WO2022127943A1 - 一种高产多糖的灵芝少孢品种及其人工栽培方法 - Google Patents

一种高产多糖的灵芝少孢品种及其人工栽培方法 Download PDF

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WO2022127943A1
WO2022127943A1 PCT/CN2022/076139 CN2022076139W WO2022127943A1 WO 2022127943 A1 WO2022127943 A1 WO 2022127943A1 CN 2022076139 W CN2022076139 W CN 2022076139W WO 2022127943 A1 WO2022127943 A1 WO 2022127943A1
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ganoderma lucidum
mycelium
cultivation
culture
cultivate
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PCT/CN2022/076139
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French (fr)
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吴清平
唐健
寇秀颖
胡惠萍
梁晓薇
谢意珍
刘远超
蔡曼君
肖春
李向敏
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广东省科学院微生物研究所(广东省微生物分析检测中心)
无限极（中国）有限公司
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Publication of WO2022127943A1 publication Critical patent/WO2022127943A1/zh

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G18/00—Cultivation of mushrooms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi

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  • the invention belongs to the technical field of agricultural microorganisms, and in particular relates to a high-yielding polysaccharide-few spore variety of Ganoderma lucidum and an artificial cultivation method thereof.
  • Ganoderma lucidum polysaccharides have immune regulation, anti-tumor, antioxidant, anti-inflammatory, antibacterial, hypoglycemic and anti-diabetic and its complications, and liver protection effects (Zhao Xiaoping). .
  • the medicinal materials, medicines and health care products with Ganoderma lucidum polysaccharide as the main component have broad market space and huge commercial value, and related enterprises are gradually focusing on the target strains with high polysaccharide production to improve the quality of their products.
  • the polysaccharide content of Ganoderma lucidum varieties in the market is generally 0.9-1.5%, and a small part of Ganoderma lucidum cultivated by basswood can reach 1.8-2.5%.
  • the damage caused by basswood cultivation to the environment is very large, which is not conducive to the sustainable development of cultivation and production.
  • Substitute cultivation is the inevitable trend of large-scale production of edible fungi.
  • Ganoderma lucidum varieties face the risk of degradation after 1-2 years of use, which is not only reflected in the decline in yield, but more importantly, the main components representing their utility, such as Ganoderma lucidum polysaccharides, Ganoderma lucidum triterpenes, etc. have dropped significantly.
  • the selection and breeding of Ganoderma lucidum varieties with high and stable yield and suitable for factory-based substitute material cultivation is very important and urgent.
  • the first object of the present invention is to provide a new variety of Ganoderma lucidum with high polysaccharide yield and less sporulation - Ganoderma lucidum M624.
  • GDMCC Guangdong Microbial Culture Collection Center
  • the second object of the present invention is to provide a kind of artificial cultivation method of the above-mentioned Ganoderma lucidum M624, including the mycelium culture of Ganoderma lucidum M624, the management of lucid lucidum, and the harvesting of fruit bodies.
  • Mycelium culture Ganoderma lucidum M624 bacterial mass is inoculated on the mother seed medium, until the mycelium is covered with slanted surface, the mother seed mycelium is transferred to the production seed medium for expanded culture, and cultured until the mycelium is overgrown bag, take the production seed mycelium and inoculate it on the cultivation material, and after the mycelium is full, continue to cultivate for post-maturity cultivation;
  • Ganoderma lucidum management After the post-ripening period of the mycelium is over, the buds are stimulated. When the caps start to differentiate, the fresh air is changed. When the caps of the fruiting bodies stop growing flat, the fruiting bodies of Ganoderma lucidum are harvested.
  • Described mycelium culture is specifically preferably:
  • the mother seed culture medium is: in terms of mass fraction, it includes 20% of potato, 2% of glucose, 1% of peptone, 2% of agar, 0.3% of potassium dihydrogen phosphate, 0.15% of magnesium sulfate, 0.001% of vitamin B1, and the balance for water;
  • the production seed medium is: in terms of mass fraction, it comprises 98-99% sorghum, 1-2% calcium carbonate, and has a natural pH;
  • the cultivating material is: in terms of mass fraction, it includes 50% sawdust, 38% cottonseed husk, 10% wheat bran, 2% calcium carbonate, 60-65% water content, and natural pH.
  • the described management and harvesting fruit bodies are:
  • the buds are stimulated, the lid of the cultivation bag is opened, and the cultivation bag is placed in a vertical row at 25-28°C, the relative air humidity is kept at 75%-90%, and the scattered light is illuminated for 10h/d, and the sesame seeds are released.
