WO2020038296A1 - 微生物油脂及其提取方法 - Google Patents

微生物油脂及其提取方法 Download PDF

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WO2020038296A1
WO2020038296A1 PCT/CN2019/101034 CN2019101034W WO2020038296A1 WO 2020038296 A1 WO2020038296 A1 WO 2020038296A1 CN 2019101034 W CN2019101034 W CN 2019101034W WO 2020038296 A1 WO2020038296 A1 WO 2020038296A1
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oil
microbial
enzymolysis
sterile
lye
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PCT/CN2019/101034
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French (fr)
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瞿瀚鹏
曹晟
王身健
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梁云
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    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B1/00—Production of fats or fatty oils from raw materials
    • C11B1/02—Pretreatment
    • C11B1/025—Pretreatment by enzymes or microorganisms, living or dead
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B1/00—Production of fats or fatty oils from raw materials

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  • the invention relates to the field of extraction of microbial oils and fats, in particular to a method for extracting microbial oils and fats and microbial oils obtained by the method.
  • Microbial oils and fats are produced by oil-producing microorganisms such as fungi and bacteria by biosynthesis and transformation of nitrogen sources, carbon sources, and trace elements in the medium.
  • the resulting oils are all enveloped by cell walls, and microbial oils are commonly referred to as intracellular oils.
  • intracellular oils In order to obtain such intracellular lipids, the cell walls of these microorganisms must be cracked, so the degree of cell wall cracking has become a key factor in the efficiency of oil extraction.
  • the dry extraction process has the problems of high oxidation loss caused by high-temperature dehydration, the need for organic solvent extraction, and high energy consumption. Therefore, more and more microbial oils are extracted by a wet extraction process.
  • the cell wall is usually broken by enzymatic hydrolysis, mechanical disruption, and thermal dissolution.
  • the extraction efficiency of microbial fats and oils by the wet process is low, and the content of extracellular toxins in the microbial fats and oils is relatively high, while the content of DHA (docosahexaenoic acid) and ARA (arachidonic acid) is low.
  • An object of the present invention is to overcome the above-mentioned problems in the prior art, and provide a method for extracting microbial oils and fats and microbial oils obtained thereby.
  • the method for extracting microbial oils and oils has high extraction efficiency for microbial oils and fats and the obtained microbial oils and fats. Low extracellular toxin content, high DHA or ARA content, and high safety.
  • the inventors of the present invention discovered during the research that the main reasons for the low extraction efficiency, high extracellular toxin, and low DHA or ARA content of microbial oils and fats obtained by the existing wet extraction process are that in the current process methods, it is necessary to concentrate the fermentation Substances such as enzyme preparations and pH adjusters are introduced into the solution, and the preparations of enzyme preparations and pH adjusters are usually added directly to the fermentation broth after concentration, which will cause contamination of the fermentation broth by foreign bacteria.
  • the entire process takes more than 20 hours from the adjustment of pH, the preparation and dosing of enzyme preparations, the implementation of enzymolysis, to the post-heating treatment.
  • Bacterial microorganisms multiply very quickly, usually one generation every 20 minutes.
  • the amount of bacteria in the fermentation broth rich in carbon and nitrogen is very large.
  • the enzymatic hydrolysis at the later stage can kill a certain amount of microorganisms, microorganisms such as bacteria will secrete a large amount of exotoxin in the process.
  • Thermal heating can only kill living organisms of bacterial microorganisms, and exotoxin is a type Protein, heat has a limited effect on it, and spore exotoxin enters the fat as a protein fragment or peptide, which becomes a factor affecting the safety of the fat.
  • exotoxin is a type Protein
  • spore exotoxin enters the fat as a protein fragment or peptide, which becomes a factor affecting the safety of the fat.
  • due to the presence of a large number of foreign bacteria it will compete with oil-producing microorganisms during the treatment process, thereby weakening the treatment effect on oil-producing microorganisms, causing a large amount of foreign bacteria's intracellular substances to enter the obtained microbial oils, resulting in
  • the extraction efficiency of microbial oils and fats is low, and the content of main components such as DHA and ARA is reduced.
  • the fermentation broth produces a malodor during the treatment process, which affects the production environment.
  • one aspect of the present invention provides a method for extracting microbial oils and fats, which method includes:
  • step (2) The three-phase separation of the oil phase, the water phase, and the solid phase of the mixed liquid containing the microbial oil and fat obtained in step (1) to obtain a microbial oil and fat.
  • the extracellular matrix of an animal is a cell wall in a sense, and its chemical composition is collagen, adhesion protein, amino polysaccharide, and proteoglycan.
  • the main component of bacterial cell wall is peptidoglycan.
  • the main components in the fungal cell wall are chitin, cellulose, dextran, mannan, etc. These polysaccharides are all polymers of monosaccharides.
  • the plant cell wall is mainly cellulose, hemicellulose and pectin, and there is also a large amount of lignin in the secondary cell wall.
  • the oil-producing microorganisms are bacteria, fungi, yeast, and algae.
  • the enzymatic hydrolysis of the cell walls of oil-producing microorganisms is usually limited to cellulase, hemicellulase, pectinase, snail enzyme, and chitin. Enzymes, ligninase, etc.
  • the breaking performance of the enzyme preparations is not stable. Whether liquid or solid enzymes have large fluctuations in performance, which makes it difficult to grasp the production operation.
  • the inventor of the present invention unexpectedly discovered during the research that by introducing an alkaline protease into the enzyme preparation, not only the excellent enzymatic hydrolysis efficiency can be ensured, but also the enzymatic hydrolysis process can be performed stably, thereby further improving the extraction efficiency of microbial oils and fats. Decrease the content of extracellular toxins and increase the content of DHA or ARA.
  • the cell wall lyase includes alkaline protease and optionally other enzymes, the other enzymes are cellulase, hemicellulase, pectinase, snail enzyme, chitinase and ligninase. At least one of.
  • the second aspect of the present invention provides a microbial fat prepared by the method as described above, the microbial fat is hair oil, and the DHA (docosahexaenoic acid) content of the hair oil is greater than 35% by weight or Tetraenoic acid) content is greater than 35% by weight, extracellular toxin content is less than 50 ⁇ g / kg, anisidine value is less than 25, and peroxide value is less than 20 meq / kg.
  • DHA docosahexaenoic acid
  • Tetraenoic acid Tetraenoic acid
  • FIG. 1 is a flowchart of a specific embodiment of the present invention.
  • the present invention provides a method for extracting microbial oils and fats, the method comprising:
  • step (2) The three-phase separation of the oil phase, the water phase, and the solid phase of the mixed liquid containing the microbial oil and fat obtained in step (1) to obtain a microbial oil and fat.
