WO2020088458A1 - 一种液体培养柴达木大肥菇生产菌丝体的方法 - Google Patents

一种液体培养柴达木大肥菇生产菌丝体的方法 Download PDF

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WO2020088458A1
WO2020088458A1 PCT/CN2019/114025 CN2019114025W WO2020088458A1 WO 2020088458 A1 WO2020088458 A1 WO 2020088458A1 CN 2019114025 W CN2019114025 W CN 2019114025W WO 2020088458 A1 WO2020088458 A1 WO 2020088458A1
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medium
liquid
seed
barley
fermentor
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焦迎春
袁木荣
袁芳廷
李文霞
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青海伊纳维康生物科技有限公司
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N1/00Microorganisms, 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
    • C12N1/14Fungi; Culture media therefor

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  • the invention relates to the technical field of biological cultivation, in particular to a method for cultivating the mycelium of the large mushroom of Chaidamu.
  • Edible and medicinal fungi referred to as edible fungus
  • edible fungus has a long research history in China.
  • the world's first drug monograph "Shen Nong's Materia Medica” recorded the medicinal effects of fungi such as Ganoderma lucidum, Huoquan and Zhuquan. More than 20 kinds of fungi are contained in the book.
  • Existing research results show that there are nearly 300 species of fungi known to have a 60% to 100% inhibition rate of sarcoma and Ehrlich cancer, belonging to more than 70 genera in 51 families, and the antitumor activity of most fungi has a specific structure
  • the polysaccharides and proteins bind polysaccharides.
  • Edible and medicinal fungi are rich in resources in China, and are a valuable resource library for finding new drugs.
  • the development and utilization of polysaccharides for medicinal and medicinal fungi has developed rapidly at home and abroad.
  • research on polysaccharides has mainly focused on the development of traditional resources, separation and purification methods Discussion and physiological activity experiments.
  • its hotspots mainly focus on exploring the relationship between polysaccharide activity and structure activity, modifying natural polysaccharides to enhance their original activity; studying the immune mechanism of fungal polysaccharides, expanding clinical application areas; breeding and deep-seated high-polysaccharide producing strains Fermentation regulation.
  • Edible and medicinal fungal polysaccharide is a living active substance that can enhance the immune function of the human body, and is called Biological Response Modifier (BRM) in the world.
  • BRM Bio Response Modifier
  • Benacerraf and Sebestyn discovered that intravenous injection of Zymosan from the yeast cell wall had an effect on phagocytic cell activity. Subsequently, people separated and purified zymosan.
  • Riggi determined that the active ingredient in zymosan was dextran, which created a new era of glucan as an immunologically active substance.
  • Chihara et al Isolated biologically active substances from edible and medicinal fungal fruiting bodies, and proved that Q- (1-3) -D-glucan is the main active ingredient for tumor inhibition. Since then, people have successively isolated polysaccharides from various edible and medicinal fungi such as Yunzhi, Huoquan, Zhuquan, and Ganoderma lucidum, and evaluated their immune activity and anti-tumor
  • the Qinghai-Tibet Plateau is the region with the largest distribution of large-scale fungi in the world, suitable for the growth of large-scale fungi resistant to hypoxia and cold. According to preliminary statistics, there are more than 300 species of large-scale fungi in the Himalayas at an altitude of 3,000 to 5,800 meters, and the distribution of Flammulina velutipes, Armillaria chrysosporium, and Pseudomonas aeruginosa exceeds 4000 meters in altitude, which is rare in the world. Therefore, the Qinghai-Tibet Plateau is home to the world's highest distribution of large fungi, and is called the world's "alpine fungal treasure house”.
  • Chaidamu Mushroom Agaricus bitorquis (Quél.) Sacc. Is regarded as one of the most valuable wild edible fungi in the treasure trove of fungi resources on the Vietnamese Plateau. It has strong underground, huge fruiting bodies and resistance It is characterized by strong inversion and rich nutrition, and it is a special plateau strain in the large fat mushroom (Gao Shumin, 2010). It is mainly distributed in the Qaidam Basin from an altitude of 4000m around to 2600m in the middle. It grows under 50-60cm of the surface salt layer in the Gobi area of the basin. It is rich in a variety of anti-anoxic active substances and is known as one of the "four treasures of the basin”.
