WO2021104165A1 - 裂殖壶菌及其应用、富含Sn-2位DHA的微生物油脂及其制备方法和应用 - Google Patents

裂殖壶菌及其应用、富含Sn-2位DHA的微生物油脂及其制备方法和应用 Download PDF

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WO2021104165A1
WO2021104165A1 PCT/CN2020/130385 CN2020130385W WO2021104165A1 WO 2021104165 A1 WO2021104165 A1 WO 2021104165A1 CN 2020130385 W CN2020130385 W CN 2020130385W WO 2021104165 A1 WO2021104165 A1 WO 2021104165A1
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schizochytrium
dha
microbial
fermentation
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French (fr)
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梁云
曹晟
王身健
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Priority to KR1020227017602A priority Critical patent/KR20220088912A/ko
Priority to CA3149969A priority patent/CA3149969C/en
Priority to EP20894525.3A priority patent/EP3896167A4/en
Priority to NZ778375A priority patent/NZ778375B2/en
Publication of WO2021104165A1 publication Critical patent/WO2021104165A1/zh
Priority to US17/375,551 priority patent/US12291737B2/en
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Priority to US19/170,018 priority patent/US20250230475A1/en
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/115—Fatty acids or derivatives thereof; Fats or oils
    • 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
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/03—Organic compounds
    • A23L29/035—Organic compounds containing oxygen as heteroatom
    • A23L29/04—Fatty acids or derivatives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/065—Microorganisms
    • 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; 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
    • C12N1/12—Unicellular algae; Culture media therefor
    • 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; 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
    • C12N1/12—Unicellular algae; Culture media therefor
    • C12N1/125—Unicellular algae isolates
    • 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; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C12P7/6434—Docosahexenoic acids [DHA]
    • 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
    • C12P7/6472—Glycerides containing polyunsaturated fatty acid [PUFA] residues, 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
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/89—Algae ; Processes using algae

Definitions

  • the invention relates to the technical field of microorganisms, in particular to a strain of Schizochytrium sp. and its application, a microbial grease rich in Sn-2 DHA, and a preparation method and application thereof.
  • Docosahexaenoic acid is a major structural fatty acid in the brain and eyes. It accounts for 97% of the n-3 fatty acids in the brain and 93% in the eyes. Studies have shown that compared with triglycerides with random structures, the special structure of DHA at the Sn-2 position is more conducive to body absorption. Another study pointed out that eating special-structured oils will make the highest DHA content in the brain, while eating random-structured fats will have the highest DHA content in the liver. This indicates that eating triglycerides with different structures will lead to differences in fatty acid metabolism, namely fatty acids. The absorption and utilization at the Sn-2 position is more effective than the Sn-1 and Sn-3 positions.
  • Pancreatic lipase the main fat-digesting enzyme, works by adsorbing on the oil-water interface. At the same time, it has a specific ester bond decomposition effect, that is, it only focuses on hydrolyzing the ester bonds of Sn-1 and Sn-3. Therefore, triglyceride After ester structure oil is decomposed by pancreatic lipase, Sn-1 and Sn-3 fatty acids form free fatty acids, Sn-2 fatty acids form monoglycerides, and free fatty acids are difficult to penetrate into bile salt micelles and be absorbed by the body.
  • Sn-2 fatty acids form monoglycerides, which can easily penetrate into bile salt micelles and be absorbed by the human body. Therefore, Sn-2 fatty acids are better than Sn-1 , Sn-3 fatty acid has a higher human absorption rate.
  • microbial oils and fats have been widely used in infant foods and health foods with the strengthening of people’s health awareness, and their nutritional effects have been increasingly recognized and recognized by the public.
  • the absorption rate has also become more and more of public concern.
  • More than 90% of the fatty acids in microbial oils are in the form of triglycerides, and the current DHA oils derived from microorganisms are fermented by microorganisms such as Ukenella, Schizochytrium, Thraustochytrium, Cryptodinium, and yeast.
  • the distribution ratio of the obtained triglyceride-type DHA on the glycerol skeleton is that the content of Sn-2 position is much lower than the content of Sn-1 and Sn-3 positions, and a large amount of Sn-1 and Sn-3 position DHA are in Soap salts are formed and lost during human digestion, and the health benefits of microbial oils are limited.
  • the purpose of the present invention is to overcome the problem that the high proportion of Sn-1 and Sn-3 DHA of triglycerides in microbial oils in the prior art leads to the low absorption rate of DHA by the human body, and to provide a Schizochytrium strain (Schizochytrium). sp.) and its application, a microbial oil rich in Sn-2 DHA and its preparation method and application.
  • the microbial oil is produced by fermentation of Schizochytrium and is rich in DHA and contains the triglyceride Sn-2 in the oil.
  • the proportion of DHA is not less than 23% by weight, which effectively improves the absorption rate of DHA in the human body.
  • the first aspect of the present invention provides a microbial oil, the microbial oil contains triglycerides, and the ratio of DHA at the Sn-2 position to the total fatty acids at the Sn-2 position is not less than 23 by weight. %.
  • the total DHA content of the microbial grease is not less than 38% by weight.
  • the second aspect of the present invention provides a method for preparing microbial grease, which comprises inoculating a Schizochytrium strain into a fermentation medium and fermenting it.
  • the Schizochytrium strain is the deposit number GDMCC No. 60733.
  • the third aspect of the present invention provides a microbial oil prepared by the above method, the microbial oil comprises triglycerides, and the ratio of the DHA at the Sn-2 position of the triglyceride to the total fatty acids at the Sn-2 position is not less than 23 by weight %.
  • the fourth aspect of the present invention provides the application of the above-mentioned microbial oils and fats in foods, and the foods are preferably formula foods for infants and young children, health foods and health foods.
  • the fifth aspect of the present invention provides a strain of Schizochytrium (Schizochytrium sp.), which is Schizochytrium (Schizochytrium sp.) with a deposit number of GDMCC No. 60733.
  • the sixth aspect of the present invention provides the application of the aforementioned Schizochytrium in the production of microbial oils.
  • the microbial oil produced by the fermentation of Schizochytrium strains provided by the present invention is not only rich in DHA, but the proportion of DHA at the Sn-2 position of triglyceride is not less than 23%, which improves the human body’s microbial oil
  • the absorption rate of DHA can effectively promote the absorption and utilization of the functional health factor DHA by the human body.
  • strains of the present invention are as follows:
  • the present invention provides a microbial oil containing triglycerides, and the ratio of DHA at the Sn-2 position of the triglyceride to the total fatty acids at the Sn-2 position is not less than 23% by weight.
  • DHA refers to docosahexaenoic acid.
  • the microbial oil contains triglycerides, and the content of triglycerides in the microbial oil is 90% by weight or more.
  • the total DHA content of the microbial grease is not less than 38% by weight.
  • total DHA refers to the total amount of DHA contained in the measured microbial oils and fats, please refer to the method of GB26400-2011, and the method of determination of other fatty acid content, refer to GB5009.168-2016.
  • Triglyceride DHA means that DHA is connected to the glycerol backbone through ester bonds.
  • the proportion of DHA at the Sn-2 position of the triglyceride in the microbial oil is not less than 23%.
  • the proportion of DHA at the Sn-2 position of the triglyceride in the microbial oil is not less than 23%.
  • the total DHA content is not less than 38%.
  • the present invention provides a method for preparing microbial oil, which comprises inoculating a Schizochytrium strain into a fermentation medium and fermenting it.
  • the Schizochytrium strain has a deposit number of GDMCC No. 60733.
  • Schizochytrium sp. (Schizochytrium sp.).
  • the strain for preparing the microbial oil can be obtained by a method conventionally used in this field.
  • the Schizochytrium provided by the present invention is obtained by screening after mutagenesis.
  • the mutagenesis method may be a method conventionally used in the art, such as physical mutagenesis (such as ultraviolet mutagenesis, ARTP mutagenesis, etc.) and chemical mutagenesis.
  • the method of mutagenesis is ARTP mutagenesis.
  • ARTP mutagenesis can be carried out in a conventional mutagenesis system, for example, it can be the MPMS-a multifunctional mutagenesis system produced by Beijing Adhoc International Technology Co., Ltd.
  • the specific operation mode of the mutagenesis can be referred to the operation mode conventionally adopted in the art.