  • the primordium begins to form at the opening of the cultivation material, and the humidity is adjusted to 80-90%.
  • the fungus cap begins to differentiate, the fresh air is changed twice a day for 1 hour each time.
  • the fruiting body fungus cap stops growing flat, Harvest fruit bodies.
  • the present invention comprehensively evaluates the agronomic characters and polysaccharide content of multiple wild Ganoderma lucidum strains, and obtains a Ganoderma lucidum oligospore variety-Ganoderma lucidum M624 with high polysaccharide yield. It is higher than the 0.9% required by the pharmacopoeia, and the crude polysaccharide content of Ganoderma lucidum in the industry is generally 0.9 to 1.5%; and the patented Ganoderma lucidum M624 has a low sporulation yield, which can reduce the impact on the cultivation environment and machinery, and meet the requirements of harvesting the fruiting body. Targeted Ganoderma lucidum cultivation.
  • the method can not only increase the crude polysaccharide content of Ganoderma lucidum raw materials at the source, enrich the varieties of Ganoderma lucidum currently on the market, but also be suitable for industrial scale production, avoid the impact of a large amount of spore powder on the environment and machines during the spraying period of Ganoderma lucidum, and meet the requirements of the Ganoderma lucidum industry. development needs.
  • the invention improves the quality of Ganoderma lucidum products at the level of bacterial species, and is beneficial to the processing and application of Ganoderma lucidum polysaccharides.
  • GDMCCNo Guangdong Provincial Microbial Culture Collection Center
  • Fig. 1 is the fruiting body growth diagram of Ganoderma lucidum M624;
  • Fig. 2 is ML method to construct phylogenetic tree
  • Figure 3 is a phylogenetic tree constructed by NJ method
  • Fig. 4 is the domestication and cultivation yield situation of Ganoderma lucidum, wherein, M624 is wild Ganoderma lucidum strain; MC-GL-0046, MC-GL-0054 are market species;
  • Fig. 5 is different concentration glucose standard standard curve figure
  • Figure 6 is the crude polysaccharide content of artificially cultivated fruit bodies of wild Ganoderma lucidum strains, among which, A532, Z381, M311, Z112, W201, M624 are wild Ganoderma lucidum strains; MC-GL-0046, MC-GL-0054 are market species; the differences are significant sexual analysis ** is P>0.01, * is P>0.05;
  • Figure 7 shows the artificial cultivation of Ganoderma lucidum M624, MC-GL-0046 and MC-GL-0054.
  • Example 1 Acquisition of Ganoderma lucidum cultivar with high polysaccharide and few spores-Ganoderma lucidum M624
  • Ganoderma lucidum is mainly distributed in Guangdong, Hunan, Anhui, Henan and other places.
  • the strain HMGIM-M624 mycelium was collected, dried at low temperature, ground at room temperature, and the DNA genome was extracted by using the Ezup column fungal genome DNA extraction kit (Sangon Bioengineering (Shanghai) Co., Ltd.), and the obtained DNA solution (DNA template) refrigerated at -20°C for later use.
  • the ITS-PCR experiment was carried out by using the universal primers ITS1/ITS4 (ITS1: TCCGTAGGTGAACCTGCGG, ITS4: TCCTCCGCTTATTGAT ATGC, synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.) of the fungal ribosomal intergenic region, and the amplification was carried out on a Biometra PCR machine.
  • the composition of the PCR reaction solution (50 ⁇ l in total) is shown in Table 1, and the reaction program is shown in Table 2:
  • the PCR products were directly sent for inspection for bidirectional sequencing, which was completed by BGI.
  • HMGIM-M624 and Ganoderma lingzhi are both in the same branch, clustered into one class, and the relationship is close. It can be determined that HMGIM-M624 is a Ganoderma lingzhi strain, named Ganoderma lingzhi (Ganoderma lingzhi). Ganoderma lingzhi) M624.
  • the Ganoderma lucidum recorded in the Chinese Pharmacopoeia is the dried fruiting body of the Polyporaceae fungus G. lucidum or G. sinense.
  • the research by Professor Dai Yucheng, a Chinese taxonomy expert in 2013, showed that my country is widely distributed and Cultivated Ganoderma lucidum is different from G. lucidum produced in Europe, and Wasser et al. (2006) and Wasser (2011) also pointed out that the name G. lucidum was erroneously used for the medicinal fungus Ganoderma lucidum.
