CN117604046A - 一种单细胞油脂生产工艺 - Google Patents
一种单细胞油脂生产工艺 Download PDFInfo
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- CN117604046A CN117604046A CN202311563458.3A CN202311563458A CN117604046A CN 117604046 A CN117604046 A CN 117604046A CN 202311563458 A CN202311563458 A CN 202311563458A CN 117604046 A CN117604046 A CN 117604046A
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Classifications
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C12P7/6409—Fatty acids
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- C12P7/6434—Docosahexenoic acids [DHA]
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- 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
- C12N1/16—Yeasts; Culture media therefor
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- 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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P39/00—Processes involving microorganisms of different genera in the same process, simultaneously
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C12P7/6409—Fatty acids
- C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
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- C12P7/6431—Linoleic acids [18:2[n-6]]
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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Abstract
本发明公开了一种单细胞油脂生产工艺。本发明属于生物技术领域,所要解决的技术问题是提高单细胞油脂产品的油脂含量。本发明公开的单细胞油脂生产工艺包括将产油脂微生物进行发酵培养,在菌体繁殖前期维持一定碳氮比,以利于菌体快速生长;当菌体生长中后期时,调整碳氮比及添加诱导剂;发酵结束后,将发酵液干燥得到单细胞油脂产品的步骤。本发明得到的单细胞油脂产品中油脂的含量高,DHA、DPA、EPA、亚油酸、γ–亚麻酸和花生四烯酸的含量达到90%以上。
Description
技术领域
本发明属于生物技术领域,涉及一种单细胞油脂生产工艺。
背景技术
微生物油脂也称单细胞油脂,是微生物以碳水化合物、碳氢化合物和普通油脂为碳源,结合无机盐生产出的一些含有特殊脂肪酸或对人体有益的营养物质的功能性油脂。微生物油脂在食用油方面主要是能生产出一些功能性多不饱和脂肪酸,主要包括亚油酸、γ–亚麻酸、花生四烯酸、二十碳五烯酸(EPA)、二十二碳六烯酸(DHA)和二十二碳五烯酸(DPA)等。这些特殊脂肪酸大多是生物活性物质的前体,具有抗衰老、抗氧化、抗炎症、抑制肿瘤细胞形成、扩散和转移、防治心脏病、高血压等作用,其中EPA、DHA、DPA能促进婴儿神经系统发育,提高儿童智力。
