Methods for increasing production of polyunsaturated fatty acids in microorganisms and methods for producing microbial oils

Abstract

The present application relates to microbial fermentation technology, disclose a kind of method for increasing the production of polyunsaturated fatty acid in microorganism and the preparation method of microbial oil. The method for increasing the production of polyunsaturated fatty acid in microorganism includes: the oil-producing microorganism is accessed to the culture medium added with tetracycline and is cultured.The preparation method of microbial oil includes: the oil-producing microorganism is cultured using the above-mentioned method for increasing the production of polyunsaturated fatty acid in microorganism to obtain culture solution, the culture solution is broken wall, and extraction is obtained to obtain extract, and the extract is dried.The method provided by the present application can effectively increase the content of polyunsaturated fatty acid in microbial oil, simple, efficient and low cost.

Description

TECHNICAL FIELD

[0001] The present application relates to the microbial fermentation technology, specifically, to a method for improving the yield of polyunsaturated fatty acids in microorganisms and a method for preparing microbial oil. BACKGROUND

[0002] Polyunsaturated fatty acids (PUFAs) refer to straight-chain fatty acids containing two or more double bonds and having a carbon chain length of 18-22 carbon atoms. At present, the main source of traditional PUFA products (such as docosahexaenoic acid DHA and eicosapentaenoic acid EPA) is deep-sea fish oil, but in recent years, the problems of heavy metal pollution in the ocean and the decrease of marine fish are increasingly serious, which makes it difficult for the quality and yield of fish oil to meet the needs of the commercial development of PUFA products, and the fatty acid composition of fish oil is complex, which may contain high cholesterol and other components that are not conducive to human absorption and affect efficacy. Studies have found that marine microorganisms are rich in PUFAs, which are accumulated in fish oil through the feeding of fish, so that fish oil contains PUFA components. Therefore, marine microorganisms are the real source of PUFAs, and the production of oil containing PUFAs by microorganisms is not affected by site, season and raw material production, and can be continuously produced throughout the year except for equipment maintenance, which can effectively meet the demand for PUFAs.

[0003] In recent years, PUFAs, especially docosahexaenoic acid (DHA), have been widely used in the fields of food and medicine, and the products have gradually diversified, and the market demand has gradually increased. At present, among the microorganisms that produce high-yield DHA and other PUFAs, the most studied is Schizochytrium, which has a fast growth rate, is easy to cultivate, and has a high oil content. Although the industrial production of DHA has been realized by using Schizochytrium, the productivity of PUFAs in the fermentation process of Schizochytrium still needs to be improved to further reduce the production cost of PUFAs.

[0004] Therefore, it is of great significance to find a production method for improving the yield of PUFAs in Schizochytrium. SUMMARY

[0005] The purpose of the present application is to overcome the problem that the productivity of PUFAs needs to be improved in the prior art, and to provide a method for improving the yield of polyunsaturated fatty acids in microorganisms and a method for preparing microbial oil, which can effectively increase the content of polyunsaturated fatty acids in microbial oil and reduce the production cost.

[0006] In order to achieve the above-mentioned purpose, the first aspect of the present application provides a method for improving the yield of polyunsaturated fatty acids in microorganisms, which comprises: culturing an oil-producing microorganism in a culture medium added with tetracycline.

[0007] Preferably, the amount of tetracycline added to the culture medium is 1-5 mg / L; more preferably, it is 1.5-2.5 mg / L.

[0008] Preferably, the cultivation process includes: inoculating the oil-producing microorganism into a seed culture medium for at least one subculture to obtain a seed culture solution, and inoculating the seed culture solution into a fermentation culture medium for fermentation culture, wherein the tetracycline is added to the seed culture medium and / or the fermentation culture medium.

[0009] Preferably, the tetracycline is added to the seed culture medium.

[0010] Preferably, the number of subcultures is 5-15 times, more preferably 8-12 times.

[0011] Preferably, the seed culture medium contains: a carbon source, a nitrogen source, inorganic salts, and amino acid salts.

[0012] Preferably, the seed culture medium contains: 30-60 g / L glucose, 1-5 g / L KH2PO4, 10-14 g / L Na2SO4, 2-5 g / L MgSO4, 1-3 g / L KCl, 3-5 g / L (NH4)2SO4, 0.06-1 g / L CaCl2, 0.1-1 g / L K2SO4, 8-12 g / L sodium glutamate and 9-15 g / L yeast extract.

[0013] Preferably, the fermentation medium contains: a carbon source, a nitrogen source, inorganic salts, organic acids, and amino acid salts.

[0014] Preferably, the fermentation medium contains: 70-90 g / L glucose, 0.5-2 g / L KH2PO4, 3-5 g / L (NH4)2SO4, 0.5-2 g / L malic acid, 8-12 g / L monosodium glutamate, 8-12 g / L yeast extract, 4-6 g / L corn steep liquor powder, and 30-35 g / L sea salt.

[0015] Preferably, the conditions for each subculture include: an inoculum size of 0.5-2% by volume, an initial pH of 6-7, a rotation speed of 150-200 rpm, a temperature of 25-30°C, and a time of 20-30 h.

[0016] Preferably, the fermentation culture conditions include: an inoculum size of 5-15% by volume, an initial pH of 6-7, a rotation speed of 150-200 rpm, a temperature of 25-30°C, and a time of 100-150 h.