  • the technical problem to be solved by the present invention is to provide a method for cultivating the mycelium of Pleurotus ostreatus through liquid, which is simple, easy to operate, and can be quickly and effectively.
  • the present invention adopts the following technical solution: a method for liquid culture of Mycelia of Lactobacillus gigantea, according to the following steps,
  • Solid slant medium is PDA medium
  • first-class liquid seed medium is modified PDA medium
  • Seed bevel activation take a piece of bacteria from the original seed to a solid bevel culture medium as PDA medium, and cultivate at a temperature of 22 ⁇ 1 °C in the dark for 12 to 15 days, until the hyphae are 2/3 and dense , Refrigerate in a refrigerator at 4 °C until use;
  • first-class liquid seeds place the activated slant strains at room temperature for 48 hours, prepare a liquid medium according to the improved PDA, sterilize and cool, and use them; use a spatula to pick up several slant strains and inoculate to the first grade
  • the liquid seed culture medium, modified PDA medium was incubated at 23 ⁇ 1 °C for 48h, and then cultured on a shaker at 120r / min in the dark for 6-10 days to grow dense hyphae;
  • The% inoculation volume is transferred to a triangular flask after sterilization and cooling, and cultured at 23 ⁇ 1 °C, 120r / min in the dark for 3 to 4 days, which is the first-class liquid seeds;
  • Second-level seeds Preparation of second-level seeds: first sterilize the seed tank, after cooling, prepare liquid medium according to the seed tank medium formula, adjust the pH to 7.3, perform high temperature steam sterilization, and cool to 23 ⁇ 1 °C, Put the prepared first-level liquid seeds into the seed tank with a 15% inoculation amount, use the flame inoculation method, control the temperature of the seed tank online to 23 ⁇ 1 °C, and the stirring speed to 100rpm, and cultivate for 2 to 4 days;
  • Fermentation culture first sterilize the fermentor in an empty tank, after cooling, prepare a liquid medium according to the fermentor medium formula, adjust the pH to 7.3, perform high-temperature steam sterilization, and cool to 23 ⁇ 1 °C.
  • the prepared secondary seed liquid is connected to the fermentor with a 15% inoculation amount, using a pressure difference method, the temperature of the fermenter is controlled online at 23 ⁇ 1 ° C, the stirring speed is 110 rpm, and the cultivation is carried out for 5 to 7 days.
  • step 3 the cell area taken from the original seed is about 0.5 cm 2 , and the solid bevel medium is prepared 15 days in advance.
  • a modified PDA liquid culture medium is prepared in a triangular flask with a filling volume of 80 mL / 250 mL; when shoveling slant bacteria, 3 to 5 spores with an area of 0.5 to 1 cm 2 are inoculated with an inoculation shovel Piece.
  • the seed tank is 15L, and the empty tank is sterilized at 121 ° C for 30min.
  • a 5L liquid culture medium is prepared according to the seed tank medium formula, adjusted to pH 7.3 and steam sterilized at high temperature of 121 ° C for 30min;
  • the seed tank medium formula is: barley filtrate 400g / L, soybean protein 5g / L, MgSO 4 0.1g / L, CaCl 2 0.14g / L, compound V B 1g / L, pH 6.5-7 ; Sterilize at 121 °C for 30 minutes and use it.
  • the fermentor is 50L, and the empty tank is sterilized at 121 ° C for 30min.
  • a 30L liquid medium is prepared according to the fermentor culture medium formula, adjusted to pH 7.3 and steam sterilized at high temperature of 121 ° C for 30min;
  • the medium formula of the fermentor is: barley filtrate 400g / L, soybean protein 5g / L, MgSO 4 0.1g / L, CaCl 2 0.14g / L, compound V B 1g / L, pH 6.5-7 ; Sterilize at 121 °C for 30 minutes and use it.
  • the present invention designs a method for producing mycelia from liquid culture of Chaidamu Mushroom by liquid bevel activation, first-level liquid seed preparation, second-level seed preparation, fermentation culture and other steps, which only takes about one month in total
  • the cultivation of Mycelia of Chaidamu Mushroom is not only fast, but the method is simple and easy to operate.
  • the mycelium can replace the wild Chaidamu Mushroom fruiting body, so that the wild Chaidamu Mushroom Resources play a protective role.
  • Figure 1 is the change curve of biomass and cultivation time in the seed tank
  • Figure 2 is a graph showing the change of biomass and cultivation time in a seed tank
  • Figure 3 is the fermentation curve of Chaidamu Mushroom in 50L tank.