  • the mutagenesis conditions include: plasma mutagenesis power of 80-120W, gas flow rate of 8-12SLM, and treatment distance of 1-3mm.
  • the mutagenesis time is preferably 5-60s, more preferably 15-30s.
  • the OD 600 value is preferably 0.6-0.8 or the bacterial concentration is preferably 10 6 -10 8 cfu/mL.
  • the operating conditions of the mutagenesis are such that the lethality of Schizochytrium is 90-95%.
  • the content of Sn-2 fatty acid is determined by the method of "GB/T24894-2010/ISO6800: 1997 Determination of 2-position fatty acid composition of triglyceride molecules of animal and vegetable oils and fats".
  • the genetic stability of the strain obtained by the screening can also be evaluated.
  • the genetic stability verification theory of modern breeding believes that for mutation breeding, after 5 generations of transfer, if its performance is stable and meets the expected requirements, it is considered that the performance of the induced strain is stable.
  • the Schizochytrium sp. of the present invention was preserved at the Guangdong Provincial Microbial Culture Collection on August 8, 2019 (Address: 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou, Guangdong province, China.
  • the postal code: 510070) (the abbreviation of the depository unit is GDMCC), and the deposit number is GDMCC No. 60733.
  • the Schizochytrium provided by the present invention is fermented to produce microbial oils rich in Sn-2 DHA, and the fermentation method has no special requirements, as long as the Schizochytrium can proliferate.
  • the fermentation conditions of the Schizochytrium fermentation have no special requirements, and may be a common choice for obtaining Schizochytrium fermentation products.
  • the fermentation conditions include: pH 6 7.5, the temperature is 27-31°C, the time is 4-8 days, and the ventilation ratio is 0.5-1.1VVM.
  • the inoculum amount can be selected in a wide range, and preferably, the inoculum amount is 5-10% by volume.
  • the medium used for the fermentation of Schizochytrium may be a medium conventionally used for the fermentation of Schizochytrium in the art.
  • the fermentation medium contains a carbon source, a nitrogen source, inorganic salts, trace elements and vitamins.
  • the carbon source can be glucose, sucrose, or any other substance that can provide a carbon source or a combination thereof
  • the nitrogen source can be sodium glutamate, yeast powder, yeast extract or any other substance that can provide a nitrogen source Or a combination.
  • the carbon source is glucose and/or sucrose.
  • the nitrogen source is one or more of sodium glutamate, yeast powder and yeast extract.
  • the inorganic salt is one or more of calcium salt, phosphate, potassium salt, sodium salt, magnesium salt and ammonium salt.
  • the trace element is one or more of nickel, copper, molybdenum, cobalt, zinc, iron and manganese.
  • the vitamin is one or more of vitamin B 1 , vitamin B 12 , vitamin B 6 , calcium pantothenate and biotin.
  • the carbon source, nitrogen source and inorganic salt can be added directly.
  • the mother liquor is often prepared and the mother liquor is used. To add.
  • the content of each component in the trace element mother liquor can be selected within a wide range.
  • the formula of the trace element mother liquor is: nickel sulfate 1-3g/L, copper sulfate 1-3g/L , Sodium molybdate 0.02-0.08g/L, manganese chloride 2-4g/L, cobalt chloride 0.02-0.08g/L, zinc sulfate 2-4g/L, ferrous sulfate 8-10g/L.
  • the content of each component in the microbiotic mother liquor can be selected within a wide range.
  • the formulation of the microbiotic mother liquor is: vitamin B1 9-11g/L, vitamin B12 0.1-0.3 g/L, calcium pantothenate 2-4g/L, biotin 0.005-0.01g/L.
  • the nitrogen source in the fermentation medium contains glutamic acid.
  • the concentration of glutamic acid can be selected in a wide range.
  • the concentration of glutamic acid in the fermentation medium is 5-15 g/L.
  • the concentration of the carbon source in the fermentation medium can also be selected within a wide range.
  • the concentration of the carbon source in the fermentation medium is 5-70 g/L.
  • the content of carbon source is 5-70g/L
  • the content of nitrogen source is 15-45g/L
  • the content of inorganic salt is 5-40g/L.
  • the content of trace elements is 0.01-0.04g/L
  • the content of vitamins is 0.01-0.04g/L.
  • the content of carbon source is 40-60g/L
  • the content of nitrogen source is 20-35g/L
  • the content of inorganic salts is 10-25g/L
  • the content of trace elements is 0.015- 0.035g/L
  • the vitamin content is 0.01-0.03g/L.
  • the fermentation medium further contains an antifoaming agent, and the content of the antifoaming agent is preferably 0.1-0.5 g/L.
  • the Schizochytrium is inoculated into the fermentation medium during the fermentation process, part of the carbon source and part of the nitrogen source are added to the fermentation medium, and the carbon source is continuously added during the fermentation process according to the regulation process of the carbon to nitrogen ratio , Nitrogen source, no more carbon source is added until the residual sugar content in the medium is 0 when the fermentation terminal is approaching, so as to adjust the carbon-nitrogen ratio to form auxotrophic nutritional conditions to ensure the level of oil production by Schizochytrium fermentation .
  • the method preferably includes: the Schizochytrium strain is activated and made into a shake flask seed solution, inoculated into the seed medium for cultivation, and then transferred to the fermentation medium for fermentation cultivation .
  • the method further comprises: inoculating the Schizochytrium strain into an activation medium to obtain an activation solution after activation; inoculating the activation solution into a seed culture medium for expansion to obtain a seed solution; It is inoculated into the fermentation medium and fermented to prepare microbial oil.
  • the Schizochytrium strain can be a strain preserved in an ampoule or a glycerin tube.
  • the activation process includes thawing the frozen glycerol tube strain of the Schizochytrium strain and inoculating it into an activation medium for activation.
  • the activation solution can be prepared by one or more activations.
  • the activation conditions can be selected in a wide range.
  • the activation conditions include: a temperature of 27.5-28.5°C, a rotation speed of 150-200 r/min, and a time of 48-72h.
  • the activation medium contains carbon sources, nitrogen sources, inorganic salts, trace elements and vitamins; wherein, in the activation medium, the content of the carbon source is 30-50 g/L, and the content of the nitrogen source is 25. -45g/L, the content of inorganic salts is 25-40g/L, the content of trace elements is 0.015-0.03g/L, and the content of vitamins is 0.01-0.02g/L.
  • the process of the strain activation culture includes: thawing the frozen glycerol tube strain of the Schizochytrium strain, and inoculating the glycerol tube strain with a sterile straw
  • culture for 48-72h at a temperature of 27.5-28.5°C and a rotation speed of 150-200r/min.
  • the content of carbon source is 30-50g/L
  • the content of nitrogen source is 25-45g/L
  • the content of inorganic salts is 25-40g/L
  • the content of trace elements is 0.015-0.025 g/L
  • the vitamin content is 0.01-0.02g/L.
  • the amount of inoculation of the glycerol tube strain is based on the way that one glycerol tube is transferred to 4-6 Erlenmeyer flasks.
  • the size of the Erlenmeyer flask is 500 mL
  • the volume of the seed culture medium in each Erlenmeyer flask is 200- 300mL.
  • the method for expanding culture preferably includes: connecting the seed liquid to a seed culture medium for expanding culture to obtain the seed liquid.
  • the conditions for the expansion include: a temperature of 27-28.5°C; a time of 48-72h; and a ventilation ratio of 0.5-0.8VVM.
  • the seed culture medium contains carbon sources, nitrogen sources, inorganic salts, trace elements and vitamins; wherein, in the seed culture medium, the content of the carbon source is 25-60 g/L, and the content of the nitrogen source is 10 -30g/L, the content of inorganic salts is 15-35g/L, the content of trace elements is 0.01-0.02g/L, and the content of vitamins is 0.01-0.02g/L.
  • the seed culture medium also contains an antifoaming agent, and the content of the antifoaming agent is preferably 0.1-0.5 g/L.
  • the method of expansion culture includes: after the seed liquid is connected to the first-level seed culture medium for the first-level expansion, and then the seed liquid is connected to the second-level seed culture medium for the second-level expansion. Cultivate to obtain seed liquid.
  • the conditions for the first-level expansion training include: a temperature of 27-28.5° C.; a time of 48-60 h; and a ventilation ratio of 0.5-0.8 VVM.