  • the sterile environment means that the enzymatic system is in a sterile environment except for the oil-producing microorganisms contained in the fermentation broth.
  • the fermentation broth of the oil-producing microorganism is a fermentation broth obtained directly after the fermentation of the oil-producing microorganism, and does not need to undergo any further treatment. Therefore, compared with the prior art, the method of the present application also saves the fermentation broth Concentration, oil-producing microorganism inactivation steps, etc.
  • a method for preparing a fermentation broth of an oil-producing microorganism is well known to those skilled in the art, for example, inoculating an oil-producing microorganism into a fermentation medium for fermentation, thereby obtaining a fermentation broth of an oil-producing microorganism.
  • the oil-producing microorganisms can be various existing oil-producing microorganisms.
  • the oil-producing microorganisms can be any one of bacteria, molds, yeasts, and algae.
  • the oil-producing microorganisms are molds, yeasts, and algae. Any of them.
  • Examples of the molds may include, but are not limited to, Asoergullus terreus, Clavicepspurpurea, Tolyposporium, Mortierella alpina, and Mortierella Mortierella isabibellina; examples of the yeast may include, but are not limited to, Cryptococcus albidus, Cryptococcus albidun, Lipomyces, and Mycelium Trichospiron pullulans, Lipomy slipperer, Rhodotorula giutinis and Rhodosporidium tortorides; examples of the algae may include, but are not limited to, thraustochytrids (Thraustochytriales), Schizochytrium, Crypthecodinium, diatom, Spirulina, and Wokenella.
  • the cell wall lyase comprises an alkaline protease.
  • the cell wall lyase may further include at least one of a cellulase, a hemicellulase, a pectinase, a snail enzyme, a chitinase, and a ligninase.
  • the amount of the cell wall lyase can be selected in a wide range, as long as the cell wall of the oil-producing microorganism can be fully lysed, thereby releasing the microbial oil and fat.
  • the amount of the cell wall lyase is 1-5 g per liter of the fermentation broth.
  • the conditions for the enzymolysis may be those conventionally used for lysing the cell wall of an oil-producing microorganism by enzymatic hydrolysis, but the inventors of the present invention have found that by performing the enzymolysis under aerobic conditions, The enzymolysis efficiency is further improved, thereby improving the extraction efficiency of microbial oils and fats, and the DHA or ARA of the obtained microbial oils and fats.
  • the conditions for the enzymolysis include: a pH value of 8-10 (for example, it can be 8, 8.5, 9, 9.5, and 10), and a temperature of 40-60 ° C (for example, it can be 40 ° C, 45 °C, 50 °C, 55 °C, 60 °C), the pressure is 0.02-0.05MPa (for example, it can be 0.02MPa, 0.03MPa, 0.04MPa, 0.05MPa), and the ventilation volume is 0.2-0.6VVM (per unit volume per minute per minute The amount of gas passed through the fermentation broth is 0.2-0.6 volume) (for example, it can be 0.2VVM, 0.3VVM, 0.4VVM, 0.5VVM, 0.6VVM), and the time is 4-15 hours (for example, it can be 4 hours, 5 hours) , 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours).
  • a pH value of 8-10 for example, it can be 8, 8.5, 9,
  • the enzymatic hydrolysis is performed under the condition of stirring, and the stirring speed may be 8-30 rpm.
  • the pH value of the enzymatic hydrolysis system can be controlled to 8-10 by adding lye to the fermentation broth.
  • the selection of the lye is not particularly limited, as long as it can complete the adjustment of the pH and does not cause side reactions with the generated microbial oils and fats.
  • the lye may include, but are not limited to, at least one of a sodium oxide solution, a potassium hydroxide solution, a sodium carbonate solution, a sodium bicarbonate solution, a potassium carbonate solution, a potassium bicarbonate solution, and ammonia water.
  • the concentration of the lye is not particularly limited in the present invention, as long as the pH can be adjusted. For example, the concentration of the lye can be 15-25% by weight.
  • the lye is a food-grade lye.
  • the enzymolysis can be carried out directly in a fermentation tank.
  • the lye is preferably transported to the fermentation tank through a sterile pipeline after being sterilized, and the cell wall is lysed.
  • the enzyme is formulated into an enzyme solution and then sterilized and then transferred to the fermentation tank through a sterile pipeline.
  • the enzyme solution can be prepared by mixing the enzyme with water in a ratio of 1: 5-10.
  • the method for sterilizing the lye and the pipeline for transmitting the lye and cell wall lyase can be various sterilization methods known in the art, for example, a filtration method, an ozone disinfection method, and a high temperature saturation method. Steam method.
  • the lye and the pipeline for delivering the lye and cell wall lyase are preferably sterilized by a method of high temperature saturated steam.
  • the pressure of the high-temperature saturated steam may be 0.1-0.35 MPa
  • the temperature may be 121-145 ° C
  • the sterilization time may be 40-80 min.
  • the conventional method of aseptic filtration of the present invention can be adopted for the method of the cell wall lyase, for example, the bacteria can be sterilized by using a filtration system for filtering the sterile liquid of the filtration system.
  • the aperture of the device can be 0.15-0.25 ⁇ m.
  • the filtering system further includes a step of sterilizing the filter system before use.
  • the sterilization method may be a method of ozone disinfection, a method of high temperature saturated steam, and other methods known in the art.
  • the filtering system is preferably sterilized by a method of high temperature saturated steam.
  • the pressure of the high-temperature saturated steam may be 0.1-0.35 MPa
  • the temperature may be 121-145 ° C
  • the sterilization time may be 40-80 min.
  • the enzymolysis can also be performed in an enzymolysis tank.
  • the lye is preferably transported to the enzymolysis tank through a sterile pipeline after being sterilized.
  • the cell wall lyase is formulated into an enzyme solution, sterilized by a sterile liquid filter, and then transferred to the enzymolysis tank through a sterile pipeline, and the fermentation broth is transferred to the enzymolysis tank through a sterile pipeline.
  • the enzyme solution can be prepared by mixing the enzyme with water in a ratio of 1: 5-10.
  • the method for sterilizing the lye and the pipeline for conveying the lye, the cell wall lyase and the fermentation broth may be various sterilization methods known in the art, for example, a filtration method, an ozone disinfection method , Method of high temperature saturated steam.
  • the lye and the pipeline for conveying the lye, the cell wall lyase and the fermentation broth are preferably sterilized by a method of high temperature saturated steam.
  • the pressure of the high-temperature saturated steam may be 0.1-0.35 MPa
  • the temperature may be 121-145 ° C
  • the sterilization time may be 40-80 min.
