WO2019041338A1 - Procédé de séparation d'huile microbienne - Google Patents

Procédé de séparation d'huile microbienne Download PDF

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WO2019041338A1
WO2019041338A1 PCT/CN2017/100344 CN2017100344W WO2019041338A1 WO 2019041338 A1 WO2019041338 A1 WO 2019041338A1 CN 2017100344 W CN2017100344 W CN 2017100344W WO 2019041338 A1 WO2019041338 A1 WO 2019041338A1
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powder
fermenter
fermentation
freeze
medium
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PCT/CN2017/100344
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English (en)
Chinese (zh)
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李国斌
瞿运来
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临沂友康生物科技有限公司
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Priority to CN201780094619.5A priority Critical patent/CN111448298B/zh
Priority to PCT/CN2017/100344 priority patent/WO2019041338A1/fr
Publication of WO2019041338A1 publication Critical patent/WO2019041338A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B1/00—Production of fats or fatty oils from raw materials
    • C11B1/02—Pretreatment
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B1/00—Production of fats or fatty oils from raw materials
    • C11B1/06—Production of fats or fatty oils from raw materials by pressing
    • 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
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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 present invention relates to the field of microbial fermentation and separation, and in particular to a method for separating microbial oils and fats.
  • Microbial oil also known as single-cell oil, is a fat produced by microorganisms such as yeast, mold, bacteria and algae under certain conditions using carbohydrates, hydrocarbons and common fats and oils as carbon and nitrogen sources, supplemented by inorganic salts. Some commercially valuable lipids. Under suitable conditions, certain microorganisms produce and store more than 20% of the total biomass, and strains with such phenotypes are called oil-producing microorganisms.
  • Microbial oil is another new human edible oil resource developed after plant oils and animal fats.
  • research on oil-producing microorganisms mainly focuses on yeast, algae and mold, and the oil produced by these microorganisms is an intracellular product.
  • problems in the separation and extraction process of microbial oils For example, the cell walls of yeast and algae are thick and not easy to break, resulting in high energy consumption and high cost.
  • the microbial oil separation method mainly includes an organic solvent extraction method and physical separation.
  • the organic solvent separates the fat and oil, it involves a large amount of solvent, which has a serious safety hazard, and there is a solvent residue in the refined fat and oil, which affects the product quality.
  • the method of physical separation mainly adopts enzymatic hydrolysis and physical methods to break the cells of the fermentation broth, and centrifuges the fermentation broth after the wall is broken.
  • the process of breaking the wall there are various acids, alkalis and others.
  • the addition of chemicals has the risk of introduction of foreign contamination.
  • the main object of the present invention is to provide a method for separating microbial oils and fats to solve the risk of external contamination in the separation of microbial oils and fats in the prior art.
  • the present invention provides a method for separating microbial oil and fat, which comprises physically concentrating a fermenting bacterial liquid of a microorganism to obtain a concentrated bacterial liquid; freeze-drying and dehydrating the concentrated bacterial liquid to obtain lyophilization Bacterial powder; and the lyophilized powder is pressed and extracted to obtain microbial oil.
  • the step of concentrating the fermentation broth of the microorganism to obtain the concentrated bacterium solution comprises centrifuging the fermentation broth of the microorganism to obtain a centrifuge liquid having a water content of less than 60%; and filtering the bacterium by centrifugation A concentrated bacterial solution having a moisture content of less than 10% is obtained.
  • the fermentation broth of the microorganism is centrifuged at a rotation speed of 1000 to 2000 rpm/min to obtain a centrifuge liquid having a water content of less than 60%.
  • the centrifugal bacterial liquid is filtered using a ceramic filter; preferably, the ceramic filter has a pore diameter of ⁇ 200 nm.
  • the step of freeze-drying and dehydrating the concentrated bacterial liquid to obtain the freeze-dried bacterial powder comprises placing the concentrated bacterial liquid at a freezing temperature of -30 to -40 ° C, a vacuum degree of 1.3 to 2 Pa, and a sublimation drying temperature of 60.
  • the lyophilized powder was obtained by vacuum freeze-drying and dehydration under conditions of -80 °C.
  • the step of extracting the freeze-dried powder to obtain the microbial oil comprises: grinding the freeze-dried powder to obtain a powder; and pressing and extracting the powder to obtain a microbial oil; preferably, sanding
  • the freeze-dried powder is ground by a machine; preferably, the press-pushing step has a press pressure of 20 to 35 MPa and a press temperature of 50 to 60 °C.
  • the separation method further comprises the step of culturing the microorganism to obtain a fermentation broth.
  • the step of cultivating the microorganism to obtain the fermentation broth comprises cultivating the microorganism; and cultivating the strain in the medium to obtain a fermentation broth; preferably, the mass ratio of the strain to the medium is 1 ⁇ 10:100.
  • the medium includes: 45% to 50% of MSG, 15% to 20% of yeast extract, 10% to 15% of dry powder of corn syrup, 4% to 8% of potassium sulfate, and 1% of malic acid. ⁇ 2%, citric acid 0.5% to 1% and the balance of water.
  • the temperature of the fermentation is 26 to 30 ° C, preferably, the pressure of the fermentation is 0.03 to 0.05 MPa; preferably, the fermentation is carried out in a fermenter, and during the fermentation, the fermenter is also included a step of stirring the strain and the medium; more preferably, the stirring speed is 80 ⁇ 10 r / min; more preferably, during the fermentation, further comprising the step of introducing air into the fermenter, further preferably The amount of air introduced is 0.2 to 0.8 VVM; preferably, the fermentation time is 96 to 144 hours.
  • the method for separating microbial oil and fat obtains a high concentration of microbial cells by physically concentrating the microbial liquid, and then freeze-drying and dehydrating, so that free water in the microbial cells is lyophilized and sublimated. Removal, not only achieves wall dehydration, but also reduces cell damage and the addition of foreign chemicals. Finally, the freeze-dried powder is extracted by pressing, which not only avoids unnecessary protein and sugar under solvent extraction. Unwanted materials such as the class are extracted into the hair oil, and since the microorganisms are not treated with a chemical agent such as an organic solvent or an enzyme during the entire separation process, there is no solvent or the like in the obtained microbial oil. Material pollution.
  • FIG. 1 is a schematic flow chart showing a method for separating microbial oil and fat provided in a preferred embodiment of the present invention
  • Fig. 2 is a flow chart showing a method of separating microbial oils and fats provided in another preferred embodiment of the present invention.
  • VVM indicates the manner in which the amount of gas introduced into the fermenter during the fermentation is expressed.
  • VVM is short for air volume/culture volume/min and represents the ratio of the ventilation per minute to the actual volume of the tank.
  • a method for separating microbial oil and fat comprises: physically concentrating a fermentation broth of a microorganism to obtain a concentration The bacterial liquid; the concentrated bacterial liquid is freeze-dried and dehydrated to obtain a freeze-dried powder; and the freeze-dried powder is pressed and extracted to obtain a microbial oil.
  • the method for separating microbial oil and fat obtained by physically concentrating the microbial liquid, and then freeze-drying and dehydrating, so that free water in the microbial cells can be sublimed and dried by breaking the wall. The removal not only achieves the dehydration of the broken wall, but also reduces the addition of foreign chemicals. Finally, the freeze-dried powder is extracted by means of pressing, which not only avoids the protein, sugar, etc. under the chemical solvent extraction method. The necessary substances are extracted together into the hair oil, and since the microorganisms are not treated with a chemical agent such as an organic solvent or an enzyme during the entire separation process, there is no chemical solvent contamination in the obtained microorganism oils and fats.
