Preparation and the antitumor activity in vitro of polysaccharides from Tricholoma matsutake

Tricholoma matsutake is the most valuable mushroom exhibiting a characteristic and delicate flavor as well as several biological activities among the various mushroom species. Extraction procedure and antitumor activity of polysaccharides from Tricholoma matsutake mycelium were studied in this work. Single factor tests and Response Surface Methodology (RSM) based on a three-level three-factor Box-Behnken design were applied to optimize the water extraction conditions. The polysaccharides yields increased when the ratio of water to sample increased from 10:1 to 30:1, extraction time from 1 to 4 h and temperature from 60 to 90°C as invested in single factor tests. To enhance further polysaccharides extraction, the extraction conditions were studied with RSM. The optimum extraction conditions were confirmed as follows:extraction time 4.3 h, the ratio of water to sample 29.2:1, extraction temperature 93.8°C. The extraction rate of polysaccharides under this condition could reach the highest value of 18.43% with one time extraction, and, 21.63% with two times extraction. Furthermore, the polysaccharides presented significant antitumor activity against B16 human melanoma cells in vitro in a dose-dependent manner. At the concentration of 10 mg/ml, the inhibition rate of polysaccharides got 67%.     

In vitro antioxidant activities of the polysaccharides from Tricholoma lobayense

The antioxidant activities of three polysaccharide components (TLH-1, TLH-2, TLH-3) extracted from Tricholoma lobayense were evaluated by three different in vitro methods, namely superoxide radical (O(2)(-)) scavenging activity, inhibition of mice erythrocyte hemolysis (MEH) and malondialdehyde (MDA) mediated by hydrogen peroxide (H(2)O(2)) and investigation of oxidative modification of human serum albumin (HSA) induced by 2,2-azobis(2-amidinopropane)dihydrochloride (AAPH) through fluorescence spectroscopy. The antioxidant experiments showed that the polysaccharides had a notable activity in scavenging O(2)(-) in a concentration-dependent manner; H(2)O(2)-induced MEH and formation of MDA were effectively inhibited; by fluorescence spectroscopy, it was demonstrated that the polysaccharides could obviously inhibit AAPH-induced oxidative modification of HSA. The experimental data obtained from the in vitro models clearly revealed that TLH-3 had stronger antioxidant potency than TLH-1 and TLH-2, which indicated that TLH-3 might be exploited as effective natural antioxidant to alleviate oxidative stress.     

松口蘑(Tricholoma matsutake)菌塘土壤可培养细菌多样性

松口蘑(Tricholoma matsutake)也称松茸,是具有重要经济和药用价值的野生食用菌,菌塘是其子实体发生发育的场所。本文采用土壤平板稀释技术研究了云南省6份松口蘑菌塘土壤可培养细菌,共获得了178条细菌的16S r DNA碱基序列,经分析分别属于4个门、18个属、38个OTUs,变形菌门(Proteobacteria)序列占58.43%,厚壁菌门(Firmicutes)占26.40%,拟杆菌门(Bacteroidetes)占10.67%,放线菌门(Actinobacteria)占4.49%,其中厚壁菌门的芽孢杆菌属(Bacillus)占24.72%,变形菌门的伯克氏菌属(Burkholderia)占21.34%,假单胞菌属(Pseudomonas)占11.24%,拟杆菌门的金黄杆菌属(Chryseobacterium)占10.67%。云南松口蘑菌塘土壤可培养细菌的多样性较为丰富。     

松茸液态发酵菌丝生长量因子的研究

研究了液态发酵条件下通气量、转速、pH、可溶性淀粉浓度对松茸菌丝生长量的影响,探讨了可溶性淀粉稳定pH的作用机理。试验结果表明：通气量≤0.4vvm时松茸菌丝产量随通气量的增大而增长,当通气量＞0.5vvm时,菌丝产量增加不明显;转速增大菌丝产量增加,转速＞80r/min,菌丝产量下降;松茸菌丝发酵的最适pH为5.5,初始pH不同,发酵罐中培养基最终的pH趋于6.0;可溶性淀粉是影响松茸菌丝量的重要因子,可溶性淀粉浓度≤4g/100mL时菌丝量随可溶性淀粉浓度的增加而增大,当其浓度为4g/100mL时,菌     

