WO2020238010A1 - 一种使用微生物发酵生产特种油脂opo的方法 - Google Patents

一种使用微生物发酵生产特种油脂opo的方法 Download PDF

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WO2020238010A1
WO2020238010A1 PCT/CN2019/114911 CN2019114911W WO2020238010A1 WO 2020238010 A1 WO2020238010 A1 WO 2020238010A1 CN 2019114911 W CN2019114911 W CN 2019114911W WO 2020238010 A1 WO2020238010 A1 WO 2020238010A1
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opo
medium
oil
microbial fermentation
culture
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French (fr)
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娄文勇
张林尚
宗敏华
倪子富
杨继国
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South China University of Technology SCUT
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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
    • 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
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6463—Glycerides obtained from glyceride producing microorganisms, e.g. single cell oil

Definitions

  • the invention belongs to the field of microorganisms, and specifically relates to a method for producing special oil OPO by using microorganism fermentation.
  • OPO structural fat is the first-line fat of infant formula milk powder (human milk fat substitute). It is easy to combine with sodium cholate, easy to digest and absorb, does not form high melting point soap, and facilitates the absorption of metal ions such as calcium and magnesium.
  • the preparation methods of OPO include enzymatic method, fractional extraction method and chemical synthesis method. Among them, the enzymatic method mainly uses 1,3-position specific lipase, uses palm stearin as a substrate, adds oleic acid or ethyl oleate or high oleic oil, carries out acid hydrolysis or transesterification to prepare rich A method of OPO grease.
  • CN109082447A discloses a process for preparing OPO by enzymatic method with high oleic peanut oil as the source of oleic acid, which is characterized by omitting the step of oleic acid purification, and oleic acid or ethyl oleate prepared by hydrolysis or alcoholysis of peanut oil The ester directly reacts with palm stearin, and the reaction product is purified by molecular distillation.
  • CN105219811A discloses a microwave-assisted enzymatic acid hydrolysis method for preparing OPO. The method is characterized in that microwave technology is applied in the reaction, and 15% of algae oil is mixed into the raw materials, and the OPO produced contains a small amount of DHA.
  • CN108913725A discloses a method for preparing OPO by enzymatic acid hydrolysis, which is characterized by applying electron beam irradiation to the enzymatic reaction. The electron beam dose is 20Gy and the time is 2h. The OPO yield of the enzymatic reaction system after treatment is increased to 61.25 %.
  • Fractionation is a method of enriching OPO by fractionation technology using natural oil (lard) containing OPO.
  • the fractionation method is a purely physical operation process with minimal health risks.
  • the fractionation process is complex, technically difficult, and solvents are used, resulting in high safety risks.
  • the raw materials are limited to lard, which cannot meet religious requirements and cannot be large-scale industrialized production.
  • CN103305332A discloses a method for dry fractionation coupled with solvent fractionation and enrichment of OPO in lard.
  • the oil with an OPO content of more than 40% is obtained.
  • the main process is to melt the oil at 60°C, quickly lower the temperature to 28°C, then slowly lower the temperature to 16°C, maintain it for 10 hours, and then filter to obtain liquid and solid components.
  • the liquid component is mixed with acetone, and the temperature is rapidly cooled from 40°C to 5°C and maintained for 20h.
  • the liquid oil obtained by vacuum filtration is high OPO grease.
  • CN107473963A discloses a two-step process for preparing OPO with 1,3-dichloropropanol, methyl hexadecanoate and sodium oleate as substrates.
  • 1,3-dichloropropanol and methyl hexadecanoate are heated and refluxed in an organic solvent to prepare 1,3-dichloro-2-hexadecanoate.
  • 1,3-dichloro-2-hexadecanoate is reacted with sodium oleate under nitrogen protection at 50-100°C for 2-5 hours, and the purity of the prepared OPO is 96%.
  • the purpose of the present invention is to provide a method for producing special oil OPO using microbial fermentation.
  • the method only needs to carry out fermentation, crushing, and oil extraction to obtain high-purity OPO grease.
  • the process is simple, the cost is low, and the industrialization prospect is huge.
  • the invention provides a method for producing OPO by fermentation with microorganisms of the genus Rhodococcus.
  • the Rhodococcus genus is specifically Rhodococcus opacus PD630.
  • the steps of fermentation and production of OPO are: 1. Activating strains; 2. Culturing in MSM medium, which is an inorganic salt medium, and the carbon source contains high content of oleate and palmitate; 3 , Collect the bacteria after the culture; 4. Break the bacteria and extract the oil, the obtained oil is the oil rich in OPO.
  • the present invention provides a method for producing special oil OPO by using microbial fermentation, including the following steps:
  • step (3) Inoculating the activated strains described in step (1) into the optimized medium described in step (2), and culturing on a shaker to obtain a culture solution;
  • step (3) Centrifuging the culture broth described in step (3), taking the precipitate, washing, washing, and freeze-drying to obtain a lyophilized powder, and adding hydrochloric acid solution to obtain a mixture;
  • the organic solvent is added to the mixture described in step (4), and the extraction process is performed to remove the solvent to obtain a grease mixture.
  • the grease mixture contains special grease OPO.
  • the bacterial species in step (1) includes Rhodococcus opacus; the activation medium includes broth medium and LB medium.
  • the microorganisms in step (1) include Rhodococcus turbidus; the temperature of the shaker culture treatment is 20-40 degrees Celsius, and the rotation speed of the shaker culture treatment is 80-250 rpm, further optimized to 120-200 rpm, and further optimized to 150- 180 rpm; the time for the shaker culture treatment is 12-60h, further optimized for 18-48h, and further optimized for 20-36h.
  • the organic matter in step (2) includes palmitic acid, oleic acid, palmitate, oleate, cetane, octadecane, palm oil, palm oil fractionation products, high oleic triglycerides, and A mixture of these organic substances; the added amount of the organic substances is 0.1-10% (w/w) of the quality of the culture medium, further optimized to 0.3-7%, and further optimized to 0.5-4%.
  • the method of sterilization in step (2) includes high temperature and high pressure sterilization (121 degrees Celsius, 20 minutes) and medium and low pressure sterilization (115 degrees Celsius, 15 minutes).