  • a chemical agent such as an organic solvent or an enzyme during the entire separation process
  • the step of concentrating the fermentation broth may be concentrated by an existing concentration method, or may be concentrated by optimizing and improving the existing concentrating method.
  • the step of concentrating the fermentation broth of the microorganism to obtain the concentrated bacterial solution comprises: centrifuging the fermentation broth of the microorganism to obtain a water content of less than 60%.
  • the bacteria solution is centrifuged; and the centrifuge liquid is filtered through a filter to obtain a concentrated bacterial solution having a water content of less than 10%.
  • the step of centrifugally concentrating the fermentation broth may be carried out using a universal centrifugal rotation speed.
  • the fermentation broth of the microorganism is placed at 1000 to 2000 rpm/ Centrifugal separation was carried out at a speed of min to obtain a centrifuge liquid having a water content of less than 60%. Controlling the centrifugal rotation speed within the above range can effectively prevent the rotation speed from being too fast, so that the microbial cells are broken by the centrifugal force to cause oil loss.
  • the filter membrane in the filtration step of the centrifuge liquid may be a ceramic membrane, a metal membrane or an organic membrane.
  • the centrifuge liquid is filtered using a ceramic filter; more preferably, the ceramic filter has a pore size of ⁇ 200 nm.
  • Ceramic filter membrane has the advantages of good stability, high strength and high filtration efficiency.
  • the step of freeze-drying and dehydrating the concentrated bacterial liquid comprises: placing the concentrated bacterial liquid at a freezing temperature of -30 to -40 ° C, a vacuum degree of 1.3 to 2 Pa, and a sublimation drying temperature.
  • the lyophilized powder was obtained by vacuum freeze-drying and dehydration under conditions of 60 to 80 °C.
  • the vacuum freeze-drying speed can be accelerated, the vacuum freeze-drying time can be reduced, and the cell wall breaking efficiency can be improved;
  • the microalgae cells have double-layer cell walls, wherein the inner wall is mainly fatty acid, and the inner and outer walls are mainly water.
  • the moisture between the inner and outer walls first becomes ice, and then sublimates.
  • the water will puncture the outer wall of the cell, and the inner wall will not be damaged, so that it can play a very good role in the fatty acids inside. Good protection and at the same time achieve the function of breaking the wall. Rapid lyophilization ensures that the cell inner wall integrity is over 95%.
  • the step of extracting and liquefying the lyophilized bacterial powder to obtain microbial fats and oils can be carried out by using a conventional pressing and oiling operation step.
  • the freeze-dried powder is subjected to pressing and oil extraction
  • the step of obtaining microbial oil and fat comprises: grinding the freeze-dried powder to obtain a powder. And pressing and extracting the powder to obtain microbial oil; preferably, the freeze-dried powder is ground by a sand mill; preferably, the pressing pressure of the pressing and oiling step is 20 to 35 MPa, and the pressing temperature is 50 to 60. °C.
  • the freeze-dried powder is ground to obtain the powder, and then the powder is pressed and extracted, and the oil extraction rate is higher than that of directly pressing the freeze-dried powder.
  • the method for culturing the concentrated fermentation broth may be carried out by a conventionally known method.
  • the separation method further comprises the step of culturing the microorganism to obtain a fermentation broth before concentrating the fermentation broth of the microorganism.
  • the step of culturing the microorganism to obtain the fermentation broth comprises: preparing a strain of the microorganism; and placing the strain in the medium for fermentation to obtain a fermentation broth; preferably, the strain and the culture
  • the mass ratio of the base is from 1 to 10:10.
  • the above step of preparing the microorganism species may be carried out by using the existing strain preparation step.
  • the specific strain preparation steps vary depending on the type of microorganism to be used.
  • the microbial strain may be a microorganism of the genus Mortierella, Crypthecocci or Thraustochytrium, or a microorganism of the genus Thraustochytrium, Schizochytrium (such as microalgae), or may be a mountain quilt. Mortierella.
  • the culture medium used in the above fermentation process may be the same or different.
  • the medium comprises, by mass percent, MS% 45%-50%, yeast paste 15%-20%, corn syrup dry powder 10%-15%, potassium sulfate 4%-8%. , malic acid 1% -2% and citric acid 0.5% -1%.
  • the above-mentioned content and ratio of the medium of the present application have the advantages of high productivity and high content compared with the culture in the prior art, and DHA and ARA in the oil of the two types of oil obtained when producing DHA and ARA. The content can reach more than 50%.
  • the temperature of the fermentation is 26 to 30 ° C, preferably, the pressure of the fermentation is 0.03 to 0.05 MPa; preferably, the fermentation is carried out in a fermenter, during the fermentation, Also included is a step of agitating the strain and the culture medium in the fermenter; more preferably, the stirring speed is 80 ⁇ 10 r/min; more preferably, during the fermentation, the fermentation is further included.
  • the above fermentation pressure, flow rate, rotation speed and time are the best suitable conditions for the rapid growth of DHA content.
  • the fermentation process is carried out in a fermentor at a rate that is a rate at which the material in the fermentor is agitated.
  • the fermenter is closed and requires air to be introduced into it.
  • the flow refers to the flow of sterile air.
  • the temperature of the fermentation is controlled within the above range, and the strain grows faster. Fermentation at a pressure of 0.03 to 0.05 MPa has the optimum conditions for rapidly increasing the DHA or ARA content.
  • the flow rate and the rotation speed of the sterile air are controlled within the above range, so that the strain can be uniformly contacted with the medium, and the nutrient substance can be more fully absorbed, so that the target oil content can be rapidly increased.
  • the fermentation time can reach the maximum reproduction amount within the above range, and the problem that the bacterial biomass is increased and the oil biosynthesis and conversion efficiency are low is avoided.
  • the microalgae cells and the culture medium were placed in a fermenter at a temperature ratio of 1:100 at a temperature of 26 ° C and a pressure of 0.03 MPa, and the materials in the fermenter were stirred at a stirring speed of 200 r. /min, and according to the 0.2VVM (air volume/culture volume/min, the ratio of the ratio of the ventilation per minute to the actual volume of the tank) to the fermenter through sterile air, cultured for 24h, to obtain the first seed
  • the medium in the fermenter in terms of mass percentage, includes: 45% MSG, 15% yeast extract, 15% dry corn flour, 8% potassium sulfate, 1% malic acid, 1% citric acid, and The amount of water.
  • the first-stage seed and the culture medium were placed in a fermenter having a temperature of 26 ° C and a pressure of 0.03 MPa in a mass ratio of 1:100, and the medium was stirred at a stirring speed of 130 r/min. And according to the ventilation of 0.4VVM, the sterile seed is passed through the fermenter for 24 hours to obtain the secondary seed; wherein the medium in the fermenter is, in terms of mass percentage, including: 0% MSG, 20% yeast extract , corn syrup dry powder 10%, potassium sulfate 4%, malic acid 2%, citric acid 0.5% and the balance of water.
  • the secondary seed was placed in a fermenter at 26 ° C and a pressure of 0.03 MPa for fermentation, while the medium was stirred, the stirring speed was 70 r / min, and the sterilizing was carried out to the fermenter according to the aeration of 0.7 VVM.
  • the air was cultured for 144 hours to obtain a fermentation broth.
  • the fermenting bacterial solution of the microorganism was centrifuged at 1000 rpm/min to obtain a centrifuge liquid having a water content of 58%; the centrifuged liquid was filtered through a ceramic filter (pore size: 200 nm) to obtain a low water content. 10% concentrated bacteria solution.
  • the concentrated bacterial solution is subjected to vacuum freeze-drying and dehydration under the conditions of a freezing temperature of -30 ° C, a vacuum degree of 1.3 Pa, and a sublimation drying temperature of 60 ° C to obtain a freeze-dried bacterial powder;
  • the lyophilized powder is ground by a sand mill to obtain a powder; and the powder is pressed and oiled to obtain microbial oil; wherein the pressing pressure of the pressing step is 20 MPa, the pressing temperature is 50 ° C, and the microbial oil is The yield was 75%.