  • Chaidamu Mushroom (Agaricusbitorquis (Quél.) Sacc.) Slant strain: provided by the Food Laboratory of Qinghai University.
  • Reagents glucose, agar powder, peptone, magnesium sulfate (MgSO 4 ), calcium chloride (CaCl 2 ), etc. are all analytically pure; water is deionized water.
  • Solid slant medium potato (peeled) 200g / L, glucose 20g / L, agar powder 20g / L, peptone 5g / L, natural pH; 121 ° C, 30min, ready for use after sterilization.
  • First-class liquid seed medium potato (peeled) 200g / L, fructose 40g / L, peptone 5g / L, MgSO 4 0.1g / L, CaCl 2 0.14g / L, compound V B is 1g / L, pH is natural; 115 °C, 15min, use after sterilization.
  • Secondary seed tank culture medium barley (filtrate) 400g / L, soybean protein 5g / L, MgSO 4 0.1g / L, CaCl 2 0.14g / L, compound V B 1g / L, pH 6.5-7; 121 °C, 30min, after sterilization.
  • FA1004 electronic balance (Shanghai Jingke Balance), HWY-111 constant temperature culture shaker (Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd., vacuum freeze dryer (product of American Svant company), portable pressure steam sterilizer (Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory), low LXJ-IIB speed large capacity multi-tube centrifuge (Shanghai Anting Scientific Instrument General Factory), 101-3B electric heating blast dry heat box (Shanghai Experimental Instrument General Factory), CARY50 purple Visible spectrophotometer (American Varian Co., Ltd.), GUJS 20L mechanically stirred fermentation tank (Zhenjiang Dongfang Biological Equipment Engineering Co., Ltd.).
  • the activated slant strains were placed at room temperature for 48h, and the liquid culture medium was prepared according to the modified PDA.
  • the liquid volume was 80mL / 250mL Erlenmeyer flask, which was sterilized, cooled and used.
  • Take 0.5 ⁇ 1cm 2 (3 ⁇ 5 pieces) with an inoculation shovel inoculate the above liquid medium, 23 ⁇ 1 °C, and culture it for 48h after standing for 48h, then shake and shake at 120r / min shaker for 6-10 days (protect from light) , Until more hyphae grow out; transfer to 10 mL inoculation volume to 300mL / 1000mL Erlenmeyer flask after sterilization and cooling, 23 ⁇ 1 °C, 120r / min continuous shaking culture for 3 to 4 days (protect from light), namely For the first-level seeds.
  • the seed fermentation tank (15L) was sterilized in an empty tank (121 °C, 30min). After cooling, a 5L liquid medium was prepared according to the seed tank medium formula. After adjusting the pH to 7.3, high temperature steam sterilization (121 °C, 30min), cool to 23 ⁇ 1 °C, connect the prepared first-level seed liquid with 15% inoculation volume, use flame inoculation method to connect to the seed tank, control the seed tank online 23 ⁇ 1 °C, stirring speed 100rpm, culture 2 ⁇ 4 days.
  • Biomass determination-dry weight method amino acid determination-ninhydrin method; reducing sugar determination-3,5-dinitrosalicylic acid method; extracellular polysaccharide determination-alcohol precipitation method.
  • the biomass of Chaidamu Mushroom Mycelium in the seed tank reached the highest on the third day, so the optimal cultivation time of the seed tank was determined as 3 days, as shown in Figures 1 and 2 It shows that at this time, the pH change in the seed tank is also appropriate, and the fermentation tank cultivation time is shown in the curve of FIG. 3.