[0017] Preferably, the oil-producing microorganism is selected from at least one of Schizochytrium, Alpine Monascus, and microalgae, with Schizochytrium being the most preferred.

[0018] A second aspect of the present invention provides a method for preparing microbial oil, the method comprising: culturing oil-producing microorganisms using the method provided in the aforementioned technical solution to obtain a culture medium, breaking the cell walls of the culture medium and extracting it to obtain an extract, and drying the extract.

[0019] Preferably, the cell wall disruption is achieved by enzymatic hydrolysis using cell wall disrupting enzymes, wherein the cell wall disrupting enzymes are cellulase and / or alkaline protease.

[0020] Preferably, the amount of the cell-wall-breaking enzyme used is 3-6g relative to 1L of the culture medium, and the enzymatic hydrolysis conditions of the cell-wall-breaking enzyme include: initial pH of 10-12, rotation speed of 150-200rpm, temperature of 25-30℃, and time of 4-6h.

[0021] Preferably, the extraction solvent is n-hexane.

[0022] The beneficial effects of the present invention through the above technical solution are as follows:

[0023] The technical solution provided by this invention involves the exogenous addition of a certain proportion of tetracycline during the cultivation of oil-producing microorganisms, especially the addition of tetracycline to the seed culture medium. When applied to the fermentation process of Schizochytrium, it can exert a positive regulatory effect on the growth process of microorganisms, causing carbon to flow towards the biosynthesis of polyunsaturated fatty acids. This effectively increases the yield of polyunsaturated fatty acids in oil-producing microorganisms, altering the fatty acid composition of microbial oils. Consequently, the content of docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), eicosapentaenoic acid (ARA), and total unsaturated fatty acids in microbial oils are significantly increased. Moreover, this method is simple to operate, has low production costs, and has significant development value. Detailed Implementation

[0024] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0025] In a first aspect, the present invention provides a method for increasing the yield of polyunsaturated fatty acids in microorganisms, the method comprising: inoculating oil-producing microorganisms into a culture medium supplemented with tetracycline for cultivation.

[0026] During their research, the inventors of this invention unexpectedly discovered that adding a certain proportion of tetracycline exogenously during the cultivation of oil-producing microorganisms can have a positive regulatory effect on the microbial growth process, causing carbon to flow towards the biosynthesis of polyunsaturated fatty acids, effectively increasing the yield of polyunsaturated fatty acids in oil-producing microorganisms, thus changing the fatty acid composition in microbial oils, and consequently significantly increasing the content of docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), eicosapentaenoic acid (ARA), and total unsaturated fatty acids in microbial oils.

[0027] According to the present invention, the content of tetracycline in the culture medium is not particularly limited, as long as it can play a regulatory role and enhance the synthesis of lipids and polyunsaturated fatty acids by oleogenous microorganisms during their growth. To further increase the oil content and polyunsaturated fatty acid content after culturing the oleogenous microorganisms, preferably, the amount of tetracycline added to the culture medium is 1-5 mg / L, specifically 1 mg / L, 2 mg / L, 3 mg / L, 4 mg / L, 5 mg / L, or any value within the range of any two of the above values. More preferably, the amount of tetracycline added to the culture medium is 1.5-2.5 mg / L.

[0028] According to the present invention, the oil-producing microorganism can be any microorganism capable of synthesizing oils containing polyunsaturated fatty acids through cultivation. Preferably, the oil-producing microorganism is selected from at least one of Schizochytrium, Algalocarpus, and microalgae, more preferably Schizochytrium. The inventors have found that, under this preferred embodiment, tetracycline can exert a better promoting effect on the growth of Schizochytrium and the synthesis of polyunsaturated fatty acids. Exemplarily, the Schizochytrium used in this invention is Schizochytrium sp. HX-308, which was independently isolated and screened by the inventors' laboratory and is now deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M209059, and has been published in patent application with publication number CN101575584A.

[0029] According to the present invention, preferably, the cultivation process includes: inoculating the oil-producing microorganism into a seed culture medium for at least one subculture to obtain a seed culture solution, and inoculating the seed culture solution into a fermentation culture medium for fermentation culture, wherein the tetracycline is added to the seed culture medium and / or the fermentation culture medium.

[0030] According to the present invention, tetracycline can be added to the seed culture medium, the fermentation culture medium, or both. Preferably, the tetracycline is added to the seed culture medium. The inventors have found that, under this preferred embodiment, it is beneficial to enhance the effect of tetracycline in promoting the synthesis of polyunsaturated fatty acids (DHA, EPA, ARA) during microbial growth.

[0031] According to the present invention, before the oil-producing microorganisms are inoculated into the seed culture medium for subculture, they need to be activated. Specifically, the activation treatment can be to inoculate the oil-producing microorganism strains into the seed culture medium for activation culture to obtain an activated culture solution.

