Method for domesticating oil-producing microorganism, application and method for producing polyunsaturated fatty acid

Abstract

The invention relates to the field of microbial fermentation, and discloses a method for domesticating oil-producing microorganisms, application of the oil-producing microorganisms and a method for producing polyunsaturated fatty acid. The method for domesticating the oleaginous microorganisms comprises the step of domesticating the oleaginous microorganisms by using a domesticating culture medium containing gibberellin and / or vitamin B12. The invention also provides application of the oil-producing microorganism domestication strain obtained by domestication of the oil-producing microorganism domestication method in production of polyunsaturated fatty acid. The invention also provides a method for producing the polyunsaturated fatty acid. The method comprises the following step: fermenting the oil-producing microorganism domesticated strain obtained by domesticating according to the method. According to the method, directional domestication and transformation of high-yield polyunsaturated fatty acid of the oleaginous microorganisms are realized, the method has strong target property, the domestication method is simple and convenient, the efficiency is high, and the obtained domesticated strain of the oleaginous microorganisms is high in growth speed and high in yield of polyunsaturated fatty acid.

Description

Technical Field

[0001] The present invention relates to microbial fermentation technology, and in particular to a method and application of domesticating oil-producing microorganisms. In addition, the present invention also relates to a method for producing polyunsaturated fatty acids. Background Art

[0002] At present, the wide application of polyunsaturated fatty acids (PUFAs) in the fields of food and medicine has gradually increased their demand, especially the industrial production of docosahexaenoic acid (DHA) has made important progress. Traditionally, fish oil has been the main source of PUFAs. However, problems such as heavy metal pollution in the ocean and the reduction of fish have limited the quality and output of fish oil, which is insufficient to meet the needs of commercial development. In addition, the fatty acids in fish oil are complex and contain components that are not conducive to human absorption, which has forced the urgent need to find alternative methods to produce high-quality and pure PUFAs.

[0003] Marine microorganisms, especially algae, marine bacteria and fungi, are fish food, making marine fish rich in PUFAs, and are the real source of PUFAs. However, algae have strict requirements on conditions such as light and culture cycle, and the production of PUFAs by marine bacteria is limited. Marine fungi are represented by Schizochytrium, which has a fast growth rate, is easy to culture, and has a high oil content, but the content of PUFAs in the oil it produces still needs to be improved to meet market demand. Summary of the invention

[0004] The purpose of the present invention is to overcome the problem that the content of polyunsaturated fatty acids in the oil produced by marine fungi in the prior art needs to be improved, and to provide a method and application of domesticating oil-producing microorganisms and a method for producing polyunsaturated fatty acids. The method can effectively increase the content of polyunsaturated fatty acids in the oil of oil-producing microorganisms and reduce production costs.

[0005] In addition, the present invention also provides a method for producing polyunsaturated fatty acids, which has high yield and low production cost.

[0006] The inventors of the present application found in the process of research on the production of polyunsaturated fatty acids by fermentation of oleaginous microorganisms that adding a certain concentration of gibberellins and / or vitamin B12 to the fermentation medium 12 After domesticating oil-producing microorganisms, the production of polyunsaturated fatty acids can be increased, which indicates that the addition of gibberellins can promote the production of polyunsaturated fatty acids during the fermentation process of oil-producing microorganisms.

[0007] In order to achieve the above object and based on the above discovery, the present invention provides a method for domesticating oil-producing microorganisms in the first aspect, the method comprising: domesticating the oil-producing microorganisms with a mixture containing gibberellins and / or vitamin B 12The culture medium was used for acclimatization.

[0008] Preferably, the oil-producing microorganism is selected from at least one of Schizochytrium, Saccharomyces lipolytica and Mortierella alpina, preferably Schizochytrium.

[0009] Preferably, the domestication process comprises: using a mixture containing the gibberellin and the vitamin B 12 The oil-producing microorganism is domesticated using an domestication medium.

[0010] Further preferably, the added amount of the gibberellin is 0-10 mg / L.

[0011] More preferably, the vitamin B 12 The addition amount is 0-6g / L.