  • the minimal medium in step (2) may be MSM medium, and the carbon source of the medium contains a high proportion of palmitic acid and oleic acid, or palmitate and oleate, or cetane and oleic acid and Oleic acid ester, wherein the ratio (mass ratio) of palmitoyl and oleoyl is 1:1.4 to 1:3.
  • the inoculation amount of the inoculation in step (3) is 0.1-10% (v/v) of the optimized medium volume, further optimized to 0.2-6%, and further optimized to 0.5-3%.
  • the temperature of the shaker culture treatment in step (3) is 20-40 degrees Celsius, further optimized to 25-35 degrees Celsius, and further optimized to 26-33 degrees Celsius;
  • the rotation speed of the shaker culture treatment is 80-250 rpm, which is further optimized It is 120-200rpm, further optimized to 150-180rpm;
  • the time of shaker culture treatment is 48-216h, further optimized is 60-192h, and further optimized is 72-144h.
  • the rotation speed of the centrifugal treatment in step (4) is 3000-12000 rpm, further optimized to 4000-10000 rpm, and further optimized to 4500-8000 rpm; the centrifugal treatment time is 1-20 min, further optimized to 2-15 min, and further optimized For 3-10min.
  • the washing in step (4) is first washing with PBS buffer, and then washing with n-hexane, so as to remove the fat-soluble compounds remaining on the bacterial cells.
  • the concentration of the hydrochloric acid solution in step (4) is 1-3 mol/L, and the mass-volume ratio of the freeze-dried powder to the hydrochloric acid solution is 0.1:2-1:2 g/mL.
  • the organic solvent in step (5) includes n-propane, butane, pentane, hexane, heptane, acetone, chloroform, dichloromethane, ether, methanol and carbon dioxide.
  • the present invention provides a method for using microorganisms to ferment to produce special oil OPO.
  • the microorganisms may be Rhodococcus opacus PD630.
  • the palmitate and oleate include, but are not limited to, monoglycerides, diglycerides, triglycerides, polyglycerides, sterol esters, and sorbitol esters of palmitic acid and oleic acid.
  • the carbon source in the optimized medium in step (2) includes organic carbon sources and inorganic carbon sources that can provide for the growth of microorganisms
  • the carbon sources in the optimized medium include, but are not limited to, glucose, xylose, Fructose, sucrose, dextrin, starch, cellulose, lignin, polysaccharides and other monosaccharides, oligosaccharides and polymer carbohydrates; various alcohols, such as methanol, ethanol, propanol, butanol, pentanol, hexanol, etc.
  • Alcohols such as propane, butane, pentane, hexane, heptane, octane, nonane, decane, undecane, dodecane, tridecane, tetradecane, pentadecane Alkane, hexadecane, heptadecane, octadecane, nonadecane, eicosan, etc.; various fatty acids, such as caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, Linoleic acid, etc.; various oils, such as peanut oil, soybean oil, rapeseed oil, corn oil, palm oil, sunflower oil, sesame oil, peony seed oil, rice oil, camellia oil, olive oil, coconut oil, linseed Oil and algae oil, etc.
  • various hydrocarbons such as propane, butane, pentane, hexane,
  • the optimized medium in step (2) contains carbon source, nitrogen source, phosphate, inorganic salt and water, wherein the ratio of carbon to nitrogen substances is ⁇ 10, further optimized to ⁇ 20, and further optimized to ⁇ 30.
  • the present invention has the following advantages and beneficial effects:
  • the method for producing special oil OPO using microbial fermentation provided by the present invention adopts microbial fermentation technology, which is a brand-new technology; the process is simple, the cost is low, and OPO grows in bacteria, which is safer and healthier.
  • Figure 1 is a histogram of biomass and oil content measured in Example 1, Example 2 and Comparative Example 1;
  • Figure 2 is a gas chromatogram of the fatty acid composition of the fat and oil in Example 1;
  • Figure 3 is a gas chromatogram of the fatty acid composition of the fat of Comparative Example 2;
  • Fig. 4 is a gas chromatogram of the fatty acid composition of the fats and oils of Example 2.
  • the Rhodococcus was added to the LB medium and cultured at 30°C and 160 rpm for 24 hours to activate the strain.
  • the strain obtained by activation is a primary fermentation strain.
  • the medium is prepared with reference to Table 1 below (Table 1 is the composition table of MSM medium).
  • Table 1 is the composition table of MSM medium.
  • the volume of the medium is 1 liter.
  • MSM medium with reference to Table 1.
  • the obtained MSM medium was sterilized at 121°C, and the sterilization time was 12 minutes; the medium was divided into 250 mL conical flasks, and step (1) was activated.
  • the strains were inoculated into a 250mL Erlenmeyer flask with an inoculum amount of 1% (v/v), cultured in a shaker with a temperature of 30°C and a rotation speed of 160 rpm for 4 days, and then collected the bacteria by centrifugation at a rotation speed of 6000 rpm , The time of centrifugation is 2min, and the bacterial pellet is obtained.
  • step (3) Wash the bacterial pellet in step (3) three times with 0.1% (w/w) PBS buffer to remove the residual culture medium on the bacterial pellet, and then wash three times with n-hexane to remove the fat-soluble compounds in the culture medium; After freeze-drying, the freeze-dried bacterial powder is obtained, weighed, and the biomass is calculated.
  • step (4) Weigh 0.1g of the freeze-dried bacterial powder described in step (4), add 2mL of HCl solution with a concentration of 3mol/L, mix well, and then in a water bath at 80°C for 30min, then add 2mL of chloroform for extraction, and extract three times. Combine the three extracts, dry the chloroform in the extract with nitrogen to obtain a mixture of fats and oils, and calculate the oil content by weighing.
  • step (5) Take 0.02g of the fats and oils mixture described in step (5), add 2mL of n-hexane to dissolve the fats and oils mixture, stir evenly, then add 2mL of methanol solution with a concentration of 0.5mol/L sodium hydroxide, and water bath at 60°C After 20 minutes, the methyl esterification reaction was completed, and the supernatant (n-hexane) was taken to analyze the fatty acid composition by gas chromatography.
  • the triglycerides were scraped off and extracted three times with n-hexane to obtain pure triglycerides. Put triglycerides into a 15mL centrifuge tube, add tri-HCl hydrochloric acid, sodium cholate, CaCl 2 and lipase, then water bath for 1 min, then add 1mL of 6mol/L hydrochloric acid solution to inactivate the lipase, add ether to extract the lipid quality. The ether extract was spread on a thin layer chromatography plate, the monoglyceride part was scraped off, methyl esterification was carried out, and the fatty acid composition was analyzed by gas chromatography.
