WO2014101857A1 - 一种固料培养裂殖壶菌液体发酵生产dha的方法 - Google Patents

一种固料培养裂殖壶菌液体发酵生产dha的方法 Download PDF

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WO2014101857A1
WO2014101857A1 PCT/CN2013/090839 CN2013090839W WO2014101857A1 WO 2014101857 A1 WO2014101857 A1 WO 2014101857A1 CN 2013090839 W CN2013090839 W CN 2013090839W WO 2014101857 A1 WO2014101857 A1 WO 2014101857A1
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solid
seed
liquid
medium
fermentation
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PCT/CN2013/090839
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English (en)
French (fr)
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陈金卿
陈芳芳
陈俊煌
林超
吴美琼
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厦门金达威集团股份有限公司
内蒙古金达威药业有限公司
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Priority to US14/654,897 priority Critical patent/US10006067B2/en
Publication of WO2014101857A1 publication Critical patent/WO2014101857A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
    • C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
    • C12P7/6434—Docosahexenoic acids [DHA]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/12—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
    • 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 a method for fermentatively producing DHA, in particular to a method for producing DHA by liquid fermentation of solid culture cultured Schizochytrium. Background technique
  • Docosahexenoic acid (22:6 ⁇ 4,7,10,13,16,19, abbreviated as DHA), commonly known as "brain gold”, is an omega-3 polyunsaturated fatty acid with brain and brain , to prevent senile dementia; improve vision, prevent and treat myopia; prevent high blood pressure, arteriosclerosis, arthritis and cancer treatment and other physiological functions. Therefore, DHA has broad market prospects as a new generation of functional health care factors. With the expansion of market demand, the requirements for DHA quality are getting higher and higher. DHA production research is gradually developing towards the use of biotechnology synthesis. DHA of traditional fish oil source has gradually been replaced by algae oil DHA. Algae oil DHA refers to a long-chain polyunsaturated fatty acid such as DHA obtained by microbial fermentation.
  • the DHA-producing bacteria mainly include Schizochytrium, Thraustochytrium, and Cryptophyceae.
  • Schizochyirium also known as Schizochytrium, is a marine fungus belonging to the genus Fungi, Oomycetes, Mycorrhizal, and Thraustochytrium, single-cell, spherical.
  • the cells accumulate a large number of active substances that are useful to the human body, such as oils, pigments (carotenoids, lutein, astaxanthin), squalene, etc., wherein the total fatty acid content of DHA is as high as 35% to 45%, and 90 in the cells. More than % of the oil is in the form of a triglyceride, a neutral oil that is easily absorbed by the human body. It is an ideal production strain of DHA.
  • Chinese patent CN101575584 discloses a method of producing Vibrio cholerae and using the same to produce DHA oil, which provides an optimized fermentation medium for bacteriostasis from the perspective of osmotic pressure and element supply, and combined with a flow-adding strategy to achieve high schizophyllum.
  • Density fermentation using traditional fermentation methods, from glycerol storage to shake flask activation ⁇ first-class seed liquid ⁇ shake bottle secondary seed liquid ⁇ first-class seed tank ⁇ second-level seed tank ⁇ fermentation, the final cell dry weight reaches 70g / L, the oil content reached 31.5g / L, DHA accounted for more than 35% of total fatty acid content.
  • Chinese patent CN101519676 discloses a fermentation cultivation using trace elements. Fermentation production process of DHA by fermentation, using the traditional method to culture the strain Schizochytrium, that is, the bevel-activated culture, then transfer to the shake flask to expand the culture, and then the first-stage expansion culture to obtain the fermentation seed liquid; or the strain The slope is activated and cultured, then transferred to a shake flask to expand the culture, and then subjected to the first-stage expansion culture and the second-stage expansion culture to obtain the fermentation seed liquid.
  • the 10T tank is fermented for 95 hours, the biomass is 53 g/L, the oil content is 72%, and the DHA content is 50. %.
  • the object of the present invention is to provide a simplification of the seed expansion process, shorten the seed culture and fermentation cycle, reduce investment, and save cost.
  • the solid medium is rich in nutrients, rich in various growth factors, is more conducive to seed growth, and grows the cells of the strain. It has strong vitality and good synchronism. It is a method for producing DHA by liquid fermentation of solid biomass culture Schizochytrium with less waste water and less waste residue in the solid seed stage.
  • a first aspect of the invention relates to a method for liquid fermentation of DHA by solid culture of Schizochytrium, which comprises the following steps:
  • the Schizochytrium strain is inoculated and cultured in a medium (for example, a slant medium) to prepare a bacterial suspension;
  • (2)-stage seed preparation the bacterial suspension obtained in the step (1) is inoculated to a solid medium to be cultured to prepare a first-stage solid seed;
  • the bacterial suspension obtained in the step (1) is inoculated into a liquid seed culture medium (for example, shake flask culture) to prepare a first-stage liquid seed;
  • a liquid seed culture medium for example, shake flask culture
  • the primary solid seed is made into a first-stage solid seed liquid, and is added to the solid-state seed liquid in the solid medium;
  • the secondary solid seed is made into a secondary solid seed liquid, and inoculated into the fermenter liquid medium to expand the culture; (5) After the fermentation is completed, the bacterial cells are collected and DHA is extracted.
  • the Schizochytrium is a Schizochytrium known in the art.
  • the Schizochytrium is selected from the group consisting of Schizochytrium sp.; in an embodiment of the invention, the Schizochytrium sp. is selected from the group consisting of Schizochytrium sp. KDW-12, Schizochytrium Schizochytrium sp. S31, Schizochytrium aggregatum and Schizochytrium sp.
  • step (1) is characterized by one or more of the following:
  • the temperature of the culture is 25 - 28 ° C, and the concentration of the cells and the morphology of the cells are good, for example, the culture time is 2 to 3 days;
  • the cell concentration of the bacterial suspension 1.0 ⁇ 2.5xl0 6 th / mL.
  • step (2) is characterized by one or more of the following:
  • the inoculum amount is 0.5 ⁇ 2%;
  • the culture temperature is 25 ⁇ 28 ° C, the culture to the bacterial concentration and the bacterial morphology is good, for example, the culture time is 20 ⁇ 28h;
  • the liquid culture e.g., shake flask culture
  • the liquid culture has a rotational speed of 180 - 220 rpm.
  • step (3) is characterized by one or more of the following:
  • a solution of said solid feed a seed cell concentration may be 1.0 - 2.5xl0 6 th / mL;
  • the first solid stock seed liquid is fed into the solid medium
  • the culture temperature is 25 ⁇ 28 ° C, and the culture medium concentration and the cell morphology are good, for example For example, the culture time is 2 ⁇ 3 days.
  • step (4) is characterized by one or more of the following:
  • the temperature of the first 48 hours of the fermentation process is controlled at 25 ⁇ 28. C, using a stirring speed to maintain 30% ⁇ 50% dissolved oxygen;
  • the temperature from the 48th to the end of fermentation is controlled at 18 ⁇ 22. C, using the stirring speed to maintain 8% - 12% dissolved oxygen;
  • the fermentation is terminated, for example, fermentation for 3 to 5 days;
  • the glucose concentration is controlled by the automatic addition of the glucose solution during the fermentation to 4-8.
  • the concentration of the aqueous ammonia is 28%.
  • the glucose solution has a concentration of 580 g/L.
  • the slant medium in the step (1) comprises: glucose 20 - 30 g / L, peptone 10 - 15 g / L, yeast powder 3 - 5 g / L, sea crystal 15 - 20g / L, agar powder 15 ⁇ 25g / L, pH 6.0 ⁇ 7.0.
  • the solid medium in the step (2) or the step (3) comprises a solid component and a liquid nutrient solution component, the solid component being a cereal, 250 ⁇ 400g / L, the cereal is selected from at least one of rice, wheat and corn, the rice is selected from at least one of rice, millet, brown rice, etc., the wheat is selected from the group consisting of barley, wheat, At least one of oats;
  • the liquid nutrient solution component comprises component 1 and component 2.
  • the composition of the component 1 is: glucose 2 ⁇ 5g/L, seven: magnesium sulfate 0.2 ⁇ 0.6g/L, dipotassium hydrogen phosphate 1.0- 5.0g / L, sea salt 0.1 - 0.8g / L, glycine 0.2 - 0.6g / L, threonine 1.5 - 1.8g / L, methionine 2 - 3.5g / L, malic acid 3 ⁇ 6g / L, add water to volume Adjust pH to 6 ⁇ 7;
  • composition of component 2 is: La 3+ 10 ⁇ 15mg/L, Ce 3+ l ⁇ 4mg/L, Sm 3+ 2 - 6mg/L, Nd 3+ 8 ⁇ 12mg/L, Mn 2+ 0.6 - 1.2mg/L, Co 2+ 0.05 - O.lmg/L, biotin 0.2 - 0.6mg/L, cerulenin 0.1 -
  • the preparation method of the solid medium is as follows: the solid component is uniformly mixed with the liquid nutrient ⁇ a 1 , the cereal is steamed or boiled until the center of the grain has no white heart, and the water content is 50% to 65%, and the solid material culture is obtained. Base material; mixing the solid material of the solid medium with the liquid nutrient solution component 2, and loading it in a solid fermentation bottle, having a thickness of 0.2 to 1.0 cm, and sterilizing to obtain a solid medium;
  • the ratio of the solid component to the liquid nutrient ⁇ ia is 1 by volume/volume
  • the liquid seed culture medium comprises: glucose 20-25 g/L, sodium chloride 20-25 g/L, potassium dihydrogen phosphate 0.2-0.5 g/L, yeast powder 3 - 5g / L, seven: magnesium sulfate 8 - 12g / L, sea salt 0.1 - 0.8g / L, glutamic acid 0.2 ⁇ 0.6g / L, pH 6.0 ⁇ 7.0.
  • the fermenter liquid medium in the step (4) comprises: glucose 30-60 g/L, NaCl 15-20 g/L, MgS0 4 .7H 2 04-8 g/ L, yeast powder 3 ⁇ 8g/L, KH 2 P0 4 0.5-1.0 g/L, (NH 4 ) 2 S0 4 0.2-0.5 g/L, NaHC0 3 0.8-1.2 g/L, CoCl 2 1.0 ⁇ 2.0 g /L, MnSO 4 0.5 ⁇ 1.0 g / L, VBi 0.05 - O.lmg / L, VB 12 0.001 ⁇ 0.01mg / L, initial pH 6.0 ⁇ 7.0.
  • a second aspect of the invention relates to a medium for culturing a Schizochytrium fermentatively produced DHA, the medium being selected from one or more of the following:
  • slant medium comprising: glucose 2 0 ⁇ 3 0g / L, peptone 10 ⁇ l 5 g / L, yeast extract 3 - 5g / L, sea crystal 15 - 20g / L, agar 15 - 25g / L ( For example 20 g / L), pH 6.0 - 7.0;
  • a solid medium comprising a solid component and a liquid nutrient solution component, the solid component being a cereal having a content of 250 to 400 g/L, the cereal being selected from at least one of rice, wheat, and corn.
  • the rice is selected from at least one of rice, millet, brown rice, etc., the wheat At least one selected from the group consisting of barley, wheat, and oats;
  • the liquid nutrient solution component comprises component 1 and component 2, and the composition of the component 1 is: glucose 2 ⁇ 5g/L, magnesium sulphate 0.2 ⁇ 0.6g/L, dipotassium hydrogen phosphate 1.0- 5.0g/L, sea salt 0.1 ⁇ 0.8g / L, chelating acid 0.2 ⁇ 0.6g / L, threonine 1.5 ⁇ 1.8g / L, methionine 2 ⁇ 3.5g / L, malic acid 3 - 6g / L, add water Tolerance, adjust the pH to 6 ⁇ 7; the composition of the component 2 is: La 3+ 10 ⁇ 15mg / L, Ce 3 + l ⁇ 4mg / L, Sm 3 + 2 - 6mg / L, Nd 3 + 8 ⁇ 12mg/L, Mn 2+ 0.6 - 1.2mg/L, Co 2+ 0.05 - O.lmg/L, biotin 0.2 - 0.6mg/L, cerulenin
  • the preparation method of the solid medium is as follows: the solid component is uniformly mixed with the liquid nutrient ⁇ a 1 , the cereal is steamed or boiled until the center of the grain has no white heart, and the water content is 50% to 65%, and the solid material culture is obtained. Base material; mixing the solid material of the solid medium with the liquid nutrient solution component 2, and loading it in a solid fermentation bottle, having a thickness of 0.2 to 1.0 cm, and sterilizing to obtain a solid medium;
  • the ratio of the solid component to the liquid nutrient solution component 1 in mass/volume is (5-10) g: (2-5) mL, the solid medium and the liquid nutrient solution component 2 According to the shield volume/volume ratio (10 ⁇ 15 ) g: (3 ⁇ 6) mL, where the solid component is calculated by the shield amount, and the liquid nutrient solution component is calculated by volume.
  • Liquid seed culture medium containing: glucose 20 ⁇ 25g/L, sodium chloride 20 ⁇ 25g/L, potassium dihydrogen phosphate 0.2 ⁇ 0.5g/L, yeast powder 3 ⁇ 5g/L, seven: magnesiumate 8 ⁇ 12g / L, sea salt 0.1 - 0.8g / L, glutamic acid 0.2 ⁇ 0.6g / L, pH value of 6.00 ⁇ 7.0;
  • Fermentation tank liquid medium containing: glucose 30 - 60 g / L, NaCl 15 - 20g / L, MgS0 4 .7H 2 0 4 ⁇ 8 g / L, yeast powder 3 ⁇ 8 g / L, KH 2 P0 4 0.5 - 1.0 g/L, (NH 4 ) 2 S0 4 0.2 ⁇ 0.5 g/L, NaHC0 3 0.8 - 1.2 g/L, CoCl 2 1.0 - 2.0 g/L, MnS0 4 0.5 ⁇ 1.0 g/L, VBi 0.05 ⁇ O.lmg / L, VB 12 0.001 - O.Olmg / L, initial pH 6.0 ⁇ 7.0.
  • the invention also relates to a method for producing DHA by liquid fermentation of solid cultured Schizochytrium, characterized in that it comprises the following steps:
  • the bacterial suspension obtained in the step 1) is inoculated to a solid medium by an inoculation amount of 1% of the shield fraction to prepare a first-stage solid seed; or
  • the bacterial suspension obtained in the step 1) is inoculated into a liquid seed culture medium by a seeding amount of 1% by volume, and shake flask cultured to prepare a first-stage liquid seed;
  • Inoculation amount of 8% to 15% of the product/shield is added to the first-stage liquid seed in the solid medium, and the solid fermentation bottle is shaken evenly, 25 ⁇ 28°C, and cultured for 2 ⁇ 3 days;
  • the formulation of the slant medium is: glucose 20 ⁇ 30g/L, peptone 10 ⁇ 15g/L, yeast powder 3 ⁇ 5g/L, sea crystal 15 ⁇ 20g/ L, agar powder 15 - 25g / L (for example, 20 g / L), pH 6 ⁇ 7, 121.
  • Sterilization C for 30 min; the activation temperature of the culture may be 25 ⁇ 28'C, the culture time may be activated for 2-3 days; cell concentration of the bacterial suspension may be 1.0 ⁇ 2.5xl0 6 th / mL.