  • the microalgae cells and the culture medium were placed in a fermenter at a temperature of 30 ° C and a pressure of 0.05 MPa at a mass ratio of 10:100, and the materials in the fermenter were stirred at a stirring speed of 240 r. /min, and according to the ventilation of 0.4VVM, the culture medium is incubated for 20 hours in the fermenter to obtain the first-stage seed; wherein the medium in the fermenter is, in terms of mass percentage, including: monosodium glutamate 50%, yeast 20% cream, 10% corn syrup powder, 8% potassium sulfate, 2% malic acid, 0.5% citric acid and the balance of water.
  • the first-stage seed and the culture medium were placed in a fermenter at a temperature of 30 ° C and a pressure of 0.05 MPa in a mass ratio of 10:100, and the medium was stirred at a stirring speed of 170 r/min. And the medium is cultured for 20 hours according to the ventilation of 0.5VVM, and the second stage seed is obtained.
  • the medium in the fermenter is, in terms of mass percentage, including: MSG 45%, yeast paste 15%. , corn syrup dry powder 10%, potassium sulfate 4%, malic acid 1%, citric acid 0.5% and the balance of water.
  • the secondary seed was placed in a fermenter at 30 ° C and a pressure of 0.05 MPa for fermentation, while the medium was stirred, the stirring speed was 90 r / min, and the fermentation was carried out according to the aeration of 0.8 VVM.
  • the air was cultured for 96 hours to obtain a fermentation broth.
  • the microbial fermentation broth was centrifuged at 2000 rpm/min to obtain a centrifuge liquid having a water content of less than 60%; the centrifuged liquid was filtered through a ceramic filter (pore size 200 nm) to obtain a low water content. 10% concentrated bacteria solution.
  • the concentrated bacterial solution is subjected to vacuum freeze-drying and dehydration under the conditions of a freezing temperature of -40 ° C, a vacuum of 2 Pa, and a sublimation drying temperature of 80 ° C to obtain a freeze-dried bacterial powder;
  • the lyophilized powder is ground by a sand mill to obtain a powder; and the powder is pressed and oiled to obtain microbial oil; wherein the pressing pressure of the pressing step is 35 MPa, the pressing temperature is 60 ° C, and the microbial oil is The yield was 78%.
  • microalgae cells and the culture medium were placed in a fermentation tank at a temperature of 28 ° C and a pressure of 0.04 MPa in a mass ratio of 5:100, and the materials in the fermenter were stirred at a rotation speed of 220 r. /min, and according to the flow rate of 0.35VVM ventilation to the fermenter through sterile air, cultured for 24h, to obtain first-grade seeds; wherein the medium in the fermenter, in terms of mass percentage, including: MSG 48% Yeast extract 18%, corn syrup dry powder 13%, potassium sulfate 6%, malic acid 1.5%, citric acid 0.75% and the balance of water.
  • the first-stage seed and the medium are placed in a fermenter at a temperature of 28 ° C and a pressure of 0.04 MPa in a mass ratio of 5:100, and the medium is stirred, and the stirring speed is 150 r/min. And according to the flow rate of 0.45VVM ventilation to the fermenter through sterile air, cultured for 24h, to obtain secondary seeds; wherein the medium in the fermenter, in terms of mass percentage, including: MSG 48%, yeast paste 18%, corn syrup dry powder 13%, potassium sulfate 6%, malic acid 1.5%, citric acid 0.75% and the balance of water.
  • the secondary seed was placed in a fermenter at 28 ° C and a pressure of 0.04 MPa for fermentation culture, while the medium was stirred, the stirring speed was 80 r / min, and the flow rate was passed to the fermenter according to the flow rate of 0.55 VVM. Sterile air was incubated for 120 h to obtain a fermentation broth.
  • the microbial fermentation broth was centrifuged at 1500 rpm/min to obtain a centrifuge liquid having a water content of less than 60%; the centrifuged liquid was filtered through a ceramic filter (pore size 100 nm) to obtain a low water content. 10% concentrated bacteria solution.
  • the concentrated bacterial solution is subjected to vacuum freeze-drying and dehydration under the conditions of a freezing temperature of -35 ° C, a vacuum degree of 1.5 Pa, and a sublimation drying temperature of 70 ° C to obtain a freeze-dried bacterial powder;
  • the lyophilized powder is ground by a sand mill to obtain a powder; and the powder is pressed and oiled to obtain microbial oil; wherein the pressing pressure of the pressing step is 25 MPa, the pressing temperature is 55 ° C, and the microbial oil is The yield was 85%.
  • the microalgae cells and the culture medium were placed in a fermenter at a temperature of 26 ° C and a pressure of 0.03 MPa in a ratio of 0.5:100, and the materials in the fermenter were stirred at a stirring speed of 200 r. /min, and according to the ventilation of 0.2VVM to the fermenter through sterile air, cultured for 24h, to obtain first-grade seeds; wherein the medium in the fermenter, in terms of mass percentage, including: MSG 45%, yeast 15% cream, 15% corn syrup powder, 8% potassium sulfate, 1% malic acid, 1% citric acid and the balance of water.
  • the first-stage seed and the culture medium were placed in a fermenter having a temperature of 26 ° C and a pressure of 0.03 MPa in a mass ratio of 1:100, and the medium was stirred at a stirring speed of 130 r/min. And according to the ventilation of 0.4VVM, the sterile seed is passed through the fermenter for 24 hours to obtain the secondary seed; wherein the medium in the fermenter is, in terms of mass percentage, including: 0% MSG, 20% yeast extract , corn syrup dry powder 10%, potassium sulfate 4%, malic acid 2%, citric acid 0.5% and the balance of water.
  • the secondary seed was placed in a fermenter at 26 ° C and a pressure of 0.03 MPa for fermentation, while the medium was stirred, the stirring speed was 70 r / min, and the sterilizing was carried out to the fermenter according to the aeration of 0.7 VVM.
  • the air was cultured for 144 hours to obtain a fermentation broth.
  • the fermenting bacterial solution of the microorganism was centrifuged at 1000 rpm/min to obtain a centrifuge liquid having a water content of 58%; the centrifuged liquid was filtered through a ceramic filter (pore size: 200 nm) to obtain a low water content. 10% concentrated bacteria solution.
  • the concentrated bacterial solution is subjected to vacuum freeze-drying and dehydration under the conditions of a freezing temperature of -30 ° C, a vacuum degree of 1.3 Pa, and a sublimation drying temperature of 60 ° C to obtain a freeze-dried bacterial powder;
  • the lyophilized powder is ground by a sand mill to obtain a powder; and the powder is pressed and oiled to obtain microbial oil; wherein the pressing pressure of the pressing step is 20 MPa, the pressing temperature is 50 ° C, and the microbial oil is The yield was 75%.
  • the microalgae cells and the culture medium were placed in a fermenter at a temperature of 31 ° C and a pressure of 0.05 MPa at a mass ratio of 10:100, and the materials in the fermenter were stirred at a stirring speed of 240 r. /min, and according to the ventilation of 0.4VVM, the sterile fermented air was passed through the fermenter for 20 hours to obtain the first-stage seed; among them, the culture in the fermenter Nutrient, in terms of mass percentage, includes: 50% monosodium glutamate, 20% yeast extract, 10% corn syrup powder, 8% potassium sulfate, 2% malic acid, 0.5% citric acid, and the balance of water.
