Agaricus中提取并精制活性成分及其纯度的评价

从Agaricus中提取活性成分──过氧化物酶，并加以精制，其活性通过TMB基质测定。精制后的活性成分分别采用SDS—聚丙烯酰胺凝胶电泳、毛细管电泳和飞行时间型质谱进行评价。

Molecular Cloning and Expression of Polyphenoloxidase Genes from the Mushroom, Agaricus bisporus

The polyphenoloxidase（PPO） is the key enzyme considered to be responsible for mushroom browning.By using homology cloning and rapid amplification of cDNA ends（RACE）,two new PPO genes and the corresponding cDNA were identified from the fruit bodies of Agaricus bisporus（AbPPO3 and AbPPO4,GenBank accession nos.GU936494 and GU936493,respectively）.The genomic DNA sequences of AbPPO3 and AbPPO4 are 2 080 and 2 189 bp in length,respectively,encoding putative polypeptides of approximately 66 and 68 kDa.The deduced amino acid sequences show characteristic features of two copper-binding domains conserved in the type III copper proteins including fungal polyphenol oxidases.Sequence comparisons indicate that AbPPO3 and AbPPO4 present 55.3% similarity to each other（48% identity）.We also obtained more than 1.5-kb long sequences upstream of the start codon of the AbPPO3 and AbPPO4 and recognized them as their respective putative promoters.Analyses of the two PPO promoter regions show that they contain abundant cis-acting elements which are probably responsible for anaerobic induction,light,wound,stress,and auxin response.Semi-quantitative RT-PCR results indicate that AbPPO3 and AbPPO4 were highly expressed in the mature fruit bodies and up-regulated after 2-d storage of mushroom.These results suggest that AbPPO3 and AbPPO4 may play roles in A.bisporus browning and pigmentation during development and postharvest storage and the elements in promoters may act as regulatory elements for the inducible expression of AbPPO3 and AbPPO4.The successful cloning and expression analysis of AbPPO3 and AbPPO4 warrant a further investigation on the structure and function of A.bisporus PPO which points to the possible targets for genetic manipulation.

Identification of commercial cultivars of Agaricus bisporus in China using genome-wide microsatellite markers

Agaricus bisporus is one of the most widely cultivated mushrooms in the world. Commercial cultivars are usually phenotypically alike and easy to be copied by isolating tissue cultures. This brings great challenges to distinguish different cultivars and to protect new varieties. Thus, techniques for the accurate identification of cultivars are essentially required. In this study, we accurately identified 11 commercial cultivars of A. bisporus released in China by using microsatellite(SSR, simple sequence repeat) markers. SSR markers were developed by mining the genome sequence. A total of 3 134 SSRs were identified, of which 1 490 SSRs were distributed in gene models, and 1 644 in the intergenic regions. A total of 17 polymorphic primer pairs were developed and SSR fingerprints were constructed for all the commercial cultivars. These SSR markers generated a total of 73 alleles, with an average of 4.29 per locus. Specifically, the primer combination of AB_SSR_2341 and AB_SSR_2590 could distinguish all the 11 commercial cultivars. The similarity coefficients of the 11 commercial cultivars were between 0.56 and 0.95 indicating that some of them were close related. Our results provide an efficient technique for the identification of A. bisporus cultivars in China, which can also facilitate the marker-assisted breeding in the future.

Biodegrading Wheat Bran with Agaricus blazei and Its Effects on Intestinal Development Identified with Mice

Agaricus blazei Murill is a well known edible/medicinal mushroom used for immune-nutrient food therapies.The biodegradation of different substrates with this fungus may result in different metabolites and degraded compounds,which may enrich the function of the food therapies.In this study,soluble compounds from the culture of A.blazei grown in liquid media with whole wheat bran and its water filtrate,respectively,were compared.Total soluble sugar,arabinoxylan,protein,and amino acids were significantly higher in the fungal culture resulted from the medium with whole wheat bran （43.54,1.56,0.59,and 2.19 mg mL^-1,respectively） than that from the medium with the bran filtrate （17.28,0.37,0.13,and 1.13 mg mL^-1,respectively）.The biodegraded wheat bran with cultured mycelia was fed to Mus musculus Linnaeus as supplementation or as dietary fiber exclusive ingredient.As non-specific food therapies,feeding effects on mice intestinal development were indicated indirectly in growth performance,intestinal absorption and serum parameters.Compared to feeding uncultured wheat bran,feeding mice with culture mixes of A.blazei resulted in remarkably increase in villus height and villus height/crypt depth ratio,which were increased by 25.4 and 31.0%,respectively,when applied as supplementation,and by 44.3 and 43.4%,respectively,when applied as dietary fiber.These increases are concomitant with the higher level of D-xylose in blood serum about 16.9 and 29.2% as supplementation and dietary fiber,respectively.The results implying that culturing A.blazei with whole wheat bran enhanced extracellular metabolism of the fungus and extensive degradation of wheat bran insoluble fibrous compounds.Furthermore,feeding the culture mix including metabolites and degraded wheat bran improved intestinal villus development,proving the nutritional benefits of the A.blazei mycelial cultures.