  • the solid medium comprises a solid component and a liquid nutrient
  • the solid component is a cereal having a content of 250 to 400 g/L.
  • the cereal may be selected from at least one of rice, wheat, and corn, and the rice may be selected from at least one of rice, millet, brown rice, and the like, and the wheat may be selected from barley, wheat, and oats. At least one of them.
  • the liquid nutrient solution component comprises component 1 and component 2, and the composition of the component 1 is: glucose 2-5 g/L, magnesium sulfate heptahydrate 0.2 - 0.6g / L, dipotassium hydrogen phosphate 1.0 - 5.0g / L, sea salt 0.1 - 0.8g / L, glycine 0.2 - 0.6g / L, threonine 1.5 - 1.8g / L, methionine 2 - 3.5g / L, malic acid 3 - 6g / L, add water to volume, adjust the pH value of 6 ⁇ 7;
  • the composition of ⁇ & 2 is: La 3 + 10 - 15mg / L, Ce 3 + 1 - 4mg / L, Sm 3+ 2 - 6mg/L, Nd 3+ 8 ⁇ 12mg/L, Mn 2+ 0.6 - 1.2mg/L, Co 2+ 0.05 - O.lmg
  • the solid medium is prepared by: mixing the solid component with the liquid nutrient 3 ⁇ 4, uniformly, and steaming or boiling until the center of the grain has no white heart, and ⁇ is 50% ⁇ 65%, obtain solid-based raw materials; mix the solid medium and liquid camp 2, and install it in solid-state fermentation K bottle, thickness 0.2 ⁇ 1.0cm, 121. C was sterilized for 30 min to prepare a solid medium.
  • the ratio of the solid component to the liquid nutrient is 1 (5 ⁇ 10 ) g: ( 2 ⁇ 5 ) mL, wherein the solid component is shielded Calculation, liquid nutrition ⁇ a points 1 product calculation;
  • the ratio of the solid medium and the liquid nutrient solution component 2 according to the shield volume/volume is (10-15) ⁇ : (3 ⁇ 6) mL, wherein the solid medium is calculated according to the shield amount, liquid nutrition ⁇ a is divided into 2 product calculations.
  • the composition of the liquid seed medium is: glucose 20 - 25 g / L, sodium chloride 20 - 25 g / L, potassium dihydrogen phosphate 0.2 - 0.5 g / L , yeast powder 3 - 5g / L, seven: magnesium sulfate 8 - 12g / L, sea salt 0.1 - 0.8g / L, glutamic acid 0.2 - 0.6g / L, pH 6 ⁇ 7, 121. C sterilization for 30 min.
  • step 2) the temperature of the culture is from 25 to 28. C, the culture time is 1 day.
  • the shake flask has a rotation speed of 180 to 220 rpm; and the shake flask culture temperature is 25 to 28. C, shake flask culture time is 1 day.
  • the composition of the liquid fermentation medium used in the expanded culture is: glucose 30-60 g/L, NaCl 15-20 g/L, MgS0 4 .7H 2 0 4 ⁇ 8 g/L, yeast powder 3 ⁇ 8 g/L, KH 2 P0 4 0.5 - 1.0 g/L, (NH 4 ) 2 S0 4 0.2 - 0.5 g/L, NaHC0 3 0.8 - 1.2 g/L, CoCl 2 1.0 ⁇ 2.0 g / L, MnSO 4 0.5 ⁇ 1.0 g / L, VBi 0.05 ⁇ O.lmg / L, VB 12 0.001 ⁇ O.Olmg / L, initial pH 6.0 ⁇ 7.0; Oxygen is maintained at 8% ⁇ 12%.
  • the method for liquid fermentation of solid cultured Schizochytrium liquid to produce DHA comprises the following steps:
  • the formulation of the slant medium can be: glucose 20 ⁇ 30g/L, peptone 10 ⁇ 15g/L, yeast powder 3 ⁇ 5g/L, sea crystal 15 ⁇ 20g/L, agar powder 15 ⁇ 25g / L, pH 6 ⁇ 7, sterilization (such as 121 ° C sterilization 30min); the culture activation temperature can be 25 ⁇ 28 ° C, culture activation time can be 2 ⁇ 3 days; The cell concentration of the suspension may be 1.0 ⁇ 2.5 ⁇ 10 6 /mL;
  • Step 2) Primary seed preparation: Inoculate the bacterial suspension obtained in step 1) (for example, inoculum volume of 0.5-2% by weight, 1% inoculum) onto a solid medium to prepare a primary solid seed. ; or
  • the bacterial suspension obtained in the step 1) is inoculated (for example, 0.5-2% by weight, 1% of the seed amount) to the liquid seed culture medium, and shake flask cultured to prepare a first-stage liquid seed;
  • the solid medium comprises a solid component and a liquid nutrient
  • the solid component is a cereal
  • the content is 250-400 g/L
  • the material may be selected from the group consisting of rice, wheat and At least one of corn and the like
  • the rice may be at least one selected from the group consisting of rice, millet, brown rice, and the like
  • the wheat may be selected from at least one of barley, wheat, oats, and the like
  • the liquid nutrient solution component comprises component 1 and component 2, and the composition of the component 1 is: glucose 2 ⁇ 5g/L, seven: magnesium sulfate 0.2-0.6g/L, dipotassium hydrogen phosphate 1.0 ⁇ 5.0g / L, sea salt 0.1 - 0.8g / L, glycine 0.2 - 0.6g / L, threonine 1.5 - 1.8g / L, methionine 2 - 3.5g / L, malic acid 3 ⁇ 6g / L, add water to volume
  • the preparation method of the solid medium can be: the solid component is mixed with the liquid nutrient ⁇ a 1 , and the steam is boiled or boiled until the center of the grain has no white heart, and the water content is 50% to 65%, and the solid is obtained.
  • the material culture material is mixed with the liquid nutrient ⁇ a 2, uniformly packed in a solid-state fermentation K bottle, and the solid seed is loaded into a solid fermentation bottle with a specification of 1L ⁇ 10L according to 150g ⁇ 2500g/bottle. , thickness 0.2 - 1.0cm, 121. C sterilization for 30 min, to prepare a solid medium;
  • the solid component and the liquid nutrient ⁇ a minute 1 according to the shield volume / volume ratio can be (5 ⁇
  • the solid medium medium and the liquid nutrient ⁇ a minute 2 according to the shield volume / volume ratio can be
  • the composition of the liquid seed medium can be: glucose 20 ⁇ 25g/L, sodium chloride 20 ⁇ 25g/L, potassium dihydrogenate 0.2-0.5g/L, yeast powder 3-5g/L, seven: ⁇ Magnesium acid 8 - 12g / L, sea salt 0.1 ⁇ 0.8g / L, acid 0.2 ⁇ 0.6g / L, pH 6 ⁇ 7, sterilization (such as 121 ° C sterilization 30min);
  • the temperature of the culture may be 25 - 28 ° C, and the concentration of the cells and the morphology of the cells are good, for example, the culture time may be 20-28 h (for example, about 1 day); the shaking speed of the shake flask may be 180. - 220 rpm; shake flask culture temperature can be 25 ⁇ 28. C, culture to the concentration of the bacteria and the morphology of the bacteria is good, for example, the shaking flask culture time can be 20-28h (for example, about 1 day);
  • Inoculation amount of 8% to 15% of the product/shield is added to the first-stage liquid in the solid medium;
  • the composition of the liquid fermentation medium used for the expansion culture may be: glucose 30-60 g/L, NaCl 15-20 g/L, MgS0 4 .7H 2 04-8 g/L, yeast powder 3 ⁇ 8 g/L, KH 2 P0 4 0.5 - 1.0 g/L, (NH 4 ) 2 S0 4 0.2 - 0.5 g/L, NaHC0 3 0.8 - 1.2 g/L, C0CI21.0 - 2.0 g/L, MnS0 4 0.5 - 1.0 g / L, VBi 0.05 ⁇ O.lmg / L, VB 12 0.001 - O.Olmg / L, initial pH 6.0 ⁇ 7.0; the best dissolved oxygen of ⁇ 1 fermentation is maintained at 8% - 12%;
  • the dry weight of the cells in the fermentation broth is determined to be 105-160 g/L, and the DHA content is 20-35 g/L.
  • the Schizochytrium may be any Schizochytrium known in the art.
  • it includes a Schizochytrium strain that can be obtained from a collection of a typical culture collection center in the United States and a fermentation engineering institute in Osaka, Japan.
  • the Schizochytrium sp. is Schizochytrium sp., wherein the Schizochytrium sp. may be, for example, Schizochytrium sp. KDW. -12, S31, S8 or Schizochytrium aggregatim
  • the strain Schizochytrium sp. KDW-12 used in the present invention has been deposited with the Chinese Microbial Culture Collection Management Committee ⁇ Mini Microbiology Center on November 20, 2012, and the depository number is CGMCC No. .6843, located at Datun Road, Chaoyang District, Beijing, China Institute of Microbiology, Chinese Academy of Sciences, zip code 100101.
  • the isotonic saline solution is physiological saline, i.e., a 0.85-0.9% sodium chloride solution.
  • the determination of the secondary solid seed and the pre-stage 3 ⁇ 4t fermentation time is determined according to the optimal culture time optimization experiment carried out in the previous stage, and the cell concentration and the bacterial cell obtained in the selected culture time are determined.
  • the morphology is better; the indicator of controlling the end of fermentation on the fermenter is that the morphology of the cells begins to be autolyzed, the dry weight of the cells and the DHA yield are not increased, the growth is slow or has a downward trend.
  • the control of ammonia water and glucose is well known in the prior art, that is, the control of P H by ammonia water is mainly set by the automatic control system on the fermenter, and below this set value, the peristaltic pump will automatically
  • the ammonia is adjusted by adding ammonia;
  • the control of glucose is similar to the control of ammonia water, and the remaining sugar concentration is measured by a biosensor analyzer for each sampling, and the volume of glucose to be replenished is calculated according to the set control point, and is set in the automatic control system.
  • the corresponding opening and cycle are determined automatically by the peristaltic pump.
  • one day of the culture time is not limited to an accurate 24 hours, and may be, for example, about 20 to 28 hours, 22 to 26 hours, or 24 hours.
  • the method for preparing the activated strain or the primary or secondary solid seed into the corresponding seed liquid is as follows: For the activated strain and the primary solid seed, sterile water or the like may be added. a saline solution, for example, a 0.85%-0.9% sodium chloride solution, to wash the bacteria to prepare a bacterial suspension; for the secondary solid seed, a sterile water may be added to prepare a bacterial suspension; the bacterial suspension Corresponding The seed liquid; cell concentration of the bacterial suspension may be 1.0 ⁇ 2.5xl0 6 th / mL.
  • a saline solution for example, a 0.85%-0.9% sodium chloride solution
  • the mass in the mass/volume percentage when preparing the bacterial suspension or the seed liquid and the mass in the inoculation according to the volume/shield percentage refer to the mass of the solid medium.
  • the present invention abandons the conventional seed culture process, and the seed liquid obtained directly from the solid fermentation is subjected to liquid fermentation culture: bevel activation ⁇ first solid seed/shake liquid ⁇ second solid seed liquid-liquid fermentation.
  • the invention improves the seed preparation mode, simplifies the seed expansion process, shortens the seed culture and fermentation cycle, reduces the investment, saves the cost, and is more suitable for the production; and the solid medium is rich in nutrients and rich in various growth factors, which is more conducive to For the growth of seeds, the growth energy of the strain cells is strong and the synchrony is good; the wastewater generated during the solid seed stage is less, the waste residue is less, and the environmental pollution is small.
  • the invention overcomes the defects of the seed fermentation stage of the traditional fermentation method, and can simplify the DHA production process by improving the preparation of the DHA fermentation seed liquid; the solid medium is rich in nutrients, the seeds are full and vigorous, and the seeds can be shortened. After the tank delay period, increase DHA production, reduce production cost, maintain stable production capacity; generate waste water, less waste residue and less environmental pollution during the solid seed stage; DHA can be used as a nutrient enhancer for humans and animals Long chain polyunsaturated fatty acids are required.
  • Schizochytrium sp. KDW-12 deposited at the General Microbiology Center of the China Microbial Culture Collection Management Committee on November 20, 2012 (Address: Beijing Datun Road, Chaoyang District, Institute of Microbiology, Chinese Academy of Sciences, zip code 100101), deposit number CGMCC No.6843.
  • the Schizochytrium sp. KDW-12 strain was inoculated on a slant medium and cultured and activated at a temperature of 25 ° C for 2 days.
  • the slant medium formulation was (g/L): glucose 20, peptone 15, yeast powder 3, sea crystal (sea salt) 15, agar powder 20, pH 6.5, 121. C sterilized for 30 min.
  • the cultured expired slope was added with 0.85% physiological saline to wash the bacterial cells, and the bacterial suspension was obtained, and the control bacterial concentration was 2.5 ⁇ 10 6 /mL.
  • the obtained bacterial suspension was directly inoculated to the sterilized solid medium by the inoculum volume of 1% (V/W, bacterial suspension volume/solids culture amount) (fixed medium)
  • the amount is 1000ml of K-rich 150g/bottle), 25. C culture for 1 day, shake the K-bottle once every 8-12h, to facilitate the seeds to make full use of nutrients.
  • a first solid seed is obtained.
  • the inoculation amount of 10% (V/W) by volume/shield is applied to the solid medium (secondary solid medium is 150g/bottle in a Kml bottle of 1000ml)
  • the primary solid seed used for inoculation is 0.85% physiological salt nowadays M ⁇ according to the ratio of 1:1 (W/V) to wash the bacteria, make the bacterial suspension, control bacteria
  • the body concentration was l.OxlO 6 /mL), shake the K's bottle, mix evenly, 25. C, culture for 2.5 days, shake the K-bottle once every 8-12h during the culture, mix well, in order to facilitate the seeds to make full use of nutrient growth.
  • a secondary solid seed is obtained.
  • the second solid seed was added to the 1:4 (W/V) aseptically and the cells were washed, the concentration of the control was 1.5 ⁇ 10 6 /mL, and the inoculation amount of 15% was inoculated to 50 L fermenter to expand the culture. , after the volume is 32L.
  • the temperature was controlled at 25 °C 48 h before fermentation, and 40% of dissolved oxygen was maintained by stirring speed and aeration, and the temperature was controlled at 22 after 48 h.
  • C dissolved oxygen is maintained at about 10%, and the residual sugar and bacteria in the 3 ⁇ 41 yeast solution are measured every 8h ⁇ # times during the fermentation process.
  • Body concentration, dry cell weight and DHA content were fermented for 4 days.
  • Fermentation pH 6.5 was controlled by ammonia water with a shield percentage of 28%, and the entire fermentation process was automatically fed through 118.
  • the preparation method of the solid medium is: consisting of a solid component and a liquid nutrient solution component, wherein the solid component is millet, which is 375 g/L (solid component shield volume/liquid nutrient ⁇ & subvolume), millet and
  • the liquid nutrient ⁇ BL is divided into 8 by 8:3 (W/V) ratio, and the grain is cooked to the center of the grain without white heart, and the water content is 55%, and the raw material of the solid medium is obtained.
  • the solid medium and the liquid nutrient ⁇ a 2 are uniformly mixed in a ratio of 10: 3 (W/V), and are packed in a solid-state fermentation K-neck.
