以白桦茸为研究对象,采用平板培养及液体摇瓶培养的方式,观察、比较不同碳源(麦芽糖、葡萄糖、蔗糖、玉米淀粉、乳糖、玉米面粉、果糖、甘露醇、红薯淀粉、可溶性淀粉)对菌丝部分生物学特性及液体发酵产物活性的影响.固体培养结果表明:果糖、麦芽糖、蔗糖对菌丝生长速度影响较大,麦芽糖为碳源时,菌丝生长速度最快,达到0.302 cm/d,果糖和蔗糖可以延缓菌丝老化,而玉米面粉和葡萄糖的菌丝最为粗壮,麦芽糖和红薯淀粉的菌丝生长密度最高(密集处呈羊毛毡状);同时,液体发酵结果表明:玉米面粉总生物量最大,葡萄糖次之.对于活性物质来说,随着培养时间的延长,大部分碳源均可促进活性物质的积累,以葡萄糖为碳源,11 d菌丝体多糖含量最高,其次为乳糖.红薯淀粉、玉米淀粉、麦芽糖、乳糖为碳源时,其菌丝水提液1,1-二苯基-2-三硝基苯肼(1,1-diphenyl-2-picrylhydrazyl radical,DPPH)自由基清除率可达到70％以上.

为探讨虫草菌株胞内、胞外多糖体外抗氧化和免疫活性及其量效关系,采用水提醇沉法、sevege法和活性炭吸附法,分别对大规模发酵生产获得的虫草菌丝体及发酵液2种不同生产原料进行多糖提取,重点分析其胞内及胞外多糖体外抗氧化和免疫活性.结果表明:虫草多糖最佳提取工艺为液料比95:1(mL/g)、提取温度65℃、提取时间1.5 h;多糖中含少量的核酸和蛋白质,是以 β-糖苷键连接且具有吡喃糖环的酸性杂多糖;分离纯化自发酵液及发酵虫草菌粉中的多糖均具有较好的清除自由基能力且呈一定的量效关系;2种多糖样品均能促进RAW 264.7巨噬细胞增殖,促进细胞释放NO,且发酵液多糖活性优于胞内多糖,与LPS组具有显著性差异(P<0.001).发酵虫草菌粉及发酵液提取的多糖在体外活性研究中均呈现出较好的抗氧化活性及免疫活性,有望开发成为新型药物免疫调节剂或天然抗氧化剂等新资源功能食品,也为发酵虫草菌粉胞内多糖和发酵液利用及产品开发提供了科学依据.

该文主要研究桦褐孔菌在液体发酵过程中主要活性成分多糖、三萜甾醇的累积与代谢变化.采用液体发酵法培养桦褐孔菌获得不同发酵时间的发酵产物,分别采用苯酚硫酸法及HPTLC分析法检测不同培养时间的菌丝体中多糖、三萜和甾醇的含量,DPPH与硫酸香草醛显色法检测抗氧化活性.结果 显示,随发酵时间的延长,桦褐孔菌菌丝体生物量越来越高,经HPTLC法分析可见发酵后的菌丝体与桦褐孔菌子实体中的三萜和甾醇成分相似.且多糖、三萜甾醇的含量均呈现先增高后降低的趋势,而通过显色反应,发现菌丝体中含有子实体未发现的抗氧化三萜成分.通过研究确定了桦褐孔菌液体发酵过程中主要活性成分变化,确立最佳发酵时间,为桦褐孔菌的工业化生产提供参考.