Study on Anti-infection Effect of Polysaccharide from Agaricus blazei Murrill on Mice

In order to study the anti-infection effect of phoshporylated Agaricus blazei polysaccharide on mice, mice were drenched phoshporylated A. blazei polysaccharide for 14 d, and an A. blazei polysaccharide control group and a blank control group were also set. The mice in all the three groups were infected with Escherichia coli on the 15 th day and the 29 th day of the experiment, the body weight of the mice in each group was recorded, as well as the spleen index on the 29 th day of infection and the 36 th day of the experiment, and the anti- E. coli titer in serum was also detected. The results showed that phoshporylated A. blazei polysaccharide could promote and maintain the development of spleen, and improve the immune response of organisms, thereby resisting bacterial infection. Furthermore, phoshporylated A. blazei polysaccharide has better immunoregulation and anti-infection effects than A. blazei polysaccharide, and could play an important role in veterinary clinical treatment and prevention and control of diseases as a immunologic adjuvant or immunopotentiator.

Screening on Chemicals for Preventing Mycogone peniciosa Magn in Agaricus bisporus

[ Objective ] The aim was to screen out chemicals with good control effect against Mycogone peniciosa Magn in Agaricus bisporus. [ Method ] M. peniciosa ofA. b/sporus was taken as the detection bacteria, and nine kinds of chemicals were conducted plate efficacy test in the laboratory. Mushroom house validation was carried out for the screened chemicals with strong inhibition to M. peniciosa. [ Result] Nine kinds of chemicals had obvious difference in control effects on M. peniciosa and effects on growth ofA. bisporus; 2 400 and 3 200 times dilution of 78% Boer MZ WP and 2 400 times dilution of 69% Fulishuang WP after casing material processing had good control effects on M. peniciosa and had no apparent adverse effects on mycelial growth and fruiting development of A. bisporus. [ Conclusion] The 2 400 and 3 200 times dilution of 78% Boer MZ WP and 2 400 times dilution of 69% Fulishuang WP are worth popularizing.

Effect of Hot Water Extract from Agaricus Blazei Murill on Chemotaxis of Neutrophils

Hot water extract from the edible Brazilian mushroom, Agaricus Blazei Murill (ABM), is used for both traditional and alternative medicine. ABM is reported to stimulate anti-tumor, anti-infection, and immune activity. However, there are few reports of how ABM affects neutrophils. Therefore, in this study, we examined the effect of hot water ABM extract on neutrophil migration, phagocytosis, and reactive oxygen species production using neutrophils from guinea pig. Migratory direction and velocity as indicators of chemotactic activity of neutrophils were significantly (p 0.001) increased at concentration of 50 and 100 mg/ml in ABM extract compared with control. Phagocytic activity of neutrophil was significantly (p 0.01) increased at concentration of 5 mg/ml in ABM extract compared with control. Production of reactive oxygen species (ROS: H2O2 or ) by neutrophils was significantly (p 0.01) increased at concentration of 5 mg/ml in ABM extract compared with control. These results suggest that enhancement in neutrophil chemotactic activity, phagocytic activity and ROS production are mechanisms by which ABM extract inhibits bacterial infection in the skin and dermatitis.