  • the K-caps with a capacity of 1000 ml are dispensed in a 150 g/bottle, thickness. 0.3cm, 121. C was sterilized for 30 min to prepare a solid medium.
  • Liquid nutrition ⁇ BL points l glucose 3, magnesium pentoxide 0.2, dipotassium hydrogen phosphate 3.0, sea salt 0.6, glycine 0.6, threonine 1.5, methionine 2, malic acid 4, add water to volume Need volume, adjust pH to 6.5.
  • Component 2 (mg/L): La 3+ 15, Ce 3+ 2, Sm 3+ 3, Nd 3+ 8, Mn 2+ 1.0, Co 2+ 0.08, biotin 0.2, cerulenin 0.1, red
  • JBL in liquid fermentation glucose 30 g/L, NaCl 20 g/L, MgS0 4 .7H 2 0 5 g/L, yeast powder 3 g/L, KH2PO4 1.0 g/L, (NH 4 ) 2 S0 4 0.4 g /L, NaHC0 3 1.2 g/L, CoCl 2 2.0 g/L, MnSO 4 0.7 g/L, VBi 0.06 mg/L, VB 12 0.005 mg/L, initial pH 6.5.
  • the initial pH refers to the pH obtained by adjusting the pH meter before sterilization after the medium is prepared.
  • the cells were collected by centrifugation, and the dry weight of the cells in the fermentation broth was determined to be 125 g/L, and the DHA content in the gas phase was 25 g/L.
  • the treatment of the fermentation broth and the extraction method of DHA are carried out in accordance with the method disclosed in Chinese Patent No. CN101638361A.
  • the extracted DHA acts as a nutritional supplement to provide the long-chain polyunsaturated fatty acids needed for humans and animals.
  • Example 2 Example 2:
  • the culture was activated and the temperature was 28. C, cultured for 2 days.
  • the slant medium formulation was (g/L): glucose 25, peptone 12, yeast powder 3, sea crystal 16, agar powder 20, pH 6.0, sterilized at 121 ° C for 30 min.
  • the cultured expired slope was added with 0.85% physiological saline to wash the bacterial cells, and the bacterial suspension was obtained, and the bacterial cells were concentrated to a concentration of 6 ⁇ 4 ⁇ 1.OxlO 6 /mL.
  • the obtained bacterial suspension was directly applied to a shake flask liquid medium (500 ml of a 500 ml flask), the inoculum amount was 1%, and the shake flask speed was 220 rpm, 28. C was cultured for 1 day to obtain a first-grade liquid seed.
  • Shake flask liquid seed medium composition g/L: glucose 25, sodium chloride 20, potassium dihydrogen phosphate 0.2, yeast powder 5, seven: magnesium sulfate 10, sea salt 0.4, glutamic acid 0.4, pH 6.0, 121. C sterilization for 30 min.
  • the inoculation amount of the product/shield fraction of 10% is connected to the first-stage liquid seed in the solidification ⁇ (packed with a Kml bottle of 1000 ml size), and the K-neck is shaken. Mix evenly, 26. C, culture for 3 days, culture: shake the K's bottle once every 8-12h, mix well, in order to facilitate the seeds to fully utilize the nutrient growth. A secondary solid seed is produced.
  • the second solid seed was added to the 1:3 (W/V) aseptically and the bacteria were washed.
  • the concentration of the control was 2.5 ⁇ 10 6 /mL, and the inoculation amount of 20% by volume/shield was inoculated to 100L.
  • the fermenter was expanded and the volume was 60L.
  • the temperature was controlled at 28 hours before fermentation.
  • C 30% of dissolved oxygen is maintained by stirring speed and ventilation, and the temperature is controlled to 18 at 48 h until the end of fermentation.
  • C dissolved oxygen is maintained at around 10%.
  • the residual sugar, cell concentration, cell dry weight and DHA content in the fermentation broth were measured every 8 h during the fermentation.
  • the fermentation was carried out for 5 days.
  • Fermentation pH 6.0 was controlled by 28% aqueous ammonia, and the entire fermentation process was automatically fed through 118.
  • the glucose solution with a concentration of 580 g/L for 25 min was sterilized to control the fermentation glucose concentration at 7 g/L.
  • the preparation method of the solid medium is: consisting of a solid component and a liquid nutrient solution component, wherein the solid component is millet, the content is 300 g/L, the millet and the liquid nutrient ⁇ BL score 1 is 8:3 (W/V) The ratio is evenly mixed, the grain is cooked until the center of the grain has no white heart, and the water content is
  • the ratio of 10: 4 (W/V) is evenly mixed. It is packed in a solid-state fermentation K-bottle, and the volume is 1000 ml of K 250g/bottle, thickness 0.5cm, 121. C sterilization for 30 min, made Solid medium.
  • Liquid nutrition ⁇ ia points l glucose 5, magnesium pentoxide 0.4, dipotassium hydrogen phosphate 2.0, sea salt 0.5, glycine 0.3, threonine 1.5, methionine 3.5, malic acid 6, add water to volume Need volume, adjust pH value 6.0.
  • Component 2 (mg/L): La 3+ 10, Ce 3+ 4, Sm 3+ 5, Nd 3+ 9, Mn 2+ 0.8, Co 2+ 0.06, biotin 0.2, cerulenin 0.1, red
  • Composition of liquid fermentation medium glucose 40 g/L, NaCl 15 g/L, MgS0 4 .7H 2 0 4 g/L, yeast powder 5 g/L, KH2PO4 0.5 g/L, (NH 4 ) 2 S0 4 0.2 g/ L, NaHC0 3 0.8 g/L, CoCl 2 1.5 g/L, MnSO 4 1.0 g/L, VBi O.lmg/L, VB 12 0.01 mg/L, initial ⁇ 6 ⁇ 0.
  • the cells were collected by centrifugation, and the dry weight of the cells in the fermentation broth was determined to be 159 g/L, and the DHA content in the gas phase was 34.5 g/L.
  • the treatment of the fermentation broth and the method of extracting DHA are carried out in accordance with the method disclosed in Chinese Patent No. CN101638361A.
  • the extracted DHA acts as a nutritional supplement to provide the long-chain polyunsaturated fatty acids needed for humans and animals.
  • Example 3 Example 3:
  • Schizochytrid (Schizochytri leg sp.) KDW-12 strain was inoculated on a slant medium and cultured at a temperature of 28. C, cultured for 3 days.
  • the slant medium formulation was (g/L): glucose 30, peptone 12, yeast powder 3, sea crystal 15, agar powder 20, pH 6.5, sterilized at 121 ° C for 30 min.
  • the cultured expired slope was added with 0.85% physiological saline to wash the bacterial cells, and the bacterial suspension was obtained, and the bacterial cells were controlled to be concentrated at 3 ⁇ 4 ⁇ 2.0x10 6 cells/mL.
  • the obtained bacterial suspension was directly inoculated to a shake flask liquid medium (500 ml of a triangular liquid amount of 150 ml) at a 1% inoculation amount, and shaken at 220 rpm, 28. C was cultured for 1 day to prepare a first-stage liquid seed.
  • Shake flask liquid seed medium composition g/L: glucose 20, sodium chloride 22, potassium dihydrogen phosphate 0.2, yeast powder 3, seven: magnesium sulfate 10, sea salt 0.3, glutamic acid 0.6, pH 6.5, 121. C sterilization for 30 min.
  • the inoculum volume of 8% by volume/shield fraction is added to the first-stage liquid in the solid-supporting base (packaged 1000ml K-300g/bottle), and the K-neck is evenly mixed.
  • 26. C culture for 3 days, training: shaking K every 8-12h Once in a bottle, mix well to help the seeds make the most of the nutrients. A secondary solid seed is obtained.
  • the second solid seed was added to the 1:3 (W/V) aseptically washed cells, the control concentration was 2.0 ⁇ 10 6 /mL, and the inoculation amount of 10% was inoculated to 100 L fermenter to expand the culture. , after the volume is 60L.
  • the temperature was controlled at 25 °C 48 h before fermentation, and 30% of dissolved oxygen was maintained by stirring speed and aeration, and the temperature was controlled at 20 after 48 h. C, dissolved oxygen is maintained at around 10%.
  • the residual sugar, cell concentration, cell dry weight and DHA content in the fermentation solution were measured every 5 h ⁇ # -, and the fermentation was carried out for 5 days.
  • Fermentation pH 6.0 was controlled by 28% ammonia water, and the whole fermentation process was automatically added with a glucose solution of 580 g/L sterilized at 118 ° C for 25 min to control the fermentation glucose concentration at 8 g/L.
  • the preparation method of the solid medium is: consisting of a solid component and a liquid nutrient ⁇ >, wherein the solid component is wheat, 350 g/L, and the wheat and liquid nutrient ⁇ a minute 1 is 10:4 (W/V) The ratio is evenly mixed, the grain is cooked to the center of the grain without white heart, and the water content is
  • Liquid nutrition ⁇ iBL points l glucose 3.5, magnesium citrate 0.3, dipotassium hydrogen phosphate 3.0, sea salt 0.4, glycine 0.5, threonine 1.6, methionine 3.5, malic acid 5, add water to volume Need volume, adjust pH value 6.0.
  • Component 2 (mg/L): La 3+ 12, Ce 3+ 4, Sm 3+ 5, Nd 3+ 8, Mn 2+ 0.9, Co 2+ 0.08, biotin 0.4, cerulenin 0.1, red
  • Composition of liquid fermentation medium glucose 40 g/L, NaCl 16 g/L, MgS0 4 .7H 2 0 6 g/L, yeast powder 4 g/L, KH2PO4 1.0 g/L, (NH 4 ) 2 S0 4 0.5 g/L, NaHC0 3 1.2 g/L, CoCl 2 2.0 g/L, MnSO 4 1.0 g/L, VBi 0.05 mg/L, VB 12 0.005 mg/L, initial ⁇ 6 ⁇ 0.
  • the cells were collected by centrifugation, and the dry weight of the cells in the fermentation broth was determined to be 132 g/L, and the DHA content in the gas phase was 30.5 g/L.
  • Treatment of fermentation broth and extraction of DHA The method is carried out in accordance with the method disclosed in Chinese Patent No. CN101638361A.
  • the extracted DHA acts as a nutritional supplement to provide the long-chain polyunsaturated fatty acids needed for humans and animals.
  • Example 4 Example 4:
  • the strain of Schizochytrium sp. S31 was inoculated on a slant medium and cultured at a temperature of 28. C, cultured for 2 days.
  • the slant medium formulation was (g/L): glucose 30, peptone 12, yeast powder 3, sea crystal 15, agar powder 20, pH 6.5, 121. C sterilized for 30 min.
  • the cultured expired slope was added with 0.85% physiological saline to wash the bacterial cells, and the bacterial suspension was obtained, and the control bacterial concentration was 1.5 ⁇ 10 6 /mL.
  • the obtained bacterial suspension was directly inoculated to a shake flask liquid medium (500 ml of a triangular liquid amount of 150 ml) at a 1% inoculation amount, and shaken at 200 rpm, 28. C was cultured for 1 day to prepare a first-stage liquid seed.
  • Shake flask liquid seed medium composition g/L: glucose 20, sodium chloride 22, potassium dihydrogen phosphate 0.2, yeast powder 5, seven: magnesium sulfate 12, sea salt 0.3, glutamic acid 0.6, pH 6.5, 121. C sterilization for 30 min.
  • the inoculation amount of the product/shield fraction of 15% is added to the first-stage liquid in the solidification ⁇ (package of 1000ml K-side 170g/bottle), and the K-neck is evenly mixed.
  • 26. C culture for 2 days, shake the K's bottle once every 8-12h, mix well, in order to facilitate the seeds to fully utilize the nutrient growth. A secondary solid seed is produced.
  • the second solid seed was added to the 1:3 (W/V) aseptically and the cells were washed.
  • the concentration of the control cells was 2.0 ⁇ 10 6 /mL, and inoculated into a 100 L fermentor according to the inoculation amount of 10%.
  • the rear volume is 60L.
  • the temperature was controlled at 25 °C 48 h before fermentation, and 30% of dissolved oxygen was maintained by stirring speed and aeration, and the temperature was controlled at 20 after 48 h. C, dissolved oxygen is maintained at around 10%.
  • the residual sugar, cell concentration, cell dry weight and DHA content in the fermentation broth were measured every 8h during the fermentation process. The fermentation was carried out for 4.5 days.
  • the preparation method of the solid medium is: consisting of a solid component and a liquid nutrient ⁇ &
  • the solid component is millet, 350g/L, and the millet is mixed with liquid nutrient ⁇ & min 1 in a ratio of 10: 4 (W/V).
  • the grain is cooked to the center of the grain without white heart, and the water content is
  • the ratio of 10: 4 (W/V) is evenly mixed. It is packed in a solid-state fermentation K-neck.
  • the K-shaped bottle with a capacity of 1000ml is packed in 170g/bottle, and the thickness is 0.3cm, 121. C was sterilized for 30 minutes to prepare a solid medium.
  • Liquid nutrition ⁇ iBL points l glucose 3, magnesium citrate 0.5, dipotassium hydrogen phosphate 5.0, sea salt 0.8, glycine 0.2, threonine 1.6, methionine 3.5, malic acid 3, add water to volume Need volume, adjust pH value 6.0.
  • Component 2 (mg/L): La 3+ 15, Ce 3+ 4, Sm 3+ 5, Nd 3+ 10, Mn 2+ 0.9, Co 2+ 0.1, biotin 0.4, cerulenin 0.2, red
  • the peptide GA 2, VB 12 1.0, folic acid 0.02 is water.
  • Composition of liquid fermentation medium glucose 40 g/L, NaCl 20 g/L, MgS0 4 .7H 2 0 6 g/L, yeast powder 4 g/L, KH2PO4 1.0 g/L, (NH 4 ) 2 S0 4 0.5 g /L, NaHC0 3 0.8 g/L, CoCl 2 2.0 g/L, MnSO 4 0.5 g/L, VBi 0.05 mg/L, VB 12 0.005 mg/L, initial ⁇ 6 ⁇ 0.
  • the cells were collected by centrifugation, and the dry weight of the cells in the fermentation broth was measured to be 115 g/L, and the DHA content in the gas phase was 22.7 g/L.
  • the treatment of the fermentation broth and the method of extracting DHA are carried out in accordance with the method disclosed in Chinese Patent No. CN101638361A.
  • the extracted DHA acts as a nutritional supplement to provide the long-chain polyunsaturated fatty acids needed for humans and animals.
  • the Schizochyirium sp. S8 (ATCC 20889) strain was inoculated on a slant medium and cultured and activated at a temperature of 28 ° C for 2 days.
  • the slant medium formulation was (g/L): glucose 20, peptone 12, yeast powder 3, sea crystal 20, agar powder 20, pH 6.5, 121. C sterilized for 30 min.
  • the cultured expired slope was added with 0.85% physiological saline to wash the bacterial cells, and the bacterial suspension was obtained, and the control bacterial concentration was 2.5 ⁇ 10 6 /mL.
  • the obtained bacterial suspension was directly inoculated to a shake flask liquid medium (1 L of a triangular liquid amount of 350 ml) at a 1% inoculation amount, and shake flask il 220 rpm, 28. C was cultured for 1 day to prepare a first-stage liquid seed.
  • Shake flask liquid seed medium composition ( g / L ): glucose 25, sodium chloride 22, Potassium dihydrogen phosphate 0.5, yeast powder 5, seven: magnesium acetate 12, sea salt 0.3, glutamic acid 0.6, pH 6.5, 121. C sterilized for 30 min.