单细胞油脂是指来自于可食用微生物(如真菌(例如酵母))和海藻的油脂。据估算,1t精炼酵母油的生产成本为800~1000美元,与动植物油相比没有经济效益优势。因此,单细胞油脂商业化开发转向了动植物油脂中含量极低且人和动物必需的花生四烯酸(ARA)和二十二碳六烯酸(DHA)的生产领域中。海洋微生物具有从头合成DHA和大量积累这种脂肪酸的能力,并经食物链转移到鱼油中,但是海洋环境中的有毒有害污染物(如农药残留物、重金属等)可能进入海洋生物体内积累,成为鱼油安全食用的潜在威胁。
微生物油脂产品具有良好的安全性。大量的人体和临床试验表明,微生物油脂是人类可食用的安全食用油脂。由于微生物油脂全程在封闭、清洁的环境中生产,可以实现食品安全高度可控、可追溯。另外,由于是一个连续的生产过程,产品加工周期非常紧凑,微生物油脂中的DHA、ARA等敏感成分被采取了有效的保护措施,茴香胺值、反式脂肪酸、无良污染物得到良好的控制,因此产品具有很好的新鲜度、感官和货架期。
微生物油脂生产是一种可持续的发展方式。首先,微生物细胞具有生长周期短、繁殖速度快、易于培养,在节约劳动力的同时不受场地、气候、季节的限制,有利于连续化生产;其次,用于工业化的产油脂微生物合成的脂肪和功能性长链多不饱和脂肪酸(LCPUFA)比动植物来源的含量高很多,例如裂壶藻干物质的含油率可高达60%,其中DHA在油脂中含量高达60%;另外,微生物油脂生产一般综合利用玉米糖浆、大豆饼粕、酵母粉等大宗、低附加值农产品加工副产品作为原料,原料易得且资源集约、不消耗自然生态资源。这也是很多国家将微生物油脂作为解决供应链短缺支撑项目的重要原因之一。
经过了近80年的研究与发展,微生物油脂逐渐被市场所接受,并且随着社会环境与资源问题的日渐凸显,人们逐渐认识到发展微生物油脂的可行性与必要性。但遗憾的是微生物油脂较高的生产成本问题仍未得到有效的解决。
发明内容
本发明所要解决的技术问题是提高单细胞油脂产品的油脂含量。为此,发明提供一种单细胞油脂生产工艺。具体地,本发明通过在产油脂微生物发酵的不同阶段调控碳氮比来促进菌体的快速繁殖和积累油脂,并添加诱导剂对产油脂微生物的油脂分泌代谢网络进行人工干预,从而使其产生单细胞油脂。
本发明提供一种单细胞油脂生产工艺,包括如下步骤:
将产油脂微生物进行发酵培养,发酵20-26h时开始补料碳源和氮源,控制碳氮比为76-100:1,其中碳源的流速为每分钟流加初始发酵体积的0.02-0.03%,并根据碳氮比调节氮源流速;
发酵34-38h时,添加乙酰CoA羧化酶和甘油三酯作为诱导剂诱导油脂的合成,使得乙酰CoA羧化酶和甘油三酯的终浓度分别为0.02-0.05g/L和0.04-0.08g/L,碳氮比调整为200-232:1,其中碳源的流速为每分钟流加初始发酵体积的0.02-0.03%,并根据碳氮比调节氮源流速;
发酵至少45h,结束发酵,将发酵液离心,得到沉淀;将所述沉淀进行干燥,得到单细胞油脂产品。
在一些实施方案中,上述方法中,所述发酵培养使用的发酵培养基组成为:玉米浆干粉12-38g/L、甘油26-42g/L、酵母粉36-52g/L、硫酸镁1.8-16g/L、维生素B1 5.6-24g/L、甜菜碱6.5-18g/L、硫酸铵22-65g/L、磷酸二氢钾30-90g/L、磷酸氢二钠10-65g/L、硼酸5-7.5mL/L、MnSO4·7H2O 200-400mg/L,pH为6-8。
在一些实施方案中,上述任一所述的方法中,所述发酵培养的初始OD600为3-4,例如3.1、3.2、3.3、3.4、3.5、3.6、3.7、3.8、3.9、4.0,或这些数值中的任意二者之间的数值和范围,例如OD600为3.6-3.8。
在一些实施方案中,上述任一所述的方法中,所述发酵培养中溶氧为20%以上,例如初始转速为200rpm,初始通气量为0.2vvm,并随着溶氧的下降逐渐提高转速和通气量,保证溶氧为20%以上。
在一些实施方案中,上述任一所述的方法中,所述补料的碳源为甘油。
在一些实施方案中,上述任一所述的方法中,所述补料的氮源为200-300g/L的硫酸铵水溶液,例如200、210、220、230、240、250、260、270、280、290、300g/L的硫酸铵水溶液,或这些数值中的任意二者之间的数值和范围,例如250g/L的硫酸铵水溶液。