  • Chinese Ganoderma lucidum (commonly known as Ganoderma lucidum) is a new species: Ganoderma lingzhi S.H.Wu, Y.Cao & Y.C.Dai (Cao et al.2012), so the Latin name of Ganoderma lucidum recorded in the Chinese Pharmacopoeia should be G.lingzhi, not G .lucidum, the bacterial strain protected by this patent is Chinese native Ganoderma lucidum G.lingzhi wild strain, but because the current pharmacopoeia is still using G.lucidum as the Latin name of Ganoderma lucidum, the strain preservation species name protected by this patent is tentatively designated as Ganoderma lucidum (Ganoderma lucidum).
  • GDMCC Guangdong Provincial Microbial Culture Collection Center
  • Example 2 Domestication and cultivation of Ganoderma lucidum M624 and evaluation of crude polysaccharide content in fruiting bodies
  • Mother seed medium (enriched with comprehensive PDA): by mass fraction, including potato 20% + glucose 2% + peptone 1% + agar 2% + potassium dihydrogen phosphate 0.3% + magnesium sulfate 0.15% + vitamin B1 0.001 %, the balance is water, and its preparation method is: first wash and peel the potatoes, then weigh 200g of potatoes and cut them into small pieces, add water and boil them (boil for 20 to 30 minutes, and they can be punctured by a glass rod), Filter with eight layers of gauze, add 20g of glucose, 10g of peptone, 20g of agar, 3g of potassium dihydrogen phosphate, 1.5g of magnesium sulfate, and 0.01g of vitamin B1 to the filtrate. Sterilize with high temperature, high pressure and moist heat at 121°C for 30 minutes, and place it on an inclined surface to cool and shape.
  • Production seed medium by mass fraction, including 99% sorghum + 1% calcium carbonate, with natural pH. Weigh the sorghum in the required proportion, soak it overnight and cook it until it is through, take out and filter to discard the excess water, add calcium carbonate in proportion and mix well, put it into a 13cm ⁇ 25cm high temperature-resistant transparent polypropylene bag, equivalent to each bag Dry material 200-250g. 0.11MPa, 121°C high temperature and high pressure moist heat sterilization for 30min.
  • Cultivation materials by mass fraction, including 50% sawdust, 38% cottonseed hulls, 10% wheat bran, 2% calcium carbonate, natural pH. Take the above formula and mix thoroughly, add water to a moisture content of 60-65%, put into a 17cm ⁇ 35cm high temperature-resistant transparent polypropylene seed bag, equivalent to 400-420g of dry material per bag. After filling the material, use a punching rod to make a hole in the bag material, the hole is deep to the bottom of the bag, then put a plastic ring on the mouth of the bag, and fasten the matching cover, that is, a prepared original bag material. Sterilize at 0.147MPa atmospheric pressure and 128°C high temperature, high pressure and moist heat for 90min.
  • Mycelial culture take 0.5cm ⁇ 0.5cm of Ganoderma lucidum M624 preserved seed bacteria and inoculate it into the mother seed medium (enriched with comprehensive PDA) under aseptic operation conditions, with a relative humidity of 50%-60%, and cultivated in the dark at a constant temperature of 25°C 7-10 days until the mycelium is covered with slants.
  • the mycelium of the parent seed is transferred to the production seed medium for expanded cultivation, the relative humidity is 50%-60%, and the dark culture at a constant temperature of 25 DEG C is carried out until the mycelium is full of bags to obtain the production seed.
  • Mushroom management After the post-mature period of the mycelium is over, the buds are stimulated, the lid of the cultivation bag is opened, and the cultivation bag is placed in a vertical row (15cm apart), 25-28°C, and the relative air humidity is kept at 75% Under the condition of ⁇ 90%, scattered light illumination for 10h/d, after 8-10 days of turmeric management, the primordium began to form at the opening of the cultivation material, and the relative humidity was adjusted to 80-95%. times, 1h each time.
  • the average bag yield of artificially cultivated Ganoderma lucidum M624 was 34.91 ⁇ 1.15 g (Fig. 4), and the cap diameter was 10.73 ⁇ 0.34 cm, which was comparable to the yield of control strains MC-GL-0054 and MC-GL-0046. It has obvious advantages, and the yield of Ganoderma M624 can be improved by formulating suitable cultivation formulas. As shown in Figure 7, the spore production of Ganoderma lucidum M624 cultivated with the substitute material was small, and the data analysis could not be carried out, so there was no spore data.
  • the spore projectile ability is weak, and only a small number of spores are ejected when mature, but the lack of its spore formation mechanism will not affect the polysaccharide. Synthesis of metabolites.
  • the fruiting body of Ganoderma lucidum has a thicker cap, and more active ingredients will be accumulated during the growth and development process, and its biological activity is also stronger, which meets the product demand of Ganoderma lucidum market with fruiting bodies as raw materials.