松茸多糖提取方法的研究

利用不同温度 ,不同酸碱盐介质以及微波提取松茸多糖。结果表明 :用乙醚、甲醇将松茸子实体粉末进行回流提取后 ,再用质量浓度为 2 0g·L- 1 的Na2 CO3 溶液微波浸提 3次 ,可将松茸中的多糖及其它有效成分充分提取出来 ;其中松茸菌盖中提取的多糖质量分数为 3.6 5 %,菌柄中提取的多糖质量分数为 3.0 2 %。     

Artificial shiro formation of Tricholoma matsutake

One type of soil collected from Maoer-shan in Heilongjiang Province,China was selected to induce hyphal growth of Tricholoma matsutake by a soil screening experiment.It was confirmed that hyphal growth of all the tested T.matsutake isolates was significantly stimulated in soil by supplemented with 0.5%～2.0% olive oil.The aggregation of hyphae and soil resembled natural Shiro.The biomass of hyphae in the soil increases with increasing olive oil concentrations.Moreover,seedlings of Pinus densiflora grew well in the soil containing 0.5%～1.0% olive oil and were also successfully infected by T.matsutake isolate A in the soil containing 1.0% olive oil.This study established a culture system of artificial Shiro formation and also provided a premise for formulation of culture substratum for fruit body formation of T.matsutake.     

Physiological variation among Tricholoma matsutake isolates generated from basidiospores obtained from one basidioma

Matsutake (Tricholoma matsutake) is a commercially valuable edible ectomycorrhizal mushroom. The physiological traits of T. matsutake have been previously assessed using mycelial isolates isolated from basidiomata; however, few studies have focused on basidiospores. Here, we report that sibling T. matsutake isolates generated from basidiospores on a single basidioma show distinct physiological variation. We first established 145 isolates of T. matsutake on modified Norkrans' C (MNC) agar medium and found that their radial growth varied significantly. The mycelial biomasses of nine isolates with different growth rates were reduced on low-carbon and low-nitrogen MNC media. However, the colony diam of one isolate was significantly elevated on low-carbon medium, and the colony diam of two isolates were significantly elevated on low-nitrogen medium. In co-cultures of two or three isolates, commensal and amensal interactions were observed. The physiological variation induced by low carbon and nitrogen levels and the mycelial interactions between sibling isolates imply mechanisms for the genetic and functional characteristics of mycelia of T. matsutake.     

Purification, chemical characterization and radical scavenging activities of alkali-extracted polysaccharide fractions isolated from the fruit bodies of Tricholoma matsutake

Tricholoma matsutake, a high-class edible mushroom in China, has been regarded as famous foods and biopharmaceutical materials with a great deal of interest. In the previous investigations, researchers believe the water-soluble polysaccharide β-glucan is the major active component of T. matsutake, which displays various biological activities. In the present study, two novel alkali-extracted polysaccharide fractions, TM-APS-1 and TM-APS-2, were isolated from the fruit bodies of T. matsutake by DEAE-cellulose and Sepharose CL-6B columns on ÄTKA explorer chromatography system. Their chemical and physical characteristics and radical scavenging capacity were valuated, including chemical methods, GC, HPLC, scavenging activity against DPPH radicals, superoxide radicals, hydroxyl radicals, and chelating ability. The results showed that TM-APS-1 and TM-APS-2 exhibited significantly antioxidant activity at a concentration-dependent manner. The alkali-extracted polysaccharide fractions from T. matsutake can be developed to be novel functional food or pharmaceutical products with antioxidant effects.     

Successful inoculation of mature pine with Tricholoma matsutake

Our finding demonstrates, for the first time, that the roots of mature pine trees can be successfully inoculated with a symbiotic ectomycorrhizal fungus, the valuable matsutake mushroom. Long root segments (ca. 5–10 mm in diameter, ca. 50 cm in length) of 50-year-old Pinus densiflora trees were excavated, washed, auxin-treated (2–5 mg indole butyric acid, IBA, per root) and incubated in moist Spagnum moss. Twelve months later, short roots were regenerated, of which approximately 90% were free of mycorrhizae. Mycorrhiza-free short roots were inoculated with mycelial pieces of Tricholoma matsutake and incubated further in a sterilized substrate. Four-and-a-half months later, roots putatively colonized by Matsutake were sampled near the inoculation points. A T. matsutake-specific ITS-rDNA fragment was amplified by nested PCR from approximately 80% of the root samples analyzed, whereas approximately 66% of the root samples processed for staining with Chlorazol black E displayed characteristic T. matsutake Hartig net structures. These results confirm the symbiotic infection of mature P. densiflora roots by matsutake.     