  • the inoculum volume with a 12% accumulation/shield fraction is added to the first-stage liquid seed in the solidification ⁇ (packaged at 2250g/bottle of the KL bottle of 10L size), and the K-neck is shaken. Mix evenly, 28. C, culture for 3 days, culture: shake the K's bottle once every 8-12h, mix well, in order to facilitate the seeds to make full use of nutrients to grow. A secondary solid seed is obtained.
  • the second solid seed was added to the 1:4 (W/V) aseptically and the bacteria were washed.
  • the concentration of the control cells was 2.0 ⁇ 10 6 /mL, and the inoculation amount was inoculated to 1000 L fermenter according to the 18% inoculation amount.
  • the rear volume is 600L.
  • the temperature was controlled at 28 °C 48 h before fermentation, and 30% of dissolved oxygen was maintained by stirring speed and aeration, and the temperature was controlled at 20 after 48 h. C, dissolved oxygen is maintained at around 10%.
  • the residual sugar, cell concentration, cell dry weight and DHA content in the fermentation broth were measured every 8 h during the fermentation. The fermentation was carried out for 5 days.
  • the preparation method of the solid medium is: consisting of a solid component and a liquid nutrient, wherein the solid component is rice, the content is 300 g/L, and the rice and the liquid nutrient ⁇ BL are divided into 8:
  • Liquid nutrition ⁇ iBL points l glucose 3, magnesium citrate 0.5, dipotassium hydrogen phosphate 5.0, sea salt 0.8, glycine 0.2, threonine 1.6, methionine 3.5, malic acid 3, add water to volume Need volume, adjust pH value 6.0.
  • Component 2 (mg/L): La 3+ 15, Ce 3+ 4, Sm 3+ 5, Nd 3+ 10, Mn 2+ 1.2, Co 2+ 0.1, biotin 0.4, cerulenin 0.2, red
  • the peptide GA2, VB 12 1.0, folic acid 0.02, the balance is water.
  • Liquid fermentation medium composition glucose 40 g / L, NaCl 20 g / L, MgS0 4 .7H 2 0
  • the cells were collected by centrifugation, and the dry weight of the cells in the fermentation broth was determined to be 138 g/L, and the DHA content in the gas phase was 31.6 g/L.
  • the treatment of the fermentation broth and the method of extracting DHA are carried out in accordance with the method disclosed in Chinese Patent No. CN101638361A.
  • the extracted DHA acts as a nutritional supplement to provide the long-chain polyunsaturated fatty acids needed for humans and animals.
  • Example 6 Example 6:
  • the Schizochytrium aggregatum (ATCC 28209) strain was inoculated on a slant medium and cultured at a temperature of 25. C, cultured for 2 days.
  • the slant medium formulation was (g/L): glucose 20, peptone 15, yeast powder 3, sea crystal 15, agar powder 20, pH 6.5, 121. C sterilized for 30 min.
  • the cultured expired slope was added with 0.85% physiological saline to wash the bacterial cells, and the bacterial suspension was obtained, and the control bacterial concentration was 2.5 ⁇ 10 6 /mL.
  • the obtained bacterial suspension was directly inoculated to a sterilized solid medium (packaged at 225 g/bottle of a KOO bottle of 100 mL) on the inoculation amount of 1% by volume/shield. C Incubate for 1 day. Shake the K-Bottle once every 8-12 h during the culture and mix it to help the seeds make full use of nutrients. A first solid seed is obtained.
  • the inoculum volume with a 20% accumulation/shield fraction is added to the primary solid seed in the solidification ⁇ (package of 10L K 2 ⁇ 2250g/bottle) (for inoculation)
  • the first-stage solid seed is washed with 0.85% physiological saline according to the ratio of 1:1 (W/V), and the bacterial suspension is prepared to control the concentration of the bacteria to be 1.5 ⁇ 10 6 /mL), and the K-neck is evenly mixed.
  • 25. C culture for 3 days, culture: shake the K-bottle once every 8-12h, mix well, in order to facilitate the seeds to make full use of nutrient growth.
  • a secondary solid seed is obtained.
  • the second solid seed was added to the 1:4 (W/V) aseptically and the bacteria were washed.
  • the concentration of the control cells was 2.0 ⁇ 10 6 /mL.
  • the inoculum was inoculated to the 1000 L fermentor according to the inoculation amount of 12%.
  • the rear volume is 600L.
  • the temperature was controlled at 28 °C 48 h before fermentation, and 40% of dissolved oxygen was maintained by stirring speed and aeration, and the temperature was controlled at 18 after 48 h. C, dissolved oxygen is maintained at around 10%.
  • the residual sugar, the concentration of the cells, the dry weight of the cells and the DHA content in the fermentation broth were measured once every 8 hours.
  • the preparation method of the solid medium is: consisting of a solid component and a liquid nutrient solution component, wherein the solid component is barley, the content is 375 g/L, and the barley and liquid nutrient ⁇ BL is 1 by 8:3 (W/V) The ratio is evenly mixed, the grain is cooked until the center of the grain has no white heart, and the water content is
  • Liquid nutrition ⁇ iBL points l glucose 5, magnesium pentoxide 0.2, dipotassium hydrogen phosphate 3.0, sea salt 0.6, glycine 0.6, threonine 1.5, methionine 2, malic acid 4, add water to volume Need volume, adjust pH 6.5.
  • Component 2 (mg/L): La 3+ 15, Ce 3+ 2, Sm 3+ 3, Nd 3+ 8, Mn 2+ 1.2, Co 2+ 0.08, biotin 0.2, cerulenin 0.1, red
  • Liquid fermentation culture glucose 30 g / L, NaCl 20 g / L, MgS0 4 .7H 2 0 5 g / L, yeast powder 3 g / L, KH2PO4 1.0 g / L, (NH 4 ) 2 S0 4 0.4 g / L, NaHC0 3 1.2 g/L, CoCl 2 2.0 g/L, MnSO 4 0.7 g/L, VBi 0.06 mg/L, VB 12 0.005 mg/L, initial ⁇ 6 ⁇ 5.
  • the cells were collected by centrifugation, and the dry weight of the cells in the fermentation broth was determined to be 142 g/L, and the DHA content in the gas phase was 32.3 g/L.
  • the treatment of the fermentation broth and the extraction method of DHA are carried out in accordance with the method disclosed in Chinese Patent No. CN101638361A.
  • the extracted DHA acts as a nutritional supplement to provide the long-chain polyunsaturated fatty acids needed for humans and animals.

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Abstract

提供了一种发酵生产DHA的方法，具体为一种固料培养裂殖壶菌液体发酵生产DHA的方法。所述方法包括菌种活化与菌悬液制备、一级种子制备、二级固料种子的制备、二级固料种子发酵罐扩大培养；以及发酵结束后，收集菌体细胞，提取DHA油脂的步骤。该发酵方法改进了种子制备方式，简化了扩种工艺，缩短了种子培养和发酵周期，可减少投资，节约成本；同时固料培养基营养丰富，富含多种生长因子，更利于种子的生长，菌种细胞的生长活力强、同步性较好；培养固料种子阶段产生的废水、废渣少，环境污染小。

Description

一种固料培养裂殖壶菌液体发酵生产 DHA的方法 技术领域
本发明涉及一种发酵生产 DHA的方法， 具体涉及一种固料培养裂 殖壶菌液体发酵生产 DHA的方法。 背景技术
二十二碳六烯酸 (Docosahexenoic acid, 22:6 Δ4,7,10,13,16,19, 简 称 DHA)俗称"脑黄金"，属于 ω-3 型多不饱和脂肪酸，具有补脑健脑， 预防老年性痴呆症； 提高视力， 防治近视； 预防高血压、 动脉硬化、 关 节炎及治疗癌症等生理功能。 因此， DHA作为新一代功能性保健因子 具有广阔的市场前景。 随着市场需求量的扩大， 对 DHA品质的要求也 越来越高， DHA生产研究逐渐向利用生物技术合成方向发展， 传统鱼 油来源的 DHA已逐渐被藻油 DHA所取代。 藻油 DHA是指通过微生 物发酵获得的含有 DHA等长链多不饱和脂肪酸的油脂， 其中 DHA生 产菌主要有裂殖壶菌、 破囊壶菌、 隐甲藻等。