  • the conditions for the secondary expansion training include: a temperature of 27-28.5°C; a time of 12-24h; and a ventilation ratio of 0.5-0.8VVM.
  • the content of carbon source is 25-35g/L
  • the content of nitrogen source is 10-30g/L
  • the content of inorganic salts is 15-35g/L
  • the content of trace elements is 0.01-0.02g/L
  • the content of vitamins is 0.01-0.02g/L.
  • the carbon source content is 40-60 g/L
  • the nitrogen source content is 10-15 g/L
  • the inorganic salt content is 15-20 g/L
  • the trace element content is 0.01-0.02g/L
  • the content of vitamins is 0.01-0.02g/L.
  • the present invention can further process the above-mentioned fermentation products to obtain microbial oils.
  • the processing method is not particularly limited, as long as the microbial oils can be separated from the fermentation products, in order to obtain higher microorganisms.
  • the oil yield, the treatment includes breaking the wall of the fermented product, and extracting to obtain the oil product in the fermented product.
  • the present invention provides a microbial oil prepared by the above method, the microbial oil contains triglycerides, and the ratio of DHA at the Sn-2 position of the triglyceride to the total fatty acids at the Sn-2 position is not less than 23. weight%.
  • the total DHA content of the microbial grease is not less than 38% by weight.
  • the present invention provides the application of the above-mentioned microbial oil in food.
  • the food is preferably infant formula food, health food or health food.
  • the fifth aspect of the present invention provides a strain of Schizochytrium sp., which is Schizochytrium sp. with the deposit number GDMCC No. 60733.
  • the method of obtaining the Schizochytrium is described in the second aspect, and will not be repeated here.
  • the sixth aspect of the present invention provides the application of the aforementioned Schizochytrium in the production of microbial oils.
  • the microbial oil contains triglycerides, and the ratio of the DHA at the Sn-2 position of the triglyceride to the total fatty acids at the Sn-2 position is not less than 23% by weight.
  • the total DHA content of the microbial grease is not less than 38% by weight.
  • the content of DHA in microbial oils and fats is measured by the method in GB26400-2011, and the fatty acid composition is measured by the method in GB5009.168-2016;
  • the method for determining the human body’s absorption rate of DHA is the “human body efficacy test method”: male and female volunteers are recruited to consume the DHA oil produced by this patented method and contrast DHA oil, and then blood is drawn to determine the DHA content in the blood and the Sn-2 DHA fatty acid content To calculate the absorption rate.
  • the conventional strains of Schizochytrium come from the China Industrial Microorganism Collection and Management Center, and the number is CICC 11091s;
  • Glucose sucrose, yeast powder, sodium glutamate, yeast extract, sodium chloride, magnesium sulfate, calcium chloride, potassium dihydrogen phosphate, nickel sulfate, copper sulfate, sodium molybdate, cobalt chloride, zinc sulfate, sulfuric acid Ferrous iron, manganese chloride, vitamin B 1 , vitamin B 12 , vitamin B 6 , calcium pantothenate, biotin, sodium bicarbonate, sodium sulfate, ammonium sulfate, and potassium chloride are all commercially available products.
  • the formula of the mother liquor of trace elements is: nickel sulfate 2g/L, copper sulfate 1.9g/L, sodium molybdate 0.04g/L, manganese chloride 2.8g/L, cobalt chloride 0.04g/L , Zinc sulfate 3.2g/L, Ferrous sulfate 9g/L;
  • the formula of the vitamin mother liquor is: vitamin B 1 10.3 g/L, vitamin B 12 0.16 g/L, calcium pantothenate 3.2 g/L, and biotin 0.008 g/L.
  • This preparation example is used to illustrate the method for obtaining Schizochytrium sp. according to the present invention.
  • the activation medium formula is: glucose 40g/L, sodium glutamate 31g/L, sodium chloride 19g/L, yeast extract 5.8g/L, magnesium sulfate 8g/L, potassium dihydrogen phosphate 5.7g/L, trace amount Element 1g/L, vitamin 1g/L. In the case of solid medium, it also contains 18g/L agar.
  • the hair strain stored in the ampoule inoculate it into the activation medium, and activate and culture it at 28°C for 2 days.
  • the plasma mutagenesis power is 100W
  • the gas flow rate is 100W.
  • the treatment distance is 2mm
  • the DHA bacterial suspension is prepared with an OD 600 value of 0.6-0.8 or a bacterial concentration of 10 6 -10 8
  • the mutagenesis time is 25 s
  • the lethality rate is 92.54%.
  • the Schizochytrium sp. provided by the present invention was obtained, which was deposited in the Guangdong Provincial Microbial Culture Collection on August 8, 2019 (Address: 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou , Guangdong Institute of Microbiology, Zip Code: 510070) (abbreviation of the depository is GDMCC), and the deposit number is GDMCC No. 60733.
  • This example is used to illustrate the 30-L fermentation production of Schizochytrium according to the present invention.
  • the activation medium formula in this example is: glucose 40g/L, sodium glutamate 31g/L, yeast extract 5.8g/L, sodium chloride 19g/L, magnesium sulfate 8g/L, potassium dihydrogen phosphate 5.7g /L, trace element 1g/L, vitamin 1g/L;
  • the seed culture medium formula is: glucose 30g/L, sodium glutamate 6.3g/L, yeast extract 8.3g/L, yeast powder 8.3g/L, sodium chloride 1.45g/L, magnesium sulfate 5.18g/L, Potassium dihydrogen phosphate 1.66g/L, calcium chloride 0.25g/L, sodium bicarbonate 0.25g/L, sodium sulfate 9.34g/L, ammonium sulfate 1.04g/L, potassium chloride 0.83g/L, trace elements 1g /L, vitamin 1g/L, antifoaming agent 0.3g/L;
  • the fermentation medium formula is: glucose 50g/L, sodium glutamate 15g/L, yeast extract 10.9g/L, sodium chloride 2.6g/L, magnesium sulfate 5.8g/L, potassium dihydrogen phosphate 2.4g/L , Calcium chloride 0.25g/L, sodium bicarbonate 0.22g/L, sodium sulfate 3.62g/L, ammonium sulfate 1.13g/L, potassium chloride 0.94g/L, trace element mother liquor 1.1g/L, vitamin mother liquor 1.1 g/L, defoamer 0.19g/L.
  • step (2) Inoculate the activation solution obtained in step (1) into a shake flask containing 200 mL of activation medium for expansion culture, the inoculation amount is 3%, and culture for 72 hours on a shaker with a rotation speed of 180r/min and a temperature of 28°C ;
  • the seed tank is filled with 3L of seed culture medium, and 200mL of the activation solution obtained in step (2) is connected to the seed tank.
  • the temperature is 28°C
  • the stirring speed is 180r/min
  • the aeration rate is 0.6vvm
  • the fermenter is equipped with 14L fermentation medium, and all the seed liquid obtained in step (3) is transferred to the fermenter, and the temperature is 29°C, the stirring speed is 140r/min, the aeration rate is 0.95vvm, and the tank Cultivate for 5-6 days at a pressure of 0.03Mpa and a pH of 6.8.
  • a sterile solution with a glucose concentration of 250g/L and a sterile solution with a sodium glutamate concentration of 250g/L are added to maintain glutamic acid.
  • the concentration is 5-8g/L
  • the carbon source concentration is 10-23g/L.
  • step (4) After 5L of the fermentation broth obtained in step (4) is broken by enzymolysis, the water phase, the residue phase and the oil phase are separated by a laboratory high-speed centrifuge to obtain the microbial oil ester;
  • step (5) The microbial oil obtained in step (5) was subjected to analysis and determination of the DHA content, fatty acid composition and the ratio of DHA at the Sn-2 position of triglyceride in the oil. The results are shown in Table 1.
  • This example is used to illustrate the 100L fermentation production of Schizochytrium in the present invention.