[0012] Preferably, during the acclimation process, the concentration of the gibberellin in the acclimation medium is increased in a gradient of 1-3 mg / L each time, and the vitamin B 12 The concentration was decreased in a gradient of 2-3 g / L each time.

[0013] More preferably, the acclimation time is 5-20 days.

[0014] More preferably, the number of generations of domestication is 3-20 generations.

[0015] Preferably, the acclimation medium also contains: 30-60 g / L glucose, 1-5 g / L KH 2 PO 4 、10-14g / LNa 2 SO 4 、2-5g / L MgSO 4 , 1-3g / L KCl, 3-5g / L (NH 4 ) 2 SO 4 , 0.06-1g / LCaCl 2 , 0.1-1g / LK 2 SO 4 , 8-12g / L MSG, 9-15g / L yeast powder.

[0016] Preferably, the conditions of each subculture include: inoculation volume of 0.5-2% by volume, initial pH of 6-7, rotation speed of 150-200 rpm, temperature of 25-30° C., and time of 20-30 h.

[0017] The second aspect of the present invention provides the use of the domesticated strain of oil-producing microorganisms obtained by the method described in the first aspect in the production of polyunsaturated fatty acids.

[0018] The third aspect of the present invention provides a method for producing polyunsaturated fatty acids, comprising the following steps: fermenting the domesticated strain of oil-producing microorganisms obtained by domestication according to the method described in the first aspect.

[0019] Preferably, the fermentation process comprises: culturing the domesticated strain of oil-producing microorganism in a fermentation medium containing gibberellin.

[0020] Further preferably, the content of gibberellin in the fermentation medium is 1-10 mg / L.

[0021] Further preferably, the fermentation medium further contains: 70-90 g / L glucose, 0.5-2 g / L KH 2 PO 4 、3-5g / L(NH 4 ) 2 SO 4 , 0.5-2g / L malic acid, 8-12g / L MSG, 8-12g / L yeast extract powder, 4-6g / L corn steep liquor powder, 30-35g / L sea salt.

[0022] Further preferably, the culture conditions include: inoculation amount of 5-15% by volume, initial pH of 6-7, rotation speed of 350-500 rpm, temperature of 25-30° C., and time of 100-150 h.

[0023] Preferably, the method further comprises: breaking the cell wall of the fermentation broth obtained by the fermentation with a cell wall breaking enzyme, mixing the fermentation broth with an extraction solvent, and extracting the polyunsaturated fatty acids by a subcritical fluid extraction method.

[0024] More preferably, the wall-breaking enzyme is selected from at least one of wall-lytic enzyme, snail enzyme and alkaline protease.

[0025] Further preferably, the amount of the cell wall-breaking enzyme used is 2-4 g relative to 1 L of the fermentation medium.

[0026] Further preferably, the conditions for enzymatic hydrolysis with the wall-breaking enzyme include: pH 10-12, rotation speed 120-150 rpm, temperature 50-60°C, and time 6-8h.

[0027] Further preferably, the extraction solvent is selected from at least one of propane, butane and dimethyl ether.

[0028] Through the above technical solution, the beneficial effects of the present invention are:

[0029] The domestication method of the oil-producing microorganism provided by the present invention is to produce oil-producing microorganisms by using gibberellins and vitamin B 12The invention has a domestication induction effect on oil-producing microorganisms, realizes the directional domestication transformation of oil-producing microorganisms with high polyunsaturated fatty acid production, has a strong target, and the domestication method is simple and efficient. The domesticated strain of oil-producing microorganisms obtained has a fast growth rate and a high polyunsaturated fatty acid production. Furthermore, the invention uses gibberellins and gradually reduces vitamin B 12 The oil-producing microorganisms are domesticated by adding a certain amount of oil, which changes the fatty acid composition of the oil-producing microorganism oil, thereby increasing the content of docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), eicosatetraenoic acid (ARA) and total unsaturated fatty acids in the oil-producing microorganism oil, promoting the anabolism of polyunsaturated fatty acids in the oil-producing microorganisms, and enabling the domesticated strains of oil-producing microorganisms to continuously and efficiently produce polyunsaturated fatty acids during the fermentation process, thereby achieving the purpose of high yield of polyunsaturated fatty acids. DETAILED DESCRIPTION

[0030] The endpoints and any values ​​of the ranges disclosed in this article 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 each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0031] In a first aspect, the present invention provides a method for taming an oil-producing microorganism, the method comprising: taming the oil-producing microorganism with a mixture containing gibberellins and / or vitamin B 12 The culture medium was used for acclimatization.