  • the first-stage seed and the culture medium were placed in a fermenter at a temperature of 31 ° C and a pressure of 0.05 MPa in a mass ratio of 10:100, and the medium was stirred at a stirring speed of 170 r/min. And the medium is cultured for 20 hours according to the ventilation of 0.5VVM, and the second stage seed is obtained.
  • the medium in the fermenter is, in terms of mass percentage, including: MSG 45%, yeast paste 15%. , corn syrup dry powder 10%, potassium sulfate 4%, malic acid 1%, citric acid 0.5% and the balance of water.
  • the secondary seed was placed in a fermenter at 31 ° C and a pressure of 0.05 MPa for fermentation, while the medium was stirred, the stirring speed was 90 r / min, and the flow rate was 0.8 VVM to the fermenter.
  • the bacteria air was cultured for 96 hours to obtain a fermentation broth.
  • the microbial fermentation broth was centrifuged at 2000 rpm/min to obtain a centrifuge liquid having a water content of less than 60%; the centrifuged liquid was filtered through a ceramic filter (pore size 200 nm) to obtain a low water content. 10% concentrated bacteria solution.
  • the concentrated bacterial solution is subjected to vacuum freeze-drying and dehydration under the conditions of a freezing temperature of -40 ° C, a vacuum of 2 Pa, and a sublimation drying temperature of 80 ° C to obtain a freeze-dried bacterial powder;
  • the lyophilized powder is ground by a sand mill to obtain a powder; and the powder is pressed and oiled to obtain microbial oil; wherein the pressing pressure of the pressing step is 35 MPa, the pressing temperature is 60 ° C, and the microbial oil is The yield was 76%.
  • the microalgae cells and the culture medium were placed in a fermenter at a temperature of 30 ° C and a pressure of 0.025 MPa in a mass ratio of 10:100, and the materials in the fermentor were stirred at a stirring speed of 240 r. /min, and according to the ventilation of 0.4VVM, the culture medium is incubated for 20 hours in the fermenter to obtain the first-stage seed; wherein the medium in the fermenter is, in terms of mass percentage, including: monosodium glutamate 50%, yeast 20% cream, 10% corn syrup powder, 8% potassium sulfate, 2% malic acid, 0.5% citric acid and the balance of water.
  • the first-stage seed and the culture medium are placed in a fermenter at a temperature of 30 ° C and a pressure of 0.025 MPa in a mass ratio of 10:100, and the medium is stirred, and the stirring speed is 170 r/min. And the medium is cultured for 20 hours according to the ventilation of 0.5VVM, and the second stage seed is obtained.
  • the medium in the fermenter is, in terms of mass percentage, including: MSG 45%, yeast paste 15%. , corn syrup dry powder 10%, potassium sulfate 4%, malic acid 1%, citric acid 0.5% and the balance of water.
  • the secondary seed was placed in a fermenter at 30 ° C and a pressure of 0.025 MPa for fermentation, while the medium was stirred, the stirring speed was 90 r / min, and the fermentation was carried out according to the aeration of 0.8 VVM.
  • the air was cultured for 96 hours to obtain a fermentation broth.
  • the microbial fermentation broth was centrifuged at 2000 rpm/min to obtain a centrifuge liquid having a water content of less than 60%; the centrifuged liquid was filtered through a ceramic filter (pore size 200 nm) to obtain a low water content. 10% concentrated bacteria solution.
  • the concentrated bacterial solution is subjected to vacuum freeze-drying and dehydration under the conditions of a freezing temperature of -40 ° C, a vacuum of 2 Pa, and a sublimation drying temperature of 80 ° C to obtain a freeze-dried bacterial powder;
  • the lyophilized powder is ground by a sand mill to obtain a powder; and the powder is pressed and oiled to obtain microbial oil; wherein the pressing pressure of the pressing step is 35 MPa, the pressing temperature is 60 ° C, and the microbial oil is The yield was 77%.
  • the microalgae cells and the culture medium were placed in a fermenter at a temperature of 30 ° C and a pressure of 0.05 MPa at a mass ratio of 10:100, and the materials in the fermenter were stirred at a stirring speed of 240 r. /min, and according to the ventilation of 0.4VVM, the culture medium was incubated for 20 hours through sterile air, and the first-stage seed was obtained; wherein the medium in the fermenter was based on mass percentage, including: monosodium glutamate 40%, yeast Cream 12%, corn syrup dry powder 9%, potassium sulfate 10%, malic acid 3%, citric acid 1.5% and the balance of water.
  • the first-stage seed and the culture medium were placed in a fermenter at a temperature of 30 ° C and a pressure of 0.05 MPa in a mass ratio of 10:100, and the medium was stirred at a stirring speed of 170 r/min. And the medium is cultured for 20 hours according to the ventilation of 0.5VVM, and the second stage seed is obtained.
  • the medium in the fermenter is, in terms of mass percentage, including: MSG 45%, yeast paste 15%. , corn syrup dry powder 10%, potassium sulfate 4%, malic acid 1%, citric acid 0.5% and the balance of water.
  • the secondary seed was placed in a fermenter at 30 ° C and a pressure of 0.05 MPa for fermentation, while the medium was stirred, the stirring speed was 90 r / min, and the fermentation was carried out according to the aeration of 0.8 VVM.
  • the air was cultured for 96 hours to obtain a fermentation broth.
  • the microbial fermentation broth was centrifuged at 2000 rpm/min to obtain a centrifuge liquid having a water content of less than 60%; the centrifuged liquid was filtered through a ceramic filter (pore size 200 nm) to obtain a low water content. 10% concentrated bacteria solution.
  • the concentrated bacterial solution is subjected to vacuum freeze-drying and dehydration under the conditions of a freezing temperature of -40 ° C, a vacuum of 2 Pa, and a sublimation drying temperature of 80 ° C to obtain a freeze-dried bacterial powder;
  • the lyophilized powder is ground by a sand mill to obtain a powder; and the powder is pressed and oiled to obtain microbial oil; wherein the pressing pressure of the pressing step is 35 MPa, the pressing temperature is 60 ° C, and the microbial oil is The yield was 75%.
  • the microalgae cells and the culture medium were placed in a fermenter at a temperature of 30 ° C and a pressure of 0.05 MPa at a mass ratio of 10:100, and the materials in the fermenter were stirred at a stirring speed of 240 r. /min, and according to the ventilation of 0.4VVM, the sterile fermented air was passed through the fermenter for 20 hours to obtain the first-stage seed; among them, the culture in the fermenter Nutrient, in terms of mass percentage, includes: 50% monosodium glutamate, 20% yeast extract, 10% corn syrup powder, 8% potassium sulfate, 2% malic acid, 0.5% citric acid, and the balance of water.
  • the first-stage seed and the culture medium were placed in a fermenter at a temperature of 30 ° C and a pressure of 0.05 MPa in a mass ratio of 10:100, and the medium was stirred at a stirring speed of 170 r/min. And the medium is cultured for 20 hours according to the ventilation of 0.5VVM, and the second stage seed is obtained.
  • the medium in the fermenter is, in terms of mass percentage, including: MSG 45%, yeast paste 15%. , corn syrup dry powder 10%, potassium sulfate 4%, malic acid 1%, citric acid 0.5% and the balance of water.
  • the secondary seed was placed in a fermenter at 30 ° C and a pressure of 0.05 MPa for fermentation, while the medium was stirred, the stirring speed was 60 r / min, and the sterilizing was carried out to the fermenter according to the aeration of 0.8 VVM.
  • the air was cultured for 96 hours to obtain a fermentation broth.
  • the microbial fermentation broth was centrifuged at 2000 rpm/min to obtain a centrifuge liquid having a water content of less than 60%; the centrifuged liquid was filtered through a ceramic filter (pore size 200 nm) to obtain a low water content. 10% concentrated bacteria solution.