  • the conventional method of sterilization at normal temperature of the present invention can be used for the method of the cell wall lyase, for example, the bacteria can be sterilized by using a filtration system, which is a liquid sterile filter.
  • the pore size can be 0.15-0.25 ⁇ m.
  • the filtering system further comprises a step of sterilizing the filter system before use.
  • the sterilization method may be a method of ozone sterilization, a method of high temperature saturated steam, and other methods known in the art.
  • the filtering system is preferably sterilized by a method of high temperature saturated steam.
  • the pressure of the high-temperature saturated steam may be 0.1-0.35 MPa
  • the temperature may be 121-145 ° C
  • the sterilization time may be 40-80 min.
  • Preparation of lye add a certain amount of water to the alkali tank, weigh NaOH according to the concentration of 20% by weight, dissolve it in the alkali tank, and sterilize the prepared lye in high-temperature saturated steam for 1 h. Pass the tank with sterile compressed air to make the tank pressure of the alkali tank be 0.02-0.05 MPa, and pass cooling water into the jacket of the alkali tank to cool the alkali solution to 35-45 ° C and reserve.
  • the conditions for the solid-liquid separation may include: a temperature of 80-95 ° C, a rotation speed of 5000-8000 rpm, and a time of 15-20 hours.
  • the present invention provides a microbial oil and fat prepared by the method described above, the microbial oil and fat is a hair oil, the DHA content of the hair oil is greater than 35% by weight, or the ARA content is greater than 35% by weight, extracellular
  • the toxin content is less than 50 ⁇ g / kg, the anisidine value is less than 25, and the peroxide value is less than 20 meq / kg.
  • Alkaline protease was purchased from Danisco, PD 216661-7.0CHN;
  • Pectinase was purchased from Dongheng Huadao Biotechnology Co., Ltd., P128776;
  • Cellulase was purchased from Jiangsu Yihaotian Biotechnology Co., Ltd., article number 232-734-4;
  • Oil, water, solid three-phase separation system was purchased from Jiangsu Yixing Huading Grain and Oil Machinery Co., Ltd., article number BTSD95
  • the oil-producing microbial fermentation broth 1 is a fermented broth obtained by fermenting mildew in high mountains, mainly containing ARA;
  • the oil-producing microorganism fermentation broth 2 is a fermentation broth obtained by Schizochytrium fermentation, which mainly contains DHA;
  • the DHA content in the obtained crude oil was determined by gas chromatography according to the method of GB26400-2011;
  • EPA content was determined by gas chromatography in accordance with the method of GB5009.168-2016;
  • ARA content was measured by gas chromatography according to GB26401-2011 method
  • the extracellular toxin content was determined by high performance liquid chromatography according to the method of GB5009.22-2016;
  • Anisidine value was measured by GB / T 24304-2009 method
  • Peroxide value is measured by ultraviolet spectrophotometer according to GB / T24304-2009 method
  • the yield of microbial oils and fats is measured by a method of extraction and weighing with a rotary evaporator;
  • the residual oil ratio in the solid residue was measured by a rotary evaporator extraction weighing method.
  • Preparation of lye Add 200L of demineralized water to the alkali tank, weigh out food-grade NaOH at a concentration of 20% by weight, and dissolve it in the alkali tank. Place the prepared lye at a pressure of 0.14MPa and temperature Sterilize in saturated steam at 145 ° C for 1 hour, then pass sterile compressed air into the alkali tank to make the tank pressure of the alkali tank be 0.02-0.05MPa, and pass cooling water into the jacket of the alkali tank to cool the alkali solution to 35-45 °C, spare;
  • the solid phase, oil phase and water phase are separated through a three-phase separation system.
  • the feed liquid is heated to 90 ° C, the centrifuge speed is 6700 rpm, and the centrifugation time is 18 hours. See Table 1 for microbial oils and fats, extraction efficiency of oils and fats, residual oil rate in solid phase residues, ARA content in oils and fats, EPA content, extracellular toxin content, anisidine value, and peroxide value.
  • Preparation of lye Add 200L of demineralized water to the alkali tank, weigh food-grade sodium carbonate at a concentration of 15% by weight, and dissolve it in the alkali tank. Place the prepared lye at a pressure of 0.14MPa. Sterilize in saturated steam at 145 ° C for 1 hour, then pass sterile compressed air into the alkali tank to make the tank pressure of 0.02-0.05MPa, and pass cooling water into the jacket of the alkali tank to cool the alkali solution. To 35-45 °C, spare;
  • the solid phase, oil phase and water phase are separated through a three-phase separation system.
  • the feed liquid is heated to 95 ° C, the centrifuge speed is 6000 rpm, and the centrifugation time is 13 hours. See Table 1 for microbial oils and fats, extraction efficiency of oils and fats, residual oil rate in solid phase residues, ARA content in oils and fats, EPA content, extracellular toxin content, anisidine value, and peroxide value.
  • Preparation of lye Add 200L of demineralized water to the alkali tank, weigh out food-grade sodium bicarbonate at a concentration of 25% by weight, and dissolve it in the alkali tank. Place the prepared lye at a pressure of 0.14MPa , Sterilize in saturated steam at 145 °C for 1h, then pass sterile compressed air into the alkali tank to make the tank pressure of the alkali tank be 0.02-0.05MPa, and pass cooling water into the jacket of the alkali tank to lye Cool to 35-45 ° C and reserve;
  • the solid phase, oil phase and water phase are separated by a three-phase separation system.
  • the feed liquid is heated to a temperature of 85 ° C, the centrifuge speed is 7500 rpm, and the centrifugation time is 20 hours.
  • Table 1 for the microbial oils and fats of crude oil, extraction efficiency of oils and fats, residual oil rate in solid phase residues, ARA content in oils and fats, EPA content, extracellular toxin content, anisidine value, and peroxide value.
  • the microbial oil and fat preparation was performed according to the method of Example 1, except that the oil-producing microorganism fermentation broth 1 in step 3 was replaced with the oil-producing microorganism fermentation broth 2.
  • the extraction efficiency of oils and fats, the residual oil rate in solid residues, the DHA content in oils and fats, the EPA content, the extracellular toxin content, the anisidine value, and the peroxide value are shown in Table 1.
  • the microbial oil and fat preparation was performed according to the method of Example 2, except that the oil-producing microbial fermentation broth 1 in step 3 was replaced with the oil-producing microbial fermentation broth 2.
  • the extraction efficiency of oils and fats, the residual oil rate in solid residues, the DHA content in oils and fats, the EPA content, the extracellular toxin content, the anisidine value, and the peroxide value are shown in Table 1.