裂殖壶菌 ( Schizochyirium)又称裂壸藻, 属于真菌门、 卵菌纲、 水 霉目、 破囊壶菌科的一类类藻的海洋真菌， 单细胞、 球形。 细胞累积大 量对人体有用的活性物质， 如油脂、 色素（类胡萝卜素、 叶黄素、 坏青 素）、 角鲨烯等， 其中总脂肪酸中 DHA含量高达 35% ~ 45%, 且细胞 中 90%以上的油脂以人体易吸收的中性油脂——甘油三酯形式存在，是 DHA的理想生产菌林。
中国专利 CN101575584公开一种裂殖弧菌及利用其生产 DHA油脂 的方法， 该方法提供一种从渗透压和元素供应角度优化菌林发酵培养 基，并结合流加策略， 实现裂殖壶菌高密度发酵，采用传统的发酵方法， 从甘油保存种接入摇瓶活化→一级种子液→摇瓶二级种子液→一级种 子罐→二级种子罐→发酵， 最终细胞干重达到 70g/L， 油脂含量达 31.5g/L, DHA占总脂肪酸含量高于 35%。
中国专利 CN101519676公开一种采用含微量元素的发酵培养羞 J£ 行发酵生产 DHA的发酵生产工艺， 采用传统方法培养菌种裂殖壶菌， 即斜面活化培养、 然后转接入摇瓶扩大培养、 再经一级扩大培养得到发 酵用种子液； 或菌种经斜面活化培养、 然后转接入摇瓶扩大培养、 再经 一级扩大培养和二级扩大培养后得到发酵用种子液， 10T罐发酵 95h生 物量为 53g/L， 油脂含量 72%, DHA含量 50%。
但上述的传统的种子培养和发酵方法涉及的发酵级数较多， 工艺较 繁瑣，培养周期长，投资成本高。 因此，本领域亟需改进种子制备方式， 简化扩种工艺， 降低生产成本， 以更有利于 DHA的工业化生产。 发明内容
本发明的目的在于提供可简化扩种工艺， 缩短种子培养和发酵周 期， 减少投资， 节约成本， 固料培养基营养丰富， 富含多种生长因子， 更利于种子的生长， 菌种细胞的生长活力强、 同步性较好， 培养固料种 子阶段产生的废水、 废渣少， 环境污染小的一种固料培养裂殖壶菌液体 发酵生产 DHA的方法。
本发明第一方面涉及一种固料培养裂殖壶菌液体发酵生产 DHA 的方法， 其包括以下步骤：
(1)菌种活化： 将裂殖壶菌菌种接种于培养基 (例如斜面培养基)培 养活化， 制备获得菌悬液；
(2)—级种子制备：将步骤 (1)获得的菌悬液接种至固料培养基上培 养， 制成一级固料种子； 或者
将步骤 (1)获得的菌悬液接种到液体种子培养基培养 (例如摇瓶培 养)， 制成一级液体种子；
(3)二级固料种子制备： 将一级固料种子制成一级固料种子液， 在 固料培养基中接入一级固料种子液培养； 或者
在固料培养基中接入一级液体种子液培养；
制得二级固料种子；
(4)二级固料种子发酵罐扩大培养： 将二级固料种子制成二级固料 种子液， 接种至发酵罐液体培养基中扩大培养； (5)发酵结束后， 收集菌体细胞， 提取 DHA。
根据本发明第一方面任一项的方法， 其中所述裂殖壶菌为本领域 公知的裂殖壶菌。 在本发明的实施方案中， 所述裂殖壶菌选自 Schizochytrium sp.; 在^发明的实施方案中， 所述 Schizochytrium sp. 选自裂殖壶菌 Schizochytrium sp. KDW-12、裂殖壶菌 Schizochytrium sp. S31、 裂殖壶菌 Schizochytrium aggregatum和裂殖壶菌 Schizochytrium sp. S8, 其中所述裂殖壶菌
Figure imgf000004_0001
sp.KDW-12已于 2012年 11 月 20日保藏于中国微生物菌种保藏管理委员^ 通微生物中心 (地址： 北京市朝阳区大屯路， 中国科学院微生物研究所， 邮编为 100101)， 保 藏编号 CGMCC No.6843。
根据本发明第一方面任一项的方法， 其中步骤 (1)的特征在于以下 几项中的一项或数项：
a.所述培养的温度为 25 - 28 °C，培养至菌体浓度及菌体形态较好， 例如培养的时间为 2 ~ 3天；
b.所述菌悬液的菌体浓度为 1.0 ~ 2.5xl06个 /mL。
根据本发明第一方面任一项的方法， 其中步骤 (2)的特征在于以下 几项中的一项或数项：
a.所述接种量为 0.5 ~ 2%；
b.所述培养的温度为 25 ~ 28°C , 培养至菌体浓度和菌体形态较好， 例如培养的时间为 20 ~ 28h;
c.所述液体培养 (例如摇瓶培养)的转速为 180 - 220 rpm。
根据本发明第一方面任一项的方法， 其中步骤 (3)的特征在于以下 几项中的一项或数项：
a.所述一级固料种子液的菌体浓度可为 1.0 - 2.5xl06个 /mL;
b.按照体积 /盾量分数为 10% - 20%的接种量在固料培养基中接 入一级固料种子液；
c.按照体积 /盾量分数为 8% - 15%的接种量在固料培养基中接入 一级液体种子；
d.所述培养温度为 25 ~ 28°C，培养至菌体浓度和菌体形态较好，例 如培养的时间为 2~3天。
才艮据本发明第一方面任一项的方法， 其中步骤 (4)的特征在于以下 几项中的一项或数项：
a.将二级固料种子液按体积分数为 10% - 20%的接种量接种至发 酵罐扩大培养；
b.发酵过程的前 48h 温度控制在 25~28。C， 利用搅拌转速保持 30% ~ 50%的溶氧；
c.发酵过程的第 48h至发酵结束温度控制在 18~22。C， 利用搅拌转 速保持 8% - 12%的溶氧；
d.在菌体形态开始自溶、 菌体细胞干重和 DHA产量均无增加、 增 加较慢或有下降趋势时， 结束发酵， 例如发酵 3~5天；
e.发酵过程中通过氨: ^制发酵液 pH为 6.0 ~ 6.5;
f.发酵过程中通过自动流加葡萄糖溶液控制葡萄糖浓度在 4~8 在本发明的实施方案中， 所述氨水的浓度为 28%。
在本发明的实施方案中， 所述葡萄糖溶液的浓度为 580g/L。
根据本发明第一方面任一项的方法， 其中步骤 (1)中的斜面培养基 含有： 葡萄糖 20 - 30g/L, 蛋白胨 10 - 15g/L, 酵母粉 3 - 5g/L, 海水晶 15 - 20g/L, 琼脂粉 15~25g/L， pH值 6.0~7.0。
根据本发明第一方面任一项的方法，其中步骤 (2)或步骤 (3)中的固 料培养基包括固态组分和液体营养液组分， 所述固态组分为谷物， 为 250~400g/L， 所述谷物选自米类、 麦类和玉米中的至少一种， 所述 米类选自大米、 小米、糙米等中的至少一种， 所述麦类选自大麦、 小麦、 燕麦中的至少一种；
所述液体营养液组分包括组分 1和组分 2， 所述组分 1的组成为： 葡萄糖 2~5g/L， 七:^酸镁 0.2~0.6g/L, 磷酸氢二钾 1.0 - 5.0g/L, 海盐 0.1 - 0.8g/L,甘氨酸 0.2 - 0.6g/L,苏氨酸 1.5 - 1.8g/L,蛋氨酸 2 - 3.5g/L, 苹果酸 3~6g/L， 加水定容， 调节 pH值为 6~7； 所述组分 2 的组成为： La3+ 10 ~ 15mg/L， Ce3+l~4mg/L, Sm3+ 2 - 6mg/L, Nd3+8~ 12mg/L, Mn2+ 0.6 - 1.2mg/L, Co2+ 0.05 - O.lmg/L,生物素 0.2 - 0.6mg/L, 浅蓝菌素 0.1 - 0.3mg/L, 赤霉素 GA 2 ~ 5mg/L, VB120.5 - l.Omg/L, 叶 酸 0.01~0.05mg/L， 余量为水；
所述固料培养基的制备方法为： 固态组分与液体营养 ^ a分 1混合 均匀， 将谷物蒸或煮至谷物中心无白心， 且含水率为 50%~65%， 获得 固料培养基原料； 将固料培养基原料与液体营养液组分 2混合均匀， 装 于固态发酵瓶中， 厚度 0.2~1.0cm， 灭菌， 制得固料培养基；
优选地， 所述固态组分与液体营养 ^ia分 1按盾量 /体积的配比为
(5~10) §： (2-5) mL, 所述固料培养 料与液体营养 分 2 按盾量 /体积的配比为 （10~15) g: (3~6) mL, 其中固态组分按盾 量计算， 液体营养液组分按体积计算。
根据本发明第一方面任一项的方法， 其中在步骤 2)中， 所述液体 种子培养基含有： 葡萄糖 20~25g/L， 氯化钠 20 - 25g/L, 磷酸二氢钾 0.2 - 0.5g/L,酵母粉 3 - 5g/L,七:^酸镁 8 - 12g/L,海盐 0.1 - 0.8g/L, 谷氨酸 0.2~0.6g/L， pH值 6.0~7.0。
根据本发明第一方面任一项的方法， 其中步骤 (4)中的发酵罐液体 培养基含有： 葡萄糖 30~60g/L， NaCl 15 - 20g/L, MgS04.7H204 ~ 8 g/L, 酵母粉 3~8g/L， KH2P040.5-1.0 g/L, (NH4)2S040.2-0.5 g/L, NaHC030.8-1.2 g/L, CoCl21.0 ~ 2.0 g/L， MnSO40.5 ~ 1.0 g/L， VBi 0.05- O.lmg/L, VB120.001 ~0.01mg/L, 初始 pH6.0~7.0。 本发明第二方面涉及用于培养裂殖壶菌发酵生产 DHA的培养基， 所述培养基选自以下几种培养基中的一种或几种：
1) 斜面培养基， 含有： 葡萄糖 20~30g/L， 蛋白胨 10~l5g/L， 酵 母粉 3 - 5g/L, 海水晶 15 - 20g/L, 琼脂粉 15 - 25g/L (例如 20 g/L ) ， pH值 6.0 - 7.0；
2)固料培养基， 包括固态组分和液体营养液组分， 所述固态组分 为谷物， 含量为 250 ~ 400g/L， 所述谷物选自米类、 麦类和玉米中的至 少一种， 所述米类选自大米、 小米、 糙米等中的至少一种， 所述麦类 选自大麦、 小麦、 燕麦中的至少一种；
所述液体营养液组分包括组分 1和组分 2， 所述组分 1的组成为： 葡萄糖 2~5g/L， 七?^酸鎂 0.2~0.6g/L, 磷酸氢二钾 1.0 - 5.0g/L, 海盐 0.1 ~ 0.8g/L,甘氛酸 0.2 ~ 0.6g/L,苏氨酸 1.5 ~ 1.8g/L,蛋氨酸 2 ~ 3.5g/L, 苹果酸 3 - 6g/L, 加水定容， 调节 pH值为 6 ~ 7; 所述组分 2 的组成为： La3+ 10 ~ 15mg/L， Ce3+l~4mg/L, Sm3+ 2 - 6mg/L, Nd3+8~ 12mg/L, Mn2+ 0.6 - 1.2mg/L, Co2+ 0.05 - O.lmg/L,生物素 0.2 - 0.6mg/L, 浅蓝菌素 0.1 ~ 0.3mg/L, 赤霉素 GA 2 ~ 5mg/L, VB120.5 - l.Omg/L, 叶 酸 0.01~0.05mg/L， 余量为水；
所述固料培养基的制备方法为： 固态组分与液体营养 ^ a分 1混合 均匀， 将谷物蒸或煮至谷物中心无白心， 且含水率为 50%~65%， 获得 固料培养基原料； 将固料培养基原料与液体营养液组分 2混合均匀， 装 于固态发酵瓶中， 厚度 0.2~1.0cm， 灭菌， 制得固料培养基；
优选地， 所述固态组分与液体营养液组分 1 按质量 /体积的配比为 (5~10) g: (2-5) mL, 所述固料培养基原料与液体营养液组分 2 按盾量 /体积的配比为（ 10 ~ 15 ) g： (3~6) mL, 其中固态组分按盾量 计算， 液体营养液组分按体积计算
3) 液体种子培养基，含有： 葡萄糖 20~25g/L， 氯化钠 20~25g/L， 磷酸二氢钾 0.2 ~ 0.5g/L， 酵母粉 3~5g/L， 七:^酸镁 8 ~ 12g/L， 海盐 0.1 - 0.8g/L, 谷氨酸 0.2~0.6g/L， pH值 6·0~7.0;
4) 发酵罐液体培养基，含有：葡萄糖 30 - 60 g/L, NaCl 15 - 20g/L, MgS04.7H20 4~8 g/L, 酵母粉 3~8 g/L, KH2P04 0.5 - 1.0 g/L, (NH4)2S040.2 ~ 0.5 g/L, NaHC030.8 - 1.2 g/L, CoCl21.0 - 2.0 g/L， MnS040.5 ~ 1.0 g/L， VBi 0.05 ~ O.lmg/L, VB120.001 - O.Olmg/L, 初 始 pH6.0~7.0。 本发明还涉及一种固料培养裂殖壶菌液体发酵生产 DHA的方法， 其特征在于包括如下步骤：
1)菌种活化与菌悬液制备： 将裂殖壶菌菌种接种于斜面培养基上 培养活化， 培养到期斜面加入 0.85%的生理盐水洗涤菌体， 获得菌悬 液；
2)一级种子制备： 将步骤 1)获得的菌悬液按盾量分数为 1%的 接种量接种至固料培养基上培养， 制成一级固料种子； 或
将步骤 1 )获得的菌悬液按体积分数为 1%的接种量接种至液体种 子培养基， 摇瓶培养， 制成一级液体种子；
3 )二级固料种子的制备： 一级固料种子以 0.85%的生理盐?! M艮据 1:1(W/V)比例洗涤菌体， 制成菌悬液；
在无菌条件下：
积 /盾量分 10% - 20%的接种量在固 ^^养基中接入一级 固料种子液； 或
积 /盾量分 8% ~ 15%的接种量在固料培养基中接入一级 液体种子， 摇动固态发酵瓶混合均匀， 25~28°C， 培养 2~3天；
制得二级固料种子；
4)二级固料种子发酵罐扩大培养： 二级固料种子加入 1:3 ~ 5 (W/V)无菌水振荡洗下菌体， 按体积分数为 10% ~ 20%的接种量接 种至发酵罐扩大培养， 发酵前 48h温度控制在 25~28°C， 利用搅拌转 速保持 30% ~ 50%的溶氧， 48h至发酵结束温度控制在 18~22。C， 发 酵 3 ~ 5天，通 it*量百分比为 28%的氨:^制发酵 pH6.0 - 6.5,整个 发酵过程自动流加经 118。C灭菌 25min的浓度为 580g/L的葡萄糖溶液 控制发酵葡萄糖浓度在 4~8 g/L;
5)发酵结束后， 收集菌体细胞， 提取 DHA油脂。
在本发明的实施方案中， 步骤 1) 中， 所述斜面培养基的配方为： 葡萄糖 20~30g/L， 蛋白胨 10~15g/L， 酵母粉 3~5g/L， 海水晶 15 ~ 20g/L,琼脂粉 15 - 25g/L(例如 20 g/L )， pH值 6 ~ 7， 121。C灭菌 30min; 所述培养活化的温度可为 25~28'C，培养活化的时间可为 2~3天； 所 述菌悬液的菌体浓度可为 1.0~2.5xl06个 /mL。
在本发明的实施方案中， 在步骤 2)中， 所述固料培养基包括固态 组分和液体营养 ^ BL分， 所述固态组分为谷物， 含量为 250 ~ 400g/L， 所述谷物可选自米类、 麦类和玉米中的至少一种， 所述米类可选自大 米、 小米、 糙米等中的至少一种， 所述麦类可选自大麦、 小麦、 燕麦 中的至少一种。
在本发明的实施方案中， 在步骤 2)中， 所述液体营养液组分包括 组分 1和组分 2， 所述组分 1的组成为： 葡萄糖 2~5g/L， 七水硫酸镁 0.2 - 0.6g/L,磷酸氢二钾 1.0 - 5.0g/L,海盐 0.1 - 0.8g/L,甘氨酸 0.2 - 0.6g/L, 苏氨酸 1.5 - 1.8g/L, 蛋氨酸 2 - 3.5g/L, 苹果酸 3 - 6g/L, 加 水定容，调节 pH值为 6 ~ 7; 所^ &分 2的组成为： La3+ 10 - 15mg/L, Ce3+ 1 - 4mg/L, Sm3+ 2 - 6mg/L, Nd3+8~ 12mg/L, Mn2+ 0.6 - 1.2mg/L, Co2+ 0.05 - O.lmg/L, 生物素 0.2 - 0.6mg/L, 浅蓝菌素 0.1 - 0.3mg/L, 赤霉素 GA 2 ~ 5mg/L， VB120.5 - l.Omg/L, 叶酸 0.01 ~ 0.05mg/L， 余 量为水。