Objective: The present study intended to explore the anti-fatigue and anti-hypoxia efficacy of Tremella extract in mouse model. Methods: Kunming mice were randomized into 5 groups randomly (n = 12/group; half male and female), and given 0.9%Nacl (10 mL/Kg Serving as blank control group)orally, 0.6 g/kg rhodiola capsule (Serving as positive group) and Tremella extract at doses of high (3.0 g/kg), middle (1.5 g/kg) and low (0.5 g/kg) once a day for 14 days. At the end of drug intragastric gavage, following endurance running test, rotating rod test, normobarie hypoxia test and sodium nitrite poisoning test were performed. Result: 14-day Tremella extract administration fails to influence mouse horizontal and vertical movement indicating little neurotoxicity at chosen doses. Through rotating rod, forced running test, the anti-fatigue activity of Tremella extract was demonstrated. Via normobarie hypoxia test, sodium nitrite poisoning test, Tremella extract was confirmed to possess anti-hypoxia effect. Tremella extract treatment significantly enhanced the liver levels of superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px), increased the expressions of ATP and hepatic glycogen, muscle glycogen in tissue. Conclusion: We conclude that Tremella extract efficiently enhances mouse fatigue endurance and anti-hypoxia capability partly through energy reserves and antioxidant enzyme activity. (C) 2019 Production and hosting by Elsevier B.V.

Background: Cultured Cordyceps militaris is very popular. Objective: To gain dynamic insight into activity markers in fruiting body of Cordyceps militaris (C. militaris) in Bombyxmori (B. mori), also named silkworm. Methods: The development stages of samples at 3, 9, 12, 19, 27, and 33 days after inoculation (DAI) were collected. HPLC coupled with diode array detection and evaporative light-scattering detection method (HPLC-DAD-ELSD) was used to determine eight makers, including six nucleosides and two carbohydrates from the samples. Results: C. militaris cultured 33 DAI with fifth star silkworm larva could accumulate higher levels of cordycepin (13.43 mg/g) than the highest reported cordycepin (8.57 g/L). The contents of cordycepin, adenosine, and trehalose were gradually increased with the formation of C. militaris fruiting bodies on silkworm larva, while mannitol was decreased. The change of guanosine was similar to uracil. Conclusions: Results suggested that mannitol could be accumulated in a short period during mycelium growth and could metabolize and transform into energy store and trehalose during fruit body formation. The inosine in the insect was completely utilized and transformed. The synergistic formation of cordycepin and adenosine or differences in metabolized pathways are a great possibility according to the same trend. Highlights: This research offered some reference to further find a certain regularity or metabolic mechanism.

Objective:To compare the composition and ratio of monosaccharides in polysaccharides of Cordyceps spp.from different origins or habitats.Methods:The hydrolysates of polysaccharides were separated on a cellulose plate.The plate was developed with ethyl acetate-pyridine-acetic acid-water (7.0 ∶ 3.0 ∶ 0.2 ∶ 1.4),and colorized with aniline-O-phthalic acid reagent.The quantification of monosaccharides was performed by using thin layer chromatography densitometry method at 410 nm.Results:Six monosaccharides (galactose,glucose,mannose,xylose,ribose and rhamnose) had good linearity (r＞0.995 7) in given ranges.The LOD and LOQ were in the range of 0.02-0.10 μg and 0.08-0.40 μg,respectively.Their RSDs for precision,repeatability and stability were 3.1％-8.4％,2.3％-6.3％ and 0.66％-2.4％,respectively.The average recoveries were between 94.0％ and 103.8％.The investigated Cordyceps spp.samples showed that galactose,glucose and mannose were main compositional monosaccharides.Higher contents of glucose were found in most of natural Cordyceps sinensis and cultured Cordyceps militaris fruiting-body samples,the average contents ratio of glucose to galactose were 5.2 ∶ 1 and 1.9 ∶ 1,and the glucose to mannose were 16.9 ∶ 1 and 5.9 ∶ 1,respectively.While the average contents ratio of glucose to galactose and glucose to mannose were 0.96 ∶ 1 and 2.0 ∶ 1,respectively,in fermented Cordyceps samples.However,the average contents of glucose in C.gunnii,C.liangshanensis,C.hawkesii,C.gracilis and C.cicadae were low,and the average contents ratio of glucose to galactose and glucose to mannose were 0.31 ∶ 1 and 0.81 ∶ 1,respectively.Conclusion:The compositional monosaccharides contents in polysaccharides from Cordyceps spp.were found to be different,which should be helpful to the quality control of Cordyceps spp.