  • the microbial oil and fat preparation was carried out according to the method of Example 3, except that the oil-producing microorganism fermentation broth 1 in step 3 was replaced with the oil-producing microorganism fermentation broth 2.
  • the extraction efficiency of oils and fats, the residual oil rate in solid residues, the DHA content in oils and fats, the EPA content, the extracellular toxin content, the anisidine value, and the peroxide value are shown in Table 1.
  • the microbial oil was prepared according to the method of Example 1. The difference was that the enzyme used for enzymolysis was replaced with snail enzyme, cellulase and pectinase. The amount of enzyme used per liter of fermentation broth was 2.5g of cellulase. The results are shown in Table 1 with 1.5 g of pectinase and 1 g of snail enzyme.
  • This comparative example is used to explain the reference microbial oil and fat and its preparation method
  • the preparation of microbial oils and fats was performed according to the method of Example 1. The difference is that the lye, the enzymatic hydrolysis solution, and the pipelines that transport the lye and the enzymatic hydrolysis solution did not pass the sterilization. The results are shown in Table 1.
  • This comparative example is used to explain the reference microbial oil and fat and its preparation method
  • the microbial oil and fat preparation was performed according to the method of Example 4, except that the lye, the enzymatic hydrolysis solution, and the pipelines for conveying the lye and the enzymatic hydrolysis solution did not pass the sterilization.
  • the results are shown in Table 1.
  • the degree of enzymatic hydrolysis is usually measured by the following two methods:

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Abstract

微生物油脂及其提取方法，具体步骤是：(1)在无菌环境下，将产油微生物的发酵液与细胞壁裂解酶接触，以对发酵液中的产油微生物进行酶解，获得含有微生物油脂的混合液；(2)将含有微生物油脂的混合液进行油相、水相和固相的三相分离，得到微生物油脂。通过将整个工艺过程控制在无菌环境下操作，可以杜绝微生物油脂提取过程中杂菌污染，避免杂菌毒素迁移到产品中，增加微生物油脂的安全性，且所得作为毛油的微生物油脂的收率可提高到97-98％；毛油中的DHA含量或ARA也含量得到了显著的提高。

Description

微生物油脂及其提取方法 技术领域
本发明涉及微生物油脂的提取领域，具体涉及一种微生物油脂的提取方法，以及由此方法提取得到的微生物油脂。
背景技术
微生物油脂是由产油微生物如真菌、细菌等将培养基中氮源、碳源、微量元素等进行生物合成与转化而来。产生的油脂均被细胞壁所包裹，通常称微生物油脂为胞内油脂。为得到该类胞内油脂，必须将这些微生物的细胞壁予以破解，所以细胞壁破解的程度高低成了油脂提取效率高低的关键因素。
目前，微生物油脂提取主要有湿法、干法两种提取工艺。干法提取工艺存在高温脱水导致油脂氧化损失大、需要采用有机溶剂萃取、能耗高等问题。因此，越来越多的微生物油脂的提取采用湿法的提取工艺，在湿法工艺中，通常是将细胞壁采用酶解、机械破碎、热力溶解等方法予以破壁。但目前采用湿法工艺微生物油脂提取效率较低，且得到的微生物油脂中胞外毒素含量较高、而DHA(二十二碳六烯酸)、ARA(花生四烯酸)含量较低。
发明内容
本发明的目的是为了克服现有技术存在的上述问题，提供一种微生物油脂的提取方法及由此得到的微生物油脂，该微生物油脂的提取方法对微生物的油脂提取效率高，且得到的微生物油脂胞外毒素含量低、DHA或者ARA含量较高，具有较高的安全性。
本发明的发明人在研究的过程中发现，现有湿法提取工艺得到的微生物油脂提取效率低、胞外毒素高、DHA或者ARA含量低的主要原因在于，目前工艺方法中，需要向发酵浓缩液中引进酶制剂、pH调节剂等物质，而酶制剂、pH调节剂等物质配制后通常会直接加入浓缩处理的发酵液中，因此会导致外来细菌对发酵液的污染。