  • the polysaccharide content of the fruiting bodies of the above-mentioned 8 strains of Ganoderma lucidum was measured, according to the "sulfuric acid-anthrone colorimetric method" in "Chinese Pharmacopoeia” Ganoderma lucidum, the method is as follows:
  • the results of the evaluation of the polysaccharide content of the artificially cultivated fruit bodies of wild Ganoderma lucidum strains are shown in Figure 6.
  • the crude polysaccharide content is ranked in order: Ganoderma lucidum M624>HMGIM-M311>MC-GL-0054>HMGIM-W201>HMGIM-A532>MC-GL-0046>HMGIM -Z381>HMGIM-Z112;
  • the polysaccharide content of Ganoderma lucidum M624 was 2.40 ⁇ 0.09%, which was significantly higher than that of other wild strains, and more than 1.5 times that of the control group MC-GL-0046 and MC-GL-0054.
  • the crude polysaccharide content of Ganoderma lucidum in the industry is generally 0.9 to 1.5%.
  • Ganoderma lucidum M624 can be determined as a high-quality Ganoderma lucidum strain with high polysaccharide content and low sporulation.

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Abstract

公开了一种高产多糖的灵芝少孢品种及其人工栽培方法。灵芝（ganoderma lucidum）M624，其保藏编号为：GDMCC No：60889。人工栽培方法，包括灵芝M624的菌丝培养、出芝管理和采收子实体。该灵芝少孢品种-灵芝M624，代料栽培中，其子实体中粗多糖含量高达2.40±0.09％；而且灵芝M624产孢量低，能减少对栽培环境和机器的影响，满足以子实体为采收目标的灵芝栽培。

Description

一种高产多糖的灵芝少孢品种及其人工栽培方法 技术领域：
本发明属于农业微生物技术领域，具体涉及一种高产多糖的灵芝少孢品种及其人工栽培方法。
背景技术：
灵芝的功效及其相关产品在国内外均倍受认可，早在2010年被列入美国药典，目前其栽培及加工产业(包括保健食品、护肤品等)年产值达1000亿元以上。我国认定的灵芝品种有沪农1号等19个，其中1个是国外引进，3个是通过原生质体融合获得的。但根据广东省微生物研究所食用菌研究发展中心科研人员2016年的菌种调查，全国的灵芝主产区正在使用的主产菌种：韩芝7个，美国灵芝2个，龙芝2个，沪农灵芝2个，日本灵芝1个，不明确品种1个。虽然本土选育的沪农、龙芝等系列品种有一定的推广，但是我国拥有自主知识产权的灵芝品种数量和普及度远不如引进种，如适合工厂化栽培、高产多糖或高产三萜等品种仍然是非常缺乏，因此灵芝菌种的选育是目前灵芝产业发展的卡脖子问题。随着灵芝研究的深入，其多糖药理活性得到被证实，研究表明灵芝多糖具有免疫调节、抗肿瘤、抗氧化、抗炎、抗菌、降血糖和抗糖尿病及其并发症、保护肝脏等作用(赵小平.药用真菌灵芝研究与栽培现状.中国食用菌,2019,38(7):1-5)。以灵芝多糖为主要成分的药材、药物和保健品具有广阔的市场空间和巨大的商业价值，相关企业也逐步将重心投放在高产多糖的目标菌株以提高自身产品的质量。据文献报道，市场灵芝品种多糖含量一般为0.9-1.5％，椴木栽培的灵芝少部分可达1.8-2.5％，但是椴木栽培对环境造成的破坏非常大，不利于栽培生产的可持续发展，代料栽培是食用菌规模化生产的必然趋势。而且据国内灵芝栽培企业反映，灵芝品种在使用1-2 年后就面临退化风险，不仅表现在产量下降，更重要的是代表其效用的主要成分，如灵芝多糖、灵芝三萜等大幅下降，严重影响产品质量，由此可见高产稳产且适应工厂化代料栽培的灵芝品种选育工作是非常重要和迫切的。