  • the activation medium formula in this example is: glucose 30g/L, sodium glutamate 20g/L, yeast extract 5g/L, sodium chloride 15g/L, magnesium sulfate 6g/L, potassium dihydrogen phosphate 4g/L , Trace element 0.8g/L, vitamin 0.75g/L;
  • the seed medium formula is: glucose 25g/L, sodium glutamate 5.5g/L, yeast extract 7g/L, yeast powder 7g/L, sodium chloride 1.1g/L, magnesium sulfate 4g/L, dihydrogen phosphate Potassium 1.2g/L, calcium chloride 0.15g/L, sodium bicarbonate 0.15g/L, sodium sulfate 7g/L, ammonium sulfate 0.8g/L, potassium chloride 0.6g/L, trace elements 0.8g/L, Vitamin 0.75g/L, defoamer 0.2g/L;
  • the fermentation medium formula is: glucose 30g/L, sucrose 10g/L, sodium glutamate 12g/L, yeast extract 9g/L, sodium chloride 2g/L, magnesium sulfate 4.5g/L, potassium dihydrogen phosphate 2g /L, calcium chloride 0.2g/L, sodium bicarbonate 0.2g/L, sodium sulfate 2.8g/L, ammonium sulfate 1g/L, potassium chloride 0.8g/L, trace elements 0.9g/L, vitamin 0.9g /L, defoamer 0.15g/L.
  • the inoculation amount is: inoculate 5 bottles of the activation solution obtained in step (1) from 2 bottles In the seed culture medium, cultivate for 48h on a shaker with a rotation speed of 150r/min and a temperature of 28°C;
  • the seed tank is equipped with 6L of seed culture medium, and 400mL of the activation solution obtained in step (2) is connected to the seed tank.
  • the temperature is 28°C
  • the stirring speed is 150r/min
  • the aeration rate is 0.5vvm
  • the fermentation tank is equipped with 45L fermentation medium, and all the seed liquid obtained in step (3) is transferred to the fermentation tank, with a temperature of 28-29°C, a stirring speed of 90-120r/min, and aeration volume Cultivate for 5-6 days under the conditions of 0.5-0.8vvm and tank pressure of 0.03Mpa.
  • sterile liquid with a glucose concentration of 250g/L and a sterile liquid with a sodium glutamate concentration of 250g/L to maintain The concentration of glutamic acid is 8-12g/L, the concentration of carbon source is 40-65g/L, the supplement of carbon source and glutamic acid is stopped for 96h, and the fermentation broth is finally obtained;
  • step (5) After enzymatic hydrolysis of 10L of the fermentation broth obtained in step (5), use a laboratory centrifuge to separate the microbial oil ester;
  • step (5) The microbial oil obtained in step (5) was subjected to analysis and determination of the DHA content, fatty acid components and the ratio of DHA at the Sn-2 position of triglyceride in the oil. The results are shown in Table 2.
  • This example is used to illustrate the 45M 3 industrial fermentation production of Schizochytrium according to the present invention.
  • the activation medium formula in this example is: glucose 50g/L, sodium glutamate 35g/L, yeast extract 10g/L, sodium chloride 22g/L, magnesium sulfate 10g/L, potassium dihydrogen phosphate 8g/L , Trace elements 1.4g/L, vitamin 1.5g/L;
  • the first-level seed culture medium formula is: glucose 35g/L, sodium glutamate 8g/L, yeast extract 10g/L, yeast powder 10g/L, sodium chloride 1.9g/L, magnesium sulfate 6g/L, diphosphate Potassium hydrogen 2.1g/L, calcium chloride 0.4g/L, sodium bicarbonate 0.4g/L, sodium sulfate 12g/L, ammonium sulfate 1.3g/L, potassium chloride 1.1g/L, trace elements 1.2g/L , Vitamin 1.2g/L, defoamer 0.4g/L;
  • the secondary seed culture medium formula is: glucose 50g/L, sodium glutamate 5.65g/L, yeast extract 5.65g/L, sodium chloride 1.3g/L, magnesium sulfate 4.65g/L, potassium dihydrogen phosphate 1.49 g/L, calcium chloride 0.22g/L, sodium bicarbonate 0.22g/L, sodium sulfate 8.39g/L, ammonium sulfate 0.93g/L, potassium chloride 0.74g/L, trace elements 0.93g/L, vitamins 0.93g/L, defoamer 0.3g/L;
  • the fermentation medium formula is: glucose 40g/L, sucrose 20g/L, sodium glutamate 20g/L, yeast extract 12g/L, sodium chloride 3.2g/L, magnesium sulfate 7g/L, potassium dihydrogen phosphate 3g /L, calcium chloride 0.4g/L, sodium bicarbonate 0.3g/L, sodium sulfate 4.5g/L, ammonium sulfate 1.5g/L, potassium chloride 1.2g/L, trace elements 1.6g/L, vitamin 1.6 g/L.
  • step (2) Inoculate the 2 bottles of activation solution obtained in step (1) into 5 Erlenmeyer flasks containing 200 mL of activation medium for expansion culture, and incubate on a shaker with a rotation speed of 180r/min and a temperature of 28 ⁇ 0.5°C for 60 hours ;
  • the first-level seed tank is filled with 500L first-level seed culture medium, sterilized and cooled to below 40°C, and then 1L of the activation solution obtained in step (2) is transferred to the first-level seed tank, and the temperature is 28 ⁇ Cultivate for 55h under the conditions of 0.5°C, stirring speed of 150r/min and pH of 6.8;
  • the secondary seed tank is equipped with 6M 3 secondary seed culture medium, sterilized and cooled to below 40°C, and then all the primary seed liquid obtained in step (3) is transferred to the secondary seed under aseptic conditions In the tank, incubate for 18 hours at a temperature of 28 ⁇ 0.5°C, stirring at 150r/min, and pH 6.8;
  • the main fermentation tank is equipped with 22M 3 fermentation medium, sterilized and cooled to below 40°C, and all the secondary seed liquid obtained in step (4) is transferred to the main fermentation tank under aseptic conditions. Cultivate for 5 days under the conditions of 28 ⁇ 0.5°C, stirring 100r/min, aeration ratio of 1.0VVM, and pH7.5. During the fermentation process, sterile liquid with a glucose concentration of 250g/L and sodium glutamate with a concentration of 250g/L are added. Sterile liquid, to keep the glutamic acid concentration at 12-15g/L and the carbon source concentration at 55-70g/L, stop supplementing the carbon source and glutamic acid at 96h, and finally obtain the fermentation broth;
  • the fermentation broth is preheated to 85-90°C and separated by a three-phase centrifuge to obtain microbial oil esters
  • step (6) The microbial oil obtained in step (6) was subjected to analysis and determination of DHA content, fatty acid components and the ratio of DHA at the Sn-2 position of triglyceride in the oil. The results are shown in Table 3.