[0032] According to the present invention, the acclimation medium can be a liquid medium or a solid medium. In the process of preparing the acclimation medium, the corresponding gibberellin and / or vitamin B12 are added. 12 That's it.

[0033] According to the present invention, the oil-producing microorganisms can be domesticated using a domestication medium containing gibberellins, or vitamin B 12 Domestication of oil-producing microorganisms can also be achieved by using a mixture containing gibberellins and vitamin B 12 The oil-producing microorganisms are domesticated using a domestication medium containing gibberellins and a domestication medium containing vitamin B 12 The oil-producing microorganisms were domesticated using the domestication medium alone or alternately.

[0034] The inventors found that in this preferred embodiment, first use a mixture containing vitamin B 12 The acclimation medium can acclimate oil-producing microorganisms to gradually lack vitamin B 12 Under the conditions of normal growth, and then using gibberellins and no vitamin B12 The liquid domestication medium enables the domesticated oil-producing microorganisms to grow normally under conditions containing a certain concentration of gibberellins, so as to achieve high oil production during the fermentation process of the oil-producing microorganisms.

[0035] According to the present invention, preferably, the oil-producing microorganism is selected from at least one of Schizochytrium, Saccharomyces lipolytica and Mortierella alpina, and more preferably, the oil-producing microorganism is Schizochytrium. The inventors have found that when the oil-producing microorganism is Schizochytrium, the growth state after acclimation is the best, and the amount of oil produced by Schizochytrium fermentation can be increased.

[0036] According to the present invention, preferably, the domestication process comprises: using a mixture containing the gibberellin and the vitamin B 12 The oil-producing microorganism is domesticated with an acclimation medium. Further preferably, the amount of gibberellin added is 0-10 mg / L, specifically 0 mg / L, 1 mg / L, 3 mg / L, 5 mg / L, 7 mg / L, 9 mg / L, 10 mg / L, or any value between any two of the above values. Further preferably, the vitamin B 12 The addition amount is 0-6 g / L, specifically 0 g / L, 2 g / L, 4 g / L, 6 g / L, or any value between any two of the above values. The inventors found that under this preferred embodiment, the obtained domesticated strain of oil-producing microorganisms has the fastest growth rate and has a higher bacterial yield and oil content.

[0037] According to the present invention, preferably, during the acclimation process, the concentration of the gibberellin in the acclimation medium is increased in a gradient of 1-3 mg / L each time, specifically 1 mg / L, 2 mg / L, 3 mg / L, or any value between any two of the above values; the vitamin B 12 The concentration is decreased in a gradient of 2-3 mg / L each time, and can be 2 mg / L, 3 mg / L, or any value between any two of the above values. The inventors found that under this preferred embodiment, the obtained domesticated strain of oil-producing microorganisms has the fastest growth rate and has a higher bacterial yield and oil content.

[0038] According to the present invention, further preferably, the domestication time is 5-20 days, specifically 5 days, 10 days, 15 days, 20 days, or any value between any two of the above values. The inventors found that under this preferred embodiment, the domesticated strain of oil-producing microorganisms obtained has the fastest growth rate and has a higher bacterial yield and oil content.

[0039] According to the present invention, further preferably, the number of passages of the domestication is 3-20 generations, specifically 3 generations, 5 generations, 7 generations, 9 generations, 11 generations, 13 generations, 15 generations, 20 generations, or any value between any two of the above values. The inventors found that under this preferred embodiment, the obtained domesticated strain of oil-producing microorganisms has a better growth state, with higher bacterial yield and oil content.