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Abstract

一种液体培养柴达木大肥菇生产菌丝体的方法,以柴达木大肥菇斜面菌种、青稞为原料配合去离子水以及作为分析纯的葡萄糖、琼脂粉、蛋白胨、硫酸镁和氯化钙,准备好培养基,然后按以下流程进行:种子斜面活化、一级液体种子制备、二级种子制备、发酵培养。通过设计由种子斜面活化、一级液体种子制备、二级种子制备、发酵培养等步骤构成的由液体培养柴达木大肥菇生产菌丝体的方法,总共仅需一个月左右即可培养得到柴达木大肥菇生产菌丝体。

Description

一种液体培养柴达木大肥菇生产菌丝体的方法
本申请要求于2018年10月31日提交中国专利局、申请号为201811283905.9、发明名称为“一种液体培养柴达木大肥菇生产菌丝体的方法”的中国专利申请的优先权,其全部内容通过引用结合在本申请中。
技术领域
本发明涉及生物培养技术领域,具体涉及一种培养柴达木大肥菇生产菌丝体的方法。
背景技术
食药用真菌简称食用菌,在我国己有悠久的研究历史。早在两千多年前的东汉末期,世界上第一部药物专著《神农本草经》中就记载了灵芝、获荃、猪荃等真菌的药效;明代著名医学家李时珍在《本草纲目》中收载了20多种真菌。现有的研究结果表明,己知对肉瘤和艾氏癌抑制率达60%~100%的真菌有近300种,分属51科70多个属,大多数真菌的抗肿瘤活性是具有特定结构的多糖和蛋白质结合多糖体。食药用真菌在我国资源丰富,是寻找新药物的宝贵资源库,国内外对食药用真菌多糖的开发利用发展很快,过去对多糖的研究主要集中在对传统资源的开发、分离纯化方法探讨以及生理活性实验方面。目前以至将来,其热点主要集中在探究多糖活性与构效之间的关系,修饰天然多糖增强其原有活性;研究真菌多糖的免疫作用机理,扩大临床应用领域;多糖高产菌株的选育以及深层发酵调控。
食药用真菌多糖是一种能够增强人体免疫功能的生活活性物质,在国际上被称为生物反应调节剂(Biological Response Modifier,BRM)。早在1957年,Benacerraf和Sebestyn发现了静脉注射来自酵母细胞壁的酵母聚糖(Zymosan)对吞噬细胞活性有影响。随后人们对酵母聚糖进行了分离纯化,1961年Riggi确定了酵母聚糖中的这种活性成分是葡聚糖,开创了葡聚糖作为免疫活性物质的新纪元。1969年,Chihara等人分别从食药用真菌子实体中分离得到了生物活性物质,并证明了Q-(1-3)-D-葡聚糖是抑瘤作用的主要活性成分。此后,人们陆续从云芝、获荃、猪荃、灵芝等多种食药用真菌中分离出多糖,并对它们的免疫活性和抗肿瘤能力进行了评价。
20世纪70年代以来,随着免疫物质、生物膜以及多种生物活性物质的研究进展表明,糖类不仅是所有生命有机体的重要能量来源以及维持生命所必需的结构材料,更重要的是它参与了生命科学中细胞的各种活动,如参与免疫功能的调节、细胞与细胞的识别、细胞间物质的运输、癌症的诊断与治疗,细胞的分裂和分化、细胞的生长和衰老等。由于食药用真菌多糖及其复合物的多种多样生物活性功能以及在功能食品和临床上的广泛使用,使其成为近年生命科学、生物学、医药和食品科学等研究领域的热点之一。
青藏高原是世界上大型真菌分布最高的区域,适于耐缺氧、耐寒大型真菌生长。据初步统计,喜马拉雅海拔3000~5800m,就有大型真菌300余种,金针菇、黄绿蜜环菌、铅色灰球菌等分布超过海拔4000m,为世界所罕见。所以,青藏高原汇集了全球分布最高的大型真菌,被称为世界“高山真菌宝库”。柴达木大肥蘑菇Agaricus bitorquis(Quél.)Sacc.作为青藏高原真菌资源宝库中的代表种被公认为最有开发价值的名贵野生食用菌类之一,其具有地下结实、子实体巨大、抗逆性强、营养丰富等特点,为大肥蘑菇中的高原特殊菌种(高淑敏,2010)。主要分布于海拔从四周4000m到中部2600m的柴达木盆地,生长在盆地戈壁地区表面盐层50~60cm下,富含多种抗缺氧活性物质,被誉为“盆地四宝”之一,一直以来被当地牧民作为抗缺氧、提高抵抗力的食材广泛食用,连年采挖导致草原土壤植被和野生柴达木大肥菇生长环境的严重被破坏。有研究表明其菌丝体具有与大肥菇子实体(蘑菇)一致的营养成分,采用液体培养柴达木大肥菇菌丝细胞,生产菌丝体代替子实体,不仅可以有效保护这一重要资源,还可以为进一步开发柴达木大肥菇胞内多糖、三萜等活性成分,奠定物质基础。研究表明,通过比较柴达木大肥菇子实体多糖、发酵液多糖和菌丝体多糖对小鼠力竭游泳时间及力竭游泳结束后小鼠血液中Hb、血清中BUN、LDH和LA含量、肝脏中HG含量变化的差异性,结果表明柴达木大肥菇子实体多糖、发酵液多糖和菌丝体多糖通过延长小鼠力竭游泳时间、提高小鼠血液中Hb、HG和血清中LDH含量、降低小鼠血清中BUN和LA含量,从而提高小鼠的运动耐力、缓解运动疲劳。说明该3种多糖具有抗疲劳作用,且菌丝体多糖的抗疲劳效果优于子实体多糖和发酵液多糖。