发明内容：
本发明的第一个目的是提供一种高产多糖、产孢量少的灵芝新品种-灵芝(ganoderma lucidum)M624。
本发明的灵芝(ganoderma lucidum)M624，其于2019年11月7日保藏于广东省微生物菌种保藏中心(GDMCC)，地址：广州市先烈中路100号大院59号楼5楼，其保藏编号为：GDMCCNo：60889。
本发明的第二个目的是提供一种上述灵芝M624的人工栽培方法，包括灵芝M624的菌丝培养、出芝管理和采收子实体。
具体优选为：
a、菌丝培养：灵芝M624菌块接种于母种培养基上，直至菌丝体长满斜面，将母种菌丝体转接至生产种培养基上扩大培养，培养至菌丝体长满袋，取生产种菌丝体接种于栽培料，至菌丝长满带后，继续培养进行后熟期培养；
b、出芝管理：菌丝后熟期满后进行催蕾，待菌盖开始分化时，换新风，当子实体菌盖停止平展生长时，采收灵芝子实体。
所述的菌丝培养具体优选为：
在无菌操作条件下取灵芝M624接种于母种培养基，相对湿度50％-60％，25℃恒温黑暗培养7-10天至菌丝体长满斜面，将母种菌丝体转接至生产种培养基上扩大培养，25℃恒温黑 暗培养至菌丝体长满袋，取生产种菌丝体接种于栽培料，25℃恒温黑暗培养，相对湿度50％-60％，定时换气以保持二氧化碳浓度4000ppm以下，培养20-25天至菌丝长满带后，同条件培养23-25天进行后熟期培养；
所述的母种培养基为：按质量分数计，包括马铃薯20％、葡萄糖2％、蛋白胨1％、琼脂2％、磷酸二氢钾0.3％、硫酸镁0.15％、维生素B1 0.001％、余量为水；
所述的生产种培养基为：按质量分数计，包括98-99％高粱、1-2％碳酸钙，pH自然；
所述的栽培料为：按质量分数计，包括50％杂木屑，38％棉籽壳，10％麦麸，2％碳酸钙，含水量60-65％，pH自然。
所述的出芝管理和采收子实体为：
菌丝后熟期满后进行催蕾，打开栽培袋盖子，把栽培袋竖排放置，25～28℃，空气相对湿度保持在75％～90％条件下，散射光光照10h/d，出芝管理8-10天后在栽培料开口位置开始形成原基，调整湿度为80～90％，待菌盖开始分化时，每天换新风2次，每次1h，当子实体菌盖停止平展生长时，采收子实体。
本发明综合评价多株野生灵芝菌株的农艺性状和多糖含量，筛选获得一种高产多糖的灵芝少孢品种-灵芝M624，代料栽培中，其子实体中粗多糖含量达2.40±0.09％，远高于药典要求的0.9％，行业中代栽灵芝粗多糖含量一般为0.9～1.5％；而且本专利灵芝M624产孢量低，能减少对栽培环境和机器的影响，满足以子实体为采收目标的灵芝栽培。本发明既能在源头处提高灵芝原材料粗多糖含量，丰富目前市场灵芝品种，也适合工厂化规模化生产，避免灵芝喷孢时期产生大量的孢子粉对环境和机器带来的影响，满足灵芝产业化发展需求。本发明在菌种的层面提高灵芝产品的品质，有利于灵芝多糖的加工应用。
Ganoderma lucidum M624于2019年11月7日保藏于广东省微生物菌种保藏中心(GDMCC)，地址：广州市先烈中路100号大院59号楼5楼，其保藏编号为：GDMCCNo：60889。
附图说明：
图1是灵芝M624的子实体生长图；
图2是ML法构建系统发育树；
图3是NJ法构建系统发育树；
图4是灵芝驯化栽培产量情况，其中，M624为野生灵芝菌株；MC-GL-0046、MC-GL-0054为市场种；
图5是不同浓度葡萄糖标准品标准曲线图；
图6是野生灵芝菌株人工栽培子实体粗多糖含量情况，其中，A532、Z381、M311、Z112、W201、M624为野生灵芝菌株；MC-GL-0046、MC-GL-0054为市场种；差异显著性分析**为P﹥0.01，*为P﹥0.05；
图7是灵芝M624、MC-GL-0046、MC-GL-0054的人工栽培情况。
