【作者】 陈新华；

【导师】 邱冠周； 周洪波；

【作者基本信息】 中南大学 ， 生物工程， 2010， 博士

【摘要】 广东梅州商品红菇属于红菇目红菇科红菇属,是一种美味的野生菌根性食用菌。该菇味道香甜,口感脆滑,在广东梅州地区有多年食用的历史,市场交易规模较大。红菇属真菌形态变异很大,以至于该属的分类一直比较混乱,广东梅州商品红菇的分类长期存在着争议。本论文通过野外生态因子调查,子实体、菌根、孢子等形态显微观察,结合DNA中内部转录间隔区(ITS)及大亚基(LSU)序列分析,首次对广东梅州商品红菇进行形态学与分子学鉴定；进一步系统测定和分析了该商品红菇营养成分及功能成分；运用滤纸片扩散法评价了该红菇抗菌活性并通过GC-MS联用分析了该红菇挥发油及石油醚萃取物的化学成分；然后采用四种抗氧化测定方法评价了该红菇的抗氧化作用,检测了其抗氧化活性成分,研究了该商品红菇的抗氧化活性与黄酮和多酚含量的相关性,以及初步探讨其清除自由基的作用机理。主要研究内容和结果如下：1)广东梅州商品红菇形态显微观察通过野外生态调查和显微镜技术分别对广东梅州商品进行了生态学和组织学研究。结果表明,广东梅州商品是一种外生菌根菌,在相对稳定的生态环境条件下,出菇的最适气温是24-32℃,相对空气湿度达80%-100%,土壤含水量达到40%以上,湿度是主导因子。每年在夏末秋初高温阵雨之后为发生盛期,发生地林间郁闭度0.9左右,土壤pH值3-4,其共生树种为壳斗科植物。广东梅州商品红菇颜色以红色为主,囊状体特别发达,菌肉灰色,菌髓为异型菌髓,担孢子近球形,孢子大小为8.55-10.29×10.65-11.01μm,孢子表面有脊状突起,其高度约1.14μm,宽0.57μm,相邻脊间最宽距离2.14μm。确证该商品红菇菌丝细胞与细胞连接处没有锁状联合。2)广东梅州商品红菇分子鉴定实验采用核酸序列分析法,对采自广西浦北、福建上杭和广东梅州的8个商品红菇子实体的rDNA ITS(Internal Transcribed Spacer)以及28S rDNA区段进行克隆测序和序列特征比较分析,并对rDNA ITS序列进行核酸序列数据库GenBank同源性检索比对,将从GeneBank检索获得的相似物种rDNA ITS以及28S rDNA区段序列构建系统发育树。rDNAITS序列比较结果表明,8个红菇属菌株的rDNAITS序列长度为681bp-699bp, GC含量为48.56%-49.64%,子实体R1、R2、R3、R6和R8的rDNA ITS区段与GenBank中已知灰肉红菇(Russula griseocarnosa)的相似率达99%,其余几株与灰肉红菇的相似率低于95%。由此推断,从广西浦北和广东梅州隆文采集的商品红菇子实体是灰肉红菇,但在广东梅州、福建上杭产区可能存在多种食用红菇共同生长。对多种红菇属真菌的ITS序列和LSU序列系统发育关系的研究表明,8株不同产地(来源)的红菇聚在一起,表明它们之间有相对较密切的亲缘关系。从ITS序列构成的系统发育树看,R1、R2、R6、R7与R. griseocarnosa聚成一支,R3、R4、R5、R8与R. emetica聚在一起。从LSU序列构成的系统发育树看,8个红菇属菌株与Russula vinosa聚成一支。无论是ITS序列还是LSU序列构造的发育树均证明所测的各菌株之间大多存在一定的变异。3)灰肉红菇营养成分分析按照国家标准方法对采自广东梅州隆文灰肉红菇的一般营养成分进行分析,结果表明：菌盖和菌柄中粗蛋白,粗脂肪,粗纤维,灰分,还原糖,碳水化合物含量分别为32.31%,19.10%；7.17%,5.18%；8.85%,11.72%；7.84%,6.59%；2.11%,1.97%;49.10%,62.68%。采用氨基酸分析仪、原子吸收分光光度计、HPLC、GC/MS等方法对灰肉红菇的营养成分进一步分析结果表明：灰肉红菇子实体中含有磷、钾、铁、钠、钙、镁、锌等多种具有生理活性的矿物质元素。子实体菌盖和菌柄中K/Na比分别高达14.78和31.7。菌盖和菌柄中都含有7种脂肪酸,主要是油酸、亚油酸和棕榈酸,子实体菌盖中油酸含量最高,为47.92%；菌柄中亚油酸含量最高,为50.22%。全氨基酸分析表明含有17种氨基酸,菌盖和菌柄中氨基酸总量不同,分别占子实体干重的12.75%和8.98%。人体必需的7种氨基酸齐全(色氨酸未测),菌盖和菌柄中必需氨基酸分别占氨基酸总量的46.04%和52.18%。4)灰肉红菇抗菌活性利用滤纸片扩散法对灰肉红菇抗菌活性进行了研究并通过GC-MS联用分析了灰肉红菇挥发油及石油醚萃取物的化学成分。结果表明：灰肉红菇中挥发油对大肠杆菌、枯草芽孢杆菌、金黄色葡萄球菌、藤黄微球菌以及沙门氏菌均有明显的抑制作用。石油醚萃取物对金黄色葡萄球菌、藤黄微球菌有明显的抑制作用。子实体乙醇提取物总体抑菌作用不明显。灰肉红菇挥发油中的主要化学成分为酮、烷烃、醛、烯烃等化合物,而石油醚萃取物主要成分为酸、酯、烯烃、醇等化合物。5)灰肉红菇抗氧化活性的测定从还原能力、DPPH清除率等和对不同体系产生的活性氧自由基清除效果等方面对灰肉红菇提取物的抗氧化活性进行了试验研究和评价。结果表明灰肉红菇提取物具有较强的还原能力,对DPPH、Fenton反应产生的羟自由基的抑制作用或清除作用,以及通过螯合金属来抑制金属对脂质氧化的催化作用。通过抗氧化性与总酚和黄酮相关性分析表明：还原力与总酚含量相关(R2=0.7831),清除DPPH自由基与黄酮含量相关(R2=0.816),还原力与黄酮含量相关(R2=0.8527),可知黄酮的抗氧化能力主要体现在还原力上。6)抗氧化活性成分的检测采用依利特Hypersil BDS-C18色谱柱(200 mm×4.6 mm,5μm)),甲醇-0.1%磷酸溶液为流动相,梯度洗脱,流速为1.0 mL/min, SPM-M10AVP紫外检测器,25℃柱温,检测波长273 nm的高效液相色谱法对灰肉红菇提取物抗氧化活性成分—酚类物质进行定性定量检测。以5种不同黄酮类化合物和8种酚酸为对照品,检测出灰肉红菇中咖啡酸、槲皮素以及原儿茶酸3种抗氧化活性物质,其中以槲皮素的含量最高,达95.82μg/g。更多还原