因此,研究可代替野生柴达木大肥菇子实体的人工培养柴达木大肥菇菌丝体的方法,成为业界当前亟待解决的问题。
发明内容
本发明要解决的技术问题是,提供一种方法简单、易于操作、可快速、有效的通过液体培养柴达木大肥菇菌丝体的方法。
为解决上述技术问题,本发明采用如下技术方案:一种液体培养柴达木大肥菇生产菌丝体的方法,按以下步骤进行,
1)原料准备:柴达木大肥菇斜面菌种、青稞、去离子水以及作为分析纯的葡萄糖、琼脂粉、蛋白胨、硫酸镁和氯化钙;
2)培养基:固体斜面培养基为PDA培养基、一级液体种子培养基为改良PDA培养基、二级种子罐培养基和酵罐培养基;
3)种子斜面活化:从原种中取一块菌体到固体斜面培养基为PDA培养基,以22±1℃的温度避光培养12~15天,待菌丝长满斜面2/3且密集,于4℃的冰箱中冷藏待用;
4)一级液体种子制备:将活化好的斜面菌种置于室温下48h,按改良PDA配制液体培养基,灭菌、冷却后备用;用接种铲取若干块斜面菌种,接种到一级液体种子培养基即改良PDA培养基中,以23±1℃的温度静置培养48h,然后于120r/min的摇床避光振荡培养6~10天,至长出密集菌丝球;以10%接种量转接至灭菌冷却后的三角瓶中,以23±1℃、120r/min连续避光振荡培养3~4天,即为一级液体种子;
5)二级种子制备:先将种子罐进行空罐灭菌,冷却后,按种子罐培养基配方配制液体培养基,调节pH至7.3后,进行高温蒸汽灭菌,冷却至23±1℃,将已制备好的一级液体种子以15%的接种量,采用火焰接种法接入种子罐,在线控制种子罐温度为23±1℃、搅拌转速为100rpm,培养2~4天;
6)发酵培养:先将发酵罐进行空罐灭菌,冷却后,按发酵罐培养基配方配制液体培养基,调节pH至7.3后,进行高温蒸汽灭菌,冷却至23±1℃,将已制备好的二级种子液以15%的接种量,采用压差法接入发酵罐,在线控制发酵罐温度为23±1℃、搅拌转速110rpm,培养5~7天。
进一步地,在步骤3)中,从原种中取的菌体面积大约为0.5cm 2,而固体斜面培养基提前15天制备。
进一步地,在步骤4)中,以装液量为80mL/250mL的三角瓶配制改良PDA液体培养基;铲取斜面菌种时用接种铲取3~5块面积为0.5~1cm 2的菌种块。
进一步地,在步骤5)中,种子罐为15L,空罐121℃灭菌30min,冷却后,按种子罐培养基配方配制5L液体培养基,调节pH至7.3后高温121℃蒸汽灭菌30min;种子罐培养基配方为:青稞滤液为400g/L、大豆蛋白为5g/L、MgSO 4为0.1g/L、CaCl 2为0.14g/L、复合V B为1g/L,pH为6.5~7;以121℃的温度灭菌30min后备用。
进一步地,在步骤6)中,发酵罐为50L,空罐121℃灭菌30min,冷却后,按发酵罐培养基配方配制30L液体培养基,调节pH至7.3后高温121℃蒸汽灭菌30min;发酵罐培养基配方为:青稞滤液为400g/L、大豆蛋白为5g/L、MgSO 4为0.1g/L、CaCl 2为0.14g/L、复合V B为1g/L,pH为6.5~7;以121℃的温度灭菌30min后备用。
进一步地,按青稞:水=400g:1000mL的比例,加水浸没煮沸20min后,过滤得到青稞滤液;以青稞滤液为基液,加入各营养成分,配制成种子罐培养基。
进一步地,按青稞:水=400g:1000mL的比例,加水浸没煮沸20min后,过滤得到青稞滤液;以青稞滤液为基液,加入各营养成分,配制成发酵罐培养基。
虽然柴达木大肥菇子实体、发酵液和菌丝体多糖都可以延长小鼠力竭游泳时间,即具有抗疲劳活性,但是菌丝体的抗疲劳活性要优于发酵液多糖和子实体多糖,因此以菌丝体代替柴达木大肥菇子实体是完全可行的(请参照论文《柴达木大肥菇多糖对小鼠的抗疲劳作用》,现代食品科技,2018,Vol.34,No.8,文章篇号:1673-9078(2018)08-24-30,焦迎春、旷慧、吴嘉南、何成日、陈启和)。