[0032] According to the present invention, during activation culture and subculture, the inoculation amount of oil-producing microorganisms in the seed culture medium is 0.5-2% by volume; if microbial strains preserved in glycerol tubes are used for inoculation, the strains in each glycerol tube are inoculated into 100 mL of the first culture medium. The parameters such as temperature, pH, rotation speed, and time used for activation culture and subculture can be conventional settings in the art. Preferably, the conditions for activation culture and each subculture include: an initial pH of 6-7, specifically 6, 6.2, 6.4, 6.6, 6.8, 7, or any value within the range of any two of the above values; a rotation speed of 150-200 rpm, specifically 150 rpm, 160 rpm, 170 rpm, 180 rpm, 190 rpm, 200 rpm, or any value within the range of any two of the above values; a temperature of 25-30℃, specifically 25℃, 26℃, 27℃, 28℃, 29℃, 30℃, or any value within the range of any two of the above values; and a time of 20-30 h, specifically 20 h, 22 h, 24 h, 26 h, 28 h, 30 h, or any value within the range of any two of the above values. The inventors have found that under this preferred embodiment, it is beneficial to promote the growth of oil-producing microorganisms and increase the biomass of fermentation.

[0033] According to the present invention, the subculture of oil-producing microorganisms refers to the process of transferring a portion of the culture of oil-producing microorganisms to a new culture medium and then culturing it again. There is no particular limitation on the number of subcultures, as long as tetracycline can promote the synthesis of polyunsaturated fatty acids by the oil-producing microorganisms. Preferably, the number of subcultures is 5-15 times, specifically 5, 7, 9, 11, 13, or 15 times, or any value within the range of any two of the above values. More preferably, the number of subcultures is 8-12 times.

[0034] According to the present invention, preferably, the seed culture medium contains: a carbon source, a nitrogen source, inorganic salts, and amino acid salts, wherein the carbon source, nitrogen source, inorganic salts, and amino acid salts can be any one of the corresponding substances used for microbial fermentation. For example, the carbon source can be glucose, the nitrogen source can be yeast extract powder and / or corn steep liquor powder, the inorganic salts can be any one or more of phosphates, sulfates, and chlorides, and the amino acid salts can be any one or more of glutamate, lysine, and alanine.

[0035] To further increase the content of polyunsaturated fatty acids in the oil produced by oil-producing microorganisms, preferably, the seed culture medium contains: glucose, KH2PO4, Na2SO4, MgSO4, KCl, (NH4)2SO4, CaCl2, K2SO4, monosodium glutamate, and yeast extract. More preferably, the seed culture medium contains: 30-60 g / L glucose, 1-5 g / L KH2PO4, 10-14 g / L Na2SO4, 2-5 g / L MgSO4, 1-3 g / L KCl, 3-5 g / L (NH4)2SO4, 0.06-1 g / L CaCl2, 0.1-1 g / L K2SO4, 8-12 g / L monosodium glutamate, and 9-15 g / L yeast extract.

[0036] According to the present invention, preferably, the fermentation medium contains: a carbon source, a nitrogen source, inorganic salts, organic acids, and amino acid salts. The carbon source, nitrogen source, inorganic salts, organic acids, and amino acid salts can be any one of the corresponding substances used for microbial fermentation. For example, the carbon source can be glucose, the nitrogen source can be yeast extract powder and / or corn steep liquor powder, the inorganic salts can be any one or more of phosphates, sulfates, and chlorides, the organic acids can be any one or more of malic acid, citric acid, and alanine, and the amino acid salts can be any one or more of glutamate, lysine, and alanine.

[0037] According to the present invention, in order to further increase the content of polyunsaturated fatty acids in the oil produced by oil-producing microorganisms, preferably, the fermentation medium contains: glucose, KH2PO4, (NH4)2SO4, malic acid, monosodium glutamate, yeast extract powder, corn steep liquor powder, and sea salt. Preferably, the fermentation medium contains: 70-90 g / L glucose, 0.5-2 g / L KH2PO4, 3-5 g / L (NH4)2SO4, 0.5-2 g / L malic acid, 8-12 g / L monosodium glutamate, 8-12 g / L yeast extract powder, 4-6 g / L corn steep liquor powder, and 30-35 g / L sea salt.

[0038] According to the present invention, the parameters used in the fermentation culture, such as inoculum size, temperature, pH, rotation speed, and time, can be conventional settings within the art. Preferably, the fermentation culture conditions include: an inoculum size of 5-15% by volume; an initial pH of 6-7, specifically 6, 6.2, 6.4, 6.6, 6.8, or 7, or any value within the range formed by any two of the above values; a rotation speed of 150-200 rpm, specifically 150 rpm, 160 rpm, 170 rpm, 180 rpm, 190 rpm, or 200 rpm, or any value within the range formed by any two of the above values; a temperature of 25-30°C, specifically 25°C, 26°C, 27°C, 28°C, 29°C, or 30°C, or any value within the range formed by any two of the above values; and a time of 100-150 h, specifically 100 h, 110 h, 120 h, 130 h, 140 h, or 150 h, or any value within the range formed by any two of the above values. The inventors discovered that, under this preferred embodiment, it is beneficial to promote the growth of oil-producing microorganisms and increase the biomass of fermentation.

[0039] According to the present invention, subculturing and fermentation can be carried out separately in shake flasks or in fermenters, depending on the scale of the fermentation culture of the oil-producing microorganisms. Accordingly, when using fermenters, the conditions for subculturing and fermentation can be optimized according to the actual situation.