Effect of ^( 60)Co γ Ray Irradiation on Contents of La, Pr, Yb and Mineral Elements in Fruitbodies of Agaricus biazei Murvill

The effects of 60Co γ ray irradiation on the contents of rare earth and mineral elements and their relativities in the fruitbodies of Agaricus biazei Murvill and their relativities were studied. The results show that there are significant difference at the 0.01 level in the contents of rare earth and mineral elements in the fruitbodies among different treatments, and that the variational laws of different elements contents are different with the varieties of the dosages accordingly. Thereinto, the contents of Pr and Mo increase with the increase of the dosage, but they decrease when the dosage are in a certain degree(1.5 KGy). But the Yb content decreases with the increase of the dosage. The variational laws of the contents of K and Cu were similar along with the variety of the dosage, and they all decrease with the increase of the dosage. But the contents of them increase and are higher than that of the three anterior treatments(0, 0.5, 1.0 KGy) when the dosage are 1.5 KGy. It can be seen that the contents of rare earth and mineral elements in the fruitbodies have something to do with the dosages in different degrees, and that rare earth also has something to do with mineral elements in different degrees. Thereinto there lied good linear relationship between the content of Yb and the dosage.

Effect of protease inhibitor from Agaricus bisporus on glucose uptake and oxidative stress in 3T3-L1 adipocytes

Objective:To explore the effect of the protease inhibitor from Agaricus bisporus(J.E.Lange)Imbach(AbPI)on glucose uptake and oxidative stress in 3 T3-L1 adipocytes.Methods:Adipocytes were differentiated and stained with OilRed-O staining to confirm adipogenesis.The toxic/protective effect of AbPI on the adipocytes was determined by MTT assay,intracellular reactive oxygen species generation through flow cytometry,and morphologically through confocal microscopy using propidium iodide,4,6-diamino-2-phenylindol dihydrochloride,and 2’,7’-dichlorofluorescein diacetate dyes.The uptake of fluorescent glucose analog,2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose by adipocytes was also studied through confocal microscopy.Results:MTT assay showed that the cell survival rate was(28.00±3.00)%,(92.33±2.60)%,and(71.34±2.10)%in the presence of 2 mM H2O2,AbPI alone,and AbPI and H2O2 both,respectively,in comparison to the control.Oil-Red-O staining indicated that Ab PI enhanced adipogenesis.AbPI stimulated the glucose uptake by adipocytes similar to the drug rosiglitazone,and showed insulinsensitizing effect in the presence of insulin,but failed to stimulate the uptake in the absence of insulin.Intracellular reactive oxygen species generation was reduced in differentiating adipocytes upon Ab PI treatment.Confocal microscopy showed that the damaged cell population rose to 3.50%,117.84%,and 261.50%in the presence of Ab PI alone,AbPI with H2O2,and H2O2 alone,respectively.Conclusions:The protease inhibitor enhances glucose uptake by adipocytes and exhibits a cytoprotective effect on them.

Two Routes to Produce Chitosan from Agaricus bisporus

Two methods were used to produce chitosan by deacetylation of chitin which was extracted from Agaricus bisporus stipes.The first one gives chitosan 1 with low yield of 2.5%,degree of acetylation(DA)of 4%,molecular weight(MW)of 2.973×10^(5)(g/mol).The second route produces chitosan 2 with higher yield of 41%,degree of acetylation(DA)of 17.23%,molecular weight(MW)of 2.939×10^(5)(g/mol).Both chitosans were characterized by XRD,FTIR,^(1)H-NMR spectroscopy nuclear magnetic resonance of proton.The molecular weight(MW)was determined by size exclusion chromatography(SEC).Thermal analysis shows that both chitosans have moisture content lower than 10%.However chitosan 2 has the less ash%which is the quality grade for chitosan medical applications.Accordingly,fungal chitosan 2 could have potential medical and agricultural applications.

Study on Several Characteristics of Agaricus blazei MurrilStrain J_3 Irradiated by ^(60)Co

A new mutant strain J3 from Agaricus blazei Murril was obtained by 60Co irradiation. The yields of successive generations were increased more than 70% to compare with their original strain. The component analysis on amino acids and fatty acid illuminated that the nutrient value of strain J3 fruiting body was better than the original strain. The apparent nodule structure was found in the hyphea of J3 strain through the observation by scanning electron microscope. RAPD analysis showed the great difference of PCR fingerprints between J3 and its original strain. It is a promising mutant strain for further commercial development in the future.

Morphological Features of Agaricus bisporus and Indoor Toxicity Test of Chemicals

Through separating and purifying diseased mushrooms infected by Mycogone perniciosa in Gaochun Area, Nanjing City, morphological features of the bacteria were identified, and toxicity of the bacterial strains was tested. The results showed that five bactericides in the experiment had obvious inhibitory effect on the growth of M. perniciosa, especially 50% Prochloraz manganese chloride complex WP 5 g/L had the best efficiency.