利用松茸ITS特异性引物对松茸分离物进行鉴定

利用一对ITS通用引物(YIS4-ITS5)和一对松茸物ITS特异性引物(TMF-TMR)对来源于云南省不同地区的6个松茸(Tricholoma matsutake)子实体及其6株分离物,3个假松茸(Tricholoma bakamatsutake)子实体及其3株分离物、侧耳(Pleurotus astreatus)、金针菇(Flammulina velutipes)和双孢蘑菇(Agaricus bisporus)的子实体进行了PCR扩增,琼脂糖凝胶电泳分析,结果表明ITS4-ITS5能将所有的样品扩增,并得到600bp左右的DNA扩增条带,TMFTMR扩增时,只有松茸子实体及其对应分离物有扩增条带,DNA片段大小在500bp左右。进一步对松茸子实体(TG25)及其分离物(TM25112．2)进行WS序列测定表明两者的DNA同源性为100％,从而证明所分离到的6个菌株确为松茸的纯培养物。     

Regeneration and Fusion of Mycelial Protoplasts of Tricholoma matsutake

The Hill-reaction inhibitory activity of 30 derivatives of 2-difluoromethylthio-1,3,5-triazine was determined using chlorella. The structure-activity relationships were analyzed using the hydrophobic parameter (log P), the electronic substituent constant (c) and the steric substituent constant (Es). The Hill-reaction inhibitory activity showed a parabolic relationship to log P and the steric substituent parameters at 4,6-dialkylamino- positions in the triazine ring. The equation revealed that optimum hydrophobicity and size of the substituents were necessary for high activity.     

Agrobacterium-mediated transformation of the ectomycorrhizal basidiomycete Tricholoma matsutake that produces commercially valuable fruit bodies, matsutake

Using agroinfection with a T-DNA vector carrying a hygromycin resistance marker, the recombinants were generated for the first time from the ectomycorrhizal basidiomycete Tricholoma matsutake, which produces commercially valuable fruit bodies, matsutake, during association with Pinus sp. plants. The transformation system may be useful in the genetic analysis of T. matsutake.     

松茸利用淀粉的初步研究

目的：研究松茸在以淀粉为碳源的人工培养基上生长的可能性,初步了解共生菌根真菌松茸离体培养下的营养生长习性。方法：采用改良真菌合成培养基和单因子试验方法,比较了葡萄糖、麦芽糖、蔗糖、可溶性淀粉和普通玉米淀粉等不同碳源对松茸纯培养菌丝体生长的影响,描述了菌丝体萌动生长速度与菌落扩展的态势,定期收获并称量菌丝体生物量（干重）,然后进行统计分析。结果：当查氏（Czapek）培养基以KNO3为供试氮源,供试碳源是普通玉米淀粉时,移植的松茸菌块萌发菌丝体相对延迟,长势较弱,松茸菌落扩展速度为0.13～0.2mm·d-1之间,生物量积累较少;以NH4Cl为优化氮源,松茸菌落扩展速度提高至0.37mm·d-1,1个月生物量积累27.3mg;进一步添加1‰（W/V）马铃薯汁,菌落扩展速度达到0.63mm·d-1,生物量积累达73.4mg。结论：共生真菌松茸可以利用淀粉作为单一碳源进行营养生长,但菌丝体扩展速度和生长态势较其它供试碳源条件下弱。而普通淀粉经改性加工成可溶性淀粉后,松茸长势有所改善。优化氮源,补充生长因子,则显著促进了松茸利用普通玉米淀粉的生长水平。     

松茸组织分离物的rDNA-ITS序列鉴定

以采自云南丽江的松茸子实体为材料,进行组织分离后,利用一对ITS引物(ITS1-ITS4)对子实体(SR176B,SR172B)和分离物(SR176H,SR172H)进行了PCR扩增、琼脂糖凝胶电泳分析,得到了700bp左右的扩增条带,进一步对ITS序列进行同源性检索比对,结果表明SR176H与SR176B,SR172H与SR172B序列同源性均为100%,鉴定出该分离物就是松茸的纯培养物。     

Immunostimulatory activity of polysaccharides isolated from Caulerpa lentillifera on macrophage cells