【Abstract】 Belonging to the genus of Russula, Russulaceae family and Russulales, commercial Russula sp. produce in Guangdong, a precious indigenous mushroom, is an edible ectomycorrhizal fungi with unique natural zest and delicious taste and with high economical value. Because of long history of using this delicious food in the distribution areas, a large scale of consuming market is formed locally. The taxonomy of Russula however has been confusing because of the variable morphology. It is very important to clarify the taxonomy of the commercial Russula sp. The biological characters of the commercial Russula sp. were studied systematically through investigation of ecological factors in the field, microscopic observation of its shapes of fruiting bodies, ectomycorrhizae, spores and, and its DNA sequences of internal transcribed spacers (ITS) and large subunit (28 S) of ribosomal RNA genes were PCR amplified, sequenced and phylogenetically analysed along with their GenBank allies. Antimicrobial activity of the different extracts of R. griseocarnosa by the disc diffusion was assayed and the main antimicrobial components of the petroleum ether and essential oil of R. griseocarnosa were analyzed by the GC-MS. Furthermore, the four methods of reducing powers, chelating effect on ferrous ions, scavenging effect on hydroxyl free radicals, and 2,2-diphenly-l-picrylhydrazyl (DPPH) radical scavenging activity were used to assay antioxidant activities of the commercial Russula sp. and antioxidant components were determined. The correlation coefficients for the correlation between antioxidant activity and flavone and total phenolic contents of R. griseocarnosa extracts were also studied.The major research results are showed as follows:1) Morphological and structural characteristics of commercial Russula sp. produce in GuangdongBased on ecological observation of commercial Russula sp. produce in Guangdong, its ecological environment was investigated in order to simulate its ecological environment when they are cultivated. Morphological and structural characteristics of the fruit body, basidiospore and ectomycorrhizae were examined using light and scanning electron microscopy. The results showed that it is a kind of ecotomycorrhizal fungi, it emerged and developed in the relatively steady ecological environment where average temperature, humidity and soil water content were 24-32℃,80-100% and 40% respectively. Among the three environmental factors, soil water content was the leadig factor. The ommercial Russula sp. grows fast after shower in the summer and autumn when it was hot. They emerged in the forest stand in which crown density was 0.9 or so. Soil pH was 3-4, and Fagaceae species dominated. The pileus was dark red in colour and context was greyish in pileus and stipe. Cystidia were highly developed. Basidiospores were 8.55-10.29×10.65-11.01μm and had large conical spines 1.14μm high and 0.57μm wide; the widest space between two adjacent ridges was 2.14μm. The trama and context are all made up of both the mycelia and vesicular tissue. It was ascertained that there was no clamp between adjacent hypha cells of the fungal mycelium irrespective of whether the stipe, or context or trama.2) Molecular identification of commercial