  • the microorganism (Rhodococcus turbidus) was inoculated into the activation medium (LB medium), and the temperature of the shaking culture was 30 degrees Celsius, the rotation speed of the shaking culture was 160 rpm, and the time of the shaking culture was 24h, get the activated strain;
  • step (3) Inoculate the activated strain described in step (1) into the optimized medium described in step (2), the inoculum amount is 1% (v/v), and the temperature of the shaker culture treatment is 30 degrees Celsius, the time of the shaker culture treatment is 96h, and the rotation speed of the shaker culture treatment is 160rpm to obtain the culture solution;
  • step (3) Centrifuge the culture broth described in step (3) at a rate of 6000 rpm and a time of 2 min. Take the bacterial pellet, wash, and precipitate the bacterial cell with 0.1% (w/ w) Wash three times with PBS buffer to remove the residual medium on the bacterial pellet, and then wash three times with n-hexane to remove the fat-soluble compounds in the residual medium; then freeze-dry to obtain freeze-dried bacterial powder, weigh, and calculate the biomass ;
  • step (4) Weigh 0.1g of the freeze-dried bacterial powder described in step (4), add 2mL of HCl solution with a concentration of 3mol/L, mix well, and then in a water bath at 80°C for 30min, then add 2mL of chloroform for extraction.
  • Extract three times combine the three extracts, blow dry the chloroform in the extract with nitrogen to obtain a mixture of fats and oils, weigh and calculate the oil content; take 0.02g of the mixture of fats and oils, and add the mixture of fats and oils to 2mL of n-hexane to dissolve , Stir evenly, then add 2mL of 0.5mol/L sodium hydroxide methanol solution, water bath at 60°C for 20min, complete the methyl esterification reaction, take the supernatant (n-hexane) and analyze the fatty acid composition by gas chromatography ;
  • a method of using microbial fermentation to produce special oil OPO includes the following steps:
  • the microorganism (Rhodococcus turbidus) was inoculated into the activation medium (LB medium), and the temperature of the shaking culture was 30 degrees Celsius, the rotation speed of the shaking culture was 160 rpm, and the time of the shaking culture was 24h, get the activated strain;
  • MSM medium according to the formula in Table 1. During the preparation, add 0.8 g ethyl oleate and 0.4 g ethyl palmitate (the mass ratio of ethyl oleate to ethyl palmitate is 2:1). , Mix well and sterilize (high temperature and high pressure sterilization) to obtain optimized culture medium;
  • step (3) Inoculate the activated strain described in step (1) into the optimized medium described in step (2), the inoculum amount is 1% (v/v), and the temperature of the shaker culture treatment is 30 degrees Celsius, the time of the shaker culture treatment is 96h, and the rotation speed of the shaker culture treatment is 160rpm to obtain the culture solution;
  • step (3) Centrifuge the culture broth described in step (3) at a rate of 6000 rpm and a centrifugal treatment time of 5 min to obtain bacterial pellets.
  • the bacterial pellets were deposited with 0.1% (w/w) Wash three times with PBS buffer to remove the residual medium on the bacterial pellet, and then wash three times with n-hexane to remove the fat-soluble compounds in the residual medium; then freeze-dry to obtain freeze-dried bacterial powder, weigh, and calculate the biomass;
  • step (4) Weigh 0.1g of the freeze-dried bacterial powder described in step (4), add 2mL of HCl solution with a concentration of 3mol/L, mix well, and then in a water bath at 80°C for 30min, then add 2mL of chloroform for extraction. Extract three times, combine the three extracts, and blow dry the chloroform in the extract with nitrogen to obtain an oil mixture (the oil mixture contains special oil OPO);
  • Test step (5) The fatty acid composition, sn-2 fatty acid composition, and oil content of the whole sample of the oil and fat mixture (determined in accordance with GB 5009.168-2016 National Food Safety Standard).
  • a method of using microbial fermentation to produce special oil OPO includes the following steps:
  • the microorganism (Rhodococcus turbidus) was inoculated into the activation medium (LB medium), and the temperature of the shaking culture was 30 degrees Celsius, the rotation speed of the shaking culture was 160 rpm, and the time of the shaking culture was 24h, get the activated strain;
  • step (3) Inoculate the activated strain described in step (1) into the optimized medium described in step (2), the inoculum amount is 1% (v/v), and the temperature of the shaker culture treatment is 30 degrees Celsius, the time of the shaker culture treatment is 96h, and the rotation speed of the shaker culture treatment is 160rpm to obtain the culture solution;
  • step (3) Centrifuge the culture broth described in step (3) at a rate of 6000 rpm and a centrifugal treatment time of 5 min. Take the precipitate to obtain a bacterial pellet.
  • the bacterial pellet is precipitated with 0.1% (w /w) Wash three times with PBS buffer to remove the residual medium on the bacterial pellet, and then wash three times with n-hexane to remove the fat-soluble compounds in the residual medium; then freeze-dry to obtain freeze-dried bacterial powder, weigh, and calculate the organism the amount;
  • step (4) Weigh 0.1g of the freeze-dried bacterial powder described in step (4), add 2mL of HCl solution with a concentration of 3mol/L, mix well, and then in a water bath at 80°C for 30min, then add 2mL of chloroform for extraction. Extract three times, combine the three extracts, and blow dry the chloroform in the extract with nitrogen to obtain an oil mixture (the oil mixture contains special oil OPO);
  • Test step (5) The fatty acid composition, sn-2 fatty acid composition, and oil content of the whole sample of the oil and fat mixture (determined in accordance with GB 5009.168-2016 National Food Safety Standard).
  • a method of using microbial fermentation to produce special oil OPO includes the following steps:
  • the microorganism (Rhodococcus turbidus) was inoculated into the activation medium (LB medium), and the temperature of the shaker culture was 20 degrees Celsius, the rotation speed of the shaker culture was 250 rpm, and the time of the shaker culture was 12h, get the activated strain;
  • the MSM medium was prepared according to the formula in Table 1. During the preparation process, 1.6 g ethyl oleate and 0.8 g ethyl palmitate (the mass ratio of ethyl oleate to ethyl palmitate was 1:2) were added to the medium. , Mix well and sterilize (high temperature and high pressure sterilization) to obtain optimized culture medium;
  • step (3) Inoculate the activated strain described in step (1) into the optimized medium described in step (2), the inoculum amount is 10% (v/v), and the temperature of the shaker culture treatment is 40 degrees Celsius, the time of the shaker culture treatment is 216h, and the rotation speed of the shaker culture treatment is 80 rpm to obtain the culture solution;
  • step (3) Centrifuge the culture broth described in step (3) at a rate of 12000 rpm and a centrifugal time of 1 min to obtain bacterial pellets.