本发明通过设计由种子斜面活化、一级液体种子制备、二级种子制备、发酵培养等步骤构成的由液体培养柴达木大肥菇生产菌丝体的方法,总共仅需一个月左右即可培养得到柴达木大肥菇生产菌丝体,不仅速度快,而且方法简单、容易操作,其菌丝体可以代替野生柴达木大肥菇子实体,从而可以对野生柴达木大肥菇资源起保护作用。
附图说明
图1为种子罐中生物量与培养时间的变化曲线图;
图2为种子罐中生物量与培养时间的变化曲线图;
图3为柴达木大肥菇50L罐发酵曲线图。
具体实施方式
下面结合具体实施例对本发明做进一步说明,但这并不意味着对本发明的任何限制。
1.材料与方法
1.1原材料与培养基
柴达木大肥菇(Agaricusbitorquis(Quél.)Sacc.)斜面菌种:由青海大学食品实验室提供。
青稞(Hordeum vulgare Linn.Var.nudum Hook.F.):青海西宁购买。
试剂:葡萄糖、琼脂粉、蛋白胨、硫酸镁(MgSO 4)、氯化钙(CaCl 2)等均为分析纯;水为去离子水。
固体斜面培养基(PDA培养基):马铃薯(去皮)200g/L,葡萄糖20g/L,琼脂粉20g/L,蛋白胨5g/L,pH自然;121℃,30min,灭菌后备用。
一级液体种子培养基(改良PDA培养基):马铃薯(去皮)200g/L、果糖40g/L,蛋白胨5g/L,MgSO 4为0.1g/L,CaCl 2为0.14g/L,复合V B为1g/L,pH自然;115℃,15min,灭菌后备用。
二级种子罐培养基:青稞(滤液)400g/L,大豆蛋白5g/L,MgSO 4为0.1g/L,CaCl 2为0.14g/L,复合V B为1g/L,pH 6.5~7;121℃,30min,灭菌后备用。
发酵罐培养基:同上。
按青稞:水=400:1000(g:mL),加水浸没煮沸20min后,过滤得到青稞滤液;以青稞滤液为液体,加入上述营养成分,配制成种子罐培养基与发酵罐培养基。
1.2试验仪器
FA1004型电子天平(上海精科天平)、HWY-111恒温培养摇床(上海智城分析仪器制造有限公司、真空冷冻干燥机(美国Svant公司产品)、手提式压力蒸汽灭菌器(上海博迅实业有限公司医疗设备厂)、低LXJ-IIB速大容量多管离心机(上海安亭科学仪器总厂)、101-3B型电热鼓风干热箱(上海市实验仪器总厂)、CARY50紫个可见分光光度计(美国瓦里安有限公司)、GUJS 20L机械搅拌发酵罐(镇江东方生物装备工程有限公司)。
2.方法
2.1试验方法
2.1.1种子斜面活化
从原种中取一小块菌体(面积约0.5cm 2大小)到PDA斜面培养基(提前15天制备),22±1℃避光培养12~15天,待菌丝长满斜面2/3且密集,于4℃冰箱中冷藏待用。
2.1.2一级(液体)种子制备
将活化好的斜面菌种置于室温下48h,按改良PDA配制液体培养基,装液量为80mL/250mL三角瓶,灭菌、冷却后备用。用接种铲取0.5~1cm 2(3~5块),接种到上述液体培养基中,23±1℃,静置培养48h后,于120r/min摇床振荡培养6~10天(避光),至长出较多菌丝球;以10%接种量转接至灭菌冷却后300mL/1000mL三角瓶中,23±1℃,120r/min连续振荡培养3~4天(避光),即为一级种子。
2.1.3二级(种子罐)种子制备
先将种子发酵罐(15L)进行空罐灭菌(121℃,30min),冷却后,按种子罐培养基配方配制5L液体培养基,调节pH至7.3后,进行高温蒸汽灭菌(121℃,30min),冷却至23±1℃,将已制备好的一级种子液以15%的接种量,采用火焰接种法接入种子罐,在线控制种子罐23±1℃、搅拌转速100rpm,培养2~4天。
2.1.4发酵(发酵罐)培养
先将发酵罐(50L)进行空罐灭菌(121℃,30min),冷却后,按发酵罐培养基配方配制30L液体培养基,调节pH至7.3后,进行高温蒸汽灭菌(121℃,30min),冷却至23±1℃,将已制备好的二级种子液以15%的接种量,采用压差法接入发酵罐,在线控制发酵罐23±1℃,搅拌转速110rpm,培养5~7天。
2.2测定方法
