.56% for immobilized and raw L. edodes, respectively. The Cd^2＋ adsorption isotherms in singlemetal and dual-metal solutions were analyzed by using Langmuir, Freuxldlich, and Dubinin-Radushkevich models. The Cd^2＋ adsorption capacities （qm） in single-metal solution were 6.448 mg.L^-1 and 2.832 mg.L^-1 for immobilized and raw L. edodes, respectively. The qm of immobilized L. edodes were 1.850 mg Cd. gl in Cd^2＋ ＋ Pb^2＋ solution and 3.961mg Cd.g^-1 in Cd^2＋ ＋ Cu^2＋ solution, respectively. The Cd^2＋ adsorption processes subjected to both adsorbents follow pseudo-second-order model. Mechanism study showed the functional group of L. edodes was -OH, -NH, -CO, and PVA played an important role in metal adsorbing. Mining wastewater treatment test showed that PVA-SA-immobilized L. edodes was effective in mixed pollutant treatment even for wastewater containing metal ions in very low concentration.

Identification of phytochemicals from Lentinus edodes and Auricularia auricula with UPLC-Q-Exactive Orbitrap MS

Lentinus edodes and Auricularia auricula are the first and second largest edible fungi in China with high nutritional and medicinal values.Research about phytochemicals of L.edodes and A.auricula mainly focuses on polysaccharides,other phytochemicals are not fully studied.Phytochemicals of L.edodes and A.auricula were investigated by ultra-high performance liquid chromatography tandem hybrid quadrupole-orbitrap mass spectrometry(UPLC-Q-Exactive-Orbitrap MS).Twenty-three compounds were identified in L.edodes,mainly including amino acids and analogs,flavonoids,nucleosides and analogs,phenols and steroids.Twenty-nine compounds were identified in A.auricula,mainly including amino acids and analogs,nucleosides and analogs,flavonoids and phenols.There are 9 common ingredients of L.edodes and A.auricula,including 4 amino acids and analogs,2 flavonoids,2 nucleosides and analogs and 1 phenol.

香菇柄复合酶解工艺研究

采用纤维素酶(A)和中性蛋白酶(B)复合酶系水解香菇柄,确定香菇(Lentinus edodes)柄中风味成分最佳溶出条件。以可溶性蛋白质溶出率和氨基酸释放率为评价指标,分别考察酶A水解时料液比、p H、添加量、酶解温度及酶解时间和酶B水解时p H、添加量、酶解温度和酶解时间对提取率的影响;以游离氨基酸、呈味核苷酸、多糖的溶出率和酶解液感官品质为评价指标,比较了热水浸提法、纤维素酶单酶解法、复合酶解法3种方法的优劣。结果表明,最适酶解工艺条件为先以0.2%食用菌水解酶A,料液比(m∶V)为1∶6,p H 4.5,温度55℃的条件酶解2 h,在此条件下可溶性蛋白质溶出率为4.12%,然后在酶解液中加入0.15%酶B,于p H 6.5,温度50℃酶解6 h,在此条件下总氨基酸释放率为2.16%;复合酶法水解香菇柄所得游离氨基酸、呈味核苷酸、多糖含量、感官评分分别为2.16%、1.57%、7.25%、82分,均优于另外2种方法。

鲜香菇渗透脱水与真空油炸联合干燥动力学分析

研究了鲜香菇[Lentinus edodes(Berk.)sing]在渗透脱水与真空油炸联合干燥过程中水分含量及脂肪含量的变化,建立干燥动力学方程,渗透脱水采用40%混合糖溶液,温度30℃;真空油炸温度为90℃,真空度-0.1 MPa。结果表明,随着渗透时间的延长,鲜香菇的水分含量逐渐降低,香菇脆片脂肪含量及脂肪吸收率也逐渐减少;根据K Pearson积差相关分析得出,脂肪含量与其初始水分含量具有极显著的相关性,结合真空油炸一级反应动力学规律和渗透脱水费克水分扩散规律,得到鲜香菇联合干燥过程中水分与脂肪含量变化的动力学方程。

四种杀菌剂对鲜切香菇贮藏特性的影响

为提高鲜切香菇[Lentinus edodes(Berk.)sing]品质,研究了次氯酸钠、二氧化氯、过氧化氢、乙醇等4种杀菌剂对鲜切香菇贮藏期间品质的影响,系统分析了贮藏期鲜切香菇的菌落总数、白度、呼吸强度、失重率等变化规律。结果表明,4种杀菌剂对鲜切香菇均有抑菌效果,以150 mg/L Na Cl O抑菌效果最佳;Cl O2、低浓度Na Cl O和H2O2可抑制鲜切香菇发生褐变;50、100 mg/L Na Cl O均能降低鲜切香菇呼吸强度;4种杀菌剂对鲜切香菇失重均有较明显的抑制效果,以200 mg/L Na Cl O最佳。综合考虑,Na Cl O宜作为鲜切香菇的杀菌剂。

真空油炸香菇脆片等温吸湿规律的研究

研究了真空油炸香菇（Lentinus edodes）脆片在10、25、40℃3种温度下,水分活度为0.06~0.982条件下的吸湿等温线。根据5种常用的等温吸附模型对香菇脆片的水分活度（aw）、平衡相对湿度（ERH）的试验数据进行拟合,以模型的确定系数、标准误差以及平均相对预测误差作为评价指标进行模拟比较,得出模型Peleg为香菇脆片最适用的吸湿模型,并确定了模型系数。