[0040] According to the present invention, the growth state of the domesticated strain can be evaluated by biomass. The measurement of biomass can be measured by conventional test methods in the art. Exemplarily, the biomass of the oil-producing microorganism can be obtained by the following method: take 1 mL of fermentation broth in a centrifuge tube, centrifuge it at 4000-25000 rpm for 5-10 min for solid-liquid separation, remove the supernatant, wash the precipitate once with 2-4wt% sodium chloride solution, wash it once with distilled water, then put the precipitate in an oven, dry it at 100-110°C for 6-12 hours to constant weight, and weigh the precipitate weight as the biomass.

[0041] According to the present invention, preferably, the acclimation medium further contains: 30-60 g / L glucose, specifically 30 g / L, 40 g / L, 50 g / L, 60 g / L, or any value between any two of the above values; 1-5 g / L KH 2 PO 4 , specifically 1g / L, 2g / L, 3g / L, 4g / L, 5g / L, or any value between any two of the above values; 10-14g / L Na 2 SO 4 , specifically 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, or any value between any two of the above values; 2-5 g / L MgSO 4 , specifically 2g / L, 3g / L, 4g / L, 5g / L, or any value between any two of the above values; 1-3g / L KCl, specifically 1g / L, 2g / L, 3g / L, or any value between any two of the above values; 3-5g / L (NH 4 ) 2 SO 4 Specifically, it can be 3g / L, 4g / L, 5g / L, or any value between any two of the above values; 0.06-1g / L CaCl 2 , specifically 0.06g / L, 0.3g / L, 0.5g / L, 0.7g / L, 0.9g / L, 1g / L, or any value between any two of the above values; 0.1-1g / LK 2 SO 4, specifically 0.1g / L, 0.3g / L, 0.5g / L, 0.7g / L, 0.9g / L, 1g / L, or any value between any two of the above values; 8-12g / L monosodium glutamate, specifically 8g / L, 9g / L, 10g / L, 11g / L, 12g / L, or any value between any two of the above values; 9-15g / L yeast extract, specifically 9g / L, 11g / L, 13g / L, 15g / L, or any value between any two of the above values. The inventors have found that under this preferred embodiment, it is more conducive to the domestication of oil-producing microorganisms to produce high oils.

[0042] According to the present invention, preferably, the conditions of each subculture include: the inoculation amount is 0.5-2 volume%, specifically 0.5 volume%, 1 volume%, 1.5 volume%, 2 volume%, or any value between any two of the above values; the initial pH is 6-7, specifically 6, 7, or any value between any two of the above values; the speed is 150-200rpm, specifically 150rpm, 160rpm, 170rpm, 180rpm, 190rpm, 200rpm, or any value between any two of the above values; the temperature is 25-30℃, specifically 25℃, 26℃, 27℃, 28℃, 29℃, 30℃, or any value between any two of the above values; the time is 20-30h, specifically 20h, 25h, 30h, or any value between any two of the above values. The inventor found that under this preferred specific embodiment, the obtained oil-producing microbial domesticated strain grows faster and has a higher bacterial yield and oil content.

[0043] According to the present invention, the above-mentioned oil is the main source of polyunsaturated fatty acids (PUFAs), and the polyunsaturated fatty acids (PUFAs) include docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), eicosatetraenoic acid (ARA) and total unsaturated fatty acids. The inventors found that the content of polyunsaturated fatty acids in the oil produced by domesticated oil-producing microorganisms is significantly increased.