  • the bacterial cells are precipitated with 0.1% (w/w) Wash three times with PBS buffer to remove the residual medium on the bacterial pellet, and then wash three times with n-hexane to remove the fat-soluble compounds in the residual medium; then freeze-dry to obtain freeze-dried bacterial powder, weigh, and calculate the biomass;
  • step (4) Weigh 0.5g of the freeze-dried bacterial powder described in step (4), add 2mL of 1mol/L HCl solution, mix well, and then in a water bath at 80°C for 30min, then add 2mL of chloroform for extraction. Extract three times, combine the three extracts, and blow dry the chloroform in the extract with nitrogen to obtain an oil mixture (the oil mixture contains special oil OPO);
  • Test step (5) The fatty acid composition, sn-2 fatty acid composition, and oil content of the whole sample of the oil and fat mixture (determined in accordance with GB 5009.168-2016 National Food Safety Standard).
  • a method of using microbial fermentation to produce special oil OPO includes the following steps:
  • the microorganism (Rhodococcus turbidus) was inoculated into the activation medium (LB medium), and the temperature of the shaking culture was 40 degrees Celsius, the rotation speed of the shaking culture was 80 rpm, and the time of the shaking culture was 60h, get activated strain;
  • step (3) Inoculate the activated strains described in step (1) into the optimized medium described in step (2), the inoculum amount is 0.1% (v/v), the temperature of the shaker culture treatment is 20 degrees Celsius, the time of the shaker culture treatment is 48h, and the rotation speed of the shaker culture treatment is 250 rpm to obtain the culture solution;
  • step (3) Centrifuge the culture broth described in step (3) at a speed of 3000 rpm and a time of 20 min to obtain a bacterial pellet.
  • the bacterial pellet was deposited with 0.1% (w/w) Wash three times with PBS buffer to remove the residual medium on the bacterial pellet, and then wash three times with n-hexane to remove the fat-soluble compounds in the residual medium; then freeze-dry to obtain freeze-dried bacterial powder, weigh, and calculate the biomass;
  • Test step (5) The fatty acid composition, sn-2 fatty acid composition, and oil content of the whole sample of the oil and fat mixture (determined in accordance with GB 5009.168-2016 National Food Safety Standard).
  • Table 2 is a data table (%) of the main fatty acid composition of the oil sample.
  • the fat fatty acid composition in Comparative Example 1 has a relatively low oleic acid content, and the ratio of palmitic acid to oleic acid is 1:0.7.
  • the whole fatty acids are consistent with sn-2 fatty acids, indicating that the fatty acids are distributed randomly on the glycerol backbone.