  • the concentrated bacterial solution is subjected to vacuum freeze-drying and dehydration under the conditions of a freezing temperature of -40 ° C, a vacuum of 2 Pa, and a sublimation drying temperature of 80 ° C to obtain a freeze-dried bacterial powder;
  • the lyophilized powder is ground by a sand mill to obtain a powder; and the powder is pressed and oiled to obtain microbial oil; wherein the pressing pressure of the pressing step is 35 MPa, the pressing temperature is 60 ° C, and the microbial oil is The yield was 76%.
  • the microalgae cells and the culture medium were placed in a fermenter at a temperature of 30 ° C and a pressure of 0.05 MPa at a mass ratio of 10:100, and the materials in the fermenter were stirred at a stirring speed of 240 r. /min, and according to the flow rate of 4.2m 3 /h into the fermenter through sterile air, cultured for 20h, to obtain a first-grade seed; wherein the medium in the fermenter, in terms of mass percentage, including: monosodium glutamate 50% , yeast extract 20%, corn syrup dry powder 10%, potassium sulfate 8%, malic acid 2%, citric acid 0.5% and the balance of water.
  • the first-stage seed and the culture medium were placed in a fermenter at a temperature of 30 ° C and a pressure of 0.05 MPa in a mass ratio of 10:100, and the medium was stirred at a stirring speed of 170 r/min. And the medium is cultured for 20 hours according to the flow rate of 30 m 3 /h through the sterile air, and the second stage seed is obtained; wherein the medium in the fermenter is, in terms of mass percentage, including: MSG 45%, yeast paste 15 %, corn syrup dry powder 10%, potassium sulfate 4%, malic acid 1%, citric acid 0.5% and the balance of water.
  • the secondary seed was placed in a fermenter at 30 ° C and a pressure of 0.05 MPa for fermentation culture, while the medium was stirred, the stirring speed was 90 r / min, and the flow rate was passed to the fermenter at a flow rate of 500 m 3 /h.
  • the bacteria air was cultured for 96 hours to obtain a fermentation broth.
  • the microbial fermentation broth was centrifuged at 2000 rpm/min to obtain a centrifuge liquid having a water content of less than 60%; the centrifuged liquid was filtered through a ceramic filter (pore size 200 nm) to obtain a low water content. 10% concentrated bacteria solution.
  • the concentrated bacterial solution is subjected to vacuum freeze-drying and dehydration under the conditions of a freezing temperature of -40 ° C, a vacuum of 2 Pa, and a sublimation drying temperature of 80 ° C to obtain a freeze-dried bacterial powder;
  • the lyophilized powder is ground by a sand mill to obtain a powder; and the powder is pressed and oiled to obtain microbial oil; wherein the pressing pressure of the pressing step is 35 MPa, the pressing temperature is 60 ° C, and the microbial oil is The yield was 77%.
  • the microalgae cells and the culture medium were placed in a fermenter at a temperature of 30 ° C and a pressure of 0.05 MPa at a mass ratio of 10:100, and the materials in the fermenter were stirred at a stirring speed of 240 r. /min, and according to the ventilation of 0.4VVM, the culture medium is incubated for 20 hours in the fermenter to obtain the first-stage seed; wherein the medium in the fermenter is, in terms of mass percentage, including: monosodium glutamate 50%, yeast 20% cream, 10% corn syrup powder, 8% potassium sulfate, 2% malic acid, 0.5% citric acid and the balance of water.
  • the first-stage seed and the culture medium were placed in a fermenter at a temperature of 30 ° C and a pressure of 0.05 MPa in a mass ratio of 10:100, and the medium was stirred at a stirring speed of 170 r/min. And the medium is cultured for 20 hours according to the ventilation of 0.5VVM, and the second stage seed is obtained.
  • the medium in the fermenter is, in terms of mass percentage, including: MSG 45%, yeast paste 15%. , corn syrup dry powder 10%, potassium sulfate 4%, malic acid 1%, citric acid 0.5% and the balance of water.
  • the secondary seed was placed in a fermenter at 30 ° C and a pressure of 0.05 MPa for fermentation, while the medium was stirred, the stirring speed was 90 r / min, and the fermentation was carried out according to the aeration of 0.8 VVM.
  • the air was cultured for 96 hours to obtain a fermentation broth.
  • the microbial fermentation broth was centrifuged at 2500 rpm/min to obtain a centrifuge liquid having a water content of less than 60%; the centrifuged liquid was filtered through a ceramic filter (pore size 200 nm) to obtain a low water content. 10% concentrated bacteria solution.
  • the concentrated bacterial solution is subjected to vacuum freeze-drying and dehydration under the conditions of a freezing temperature of -40 ° C, a vacuum of 2 Pa, and a sublimation drying temperature of 80 ° C to obtain a freeze-dried bacterial powder;
  • the lyophilized powder is ground by a sand mill to obtain a powder; and the powder is pressed and oiled to obtain microbial oil; wherein the pressing pressure of the pressing step is 35 MPa, the pressing temperature is 60 ° C, and the microbial oil is The yield was 74%.
  • the microalgae cells and the culture medium were placed in a fermenter at a temperature of 30 ° C and a pressure of 0.05 MPa at a mass ratio of 10:100, and the materials in the fermenter were stirred at a stirring speed of 240 r. /min, and according to the ventilation of 0.4VVM, the sterile fermented air was passed through the fermenter for 20 hours to obtain the first-stage seed; among them, the culture in the fermenter Nutrient, in terms of mass percentage, includes: 50% monosodium glutamate, 20% yeast extract, 10% corn syrup powder, 8% potassium sulfate, 2% malic acid, 0.5% citric acid, and the balance of water.
  • the first-stage seed and the culture medium were placed in a fermenter at a temperature of 30 ° C and a pressure of 0.05 MPa in a mass ratio of 10:100, and the medium was stirred at a stirring speed of 170 r/min. And the medium is cultured for 20 hours according to the ventilation of 0.5VVM, and the second stage seed is obtained.
  • the medium in the fermenter is, in terms of mass percentage, including: MSG 45%, yeast paste 15%. , corn syrup dry powder 10%, potassium sulfate 4%, malic acid 1%, citric acid 0.5% and the balance of water.
  • the secondary seed was placed in a fermenter at 30 ° C and a pressure of 0.05 MPa for fermentation, while the medium was stirred, the stirring speed was 90 r / min, and the fermentation was carried out according to the aeration of 0.8 VVM.
  • the air was cultured for 96 hours to obtain a fermentation broth.
  • the microbial fermentation broth was centrifuged at 2000 rpm/min to obtain a centrifuge liquid having a water content of less than 60%; the centrifugal filter was filtered through a ceramic filter (pore size: 250 nm) to obtain a low water content. 10% concentrated bacteria solution.
  • the concentrated bacterial solution is subjected to vacuum freeze-drying and dehydration under the conditions of a freezing temperature of -40 ° C, a vacuum of 2 Pa, and a sublimation drying temperature of 80 ° C to obtain a freeze-dried bacterial powder;
  • the lyophilized powder is ground by a sand mill to obtain a powder; and the powder is pressed and oiled to obtain microbial oil; wherein the pressing pressure of the pressing step is 35 MPa, the pressing temperature is 60 ° C, and the microbial oil is The yield was 75%.
  • the microalgae cells and the culture medium were placed in a fermenter at a temperature of 30 ° C and a pressure of 0.05 MPa at a mass ratio of 10:100, and the materials in the fermenter were stirred at a stirring speed of 240 r. /min, and according to the ventilation of 0.4VVM, the culture medium is incubated for 20 hours in the fermenter to obtain the first-stage seed; wherein the medium in the fermenter is, in terms of mass percentage, including: monosodium glutamate 50%, yeast 20% cream, 10% corn syrup powder, 8% potassium sulfate, 2% malic acid, 0.5% citric acid and the balance of water.