在本发明的实施方案中， 在步骤 2)中， 所述固料培养基的制备方 法为： 固态组分与液体营养 ¾ 分 1 混合均匀， 将 蒸或煮至谷物 中心无白心， 且含^为 50%~65%， 获得固 养基原料； 将固料 培养基原料与液体营 分 2混合均匀， 装于固态发酵 K瓶中， 厚 度 0.2~1.0cm， 121。C灭菌 30min， 制得固料培养基。
在本发明的实施方案中， 所述固态组分与液体营养 ^ a分 1按盾 量 /体积的配比为（ 5 ~ 10 ) g： ( 2 ~ 5 ) mL，其中固态组分按盾量计算， 液体营养^ a分 1 积计算；
所述固料培养基原料与液体营养液组分 2按盾量 /体积的配比为 (10-15) §： (3~6) mL, 其中固料培养基原料按盾量计算， 液体 营养 ^ a分 2 积计算。
在本发明的实施方案中， 在步骤 2)中， 所述液体种子培养基的组 成为： 葡萄糖 20 - 25g/L,氯化钠 20 - 25g/L,磷酸二氢钾 0.2 - 0.5g/L, 酵母粉 3 - 5g/L,七:^酸镁 8 - 12g/L,海盐 0.1 - 0.8g/L,谷氨酸 0.2 - 0.6g/L, pH值 6~7， 121。C灭菌 30min。
在本发明的实施方案中， 在步骤 2) 中， 所述培养的温度为 25~ 28。C， 培养的时间为 1天。 在本发明的实施方案中， 在步骤 2) 中， 所述摇瓶的转速为 180 ~ 220 rpm; 摇瓶培养的温度为 25 ~ 28。C， 摇瓶培养的时间为 1天。
在本发明的实施方案中， 在步骤 4)中， 所述扩大培养采用的液体 发酵培养基的组成为： 葡萄糖 30~60 g/L, NaCl 15 - 20g/L, MgS04.7H20 4~8 g/L, 酵母粉 3~8 g/L, KH2P04 0.5 - 1.0 g/L, (NH4)2S040.2 - 0.5 g/L, NaHC030.8 - 1.2 g/L, CoCl21.0 ~ 2.0 g/L， MnSO40.5~1.0 g/L， VBi 0.05 ~ O.lmg/L, VB120.001 ~ O.Olmg/L, 初 始 pH6.0~7.0; 所^ C酵最好溶氧维持在 8% ~ 12%。 在本发明的具体实施方案中， 固料培养裂殖壶菌液体发酵生产 DHA的方法包括以下步骤：
1)菌种活化与菌悬液制备： 将裂殖壶菌菌种接种于斜面培养基上 培养活化，培养到期斜面加入无菌水或等渗盐溶液 (例如 0.85% - 0.9% 的生理盐水)洗涤菌体， 获得菌悬液；
在步骤 1) 中， 所述斜面培养基的配方可为： 葡萄糖 20~30g/L， 蛋白胨 10~15g/L， 酵母粉 3~5g/L， 海水晶 15~20g/L， 琼脂粉 15 ~ 25g/L, pH值 6~7， 灭菌 (例如 121。C灭菌 30min); 所述培养活化的温 度可为 25~28°C，培养活化的时间可为 2 ~3天； 所述菌悬液的菌体浓 度可为 1.0~2.5xl06个 /mL;
2 )一级种子制备： 将步骤 1 )获得的菌悬液接种 (例如按盾量分数 为 0.5-2%、 1%的接种量)至固料培养基上培养， 制成一级固料种子； 或
将步骤 1)获得的菌悬液接种 (例如按盾量分 0.5-2%、 1%的接 种量)至液体种子培养基， 摇瓶培养， 制成一级液体种子；
在步骤 2) 中， 所述固料培养基包括固态组分和液体营养 ¾ 分， 所述固态组分为谷物， 含量为 250~400g/L， 所^物可选自米类、 麦 类和玉米等中的至少一种， 所述米类可选自大米、 小米、 糙米等中的 至少一种， 所述麦类可选自大麦、 小麦、 燕麦等中的至少一种； 所述液体营养液组分包括组分 1和组分 2， 所述组分 1的组成为： 葡萄糖 2~5g/L， 七:^酸镁 0.2 - 0.6g/L, 磷酸氢二钾 1.0 ~ 5.0g/L， 海盐 0.1 - 0.8g/L,甘氨酸 0.2 - 0.6g/L,苏氨酸 1.5 - 1.8g/L,蛋氨酸 2 - 3.5g/L, 苹果酸 3~6g/L， 加水定容， 调节 pH值为 6~7； 所述组分 2 的组成为： La3+ 10 - 15mg/L, Ce3+ 1 - 4mg/L, Sm3+ 2 - 6mg/L, Nd3+ 8 - 12mg/L, Mn2+ 0.6~ 1.2mg/L, Co2+ 0.05 ~ O.lmg/L , 生物素 0.2 ~ 0.6mg/L, 浅蓝菌素 0.1 ~ 0.3mg/L， 赤霉素 GA 2 ~ 5mg/L， VB120.5 - l.Omg/L, 叶酸 0.01 ~ 0.05mg/L， 余量为水；
所述固料培养基的制备方法可为： 固态组分与液体营养 ^ a分 1 混合均匀，将^^蒸或煮至谷物中心无白心，且含水率为 50%~65%， 获得固料培养 料； 将固^^养 料与液体营养 ^ a分 2混合均 匀， 装于固态发酵 K瓶中， 将固料种子按照 150g~2500g/瓶分装进规 格为 1L ~ 10L的固态发酵瓶中， 厚度 0.2 - 1.0cm, 121。C灭菌 30min， 制得固料培养基；
所述固态组分与液体营养 ^ a分 1按盾量 /体积的配比可为 （5~
10 ) g： ( 2 ~ 5 ) mL, 其中固态组分按盾量计算， 液体营养^&分 1 积计算；
所述固料培养基原料与液体营养 ^ a分 2按盾量 /体积的配比可为
(10-15) §： (3~6) mL, 其中固料培养基原料按盾量计算， 液体 营养 ^ a分 2 积计算；
所述液体种子培养基的组成可为： 葡萄糖 20~25g/L，氯化钠 20 ~ 25g/L,磚酸二氢钾 0.2 - 0.5g/L,酵母粉 3 - 5g/L,七:^酸镁 8 - 12g/L, 海盐 0.1 ~ 0.8g/L， 酸 0.2~0.6g/L， pH值 6~7， 灭菌 (例如 121。C 灭菌 30min);
所述培养的温度可为 25 - 28°C ,培养至菌体浓度及菌体形态较好， 例如培养的时间可为 20-28h (例如 1 天左右)； 所述摇瓶的转速可为 180 - 220 rpm; 摇瓶培养的温度可为 25 ~ 28。C， 培养至菌体浓度及菌 体形态较好， 例如摇瓶培养的时间可为 20-28h (例如 1天左右)；
3)二级固料种子的制备：一级固料种子以无菌水或等渗盐溶液 (例 如为 0.85%的生理盐水)洗涤菌体 (例如根据 1:1(W/V)比例)， 制成菌悬 液；
在无菌条件下：
积 /盾量分 10% - 20%的接种量在固 ^^养基中接入一级 固料种子液； 或
积 /盾量分 8% ~ 15%的接种量在固料培养基中接入一级 液 子；
摇动 K瓶混合均匀， 25 ~ 28°C，培养至菌体浓度及菌体形态较好， 例如培养 2 ~ 3天；
制得二级固料种子；
4)二级固料种子发酵罐扩大培养： 二级固料种子加入 1:3 ~ 5 (W/V)无菌水振荡洗下菌体， 按体积分数为 10% ~ 20%的接种量接 种至发酵罐扩大培养， 发酵前 48h温度控制在 25~28°C， 利用搅拌转 速保持 30% ~ 50%的溶氧， 48h至发酵结束温度控制在 18~22。C， 发 酵至菌体形态开始自溶、 菌体细胞干重和 DHA产量均无增加、增加较 慢或有下降趋势， 例如发酵 3~5天， 通过氨水 (例如为 28%的氨水)^ 制发酵 pH6.0~6.5， 整个发酵过程自动 葡萄糖溶液 (例如经 118。C 灭菌 25min 的浓度为 580g/L 的葡萄糖溶液)控制发酵葡萄糖浓度在 4~8 g/L;
在步骤 4) 中， 所述扩大培养采用的液体发酵培养基的组成可为： 葡萄糖 30~60g/L， NaCl 15 - 20g/L, MgS04.7H204 ~ 8 g/L, 酵母粉 3~8 g/L, KH2P040.5 - 1.0 g/L, (NH4)2S040.2 - 0.5 g/L, NaHC030.8 - 1.2 g/L, C0CI21.0 - 2.0 g/L, MnS040.5 - 1.0 g/L, VBi 0.05 ~ O.lmg/L, VB120.001 - O.Olmg/L, 初始 pH6.0~7.0; 所^1酵最好溶 氧维持在 8% - 12%;
5)发酵结束后， 收集菌体细胞， 提取 DHA。
在本发明的实施方案中， 经测定， 发酵液中细胞干重达 105 ~ 160g/L, DHA含量达 20~35g/L。
在本发明中， 所述裂殖壶菌可以为本领域公知的任何裂殖壶菌， 例如包括可以从美国典型培养物收集中心、 日本大阪发酵工程研究所 等保藏机构获得的裂殖壶菌菌种。 在本发明的实施方案中， 所述裂殖 壶菌为裂殖壶菌 ( Schizochytrium sp. ) , 其中所述裂殖壶菌 ( Schizochytrium sp. )例如可以为裂殖壶菌 ( Schizochytrium sp. ) KDW-12, S31、 S8或裂殖壶菌 Schizochytrium aggregatim
本发明采用的菌种裂殖壶菌（ Schizochytrium sp. ) KDW-12, 已于 2012年 11月 20日保藏在中国微生物菌种保藏管理委员^ 通微生物 中心，保藏中心登记入册编号为 CGMCC No.6843,地址为北京市朝阳 区大屯路， 中国科学院微生物研究所， 邮编为 100101。 在本发明的实施方案中， 所述等渗盐溶液为生理盐水， 即 0.85-0.9%的氯化钠溶液。
在本发明中， 二级固料种子及前期各阶 ¾t酵培养时间的确定，是 根据前期开展的最佳培养时间优化实验而定，在选定的培养时间内获得 的菌体浓度及菌体形态较好； 发酵罐上控制发酵结束的指标为菌体形态 开始自溶、 菌体细胞干重和 DHA产量均无增加、 增加较慢或有下降趋 势。
在本发明中， 氨水和葡萄糖的控制均为公知的现有技术， 即利用氨 水控制 PH主要是通过发酵罐上的自动控制系统设定控制值， 低于此设 定值， 蠕动泵会自动流加氨水进行调节； 而葡萄糖的控制类似氨水的控 制， 每次取样通过生物传感分析仪测定剩余的糖浓度， 根据设定的控制 点计算需补入的葡萄糖体积， 在自动控制体统中设定相应的开度和周 期， 通过蠕动泵自动调节。
在本发明中， 所述培养时间中的 1天并不局限于准确的 24小时， 例如可约为 20 ~ 28小时、 22 ~ 26小时、 24小时。
在本发明中， 将活化后的菌种或一级、 二级固料种子制成相应的种 子液的方法为： 对于活化后的菌种和一级固料种子， 可以加入无菌水或 等渗盐溶液，例如 0.85% - 0.9%的氯化钠溶液，洗涤菌体，制成菌悬液； 对于二级固料种子， 可以加入无菌水， 制成菌悬液； 所述菌悬液即相应 的种子液； 所述菌悬液的菌体浓度可为 1.0 ~ 2.5xl06个 /mL。
在本发明中，所述制备菌悬液或种子液时质量 /体积百分数中的质量 以及按照体积 /盾量百分数接种中的质量均指固料培养基的质量。 发明的有益效果
本发明摒弃传统的种子培养工艺¾½， 直接由固料发酵获得的种子 液进行液体发酵培养:斜面活化→一级固料种子 /摇瓶液 子→二级固 料种子液—液体发酵。 本发明改进种子制备方式， 简化扩种工艺， 缩短 种子培养和发酵周期， 可减少投资， 节约成本， 更适合 ^^化生产； 同 时固料培养基营养丰富， 富含多种生长因子， 更利于种子的生长， 菌种 细胞的生长活力强、 同步性较好； 培养固料种子阶段产生的废水、 废渣 少， 环境污染小。
本发明克服了传统发酵方法种子扩培级数较多的缺陷， 通过改进 DHA发酵用种子液的制备， 可简化 DHA生产工艺过程； 固料培养基 营养丰富， 种子饱满活力强， 可缩短种子上罐后延迟期， 提高 DHA产 量， 降低生产成本， 保持稳定的生产能力； 培养固料种子阶段产生的 废水、 废渣少， 环境污染小； 发酵得到的 DHA可作为营养强化剂， 为 人类和动物提供所需长链多不饱和脂肪酸。 具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述， 但是本领 域技术人员将会理解， 下列实施例仅用于说明本发明， 而不应视为限 定本发明的范围。 实施例中未注明具体条件者， 按照常规条件或制造 商建议的条件进行。 所用试剂或仪器未注明生产厂商者， 均为可以通 过市购获得的常规产品。 实施例中用到的菌种为：
裂殖壶菌 ( Schizochytrium sp. ) KDW-12, 已于 2012年 11月 20 日保藏于中国微生物菌种保藏管理委员会普通微生物中心 (地址： 北京 市朝阳区大屯路， 中国科学院微生物研究所， 邮编为 100101)， 保藏编 号 CGMCC No.6843。
裂殖壶菌 （ Schizochytrium sp. ) S31 (ATCC No. 20888)
裂殖壶菌 （ Schizochytrium sp. ) S8 (ATCC No. 20889)
裂殖壶菌 Schizochytrium aggregatum (ATCC No. 28209) 实施例 1:
将裂殖壶菌（ Schizochytrium sp. ) KDW-12菌种接种于斜面培养基 上培养活化， 温度为 25°C , 培养 2天。 斜面培养基配方为 （g/L ) ： 葡 萄糖 20，蛋白胨 15，酵母粉 3， 海水晶 (海盐 )15， 琼脂粉 20， pH值 6.5， 121。C灭菌 30min。 培养到期斜面加入 0.85%的生理盐水洗涤菌体， 获 得菌悬液， 控制菌体浓度为 2.5X106个 /mL。
获得的菌悬液按体积 /盾量分数为 1%(V/W,菌悬液体积 /固料培养 量)的接种量直 种至灭菌的固料培养基上（固料培养基的装量 为 1000ml麟的 K氏餘 150g/瓶分装）， 25。C培养 1天，培^间每 隔 8-12h摇动 K氏瓶一次混匀， 以利于种子充分利用营养成分生长。 制得一级固料种子。
在无菌条件下， 按体积 /盾量分数为 10%(V/W)的接种量在固料培 养基 (二级固料培养基按 150g/瓶分装于规格为 1000ml的 K氏瓶） 中 接入一级固料种子（用于接种的一级固料种子以 0.85%的生理盐?! M艮 据 1:1(W/V)比例洗涤菌体， 制成菌悬液， 控制菌体浓度为 l.OxlO6个 /mL )，摇动 K氏瓶，混合均匀， 25。C，培养 2.5天，培养期间每隔 8-12h 摇动 K氏瓶一次， 混匀， 以利于种子充分利用营养成分生长。 制得二 级固料种子。
二级固料种子加入 1:4 ( W/V )无菌^荡洗下菌体， 控制菌体浓 度为 1.5X106个 /mL， 积分数为 15%的接种量接种至 50L发酵罐扩 大培养， 接后体积 32L。 发酵前 48h温度控制在 25°C， 利用搅拌转速 和通气量保持 40%的溶氧， 48h至发酵结束温度控制在 22。C， 溶氧维 持在 10%左右，发酵过程中每隔 8h ^#—次测 ¾1酵液中的残糖、菌 体浓度、 细胞干重和 DHA含量， 发酵 4天， 此时菌体形态开始自溶、 菌体细胞干重和 DHA产量均无增加、增加较慢或有下降趋势。通过盾 量百分比为 28%的氨水控制发酵 pH6.5， 整个发酵过程自动流加经 118。C灭菌 25miii的浓度为 580g/L的葡萄糖溶液控制发酵葡萄糖浓度 在 8 g/Lo
固料培养基的制备方法为： 由固态组分和液体营养液组分组成， 其中固态组分为小米， 为 375g/L (固料组分盾量 /液体营养^ &分体 积)， 小米与液体营养 ^ BL分 1按 8: 3 ( W/V )比例^^均匀， 将谷物 蒸煮至谷物中心无白心， 且含水率为 55%, 获得固料培养基原料。 固 料培养基原料与液体营养 ^ a分 2按 10: 3 ( W/V )比例混合均匀， 装 于固态发酵 K氏瓶中， 装量为 1000ml 的 K氏瓶按 150g/瓶分装， 厚度 0.3cm， 121。C灭菌 30min， 制得固料培养基。