[0040] Based on the above-mentioned method for increasing the yield of polyunsaturated fatty acids in microorganisms, a second aspect of the present invention provides a method for preparing microbial oil, the method comprising: culturing oil-producing microorganisms using the method provided in the aforementioned technical solution to obtain a culture medium, breaking the cell walls of the culture medium and extracting it to obtain an extract, and drying the extract.

[0041] According to the present invention, cell wall disruption of oil-producing microorganisms can be carried out using conventional methods in the art. Preferably, the cell wall disruption is carried out by enzymatic hydrolysis with cell wall disrupting enzymes, which can improve the cell wall disruption efficiency and reduce the damage to the metabolites in the microbial cells.

[0042] In this invention, after the culture medium is enzymatically hydrolyzed and the cell walls are broken, the resulting broken-cell solution is mixed with anhydrous ethanol to precipitate the proteins in the broken-cell solution, thereby improving the extraction efficiency and yield of microbial lipids. The amount of anhydrous ethanol used is not particularly limited, as long as it is sufficient to completely precipitate the proteins in the broken-cell solution. Preferably, the volume ratio of the broken-cell solution to anhydrous ethanol is 1:0.8-2.

[0043] According to the present invention, preferably, the cell wall-breaking enzyme is cellulase and / or alkaline protease. More preferably, the amount of the cell wall-breaking enzyme used is 3-6 g relative to 1 L of the culture medium, specifically 3 g / L, 4 g / L, 5 g / L, 6 g / L, or any value within the range formed by any two of the above values.

[0044] According to the present invention, preferably, the conditions for the enzymatic hydrolysis by the cell-wall breaking enzyme include: an initial pH of 10-12, specifically 10, 11, 12, or any value within the range formed by any two of the above values; a rotation speed of 150-200 rpm, specifically 150 rpm, 160 rpm, 170 rpm, 180 rpm, 190 rpm, 200 rpm, or any value within the range formed by any two of the above values; a temperature of 25-30℃, specifically 25℃, 30℃, 35℃, or any value within the range formed by any two of the above values; and a time of 4-6 h, specifically 4 h, 5 h, 6 h, or any value within the range formed by any two of the above values.

[0045] According to the present invention, in order to improve the extraction efficiency of microbial oils, the extraction solvent is preferably n-hexane. When using n-hexane to extract microbial oils, the amount of n-hexane used is not particularly limited, and multiple extraction and layering processes can be performed until the extract is transparent and colorless, thereby improving the extraction yield of microbial oils.

[0046] According to a particularly preferred embodiment of the present invention, a method for preparing microbial oil includes the following steps:

[0047] (1) Inoculate the Schizochytrium strain into the seed culture medium at an inoculation amount of 0.5-2% by volume, and activate it for 20-30 days under the conditions of initial pH of 6-7, rotation speed of 150-200 rpm and temperature of 25-30℃ to obtain the activated culture solution.

[0048] (2) The activated culture solution obtained in step (1) is inoculated into a seed culture medium containing 1-5 mg / L tetracycline at an inoculation amount of 0.5-2% by volume and then subcultured for 5-15 times to obtain the seed culture solution. The conditions for each subculture include: initial pH of 6-7, rotation speed of 150-200 rpm, temperature of 25-30℃, and time of 20-30 h.

[0049] (3) The seed culture obtained in step (2) is inoculated into a fermenter containing fermentation medium at an inoculation amount of 5-15% by volume. Fermentation is carried out for 100-150 hours under the conditions of initial pH of 6-7, rotation speed of 150-200 rpm and temperature of 25-30℃ to obtain fermentation culture.

[0050] (4) Add the cell wall breaking enzyme to the fermentation culture obtained in step (3) at an addition amount of 3-6 g / L, and enzymatically hydrolyze it for 4-6 h at a pH of 10-12, a rotation speed of 150-200 rpm and a temperature of 25-30℃ to obtain the cell wall breaking liquid. Mix the cell wall breaking liquid with anhydrous ethanol, and then mix it with n-hexane to extract the aqueous phase, n-hexane phase and solids. Remove the n-hexane phase by rotary evaporation and then dry it to obtain microbial oil.

[0051] The seed culture medium contains: 30-60 g / L glucose, 1-5 g / L KH2PO4, 10-14 g / L Na2SO4, 2-5 g / L MgSO4, 1-3 g / L KCl, 3-5 g / L (NH4)2SO4, 0.06-1 g / L CaCl2, 0.1-1 g / L K2SO4, 8-12 g / L monosodium glutamate, and 9-15 g / L yeast extract; the fermentation culture medium contains: 70-90 g / L glucose, 0.5-2 g / L KH2PO4, 3-5 g / L (NH4)2SO4, 0.5-2 g / L malic acid, 8-12 g / L monosodium glutamate, 8-12 g / L yeast extract, 4-6 g / L corn steep liquor powder, and 30-35 g / L sea salt.

[0052] The microbial oil prepared by the method provided in the above preferred embodiments has significantly increased contents of DHA, ARA and EPA.

[0053] The present invention will be described in detail below through embodiments.

[0054] In the following examples and comparative examples, the Schizochytrium sp. HX-308 was isolated and screened independently by our laboratory from coastal areas and is currently deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M209059, as described in patent application CN101575584A. Tetracycline was purchased from Shanghai Aladdin Reagent Co., Ltd., and other raw materials and reagents were all conventional commercially available products.