在一些实施方案中,上述任一所述的方法中,所述发酵培养过程中,添加所述诱导剂之前控制发酵温度为32-35℃,添加所述诱导剂之后控制发酵温度为26-30℃。
在一些实施方案中,上述任一所述的方法中,所述干燥为喷雾干燥,例如用水重悬沉淀控制菌体湿重为480-660g/L,然后以进料速度1.2-2.4L/小时,进风温度110-125℃,频率为160-900赫兹进行喷雾干燥,即得到单细胞油脂产品。
在一些实施方案中,上述任一所述的方法中,在所述发酵培养之前还有种子培养的步骤,所述种子培养使用的种子培养基组成为:豆粕粉20-50g/L、甘油8-22g/L、酵母粉8-31g/L、酪蛋白胨40-62g/L、无氨基酵母氮源0.8-4g/L、碘化钾0.013-0.03g/L、肌醇0.12 -0.18g/L、核黄素0.22-0.38g/L、吡哆醇0.11 -0.26g/L、硫酸亚铁0.12-0.31g/L、生物素20-60mg/L、泛酸钙400-700mg/L,pH为5-7;
所述种子培养的条件可以为:温度33℃,转速150rpm。
在一些实施方案中,上述任一所述的方法中,所述产油脂微生物选自由解脂耶氏酵母(Yarrowia lipolytica)、酿酒酵母(Saccharomyces cerevisiae)、热带假丝酵母(Candida tropicalis)组成的组。
在一些实施方案中,上述任一所述的方法中,所述单细胞油脂为DHA、DPA、EPA、亚油酸、γ–亚麻酸和花生四烯酸中的一种或多种。
本发明的原理如下:产油脂微生物的油脂积累过程能够受到环境因素的刺激调控;当环境中存在充足的碳源和氮源时,产油脂微生物处于增殖期,这一时期微生物快速增殖,自身合成的油脂类物质主要用于细胞骨架的形成,从而保持较高的菌体密度;而当环境中氮源含量降低,但碳源依然充足时,微生物转而进入油脂积累期,自身增殖速度快速减弱,并激活相应代谢途径的表达,从而将多余的碳源代谢转化为油脂储存在菌体细胞内,在此过程中甘油三酯协助乙酰CoA羧化酶进入到细胞内后,乙酰CoA羧化酶催化底物发生羧化反应,然后经多次链延长以及去饱和作用等形成相应的脂肪酸,随后经甘油三酯合成途径将合成的脂肪酸储存在体内。
本发明将产油脂微生物在特定的培养基上逐级培养;在菌体繁殖前期维持一定碳氮比,以利于菌体快速生长;当菌体生长中后期时,调整碳氮比及添加诱导剂,对微生物的油脂分泌代谢网络进行干预,激活相应代谢途径的表达,从而将多余的碳源代谢转化为油脂储存在菌体细胞内;发酵结束后,经过干燥得到单细胞油脂产品。
本发明得到的单细胞油脂产品中油脂的含量高,DHA、DPA、EPA、亚油酸、γ–亚麻酸和花生四烯酸的含量达到90%以上。另外,本发明的生产工艺稳定、简单、成本低。
具体实施方式
以下结合具体实施例,对本发明作进一步说明。应理解,以下实施例仅用于说明本发明而非用于限制本发明的范围。如非特殊说明,实施例中所用的技术手段均为本领域常规操作,或按照试剂盒及仪器设备厂商所建议的实验方法。实施例中使用的试剂和生物材料如无特殊说明均可从商业途径获得。
湿重检测方法:取适量的发酵液,测定其体积并记录为V1(单位:L);将该发酵液8000rpm离心10分钟,得到沉淀;所得沉淀加水重悬,再次8000rpm离心10分钟,称量沉淀并记录为M1(单位:g)。菌体的湿重计算公式为:湿重(g/L)=M1/V1。
干重检测方法:取适量的发酵液,测定其体积并记录为V1(单位:L);将该发酵液8000rpm离心10分钟,得到沉淀;所得沉淀加水重悬,再次8000rpm离心10分钟,称量沉淀并记录为M1(单位:g);将该沉淀按照国标GB/T 6435-2014《饲料中水分的测定》的方法在103±2℃进行烘干,得到干菌体,称其重量并记录为M2(单位:g)。菌体的干重计算公式为:干重(g/L)=M2/V1。
二十二碳六烯酸(DHA)、二十二碳五烯酸(DPA)、二十碳五烯酸(EPA)、亚油酸、γ–亚麻酸和花生四烯酸的检测均根据国标GB5009.168-2016《食品中脂肪酸的测定方法》进行,将干菌体经过碱性甲醇溶液将脂肪酸甲酯化,再通过气相色谱中的毛细管色谱柱分离,氢火焰检测器(FID)检测,得到干菌体中DHA、DPA、EPA、亚油酸、γ–亚麻酸、花生四烯酸的含量;