  • the first-stage seed and the culture medium were placed in a fermenter at a temperature of 30 ° C and a pressure of 0.05 MPa in a mass ratio of 10:100, and the medium was stirred at a stirring speed of 170 r/min. And the medium is cultured for 20 hours according to the ventilation of 0.5VVM, and the second stage seed is obtained.
  • the medium in the fermenter is, in terms of mass percentage, including: MSG 45%, yeast paste 15%. , corn syrup dry powder 10%, potassium sulfate 4%, malic acid 1%, citric acid 0.5% and the balance of water.
  • the secondary seed was placed in a fermenter at 30 ° C and a pressure of 0.05 MPa for fermentation, while the medium was stirred, the stirring speed was 90 r / min, and the fermentation was carried out according to the aeration of 0.8 VVM.
  • the air was cultured for 96 hours to obtain a fermentation broth.
  • the microbial fermentation broth was centrifuged at 2000 rpm/min to obtain a centrifuge liquid having a water content of less than 60%; the centrifuged liquid was filtered through a ceramic filter (pore size 200 nm) to obtain a low water content. 10% concentrated bacteria solution.
  • the concentrated bacterial solution is subjected to vacuum freeze-drying and dehydration under the conditions of a freezing temperature of -25 ° C, a vacuum of 2 Pa, and a sublimation drying temperature of 80 ° C to obtain a freeze-dried bacterial powder;
  • the lyophilized powder is ground by a sand mill to obtain a powder; and the powder is pressed and oiled to obtain microbial oil; wherein the pressing pressure of the pressing step is 35 MPa, the pressing temperature is 60 ° C, and the microbial oil is The yield was 75%.
  • the microalgae cells and the culture medium were placed in a fermenter at a temperature of 30 ° C and a pressure of 0.05 MPa at a mass ratio of 10:100, and the materials in the fermenter were stirred at a stirring speed of 240 r. /min, and according to the ventilation of 0.4VVM, the culture medium is incubated for 20 hours in the fermenter to obtain the first-stage seed; wherein the medium in the fermenter is, in terms of mass percentage, including: monosodium glutamate 50%, yeast 20% cream, 10% corn syrup powder, 8% potassium sulfate, 2% malic acid, 0.5% citric acid and the balance of water.
  • the first-stage seed and the culture medium were placed in a fermenter at a temperature of 30 ° C and a pressure of 0.05 MPa in a mass ratio of 10:100, and the medium was stirred at a stirring speed of 170 r/min. And the medium is cultured for 20 hours according to the ventilation of 0.5VVM, and the second stage seed is obtained.
  • the medium in the fermenter is, in terms of mass percentage, including: MSG 45%, yeast paste 15%. , corn syrup dry powder 10%, potassium sulfate 4%, malic acid 1%, citric acid 0.5% and the balance of water.
  • the secondary seed was placed in a fermenter at 30 ° C and a pressure of 0.05 MPa for fermentation, while the medium was stirred, the stirring speed was 90 r / min, and the fermentation was carried out according to the aeration of 0.8 VVM.
  • the air was cultured for 96 hours to obtain a fermentation broth.
  • the microbial fermentation broth was centrifuged at 2000 rpm/min to obtain a centrifuge liquid having a water content of less than 60%; the centrifuged liquid was filtered through a ceramic filter (pore size 200 nm) to obtain a low water content. 10% concentrated bacteria solution.
  • the concentrated bacterial liquid is subjected to vacuum freeze-drying and dehydration under the conditions of a freezing temperature of -40 ° C, a vacuum degree of 2.2 Pa, and a sublimation drying temperature of 80 ° C to obtain a freeze-dried bacterial powder;
  • the lyophilized powder is ground by a sand mill to obtain a powder; and the powder is pressed and oiled to obtain microbial oil; wherein the pressing pressure of the pressing step is 35 MPa, the pressing temperature is 60 ° C, and the microbial oil is The yield was 76%.
  • the microalgae cells and the culture medium were placed in a fermenter at a temperature of 30 ° C and a pressure of 0.05 MPa at a mass ratio of 10:100, and the materials in the fermenter were stirred at a stirring speed of 240 r. /min, and according to the ventilation of 0.4VVM, the sterile fermented air was passed through the fermenter for 20 hours to obtain the first-stage seed; among them, the culture in the fermenter Nutrient, in terms of mass percentage, includes: 50% monosodium glutamate, 20% yeast extract, 10% corn syrup powder, 8% potassium sulfate, 2% malic acid, 0.5% citric acid, and the balance of water.
  • the first-stage seed and the culture medium were placed in a fermenter at a temperature of 30 ° C and a pressure of 0.05 MPa in a mass ratio of 10:100, and the medium was stirred at a stirring speed of 170 r/min. And the medium is cultured for 20 hours according to the ventilation of 0.5VVM, and the second stage seed is obtained.
  • the medium in the fermenter is, in terms of mass percentage, including: MSG 45%, yeast paste 15%. , corn syrup dry powder 10%, potassium sulfate 4%, malic acid 1%, citric acid 0.5% and the balance of water.
  • the secondary seed was placed in a fermenter at 30 ° C and a pressure of 0.05 MPa for fermentation, while the medium was stirred, the stirring speed was 90 r / min, and the fermentation was carried out according to the aeration of 0.8 VVM.
  • the air was cultured for 96 hours to obtain a fermentation broth.
  • the microbial fermentation broth was centrifuged at 2000 rpm/min to obtain a centrifuge liquid having a water content of less than 60%; the centrifuged liquid was filtered through a ceramic filter (pore size 200 nm) to obtain a low water content. 10% concentrated bacteria solution.
  • the concentrated bacterial liquid is subjected to vacuum freeze-drying and dehydration under the conditions of a freezing temperature of -40 ° C, a vacuum of 2 Pa, and a sublimation drying temperature of 55 ° C to obtain a freeze-dried bacterial powder;
  • the lyophilized powder is ground by a sand mill to obtain a powder; and the powder is pressed and oiled to obtain microbial oil; wherein the pressing pressure of the pressing step is 35 MPa, the pressing temperature is 60 ° C, and the microbial oil is The yield was 74%.
  • the microalgae cells and the culture medium were placed in a fermenter at a temperature of 30 ° C and a pressure of 0.05 MPa at a mass ratio of 10:100, and the materials in the fermenter were stirred at a stirring speed of 240 r. /min, and according to the ventilation of 0.4VVM, the culture medium is incubated for 20 hours in the fermenter to obtain the first-stage seed; wherein the medium in the fermenter is, in terms of mass percentage, including: monosodium glutamate 50%, yeast 20% cream, 10% corn syrup powder, 8% potassium sulfate, 2% malic acid, 0.5% citric acid and the balance of water.
  • the first-stage seed and the culture medium were placed in a fermenter at a temperature of 30 ° C and a pressure of 0.05 MPa in a mass ratio of 10:100, and the medium was stirred at a stirring speed of 170 r/min. And the medium is cultured for 20 hours according to the ventilation of 0.5VVM, and the second stage seed is obtained.
  • the medium in the fermenter is, in terms of mass percentage, including: MSG 45%, yeast paste 15%. , corn syrup dry powder 10%, potassium sulfate 4%, malic acid 1%, citric acid 0.5% and the balance of water.
  • the secondary seed was placed in a fermenter at 30 ° C and a pressure of 0.05 MPa for fermentation, while the medium was stirred, the stirring speed was 90 r / min, and the fermentation was carried out according to the aeration of 0.8 VVM.