生物量测定——干重法;氨基酸测定——茚三酮法;还原糖测定——3,5-二硝基水杨酸法;胞外多糖测定——酒精沉淀法。
3结果与分析
3.1种子罐发酵情况
采用上述培养基及培养方法,在种子罐中柴达木大肥菇菌丝体生物量在第3天达到最高,故确定种子罐的最佳培养时间为3天,如图1和图2所示, 此时,种子罐中pH变化也较适宜,而发酵罐培养时间如图3的曲线所示。
以上已将本发明做一详细说明,以上所述,仅为本发明之较佳实施例而已,当不能限定本发明实施范围,即凡依本申请范围所作均等变化与修饰,皆应仍属本发明涵盖范围内。

Claims (7)

  1. 一种液体培养柴达木大肥菇生产菌丝体的方法,其特征在于:按以下步骤进行:
    1)原料准备:柴达木大肥菇斜面菌种、青稞、去离子水以及作为分析纯的葡萄糖、琼脂粉、蛋白胨、硫酸镁和氯化钙;
    2)培养基:固体斜面培养基为PDA培养基、一级液体种子培养基为改良PDA培养基、二级种子罐培养基和发酵罐培养基;
    3)种子斜面活化:从原种中取一块菌体到固体斜面培养基为PDA培养基,以22±1℃的温度避光培养12~15天,待菌丝长满斜面2/3且密集,于4℃的冰箱中冷藏待用;
    4)一级液体种子制备:将活化好的斜面菌种置于室温下48h,按改良PDA配制液体培养基,灭菌、冷却后备用;用接种铲取若干块斜面菌种,接种到一级液体种子培养基即改良PDA培养基中,以23±1℃的温度静置培养48h,然后于120r/min的摇床避光振荡培养6~10天,至长出密集菌丝球;以10%接种量转接至灭菌冷却后的三角瓶中,以23±1℃、120r/min连续避光振荡培养3~4天,即为一级液体种子;
    5)二级种子制备:先将种子罐进行空罐灭菌,冷却后,按种子罐培养基配方配制液体培养基,调节pH至7.3后,进行高温蒸汽灭菌,冷却至23±1℃,将已制备好的一级液体种子以15%的接种量,采用火焰接种法接入种子罐,在线控制种子罐温度为23±1℃、搅拌转速为100rpm,培养2~4天;
    6)发酵培养:先将发酵罐进行空罐灭菌,冷却后,按发酵罐培养基配方配制液体培养基,调节pH至7.3后,进行高温蒸汽灭菌,冷却至23±1℃,将已制备好的二级种子液以15%的接种量,采用压差法接入发酵罐,在线控制发酵罐温度为23±1℃、搅拌转速110rpm,培养5~7天。
  2. 根据权利要求1所述的方法,其特征在于:在步骤3)中,从原种中取的菌体面积为0.5cm 2,而固体斜面培养基提前15天制备。
  3. 根据权利要求1所述的方法,其特征在于:在步骤4)中,以装液量为80mL/250mL的三角瓶配制改良PDA液体培养基;铲取斜面菌种时用 接种铲取3~5块面积为0.5~1cm 2的菌种块。
  4. 根据权利要求1所述的方法,其特征在于:在步骤5)中,种子罐为15L,空罐121℃灭菌30min,冷却后,按种子罐培养基配方配制5L液体培养基,调节pH至7.3后高温121℃蒸汽灭菌30min;种子罐培养基配方为:青稞滤液为400g/L、大豆蛋白为5g/L、MgSO 4为0.1g/L、CaCl 2为0.14g/L、复合V B为1g/L,pH为6.5~7;以121℃的温度灭菌30min后备用。
  5. 根据权利要求1所述的方法,其特征在于:在步骤6)中,发酵罐为50L,空罐121℃灭菌30min,冷却后,按发酵罐培养基配方配制30L液体培养基,调节pH至7.3后高温121℃蒸汽灭菌30min;发酵罐培养基配方为:青稞滤液为400g/L、大豆蛋白为5g/L、MgSO 4为0.1g/L、CaCl 2为0.14g/L、复合V B为1g/L,pH为6.5~7;以121℃的温度灭菌30min后备用。
  6. 根据权利要求5所述的方法,其特征在于:按青稞:水=400g:1000mL的比例,加水浸没煮沸20min后,过滤得到青稞滤液;以青稞滤液为基液,加入各营养成分,配制成种子罐培养基。
  7. 根据权利要求1所述的方法,其特征在于:按青稞:水=400g:1000mL的比例,加水浸没煮沸20min后,过滤得到青稞滤液;以青稞滤液为基液,加入各营养成分,配制成发酵罐培养基。
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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102766663A (zh) * 2012-05-30 2012-11-07 陕西师范大学 桑黄活性多糖的制备方法
