【作者】 林威；

【导师】 蒋咏梅；

【作者基本信息】 福建师范大学 ， 微生物学， 2022， 硕士

【摘要】 灵芝是我国传统的药食两用真菌,具备抗肿瘤、抗炎、抗菌、抗衰老等功效。目前灵芝的培养方法主要有三种,分别是子实体栽培、液态培养与固态培养。与前两种培养方式相比较,灵芝固态发酵的相关研究较少,且目前多采用含有部分化学成分的培养基,不利于菌质的后期应用。本论文选用以麦麸为主料的天然培养基,以生物量、多糖和三萜三个指标对菌质发酵工艺进行响应面优化,并对三种不同菌质发酵工艺下灵芝菌质的活性成分及其合成关键酶基因转录水平、菌质的抗氧化特性进行了研究。结论如下:(1)固态培养基的选择:以麦麸为主料,玉米粉、籼米粉为碳源,花生粉、豆粉为氮源组合成9种固态培养基。以生物量为指标,在温度30℃、初始含水量60%、接种量10%、搅拌间隔120 h与培养天数10 d的培养条件下,进行固态培养基的选择与优化,得到最优固态培养基为麦麸+玉米粉+豆粉,质量比为2:1:1,菌丝生物量为199.04±9.58mg/g。(2)菌质发酵工艺的响应面优化:分别以菌丝生物量、菌质多糖和菌质三萜为指标,对灵芝菌质的培养条件进行响应面优化,结果如下:菌丝生物量的最佳工艺参数:初始含水量66%,培养温度26.5℃,接种量10.7%。经120 h搅拌间隔与10 d的培养时间,菌丝生物量0.260 g/g。菌质多糖的最佳工艺参数:初始含水量60%,培养温度29℃,接种量9.7%。120 h的搅拌间隔10 d的培养时间,菌质多糖最高达328.241 mg/g。菌质三萜的最佳工艺参数:初始含水量60%,培养温度30℃,接种量7.6%,经47 h的搅拌间隔与8 d培养时间,菌质三萜最高达35.717 mg/g。(3)三种菌质发酵工艺的比较:灵芝多糖合成途径中的三个关键酶基因为葡萄糖磷酸变位酶(PGM)、尿普二磷酸葡萄糖焦磷酸化酶(UGP)和葡聚糖合成酶(GLS)。灵芝三萜合成途径中的关键酶基因为3-羟基-3-甲基戊二酰Co A还原酶(HMGR)、鲨烯合酶(SQS)、鲨烯环氧酶(SE)与羊毛甾醇合酶(LS)为关键酶,这些关键酶基因的表达量与灵芝酸含量密切相关。多糖工艺下,PGM、UGP与GLS基因的相对表达量均在第6天时达到最大值,PGM基因的相对表达量是生物量和三萜工艺的1.53和1.36倍;UGP基因的相对表达量是生物量、三萜工艺的2.13和1.85倍;GLS基因的相对表达量是生物量、三萜工艺的1.83、1.31倍。三萜工艺中,HMGR、SQS基因的相对表达量在第8天时达到峰值,LS、SE基因的相对表达量在第6天时达到最大值。三萜工艺下的HMGR相对表达量整体水达到峰值时分别是生物量、多糖工艺的1.12、1.66倍。SQS基因相对表达量达到峰值时分别为生物量、多糖工艺的1.6、1.98倍。第6天时三种工艺下LS的相对表达量相差不大,在第8、10天时,三萜工艺中的LS表达量均要高于其他两种工艺。SE基因的相对表达量达到峰值时分别为生物量、多糖工艺的1.87和1.35倍。将三种菌质发酵工艺培养过程中的活性成分进行比较,培养10 d时,生物量工艺中菌丝生物量分别为多糖、三萜工艺的1.51、1.83倍;多糖工艺中菌质多糖含量分别为生物量、三萜工艺的1.49、1.28倍。培养8d时,三萜工艺中菌质三萜含量分别为生物量、多糖工艺的1.25、1.26倍。对于菌丝生物量、菌质多糖与菌质三萜都有一定需求的,则选择含量较为均衡的菌质多糖工艺进行培养。(4)三种菌质的抗氧化特性:对三种菌质的总还原能力、DPPH自由基清除能力与羟自由基清除能力进行测定,综合以上三种指标,发现生物量工艺在第8天、多糖工艺在第8天、三萜工艺在第10天时抗氧化特性最强。本论文针对灵芝的不同活性成分优化出三种菌质发酵工艺,在保证灵芝菌质中活性成分含量的前提下,在将来可以尝试对灵芝菌质进行食品化。更多还原