  • the air was cultured for 96 hours to obtain a fermentation broth.
  • the microbial fermentation broth was centrifuged at 2000 rpm/min to obtain a centrifuge liquid having a water content of less than 60%; the centrifuged liquid was filtered through a ceramic filter (pore size 200 nm) to obtain a low water content. 10% concentrated bacteria solution.
  • the concentrated bacterial solution is subjected to vacuum freeze-drying and dehydration under the conditions of a freezing temperature of -40 ° C, a vacuum of 2 Pa, and a sublimation drying temperature of 80 ° C to obtain a freeze-dried bacterial powder;
  • the freeze-dried powder is ground by a sand mill to obtain a powder; and the powder is pressed and oiled to obtain microbial oil; wherein the pressing pressure of the pressing step is 35 MPa, the pressing temperature is 70 ° C, and the microbial oil is The yield was 73%.
  • the microalgae cells and the culture medium were placed in a fermenter at a temperature of 30 ° C and a pressure of 0.05 MPa at a mass ratio of 10:100, and the materials in the fermenter were stirred at a stirring speed of 240 r. /min, and according to the ventilation of 0.4VVM, the culture medium is incubated for 20 hours in the fermenter to obtain the first-stage seed; wherein the medium in the fermenter is, in terms of mass percentage, including: monosodium glutamate 50%, yeast 20% cream, 10% corn syrup powder, 8% potassium sulfate, 2% malic acid, 0.5% citric acid and the balance of water.
  • the first-stage seed and the culture medium were placed in a fermenter at a temperature of 30 ° C and a pressure of 0.05 MPa in a mass ratio of 10:100, and the medium was stirred at a stirring speed of 170 r/min. And the medium is cultured for 20 hours according to the ventilation of 0.5VVM, and the second stage seed is obtained.
  • the medium in the fermenter is, in terms of mass percentage, including: MSG 45%, yeast paste 15%. , corn syrup dry powder 10%, potassium sulfate 4%, malic acid 1%, citric acid 0.5% and the balance of water.
  • the secondary seed was placed in a fermenter at 30 ° C and a pressure of 0.05 MPa for fermentation, while the medium was stirred, the stirring speed was 90 r / min, and the fermentation was carried out according to the aeration of 0.8 VVM.
  • the air was cultured for 96 hours to obtain a fermentation broth.
  • the microbial fermentation broth was centrifuged at 2000 rpm/min to obtain a centrifuge liquid having a water content of less than 60%; the centrifuged liquid was filtered through a ceramic filter (pore size 200 nm) to obtain a low water content. 10% concentrated bacteria solution.
  • the concentrated bacterial solution is subjected to vacuum freeze-drying and dehydration under the conditions of a freezing temperature of -40 ° C, a vacuum of 2 Pa, and a sublimation drying temperature of 80 ° C to obtain a freeze-dried bacterial powder;
  • the freeze-dried powder is ground by a sand mill to obtain a powder; and the powder is pressed and oiled to obtain microbial oil; wherein the pressing pressure of the pressing step is 15 MPa, the pressing temperature is 60 ° C, and the microbial oil is The yield was 72%.
  • the Mortierella alpina and the culture medium were placed in a fermenter at a temperature of 28 ° C and a pressure of 0.04 MPa in a mass ratio of 5:100, and the material in the fermenter was stirred at a stirring speed of 220r/min, and according to the aeration of 0.35VVM, the sterile fermented air was passed through the fermenter for 24 hours to obtain a first-class seed; wherein, in the fermenter
  • the medium in terms of mass percentage, includes: 48% monosodium glutamate, 18% yeast extract, 13% dry corn flour, 6% potassium sulfate, 1.5% malic acid, 0.75% citric acid, and the balance water.
  • the first-stage seed and the medium are placed in a fermenter at a temperature of 28 ° C and a pressure of 0.04 MPa in a mass ratio of 5:100, and the medium is stirred, and the stirring speed is 150 r/min. And according to the ventilation of 0.45VVM, the sterile seed is passed through the fermenter for 24 hours to obtain the secondary seed; wherein the medium in the fermenter is, by mass percentage, including: 48% MSG, 18% yeast extract , corn syrup dry powder 13%, potassium sulfate 6%, malic acid 1.5%, citric acid 0.75% and the balance of water.
  • the secondary seed was placed in a fermenter at 28 ° C and a pressure of 0.04 MPa for fermentation, while the medium was stirred, the stirring speed was 80 r / min, and the sterilizing was carried out to the fermenter according to the aeration of 0.55 VVM.
  • the air was cultured for 120 hours to obtain a fermentation broth.
  • the microbial fermentation broth was centrifuged at 1500 rpm/min to obtain a centrifuge liquid having a water content of less than 60%; the centrifuged liquid was filtered through a ceramic filter (pore size 100 nm) to obtain a low water content. 10% concentrated bacteria solution.
  • the concentrated bacterial solution is subjected to vacuum freeze-drying and dehydration under the conditions of a freezing temperature of -35 ° C, a vacuum degree of 1.5 Pa, and a sublimation drying temperature of 70 ° C to obtain a freeze-dried bacterial powder;
  • the lyophilized powder is ground by a sand mill to obtain a powder; and the powder is pressed and oiled to obtain microbial oil; wherein the pressing pressure of the pressing step is 25 MPa, the pressing temperature is 55 ° C, and the microbial oil is The yield was 83%.
  • the microalgae cells and the culture medium were placed in a fermentation tank at a temperature of 28 ° C and a pressure of 0.04 MPa in a mass ratio of 5:100, and the materials in the fermenter were stirred at a rotation speed of 220 r. /min, and according to the aeration of 0.35 VVM, the culture medium was incubated for 24 hours in a sterile air, and the first-stage seed was obtained.
  • the medium in the fermenter was, in terms of mass percentage, including: monosodium glutamate 48%, yeast Cream 18%, corn syrup dry powder 13%, potassium sulfate 6%, malic acid 1.5%, citric acid 0.75% and the balance of water.
  • the first-stage seed and the medium are placed in a fermenter at a temperature of 28 ° C and a pressure of 0.04 MPa in a mass ratio of 5:100, and the medium is stirred, and the stirring speed is 150 r/min. And according to the ventilation of 0.45VVM, the sterile seed is passed through the fermenter for 24 hours to obtain the secondary seed; wherein the medium in the fermenter is, by mass percentage, including: 48% MSG, 18% yeast extract , corn syrup dry powder 13%, potassium sulfate 6%, malic acid 1.5%, citric acid 0.75% and the balance of water.
  • the secondary seed was placed in a fermenter at 28 ° C and a pressure of 0.04 MPa for fermentation, while the medium was stirred, the stirring speed was 80 r / min, and the sterilizing was carried out to the fermenter according to the aeration of 0.55 VVM.
  • the air was cultured for 120 hours to obtain a fermentation broth.
  • the microbial fermentation broth was centrifuged at 1500 rpm/min to obtain a centrifuge liquid having a water content of less than 60%; the centrifuged liquid was filtered through a ceramic filter (pore size 100 nm) to obtain a low water content. 10% concentrated bacteria solution.
  • the concentrated bacterial solution is subjected to vacuum freeze-drying and dehydration under the conditions of a freezing temperature of -35 ° C, a vacuum degree of 1.5 Pa, and a sublimation drying temperature of 70 ° C to obtain a freeze-dried bacterial powder;
  • the lyophilized powder is ground by a sand mill to obtain a powder; and the powder is pressed and oiled to obtain microbial oil; wherein the pressing pressure of the pressing step is 25 MPa, the pressing temperature is 55 ° C, and the microbial oil is The yield was 82%.