具体实施方式
以下将参照附图，结合具体实施例对本发明进行进一步解释。但实施例本身对本发明不做任何形式的限定。
实施例1：高产多糖的灵芝少孢品种-灵芝(ganoderma lucidum)M624的获取
根据广东省微生物研究所食用菌研究发展中心科研人员长期调研野生大型真菌资源情况，灵芝主要分布广东、湖南、安徽、河南等地。广东省微生物研究所食用菌研究发展中心科研 人员从安徽省六安市，纬度31°33′，经度116°09′，海拔70m，散生，林相为阔叶林，腐殖土基质采集到野生菌株HMGIM-M624子实体(即灵芝(ganoderma lucidum)M624)，子实体具侧生柄，菌盖平展盖型，直径约为2.8cm，菌柄长度约为6.5cm；菌盖大部分为黄褐色，边缘为浅黄色且钝，孔口表面白色，菌肉浅褐色(图1)。
1、组织分离
将野外采集获得的菌株HMGIM-M624子实体用75％酒精擦拭表面，在无菌环境下撕开并夹取0.2-0.5mm×0.2-0.5mm的内部菌肉组织接入加富综合PDA培养基。置于25℃培养箱中恒温暗培养，待菌丝长满斜面后取尖端菌丝转接纯化培养。
2、分子鉴定
ITS鉴定方法
收集菌株HMGIM-M624菌丝，低温烘干，常温研磨，利用Ezup柱式真菌基因组DNA抽提试剂盒(生工生物工程(上海)股份有限公司)进行DNA基因组的提取，得到的DNA溶液(DNA模板)冷藏于-20℃备用。通过真菌核糖体基因间隔区通用引物ITS1/ITS4(ITS1:TCCGTAGGTGAACCTGCGG，ITS4:TCCTCCGCTTATTGAT ATGC，由生工生物工程(上海)股份有限公司合成)进行ITS-PCR实验，扩增在Biometra PCR仪上进行，PCR反应液组成(共50μl)如表1，反应程序如表2：
表1 真菌ITS PCR体系
Figure PCTCN2022076139-appb-000001
Figure PCTCN2022076139-appb-000002
表2 真菌ITS PCR扩增程序
Figure PCTCN2022076139-appb-000003
PCR产物直接送检进行双向测序，由华大基因完成。
2.1分类学地位确定
将测序结果(核苷酸序列如SEQ ID NO.1所示)在NCBI GenBank进行序列比对，ITS鉴定结果表示该菌株为灵芝(Ganoderma lingzhi)菌株。
为进一步确认其品种，应用最大似然法(ML)和邻接法(NJ)构建系统发育树，以云芝(Trametes versicolor)作为外群，分别进行了系统发育树的构建(见图2～3)。在两个算法建成的系统发育树中，HMGIM-M624与Ganoderma lingzhi均处于同一分枝内，聚为一类，亲缘关系接近，可以确定HMGIM-M624为灵芝(Ganoderma lingzhi)菌株，命名为灵芝(Ganoderma lingzhi)M624。我国药典记载的灵芝为多孔菌科真菌赤芝G.lucidum或紫芝G.sinense的干燥子实体，但随着系统发育研究的进展，2013年我国分类学专家戴玉成教授的研究表明，我国广泛分布和栽培的灵芝与产于欧洲的G.lucidum不同，Wasser et al.(2006)和Wasser(2011)也指出G.lucidum这一名称被错误地使用到药用真菌灵芝中。实际上中国灵芝(俗称赤芝) 为一个新种：Ganoderma lingzhi S.H.Wu,Y.Cao&Y.C.Dai(Cao et al.2012)，因此中国药典记载的赤芝拉丁名应为G.lingzhi，而非G.lucidum，本专利保护的菌株是中国本土的灵芝G.lingzhi野生菌株，但是由于目前药典仍在使用G.lucidum作为灵芝的拉丁名，本专利保护的菌株保藏物种名暂定为灵芝(Ganoderma lucidum)M624，待药典修改后更正。因此将菌株HMGIM-M624命名为灵芝(Ganoderma lucidum)M624，其于2019年11月7日保藏于广东省微生物菌种保藏中心(GDMCC)，地址：广州市先烈中路100号大院59号楼5楼，其保藏编号为：GDMCCNo：60889。
实施例2：灵芝M624的驯化栽培及子实体粗多糖含量评价
一、野生灵芝菌株驯化栽培研究
(一)、培养基配方及配置