液体营养 ^ BL分 l(g/L): 葡萄糖 3， 七^酸镁 0.2, 磷酸氢二 钾 3.0， 海盐 0.6， 甘氨酸 0.6， 苏氨酸 1.5， 蛋氨酸 2, 苹果酸 4， 加 水定容至所需体积， 调节 pH值至 6.5。 组分 2 ( mg/L ) : La3+15, Ce3+ 2， Sm3+ 3, Nd3+ 8, Mn2+ 1.0， Co2+0.08， 生物素 0.2， 浅蓝菌素 0.1， 赤霉素 GA 5， VB121.0, 叶酸 0.03， 余量为水。
液体发酵培养 JBL成： 葡萄糖 30 g/L， NaCl 20g/L, MgS04.7H20 5 g/L,酵母粉 3 g/L, KH2PO4 1.0 g/L, (NH4)2S04 0.4 g/L, NaHC03 1.2 g/L, CoCl22.0 g/L， MnSO40.7 g/L， VBi 0.06mg/L, VB120.005mg/L, 初始 pH6.5。 初始 pH是指培养基配制好后， 灭菌前通过 pH计调节得 到的 pH。
发酵结束， 离心收集菌体细胞， 测定发酵液中细胞干重达 125g/L， 气相检测 DHA含量达 25g/L。发酵液的处理和 DHA的提取测定方法 参照中国专利 CN101638361A公开的方法进行。提取的 DHA可作为 营养强化剂， 为人类和动物提供所需长链多不饱和脂肪酸。 实施例 2:
将裂殖壶菌（ Schizochytrium sp. ) KDW-12菌种接种于斜面培养基 上培养活化， 温度为 28。C， 培养 2天。 斜面培养基配方为 （g/L ): 葡 萄糖 25，蛋白胨 12，酵母粉 3，海水晶 16，琼脂粉 20， pH值 6.0， 121°C 灭菌 30min。培养到期斜面加入 0.85%的生理盐水洗涤菌体，获得菌悬 液， 控制菌体浓¾^ l.OxlO6个 /mL。
获得的菌悬液直 种至摇瓶液体培养基 ( 500ml三角瓶装液量为 150ml )上， 接种量 1%， 摇瓶转速 220 rpm, 28。C培养 1天， 制得一 级液体种子。摇瓶液体种子培养基组成 ( g/L ): 葡萄糖 25， 氯化钠 20, 磷酸二氢钾 0.2， 酵母粉 5， 七:^酸镁 10， 海盐 0.4， 谷氨酸 0.4， pH 值 6.0， 121。C灭菌 30min。
在无菌糾下， 积 /盾量分数为 10%的接种量在固 养^ (装 量为 1000ml规格的 K氏瓶按 250g/瓶分装） 中接入一级液体种子， 摇 动 K氏瓶混合均匀， 26。C， 培养 3天， 培:^间每隔 8-12h摇动 K氏 瓶一次， 混匀， 以利于种子充分利用营养成分生长。 制得二级固料种 子。
二级固料种子加入 1:3 ( W/V )无菌^荡洗下菌体， 控制菌体浓 度为 2.5X106个 /mL， 按体积 /盾量分数为 20%的接种量接种至 100L发 酵罐扩大培养， 接后体积 60L。 发酵前 48h温度控制在 28。C， 利用搅 拌转速和通气量保持 30%的溶氧， 48h至发酵结束温度控制在 18。C， 溶氧维持在 10%左右。发酵过程中每隔 8h取样一次测定发酵液中的残 糖、 菌体浓度、 细胞干重和 DHA含量， 发酵 5天， 此时菌体形态开始 自溶、 菌体细胞干重和 DHA产量均无增加、 增加较慢或有下降趋势。 通过 28%的氨水控制发酵 pH6.0,整个发酵过程自动流加经 118。C灭菌 25min的浓度为 580g/L的葡萄糖溶液控制发酵葡萄糖浓度在 7 g/L。
固料培养基的制备方法为： 由固态组分和液体营养液组分组成， 其中固态组分为小米， 含量为 300g/L， 小米与液体营养^ BL分 1按 8: 3 ( W/V )比例混合均匀， 将谷物蒸煮至谷物中心无白心， 且含水率为
60%, 获得固 ^^养基原料。 固^^养^料与液体营养 ^ a分 2按
10: 4 ( W/V )比例混合均匀， 装于固态发酵 K氏瓶中， 装量为 1000ml 的 K 250g/瓶分装， 厚度 0.5cm, 121。C灭菌 30min， 制得 固料培养基。
液体营养 ^ia分 l(g/L): 葡萄糖 5， 七^酸镁 0.4, 磷酸氢二钾 2.0， 海盐 0.5， 甘氨酸 0.3, 苏氨酸 1.5， 蛋氨酸 3.5， 苹果酸 6， 加水 定容至所需体积， 调节 pH值 6.0。 组分 2 ( mg/L ) : La3+ 10, Ce3+ 4, Sm3+ 5, Nd3+ 9, Mn2+ 0.8, Co2+0.06, 生物素 0.2， 浅蓝菌素 0.1， 赤霉 素 GA 2， VB120.8, 叶酸 0.03， 余量为水。
液体发酵培养基组成： 葡萄糖 40 g/L， NaCl 15g/L, MgS04.7H20 4g/L,酵母粉 5 g/L, KH2PO4 0.5 g/L, (NH4)2S04 0.2 g/L, NaHC03 0.8 g/L, CoCl21.5 g/L， MnSO41.0 g/L， VBi O.lmg/L, VB120.01mg/L, 初始 ρΗ6·0。
发酵结束， 离心收集菌体细胞， 测定发酵液中细胞干重达 159g/L， 气相检测 DHA含量达 34.5g/L。 发酵液的处理和 DHA的提取测定方 法参照中国专利 CN101638361A公开的方法进行。提取的 DHA可作 为营养强化剂， 为人类和动物提供所需长链多不饱和脂肪酸。 实施例 3:
将裂殖壶菌（ Schizochytri腿 sp. ) KDW-12菌种接种于斜面培养基 上培养活化， 温度为 28。C， 培养 3天。 斜面培养基配方为 （g/L ): 葡 萄糖 30，蛋白胨 12，酵母粉 3，海水晶 15，琼脂粉 20， pH值 6.5， 121°C 灭菌 30min。培养到期斜面加入 0.85%的生理盐水洗涤菌体，获得菌悬 液， 控制菌体浓¾^ 2.0xl06个 /mL。
获得的菌悬液按 1%的接种量直接接种至摇瓶液体培养基（ 500ml 三角 液量为 150ml )上， 摇^ il 220 rpm， 28。C培养 1天， 制得 一级液体种子。 摇瓶液体种子培养基组成 ( g/L ): 葡萄糖 20， 氯化钠 22，磷酸二氢钾 0.2， 酵母粉 3，七:^酸镁 10， 海盐 0.3，谷氨酸 0.6， pH值 6.5， 121。C灭菌 30min。
在无菌糾下，按体积 /盾量分数为 8%的接种量在固 养基（装 量为 1000ml 的 K氏 300g/瓶分装） 中接入一级液 子， 摇 动 K氏瓶混合均匀， 26。C， 培养 3天， 培:^间每隔 8-12h摇动 K氏 瓶一次， 混匀， 以利于种子充分利用营养成分生长。 制得二级固料种 子。
二级固料种子加入 1:3 ( W/V )无菌^荡洗下菌体， 控制菌体浓 度为 2.0xl06个 /mL， 积分数为 10%的接种量接种至 100L发酵罐 扩大培养， 接后体积 60L。 发酵前 48h温度控制在 25°C， 利用搅拌转 速和通气量保持 30%的溶氧， 48h至发酵结束温度控制在 20。C， 溶氧 维持在 10%左右。 发酵过程中每隔 8h ^#—次测 ¾1酵液中的残糖、 菌体浓度、细胞干重和 DHA含量，发酵 5天，此时菌体形态开始自溶、 菌体细胞干重和 DHA产量均无增加、 增加较慢或有下降趋势。 通过 28%的氨水控制发酵 pH6.0， 整个发酵过程自动流加经 118°C灭菌 25min的浓度为 580g/L的葡萄糖溶液控制发酵葡萄糖浓度在 8 g/L。
固料培养基的制备方法为： 由固态组分和液体营养^ &分组成， 其中固态组分为小麦， 为 350g/L，小麦与液体营养 ^ a分 1按 10: 4 ( W/V )比例混合均匀， 将谷物蒸煮至谷物中心无白心， 且含水率为
55%, 获得固^^养基原料。 固^^养^料与液体营养 ^ a分 2按
15: 4 ( W/V ) 比例齡均匀， 装于固态发酵 K瓶中， 装量为 1000ml 的 K 300g/瓶分装， 厚度 0.7cm, 121。C灭菌 30min， 制得 固料培养基。
液体营养 ^iBL分 l(g/L): 葡萄糖 3.5， 七^酸镁 0.3, 磷酸氢二 钾 3.0， 海盐 0.4， 甘氨酸 0.5, 苏氨酸 1.6, 蛋氨酸 3.5， 苹果酸 5， 加 水定容至所需体积，调节 pH值 6.0。组分 2 ( mg/L )： La3+ 12， Ce3+ 4， Sm3+ 5, Nd3+ 8, Mn2+ 0.9, Co2+0.08， 生物素 0.4， 浅蓝菌素 0.1， 赤霉 素 GA 3， VB12 0.8, 叶酸 0.02， 余量为水。
液体发酵培养基组成： 葡萄糖 40 g/L， NaCl 16 g/L, MgS04.7H20 6 g/L,酵母粉 4 g/L, KH2PO4 1.0 g/L, (NH4)2S04 0.5 g/L, NaHC03 1.2 g/L, CoCl22.0 g/L， MnSO41.0 g/L， VBi 0.05mg/L, VB120.005mg/L, 初始 ρΗ6·0。
发酵结束， 离心收集菌体细胞， 测定发酵液中细胞干重达 132g/L， 气相检测 DHA含量达 30.5g/L。 发酵液的处理和 DHA的提取测定方 法参照中国专利 CN101638361A公开的方法进行。提取的 DHA可作 为营养强化剂， 为人类和动物提供所需长链多不饱和脂肪酸。 实施例 4:
将 壶菌 (Schizochytrium sp. S31 ) ( ATCC No. 20888)菌种接种 于斜面培养基上培养活化， 温度为 28。C， 培养 2天。 斜面培养基配方 为 （g/L ): 葡萄糖 30， 蛋白胨 12， 酵母粉 3， 海水晶 15， 琼脂粉 20， pH值 6.5， 121。C灭菌 30min。 培养到期斜面加入 0.85%的生理盐水洗 涤菌体， 获得菌悬液， 控制菌体浓度为 1.5X106个 /mL。
获得的菌悬液按 1%的接种量直接接种至摇瓶液体培养基（ 500ml 三角 液量为 150ml )上， 摇^ il 200 rpm， 28。C培养 1天， 制得 一级液体种子。 摇瓶液体种子培养基组成 ( g/L ): 葡萄糖 20， 氯化钠 22，磷酸二氢钾 0.2， 酵母粉 5，七:^酸镁 12， 海盐 0.3，谷氨酸 0.6， pH值 6.5， 121。C灭菌 30min。
在无菌糾下， 积 /盾量分数为 15%的接种量在固 养^ (装 量为 1000ml 的 K氏麵 170g/瓶分装） 中接入一级液 子， 摇 动 K氏瓶混合均匀， 26。C， 培养 2天， 培^间每隔 8-12h摇动 K氏 瓶一次， 混匀， 以利于种子充分利用营养成分生长。 制得二级固料种 子。
二级固料种子加入 1:3 ( W/V )无菌^荡洗下菌体， 控制菌体浓 度为 2.0xl06个 /mL， 按 10%的接种量接种至 100L发酵罐扩大培养， 接后体积 60L。 发酵前 48h温度控制在 25°C， 利用搅拌转速和通气量 保持 30%的溶氧， 48h至发酵结束温度控制在 20。C， 溶氧维持在 10% 左右。 发酵过程中每隔 8h取样一次测定发酵液中的残糖、 菌体浓度、 细胞干重和 DHA含量， 发酵 4.5天， 此时菌体形态开始自溶、 菌体细 胞干重和 DHA产量均无增加、 增加较慢或有下降趋势。 通过 28%的 氨?! 制发酵 ρΗ6.0, 整个发酵过程自动 »经 118。C灭菌 25min的 浓度为 580g/L的葡萄糖溶液控制发酵葡萄糖浓度在 6g/Le
固料培养基的制备方法为： 由固态组分和液体营养^ &分组成， 其中固态组分为小米， 为 350g/L，小米与液体营养^ &分 1按 10: 4 ( W/V )比例混合均匀， 将谷物蒸煮至谷物中心无白心， 且含水率为
55%, 获得固^^养基原料。 固^^养^料与液体营养 ^ a分 2按
10: 4 ( W/V )比例混合均匀， 装于固态发酵 K氏瓶中， 装量为 1000ml 规格的 K氏瓶按 170g/瓶分装， 厚度 0.3cm, 121。C灭菌 30min， 制得 固料培养基。
液体营养 ^iBL分 l(g/L): 葡萄糖 3， 七^酸镁 0.5, 磷酸氢二钾 5.0， 海盐 0.8， 甘氨酸 0.2, 苏氨酸 1.6， 蛋氨酸 3.5， 苹果酸 3， 加水 定容至所需体积， 调节 pH值 6.0。 组分 2 ( mg/L ) : La3+ 15, Ce3+ 4, Sm3+ 5, Nd3+ 10, Mn2+ 0.9, Co2+ 0.1， 生物素 0.4， 浅蓝菌素 0.2， 赤 霉素 GA 2， VB12 1.0, 叶酸 0.02， 为水。
液体发酵培养基组成： 葡萄糖 40 g/L， NaCl 20g/L, MgS04.7H20 6 g/L,酵母粉 4 g/L, KH2PO4 1.0 g/L, (NH4)2S04 0.5 g/L, NaHC03 0.8 g/L, CoCl22.0 g/L， MnSO40.5 g/L， VBi 0.05mg/L, VB120.005mg/L, 初始 ρΗ6·0。
发酵结束， 离心收集菌体细胞， 测定发酵液中细胞干重达 115g/L， 气相检测 DHA含量达 22.7g/L。 发酵液的处理和 DHA的提取测定方 法参照中国专利 CN101638361A公开的方法进行。提取的 DHA可作 为营养强化剂， 为人类和动物提供所需长链多不饱和脂肪酸。 实施例 5:
将裂殖壶菌 (Schizochyirium sp. S8 )( ATCC 20889)菌种接种于斜面 培养基上培养活化，温度为 28°C，培养 2天。斜面培养基配方为（ g/L ): 葡萄糖 20， 蛋白胨 12， 酵母粉 3， 海水晶 20， 琼脂粉 20， pH值 6.5， 121。C灭菌 30min。 培养到期斜面加入 0.85%的生理盐水洗涤菌体， 获 得菌悬液， 控制菌体浓度为 2.5X106个 /mL。
获得的菌悬液按 1%的接种量直接接种至摇瓶液体培养基 ( 1L三 角 液量为 350ml )上， 摇瓶 il 220 rpm， 28。C培养 1天， 制得一 级液体种子。摇瓶液体种子培养基组成 ( g/L ): 葡萄糖 25， 氯化钠 22, 磷酸二氢钾 0.5， 酵母粉 5， 七:^酸镁 12， 海盐 0.3， 谷氨酸 0.6， pH 值 6.5， 121。C灭菌 30min。
在无菌糾下， 积 /盾量分数为 12%的接种量在固 养^ (装 量为 10L规格的 K氏瓶按 2250g/瓶分装） 中接入一级液体种子， 摇动 K氏瓶混合均匀， 28。C， 培养 3天， 培:^间每隔 8-12h摇动 K氏瓶 一次， 混匀, 以利于种子充分利用营养成分生长。 制得二级固料种子。
二级固料种子加入 1:4 ( W/V )无菌^荡洗下菌体， 控制菌体浓 度为 2.0xl06个 /mL， 按 18%的接种量接种至 1000L发酵罐扩大培养， 接后体积 600L。发酵前 48h温度控制在 28°C， 利用搅拌转速和通气量 保持 30%的溶氧， 48h至发酵结束温度控制在 20。C， 溶氧维持在 10% 左右。 发酵过程中每隔 8h取样一次测定发酵液中的残糖、 菌体浓度、 细胞干重和 DHA含量， 发酵 5天， 此时菌体形态开始自溶、 菌体细胞 干重和 DHA产量均无增加、 增加较慢或有下降趋势。 通过 28%的氨 水控制发酵 pH6.5, 整个发酵过程自动流加经 118。C灭菌 25min的浓 度为 580g/L的葡萄糖溶液控制发酵葡萄糖浓度在 8 g/L。
固料培养基的制备方法为： 由固态组分和液体营养^ &分组成， 其中固态组分为大米， 含量为 300g/L， 大米与液体营养^ BL分 1按 8:
5 ( W/V )比例混合均匀， 将谷物蒸煮至谷物中心无白心， 且含水率为