Russula sp. produce in GuangdongIn an attempt to elucidate the identities of 8 fruitbodies of edible Russula fungi in Guangxi Pubei, Fujian Shanghang, and Guangdong Meizhou, nucleotide sequences of ITS and 28S of ribosomal RNA genes were employed for the studies of these fungi. The phylogenetic trees were generated from ITS and 28S data sets of this study along with their GenBank alies using maximum likelihood and neighbour-joining analysis methods. rDNA ITS sequences of the eight strains ranged from 681 to 699 bp in length and between 48.56%and 49.64% in GC content. The rDNA ITS sequence similarities of four strains (R1, R2, R3, R6 and R8) and Russula griseocarnosa were up to 99% except for three (R4, R5 and R7) from Fujian Shanghang, and Guangdong Meizhou, and the sequence similarities between bodies (R4, R5 and R7) and species of Russula from GenBank database couldn’t identify to species level. The result demonstrated that edible Russula specimens obtained from Pubei county in Guangxi and Longwen town in Guangdong were Russula griseocarnosa, but up to two species of edible Russula occured potentially in Fujian Shanghang, and Guangdong Meizhou.Phylogenic relationships based on ITS and LSU sequences of species of Russula showed that Russula sp. from different areas were clustered together, indicated a closer consanguinity among them. Four ITS sequences (R1, R2, R6 and R7) were clustered in a group with R. griseocarnosa, and other four strains (R3, R4, R5 and R8) formed another cluster with R. emetica. LSU sequences of eight strains were located in the same branch. The phylogenic trees based on both ITS sequences and LSU sequences were shown that there were differences among all the fruitbodies of Russula sp. fom different areas.3) Analysis of nutritional components of R. griseocarnosa The general nutritional components of R. griseocarnosa were analysed according to National Standard analytic methods. The pileus and stipe of the mushroom contain 32.31% and 19.10% of crude protein,7.17% and 5.18% of crude fat,8.85% and 11.72% of crude fiber,7.84% and 6.59% of ash,2.11% and 1.97% of reducing sugars, 49.10% and 62.68% of carbohydrates respectively.Some nutritional components in R. griseocarnosa were studied by the means of amino acid analyer, atomic absorption spectrometer, HPLC, and GC/MS. The results revealed that and it can be regarded as good source of useful elements, such as P, K, Ca, Zn, Fe, Na, Mg. The content of potassium was especially high in comparison to sodium and K/Na ratio is 14.78 and 31.7 in pileus and stipe, respectively. There are seven kinds of fatty acid in pileus and stipe. Fatty acid compositions are mainly oleic acid, linoleic acid and palmitic. The content of oleic acid in pileus is high, which is 47.92% of the total fatty acid. Whereas the content of linoleic acid in stipe is high, which is 50.22% of the total fatty acid. The analysis of amino acids in R. griseocarnosa