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Abstract

一种裂殖壶菌及其应用、富含Sn-2位DHA的微生物油脂及其制备方法和应用。所述微生物油脂中甘油三酯Sn-2位上DHA的比例不低于23％，所述微生物油脂的制备方法包括将裂殖壶菌菌株接种到发酵培养基中发酵制得，所述裂殖壶菌菌株为保藏编号为GDMCC No.60733的裂殖壶菌(Schizochytrium sp.)。该微生物油脂中甘油三酯Sn-2位上DHA的比例不低于23％，从而使该微生物油脂中DHA的人体吸收率明显高于常规菌株生产的微生物油脂，促进人体对功能性脂肪酸DHA的吸收与利用。

Description

裂殖壶菌及其应用、富含Sn-2位DHA的微生物油脂及其制备方法和应用
相关申请的交叉引用
本申请要求2019年11月26日提交的中国专利申请201911175787.4的权益，该申请的内容通过引用被合并于本文。
技术领域
本发明涉及微生物技术领域，具体涉及一株裂殖壶菌(Schizochytrium sp.)及其应用，一种富含Sn-2位DHA的微生物油脂及其制备方法和应用。
背景技术
二十二碳六烯酸(DHA)是大脑和眼部中的一种主要结构性脂肪酸，占大脑中n-3脂肪酸的97％，在眼部中的比例达93％。研究表明与随机结构的甘油三酯相比，DHA在Sn-2位的特殊结构甘油三酯更利于人体吸收。另有研究指出进食特殊结构油脂会使DHA在大脑中含量最高，而进食随机结构的油脂，DHA则在肝脏中含量最高，这表明进食不同结构的甘油三酯会导致脂肪酸代谢的差异，即脂肪酸在Sn-2位比在Sn-1、Sn-3位的吸收利用更有效。胰腺脂肪酶这一主要的脂肪消化酶，通过吸附在油水界面来发挥作用，同时它具有专一的酯键分解作用，即只专注水解Sn-1、Sn-3的酯键，因此，甘油三酯结构的油脂经胰腺脂肪酶分解后，Sn-1、Sn-3位脂肪酸形成游离的脂肪酸、Sn-2位脂肪酸形成单甘油酯，而呈游离态的脂肪酸难以渗入胆盐微胶粒被人体吸收，从而容易与肠道中钙、镁离子结合形成不溶性的皂盐损失掉，而Sn-2位脂肪酸形成单甘油酯易渗入胆盐微胶粒被人体吸收，所以Sn-2位脂肪酸比Sn-1、Sn-3位脂肪酸具有更高的人体吸收 率。
微生物油脂作为DHA的主要来源之一，随着人们保健意识的加强，已被广泛应用于婴幼儿食品、保健食品中，其营养功效愈来愈得到公众的认识、认可，而微生物油脂中DHA的吸收率也愈来愈为公众所关注。微生物油脂中90％以上的脂肪酸是甘油三酯结构形式，而目前微生物来源的DHA油脂采用吾肯氏菌属、裂殖壶菌属、破囊壶菌目、隐甲藻属、酵母等微生物发酵所获取，得到的甘油三酯型DHA的甘油骨架上的分布比例是Sn-2位的含量远远低于Sn-1、Sn-3位的含量，大量Sn-1、Sn-3位DHA在人体消化过程中形成皂盐而损失，微生物油脂的保健功效受到了限制。
发明内容
本发明的目的是为了克服现有技术存在的微生物油脂中甘油三酯Sn-1、Sn-3位DHA的比例高导致人体对DHA的吸收率低的问题，提供一株裂殖壶菌(Schizochytrium sp.)及其应用，一种富含Sn-2位DHA的微生物油脂及其制备方法和应用，该微生物油脂通过裂殖壶菌发酵生产所得，富含DHA且油脂中甘油三酯Sn-2位DHA的比例不低于23重量％，有效提高DHA的人体吸收率。
为了实现上述目的，本发明第一方面提供了一种微生物油脂，所述微生物油脂包含甘油三酯，所述甘油三酯Sn-2位DHA占Sn-2位全脂肪酸的比例不低于23重量％。
优选地，所述微生物油脂的总DHA的含量不低于38重量％。
本发明第二方面提供了一种微生物油脂的制备方法，该方法包括将裂殖壶菌菌株接种到发酵培养基中发酵制得，所述裂殖壶菌菌株为保藏编号为GDMCC No.60733的裂殖壶菌(Schizochytrium sp.)。
本发明第三方面提供了由上述的方法制备得到的微生物油脂，所述微生物油脂包含甘油三酯，所述甘油三酯Sn-2位DHA占Sn-2位全脂肪酸的 比例不低于23重量％。
本发明第四方面提供了如上所述的微生物油脂在食品中的应用，所述食品优选为婴幼儿配方食品、保健食品以及健康食品。
本发明第五方面提供一株裂殖壶菌(Schizochytrium sp.)，所述裂殖壶菌为保藏编号为GDMCC No.60733的裂殖壶菌(Schizochytrium sp.)。
本发明第六方面提供如上所述的裂殖壶菌在生产微生物油脂中的应用。
通过上述技术方案，本发明提供的利用裂殖壶菌菌株发酵生产的微生物油脂，不仅富含DHA，而且甘油三酯Sn-2位上DHA的比例不低于23％，提高人体对该微生物油脂中DHA的吸收率，从而有效促进人体对功能保健因子DHA的吸收与利用。
本发明的其他特征和优点将在随后的具体实施方式部分予以详细说明。
生物保藏
本发明的菌株如下：
裂殖壶菌(Schizochytriumsp.)，于2019年8月8日被保藏在广东省微生物菌种保藏中心(地址：广州市先烈中路100号大院59号楼5楼，广东省微生物研究所，邮政编码：510070)(保藏单位的缩写为GDMCC)，保藏编号为GDMCC No.60733。
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，所述微生物油脂包含甘油 三酯，所述甘油三酯Sn-2位DHA占Sn-2位全脂肪酸的比例不低于23重量％。
在本发明中，DHA是指二十二碳六烯酸。
优选地，所述微生物油脂中含有甘油三酯，所述微生物油脂中甘油三酯的含量为90重量％以上。
优选地，所述微生物油脂的总DHA的含量不低于38重量％。
应当理解的是，总DHA是指测定的微生物油脂中所含的DHA总量，可参见GB26400-2011的方法，其他脂肪酸的含量测定方法，参见GB5009.168-2016。甘油三酯型DHA是指DHA通过酯键与甘油骨架连接。
在本发明的一种优选的实施方式中，所述微生物油脂中甘油三酯Sn-2位上DHA的比例不低于23％。
在本发明的一种优选的实施方式中，所述微生物油脂中甘油三酯Sn-2位上DHA的比例不低于23％。总DHA含量不低于38％。
第二方面，本发明提供了一种微生物油脂的制备方法，该方法包括将裂殖壶菌菌株接种到发酵培养基中发酵制得，所述裂殖壶菌菌株为保藏编号为GDMCC No.60733裂殖壶菌(Schizochytriumsp.)。
在本发明中，可以通过本领域常规使用的方法获得制备该微生物油脂的菌株。
本发明提供的所述裂殖壶菌为通过诱变后筛选得到。
所述诱变的方法可以是本领域常规使用的方法，比如可以为物理诱变(如紫外诱变、ARTP诱变等)和化学诱变等。
在本发明一种优选的实施方式中，所述诱变的方法为ARTP诱变。
ARTP诱变可以在常规使用的诱变系统中进行，比如可以为北京艾德豪克国际技术有限公司生产的MPMS——多功能诱变系统。
所述诱变的具体操作方式可参见本领域常规采用的操作方式，优选地，诱变条件包括：等离子诱变功率为80-120W，气体流量为8-12SLM，处理 距离为1-3mm。
诱变时间优选为5-60s，更优选为15-30s。
使用菌悬液进行诱变，其OD 600值优选为0.6-0.8或菌浓度优选为10 6-10 8cfu/mL。
优选地，所述诱变的操作条件使得裂殖壶菌的致死率为90-95％。
出发菌株经过诱变后，可以通过多次筛选得到高产油率、且Sn-2位DHA含量高的菌株。
应当理解的是，本领域技术人员知晓通过比较Sn-2位为DHA的甘油三酯的比例大小来筛选高产Sn-2位为DHA的甘油三酯的菌株。
在本发明中，Sn-2位脂肪酸的含量采用“GB/T24894-2010/ISO6800：1997动植物油脂甘三酯分子2-位脂肪酸组分的测定”的方法测定。
在本发明中，为了获得稳定性高的菌株，还可以对筛选得到的菌株进行遗传稳定性评价。现代育种的遗传稳定验证理论认为，对于诱变育种，采用5代转接后，如果其性能稳定达到预期要求，则认为该诱变得到的菌种的性能稳定。