[0044] For example, taking the domestication of Schizochytrium to produce polyunsaturated fatty acids as an example, the domestication process may specifically include the following steps:

[0045] (1) inoculating Schizochytrium species on a seed culture medium at an inoculation amount of 0.5-2% by volume, culturing for 20-30 hours at an initial pH of 6-7, a rotation speed of 150-200 rpm, and a temperature of 25-30° C. to obtain a Schizochytrium seed solution;

[0046] (2) Inoculate the Schizochytrium seed solution in step (1) with vitamin B 12The concentration of 3-6 g / L liquid acclimation medium was cultured at an initial pH of 6-7, a rotation speed of 150-200 rpm, and a temperature of 25-30 ° C to obtain vitamin B of Schizochytrium. 12 First generation domesticated seed solution;

[0047] (3) Reduce the amount of vitamin B in the liquid acclimatization medium by 2-3 g / L 12 The concentration of vitamin B in step (2) is 12 First generation acclimated seed liquid inoculated with vitamin B 12 Alternatively, reduce the concentration of vitamin B in the acclimation medium. 12 concentration, continuing to acclimate and culture in the liquid acclimate medium until the Schizochytrium grows normally to form a seed liquid after the Schizochytrium subculture;

[0048] (4) inoculating the seed liquid of Schizochytrium subcultured in step (3) into a liquid acclimation medium having a gibberellin concentration of 0-10 mg / L, with an inoculation amount of 0.5-2% by volume, and culturing the medium at an initial pH of 6-7, a rotation speed of 150-200 rpm, and a temperature of 25-30° C. until the cells grow normally, thereby obtaining a first generation acclimation seed liquid of Schizochytrium gibberellin;

[0049] (5) increasing the concentration of gibberellins in the liquid acclimation medium by a gradient of 1-3 mg / L, inoculating the first generation acclimation seed liquid of Schizochytrium gibberellins in step (4) into the acclimation medium with increased concentration of gibberellins, or increasing the concentration of gibberellins in the acclimation medium, continuing acclimation culture in the liquid acclimation medium, and culturing until the cells grow normally;

[0050] (6) Repeat the cultivation and domestication according to the method of step (4) and step (5), the domestication time is 5-20 days, and the number of domestication passages is 3-20 generations, until the domesticated strain can be grown in the absence of vitamin B 12 It grows normally in a liquid acclimation medium with a gibberellin content of 1-10 mg / L, and the cell biomass is 18-22 g / L.

[0051] The second aspect of the present invention provides the use of the domesticated strain of oil-producing microorganisms obtained by the method described in the first aspect in the production of polyunsaturated fatty acids. The inventors have found that the oil-producing microorganisms domesticated by the above method are used in the production of polyunsaturated fatty acids, which can effectively increase the content of polyunsaturated fatty acids in the oil of the oil-producing microorganisms and reduce production costs.

[0052] The third aspect of the present invention provides a method for producing polyunsaturated fatty acids, comprising the following steps: fermenting the domesticated strain of oil-producing microorganisms obtained by domestication according to the method described in the first aspect.

[0053] The present invention has no particular restrictions on the fermentation culture conditions of the domesticated oil-producing microorganism strain obtained by the domestication method, as long as the domesticated oil-producing microorganism strain can be proliferated in large quantities in the fermentation medium through the culture method.

[0054] According to the present invention, preferably, the fermentation process comprises: culturing the domesticated strain of oil-producing microorganisms in a fermentation medium containing gibberellins. Further preferably, the content of the gibberellins in the fermentation medium is 1-10 mg / L, specifically 1 mg / L, 3 mg / L, 5 mg / L, 7 mg / L, 9 mg / L, 10 mg / L, or any value between any two of the above values. The inventors have found that under this preferred specific embodiment, the domesticated strain of oil-producing microorganisms can be promoted to synthesize polyunsaturated fatty acids during the fermentation process, increase the yield of polyunsaturated fatty acids, and reduce its production cost.