  • the ratio of palmitic acid to oleic acid in the whole fatty acid composition is 1:1.87, and the palmitic acid content in sn-2 fatty acid is 74.54%, indicating that the triglyceride is OPO.
  • the ratio of the whole fatty acid palmitic acid to oleic acid is 1:2.15, and the content of sn-2 fatty acid palmitic acid is 65.67%, indicating that triglycerides are OPO.
  • Embodiment 3 and Embodiment 4 are similar to those of Embodiment 1. Refer to FIG. 1, FIG. 2, FIG. 4, and Table 2.

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Abstract

使用微生物发酵生产OPO的方法，包括如下步骤：将微生物接种至活化培养基中摇床培养，得到活化菌种；将有机物加入基本培养基制成优化培养基；菌种接种至优化培养基摇床培养，得到培养液；培养液离心，取沉淀洗涤冻干，加入盐酸溶液得混合物；利用有机溶剂萃取混合物，去除溶剂得OPO，其中所述微生物为浑浊红球菌。

Description

一种使用微生物发酵生产特种油脂OPO的方法 技术领域
本发明属于微生物领域，具体涉及一种使用微生物发酵生产特种油脂OPO的方法。
背景技术
OPO结构脂是婴幼儿配方奶粉的一线油脂(人乳脂替代品)，具有易与胆酸钠结合、易于消化吸收、不形成高熔点皂、利于钙镁等金属离子的吸收等特点。OPO的制备方法有酶法、分提法和化学合成法。其中，酶法主要是利用1，3-位专一性的脂肪酶，以棕榈硬脂为底物，添加油酸或者油酸乙酯或者高油酸油脂，进行酸解或者酯交换制备富含OPO油脂的一种方法。酶法制备OPO是目前工业操作的主流方法，但是该方法成本较高，酶的价格高，另外该方法还存在酰基位移的技术壁垒无法克服。CN109082447A公布了一种以高油酸花生油为油酸来源，通过酶法制备OPO的工艺，其特点是省去了油酸纯化的步骤，将花生油水解或者醇解制备出的油酸或者油酸乙酯直接与棕榈硬脂反应，反应产物再进行分子蒸馏纯化。该方法生产的OPO含量为46.35％-44.17％，完全可以达到GB30604-2015的要求。CN105219811A公布了一种微波辅助酶法酸解制备OPO的方法，该方法的特点是反应中应用了微波技术，同时在原料中掺入了15％的藻油，所产OPO中含有少量DHA。CN108913725A公布了一种酶法酸解制备OPO方法，其特点是将电子束辐照应用到酶促反应中，电子束剂量为20Gy，时间2h，经过处理的酶促反应体系OPO得率提高至61.25％。
分提法是利用含OPO的天然油脂(猪油)通过分提技术富集OPO的一种方法。分提法是一个纯物理操作过程，健康风险最小，但是分提工艺复杂、技术难度高，使用溶剂，安全风险大，而且原料仅限于猪油，无法满足宗教的要求，尚无法大规模工业化生产。CN103305332A公布了一种干法分提耦合溶剂分提富集猪油中OPO的方法。最终获得OPO含量大于40％油脂，其主要过程为60℃熔化油脂，迅速降温至28℃，再缓慢降温至16℃，维持10h，然后过滤得到液体组分和 固体组分。液体组分在与丙酮混合，由40℃迅速降温至5℃，并维持20h，真空抽滤得到的液油即为高OPO油脂。
化学合成法容易得到高纯度的OPO，但是食品级原料很难获得，因此化学合成的方法只能在实验室中进行，无法真正工业化生产。CN107473963A公布了一种以1，3-二氯丙醇、十六烷酸甲酯、油酸钠为底物，两步法制备OPO的过程。首先1，3-二氯丙醇与十六烷酸甲酯在有机溶剂中加热回流制备1，3-二氯-2-十六烷酸酯。反应的第二步，1，3-二氯-2-十六烷酸酯在与油酸钠在氮气保护下，50-100℃，反应2-5h，制备的OPO纯度为96％。
发明概述
技术问题
问题的解决方案
技术解决方案
为了克服现有技术存在的上述不足，本发明的目的是提供一种使用微生物发酵生产特种油脂OPO的方法。该方法只需要进行发酵、破碎、提油即可以得到高纯度的OPO油脂，工艺简单、成本低廉、工业化前景巨大。
本发明的目的至少通过如下技术方案之一实现。
本发明提供了一种利用红球菌属微生物发酵生产OPO的方法。所述红球菌属具体为浑浊红球菌(Rhodococcus opacus PD630)。发酵生产OPO的步骤为：1、菌种活化；2、在MSM培养基中进行培养，所述MSM培养基为无机盐培养基，其中碳源含有高含量的油酸酯和棕榈酸酯；3、培养结束后收集菌体；4、进行破碎菌体，提取油脂，所得到的油脂即为富含OPO的油脂。
本发明提供的一种使用微生物发酵生产特种油脂OPO的方法，包括如下步骤：
(1)菌种活化
将微生物接种至活化培养基中，摇床培养处理，得到活化的菌种；
(2)配制优化培养基
将有机物加入基本培养基中，混合均匀，灭菌处理，得到优化培养基；
(3)将步骤(1)所述活化的菌种接种到步骤(2)所述优化培养基中，摇床培养处理，得到培养液；
(4)将步骤(3)所述培养液进行离心处理，取沉淀，洗涤，洗涤，冷冻干燥得到冻干粉，加入盐酸溶液，得到混合物；
(5)将有机溶剂加入步骤(4)所述混合物中，进行萃取处理，去除溶剂得到油脂混合物，所述油脂混合物中含有特种油脂OPO。
进一步地，步骤(1)所述菌种包括浑浊红球菌；所述活化培养基包括肉汤培养基及LB培养基。
进一步地，步骤(1)所述微生物包括浑浊红球菌；摇床培养处理的温度为20-40摄氏度，摇床培养处理的转速为80-250rpm，进一步优化为120-200rpm，进一步优化为150-180rpm；摇床培养处理的时间为12-60h，进一步优化为18-48h，进一步优化为20-36h。