通过上述诱变、筛选和遗传稳定性评价，得到Sn-2位DHA含量高的菌株。
本发明的裂殖壶菌(Schizochytriumsp.)，于2019年8月8日被保藏在广东省微生物菌种保藏中心(地址：广州市先烈中路100号大院59号楼5楼，广东省微生物研究所，邮政编码：510070)(保藏单位的缩写为GDMCC)，保藏编号为GDMCC No.60733。
本发明提供的裂殖壶菌经过发酵产生富含Sn-2位DHA的微生物油脂，所述发酵的方法没有特别的要求，只要是能使所述裂殖壶菌增殖即可。
所述裂殖壶菌发酵的发酵条件没有特别的要求，可以是获得裂殖壶菌发酵产物常用的选择，为了获得较多的发酵产物，优选地，所述发酵的条件包括：pH为6-7.5，温度为27-31℃，时间为4-8天，通气比为0.5-1.1VVM。
当裂殖壶菌以种子液的形式接种时，其接种量可以在较宽的范围内选择，优选地，接种量为5-10体积％。
用于裂殖壶菌发酵的培养基可以为本领域常规用于裂殖壶菌发酵的培养基，优选地，所述发酵培养基包含碳源、氮源、无机盐、微量元素和维生素。
本发明中，所述碳源可以为葡萄糖、蔗糖或其他任何可以提供碳源的物质或其组合，氮源可以为谷氨酸钠、酵母粉、酵母浸膏或其他任何可以提供氮源的物质或其组合。
优选地，所述碳源为葡萄糖和/或蔗糖。
优选地，所述氮源为谷氨酸钠、酵母粉和酵母浸膏中的一种或多种。
优选地，所述无机盐为钙盐、磷酸盐、钾盐、钠盐、镁盐和铵盐中的一种或多种。
优选地，所述微量元素为镍、铜、钼、钴、锌、铁和锰中的一种或多种。
优选地，所述维生素为维生素B 1、维生素B 12、维生素B 6、泛酸钙和生物素中的一种或多种。
在本发明中，所述碳源、氮源和无机盐可以通过直接添加的方式添加，但是微量元素和维生素在培养基体积较小的情况下，难以直接添加，往往通过配制母液，并以母液的形式进行添加。
在本发明中，所述微量元素母液中各组分的含量可以在较宽的范围内选择，优选地，微量元素母液的配方为：硫酸镍1-3g/L，硫酸铜1-3g/L，钼酸钠0.02-0.08g/L，氯化锰2-4g/L，氯化钴0.02-0.08g/L，硫酸锌2-4g/L，硫酸亚铁8-10g/L。
在本发明中，所述微生素母液中各组分的含量可以在较宽的范围内选择，优选地，微生素母液的配方为：维生素B1 9-11g/L，维生素B12 0.1-0.3g/L，泛酸钙2-4g/L，生物素0.005-0.01g/L。
优选地，所述发酵培养基中的氮源包含谷氨酸。
所述谷氨酸的浓度可以在较宽的范围内选择，优选地，所述发酵培养基中，谷氨酸的浓度为5-15g/L。
所述发酵培养基中碳源的浓度也可以在较宽的范围内选择，优选地，所述发酵培养基中，碳源的浓度为5-70g/L。
在本发明一种优选的实施方式中，所述发酵培养基中，碳源的含量为5-70g/L，氮源的含量为15-45g/L，无机盐的含量为5-40g/L，微量元素的含量为0.01-0.04g/L，维生素的含量为0.01-0.04g/L。优选地，所述发酵培养基中，碳源的含量为40-60g/L，氮源的含量为20-35g/L，无机盐的含量为10-25g/L，微量元素的含量为0.015-0.035g/L，维生素的含量为0.01-0.03g/L。
优选地，所述发酵培养基中还含有消泡剂，所述消泡剂的含量优选为0.1-0.5g/L。
其中，所述裂殖壶菌接种到发酵培养基中发酵的过程中，在发酵培养基中投加部分碳源、部分氮源，在发酵过程中根据碳氮比的调控工艺不断流加碳源、氮源，在接近发酵终点时不再补充碳源直至培养基中残糖含量为0，以此来调控碳氮比形成营养缺陷型营养条件，以确保裂殖壶菌发酵产油量的水平。
为了获得较多的发酵产物，该方法优选包括：所述裂殖壶菌菌株经活化后制作成摇瓶种子液接种到所述种子培养基中培养，然后再转接入发酵培养基中发酵培养。优选地，该方法进一步包括：将所述裂殖壶菌菌株接种到活化培养基中，经活化得到活化液；将活化液接种到种子培养基中进行扩培，得到种子液；然后将种子液接种到所述发酵培养基中发酵培养，制备得到微生物油脂。
在本发明中，所述裂殖壶菌菌株可以是安瓿瓶保存或甘油管保存的菌株。
优选地，所述活化的过程包括将所述裂殖壶菌菌株的冷冻甘油管菌种 解冻、接种到活化培养基中进行活化。
在本发明中，可以通过一次或多次活化以制备活化液。
其中，所述活化的条件可以在较宽的范围内选择，优选地，所述活化的条件包括：温度为27.5-28.5℃，转速为150-200r/min，时间为48-72h。
优选地，所述活化培养基中包含碳源、氮源、无机盐、微量元素和维生素；其中，所述活化培养基中，碳源的含量为30-50g/L、氮源的含量为25-45g/L、无机盐的含量为25-40g/L、微量元素的含量为0.015-0.03g/L、维生素的含量为0.01-0.02g/L。
在本发明的一种优选的实施方式中，所述菌株活化培养的过程包括：将所述裂殖壶菌菌株的冷冻甘油管菌种解冻，用无菌吸管将所述甘油管菌种接种到已灭菌的活化培养基中，在温度为27.5-28.5℃、转速为150-200r/min的条件下培养48-72h。其中，所述活化培养基中，碳源的含量为30-50g/L，氮源的含量为25-45g/L，无机盐的含量为25-40g/L，微量元素的含量为0.015-0.025g/L，维生素的含量为0.01-0.02g/L。
优选地，所述甘油管菌种的接种量按一支甘油管转接4-6个三角瓶的方式，比如，三角瓶的规格为500mL，每个三角瓶中的种子培养基体积为200-300mL。
在本发明中，所述扩培的方法优选包括：将种子液接入种子培养基中进行扩培，得到种子液。
优选地，所述扩培的条件包括：温度为27-28.5℃；时间为48-72h；通气比为0.5-0.8VVM。
优选地，所述种子培养基中包含碳源、氮源、无机盐、微量元素和维生素；其中，所述种子培养基中，碳源的含量为25-60g/L，氮源的含量为10-30g/L，无机盐的含量为15-35g/L，微量元素的含量为0.01-0.02g/L，维生素的含量为0.01-0.02g/L。
优选地，所述种子培养基中还含有消泡剂，所述消泡剂的含量优选为 0.1-0.5g/L。
在本发明的一种优选的实施方式中，所述扩培的方法包括：将种子液接入一级种子培养基中进行一级扩培后，接入二级种子培养基中进行二级扩培，得到种子液。
优选地，所述一级扩培的条件包括：温度为27-28.5℃；时间为48-60h；通气比为0.5-0.8VVM。
优选地，所述二级扩培的条件包括：温度为27-28.5℃；时间为12-24h；通气比为0.5-0.8VVM。
优选地，所述一级种子培养基中，碳源的含量为25-35g/L，氮源的含量为10-30g/L，无机盐的含量为15-35g/L，微量元素的含量为0.01-0.02g/L，维生素的含量为0.01-0.02g/L。
优选地，所述二级种子培养基中，碳源的含量为40-60g/L，氮源的含量为10-15g/L，无机盐的含量为15-20g/L，微量元素的含量为0.01-0.02g/L，维生素的含量为0.01-0.02g/L。
所述活化培养基和种子培养基各组分的种类参见发酵培养基。
本发明可以进一步对上述发酵的产物进行处理，以获得微生物油脂，所述处理的方法没有特别的限制，只要是能从所述发酵的产物中将微生物油脂分离即可，为了获得较高的微生物油脂收率，所述处理包括对所述发酵的产物进行破壁、萃取得到所述发酵的产物中的油脂产物。
第三方面，本发明提供了由上述的方法制备得到的微生物油脂，所述微生物油脂包含甘油三酯，所述甘油三酯Sn-2位DHA占Sn-2位全脂肪酸的比例不低于23重量％。
优选地，所述微生物油脂的总DHA的含量不低于38重量％。
第四方面，本发明提供了如上所述的微生物油脂在食品中的应用。
所述食品优选为婴幼儿配方食品、保健食品或健康食品。
本发明第五方面提供一株裂殖壶菌(Schizochytriumsp.)，所述裂殖壶 菌为保藏编号为GDMCC No.60733的裂殖壶菌(Schizochytriumsp.)。