[0055] In the following examples and comparative examples, experimental methods without specific conditions are generally performed under conventional conditions or as recommended by the manufacturer.

[0056] The biomass was measured as follows: 5 mL of fermentation broth was placed in a pre-weighed centrifuge tube, centrifuged to remove the supernatant, dried in an oven to constant weight, and then weighed for calculation.

[0057] The test method for fatty acid composition in microbial oils is as follows:

[0058] a. Methylation of microbial oil: Add 1 mL of potassium hydroxide methanol solution to a 20 mL volumetric flask, add 50 μL of microbial oil and mix well, then add 2 mL of potassium hydroxide methanol solution to the volumetric flask, mix well, and incubate at 65 °C for 17 min, then cool to room temperature; add 2 mL of boron trifluoride diethyl ether (boron trifluoride to diethyl ether volume ratio = 3:7), mix well, and incubate at 65 °C for 7 min; then add 2 mL of saturated sodium chloride and shake well, add 3 mL of n-hexane (chromatographic grade), and allow to stand for layering;

[0059] b. After separation in step a, the upper liquid is passed through a microporous membrane to remove impurities. After sample preparation, it is tested using a Shimadzu DB-23 gas chromatograph to detect fatty acid components. The gas chromatographic test method is as follows: Refer to the chromatographic conditions for detecting docosahexaenoic acid (DHA) in Chen Lizhu's "Test Method for the Production of Docosahexaenoic Acid by Fermentation of Schizochytrium". Select a capillary column (60m*0.32nm*15um) and use programmed temperature rise: initial temperature 200℃, hold for 2min, then increase to 240℃ at 10℃ / min and hold for 40min; column pressure 200kPa, injection port temperature 250℃, detector temperature 280℃.

[0060] Example 1

[0061] (1) The seed culture medium is formulated as follows: 45 g / L glucose, 3 g / L KH2PO4, 12 g / L Na2SO4, 3 g / L MgSO4, 2 g / L KCl, 4 g / L (NH4)2SO4, 0.08 g / L CaCl2, 0.5 g / L K2SO4, 10 g / L sodium glutamate and 12 g / L yeast extract powder. It is sterilized at 121℃ for 20 min and then set aside. The fermentation culture medium is formulated as follows: 80 g / L glucose, 1 g / L KH2PO4, 4 g / L (NH4)2SO4, 1 g / L malic acid, 10 g / L monosodium glutamate, 10 g / L yeast extract powder, 5 g / L corn steep liquor powder and 32 g / L sea salt. It is sterilized at 121℃ for 20 min and then set aside.

[0062] (2) The strain of Schizochytrium HX-308 was inoculated into the seed culture medium at an inoculation amount of 1% by volume. After activation culture for 24 hours at an initial pH of 6.5, a rotation speed of 170 rpm and a temperature of 28℃, the activated culture solution was obtained.

[0063] (3) The activated culture solution obtained in step (2) was inoculated into a seed culture medium containing 2 mg / L tetracycline at an inoculation amount of 1% by volume and then subcultured 10 times to obtain the seed culture solution. The conditions for each subculture included: initial pH of 6.5, rotation speed of 170 rpm, temperature of 28℃, and time of 24 h.

[0064] (4) The seed culture obtained in step (3) was inoculated into a fermenter containing fermentation medium at an inoculation amount of 10% by volume. Fermentation was carried out for 120 hours at an initial pH of 6.5, a rotation speed of 170 rpm, and a temperature of 28°C to obtain the fermentation culture.

[0065] (5) Add the cell wall breaking enzyme to the fermentation culture obtained in step (4) at a dosage of 4 g / L. Enzymatically hydrolyze for 5 h at pH 11, rotation speed of 170 rpm and temperature of 28℃ to obtain cell wall breaking liquid. Mix the cell wall breaking liquid with anhydrous ethanol at a volume ratio of 1:1, and then mix with n-hexane for repeated extraction until the n-hexane is transparent and colorless to obtain aqueous phase, n-hexane phase and solids. Extract the n-hexane phase by rotary evaporation at 40℃. Weigh the bottle before rotary evaporation. Rotate until no more condensed water drips. After rotary evaporation, place it in an oven to dry to constant weight to obtain microbial oil.

[0066] The biomass of the fermentation culture obtained in step (4) was determined, the oil content of the microbial oil obtained in step (5) was calculated, and the fatty acid components and their contents in the microbial oil were determined. The results are shown in Table 1.

[0067] Example 2

[0068] (1) The seed culture medium is formulated as follows: 60 g / L glucose, 5 g / L KH2PO4, 14 g / L Na2SO4, 5 g / L MgSO4, 3 g / L KCl, 5 g / L (NH4)2SO4, 0.1 g / L CaCl2, 1 g / L K2SO4, 12 g / L sodium glutamate and 15 g / L yeast extract powder. It is sterilized at 121℃ for 20 min and then used for later use. The fermentation culture medium is formulated as follows: 90 g / L glucose, 2 g / L KH2PO4, 5 g / L (NH4)2SO4, 2 g / L malic acid, 12 g / L monosodium glutamate, 12 g / L yeast extract powder, 6 g / L corn steep liquor powder and 35 g / L sea salt. It is sterilized at 121℃ for 20 min and then used for later use.