其中,气相色谱分析脂肪酸的条件如下:安捷伦450气相色谱,带有氢火焰检测器和毛细管色谱柱,烘箱加热条件为:在12分钟内温度逐渐升高至23℃,在23℃保持4分钟,分流比为20:1,通过Sigma-Aldrich公司的标准品进行比较对各脂肪酸进行定性和定量。
实施例1
1、从超低温冰箱内取出一管解脂耶氏酵母(Yarrowia lipolytica)(购自中国工业微生物菌种保藏管理中心,编号:CICC 32291),放在手心快速解冻,在超净台内将解冻后的菌种液加入到装有种子培养基的摇瓶中,每100ml种子培养基接入200μl菌种液。
种子培养基:豆粕粉20g/L、甘油8g/L、酵母粉8g/L、酪蛋白胨40g/L、无氨基酵母氮源(YNB)0.8g/L、碘化钾0.013g/L、肌醇0.12g/L、核黄素0.22g/L、吡哆醇0.11g/L、硫酸亚铁0.12g/L、生物素20mg/L、泛酸钙400mg/L,余量为水,用盐酸或氢氧化钠调节pH为5。
2、接种后将摇瓶放入摇床中,33℃、150rpm培养16小时,得到种子液。
3、将种子液按照5%(v/v)的接种量接入装有2L发酵培养基的5L发酵罐中,初始OD600为3.6,在32℃进行发酵培养,初始转速为200rpm,初始通气量为0.2vvm,并随着溶氧的下降逐渐提高转速和通气量,保证溶氧为20%以上。
发酵培养基:玉米浆干粉12g/L、甘油26g/L、酵母粉36g/L、硫酸镁1.8g/L、维生素B1 5.6g/L、甜菜碱6.5g/L、硫酸铵22g/L、磷酸二氢钾30g/L、磷酸氢二钠10g/L、硼酸5mL/L、MnSO4·7H2O 200mg/L,余量为水,用盐酸或氢氧化钠调节pH为6。
4、发酵24h时开始补料碳源和氮源,其中,碳源为甘油,氮源为经过灭菌的250g/L的硫酸铵水溶液,控制碳氮比为76:1,碳源流速固定为40ml/min(即,每分钟流加初始发酵体积的0.02%),并根据碳氮比调节氮源流速。
5、发酵36h时,添加100ml浓度为0.8g/L的乙酰CoA羧化酶的水溶液和100ml浓度为1.2g/L的甘油三酯(Sigma-Aldrich,CAS号:538-24-9)的水溶液作为诱导剂,乙酰CoA羧化酶和甘油三酯的终浓度分别为0.03g/L和0.05g/L,对微生物的油脂分泌代谢网络进行干预,激活相应代谢途径的表达,从而将多余的碳源代谢转化为油脂储存在菌体细胞内。发酵温度调整为26℃,碳氮比调整为232:1,碳源流速固定为60ml/min(即,每分钟流加初始发酵体积的0.03%),并根据碳氮比调节氮源流速。
6、发酵50h结束发酵,将发酵液4℃、8000rpm离心,然后用水重悬沉淀,使得菌体湿重为660g/L,然后进行喷雾干燥,进料速度为1.2L/小时,进风温度110℃,频率为160赫兹,干燥后放出物料,即得到单细胞油脂产品。
对单细胞油脂产品取样,气相色谱分析各脂肪酸含量,检测得到:DHA含量为18.6%,DPA含量为12.7%,EPA含量为16.3%,亚油酸含量为14.2%,γ–亚麻酸含量为17.6%,花生四烯酸含量为12.3%。
实施例2
1、从超低温冰箱内取出一管解脂耶氏酵母(Yarrowia lipolytica)(购自中国工业微生物菌种保藏管理中心,编号:CICC 32291),放在手心快速解冻,在超净台内将解冻后的菌种液加入到装有种子培养基的摇瓶中,每100ml种子培养基接入200μl菌种液。
种子培养基:豆粕粉50g/L、甘油22g/L、酵母粉31g/L、酪蛋白胨62g/L、无氨基酵母氮源(YNB)4g/L、碘化钾0.03g/L、肌醇0.18g/L、核黄素0.38g/L、吡哆醇0.26g/L、硫酸亚铁0.31g/L、生物素60mg/L、泛酸钙700mg/L,余量为水,用盐酸或氢氧化钠调节pH为7。
2、接种后将摇瓶放入摇床中,33℃、150rpm培养18小时,得到种子液。
3、将种子液按照5%(v/v)的接种量接入装有2L发酵培养基的5L发酵罐中,初始OD600为3.8,在35℃进行发酵培养,初始转速为200rpm,初始通气量为0.2vvm,并随着溶氧的下降逐渐提高转速和通气量,保证溶氧为20%以上。
发酵培养基:玉米浆干粉38g/L、甘油42g/L、酵母粉52g/L、硫酸镁16g/L、维生素B124g/L、甜菜碱18g/L、硫酸铵65g/L、磷酸二氢钾90g/L、磷酸氢二钠65g/L、硼酸7.5mL/L、MnSO4·7H2O 400mg/L,余量为水,用盐酸或氢氧化钠调节pH为8。