  • the microalgae cells and the culture medium were placed in a fermentation tank at a temperature of 28 ° C and a pressure of 0.04 MPa in a mass ratio of 5:100, and the materials in the fermenter were stirred at a rotation speed of 220 r. /min, and according to the aeration of 0.35 VVM, the culture medium was incubated for 24 hours in a sterile air, and the first-stage seed was obtained.
  • the medium in the fermenter was, in terms of mass percentage, including: monosodium glutamate 48%, yeast Cream 18%, corn syrup dry powder 13%, potassium sulfate 6%, malic acid 1.5%, citric acid 0.75% and the balance of water.
  • the first-stage seed and the medium are placed in a fermenter at a temperature of 28 ° C and a pressure of 0.04 MPa in a mass ratio of 5:100, and the medium is stirred, and the stirring speed is 150 r/min. And according to the ventilation of 0.45VVM, the sterile seed is passed through the fermenter for 24 hours to obtain the secondary seed; wherein the medium in the fermenter is, by mass percentage, including: 48% MSG, 18% yeast extract , corn syrup dry powder 13%, potassium sulfate 6%, malic acid 1.5%, citric acid 0.75% and the balance of water.
  • the secondary seed was placed in a fermenter at 28 ° C and a pressure of 0.04 MPa for fermentation, while the medium was stirred, the stirring speed was 80 r / min, and the sterilizing was carried out to the fermenter according to the aeration of 0.55 VVM.
  • the air was cultured for 120 hours to obtain a fermentation broth.
  • the microbial fermentation broth was centrifuged at 1500 rpm/min to obtain a centrifuge liquid having a water content of less than 60%; the centrifuged liquid was filtered through a ceramic filter (pore size 100 nm) to obtain a low water content. 10% concentrated bacteria solution.
  • the concentrated bacterial solution is subjected to vacuum freeze-drying and dehydration under the conditions of a freezing temperature of -35 ° C, a vacuum degree of 1.5 Pa, and a sublimation drying temperature of 70 ° C to obtain a freeze-dried bacterial powder;
  • the lyophilized powder is ground by a sand mill to obtain a powder; and the powder is pressed and oiled to obtain microbial oil; wherein the pressing pressure of the pressing step is 25 MPa, the pressing temperature is 55 ° C, and the microbial oil is The yield was 81%.
  • the fermentation broth of the microalgae cells is subjected to moisture absorption by using a water absorbing resin, and then further filtered and concentrated by a microporous membrane having a pore diameter of 150 nm to obtain a concentrated bacterial liquid;
  • the concentrated bacterial liquid is placed at -30 ° C, the vacuum degree is 1.5 Pa, and the sublimation drying temperature is 70 ° C, vacuum freeze-dried and dehydrated to obtain a freeze-dried bacterial powder;
  • the freeze-dried powder is pressed and oiled to obtain microbial oil; wherein the pressing pressure of the pressing step is 25 MPa, the pressing temperature is 55 ° C, and the yield of the microbial oil is 80%.
  • the microalgae cells and the culture medium were placed in a fermenter at a temperature ratio of 1:100 at a temperature of 26 ° C and a pressure of 0.03 MPa, and the materials in the fermenter were stirred at a stirring speed of 200 r. /min, and according to the ventilation volume of 0.15VVM (air volume/culture volume/min, which represents the ratio of the ventilation per minute to the actual volume of the tank), the air is sterilized in the fermenter for 24 hours to obtain the first-class seed.
  • the medium in the fermenter in terms of mass percentage, includes: 45% MSG, 15% yeast extract, 15% dry corn flour, 8% potassium sulfate, 1% malic acid, 1% citric acid, and The amount of water.
  • the first-stage seed and the culture medium were placed in a fermenter having a temperature of 26 ° C and a pressure of 0.03 MPa in a mass ratio of 1:100, and the medium was stirred at a stirring speed of 130 r/min. And according to the ventilation of 0.2VVM, the sterile seed is passed through the fermenter for 24 hours to obtain the secondary seed; wherein the medium in the fermenter is, in terms of mass percentage, includes: 0% MSG, 20% yeast extract , corn syrup dry powder 10%, potassium sulfate 4%, malic acid 2%, citric acid 0.5% and the balance of water.
  • the secondary seed was placed in a fermenter at 26 ° C and a pressure of 0.03 MPa for fermentation, while the medium was stirred, the stirring speed was 70 r / min, and the sterilizing was carried out to the fermenter according to the aeration of 0.9 VVM.
  • the air was cultured for 144 hours to obtain a fermentation broth.
  • the fermenting bacterial solution of the microorganism was centrifuged at 1000 rpm/min to obtain a centrifuge liquid having a water content of 58%; the centrifuged liquid was filtered through a ceramic filter (pore size: 200 nm) to obtain a low water content. 10% concentrated bacteria solution.
  • the concentrated bacterial solution is subjected to vacuum freeze-drying and dehydration under the conditions of a freezing temperature of -30 ° C, a vacuum degree of 1.3 Pa, and a sublimation drying temperature of 60 ° C to obtain a freeze-dried bacterial powder;
  • the lyophilized powder is ground by a sand mill to obtain a powder; and the powder is pressed and oiled to obtain microbial oil; wherein the pressing pressure of the pressing step is 20 MPa, the pressing temperature is 50 ° C, and the microbial oil is The yield was 75%.
  • the fermentation broth of the microalgae cells is further filtered and concentrated through a microporous membrane having a pore diameter of 150 nm, and then the water is absorbed by a water absorbing resin to obtain a concentrated bacterial solution;
  • the concentrated bacterial solution is treated with a high-pressure homogenizer to break the cells, release the microbial oil, and circulate three times at a pressure of 1500 bar;
  • the oil-absorbing felt is immersed in the above-mentioned broken cell fermentation liquid to absorb the oil in the fermentation liquid, and stirred at 60 ° C for 2 hours to fully absorb the oil in the fermentation liquid;
  • the oil absorbing felt is taken out from the fermentation broth and dried, and the oil is released by extrusion to obtain microbial crude oil, which is 64% of the total oil content in the microalgae.
  • the calculation method of the oil yield is: actual quantity / (volume volume ⁇ oil extraction); DHA content is calculated by: GB 26400 gas phase detection; ARA content calculation method is: GB 26401 gas phase detection.
  • the above-mentioned embodiments of the present invention achieve the following technical effects: by physically concentrating the microbial liquid, a high concentration of the microbial cells is obtained, and then dehydrated and dried to cause microbial cells in the cells. The free water is removed by lyophilization and sublimation, which not only achieves wall dehydration, but also reduces cell damage and the addition of foreign chemicals. Finally, the freeze-dried powder is extracted by pressing, not only avoiding the solvent.
  • the extraction method removes unnecessary proteins, sugars and other undesired materials into the hair oil, and since the microorganisms are not treated with an organic solvent or an enzyme such as an enzyme during the entire separation process, There is no contamination of chemicals such as solvents in the microbial oils and fats.

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Botany (AREA)
  • Medicinal Chemistry (AREA)
  • Virology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Biomedical Technology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Coloring Foods And Improving Nutritive Qualities (AREA)

Abstract

L'invention concerne un procédé de séparation d'huile microbienne, comprenant : la concentration physique d'un liquide bactérien fermenté d'un micro-organisme pour obtenir un liquide bactérien concentré; la lyophilisation du liquide bactérien concentré pour la décomposition de paroi cellulaire, suivie d'une déshydratation pour obtenir une poudre bactérienne lyophilisée; et le pressage de la poudre bactérienne lyophilisée pour extraire de l'huile, ce qui permet d'obtenir l'huile microbienne.
PCT/CN2017/100344 2017-09-04 2017-09-04 Procédé de séparation d'huile microbienne WO2019041338A1 (fr)

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