The fruiting body formation mechanisms of Cordyceps sinensis are still unclear. To explore the mechanisms, proteins potentially related to the fruiting body formation, proteins from fruiting bodies, and mycelia of Cordyceps species were assessed by using two-dimensional fluorescence difference gel electrophoresis, and the differential expression proteins were identified by matrix-assisted laser desorption/ionisation tandem time of flight mass spectrometry. The results showed that 198 differential expression proteins (252 protein spots) were identified during the fruiting body formation of Cordyceps species, and 24 of them involved in fruiting body development in both C. sinensis and other microorganisms. Especially, enolase and malate dehydrogenase were first found to play an important role in fruiting body development in macro-fungus. The results implied that cAMP signal pathway involved in fruiting body development of C. sinensis, meanwhile glycometabolism, protein metabolism, energy metabolism, and cell reconstruction were more active during fruiting body development. It has become evident that fruiting body formation of C. sinensis is a highly complex differentiation process and requires precise integration of a number of fundamental biological processes. Although the fruiting body formation mechanisms for all these activities remain to be further elucidated, the possible mechanism provides insights into the culture of C. sinensis.

Objectives Lepista sordida (Schum.: Fr.) Sing.is a type of edible Agaricus mushroom. It exhibits several activities including immunomodulation, and antioxidant and anti-fungal properties. The objective of this investigation was to investigate the fermentation and extraction conditions of polysaccharides from the mycelium of L. sordida. Methods This single parameter experiment was performed to identify the optimal culture conditions including culture temperature, culture cycle and initial pH. Furthermore, an orthogonal experimental design L9 (34) was used to evaluate the combination effects of carbon sources, nitrogen and minerals on the extracts from mycelia. Extraction conditions optimization including the ratio of water to material (v/w), temperature and extraction time based on response surface methodology was employed to identify the optimum conditions. Results The maximum mycelium dry weight was obtained when cultured in an optimal composition of sucrose 30 g/L, yeast extract 6 g/L, KH2PO4 0.1 g/L and MgSO4 1 g/L at temperature 28° C, culture cycles of 7 days and initial pH 7.0. The optimum conditions for polysaccharide extraction from L. sordida mycelia were a ratio of water to material of 72.1:1, temperature 92.6° C and time 2.9 hours. Under these conditions, the predictive and practical extraction efficiency was 16.5% and 16.5±0.1%, respectively. Conclusions The results are helpful for building an efficient and controllable model to improve the extraction efficiency for industrial production of bioactive polysaccharides from L. sordida. Acknowledgments This study as supported by the Guangdong Provincial College Students Innovation and Entrepreneurship Training Program (Nos. DC2016062 and DC2016063).

The chemical structure, chain conformation, and immuno-modulatory activity of polysaccharide from mycelium Cordyceps sinensis fungus UM01 were investigated. The molecular weights and the intrinsic viscosity of purified polysaccharide (UM01-S4) were determined as 22,559 Da and 5.09 mL/g, respectively. Using the polymer solution theory, UM01-S4 has a sphere-like conformation in aqueous NaCl at 35 °C. The chemical structure of UM01-S4 contains an α-(1→2)-Manp core. The side chains were composed of β-(1→2)-Galf, β-(1→4)-Glcp, α-GalpA, and α-Manp units, which attached to the mannan core at the O-6 position. The immuno-modulatory assays showed that UM01-S4 exhibited good activities, including macrophages proliferation, phagocytosis, and release of NO and cytokines. The mechanism underlying the macrophage regulation of UM01-S4 might be related to the activation of the MAPK and NF-κB signalling pathways. Thus, mycelium of C. sinensis UM01 could be used as a natural immune-stimulating agent in functional food areas and pharmaceutical industries.