[0055] According to the present invention, further preferably, the fermentation medium further contains: 70-90 g / L glucose, specifically 70 g / L, 80 g / L, 90 g / L, or any value between any two of the above values; KH 2 PO 4 0.5-2 g / L, specifically 0.5 g / L, 1 g / L, 1.5 g / L, 2 g / L, or any value between any two of the above values; 3-5 g / L (NH 4 ) 2 SO 4, 0.5-2g / L malic acid, specifically 0.5g / L, 1g / L, 1.5g / L, 2g / L, or any value between any two of the above values; 8-12g / L monosodium glutamate, specifically 8g / L, 9g / L, 10g / L, 11g / L, 12g / L, or any value between any two of the above values; 8-12g / L yeast extract powder, specifically 8g / L, 9g / L, 10g / L, 11g / L, 12g / L, or any value between any two of the above values; 4-6g / L corn steep liquor powder, specifically 4g / L, 5g / L, 6g / L, or any value between any two of the above values; 30-35g / L sea salt, specifically 30g / L, 31g / L, 32g / L, 33g / L, 34g / L, 35g / L, or any value between any two of the above values. Further preferably, the culture conditions include: an inoculation amount of 5-15% by volume, specifically 5% by volume, 10% by volume, 15% by volume, or any value between any two of the above values; an initial pH of 6-7, specifically 6, 7, or any value between any two of the above values; a rotation speed of 350-500 rpm, specifically 350 rpm, 400 rpm, 450 rpm, 500 rpm, or any value between any two of the above values; a temperature of 25-30°C, specifically 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, or any value between any two of the above values; a time of 100-150h, specifically 100h, 125h, 150h, or any value between any two of the above values.

[0056] According to the present invention, the method for extracting fatty acids from the cells of oleaginous microorganisms can be ultrasonic assisted extraction, Soxhlet extraction, supercritical fluid extraction, accelerated solvent extraction, microwave assisted extraction, and subcritical fluid extraction, etc. The above methods can be extracted and separated by referring to the conventional extraction operation steps in the art.

[0057] According to the present invention, preferably, the method further comprises: the fermentation broth obtained by the fermentation is broken by a wall-breaking enzyme, and then mixed with an extraction solvent to extract the polyunsaturated fatty acids by subcritical fluid extraction. The inventors found that when the wall-breaking enzyme and the extraction solvent are added and collected by subcritical fluid extraction, the extraction rate of fatty acids is high, and the extraction process is carried out under closed, anaerobic and low-pressure conditions, which helps to protect the polyunsaturated fatty acids from oxidative damage and improve the extraction quality of polyunsaturated fatty acids. Further preferably, the wall-breaking enzyme is selected from at least one of lysozyme, snail enzyme and alkaline protease. Further preferably, relative to 1L of the fermentation medium, the amount of the wall-breaking enzyme is 2-4g, specifically 2g, 3g, 4g, or any value between any two of the above values. The inventors found that when the above preferred embodiment is adopted, the extraction rate of polyunsaturated fatty acids is higher and the product extraction quality is better. Exemplarily, the conditions of the subcritical fluid extraction method at least meet the following requirements: the temperature is 25-40°C and the pressure is 0.3-0.7MPa.

[0058] According to the present invention, further preferably, the conditions of the enzymolysis of the wall-breaking enzyme include: pH is 10-12, specifically 10, 11, 12, or any value between any two of the above values; the speed is 120-150rpm, specifically 120rpm, 130rpm, 140rpm, 150rpm, or any value between any two of the above values; the temperature is 50-60°C, specifically 50°C, 52°C, 54°C, 56°C, 58°C, 60°C, or any value between any two of the above values; the time is 6-8h, specifically 6h, 7h, 8h, or any value between any two of the above values. The inventor found that when the above preferred embodiment is adopted, the extraction rate of polyunsaturated fatty acids is higher and the product extraction quality is better.

[0059] According to the present invention, further preferably, the extraction solvent is selected from at least one of propane, butane and dimethyl ether. The extraction solvent can be one of propane, butane and dimethyl ether, can be propane and butane, can be propane and dimethyl ether, can be butane and dimethyl ether, can also be propane, butane and dimethyl ether. The inventors have found that when the above preferred embodiment is adopted, the extraction rate of polyunsaturated fatty acids is higher and the product extraction quality is better.

[0060] Exemplarily, the oil of the oleaginous microorganism in the method for producing polyunsaturated fatty acids is obtained by the following method:

[0061] (1) According to the volume of the fermentation medium, 2-4 g / L of cell wall breaking enzyme is added relative to 1 L of the fermentation medium, and then the pH of the fermentation medium is adjusted to 10-12 with 1-2 mol / L NaOH solution, and the cell wall is broken at a speed of 120-150 rpm, a temperature of 50-60° C., and a time of 6-8 h. During the cell wall breaking process, 0.1-0.5 mL of the solution can be taken and placed under a microscope to observe whether the degree of cell enzymolysis is complete, to ensure that the cells are completely enzymolyzed to obtain the feed liquid.