一般的，从pH的调整、酶制剂的准备与投加、酶解的实施、再到后期升温处理，整个过程需要耗时20多小时。而细菌微生物的繁殖速度非常快，一般每20分钟就繁殖一代，在整个提取油脂的过程中，历经20多小时，在富含碳氮源的发酵液中细菌繁殖量非常大。同时，虽然后期的升温酶解能杀灭一定量的微生物，但细菌等微生物在此过 程中将分泌大量的孢外毒素，热力升温只能杀死细菌微生物的活体，而孢外毒素是一类蛋白质，热力对其作用有限，孢外毒素作为蛋白质片段或肽的形式进入油脂中，成为影响油脂安全的因素。此外，由于外来细菌的大量存在，在处理过程中会与产油微生物形成竞争，从而削弱了对产油微生物的处理效果，导致外来细菌的胞内物大量进入得到的微生物油脂中，从而造成了微生物油脂提取效率的低下，以及DHA、ARA等主要组分含量的降低。另外，因为细菌的感染与大量繁殖，导致发酵液在处理过程中产生恶臭，影响生产环境。
基于如上的发现，为了实现上述目的，本发明一方面提供一种微生物油脂的提取方法，该方法包括：
(1)在无菌环境下，将产油微生物的发酵液与细胞壁裂解酶接触，以对发酵液中的产油微生物进行酶解，获得含有微生物油脂的混合液；
(2)将步骤(1)得到的含有微生物油脂的混合液进行油相、水相和固相的三相分离，得到微生物油脂。
本领域技术人员公知，(1)动物的细胞外基质从某种意义上说也就是细胞壁，其化学组成是胶原蛋白、粘连蛋白、氨基多糖及蛋白聚糖。(2)细菌细胞壁主要成分则是肽聚糖。(3)真菌细胞壁中主要成分为几丁质、纤维素、葡聚糖、甘露聚糖等，这些多糖都是单糖的聚合物。(4)植物细胞壁主要为纤维素、半纤维素和果胶类，次生细胞壁中还有大量木质素。而通常情况下，产油微生物为细菌、真菌、酵母和藻类，因此，目前对于产油微生物细胞壁的酶解通常局限于纤维素酶、半纤维素酶、果胶酶、蜗牛酶、几丁质酶、木质素酶等。但目前采用的酶制剂将产油微生物细胞壁破解过程中，酶制剂的破壁性能并不稳定，无论是液态还是固态酶，其性能波动很大，导致生产操作把握困难。本发明的发明人在研究的过程中意外发现，通过在酶制剂中引入碱性蛋白酶，不仅能够保证优异的酶解效率，还可以使得酶解过程稳定进行，从而进一步提高微生物油脂的提取效率，降低胞外毒素的含量、提高DHA或者ARA的含量。
因此，优选地，所述细胞壁裂解酶包括碱性蛋白酶以及可选的其他酶，所述其他酶为纤维素酶、半纤维素酶、果胶酶、蜗牛酶、几丁质酶和木质素酶中的至少一种。
本发明第二方面提供如上所述的方法制备的微生物油脂，所述微生物油脂为毛油，所述毛油的DHA(二十二碳六烯酸)含量大于35重量％或者ARA(二十碳四烯酸)含量大于35重量％、胞外毒素含量小于50μg/kg、茴香胺值小于25、过氧化值小于20meq/kg。
通过上述技术方案，通过将整个工艺过程控制在无菌环境下操作，可以杜绝微生 物油脂提取过程中杂菌污染，避免杂菌毒素迁移到产品中，增加微生物油脂的安全性，且所得作为毛油的微生物油脂的收率可提高到97-98％，与同行相比有了明显的提高；此外，毛油中的DHA含量或者ARA含量得到了显著的提高，胞外毒素的含量、过氧化值和茴香胺值得到了显著的降低。
附图说明
图1是本发明一种具体实施方式的流程图。
具体实施方式
在本文中所披露的范围的端点和任何值都不限于该精确的范围或值，这些范围或值应当理解为包含接近这些范围或值的值。对于数值范围来说，各个范围的端点值之间、各个范围的端点值和单独的点值之间，以及单独的点值之间可以彼此组合而得到一个或多个新的数值范围，这些数值范围应被视为在本文中具体公开。
第一方面，本发明提供了一种微生物油脂的提取方法，该方法包括：
(1)在无菌环境下，将产油微生物的发酵液与细胞壁裂解酶接触，以对发酵液中的产油微生物进行酶解，获得含有微生物油脂的混合液；
(2)将步骤(1)得到的含有微生物油脂的混合液进行油相、水相和固相的三相分离，得到微生物油脂。
本发明在此需要说明的是，所述无菌的环境是指除了发酵液中含有的产油微生物，所述酶解的体系处于无菌的环境中。
根据本发明，所述产油微生物的发酵液为产油微生物发酵后直接所得的发酵液，并不需要再经过任何的处理，因此，相对于现有技术，本申请的方法还节省了发酵液的浓缩、产油微生物的灭活步骤等。
根据本发明，制备所述产油微生物的发酵液的方法为本领域技术人员所公知，例如，将产油微生物接种至发酵培养基中进行发酵，从而得到产油微生物的发酵液。
其中，所述产油微生物可以为现有的各种产油微生物，例如，可以为细菌、霉菌、酵母和藻类中的任意一种，优选的，所述产油微生物为霉菌、酵母和藻类中的任意一种。其中，所述霉菌的实例可以包括但并不限于土霉菌(Asoergullus terreus)、紫瘫麦角菌(Clavicepspurpurea)、高梁褶抱黑粉菌(Tolyposporium)、高山被孢霉(Mortierella alpina)和深黄被孢霉(Mortierella isabellina)；所述酵母的实例可以包括但并不限于浅白色隐球 酵母(Cryptococcus albidus)、弯隐球酵母(Cryptococcus albidun)、斯达氏油脂酵母(Lipomyces)、茁芽丝孢酵母(Trichospiron pullulans)、产油油脂酵母(Lipomy slipofer)、胶粘红酵母(Rhodotorula giutinis)和圆红冬孢酵母菌(Rhodosporidium toruloides)；所述藻类的实例可以包括但并不限于破囊壶菌(Thraustochytriales)、裂殖壶菌(Schizochytrium)、隐甲藻(Crypthecodinium)、硅藻(diatom)、螺旋藻(Spirulina)和吾肯氏菌属。
根据本发明一种优选的实施方式，所述细胞壁裂解酶包括碱性蛋白酶。此外，优选的，所述细胞壁裂解酶还可以包括纤维素酶、半纤维素酶、果胶酶、蜗牛酶、几丁质酶和木质素酶中的至少一种。
根据本发明，所述细胞壁裂解酶的用量可以在较宽的范围内进行选择，只要能够将产油微生物的细胞壁进行充分裂解，从而将微生物油脂释放出来即可。优选的，相对于每升所述发酵液，所述细胞壁裂解酶的用量为1-5g。