1.母种培养基(加富综合PDA)：按质量分数计，包括马铃薯20％+葡萄糖2％+蛋白胨1％+琼脂2％+磷酸二氢钾0.3％+硫酸镁0.15％+维生素B1 0.001％，余量为水，其配制方法是：先将马铃薯洗净去皮，再称取200g马铃薯切成小块，加水煮烂(煮沸20～30分钟，能被玻璃棒戳破即可)，用八层纱布过滤，滤液中加入葡萄糖20g、蛋白胨10g、琼脂20g、磷酸二氢钾3g、硫酸镁1.5g、维生素B1 0.01g，搅拌均匀，稍冷却后再补足水分至1000毫升，0.11MPa、121℃高温高压湿热灭菌30min，置于斜面上冷却定型。
2.生产种培养基：按质量分数计，包括99％高粱+1％碳酸钙，pH自然。称取所需比例的高粱，浸泡过夜后煮熟至透心，取出过滤弃去多余水分，按比例加入碳酸钙拌匀，装入13cm×25cm耐高温的透明聚丙烯袋中，折合每袋装干料200-250g。0.11MPa、121℃高温高压 湿热灭菌30min。
3.栽培料：按质量分数计，包括50％杂木屑，38％棉籽壳，10％麦麸，2％碳酸钙，pH自然。取上述配方充分混合并加水至含水量为60-65％，装入17cm×35cm耐高温的透明聚丙烯菌种袋，折合每袋装干料400-420g。装好料后用打孔棒在袋料中打洞，洞深至袋底，然后在袋口套上塑料环，扣上配套的盖子，即得一个制好的原种袋料。在0.147MPa大气压、128℃高温高压湿热灭菌90min。
(二)、栽培技术参数
1.菌丝培养：在无菌操作条件下取0.5cm×0.5cm灵芝M624保藏种菌块接种于母种培养基(加富综合PDA)，相对湿度50％-60％，25℃恒温黑暗培养7-10天至菌丝体长满斜面。将母种菌丝体转接至生产种培养基扩大培养，相对湿度50％-60％，25℃恒温黑暗培养至菌丝体长满袋，得到生产种。取灵芝M624生产种菌丝体接种于栽培料中，25℃恒温黑暗培养，相对湿度50％-60％，定时换气以保持二氧化碳浓度4000ppm以下，培养20-25天至菌丝长满带后，同条件培养23-25天进行后熟期培养。
2.出芝管理：菌丝后熟期满后进行催蕾，打开栽培袋盖子，把栽培袋竖排放置(袋与袋之间相隔15cm)，25～28℃，空气相对湿度保持在75％～90％条件下，散射光光照10h/d，出芝管理8-10天后在栽培料开口位置开始形成原基，调整相对湿度为80～95％，待菌盖开始分化时，每天换新风2次，每次1h。
3.采收：原基形成后35-40天灵芝子实体成熟，采收第一潮菇。
按照上述相同的方法对6株野生灵芝菌株(HMGIM-A532、HMGIM-Z381、HMGIM-M311、HMGIM-Z112、HMGIM-W201、HMGIM-M624，图表中简写为A532、Z381、M311、Z112、 W201、M624)进行驯化栽培子实体，并且，选用了两株市场主栽的灵芝品种作为对照，其中MC-GL-0046为国外引进种韩芝二号，产孢量少；MC-GL-0054为本土筛选的优质灵芝品种沪农一号，抗逆性强、产孢量大；两株对照菌株在安徽、浙江、福建、上海等多地推广栽培多年，是目前栽培较稳定且技术成熟的灵芝菌株。
人工栽培的灵芝M624平均袋产量为34.91±1.15g(图4)，菌盖直径10.73±0.34cm，与对照菌株MC-GL-0054和MC-GL-0046产量相当，在本栽培方法下产量没有明显优势，可通过后续制定适合的栽培配方提高灵芝M624产量。如图7所示，代料栽培的灵芝M624产孢量少，无法进行数据分析，故没有孢子数据。少孢灵芝由于菌管内有一层类胶质物覆盖于菌管的网状结构上，造成孢子弹射能力弱，在成熟时仅弹射少量孢子，但其孢子形成机制的缺失并不会影响多糖等次生代谢产物的合成。与产孢灵芝相比，少孢灵芝子实体菌盖厚实，生长发育过程中会积累更多活性成分，其生物活性也更强，满足灵芝市场以子实体为原材料的产品需求。
4、灵芝栽培子实体多糖含量测定
对上述8株灵芝子实体进行多糖含量测定，按照《中国药典》灵芝中“硫酸-蒽酮比色法”，方法如下：
a)配制硫酸蒽酮溶液：精密称取蒽酮0.1g，加入硫酸100ml使溶解，摇匀备用。配制对照品溶液：精密称取葡萄糖标准品3mg，定容至25ml，浓度为0.12mg/ml。
b)制备标准曲线：精密称量对照品溶液0.2、0.4、0.6、0.8、1.0、1.2ml，分别置于10ml具塞试管中，各加水至2.0ml，迅速精密加入硫酸蒽酮溶液6ml，立即摇匀，静置15分钟，立即置于冰浴中冷却15分钟，取出，以相应的试剂为空白，照紫外-可见分光光度法，在625nm 波长处测定吸光度，以吸光度为纵坐标，浓度为横坐标，绘制标准曲线(图5)。