[0069] (2) The strain of Schizochytrium HX-308 was inoculated into the seed culture medium at an inoculation amount of 0.5% by volume. After activation culture for 30 h at an initial pH of 6, a rotation speed of 150 rpm and a temperature of 25℃, the activated culture solution was obtained.

[0070] (3) The activated culture solution obtained in step (2) was inoculated into a seed culture medium containing 2.5 mg / L tetracycline at an inoculation amount of 0.5% by volume and then subcultured 8 times to obtain the seed culture solution. The conditions for each subculture included: initial pH of 6, rotation speed of 150 rpm, temperature of 25℃, and time of 30 h.

[0071] (4) The seed culture solution obtained in step (3) was inoculated into a fermenter containing fermentation medium at an inoculation amount of 7% by volume. Fermentation was carried out for 150 hours at an initial pH of 6, a rotation speed of 150 rpm and a temperature of 25°C to obtain the fermentation culture solution.

[0072] (5) Add the cell wall breaking enzyme to the fermentation culture obtained in step (4) at a dosage of 6 g / L. Enzymatically hydrolyze for 6 h at pH 10, rotation speed of 150 rpm and temperature of 25℃ to obtain cell wall breaking liquid. Mix the cell wall breaking liquid with anhydrous ethanol at a volume ratio of 1:1.5, and then mix with n-hexane for repeated extraction until the n-hexane is transparent and colorless to obtain aqueous phase, n-hexane phase and solids. Extract the n-hexane phase by rotary evaporation at 40℃. Weigh the bottle before rotary evaporation. Rotate until no more condensed water drips. After rotary evaporation, place it in an oven to dry to constant weight to obtain microbial oil.

[0073] The biomass of the fermentation culture obtained in step (4) was determined, the oil content of the microbial oil obtained in step (5) was calculated, and the fatty acid components and their contents in the microbial oil were determined. The results are shown in Table 1.

[0074] Example 3

[0075] (1) The seed culture medium is formulated as follows: 30 g / L glucose, 1 g / L KH2PO4, 10 g / L Na2SO4, 2 g / L MgSO4, 1 g / L KCl, 3 g / L (NH4)2SO4, 0.06 g / L CaCl2, 0.1 g / L K2SO4, 8 g / L sodium glutamate and 9 g / L yeast extract powder. It is sterilized at 121℃ for 20 min and then used for later use. The fermentation culture medium is formulated as follows: 70 g / L glucose, 0.5 g / L KH2PO4, 3 g / L (NH4)2SO4, 0.5 g / L malic acid, 8 g / L monosodium glutamate, 8 g / L yeast extract powder, 4 g / L corn steep liquor powder and 30 g / L sea salt. It is sterilized at 121℃ for 20 min and then used for later use.

[0076] (2) The strain of Schizochytrium HX-308 was inoculated into the seed culture medium at an inoculation amount of 1.5% by volume. After activation culture for 20 h at an initial pH of 7, a rotation speed of 200 rpm and a temperature of 30℃, the activated culture solution was obtained.

[0077] (3) The activated culture solution obtained in step (2) was inoculated into a seed culture medium containing 1.5% by volume at an inoculation amount of 1.5 mg / L tetracycline and then subcultured 12 times to obtain the seed culture solution. The conditions for each subculture included: initial pH of 7, rotation speed of 200 rpm, temperature of 30℃, and time of 20 h.

[0078] (4) The seed culture obtained in step (3) was inoculated into a fermenter containing fermentation medium at an inoculation amount of 5% by volume. Fermentation was carried out for 100 hours at an initial pH of 7, a rotation speed of 200 rpm, and a temperature of 30°C to obtain the fermentation culture.

[0079] (5) Add the cell wall breaking enzyme to the fermentation culture obtained in step (4) at a dosage of 4 g / L. Under the conditions of pH 12, rotation speed of 200 rpm and temperature of 30℃, the cell wall breaking liquid is obtained after 4 h of enzymatic hydrolysis. The cell wall breaking liquid is mixed with anhydrous ethanol at a volume ratio of 1:0.8 and then mixed with n-hexane for repeated extraction until the n-hexane is transparent and colorless, so as to obtain the aqueous phase, the n-hexane phase and the solids. The n-hexane phase is extracted by rotary evaporation at 40℃. Before rotary evaporation, the bottle is weighed. Rotary evaporation is carried out until no more condensed water drips. After rotary evaporation, the bottle is placed in an oven to dry to constant weight to obtain microbial oil.

[0080] The biomass of the fermentation culture obtained in step (4) was determined, the oil content of the microbial oil obtained in step (5) was calculated, and the fatty acid components and their contents in the microbial oil were determined. The results are shown in Table 1.

[0081] Example 4

[0082] Microbial oil was prepared according to the method of Example 1, except that in step (3), the amount of tetracycline added to the seed culture medium was replaced with 1 mg / L.

[0083] The biomass of the fermentation culture obtained in step (4) was determined, the oil content of the microbial oil obtained in step (5) was calculated, and the contents of DHA and EPA in the microbial oil were determined. The results are shown in Table 1.

[0084] Example 5

[0085] Microbial oil was prepared according to the method of Example 1, except that in step (3), the amount of tetracycline added to the seed culture medium was replaced with 5 mg / L.