所述裂殖壶菌的获取方式在第二方面进行了说明，在此不再赘述。
本发明第六方面提供如上所述的裂殖壶菌在生产微生物油脂中的应用。
优选地，所述微生物油脂包含甘油三酯，所述甘油三酯Sn-2位DHA占Sn-2位全脂肪酸的比例不低于23重量％。
优选地，所述微生物油脂的总DHA的含量不低于38重量％。
以下将通过实施例对本发明进行详细描述。
以下实施例中，微生物油脂中DHA的含量通过GB26400-2011中的方法测定，脂肪酸组分通过GB5009.168-2016方法测得；
人体对DHA的吸收率测定方法为“人体功效试验的方法”：征集男女志愿者食用本专利方法生产的DHA油脂和对比DHA油脂，然后抽取血液测定血液中DHA含量、Sn-2位DHA脂肪酸含量来计算吸收率。
裂殖壶菌常规菌株来自于中国工业微生物菌种保藏管理中心，编号为CICC 11091s；
葡萄糖、蔗糖、酵母粉、谷氨酸钠、酵母浸膏、氯化钠、硫酸镁、氯化钙、磷酸二氢钾、硫酸镍、硫酸铜、钼酸钠、氯化钴、硫酸锌、硫酸亚铁、氯化锰、维生素B 1、维生素B 12、维生素B 6、泛酸钙、生物素、碳酸氢钠、硫酸钠、硫酸铵、氯化钾均为市售品。
本发明的实施例中，微量元素母液的配方为：硫酸镍2g/L，硫酸铜1.9g/L，钼酸钠0.04g/L，氯化锰2.8g/L，氯化钴0.04g/L，硫酸锌3.2g/L，硫酸亚铁9g/L；
维生素母液的配方为：维生素B 110.3g/L、维生素B 12 0.16g/L、泛酸钙3.2g/L、生物素0.008g/L。
制备例
本制备例用于说明本发明所述的裂殖壶菌(Schizochytriumsp.)的获取方法。
活化培养基配方为：葡萄糖40g/L，谷氨酸钠31g/L，氯化钠19g/L，酵母浸膏5.8g/L，硫酸镁8g/L，磷酸二氢钾5.7g/L，微量元素1g/L，维生素1g/L。固体培养基时，另含琼脂18g/L。
取出发菌株保存在安瓿瓶中的菌种，接种到活化培养基中并在28℃活化培养2天。将培养得到的菌液涂到活化培养基平板上，在北京艾德豪克国际技术有限公司生产的MPMS——多功能诱变系统中进行ARTP诱变处理：等离子诱变功率为100W，气体流量为10SLM，处理距离为2mm，制备DHA菌悬液，其OD 600值在0.6-0.8或菌浓度在10 6-10 8之间，诱变时间为25s，致死率为92.54％。挑选生长良好的单菌落传代，接种到装有活化培养基的摇瓶中在27℃下培养4天，进行初筛，检测培养后的产物中DHA含量、Sn-2位DHA的含量，挑选高产菌株。将初筛筛选得到的高产菌株进行摇瓶复筛，在27℃下培养4天，再次挑选高产菌株。对复筛得到的高产菌株进行遗传稳定性考察，取传代5次后，性状稳定的菌株作为生产菌株并长期保存备用。
经筛选，得到本发明提供的裂殖壶菌(Schizochytriumsp.)，于2019年8月8日被保藏在广东省微生物菌种保藏中心(地址：广州市先烈中路100号大院59号楼5楼，广东省微生物研究所，邮政编码：510070)(保藏单位的缩写为GDMCC)，保藏编号为GDMCC No.60733。
实施例1
本实施例用于说明本发明所述的裂殖壶菌的30L发酵生产。
本实施例中活化培养基配方为：葡萄糖40g/L，谷氨酸钠31g/L，酵母浸膏5.8g/L，氯化钠19g/L，硫酸镁8g/L，磷酸二氢钾5.7g/L，微量元素1g/L，维生素1g/L；
种子培养基配方为：葡萄糖30g/L，谷氨酸钠6.3g/L，酵母浸膏8.3g/L，酵母粉8.3g/L，氯化钠1.45g/L，硫酸镁5.18g/L，磷酸二氢钾1.66g/L，氯化钙0.25g/L，碳酸氢钠0.25g/L，硫酸钠9.34g/L，硫酸铵1.04g/L，氯化钾0.83g/L，微量元素1g/L，维生素1g/L，消泡剂0.3g/L；
发酵培养基配方为：葡萄糖50g/L，谷氨酸钠15g/L，酵母浸膏10.9g/L，氯化钠2.6g/L，硫酸镁5.8g/L，磷酸二氢钾2.4g/L，氯化钙0.25g/L，碳酸氢钠0.22g/L，硫酸钠3.62g/L，硫酸铵1.13g/L，氯化钾0.94g/L，微量元素母液1.1g/L，维生素母液1.1g/L，消泡剂0.19g/L。
具体步骤如下：
(1)分别将裂殖壶菌常规菌株和本发明所使用的裂殖壶菌菌株(保藏编号为GDMCC No.60733)进行活化，接种量为1支解冻后的甘油管菌种转接到4个装有200mL活化培养基的摇瓶中，在转速为180r/min、温度为28℃的摇床上培养48h；
(2)将步骤(1)所得的活化液接种到放有200mL活化培养基的摇瓶中进行扩大培养，接种量为3％，在转速为180r/min、温度为28℃的摇床上培养72h；
(3)种子罐中装有3L种子培养基，将200mL步骤(2)所得的活化液接入种子罐中，以温度为28℃、搅拌速度为180r/min、通气量为0.6vvm、罐压为0.03Mpa的条件培养50h；
(4)发酵罐中装有14L发酵培养基，将步骤(3)所得的种子液全部转接入发酵罐中，以温度为29℃、搅拌速度为140r/min、通气量为0.95vvm、罐压为0.03Mpa、pH为6.8的条件培养5-6天，发酵过程中流加葡萄糖浓度为250g/L的无菌液和谷氨酸钠浓度为250g/L的无菌液，以保持谷氨酸浓度为5-8g/L、碳源浓度为10-23g/L，96h后停止补充碳源和谷氨酸，最终获得发酵液；
(5)将5L步骤(4)所得的发酵液经酶解破壁后，用实验室高速离心 机将水相、渣相、油相分离，得到微生物油酯；
(6)将步骤(5)所得的微生物油脂进行DHA含量、脂肪酸组分和油脂中甘油三酯Sn-2位DHA的比例分析测定，结果见表1。
表1实施例1中微生物油脂中各参数的数据
Figure PCTCN2020130385-appb-000001
实施例2
本实施例用于说明本发明所述的裂殖壶菌的100L发酵生产。
本实施例中活化培养基配方为：葡萄糖30g/L，谷氨酸钠20g/L，酵母浸膏5g/L，氯化钠15g/L，硫酸镁6g/L，磷酸二氢钾4g/L，微量元素0.8g/L，维生素0.75g/L；
种子培养基配方为：葡萄糖25g/L，谷氨酸钠5.5g/L，酵母浸膏7g/L，酵母粉7g/L，氯化钠1.1g/L，硫酸镁4g/L，磷酸二氢钾1.2g/L，氯化钙0.15g/L，碳酸氢钠0.15g/L，硫酸钠7g/L，硫酸铵0.8g/L，氯化钾0.6g/L，微量元素0.8g/L，维生素0.75g/L，消泡剂0.2g/L；
发酵培养基配方为：葡萄糖30g/L、蔗糖10g/L，谷氨酸钠12g/L，酵 母浸膏9g/L，氯化钠2g/L，硫酸镁4.5g/L，磷酸二氢钾2g/L，氯化钙0.2g/L，碳酸氢钠0.2g/L，硫酸钠2.8g/L，硫酸铵1g/L，氯化钾0.8g/L，微量元素0.9g/L，维生素0.9g/L，消泡剂0.15g/L。
具体步骤如下：
(1)分别将裂殖壶菌常规菌株和本发明所使用的裂殖壶菌菌株(保藏编号为GDMCC No.60733)进行活化，接种量为1支解冻后的甘油管菌种接种到5个装有200mL活化培养基的摇瓶中，在转速为150r/min、温度为28.5℃的摇床上培养72h；
(2)将步骤(1)所得的活化液接种到5个装有200mL活化培养基的摇瓶中进行扩大培养，接种量为：将步骤(1)中得到的活化液2瓶接种到5瓶种子培养基中，在转速为150r/min、温度为28℃的摇床上培养48h；
(3)种子罐中装有6L种子培养基，将400mL步骤(2)所得的活化液接入种子罐中，以温度为28℃、搅拌速度为150r/min、通气量为0.5vvm、罐压为0.03Mpa的条件培养48h；
(4)发酵罐中装有45L发酵培养基，将步骤(3)所得的种子液全部转接入发酵罐中，以温度为28-29℃、搅拌速度为90--120r/min、通气量为0.5-0.8vvm、罐压为0.03Mpa的条件培养5-6天，发酵过程中流加葡萄糖浓度为250g/L的无菌液和谷氨酸钠浓度为250g/L的无菌液，以保持谷氨酸浓度为8-12g/L、碳源浓度为40-65g/L，96h停止补充碳源和谷氨酸，最终获得发酵液；