进一步地，步骤(2)所述有机物包括棕榈酸、油酸、棕榈酸酯、油酸酯、十六烷、十八烷、棕榈油、棕榈油的分提产物、高油酸甘油三酯以及这些有机物的混合物；所述有机物的添加量为培养基质量的0.1-10％(w/w)，进一步优化为0.3-7％，进一步优化为0.5-4％。
进一步地，步骤(2)所述灭菌处理的方式包括高温高压灭菌(121摄氏度，20min)及中低压灭菌(115摄氏度，15min)。
进一步地，步骤(2)所述基本培养基可以为MSM培养基，培养基碳源中含有高比例的棕榈酸和油酸，或者棕榈酸酯和油酸酯，或者十六烷和油酸及油酸酯，其中棕榈酰基和油酰基的比例(质量比)为1∶1.4～1∶3。
进一步地，步骤(3)所述接种的接种量为优化培养基体积的0.1-10％(v/v)，进一步优化为0.2-6％，进一步优化为0.5-3％。
进一步地，步骤(3)所述摇床培养处理的温度为20-40摄氏度，进一步优化为25-35摄氏度，进一步优化为26-33摄氏度；摇床培养处理的转速为80-250rpm，进一步优化为120-200rpm，进一步优化为150-180rpm；摇床培养处理的时间为48-216h，进一步优化为60-192h，进一步优化为72-144h。
进一步地，步骤(4)所述离心处理的转速为3000-12000rpm，进一步优化为4000-10000rpm，进一步优化为4500-8000rpm；离心处理的 时间为1-20min，进一步优化为2-15min，进一步优化为3-10min。
进一步地，步骤(4)所述洗涤为先用PBS缓冲液洗涤，再用正己烷洗涤，以便于去除残留在菌体上的脂溶性化合物。
进一步地，步骤(4)所述盐酸溶液的浓度为1-3mol/L，所述冻干粉与盐酸溶液的质量体积比为0.1∶2-1∶2g/mL。
进一步地，步骤(5)所述有机溶剂包括正丙烷、丁烷、戊烷、己烷、庚烷、丙酮、三氯甲烷、二氯甲烷、乙醚、甲醇及二氧化碳。
本发明提供的一种使用微生物发酵生产特种油脂OPO的方法，所述微生物可以为浑浊红球菌(Rhodococcus opacus PD630)。
进一步地，所述棕榈酸酯和油酸酯包括但不局限于棕榈酸和油酸的甘一酯、甘二酯、甘三酯、聚甘油酯、甾醇酯及山梨醇酯等。
进一步地，步骤(2)所述优化培养基中的碳源包括能够为微生物生长提供的有机碳源和无机碳源，所述优化培养基中的碳源包括但不局限于葡萄糖、木糖、果糖、蔗糖、糊精、淀粉、纤维素、木质素、多糖等单糖、寡糖和高分子碳水化合物；各种醇类、比如甲醇、乙醇、丙醇、丁醇、戊醇、己醇等醇类；各种烃类，比如丙烷、丁烷、戊烷、己烷、庚烷、辛烷、壬烷、癸烷、十一烷、十二烷、十三烷、十四烷、十五烷、十六烷、十七烷、十八烷、十九烷、二十烷等；各种脂肪酸，比如辛酸、癸酸、月桂酸、豆蔻酸、软脂酸、硬脂酸、油酸、亚油酸等；各种油脂，比如花生油、大豆油、菜籽油、玉米油、棕榈油、葵花籽油、芝麻油、牡丹花籽油、稻米油、山茶油、橄榄油、椰子油、亚麻籽油及藻油等。
进一步地，步骤(2)所述优化培养基包含碳源、氮源、磷酸盐、无机盐及水，其中碳氮物质的量比≥10，进一步优化为≥20，进一步优化为≥30。
发明的有益效果
有益效果
与现有技术相比，本发明具有如下优点和有益效果：
本发明提供的使用微生物发酵生产特种油脂OPO的方法，采用了微生物发酵技术，是一种全新的技术；其工艺简单，成本低，且OPO为菌体内生长，更加安 全健康。
对附图的简要说明
附图说明
图1为实施例1、实施例2及对比例1测得的生物量和含油量柱状图；
图2为实施例1油脂的脂肪酸组成的气相色谱图；
图3为对比例2油脂的脂肪酸组成的气相色谱图；
图4为实施例2油脂的脂肪酸组成的气相色谱图。
发明实施例
本发明的实施方式
以下结合附图和实例对本发明的具体实施作进一步说明，但本发明的实施和保护不限于此。需指出的是，以下若有未特别详细说明之过程，均是本领域技术人员可参照现有技术实现或理解的。所用试剂或仪器未注明生产厂商者，视为可以通过市售购买得到的常规产品。
实验方法
(1)菌种活化
将红球菌接入LB培养基中，30℃，160rpm培养24h，活化菌种。活化得到的菌种为一级发酵菌种。
(2)MSM培养基配制
参照下表1配制培养基(表1为MSM培养基的成分表)，作为举例，培养基的体积为1升。
表1
[Table 1]
  单位 质量/L
葡萄糖 g 24
KH 2PO 4 g 1.5
Na 2HPO 4·12H 2O g 9
NH 4Cl g 1.0
MgSO 4·7H 2O g 0.5
CaCl 2·2H 2O mg 20
Na 2MoO 4·2H 2O mg 2
FeNaEDTA·3H 2O mg 5
CoCl 2·6H 2O mg 0.2
ZnSO 4·7H 2O mg 0.1
MnCl 2·4H 2O mg 0.05
H 3BO 3 mg 0.3
CuCl 2·2H 2O mg 0.1
NiCl 2·6H 2O mg 0.2
(3)菌的培养
参照表1配制MSM培养基，得到的MSM培养基在121℃条件下进行灭菌处理，灭菌处理时间为12min；将培养基分装至250mL的锥形瓶中，将步骤(1)活化的菌种接种至250mL锥形瓶中，接种量为1％(v/v)，在温度为30℃、转速为160rpm的摇床中培养4天，然后采用离心的方法收菌，离心转速为6000rpm，离心的时间为2min，得到菌体沉淀。
(4)生物量测试方法
将步骤(3)所述菌体沉淀，用0.1％(w/w)PBS缓冲液洗涤三次，除去菌体沉淀上的残留培养基，再用正己烷洗涤三次除去培养基中的脂溶性化合物；之 后冻干，得到冻干菌粉，称重，计算生物量。
(5)含油量测定方法
称取0.1g步骤(4)所述冻干菌粉，加入2mL浓度为3mol/L的HCl溶液，混合均匀，然后在80℃条件下水浴30min，之后加入2mL三氯甲烷进行萃取，萃取三次，合并三次萃取液，用氮气吹干萃取液中的三氯甲烷，得到油脂类混合物，称重计算含油量。
(6)全样脂肪酸测定(GB 5009.168-2016食品安全国家标准)
取步骤(5)所述油脂类混合物0.02g，将油脂类混合物加入2mL正己烷中溶解，搅拌均匀，然后在加入2mL浓度为0.5mol/L氢氧化钠的甲醇溶液，在60℃条件下水浴20min，完成甲酯化反应，取上清液(正己烷)用气相色谱法分析脂肪酸组成。气相色谱法的条件：色谱柱选用HP-5，柱温箱的温度为180℃，进样口的温度为190℃，检测器的温度为270℃。得出结果图，根据峰的保留时间判定脂肪酸种类。
(7)Sn-2位脂肪酸测定(GB 5009.168-2016食品安全国家标准)