[0086] The biomass of the fermentation culture obtained in step (4) was determined, the oil content of the microbial oil obtained in step (5) was calculated, and the contents of DHA and EPA in the microbial oil were determined. The results are shown in Table 1.

[0087] Example 6

[0088] Microbial oil was prepared according to the method in Example 1, except that in step (3), the number of subcultures was replaced with 5.

[0089] The biomass of the fermentation culture obtained in step (4) was determined, the oil content of the microbial oil obtained in step (5) was calculated, and the contents of DHA and EPA in the microbial oil were determined. The results are shown in Table 1.

[0090] Example 7

[0091] Microbial oil was prepared according to the method in Example 1, except that in step (3), the number of subcultures was replaced with 15.

[0092] The biomass of the fermentation culture obtained in step (4) was determined, the oil content of the microbial oil obtained in step (5) was calculated, and the contents of DHA and EPA in the microbial oil were determined. The results are shown in Table 1.

[0093] Example 8

[0094] Microbial oil was prepared according to the method of Example 1, except that steps (3) and (4) were replaced with:

[0095] (3) The activated culture solution obtained in step (2) was inoculated into a seed culture medium at an inoculation amount of 1% by volume and then subcultured 10 times to obtain the seed culture solution. The conditions for each subculture included: initial pH of 6.5, rotation speed of 170 rpm, temperature of 28℃, and time of 24h.

[0096] (4) The seed culture obtained in step (3) was inoculated into a fermenter containing a fermentation medium containing 2 mg / L tetracycline at an inoculation amount of 10% by volume. Fermentation was carried out for 120 h at an initial pH of 6.5, a rotation speed of 170 rpm and a temperature of 28 °C to obtain the fermentation culture.

[0097] The biomass of the fermentation culture obtained in step (4) was determined, the oil content of the microbial oil obtained in step (5) was calculated, and the fatty acid components and their contents in the microbial oil were determined. The results are shown in Table 1.

[0098] Example 9

[0099] Microbial oil was prepared according to the method in Example 1, except that step (3) was replaced with:

[0100] The activated culture solution obtained in step (2) was inoculated into a seed culture medium containing 2 mg / L tetracycline at an inoculation amount of 1% by volume. Seed culture was carried out for 24 h at an initial pH of 6.5, a rotation speed of 170 rpm and a temperature of 28°C to obtain the seed culture solution.

[0101] The biomass of the fermentation culture obtained in step (4) was determined, the oil content of the microbial oil obtained in step (5) was calculated, and the fatty acid components and their contents in the microbial oil were determined. The results are shown in Table 1.

[0102] Example 10

[0103] Microbial oil was prepared according to the method of Example 1, except that steps (3) and (4) were replaced with:

[0104] (3) The activated culture solution obtained in step (2) was inoculated into a seed culture medium containing 2 mg / L tetracycline at an inoculation amount of 1% by volume and then subcultured 10 times to obtain the seed culture solution. The conditions for each subculture included: initial pH of 6.5, rotation speed of 170 rpm, temperature of 28℃, and time of 24 h.

[0105] (4) The seed culture obtained in step (3) was inoculated into a fermenter containing a fermentation medium containing 2 mg / L tetracycline at an inoculation amount of 10% by volume. Fermentation was carried out for 120 h at an initial pH of 6.5, a rotation speed of 170 rpm and a temperature of 28 °C to obtain the fermentation culture.

[0106] The biomass of the fermentation culture obtained in step (4) was determined, the oil content of the microbial oil obtained in step (5) was calculated, and the fatty acid components and their contents in the microbial oil were determined. The results are shown in Table 1.

[0107] Example 11

[0108] Microbial oil was prepared according to the method of Example 1, except that step (1) was replaced with:

[0109] (1) The seed culture medium is formulated as follows: 45 g / L glucose, 3 g / L KH2PO4, 12 g / L Na2SO4, 3 g / L MgSO4, 2 g / L KCl, 4 g / L (NH4)2SO4, 0.08 g / L CaCl2, 0.5 g / L K2SO4, 10 g / L sodium glutamate and 12 g / L yeast extract powder. It is sterilized at 121℃ for 20 min and then used for later use. The fermentation culture medium is formulated as follows: 80 g / L glucose, 1 g / L KH2PO4, 4 g / L (NH4)2SO4, 1 g / L citric acid, 10 g / L monosodium glutamate, 10 g / L yeast extract powder, 5 g / L corn steep liquor powder and 32 g / L sea salt. It is sterilized at 121℃ for 20 min and then used for later use.

[0110] The biomass of the fermentation culture obtained in step (4) was determined, the oil content of the microbial oil obtained in step (5) was calculated, and the fatty acid components and their contents in the microbial oil were determined. The results are shown in Table 1.