4、发酵24h时开始补料碳源和氮源,其中,碳源和氮源与实施例1相同,控制碳氮比为100:1,碳源流速固定为60ml/min(即,每分钟流加初始发酵体积的0.03%),并根据碳氮比调节氮源流速。
5、发酵36h时,添加100ml浓度为0.8g/L的乙酰CoA羧化酶的水溶液和100ml浓度为1.2g/L的甘油三酯的水溶液作为诱导剂,乙酰CoA羧化酶和甘油三酯的终浓度分别为0.03g/L和0.05g/L,对微生物的油脂分泌代谢网络进行干预,激活相应代谢途径的表达,从而将多余的碳源代谢转化为油脂储存在菌体细胞内。发酵温度调整为30℃,碳氮比调整为200:1,碳源流速固定为60ml/min(即,每分钟流加初始发酵体积的0.03%),并根据碳氮比调节氮源流速。
6、发酵50h结束发酵,将发酵液4℃、8000rpm离心,然后用水重悬沉淀,使得菌体湿重为480g/L,然后进行喷雾干燥,进料速度为2.4L/小时,进风温度125℃,频率为900赫兹,干燥后放出物料,即得到单细胞油脂产品。
对单细胞油脂产品取样,气相色谱分析各脂肪酸含量,检测得到:DHA含量为17.8%,DPA含量为11.4%,EPA含量为15.8%,亚油酸含量为13.7%,γ–亚麻酸含量为18.2%,花生四烯酸含量为14.1%。
Claims (10)
1.一种单细胞油脂生产工艺,包括如下步骤:
将产油脂微生物进行发酵培养,发酵20-26h时开始补料碳源和氮源,控制碳氮比为76-100:1,其中碳源的流速为每分钟流加初始发酵体积的0.02-0.03%,并根据碳氮比调节氮源流速;
发酵34-38h时,添加乙酰CoA羧化酶和甘油三酯作为诱导剂诱导油脂的合成,使得乙酰CoA羧化酶和甘油三酯的终浓度分别为0.02-0.05g/L和0.04-0.08g/L,碳氮比调整为200-232:1,其中碳源的流速为每分钟流加初始发酵体积的0.02-0.03%,并根据碳氮比调节氮源流速;
发酵至少45h,结束发酵,将发酵液离心,得到沉淀;将所述沉淀进行干燥,得到单细胞油脂产品。
2.根据权利要求1所述的方法,其特征在于:所述发酵培养使用的发酵培养基组成为:玉米浆干粉12-38g/L、甘油26-42g/L、酵母粉36-52g/L、硫酸镁1.8-16g/L、维生素B1 5.6-24g/L、甜菜碱6.5-18g/L、硫酸铵22-65g/L、磷酸二氢钾30-90g/L、磷酸氢二钠10-65g/L、硼酸5-7.5mL/L、MnSO4·7H2O 200-400mg/L,pH为6-8。
3.根据权利要求1或2所述的方法,其特征在于:所述发酵培养的初始OD600为3-4。
4.根据权利要求1-3任一项所述的方法,其特征在于:所述发酵培养中溶氧为20%以上。
5.根据权利要求1-4任一项所述的方法,其特征在于:所述补料的碳源为甘油;和/或
所述补料的氮源为200-300g/L的硫酸铵水溶液。
6.根据权利要求1-5任一项所述的方法,其特征在于:所述发酵培养过程中,添加所述诱导剂之前控制发酵温度为32-35℃,添加所述诱导剂之后控制发酵温度为26-30℃。
7.根据权利要求1-6任一项所述的方法,其特征在于:所述干燥为喷雾干燥。
8.根据权利要求1-7任一项所述的方法,其特征在于:在所述发酵培养之前还有种子培养的步骤,所述种子培养使用的种子培养基组成为:豆粕粉20-50g/L、甘油8-22g/L、酵母粉8-31g/L、酪蛋白胨40-62g/L、无氨基酵母氮源0.8-4g/L、碘化钾0.013-0.03g/L、肌醇0.12-0.18g/L、核黄素0.22-0.38g/L、吡哆醇0.11-0.26g/L、硫酸亚铁0.12-0.31g/L、生物素20-60mg/L、泛酸钙400-700mg/L,pH为5-7。
9.根据权利要求1-8任一项所述的方法,其特征在于:所述产油脂微生物选自由解脂耶氏酵母(Yarrowia lipolytica)、酿酒酵母(Saccharomyces cerevisiae)、热带假丝酵母(Candida tropicalis)组成的组。
10.根据权利要求1-9任一项所述的方法,其特征在于:所述单细胞油脂为DHA、DPA、EPA、亚油酸、γ–亚麻酸和花生四烯酸中的一种或多种。
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