用薄层层析板将所述油脂类混合物展开，展开剂为正己烷∶无水乙醚∶冰乙酸＝70∶30∶2(v/v/v)。将甘三酯刮下，用正己烷萃取三次，得到纯甘三酯。将甘三酯装入15mL离心管，加入tri-HCl盐酸，胆酸钠，CaCl 2和脂肪酶，然后水浴1min，然后加入1mL浓度为6mol/L的盐酸溶液灭活脂肪酶，加入乙醚萃取脂质。将乙醚萃取物在薄层层析板上展开，将甘一酯部分刮下，进行甲酯化，进气相色谱分析脂肪酸组成。
对比例1
(1)菌种活化
将微生物(浑浊红球菌)接种至活化培养基(LB培养基)中，摇床培养处理，摇床培养处理的温度为30摄氏度，摇床培养处理的转速为160rpm，摇床培养处理的时间为24h，得到活化的菌种；
(2)配制MSM培养基
按照表1配方配制MSM培养基，灭菌处理(高温高压灭菌)，得到MSM培养基；
(3)将步骤(1)所述活化的菌种接种到步骤(2)所述优化培养基中，接种量为1％(v/v)，摇床培养处理，摇床培养处理的温度为30摄氏度，摇床培养处理的时间为96h，摇床培养处理的转速为160rpm，得到培养液；
(4)将步骤(3)所述培养液进行离心处理，离心处理的速率为6000rpm，离心处理的时间为2min，取菌体沉淀，洗涤，将所述菌体沉淀，用0.1％(w/w)PBS缓冲液洗涤三次，除去菌体沉淀上的残留培养基，再用正己烷洗涤三次除去残留培养基中的脂溶性化合物；之后冻干，得到冻干菌粉，称重，计算生物量；
(5)称取0.1g步骤(4)所述冻干菌粉，加入2mL浓度为3mol/L的HCl溶液，混合均匀，然后在80℃条件下水浴30min，之后加入2mL三氯甲烷进行萃取，萃取三次，合并三次萃取液，用氮气吹干萃取液中的三氯甲烷，得到油脂类混合物，称重计算含油量；取所述油脂类混合物0.02g，将油脂类混合物加入2mL正己烷中溶解，搅拌均匀，然后在加入2mL浓度为0.5mol/L氢氧化钠的甲醇溶液，在60℃条件下水浴20min，完成甲酯化反应，取上清液(正己烷)用气相色谱法分析脂肪酸组成；
(6)测试所述油脂混合物的sn-2位脂肪酸组成(根据GB 5009.168-2016食品安全国家标准测定)。
对比例2
选取两种油脂，棕榈硬脂(高棕榈酸含量的HPO)和高油酸葵花籽油(HOSFO)，按照1∶2的比例(质量比)进行调配，然后进行酯交换，后测定全样脂肪酸组成和sn-2脂肪酸组成(根据GB 5009.168-2016食品安全国家标准测定)。
实施例1
一种使用微生物发酵生产特种油脂OPO的方法，包括如下步骤：
(1)菌种活化
将微生物(浑浊红球菌)接种至活化培养基(LB培养基)中，摇床培养处理，摇床培养处理的温度为30摄氏度，摇床培养处理的转速为160rpm，摇床培养处理的时间为24h，得到活化的菌种；
(2)配制优化培养基
按照表1配方配制MSM培养基，在配制的过程中将0.8g油酸乙酯与0.4g棕榈酸乙酯(油酸乙酯与棕榈酸乙酯的质量比为2∶1)加入培养基中，混合均匀，灭菌处理(高温高压灭菌)，得到优化培养基；
(3)将步骤(1)所述活化的菌种接种到步骤(2)所述优化培养基中，接种量为1％(v/v)，摇床培养处理，摇床培养处理的温度为30摄氏度，摇床培养处理的时间为96h，摇床培养处理的转速为160rpm，得到培养液；
(4)将步骤(3)所述培养液进行离心处理，离心处理的速率为6000rpm，离心处理的时间为5min，得到菌体沉淀，将所述菌体沉淀，用0.1％(w/w)PBS缓冲液洗涤三次，除去菌体沉淀上的残留培养基，再用正己烷洗涤三次除去残留培养基中的脂溶性化合物；之后冻干，得到冻干菌粉，称重，计算生物量；
(5)称取0.1g步骤(4)所述冻干菌粉，加入2mL浓度为3mol/L的HCl溶液，混合均匀，然后在80℃条件下水浴30min，之后加入2mL三氯甲烷进行萃取，萃取三次，合并三次萃取液，用氮气吹干萃取液中的三氯甲烷，得到油脂类混合物(所述油脂混合物中含有特种油脂OPO)；
(6)测试步骤(5)所述油脂混合物的全样脂肪酸组成、sn-2位脂肪酸组成、含油量(根据GB 5009.168-2016食品安全国家标准测定)。
实施例2
一种使用微生物发酵生产特种油脂OPO的方法，包括如下步骤：
(1)菌种活化
将微生物(浑浊红球菌)接种至活化培养基(LB培养基)中，摇床培养处理，摇床培养处理的温度为30摄氏度，摇床培养处理的转速为160rpm，摇床培养处理的时间为24h，得到活化的菌种；
(2)配制优化培养基
按照表1配方配制MSM培养基，在配制的过程中将0.4g HPO与0.8g HOSFO(HPO与HOSFO的质量比为1∶2)加入培养基中，混合均匀，灭菌处理(高温高压灭菌)，得到优化培养基；
(3)将步骤(1)所述活化的菌种接种到步骤(2)所述优化培养基中，接种量为1％(v/v)，摇床培养处理，摇床培养处理的温度为30摄氏度，摇床培养处理 的时间为96h，摇床培养处理的转速为160rpm，得到培养液；
(4)将步骤(3)所述培养液进行离心处理，离心处理的速率为6000rpm，离心处理的时间为5min，取沉淀，得到菌体沉淀，将所述菌体沉淀，用0.1％(w/w)PBS缓冲液洗涤三次，除去菌体沉淀上的残留培养基，再用正己烷洗涤三次除去残留培养基中的脂溶性化合物；之后冻干，得到冻干菌粉，称重，计算生物量；
(5)称取0.1g步骤(4)所述冻干菌粉，加入2mL浓度为3mol/L的HCl溶液，混合均匀，然后在80℃条件下水浴30min，之后加入2mL三氯甲烷进行萃取，萃取三次，合并三次萃取液，用氮气吹干萃取液中的三氯甲烷，得到油脂类混合物(所述油脂混合物中含有特种油脂OPO)；
(6)测试步骤(5)所述油脂混合物的全样脂肪酸组成、sn-2位脂肪酸组成、含油量(根据GB 5009.168-2016食品安全国家标准测定)。
实施例3
一种使用微生物发酵生产特种油脂OPO的方法，包括如下步骤：
(1)菌种活化
将微生物(浑浊红球菌)接种至活化培养基(LB培养基)中，摇床培养处理，摇床培养处理的温度为20摄氏度，摇床培养处理的转速为250rpm，摇床培养处理的时间为12h，得到活化的菌种；
(2)配制优化培养基
按照表1配方配制MSM培养基，在配制的过程中将1.6g油酸乙酯与0.8g棕榈酸乙酯(油酸乙酯与棕榈酸乙酯的质量比为1∶2)加入培养基中，混合均匀，灭菌处理(高温高压灭菌)，得到优化培养基；
(3)将步骤(1)所述活化的菌种接种到步骤(2)所述优化培养基中，接种量为10％(v/v)，摇床培养处理，摇床培养处理的温度为40摄氏度，摇床培养处理的时间为216h，摇床培养处理的转速为80rpm，得到培养液；