was made with reversed-phase high performance liquid chromatography. The result showed that seventeen kinds of amino acid (included seven kinds of essential amino acids) existed in R. griseocarnosa, except that tryptophane destroyed by hydrolysis is not checked out. The total content of amino acids in pileus and stipe is 12.75% and 8.98%, respectively. The percentage of essential amino acids was 46.04% and 52.18% in pileus and stipe, respectively.4) Antimicrobial activity of R. griseocarnosaAn experiment study on antimicrobial activity of the different extracts of R. griseocarnosa by the disc diffusion were reported and the main antimicrobial components of the petroleum ether and essential oil of R. griseocarnosa were analyzed by the GC-MS. The result showed the essential oil of R. griseocarnosa displayed significant antimicrobial activity to Escherichia coli, Bacillus subtilis, Staphalococeus aureus, Micrococcus luteus, salmonella sp. The petroleum ether of R. griseocarnosa inhibited Staphalococeus aureus and Micrococcus luteus. The ethanol extract of R. griseocarnosa almost did not show antimicrobial activity. The GC-MS analysis showed that the major antimicrobial components of essential oil were ketones, alkanes, aldehydes and alkenes, and petroleum ether extract were acid, ester, alkenes and alcohol.5) Determination of antioxidant activityThe total phenolic contents and flavonoid contents of the extracts were evaluated and the antioxidant activities of the extracts were assayed with antioxidant capacity in reducing powers, chelating effect on ferrous ions, scavenging effect on hydroxyl free radicals, and 2,2-diphenly-l-picrylhydrazyl (DPPH) radical scavenging method. The results showed that the methanol extract of R. griseocarnosa possessed strong reducing power, scavenging effect on hydroxyl free radicals, chelating effect on ferrous ions and DPPH radical scavenging activity in a concentration-dependent manner. There was a correlation coefficient for the correlation between reducing power and total phenolic contents of R. griseocarnosa extracts (R2=0.7831). Besides, flavone contents were also correlated well with reducing power (R2=0.8527) and scavenging capacity of DPPH (R2=0.816). Flanovoid and total phenolic can also be used as their antioxidant components and they can scavenge the radicals, have a better reducing power.6) Detection of antioxidant componentsA method for determination of 13 phenolic composition in R. griseocarnosa by reverse phase high performance liquid chromatography (RP-HPLC) was studied. Chromatographic separation was performed by Hypersil BDS-C18 column (4.6×200 mm,5μm) using the mobile phase gradient elution of methanol-0.1% phosphate mixture at 25℃, flow rate at 1.0 mL/min and detection wavelength at 273 nm. The results showed that three phenolic components were determined among them and quercetin (95.82μg/g) was the major component in R. griseocarnosa.更多还原