根据本发明，所述酶解的条件可以为现有的通过酶解裂解产油微生物细胞壁常规使用的条件，但本发明的发明人发现，通过在有氧的条件下进行所述酶解，能够进一步提高酶解效率，从而提高微生物油脂的提取效率，提高所得微生物油脂的DHA或ARA。因此，优选的，所述酶解的条件包括：pH值为8-10(例如，可以为8、8.5、9、9.5、10)，温度为40-60℃(例如，可以为40℃、45℃、50℃、55℃、60℃)，压力为0.02-0.05MPa(例如，可以为0.02MPa、0.03MPa、0.04MPa、0.05MPa)，通气量为0.2-0.6VVM(每分钟每单位体积的发酵液通入的气体量为0.2-0.6体积)(例如，可以为0.2VVM、0.3VVM、0.4VVM、0.5VVM、0.6VVM)，时间为4-15小时(例如，可以为4小时、5小时、6小时、7小时、8小时、9小时、10小时、11小时、12小时、13小时、14小时、15小时)。
根据本发明，为了进一步提高酶与产油微生物的接触，从而酶解效率，所述酶解在搅拌的条件下进行，所述搅拌的速度可以为8-30rpm。
其中，可以通过向发酵液中添加碱液以将酶解体系的pH值控制在8-10。本发明对于所述碱液的选择并没有特别的限定，只要能够完成pH的调节并且不与产生的微生物油脂发生副反应即可。所述碱液的实例可以包括但不限于氧化钠溶液、氢氧化钾溶液、碳酸钠溶液、碳酸氢钠溶液、碳酸钾溶液、碳酸氢钾溶液和氨水中的至少一种。其中，所述碱液的浓度本发明并没有特别的限制，只要能够实现pH值的调节即可，例如，所述碱液的浓度可以为15-25重量％。
优选的，为了保证最终所得微生物油脂的安全性，所述碱液为食品级碱液。
根据本发明，所述酶解可以直接在发酵罐中进行，为了保证体系的无菌环境，所述碱液优选经过灭菌后通过无菌管路输送至所述发酵罐中，所述细胞壁裂解酶配制成酶溶液后经过灭菌后通过无菌管路输送至所述发酵罐中。其中，所述酶溶液可以通过将酶与水按照1:5-10的比例配制。
其中，对所述碱液和输送碱液和细胞壁裂解酶的管路进行灭菌的方法可以为本领域公知的各种灭菌方法，例如，可以通过过滤的方法、臭氧消毒的方法、高温饱和蒸汽的方法。本发明优选通过高温饱和蒸汽的方法对所述碱液以及输送碱液和细胞壁裂解酶的管路进行灭菌。所述高温饱和蒸汽的压强可以为0.1-0.35MPa，温度可以为121-145℃，灭菌时间可以为40-80min。
其中，为了保证细胞壁裂解酶的活性，对于所述细胞壁裂解酶的方法可以采用本发明常规的无菌过滤方法，例如，可以通过使用过滤系统进行过滤除菌，所述过滤系统的无菌液体过滤器孔径可以为0.15-0.25μm。优选的，所述过滤系统在使用前，还包括对其进行灭菌的步骤，所述灭菌的方法可以为臭氧消毒的方法、高温饱和蒸汽的方法等本领域公知的方法。本发明优选通过高温饱和蒸汽的方法对所述过滤系统进行灭菌。所述高温饱和蒸汽的压强可以为0.1-0.35MPa，温度可以为121-145℃，灭菌时间可以为40-80min。
根据本发明，所述酶解还可以在酶解罐中进行，为了保证体系的无菌环境，所述碱液优选经过灭菌后通过无菌管路输送至所述酶解罐中，所述细胞壁裂解酶配制成酶溶液后经过无菌液体过滤器除菌后通过无菌管路输送至所述酶解罐中，所述发酵液通过无菌管路输送至所述酶解罐中。其中，所述酶溶液可以通过将酶与水按照1:5-10的比例配制。
其中，对所述碱液和输送碱液、细胞壁裂解酶和发酵液的管路进行灭菌的方法可以为本领域公知的各种灭菌方法，例如，可以通过过滤的方法、臭氧消毒的方法、高温饱和蒸汽的方法。本发明优选通过高温饱和蒸汽的方法对所述碱液以及输送碱液、细胞壁裂解酶和发酵液的管路进行灭菌。所述高温饱和蒸汽的压强可以为0.1-0.35MPa，温度可以为121-145℃，灭菌时间可以为40-80min。
其中，为了保证细胞壁裂解酶的活性，对于所述细胞壁裂解酶的方法可以采用本发明常规的常温灭菌法，例如，可以通过使用过滤系统进行过滤除菌，所述过滤系统液体无菌过滤器的孔径可以为0.15-0.25μm。优选的，所述过滤系统在使用前，还包括对其进行灭菌的步骤，所述灭菌的方法可以为臭氧消毒的方法、高温饱和蒸汽的方法等本 领域公知的方法。本发明优选通过高温饱和蒸汽的方法对所述过滤系统进行灭菌。所述高温饱和蒸汽的压强可以为0.1-0.35MPa，温度可以为121-145℃，灭菌时间可以为40-80min。
现参照图1对本发明一种具体的实施方式进行详细说明，具体的，
(1)碱液准备：在碱罐中加入定量的水，按20重量％的浓度称取NaOH投入碱罐中溶解，将配制好的碱液置于高温饱和蒸汽中灭菌1h，然后向碱罐中通入无菌压缩空气使碱罐的罐压为0.02-0.05MPa，并向碱罐的夹套中通入冷却水将碱液冷却到35-45℃，备用。
(2)细胞壁裂解液准备：将细胞壁裂解液与一定量的水混合，按酶：水＝1:(5-10)的比例配制酶解液，将无菌过滤系统用高温饱和蒸汽灭菌1h，然后用无菌压缩空气保压备用。
(3)酶解液与碱液的投加：先将发酵液的温度提升到35-55℃，然后通过无菌管道将上述碱液压入发酵罐中，使发酵液的pH为8-10；上述酶解液通过隔膜泵输进无菌过滤系统中过滤，然后通过无菌管道输送进入发酵罐中，进行发酵液的酶解，整个酶解时间为4-15h。
根据本发明，所述固液分离的条件可以包括：温度为80-95℃，转速为5000-8000rpm，时间为15-20小时。
第二方面，本发明提供了一种由上述所述的方法制备的微生物油脂，所述微生物油脂为毛油，所述毛油的DHA含量大于35重量％或者ARA含量大于35重量％、胞外毒素含量小于50μg/kg、茴香胺值小于25、过氧化值小于20meq/kg。
以下将通过实施例对本发明进行详细描述。以下实施例中，
碱性蛋白酶购自丹尼斯克，PD 216661－7.0CHN；
果胶酶购自东恒华道生物科技有限公司，P128776；
纤维素酶购自江苏宜昊添生物科技有限公司，货号232-734-4；
油、水、固的三相分离系统购自江苏宜兴华鼎粮油机械有限公司，货号BTSD95
产油微生物发酵液1为高山被饱霉发酵得到的发酵液，主要含ARA；
产油微生物发酵液2为裂殖壶菌发酵得到的发酵液，主要含DHA；
所得毛油中DHA含量通过气相色谱，按GB26400-2011方法进行测定；
EPA含量通过气相色谱，按GB5009.168-2016方法进行测定；
ARA含量通过气相色谱，按GB26401-2011方法进行测定；
胞外毒素含量通过高效液相色谱按GB5009.22-2016方法进行测定；
茴香胺值通过GB/T 24304-2009方法进行测定；
过氧化值通过紫外分光光度计，按GB/T24304-2009方法进行测定；
微生物油脂的收率通过旋转蒸发仪萃取称重的方法进行测定；
固相残渣中的残油率通过旋转蒸发仪萃取称重方法进行测定。
实施例1
本实施例用于说明本发明提供的微生物油脂及其制备方法