(4)将步骤(3)所述培养液进行离心处理，离心处理的速率为12000rpm，离心处理的时间为1min，得到菌体沉淀，将所述菌体沉淀，用0.1％(w/w)PBS缓冲液洗涤三次，除去菌体沉淀上的残留培养基，再用正己烷洗涤 三次除去残留培养基中的脂溶性化合物；之后冻干，得到冻干菌粉，称重，计算生物量；
(5)称取0.5g步骤(4)所述冻干菌粉，加入2mL浓度为1mol/L的HCl溶液，混合均匀，然后在80℃条件下水浴30min，之后加入2mL三氯甲烷进行萃取，萃取三次，合并三次萃取液，用氮气吹干萃取液中的三氯甲烷，得到油脂类混合物(所述油脂混合物中含有特种油脂OPO)；
(6)测试步骤(5)所述油脂混合物的全样脂肪酸组成、sn-2位脂肪酸组成、含油量(根据GB 5009.168-2016食品安全国家标准测定)。
实施例4
一种使用微生物发酵生产特种油脂OPO的方法，包括如下步骤：
(1)菌种活化
将微生物(浑浊红球菌)接种至活化培养基(LB培养基)中，摇床培养处理，摇床培养处理的温度为40摄氏度，摇床培养处理的转速为80rpm，摇床培养处理的时间为60h，得到活化的菌种；
(2)配制优化培养基
按照表1配方配制MSM培养基，在配制的过程中将0.8g HPO与1.6g HOSFO(HPO∶HOSFO质量比为1∶2)加入培养基中，混合均匀，灭菌处理(高温高压灭菌)，得到优化培养基；
(3)将步骤(1)所述活化的菌种接种到步骤(2)所述优化培养基中，接种量为0.1％(v/v)，摇床培养处理，摇床培养处理的温度为20摄氏度，摇床培养处理的时间为48h，摇床培养处理的转速为250rpm，得到培养液；
(4)将步骤(3)所述培养液进行离心处理，离心处理的速率为3000rpm，离心处理的时间为20min，得到菌体沉淀，将所述菌体沉淀，用0.1％(w/w)PBS缓冲液洗涤三次，除去菌体沉淀上的残留培养基，再用正己烷洗涤三次除去残留培养基中的脂溶性化合物；之后冻干，得到冻干菌粉，称重，计算生物量；
(5)称取2g步骤(4)所述冻干菌粉，加入1mL浓度为2mol/L的HCl溶液，混合均匀，然后在80℃条件下水浴30min，之后加入2mL三氯甲烷进行萃取，萃取三次，合并三次萃取液，用氮气吹干萃取液中的三氯甲烷，得到油脂类混合物 (所述油脂混合物中含有特种油脂OPO)；
(6)测试步骤(5)所述油脂混合物的全样脂肪酸组成、sn-2位脂肪酸组成、含油量(根据GB 5009.168-2016食品安全国家标准测定)。
结果分析
测试结果见于下表2、图1、图2、图3及图4，下表2为油脂样品主要脂肪酸组成数据表(％)。
表2
Figure PCTCN2019114911-appb-000001
由表2可知，对比例1中油脂脂肪酸组成油酸含量较低，且棕榈酸与油酸的比例为1∶0.7。对比例2中全样脂肪酸与sn-2脂肪酸一致，说明脂肪酸在甘油骨架上为全随机分布。实施例1中，全样脂肪酸组成棕榈酸与油酸比例为1∶1.87，sn-2脂肪酸中棕榈酸含量为74.54％，说明甘三酯为OPO。实施例2中，全样脂肪酸棕榈酸与油酸比例为1∶2.15，sn-2脂肪酸棕榈酸含量为65.67％，说明甘三脂为OPO。
实施例3与实施例4的效果与实施例1相似，可以参照图1、图2、图4与表2。
以上实施例仅为本发明较优的实施方式，仅用于解释本发明，而非限制本发明，本领域技术人员在未脱离本发明精神实质下所作的改变、替换、修饰等均应属于本发明的保护范围。

Claims (10)

  1. 一种使用微生物发酵生产特种油脂OPO的方法，其特征在于，包括如下步骤：
    (1)将微生物接种至活化培养基中，摇床培养处理，得到活化的菌种；
    (2)将有机物加入基本培养基中，混合均匀，灭菌处理，得到优化培养基；
    (3)将步骤(1)所述活化的菌种接种到步骤(2)所述优化培养基中，摇床培养处理，得到培养液；
    (4)将步骤(3)所述培养液进行离心处理，取沉淀，洗涤，冷冻干燥得到冻干粉，加入盐酸溶液，混合均匀得到混合物；
    (5)将有机溶剂加入步骤(4)所述混合物中，进行萃取处理，去除溶剂得到所述特种油脂OPO。
  2. 根据权利要求1所述的使用微生物发酵生产特种油脂OPO的方法，其特征在于，步骤(1)所述菌种包括浑浊红球菌；所述活化培养基包括肉汤培养基及LB培养基。
  3. 根据权利要求1所述的使用微生物发酵生产特种油脂OPO的方法，其特征在于，步骤(1)所述微生物包括浑浊红球菌；摇床培养处理的温度为20-40摄氏度，摇床培养处理的转速为80-250rpm，摇床培养处理的时间为12-60h。
  4. 根据权利要求1所述的使用微生物发酵生产特种油脂OPO的方法，其特征在于，步骤(2)所述有机物包括棕榈酸、油酸、棕榈酸酯、油酸酯、十六烷、十八烷、棕榈油、棕榈油的分提产物及高油酸甘油三酯；所述有机物的添加量为基本培养基质量的0.1-10％；所述基本培养基包括MSM培养基。
  5. 根据权利要求1所述的使用微生物发酵生产特种油脂OPO的方法，其特征在于，步骤(2)所述灭菌处理的方式包括高温高压灭菌及中低压灭菌。
  6. 根据权利要求1所述的使用微生物发酵生产特种油脂OPO的方法，其特征在于，步骤(3)所述接种的接种量为优化培养基体积的0.1-10％。
  7. 根据权利要求1所述的使用微生物发酵生产特种油脂OPO的方法，其特征在于，步骤(3)所述摇床培养处理的温度为20-40摄氏度，摇床培养处理的转速为80-250rpm，摇床培养处理的时间为48-216h。
  8. 根据权利要求1所述的使用微生物发酵生产特种油脂OPO的方法，其特征在于，步骤(4)所述离心处理的转速为3000-12000rpm，离心处理的时间为1-20min。
  9. 根据权利要求1所述的使用微生物发酵生产特种油脂OPO的方法，其特征在于，步骤(4)所述盐酸溶液的浓度为1-3mol/L，所述冻干粉与盐酸溶液的质量体积比为0.1∶2-1∶2g/mL。
  10. 根据权利要求1所述的使用微生物发酵生产特种油脂OPO的方法，其特征在于，步骤(5)所述有机溶剂包括正丙烷、丁烷、戊烷、己烷、庚烷、丙酮、三氯甲烷、二氯甲烷、乙醚、甲醇及二氧化碳。
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