(5)将10L步骤(5)所得的发酵液经酶解后，用实验室离心机分离得到微生物油酯；
(6)将步骤(5)所得的微生物油脂进行DHA含量、脂肪酸组分和油脂中甘油三酯Sn-2位DHA的比例分析测定，结果见表2。
表2实施例2中微生物油脂中各参数的数据
Figure PCTCN2020130385-appb-000002
实施例3
本实施例用于说明本发明所述的裂殖壶菌的45M 3工业发酵生产。
本实施例中活化培养基配方为：葡萄糖50g/L，谷氨酸钠35g/L，酵母浸膏10g/L，氯化钠22g/L，硫酸镁10g/L，磷酸二氢钾8g/L，微量元素1.4g/L，维生素1.5g/L；
一级种子培养基配方为：葡萄糖35g/L，谷氨酸钠8g/L，酵母浸膏10g/L，酵母粉10g/L，氯化钠1.9g/L，硫酸镁6g/L，磷酸二氢钾2.1g/L，氯化钙0.4g/L，碳酸氢钠0.4g/L，硫酸钠12g/L，硫酸铵1.3g/L，氯化钾1.1g/L，微量元素1.2g/L，维生素1.2g/L，消泡剂0.4g/L；
二级种子培养基配方为：葡萄糖50g/L，谷氨酸钠5.65g/L，酵母浸膏5.65g/L，氯化钠1.3g/L，硫酸镁4.65g/L，磷酸二氢钾1.49g/L，氯化钙0.22g/L，碳酸氢钠0.22g/L，硫酸钠8.39g/L，硫酸铵0.93g/L，氯化钾0.74g/L，微量元素0.93g/L，维生素0.93g/L，消泡剂0.3g/L；
发酵培养基配方为：葡萄糖40g/L、蔗糖20g/L，谷氨酸钠20g/L，酵 母浸膏12g/L，氯化钠3.2g/L，硫酸镁7g/L，磷酸二氢钾3g/L，氯化钙0.4g/L，碳酸氢钠0.3g/L，硫酸钠4.5g/L，硫酸铵1.5g/L，氯化钾1.2g/L，微量元素1.6g/L，维生素1.6g/L。
具体步骤如下：
(1)分别将裂殖壶菌常规菌株和本发明所使用的裂殖壶菌菌株(保藏编号为GDMCC No.60733)进行活化，接种量为1支解冻后的甘油管菌种接种到6个装有200mL活化培养基的摇瓶中，在转速为180r/min、温度为28℃的摇床上培养60h；
(2)将步骤(1)所得的2瓶活化液接种到5个装有200mL活化培养基的三角瓶中进行扩大培养，在转速为180r/min、温度为28±0.5℃的摇床上培养60h；
(3)一级种子罐中装有500L一级种子培养基，灭菌冷却至40℃以下，然后将1L步骤(2)所得的活化液转接入一级种子罐中，以温度为28±0.5℃、搅拌速度为150r/min、pH为6.8的条件培养55h；
(4)二级种子罐中装有6M 3二级种子培养基，灭菌冷却至40℃以下，然后将步骤(3)所得的一级种子液全部在无菌条件下转接到二级种子罐中，以温度28±0.5℃、搅拌150r/min、pH6.8的条件培养18h；
(5)主发酵罐中装有22M 3发酵培养基，灭菌冷却至40℃以下，将步骤(4)所得的二级种子液全部在无菌条件下转接到主发酵罐中，以温度28±0.5℃、搅拌100r/min、通气比为1.0VVM、pH7.5的条件培养5天，发酵过程中流加葡萄糖浓度为250g/L的无菌液和谷氨酸钠浓度为250g/L的无菌液，以保持谷氨酸浓度为12-15g/L、碳源浓度为55-70g/L，96h停止补充碳源和谷氨酸，最终获得发酵液；
(6)发酵液经酶解后，将发酵液预热到85-90℃，用三相离心机分离得到微生物油酯；
(7)将步骤(6)所得的微生物油脂进行DHA含量、脂肪酸组分和油 脂中甘油三酯Sn-2位DHA的比例分析测定，结果见表3。
表3实施例3中微生物油脂中各参数的数据
Figure PCTCN2020130385-appb-000003
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，其特征在于，所述微生物油脂包含甘油三酯，所述甘油三酯Sn-2位DHA占Sn-2位全脂肪酸的比例不低于23重量％。
  2. 根据权利要求1所述的微生物油脂，其特征在于，所述微生物油脂的总DHA的含量不低于38重量％。
  3. 一种微生物油脂的制备方法，其特征在于，该方法包括将裂殖壶菌菌株接种到发酵培养基中发酵制得，所述裂殖壶菌菌株为保藏编号为GDMCC No.60733的裂殖壶菌(Schizochytriumsp.)；
    优选地，发酵的条件包括：pH为6-7.5、温度为27-31℃、时间为4-8天、通气比为0.45-1.1VVM；和/或
    优选地，所述发酵培养基包含碳源、氮源、无机盐、微量元素和维生素；
    更优选地，所述发酵培养基中的氮源包含谷氨酸；进一步优选地，所述发酵培养基中，谷氨酸的浓度为5-15g/L
    更优选地，所述发酵培养基中，碳源的含量为5-70g/L，氮源的含量为15-45g/L，无机盐的含量为5-40g/L，微量元素的含量为0.01-0.04g/L，维生素的含量为0.01-0.04g/L。
  4. 根据权利要求3所述的方法，其特征在于，该方法进一步包括：将所述裂殖壶菌菌株接种到活化培养基中，经活化得到活化液；将活化液接种到种子培养基中进行扩培，得到种子液；然后将种子液接种到所述发酵培养基中发酵培养，制备得到微生物油脂；
    优选地，所述活化的条件包括：温度为27.5-28.5℃，转速为150-200r/min，时间为48-72h；
    优选地，所述活化培养基中包含碳源、氮源、无机盐、微量元素和维生素；其中，所述活化培养基中，碳源的含量为30-50g/L，氮源的含量为25-45g/L，无机盐的含量为25-40g/L，微量元素的含量为0.015-0.025g/L，维生素的含量为0.01-0.02g/L；
    优选地，所述扩培的条件包括：温度为27-28.5℃；时间为48-72h；通气比为0.5-0.8VVM；
    优选地，所述种子培养基中包含碳源、氮源、无机盐、微量元素和维生素；其中，所述种子培养基中，碳源的含量为25-60g/L，氮源的含量为10-30g/L，无机盐的含量为15-35g/L，微量元素的含量为0.01-0.02g/L，维生素的含量为0.01-0.02g/L。
  5. 根据权利要求3或4所述的方法，其特征在于，所述碳源为葡萄糖和/或蔗糖，和/或
    所述氮源为谷氨酸钠、酵母粉和酵母浸膏中的一种或多种，和/或
    所述无机盐为钙盐、磷酸盐、钾盐、钠盐、镁盐和铵盐中的一种或多种，和/或
    所述微量元素为镍、铜、钼、钴、锌、铁和锰中的一种或多种，和/或
    所述维生素为维生素B1、维生素B12、维生素B6、泛酸钙和生物素中的一种或多种。
  6. 根据权利要求3所述的方法，其特征在于，该方法还包括对所述发酵的产物进行萃取得到所述发酵的产物中的油脂产物。
  7. 由权利要求3-6中任意一项所述的方法制备的微生物油脂，其特征在于，所述微生物油脂包含甘油三酯，所述甘油三酯Sn-2位DHA占Sn-2位全脂肪酸的比例不低于23重量％；
    优选地，所述微生物油脂的总DHA的含量不低于38重量％。
  8. 权利要求1、2和7中任意一项所述的微生物油脂在食品中的应用，所述食品优选为婴幼儿配方食品、保健食品以及健康食品。
  9. 一株裂殖壶菌(Schizochytriumsp.)，其特征在于，所述裂殖壶菌为保藏编号为GDMCC No.60733的裂殖壶菌(Schizochytriumsp.)。
  10. 权利要求9所述的裂殖壶菌在生产微生物油脂中的应用；
    优选地，所述微生物油脂包含甘油三酯，所述甘油三酯Sn-2位DHA占Sn-2位全脂肪酸的比例不低于23重量％；
    优选地，所述微生物油脂的总DHA的含量不低于38重量％。
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