Research of Submerge Fermentation Conditions on <i>Agaricus blazei</i>Murill

Rotational speed, temperature, inoculation amount and pH are the four factors that affect the submerge fermentation of Agaricus blazei Murill. The orthogonal experiment was on the basis of single factor experiments. And the optimal submerge fermentation condition of Agaricus blazei Murill was determined through orthogonal test. The results were as follows: temperature (25℃), rotational speed (150 r/min), inoculation amount (6%) and pH (7), in this condition the cell dry weight reached 1.32 g/100mL;temperature (25℃), rotational speed (150 r/min), inoculation amount (8%) and pH (6.5) in this condition the extracellular polysaccharide reached 6.95 mg/mL.

Preliminary Study on Mixed Use of Agaricus bisporus Residue and Microbial Agent to Control Cucumber Root-knot Nematode

Single or mixed use ofAgaricus bisporus residue and microbial agent had better control effect on cucumber root-knot nematode disease in greenhouse. The results showed that the control effect of single use of A. b/sporus residue was 32.9% - 34.7%, and the yield increasing rate was 2.1% - 8.7% ; the control effect of mixed use ofA. b/sporus residue and microbial agent was 59. 1% - 63. 6%, and yield increasing rate was 2.6% - 3.5%. The experimental dose had no ad- verse effects on soil physical and chemical properties. The mixed use of A. b/sporus residue and microbial agent was one of the effective ways to control root-knot nematode disease.

Effects of Arterially Infused Hydroalcoholic <i>Agaricus blazei</i>Extracts on Perfusion Pressure and Oxygen Uptake in the Bivascularly Perfused Rat Liver

In a preceding work we have reported experiments showing that an hydroalcoholic exctract of Agaricus blazei is able to exert purinergic effects in the isolated perfused rat liver when it is infused into the portal vein in monovascular perfusion (entry: portal vein;exit: hepatic vein). In the present communication we are presenting and discussing experiments done with the bivascularly perfused rat liver (entry: portal vein + hepatic artery;exit: hepatic vein) in order to verify if the hemodynamic effects also occur in the arterial bed. It was found that the A. blazei extract is also active when infused into the hepatic arterial bed, with differences in both sensitivity and nature of the effects on either perfusion pressure or oxygen consumption. Constriction of the arterial bed required much higher concentrations of the extract than the portal bed. The kinetics of the response was also different, with a biphasic instead of a monophasic response. These results provide a promising starting point for future studies aiming to bring to light more mechanistic details about these and possibly other effects.

Effects of Agaricus blazei Murill Polysaccharides on Leadinduced Disturbance of Morphology and ASIC1a and ASIC2b Gene Expression in Hippocampus of Rats

The present study was undertaken to investigate the effects of Agaricus blazei murill polysaccharides on morphology of hippocampus tissue and expression of ASIC1a and ASIC2b mRNA in lead-poisoning rats.Forty SD rats were divided into five groups：control group,lead group,experimental group 1,2,3.After 60d of treatments,hippocampus tissues were observed by HE staining and the expression levels of ASIC1a and ASIC2b mRNA were determined by the flouoreescent quantitative real-time polymerase chain reaction（FQRT-PCR）.The results showed that after lead induction,the number of hippocampus neuron increased,cell gap increased,the chromatin was stained lightly;after administration of ABMP,the number of the hippocampus neuron increased as the dose of ABMP rose,cell gap decreased,the chromatin was stained darkly.The expression level of ASIC1a mRNA in hippocampus of rats in ABMP group decreased extremely significantly compared with that in the model group（P〈0.01）.Lead exposure and ABMP exhibited no significant effects on the expression level of ASIC2b mRNA.ABMP possesses a protective effect on nervous system of rats exposed to lead by combining with lead.