c)样品测定：灵芝子实体样品粉碎，精密称取2.00g样品加入60ml纯水，加热回流4h，趁热滤过，用少量热水洗涤滤器和滤渣，将了滤渣及滤纸置烧瓶中，加水60ml，加热回流3h，趁热过滤，合并滤液，旋蒸蒸干水分，残渣用水5ml溶解，边搅拌边缓慢滴加乙醇75ml，摇匀，在4℃放置12h，离心，弃上清，沉淀物用热水溶解并转移至50ml容量瓶中。取溶液适量，8000rpm离心，精密量取上清液3ml，定容至25ml。精密量取供试品溶液2ml，置10ml具塞试管中，迅速精密加入硫酸蒽酮溶液6ml，立即摇匀，静置15分钟，立即置于冰浴中冷却15分钟，取出，以相应的试剂为空白，照紫外-可见分光光度法，在625nm波长处测定吸光度，3个重复样，根据标准曲线计算多糖含量含量。
野生灵芝菌株人工栽培子实体多糖含量评价实验结果见图6，粗多糖含量高低排序：灵芝M624>HMGIM-M311>MC-GL-0054>HMGIM-W201>HMGIM-A532>MC-GL-0046>HMGIM-Z381>HMGIM-Z112；其中，灵芝M624多糖含量为2.40±0.09％，极显著高于其他野生菌株，比对照组MC-GL-0046和MC-GL-0054这两株市场种1.5倍以上。且远高于药典要求的0.9％，行业中代栽灵芝粗多糖含量一般为0.9～1.5％。根据多株野生灵芝菌株农艺性状和多糖含量，可确定灵芝M624为多糖含量高、产孢量少的优质灵芝菌株。
综上所述，本研究团队通过野外采集、纯化保藏、分子鉴定等系列工作建立野生灵芝种质库，从中筛选6株活力强的菌株进行驯化栽培和多糖含量评价，最终获得一种高产多糖的灵芝少孢品种-灵芝M624，常规代料栽培条件下其子实体多糖合成和积累优胜于市场主流灵芝品种等菌株，而且具有产孢量少、性状稳定遗传的特点，本专利菌株的获得将为灵芝多糖相关产品的开发提供了新的种质。
Figure PCTCN2022076139-appb-000004

Claims (5)

  1. 灵芝(ganoderma lucidum)M624，其保藏编号为：GDMCC No：60889。
  2. 一种权利要求1所述的灵芝M624的人工栽培方法，其特征在于，包括灵芝M624的菌丝培养、出芝管理和采收子实体。
  3. 根据权利要求2所述的人工栽培方法，其特征在于，具体为：
    a、菌丝培养：灵芝M624菌块接种于母种培养基上，直至菌丝体长满斜面，将母种菌丝体转接至生产种培养基上扩大培养，培养至菌丝体长满袋，取生产种菌丝体接种于栽培料，至菌丝长满带后，继续培养进行后熟期培养；
    b、出芝管理：菌丝后熟期满后进行催蕾，待菌盖开始分化时，换新风，当子实体菌盖停止平展生长时，采收灵芝子实体。
  4. 根据权利要求3所述的人工栽培方法，其特征在于，所述的菌丝培养具体为：
    在无菌操作条件下取灵芝M624接种于母种培养基，相对湿度50％-60％，25℃恒温黑暗培养7-10天至菌丝体长满斜面，将母种菌丝体转接至生产种培养基上扩大培养，25℃恒温黑暗培养至菌丝体长满袋，取生产种菌丝体接种于栽培料，25℃恒温黑暗培养，相对湿度50％-60％，定时换气以保持二氧化碳浓度4000ppm以下，培养20-25天至菌丝长满带后，同条件培养23-25天进行后熟期培养；
    所述的母种培养基为：按质量分数计，包括马铃薯20％、葡萄糖2％、蛋白胨1％、琼脂2％、磷酸二氢钾0.3％、硫酸镁0.15％、维生素B1 0.001％、余量为水；
    所述的生产种培养基为：按质量分数计，包括98-99％高粱、1-2％碳酸钙，pH自然；
    所述的栽培料为：按质量分数计，包括50％杂木屑，38％棉籽壳，10％麦麸，2％碳酸钙，含水量60-65％，pH自然。
  5. 根据权利要求3所述的人工栽培方法，其特征在于，所述的出芝管理为：
    菌丝后熟期满后进行催蕾，打开栽培袋盖子，把栽培袋竖排放置，25～28℃，空气相对湿度保持在75％～90％条件下，散射光光照10h/d，出芝管理8-10天后在栽培料开口位置开始形成原基，调整湿度为80～90％，待菌盖开始分化时，每天换新风2次，每次1h，当子实体菌盖停止平展生长时，采收子实体。
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