(1)碱液准备：在碱罐中加入200L的软化水，按照20重量％的浓度称取食品级的NaOH投入碱罐中充分溶解，将配制好的碱液置于压强为0.14MPa，温度为145℃饱和蒸汽中灭菌1h，然后向碱罐中通入无菌压缩空气使碱罐的罐压为0.02-0.05MPa，并向碱罐的夹套中通入冷却水将碱液冷却到35-45℃，备用；
(2)细胞壁裂解液准备：将串联好的二级液体无菌过滤系统用压强为0.14MPa，温度为145℃饱和蒸汽灭菌1h，然后用无菌压缩空气保压备用；所述无菌过滤系统的过滤器规格为孔径0.2μm，耐温150℃；称取碱性蛋白酶，并添加软化水按酶：水比例1:10配制酶解液。
(3)酶解液与碱液的投加：先将产油微生物发酵液1的温度提升到35-55℃，然后通过无菌碱液管道将上述碱液用压缩空气压入发酵罐中，用“梅特勒”pH探头测定发酵液的pH，直到发酵液pH为9时停止加入碱液；上述酶解液通过隔膜泵输进串联好的二级液体无菌过滤系统中过滤，然后通过无菌管道输送进入发酵罐中，相对于每升发酵液，酶的加入量为3g，进行发酵液的酶解；酶解过程中，温度控制为55℃，pH控制为9，搅拌速度控制为20rpm，通气量控制为0.45vvm，罐压控制为0.03MPa，整个酶解时间为8h。
(4)将酶解结束后，通过三相分离系统进行固相、油相和水相的分离，将料液加热至90℃，离心机转速为6700rpm，离心分离时间为18小时，得到作为毛油的微生物油脂，油脂的提取效率、固相残渣中的残油率、油脂中ARA含量、EPA含量、胞外毒素含量、茴香胺值、过氧化值见表1。
实施例2
本实施例用于说明本发明提供的微生物油脂及其制备方法
(1)碱液准备：在碱罐中加入200L的软化水，按照15重量％的浓度称取食品级的碳酸钠投入碱罐中充分溶解，将配制好的碱液置于压强为0.14MPa，温度为145℃饱和蒸汽中灭菌1h，然后向碱罐中通入无菌压缩空气使碱罐的罐压为0.02-0.05MPa，并向碱罐的夹套中通入冷却水将碱液冷却到35-45℃，备用；
(2)细胞壁裂解液准备：将串联好的二级液体无菌过滤系统用压强为0.14MPa，温度为145℃饱和蒸汽灭菌1h，然后用无菌压缩空气保压备用；所述无菌过滤系统的过滤器规格为孔径0.2μm，耐温150℃；称取碱性蛋白酶、果胶酶、纤维素酶和蜗牛酶，并添加软化水按酶：水比例1:10配制酶解液，其中，每升发酵液酶的用量为：碱性蛋白酶2g，果胶酶1g，纤维素酶1.5g，蜗牛酶0.5g。
(3)酶解液与碱液的投加：先将产油微生物发酵液1的温度提升到35-55℃，然后通过无菌碱液管道将上述碱液用压缩空气压入发酵罐中，用“梅特勒”pH探头测定发酵液的pH，直到发酵液pH为8时停止加入碱液；上述酶解液通过隔膜泵输进串联好的二级液体无菌过滤系统中过滤，然后通过无菌管道输送进入发酵罐中，进行发酵液的酶解；酶解过程中，温度控制为50℃，pH控制为8，搅拌速度控制为30rpm，通气量控制为0.2vvm，罐压控制为0.02MPa，整个酶解时间为15h。
(4)将酶解结束后，通过三相分离系统进行固相、油相和水相的分离，将料液加热至95℃，离心机转速为6000rpm，离心分离时间为13小时，得到作为毛油的微生物油脂，油脂的提取效率、固相残渣中的残油率、油脂中ARA含量、EPA含量、胞外毒素含量、茴香胺值、过氧化值见表1。
实施例3
本实施例用于说明本发明提供的微生物油脂及其制备方法
(1)碱液准备：在碱罐中加入200L的软化水，按照25重量％的浓度称取食品级的碳酸氢钠投入碱罐中充分溶解，将配制好的碱液置于压强为0.14MPa，温度为145℃饱和蒸汽中灭菌1h，然后向碱罐中通入无菌压缩空气使碱罐的罐压为0.02-0.05MPa，并向碱罐的夹套中通入冷却水将碱液冷却到35-45℃，备用；
(2)细胞壁裂解液准备：将串联好的二级液体无菌过滤系统用压强为0.14MPa，温度为145℃饱和蒸汽灭菌1h，然后用无菌压缩空气保压备用；所述无菌过滤系统的过 滤器规格为孔径0.2μm，耐温150℃；称取碱性蛋白酶，并添加软化水按酶：水＝1:10配制酶解液。
(3)酶解液与碱液的投加：先将产油微生物发酵液1的温度提升到35-55℃，然后通过无菌碱液管道将上述碱液用压缩空气压入发酵罐中，用“梅特勒”pH探头测定发酵液的pH，直到发酵液pH为10时停止加入碱液；上述酶解液通过隔膜泵输进串联好的二级液体无菌过滤系统中过滤，然后通过无菌管道输送进入发酵罐中，相对于每升发酵液，酶的加入量为5g，进行发酵液的酶解；酶解过程中，温度控制为50℃，pH控制为10，搅拌速度控制为8rpm，通气量控制为0.6vvm，罐压控制为0.05MPa，整个酶解时间为4h。
(4)将酶解结束后，通过三相分离系统进行固相、油相和水相的分离，将料液加热至温度为85℃，离心机转速为7500rpm，离心分离时间为20小时，得到作为毛油的微生物油脂，油脂的提取效率、固相残渣中的残油率、油脂中ARA含量、EPA含量、胞外毒素含量、茴香胺值、过氧化值见表1。
实施例4
按照实施例1的方法进行微生物油脂的制备，不同的是，将步骤3中的产油微生物发酵液1替换为产油微生物发酵液2。油脂的提取效率、固相残渣中的残油率、油脂中DHA含量、EPA含量、胞外毒素含量、茴香胺值、过氧化值见表1。
实施例5
按照实施例2的方法进行微生物油脂的制备，不同的是，将步骤3中的产油微生物发酵液1替换为产油微生物发酵液2。油脂的提取效率、固相残渣中的残油率、油脂中DHA含量、EPA含量、胞外毒素含量、茴香胺值、过氧化值见表1。
实施例6
按照实施例3的方法进行微生物油脂的制备，不同的是，将步骤3中的产油微生物发酵液1替换为产油微生物发酵液2。油脂的提取效率、固相残渣中的残油率、油脂中DHA含量、EPA含量、胞外毒素含量、茴香胺值、过氧化值见表1。
实施例7
本实施例用于说明本发明提供的微生物油脂及其制备方法
按照实施例1的方法进行微生物油脂的制备，不同的是，酶解所用的酶替换为蜗牛酶、纤维素酶和果胶酶，每升发酵液的酶使用量为：纤维素酶2.5g，果胶酶1.5g，蜗牛酶1g，结果见表1。
对比例1
本对比例用于说明参比的微生物油脂及其制备方法
按照实施例1的方法进行微生物油脂的制备，不同的是，碱液、酶解液以及输送碱液和酶解液的管路均不通过灭菌，结果见表1。
对比例2
本对比例用于说明参比的微生物油脂及其制备方法
按照实施例4的方法进行微生物油脂的制备，不同的是，碱液、酶解液以及输送碱液和酶解液的管路均不通过灭菌，结果见表1。
表1
Figure PCTCN2019101034-appb-000001