Polysaccharides were extracted from Caulerpa lentillifera by treating with water and then purified by size-exclusion chromatography. The purified polysaccharides, termed SP1, were found to be sulfated xylogalactans with a molecular mass of more than 100 kDa. Adding SP1 to murine macrophage RAW 264.7 cells increased the production of nitric oxide (NO) in a dose-dependent manner. NO was found by immunoblotting and RT-PCR analyses to be synthesized by an inducible NO synthase. SP1 caused the degradation of IκB-α and the nuclear translocation of nuclear factor (NF)-κB subunit p65 in macrophage cells. SP1 also increased the phosphorylation of p38 mitogen-activated protein kinase (MAPK). These results demonstrate that SP1 activated macrophage cells via both the NF-κB and p38 MAPK signaling pathways. Moreover, SP1 increased the expression of various genes encoding cytokines, and the phagocytic activity of macrophage cells. These combined results show that SP1 immunostimulated the activity of macrophage cells.     

松茸多糖抗辐射功能的初步研究

预防给予小鼠松茸多糖(PTM),测定小鼠在受到2 Gy X线照射后,脾和胸腺重量、T淋巴细胞转化能力、外周血中白细胞数和肝组织中SOD、CAT、GSH-Px活性及MDA含量的变化,结果显示PTM处理组小鼠的各项指标与辐射对照组比较均有显著性差别,提示PTM可促进机体自由基的清除,增加机体抗氧化能力,对辐射所致的免疫损伤有明显的保护作用。     

Genetic variability and bottleneck detection of four Tricholoma matsutake populations from northeastern and southwestern China

The excessive commercial collection of matsutake mushrooms can lead to extreme reduction of population size, which may cause genetic bottleneck and decrease genetic diversity of Tricholoma matsutake. Here, six polymorphic microsatellite loci markers were used to examine the genetic diversity of four natural T. matsutake populations from two main producing regions of China. The minimum combinations of four loci were able to discriminate total 86 sampled individuals with distinctive multilocus genotypes. Our analysis of molecular variance (AMOVA) revealed that about 80% and 20% of the overall genetic variation were respectively partitioned within and among populations. The principal‐coordinate analyses (PCA) distinguished the four tested populations into three genetic clusters, each of which was correlated with respective endemic host plants on a geographical basis. The AMOVA, PCA and pairwise population F_ST estimates consistently displayed the same genetic divergence patterns and spatial structure of T. matsutake mediated by host plants in China. The significant heterozygosity excesses demonstrated that a recent genetic bottleneck occurred in each population tested. The complementary M‐ratio test indicated past genetic bottleneck events over longer periods. Only four individuals were identified as putative first generation migrants within northeastern China, which implies restricted interpopulation gene flow in T. matsutake. We discuss that the significant genetic differentiation among populations of T. matsutake is most likely a function of host adaptation, host specificity, genetic bottleneck, limited dispersal and habitat fragmentation.     

滇产与日产松茸的IGS1-RFLP比较分析

应用Cfr13I限制性内切酶对采自于云南省16个市县的127个松茸子实体进行了IGS1-RFLP比较分析,发现126个松茸子实体属于A类型,来自大理白族自治州剑川县的1例(TF89)为C类型。通过对A类型的IGS1序列分析发现产自云南的松茸有一个CTTT的简单重复,滇产松茸IGS1序列差异不明显。滇产松茸的IGS1-RFLP与日产松茸的主要类型十分相似,两地松茸可能是同源的。     

Advances in the cultivation of the highly-prized ectomycorrhizal mushroom Tricholoma matsutake

Matsutake, the fruiting body of Tricholoma matsutake, is among the most economically important edible ectomycorrhizal (EM) mushrooms worldwide. This EM fungus develops “shiros”, which are mycelial aggregations that develop in association with the roots of EM coniferous trees and soil particles in well-drained and nutrient-poor forest soil. The fruiting bodies occur on the periphery of the outward-growing shiro. In spite of vast research, the cultivation of matsutake has been mostly unsuccessful. Commercial demand is therefore met by harvesting the fruiting bodies that naturally occur in forests of EM coniferous trees, mainly Pinus densiflora. Recent inoculation studies have produced mycorrhiza and shiro structures, and the host range of Tr. matsutake and associated species has been clarified. It has also become possible to identify strains of Tr. matsutake by DNA sequencing, which has also been useful to determine the origins of matsutake species in Asia and to elucidate the genetic structure of shiro. In this review, basic research and the outcomes of various trial of matsutake cultivation are discussed.