CN104277987A (zh) * 2014-10-20 2015-01-14 中山安荞生物科技有限公司 牛樟菌培养基
EP2835058A1 (en) * 2013-08-07 2015-02-11 Stichting Eco Consult Meat substitute composition and method for providing thereof
CN105505797A (zh) * 2016-01-11 2016-04-20 黄山学院 一种用于杏鲍菇菌种栽培的培养基
CN105695530A (zh) * 2016-03-14 2016-06-22 青海大学 一种高产柴达木大肥菇多糖的液体发酵培养基及应用
CN109321473A (zh) * 2018-10-31 2019-02-12 青海伊纳维康生物科技有限公司 一种液体培养柴达木大肥菇生产菌丝体的方法

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102766663A (zh) * 2012-05-30 2012-11-07 陕西师范大学 桑黄活性多糖的制备方法
EP2835058A1 (en) * 2013-08-07 2015-02-11 Stichting Eco Consult Meat substitute composition and method for providing thereof
CN104277987A (zh) * 2014-10-20 2015-01-14 中山安荞生物科技有限公司 牛樟菌培养基
CN105505797A (zh) * 2016-01-11 2016-04-20 黄山学院 一种用于杏鲍菇菌种栽培的培养基
CN105695530A (zh) * 2016-03-14 2016-06-22 青海大学 一种高产柴达木大肥菇多糖的液体发酵培养基及应用
CN109321473A (zh) * 2018-10-31 2019-02-12 青海伊纳维康生物科技有限公司 一种液体培养柴达木大肥菇生产菌丝体的方法

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
GAO, SHUMIN ET AL.: "Biological Characteristics of Wild Agaricus Bitorquis(Quél.) Sacc. Growing in Qaidam", EDIBLE FUNGI OF CHINA, vol. 29, no. 5, 31 December 2010 (2010-12-31), pages 28 - 30, XP009520742, DOI: 10.13629/j.cnki.53-1054.2010.05.007 *
GULER, P. ET AL.: "Grain Spawn Production in Agaricus bitorquis(Quel.) Sacc.", DYNAMIC BIOCHEMISTRY, PROCESS BIOTECHNOLOGY AND MOLECULAR BIOLOGY, vol. 1, no. 1, 31 December 2007 (2007-12-31), pages 74 - 78, XP009520741, ISSN: 1749-0626 *
JIAO, YINGCHUN ET AL.: "The Anti-fatigue Activities of Polysaccharides Extracted from the Agaricus Bitorquis", MODERN FOOD SCIENCE AND TECHNOLOGY, vol. 34, no. 8, 4 July 2018 (2018-07-04), pages 24 - 30,193, XP009520743, DOI: 10.13982/j.mfst.1673-9078.2018.8.004 *

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