由表1可以看出，通过将整个工艺过程控制在无菌环境下操作，可以减少微生物油脂提取过程中细菌大量繁殖，增加微生物油脂的安全性，且所得作为毛油的微生物油 脂的收率可提高到97-98％，与对照相比有了明显的提高；此外，毛油中的DHA含量或ARA含量得到了显著的提高，胞外毒素的含量、茴香胺值和过氧化物值得到了显著的降低。此外，在优选使用碱性蛋白酶以及优选的酶解条件下，效果能够得到进一步提升。
测试例
通常通过下述两种方法检测酶解程度：
(a)显微镜观察藻细胞的轮廓形态法：酶解结束后，发酵罐的取样口用锥形瓶取样200mL，用接种环挑取2-4环酶解发酵液铺展在盖玻片上，盖上观察玻片，滴1-2滴香柏油，用100倍的油镜进行观察。看是否有完整的藻细胞；是否有细胞碎片是否大片存在，结果见表2。
(b)离心观察法：酶解结束后，发酵罐的取样口用锥形瓶取样200mL，用50mL的离心试管取50mL发酵液，采用6000-10000rpm的实验室离心机进行离心处理3min，观察油相、水相的分离情况，看分层界面是否清晰，采用1-10分制进行打分，分数越高越清晰，结果见表2。
表2
Figure PCTCN2019101034-appb-000002
由表2可以看出，在优选使用碱性蛋白酶以及优选的酶解条件下，效果能够得到进一步提升。
以上详细描述了本发明的优选实施方式，但是，本发明并不限于此。在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，包括各个技术特征以任 何其它的合适方式进行组合，这些简单变型和组合同样应当视为本发明所公开的内容，均属于本发明的保护范围。

Claims (10)

  1. 一种微生物油脂的提取方法，其特征在于，该方法包括：
    (1)在无菌环境下，将产油微生物的发酵液与细胞壁裂解酶接触，以对发酵液中的产油微生物进行酶解，获得含有微生物油脂的混合液；
    (2)将步骤(1)得到的含有微生物油脂的混合液进行油相、水相和固相的三相分离，得到微生物油脂。
  2. 根据权利要求1所述的方法，其中，所述细胞壁裂解酶包括碱性蛋白酶以及可选的其他酶，所述其他酶为纤维素酶、半纤维素酶、果胶酶、蜗牛酶、几丁质酶和木质素酶中的至少一种；
    优选的，相对于每升所述发酵液，所述细胞壁裂解酶的用量为1-5g。
  3. 根据权利要求1或2所述的方法，其中，步骤(1)中，所述酶解的条件包括：pH值为8-10，温度为40-60℃，压力为0.02-0.05MPa，通气量为0.2-0.6VVM，时间为4-15小时；
    优选的，所述酶解在搅拌的条件下进行，搅拌的速度为8-30rpm。
  4. 根据权利要求1-3中任意一项所述的方法，其中，所述产油微生物为酵母、霉菌和藻类中的至少一种；
    其中，所述酵母选自浅白色隐球酵母(Cryptococcus albidus)、弯隐球酵母(Cryptococcus albidun)、斯达氏油脂酵母(Lipomyces)、茁芽丝孢酵母(Trichospiron pullulans)、产油油脂酵母(Lipomy slipofer)、胶粘红酵母(Rhodotorula giutinis)和圆红冬孢酵母菌(Rhodosporidium toruloides)中的至少一种；
    所述霉菌选自土霉菌(Asoergullus terreus)、紫瘫麦角菌(Clavicepspurpurea)、高梁褶抱黑粉菌(Tolyposporium)、高山被孢霉(Mortierella alpina)和深黄被孢霉(Mortierella isabellina)中的至少一种；
    所述藻类选自破囊壶菌(Thraustochytriales)、裂殖壶菌(Schizochytrium)、隐甲藻(Crypthecodinium)、硅藻(diatom)、螺旋藻(Spirulina)和吾肯氏菌属中的至少一种。
  5. 根据权利要求1或3所述的方法，其中，所述酶解在发酵罐中进行，通过向所 述发酵罐中加入食品级的碱液，所述碱液的加入量使得发酵液的pH值为8-10；
    所述食品级的碱液经过灭菌后通过无菌管路输送至所述发酵罐中；
    优选的，所述碱液为氢氧化钠溶液、氢氧化钾溶液、碳酸钠溶液、碳酸氢钠溶液、碳酸钾溶液、碳酸氢钾溶液和氨水中的至少一种。
  6. 根据权利要求1或3所述的方法，其中，所述酶解在酶解罐中进行，通过向所述酶解罐中加入食品级的碱液以及发酵液，所述碱液的加入量使得发酵液的pH值为8-10；
    所述食品级的碱液经过灭菌后通过无菌管路输送至所述酶解罐中，所述发酵液通过无菌管路输送至所述酶解罐中；
    优选的，所述碱液为氢氧化钠溶液、氢氧化钾溶液、碳酸钠溶液、碳酸氢钠溶液、碳酸钾溶液、碳酸氢钾溶液和氨水中的至少一种。
  7. 根据权利要求5或6所述的方法，其中，所述灭菌的方法为高温饱和蒸汽灭菌，所述高温饱和蒸汽的压强为0.1-0.35MPa，温度为121-145℃，灭菌时间为40-80min。
  8. 根据权利要求1或2所述的方法，其中，所述酶解在发酵罐中进行，所述细胞壁裂解酶经过灭菌后通过无菌管路输送至所述发酵罐中；
    优选的，所述灭菌的方法为使用无菌过滤系统进行过滤除菌，所述无菌过滤系统的液体无菌过滤器孔径为0.15-0.25μm；
    优选的，所述无菌过滤系统通过高温饱和蒸汽灭菌，所述高温饱和蒸汽的压强为0.1-0.35MPa，温度为121-145℃，灭菌时间为15-20min。
  9. 根据权利要求1或2所述的方法，其中，所述酶解在酶解罐中进行，所述细胞壁裂解酶经过灭菌后通过无菌管路输送至所述酶解罐中，所述发酵液通过无菌管路输送至所述酶解罐中；
    优选的，所述灭菌的方法为使用无菌过滤系统进行过滤除菌，所述无菌过滤系统的液体无菌过滤器的孔径为0.15-0.25μm；
    优选的，所述无菌过滤系统通过高温饱和蒸汽灭菌，所述高温饱和蒸汽的压强为0.1-0.35MPa，温度为121-145℃，灭菌时间为15-20min。
  10. 根据权利要求1-9中任意一项所述的方法制备的微生物油脂，所述微生物油脂为毛油，所述毛油的DHA含量大于35重量％或者ARA含量大于35重量％、胞外毒素含量小于50μg/kg、茴香胺值小于25、过氧化值小于20meq/kg。
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