Background Polysaccharides, one of the main bioactive compounds in natural resources such as fungi, plants and algae, have attracted lots of attention in biomedical and functional food science due to their significant pharmaceutical activity. In this study, four extraction methods were compared regarding the production, physicochemical properties and antioxidant activity of polysaccharides from Lepista sordida using hot water, an alkaline solution, a multiplex- enzyme method and an ultrasonic-assisted method. Methods Bioactive polysaccharides from L. sordida were extracted using hot water, an alkaline solution, a multiplex-enzyme method and an ultrasonic-assisted method. The anthrone-sulfuric acid method was applied to measure the crude polysaccharide content and Coomassie Brilliant Blue (CBB) G-250 staining was used to detect protein content. The scavenging effect of the hydrolysates on α, α-diphenyl-β-picrylhydrazyl (DPPH) free radical was measured. Results Polysaccharides extracted using the multiplex-enzyme method had the highest DPPH scavenging efficiency of 91.62%. The extraction efficiency of the multiplex-enzyme method was 19.61 ± 0.60%, only 1.49% lower than that of the alkaline solution method, but the polysaccharide content was 94.47 ± 3.84%, which was much higher than the 74.93 ± 5.98% obtained using the alkaline solution. The polysaccharides extracted using the multiplex-enzyme method had a low protein content. Conclusions The multiplex-enzyme method was selected to extract the antioxidant polysaccharides of L. sordida due to a higher extraction efficiency, better purity and powerful DPPH radical scavenging ability, and lower energy consumption than the other methods. The results are of great significant for developing functional food from L. sordida bioactive polysaccharides in the future. Acknowledgments This study was supported Guangdong Provincial College Students Innovation and Entrepreneurship Training Program (Nos. DC2016062 and DC2016063).

Cordyceps sinensis is a well-known tonic food with broad medicinal properties. The aim of the present study was to investigate the optimization of microwave-assisted extraction (MAE) and characterize chemical structures and chain conformation of polysaccharides from a novel C. sinensis fungus UM01. Ion-exchange and gel filtration chromatography were used to purify the polysaccharides. The chemical structure of purified polysaccharide was determined through gas chromatography-mass spectrometry. Moreover, high performance size exclusion chromatography combined with refractive index detector and multiangle laser light scattering were conducted to analyze the molecular weight (Mw ) and chain conformation of purified polysaccharide. Based on the orthogonal design L9 , optimal MAE conditions could be obtained through 1300 W of microwave power, with a 5-min irradiation time at a solid to water ratio of 1:60, generating the highest extraction yield of 6.20%. Subsequently, the polysaccharide UM01-S1 was purified. The UM01-S1 is a glucan-type polysaccharide with a (1→4)-β-d-glucosyl backbone and branching points located at O-3 of Glcp with a terminal-d-Glcp. The Mw , radius of gyration (Rg ) and hydrodynamic radius (Rh ) of UM01-S1 were determined as 5.442 × 10(6)  Da, 21.8 and 20.2 nm, respectively. Using the polymer solution theory, the exponent (ν) value of the power law function was calculated as 0.38, and the shape factor (ρ = Rg /Rh ) was 1.079, indicating that UM01-S1 has a sphere-like conformation with a branched structure in an aqueous solution. These results provide fundamental information for the future application of polysaccharides from cultured C. sinensis in health and functional food area.