【Abstract】 Ganoderma lucidum is a traditional medicinal and edible fungus in China with anti-tumor,anti-inflammatory,anti-bacterial and anti-aging effects.At present,there are three main culture methods of Ganoderma lucidum,which are fruiting bodies cultivation,liquid culture and solid-state culture.Compared with the first two culture methods,there are fewer studies related to the solid-state fermentation of Ganoderma lucidum,and currently most of the media containing some chemical components are used,which is not conducive to the later application of the fungal mass.In this thesis,a natural medium with wheat bran as the main ingredient was selected for solid-state culture of Ganoderma lucidum,and the response surface optimization of the mycoplasmic fermentation process was carried out based on three indicators: biomass,polysaccharides and triterpenes.In addition,the transcription levels of the relevant enzyme genes in the active product synthesis pathway and the antioxidant properties of Ganoderma lucidum mycoplasm were also investigated.The results are as follows:(1)Selection of solid medium:Using wheat bran as main material,corn flour and indica rice flour as carbon source,peanut flour and soybean flour as nitrogen source,nine kinds of solid-state culture media were combined.Taking biomass as the index,under the culture conditions of temperature30 ℃,initial water content 60%,inoculation amount 10%,stirring interval 120 h and culture days 10 days,the selection and optimization of solid-state medium were carried out.The optimal solid-state medium was wheat bran + corn flour + soybean flour,and the mass ratio was 2:1:1.Then the mycelial biomass in the mycelium under the medium was199.04 ± 9.58 mg / g.(2)Response surface optimization of three fungal substance processes:Taking mycelial biomass,mycelial polysaccharide and mycelial triterpene as indicators,the culture conditions of Ganoderma lucidum mycelium were optimized by response surface analysis.The results of the optimal process parameters are as follows:The optimum technological parameters of mycelial biomass were as follows: the initial water content was 66%,the culture temperature was 26.5 ℃,and the inoculation amount was 10.7%.On the basis of this condition,after 120 h stirring interval and 10 d culture time,the mycelial biomass was as high as 0.2598 g / g.The optimum technological parameters of fungal substance polysaccharide after response surface analysis were as follows: the initial water content was 60%,the culture temperature was 29 ℃,and the inoculation amount was 9.7%.On the basis of this condition,after 120 h stirring and 10 d culture time,the maximum value of fungal substance polysaccharide can be as high as 328.241 mg / g.The optimum technological parameters of fungal substance triterpenoids after response surface analysis optimization method are as follows: initial water content 60%,culture temperature 30 ℃,inoculation amount 7.6%.After 47 h stirring interval and 8 d culture time,fungal substance triterpenoids can be obtained up to 35.717 mg / g.(3)Selection and comparison of three fungal substance processes:Under the polysaccharide process,the relative expression of PGM,UGP and GLS genes reached the maximum on the 6th day.The relative expression of PGM gene was1.53 and 1.36 times higher than that of biomass and triterpene process;The relative expression of UGP gene was 2.13 and 1.85 times higher than that of biomass and triterpene technology;The relative expression of GLS gene was 1.83 and 1.31 times higher than that of biomass and triterpene technology.In the triterpene process,the relative expression of HMGR and SQS genes reached the peak on the 8th day,and the relative expression of LS and Se genes reached the maximum on the 6th day.The relative expression of HMGR in triterpene process was 1.12 and 1.66 times higher than that in biomass and polysaccharide process,respectively.When the relative expression of SQS gene reached the peak,it was 1.6 and 1.98 times higher than that of biomass and polysaccharide technology,respectively.On the 6th day,the relative expression of LS in the three processes was similar.On the 8th and 10 th day,the expression of LS in triterpene process was higher than that in the other two processes.When the relative expression of Se gene reached the peak,it was 1.87 and 1.35 times higher than that of biomass and polysaccharide technology,respectively.The active components in the three kinds of mycelial processes were compared.When cultured for 10 days,the mycelial biomass in the biomass process was 1.51 and1.83 times higher than that in the polysaccharide and triterpene processes,respectively;The content of fungal substance polysaccharide in polysaccharide process was 1.49 and1.28 times higher than that in biomass and triterpene process,respectively.When cultured for 8 days,the content of fungal substance triterpene in triterpene process was 1.25 and1.26 times higher than that in biomass and polysaccharide process,respectively.In the three processes,if there is a certain demand for mycelial biomass,fungal substance polysaccharide and fungal substance triterpene,the fungal substance polysaccharide process with relatively balanced content is selected for culture.(4)Antioxidant properties of three fungal substance:The total reducing capacity,DPPH radical scavenging capacity and hydroxyl radical scavenging capacity of the three bacteria were measured.Based on the above three indexes,it was found that the antioxidant characteristics of biomass process were the strongest on the 8th day The antioxidant properties of polysaccharide process were the strongest on the 8th day The antioxidant properties of triterpene process were the strongest on the 10 th day.In this paper,three fungal substance quality processes are optimized for different active components of Ganoderma lucidum.On the premise of ensuring the content of active components in Ganoderma lucidum,we can try to food Ganoderma lucidum in the future.更多还原