50%, 获得固^^养基原料。 固^^养^料与液体营养 ^ a分 2按
15: 3 ( W/V ) 比例齡均匀， 按 2250g/瓶分装于 10L ^¾ ^的 K氏瓶 中， 厚度 0.5cm。
液体营养 ^iBL分 l(g/L): 葡萄糖 3， 七^酸镁 0.5, 磷酸氢二钾 5.0， 海盐 0.8， 甘氨酸 0.2, 苏氨酸 1.6， 蛋氨酸 3.5， 苹果酸 3， 加水 定容至所需体积， 调节 pH值 6.0。 组分 2 ( mg/L ): La3+ 15, Ce3+ 4, Sm3+ 5, Nd3+ 10, Mn2+ 1.2, Co2+ 0.1， 生物素 0.4， 浅蓝菌素 0.2， 赤 霉素 GA2， VB12 1.0, 叶酸 0.02， 余量为水。
液体发酵培养基组成： 葡萄糖 40 g/L， NaCl 20g/L, MgS04.7H20
6 g/L,酵母粉 4 g/L, KH2PO4 1.0 g/L, (NH4)2S04 0.5 g/L, NaHC03 0.8 g/L, CoCl22.0 g/L， MnSO40.5 g/L， VBi 0.05mg/L, VB120.005mg/L, 初始 ρΗ6·5。
发酵结束， 离心收集菌体细胞， 测定发酵液中细胞干重达 138g/L， 气相检测 DHA含量达 31.6g/L。 发酵液的处理和 DHA的提取测定方 法参照中国专利 CN101638361A公开的方法进行。提取的 DHA可作 为营养强化剂， 为人类和动物提供所需长链多不饱和脂肪酸。 实施例 6:
将裂殖壶菌 (Schizochytrium aggregatum) ( ATCC 28209)菌种接种 于斜面培养基上培养活化， 温度为 25。C， 培养 2天。 斜面培养基配方 为 （g/L ): 葡萄糖 20， 蛋白胨 15， 酵母粉 3， 海水晶 15， 琼脂粉 20， pH值 6.5， 121。C灭菌 30min。 培养到期斜面加入 0.85%的生理盐水洗 涤菌体， 获得菌悬液， 控制菌体浓度为 2.5X106个 /mL。
获得的菌悬液按体积 /盾量分数为 1%的接种量直接接种至灭菌的 固料培养基 (装量为 lOOOmL规 的 K氏瓶按 225g/瓶分装 )上 28。C 培养 1天， 培养期间每隔 8-12h摇动 K氏瓶一次， 混匀， 以利于种子 充分利用营养成分生长。 制得一级固料种子。
在无菌糾下， 积 /盾量分数为 20%的接种量在固 养^ (装 量为 10L 的 K氏^^ 2250g/瓶分装 ) 中接入一级固料种子（用于 接种的一级固料种子以 0.85%的生理盐水根据 1:1(W/V)比例洗涤菌 体， 制成菌悬液， 控制菌体浓度为 1.5X106个 /mL )， 摇动 K氏瓶混合 均匀， 25。C， 培养 3天， 培:^间每隔 8-12h摇动 K氏瓶一次， 混匀， 以利于种子充分利用营养成分生长。 制得二级固料种子。
二级固料种子加入 1:4 ( W/V )无菌^荡洗下菌体， 控制菌体浓 度为 2.0xl06个 /mL， 按 12%的接种量接种至 1000L发酵罐扩大培养， 接后体积 600L。发酵前 48h温度控制在 28°C， 利用搅拌转速和通气量 保持 40%的溶氧， 48h至发酵结束温度控制在 18。C， 溶氧维持在 10% 左右。 发酵过程中每隔 8h取样一次测定发酵液中的残糖、 菌体浓度、 细胞干重、 DHA含量， 发酵 5天， 此时菌体形态开始自溶、 菌体细胞 干重和 DHA产量均无增加、 增加较慢或有下降趋势。 通过 28%的氨 水控制发酵 pH6.5, 整个发酵过程自动流加经 118。C灭菌 25min的浓 度为 580g/L的葡萄糖溶液控制发酵葡萄糖浓度在 8 g/L。
固料培养基的制备方法为： 由固态组分和液体营养液组分组成， 其中固态组分为大麦， 含量为 375g/L， 大麦与液体营养^ BL分 1按 8: 3 ( W/V )比例混合均匀， 将谷物蒸煮至谷物中心无白心， 且含水率为
55%, 获得固^^养基原料。 固^^养^料与液体营养 ^ a分 2按
10: 3 ( W/V ) 比例齡均匀， 按 2250g/瓶分装于 10L ^¾ ^的 K氏瓶 中， 或者按照 225g/瓶分装于 1L麟的 K氏瓶中， 厚度 0.5cm。
液体营养 ^iBL分 l(g/L): 葡萄糖 5， 七^酸镁 0.2, 磷酸氢二钾 3.0, 海盐 0.6， 甘氨酸 0.6, 苏氨酸 1.5， 蛋氨酸 2， 苹果酸 4， 加水定 容至所需体积， 调节 pH值 6.5。 组分 2 ( mg/L )： La3+ 15， Ce3+ 2， Sm3+ 3, Nd3+ 8, Mn2+ 1.2, Co2+ 0.08， 生物素 0.2， 浅蓝菌素 0.1， 赤 霉素 GA 5， VB121.0, 叶酸 0.03， 余量为水。
液体发酵培养 成： 葡萄糖 30 g/L， NaCl 20g/L, MgS04.7H20 5 g/L,酵母粉 3 g/L, KH2PO4 1.0 g/L, (NH4)2S04 0.4 g/L, NaHC03 1.2 g/L, CoCl22.0 g/L， MnSO40.7 g/L， VBi 0.06mg/L, VB120.005mg/L, 初始 ρΗ6·5。
发酵结束， 离心收集菌体细胞， 测定发酵液中细胞干重达 142g/L， 气相检测 DHA含量达 32.3g/L。发酵液的处理和 DHA的提取测定方法 参照中国专利 CN101638361A公开的方法进行。 提取的 DHA可作为 营养强化剂， 为人类和动物提供所需长链多不饱和脂肪酸。
尽管本发明的具体实施方式已经得到详细的描述， 本领域技术人 员将会理解。 根据已经公开的所有教导， 可以对那些细节进行各种修 改和替换， 这些改变均在本发明的保护范围之内。 本发明的全部范围 由所附权利要求及其任何等同物给出。

Claims

权 利 要 求
1. 一种固料培养裂殖壶菌液体发酵生产 DHA的方法， 其包括以 下步骤：
(1)菌种活化： 将裂殖壶菌菌种接种于培养基 (例如斜面培养基)培 养活化， 制备获得菌悬液；
(2)—级种子制备：将步骤 (1)获得的菌悬液接种至固料培养基上培 养， 制成一级固料种子； 或者
将步骤 (1)获得的菌悬液接种到液体种子培养基培养 (例如摇瓶培 养)， 制成一级液体种子；
(3)二级固料种子制备： 将一级固料种子制成一级固料种子液， 在 固料培养基中接入一级固料种子液培养； 或者
在固料培养基中接入一级液体种子液培养；
制得二级固料种子；
(4)二级固料种子发酵罐扩大培养： 将二级固料种子制成二级固料 种子液， 接种至发酵罐液体培养基中扩大培养；
(5)发酵结束后， 收集菌体细胞， 提取 DHA。
2.权利要求 1的方法，其中所述裂殖壶菌选自 Schizochytrium sp.; 优选地， 所述 Schizochytrium sp.选白裂殖壶菌 Schizochytrium sp. KDW-12、 裂殖壶菌 Schizochytrium sp. S31、 裂殖壶菌 Schizochytrium aggregatum 和裂殖壶菌 Schizochytrium sp. S8， 其中所述裂殖壶菌 Schizochytrium sp.KDW-12已于 2012年 11月 20日保藏于中国微生物 菌种保藏管理委员^ 通微生物中心 (地址：北京市朝阳区大屯路， 中国 科学院微生物研究所， 邮编为 100101)， 保藏编号 CGMCC No.6843。
3. 权利要求 1 的方法， 其中步骤 (1)的特征在于以下几项中的一 项或数项：
a.所述培养的温度为 25 - 28 °C，培养至菌体浓度及菌体形态较好， 例如培养的时间为 2 ~ 3天； b.所述菌悬液的菌体浓度为 1.0~2.5xl06个 /mL
4. 权利要求 1 的方法， 其中步骤 (2)的特征在于以下几项中的一 项或数项：
a.所述接种量为 0.5 ~ 2%；
b.所述培养的温度为 25~28C， 培养至菌体浓度和菌体形态较好， 例如培养的时间为 20~28h;
c.所述液体培养 (例如摇瓶培养)的转速为 180 - 220 rpm。
5. 权利要求 1 的方法， 其中步骤 (3)的特征在于以下几项中的一 项或数项：
a.所述一级固料种子液的菌体浓度可为 1.0~2.5xl06个 /mL;
b.按照体积 /盾量分数为 10% - 20%的接种量在固料培养基中接 入一级固料种子液；
c.按照体积 /盾量分数为 8% - 15%的接种量在固料培养基中接入 一级液体种子；
d.所述培养温度为 25~28°C，培养至菌体浓度和菌体形态较好，例 如培养的时间为 2~3天。
6. 权利要求 1 的方法， 其中步骤 (4)的特征在于以下几项中的一 项或数项：
a.将二级固料种子液按体积分数为 10% ~ 20%的接种量接种至发 酵罐扩大培养；
b.发酵过程的前 48h 温度控制在 25~28。C， 利用搅拌转速保持 30% ~ 50%的溶氧；
c.发酵过程的第 48h至发酵结束温度控制在 18~22。C;
d.在菌体形态开始自溶、 菌体细胞干重和 DHA产量均无增加、 增 加较慢或有下降趋势时， 结束发酵， 例如发酵 3~5天；
e.发酵过程中通过氛?！ 制发酵液 pH为 6.0 ~ 6.5; f.发酵过程中通过自动流加葡萄糖溶液控制葡萄糖浓度在 4~8
7. 权利要求 1的方法， 其中步骤 (1)中的斜面培养基含有： 葡萄糖 20 - 30g/L, 蛋白胨 10~15g/L， 酵母粉 3~5g/L， 海水晶 15~20g/L， 琼脂粉 15 - 25g/L, pH值 6.0 - 7.0。
8. 权利要求 1的方法，其中步骤 (2)或步骤 (3)中的固料培养基包 括固态组分和液体营养液组分， 所述固态组分为谷物， 含量为 250 ~ 400g/L, 所述谷物选自米类、 麦类和玉米中的至少一种， 所述米类选 自大米、 小米、 糙米等中的至少一种， 所述麦类选自大麦、 小麦、 燕 麦中的至少一种；
所述液体营养液组分包括组分 1和组分 2， 所述组分 1的组成为： 葡萄糖 2~5g/L， 七:^酸镁 0.2~0.6g/L, 磷酸氢二钾 1.0 - 5.0g/L, 海盐 0.1 - 0.8g/L,甘氨酸 0.2 - 0.6g/L,苏氨酸 1.5 - 1.8g/L,蛋氨酸 2 - 3.5g/L, 苹果酸 3~6g/L， 加水定容， 调节 pH值为 6~7； 所述组分 2 的组成为： La3+ 10 ~ 15mg/L， Ce3+l~4mg/L, Sm3+ 2 - 6mg/L, Nd3+8~ 12mg/L, Mn2+ 0.6 - 1.2mg/L, Co2+ 0.05 - O.lmg/L,生物素 0.2 - 0.6mg/L, 浅蓝菌素 0.1 - 0.3mg/L, 赤霉素 GA 2 ~ 5mg/L, VB120.5 - l.Omg/L, 叶 酸 0.01~0.05mg/L， 余量为水；
所述固料培养基的制备方法为： 固态组分与液体营养 ^ a分 1混合 均匀， 将谷物蒸或煮至谷物中心无白心， 且含水率为 50%~65%， 获得 固料培养基原料； 将固料培养基原料与液体营养液组分 2混合均匀， 装 于固态发酵瓶中， 厚度 0.2~1.0cm， 灭菌， 制得固料培养基；
优选地， 所述固态组分与液体营养 ^ia分 1按盾量 /体积的配比为
(5~10) §： (2-5) mL, 所述固料培养 料与液体营养 分 2 按盾量 /体积的配比为 （10~15) g: (3~6) mL, 其中固态组分按盾 量计算， 液体营养液组分按体积计算。
9. 权利要求 1的方法， 其中在步骤 2) 中， 所述液 子培养基 含有： 葡萄糖 20~25g/L， 氯化钠 20~25g/L， 磷酸二氢钾 0.2 ~ 0.5g/L， 酵母粉 3 - 5g/L,七:^酸镁 8 - 12g/L,海盐 0.1 - 0.8g/L,谷氨酸 0.2 - 0.6g/L, pH值 6·0~7·0。
10.权利要求 1的方法，其中步骤 (4)中的发酵罐液体培养基含有： 葡萄糖 30 - 60 g/L, NaCl 15 - 20g/L, MgS04.7H204~8 g/L,酵母粉 3 - 8 g/L, KH2P040.5 - 1.0 g/L, (NH4)2S040.2 - 0.5 g/L, NaHC030.8 - 1.2 g/L, C0CI21.0 - 2.0 g/L， MnS040.5 - 1.0 g/L， VBi 0.05 - O.lmg/L, VB120.001 - O.Olmg/L, 初始 pH6.0 - 7.0。
11. 用于培养裂殖壶菌发酵生产 DHA的培养基， 所述培养基选自 以下几种培养基中的一种或几种：
1) 斜面培养基， 含有： 葡萄糖 20~30g/L， 蛋白胨 10~l5g/L， 酵 母粉 3~5g/L， 海水晶 15~20g/L， 琼脂粉 15~25g/L， pH值 6.0~7.0；
2)固料培养基， 包括固态组分和液体营养液组分， 所述固态组分 为谷物， 含量为 250 ~ 400g/L， 所述谷物选自米类、 麦类和玉米中的至 少一种， 所述米类选自大米、 小米、 糙米等中的至少一种， 所述麦类 选自大麦、 小麦、 燕麦中的至少一种；
所述液体营养液组分包括组分 1和组分 2， 所述组分 1的组成为： 葡萄糖 2~5g/L， 七:^酸镁 0.2~0.6g/L, 磷酸氢二钾 1.0 - 5.0g/L, 海盐 0.1 - 0.8g/L,甘氨酸 0.2 - 0.6g/L,苏氨酸 1.5 - 1.8g/L,蛋氨酸 2 - 3.5g/L, 苹果酸 3~6g/L， 加水定容， 调节 pH值为 6~7； 所述组分 2 的组成为： La3+ 10 ~ 15mg/L， Ce3+l~4mg/L, Sm3+ 2 - 6mg/L, Nd3+8~ 12mg/L, Mn2+ 0.6 - 1.2mg/L, Co2+ 0.05 - O.lmg/L,生物素 0.2 - 0.6mg/L, 浅蓝菌素 0.1 - 0.3mg/L, 赤霉素 GA 2 ~ 5mg/L, VB120.5 - l.Omg/L, 叶 酸 0.01~0.05mg/L， 余量为水；
所述固料培养基的制备方法为： 固态组分与液体营养 ^ a分 1混合 均匀， 将谷物蒸或煮至谷物中心无白心， 且含水率为 50%~65%， 获得 固料培养基原料； 将固料培养基原料与液体营养液组分 2混合均匀， 装 于固态发酵瓶中， 厚度 0.2~1.0cm， 灭菌， 制得固料培养基；
优选地， 所述固态组分与液体营养 ^ a分 1 按盾量 /体积的配比为
(5~10) §： ( 2 ~ 5 ) mL, 所述固料培养基原料与液体营养液组分 2 按质量 /体积的配比为（10 ~ 15) g： (3~6) mL, 其中固态组分按庸量 计算， 液体营养液组分按体积计算
3) 液体种子培养基，含有： 葡萄糖 20~25g/L， 氯化钠 20~25g/L， 磷酸二氢钾 0.2 ~ 0.5g/L， 酵母粉 3~5g/L， 七:^酸镁 8 ~ 12g/L， 海盐 0.1 - 0.8g/L, 谷氨酸 0.2 - 0.6g/L, pH值 6.0~7·0;
4) 发酵罐液体培养基，含有：葡萄糖 30 ~ 60 g/L, NaCl 15 ~ 20g/L, MgS04.7H20 4~8 g/L, 酵母粉 3~8 g/L, KH2P04 0.5 - 1.0 g/L, (NH4)2S040.2 - 0.5 g/L, NaHC030.8 - 1.2 g/L, CoCl21.0 - 2.0 g/L， MnS040.5 - 1.0 g/L， VBi 0.05 - O.lmg/L, VB120.001 - O.Olmg/L, 初 始 pH6.0 - 7.0。
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