目的:建立同时测定北虫草中尿苷、肌苷、鸟苷、腺苷、虫草素和N6-(2-羟乙基)腺苷含量的高效液相色谱法.方法:采用Grace Prevail Select C18(4.6 mm×150 mm,3μm)分析柱,以水(A)-甲醇(B)为流动相二元梯度洗脱(0 ～6.0min,0→5％B;6.0～15.0 min,5％B→25％B;15.0 ～22.0 min,25％ B→100％B),流速1 mL·min-,检测波长260 nm,柱温25℃,进样量5 μL.结果:尿苷、肌苷、鸟苷、腺苷、虫草素和N6-(2-羟乙基)腺苷质量浓度分别在1.0 ～ 192.0μg· mL-1(r=0.999 9)、1.0～191.0 μg·mL-(r=0.999 9)、0.6～110.0 μg·mL-1 (r=1.000 0)、1.0～186.0 μg·mL-1(r=1.000 0)、1.0～194.0 μg·mL-1(r=0.9999)和0.8～160.0 μg·mL-1(r=0.999 9)范围内线性关系良好,加样回收率(n=6)分别为100.6％(RSD=3.2％)、98.4％ (RSD=4.8％)、99.2％ (RSD=4.6％)、99.4％ (RSD=2.5％)、101.4％ (RSD =2.1％)和99.2％ (RSD=3.2％);不同来源北虫草产品核苷含量差别较大.虫草属特征性成分N6-(2-羟乙基)腺苷的含量仅次于含量最丰富的虫草素,肌苷在所有样品中均未检出.5个核苷类成分在总含量最高的样品中分别为0.68、0.84、1.22、11.12和5.32 mg.g-1.结论:本文将虫草属的6个主要核苷类特征性成分同时作为评价指标,对市场中的北虫草样品进行了检测分析.本文建立的方法能够快速、简便地测定北虫草中主要核苷的含量,可用于北虫草的质量检测.

The optimal fermentation conditions and medium for the production of bioactive polysaccharides from the mycelium of Cordyceps sinensis fungus UM01 were investigated by using orthogonal design and high performance size exclusion chromatography coupled with multi-angel laser light scattering and refractive index detector (HPSEC-MALLS-RID). Results showed that the optimal temperature, initial pH, rotation speed, medium capacity (ratio of medium volume to the volume of flask bottle) and inoculums volume for the mycelium growth were 15°C, pH 6.0, 150rpm, 2/5 (v/v), and 3% (v/v), respectively. Furthermore, bioactive polysaccharides from the mycelium of C. sinensis fungus UM01 were determined as polysaccharide fractions with the molecular weight above 10kDa. The optimal fermentation medium was determined as a composition of glucose 30.0g/L, sucrose 30.0g/L, KH2PO4 1.0g/L, CaCl2 0.5g/L, yeast extract 3.0g/L, and MgCl2 0.1g/L according to the maximum amount of the bioactive polysaccharides (486.16±19.60mg/L) measured by HPSEC-MALLS/RID. Results are helpful to establish an efficient and controllable fermentation process for the industrial production of bioactive polysaccharides from C. sinensis UM01, and beneficial to develop a unique health and functional product in future.

Polysaccharides from seven species of natural and cultured Cordyceps were firstly investigated and compared using saccharide mapping, partially acidic/enzymatic (α-amylase, β-glucanase and pectinase) digestion followed with polysaccharide analysis by using carbohydrate gel electrophoresis (PACE) and high performance thin layer chromatography (HPTLC) analysis, respectively, to obtain the comprehensive profiles of hydrolysates of the polysaccharides and their characters. The results showed that 1,4-α-D-glucosidic, 1,4-β-D-glucosidic and 1,4-α-D-galactosidic linkages were existed in natural and cultured Cordyceps sinensis, cultured Cordyceps militaris, natural Cordyceps gracilis and Cordyceps ciecadae. The similarity of polysaccharides from cultured C. militaris to natural C. sinensis was relatively high, which might contribute to the rational use of C. militaris. Moreover, different species of natural and cultured Cordyceps can be differentiated based on the saccharide mapping, which is helpful to well understand the structural characters of polysaccharides from different species of Cordyceps and to improve the quality control of polysaccharides in natural and cultured Cordyceps.