[0111] Example 12

[0112] Microbial oil was prepared according to the method of Example 1, except that step (1) was replaced with:

[0113] (1) The seed culture medium is formulated as follows: 45 g / L glucose, 3 g / L KH2PO4, 12 g / L Na2SO4, 3 g / L MgSO4, 2 g / L KCl, 4 g / L (NH4)2SO4, 0.08 g / L CaCl2, 0.5 g / L K2SO4, 10 g / L sodium glutamate and 12 g / L yeast extract powder. It is sterilized at 121℃ for 20 min and then used for later use. The fermentation culture medium is formulated as follows: 80 g / L glucose, 1 g / L KH2PO4, 4 g / L (NH4)2SO4, 1 g / L malic acid, 10 g / L monosodium glutamate, 10 g / L yeast extract powder, 5 g / L corn steep liquor powder and 32 g / L NaCl. It is sterilized at 121℃ for 20 min and then used for later use.

[0114] The biomass of the fermentation culture obtained in step (4) was determined, the oil content of the microbial oil obtained in step (5) was calculated, and the fatty acid components and their contents in the microbial oil were determined. The results are shown in Table 1.

[0115] Comparative Example 1

[0116] Microbial oil was prepared according to the method in Example 1, except that step (3) was replaced with:

[0117] (3) The activated culture solution obtained in step (2) is inoculated into seed culture medium at an inoculation amount of 1% by volume and then subcultured 10 times to obtain seed culture solution. The conditions for each subculture include: initial pH of 6.5, rotation speed of 170 rpm, temperature of 28℃, and time of 24h.

[0118] The biomass of the fermentation culture obtained in step (4) was determined, the oil content of the microbial oil obtained in step (5) was calculated, and the fatty acid components and their contents in the microbial oil were determined. The results are shown in Table 1.

[0119] In Table 1, the units g / L for biomass and oil content represent the biomass and oil content per liter of fermentation broth; the units % for ARA content, EPA content, DHA content, and total unsaturated fatty acid content represent the weight percentage of eicosapentaenoic acid, eicosapentaenoic acid, docosahexaenoic acid, and total unsaturated fatty acids in the oil, respectively.

[0120] Table 1

[0121]

[0122] As can be seen from the results in Table 1, when the method provided by the present invention was used for the fermentation of Schizochytrium in Examples 1-12, the content of polyunsaturated fatty acids, including docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and eicosatraenoic acid (ARA), in the oil was significantly increased compared with Comparative Example 1. This indicates that the addition of tetracycline in the culture medium can direct carbon to the biosynthesis of polyunsaturated fatty acids, change the fatty acid composition in the microbial oil, and effectively increase the content of ARA, EPA, DHA, and total unsaturated fatty acids in the microbial oil fermented by oil-producing microorganisms.

[0123] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A method for increasing the production of polyunsaturated fatty acids in a microorganism, characterized in that, The method comprises: culturing the oleaginous microorganism in a culture medium added with tetracycline; wherein the oleaginous microorganism is Schizochytrium sp., and the polyunsaturated fatty acid is selected from docosahexaenoic acid, eicosapentaenoic acid or docosapentaenoic acid. The culturing process comprises: inoculating the oleaginous microorganism into a seed culture medium to obtain a seed culture solution after 8-12 times of subculture, and inoculating the seed culture solution into a fermentation culture medium to perform fermentation culture, wherein the tetracycline is added in the seed culture medium, and the fermentation culture medium contains 70-90 g / L glucose, 0.5-2 g / L KH2PO4, 3-5 g / L (NH4)2SO4, 0.5-2 g / L malic acid, 8-12 g / L monosodium glutamate, 8-12 g / L yeast extract powder, 4-6 g / L corn steep liquor dry powder and 30-35 g / L sea salt; and the tetracycline is added in the culture medium in an amount of 1.5-2.5 mg / L.

2. The method of claim 1, wherein, The seed culture medium contains a carbon source, a nitrogen source and inorganic salts.

3. The method of claim 2, wherein, The seed culture medium contains 30-60 g / L glucose, 1-5 g / L KH2PO4, 10-14 g / L Na2SO4, 2-5 g / L MgSO4, 1-3 g / L KCl, 3-5 g / L (NH4)2SO4, 0.06-0.1 g / L CaCl2, 0.1-1 g / L K2SO4, 8-12 g / L sodium glutamate and 9-15 g / L yeast extract powder.

4. The method of claim 1, wherein, The subculture conditions comprise: an inoculation amount of 0.5-1.5% by volume, an initial pH of 6-7, a rotation speed of 150-200 rpm, a temperature of 25-30 DEG C and a time of 20-30 h.

5. The method of claim 4, wherein, The fermentation culture conditions comprise: an inoculation amount of 5-10% by volume, an initial pH of 6-7, a rotation speed of 150-200 rpm, a temperature of 25-30 DEG C and a time of 100-150 h.

6. A method for producing a microbial oil, characterized by, The preparation method comprises: culturing the oleaginous microorganism by the method in any one of claims 1 to 5 to obtain a culture solution, breaking the cell wall of the culture solution, extracting the culture solution to obtain an extract, and drying the extract.

7. The production method according to claim 6, wherein The cell wall breaking is performed by using a cell wall breaking enzyme, and the cell wall breaking enzyme is cellulase and / or alkaline protease.

8. The preparation method according to claim 7, characterized in that, The amount of the cell wall breaking enzyme is 4-6 g relative to 1 L of the culture solution; and the cell wall breaking enzyme hydrolysis conditions comprise: an initial pH of 10-12, a rotation speed of 150-200 rpm, a temperature of 25-30 DEG C and a time of 4-6 h. The extraction solvent is n-hexane.

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