Bioinformatics analysis of metallothionein gene from <i>Agaricus bisporus, Ganoderma lucidum, Taiwanofungus camphorates</i>and <i>Paxillus involutus</i>

Metallothionein (MT) gene has an important role in the detoxification of toxic metals especially some heavy metals. Based on the published sequences of MT genes from Agaricus bisporus, Garnoderma lucidum, Taiwanofungus camphoratus and Paxillus involutus as a type of the edible or medical mushroom in NCBI database, their molecular structures, physiological functions and evolutionary relationship were analyzed using the bioinformatics methods. Results showed that AbMT, GlMT, TcMT and PiMT genes contained complete open reading frame (ORF) and their theoretical points of the last three encoding proteins were higher than 7.0. AbMT, GlMT, TcMT and PiMT were relatively rich in random coil and extended strand, but transmembrane helices and signal peptides were not found. 4 MTs were mainly localized in cell nucleus (over 60%) and their cellular functions might have some relation to central intermediary metabolism. Multiple sequence alignment indicated relatively high identity (more than 52%) and short genetic distances (lower than 0.900) among 4 MT nucleotide sequences. Abundant genetic diversity and strong codon bias were found based on the halotype diversity, average number of nucleotide differences, nucleotide diversity, effective number of codons, codon bias index and scaled chisquare. Simultaneously, we deduced that 4 MT genes during molecular evolution were under positive selection. The present study might provide basis for further investigation of MTs molecular mechanisms and genetic laws.

Study on the Cultivation of <i>Agaricus blazei</i>(Almond Mushroom) Grown on Compost Mixed with Selected Agro-Residues

The Agaricus blazei strain (KUMB 1221) from Forest Production Control Laboratory, Kyushu University was grown on the basal media of compost mixed with selected agro-residues (sawdust, woodchips and corncob) in the ratio of 25%, 50%, 75% and 100% (by weight). The data was collected on the colony diameter of mycelial growth, days required for spawn run (colonization), days required for primordial formation, whiteness measurement, days required for fruit body formation, number, size and fresh weight of mushrooms, biological efficiency (BE) and mushroom production (MP). It was observed that the mycelial growth showed two types of mycelia according the level of mycelial density;compact (C) and somewhat compact (SC). The fastest spawn run (17 days) was found in woodchips (50%) among all the different treatments while the least spawn run took 26 days in corncob (75%). Sawdust substrates promoted longer days for primordial formation and fruit body development, if compared with woodchips substrates and corncob substrates. With regard to yield, 100%, 75% and 50% mixture with compost were superior to 25% mixture with compost in each group of selected agro-residues and it indicated that adding more compost gave the increased yield. Interestingly, it was noted that mushroom size on compost (100%) was double to the mushroom size of other treatments. In conclusion, it was clearly showed that compost (100%), woodchips (25%) and corncob (25%) could produce better yields among all treatments, and it was also possible to obtain acceptable yields of good quality almond mushroom using main substrates of compost mixed with different agro-residues at various concentrations.

Accumulation Characteristics and Resistance to Heavy Metal Contamination of Agaricus bisporus Varieties in Shanxi Province,China

In order to evaluate the accumulation characteristics of heavy metals in the cultivars of Agaricus bisporus in Shanxi Province and to select the varieties with high resistances to heavy metal pollution, atomic absorption spectrophotometer and the atomic fluorescence spectrophotometer were used as the determination methods to compare the heavy metal contents and accumulation characteristics of 6 different varieties. The results showed that 3 heavy metal elements （lead, chromium and cadmium） were tested in the 6 varieties of A. bisporus , all of which were contaminated by heavy metals, but basically no beyond the national standards for food safety. In general, these varieties had less quality risk and could be eaten safely. The accumulation characteristics of heavy metals in these varieties were analyzed, and the varieties were screened according to the resistance to heavy metals pollution. The results showed that variety No. 1 and No. 6 were the varieties with strong comprehensive resistance to the 3 types of heavy metal pollution, and variety No. 3 was a species with weak comprehensive ability in resisting heavy metal pollution.

Study on Anti-infection Effect of Polysaccharide from Agaricus blazei Murrill on Chickens

In order to study the anti-bacterial infection effect of polysaccharide from Agaricus blazei Murrill on chickens, the experimental groups were orally administrated A. blazei polysaccharide at low dose and high dose, respectively, for 14 d continuously, and then, the chickens in various groups were infected with Escherichia coli or Pasteurella pneumotropica , so as to observe the clinical symptoms of chickens and record the change in body weight. Anatomy was performed 14 d later, and the organ indices were determined, so as to study the anti-bacterial infection effect of A. blazei polysaccharide on chickens. The results showed that after bacterial infection, the high-dose A. blazei polysaccharide group was significantly differed from other groups in changes of body weight and organ indices. It indicates that oral administration of high concentration of A. blazei polysaccharide could promote the development of poultry organs, thereby improving the immunity of organisms.