[0062] (2) extracting the feed liquid obtained in step (1) by subcritical fluid extraction, wherein the extraction solvent is selected from at least one of propane, butane, and dimethyl ether, and the extraction solvent is formed into a subcritical fluid under the conditions of a temperature of 25-40° C. and a pressure of 0.3-0.7 MPa, and is passed into an extraction device to mix with the feed liquid in reverse, and the oil is repeatedly extracted until the extract is colorless, and the extract is passed into an evaporation device to reduce the pressure and concentrate the solvent to obtain extracted oil;

[0063] (3) The extracted oil in step (2) is placed in an oven and dried at a temperature of 40-50° C. for 1-2 hours, and the oil mass is weighed.

[0064] According to the present invention, the fatty acid components in the oil can be determined by conventional testing methods in the prior art, and the determination of other indicators all adopts conventional methods in the prior art.

[0065] Exemplarily, the polyunsaturated fatty acid content in the above-mentioned oil-producing microbial oil can be determined by the following method:

[0066] 1) Perform methyl esterification on the oil: add 1-2mL of 0.8-1mol / L potassium hydroxide methanol solution to a volumetric flask, add 50-60μL of oil and mix well, then add 2-3mL of 0.8-1mol / L potassium hydroxide methanol solution to the volumetric flask, mix well, place in a water bath at 65-70℃ for 17-20min, and cool to room temperature; add 2-3mL of boron trifluoride ether (boron trifluoride: ether volume ratio is 1:2-3), mix well, and heat in a water bath at 65-70℃ for 7-10min; then add 2-3mL of saturated potassium chloride and shake well, and finally add 3-4mL of n-hexane and let stand for stratification;

[0067] 2) After stratification, the upper layer of liquid is passed through a microporous membrane to remove impurities, and after sample preparation, it is tested by gas chromatography to detect fatty acid components. The gas chromatography test method can refer to the chromatographic conditions of the detection method of docosahexaenoic acid (DHA) in "Federated Culture of Schizochytrium Fermentation to Produce Docosahexaenoic Acid" by Chen Lizhu et al.: Select a capillary column (60m×0.32nm×15μm). Use a program temperature rise: the initial temperature is 200-205℃, maintained for 2-3min, then raised to 230-240℃ at 5-10℃ / min, maintained for 35-40min, column pressure 190-200KPa, inlet temperature 245-250℃, detector temperature 275-280℃, and quantitatively measured by the internal standard method, with the internal standard being eicosanoic acid.

[0068] The present invention will be described in detail below through examples.

[0069] The glucose content in the following examples was measured by SBA-40E biosensor analyzer;

[0070] The polyunsaturated fatty acid content and composition parameters were tested by gas chromatography. The gas chromatography test method was determined using the chromatographic conditions of the docosahexaenoic acid test method in "Docosahexaenoic acid production by Schizochytrium culture fermentation" by Chen Lizhu et al. The gas chromatograph model was Shimadzu DB-23 from Japan.

[0071] The Schizochytrium used in the following examples is Schizochytrium sp. HX-308, which was provided by Professor Huang He's laboratory at Nanjing Normal University and is currently deposited in the China Center for Type Culture Collection (CCTCC) with a deposit number of CCTCCNO.M209059, which has been disclosed in CN104974944A.

[0072] Unless otherwise specified, all raw materials used were commercially available products.

[0073] Example 1

[0074] Seed culture medium: 60g / L glucose, 5g / L KH 2 PO 4 、14g / L Na 2 SO 4 、5g / L MgSO 4 , 3g / L KCl, 5g / L (NH 4 ) 2 SO 4 , 1g / L CaCl 2 , 1g / LK 2 SO 4 , 10g / L monosodium glutamate, 12g / L yeast extract powder;

[0075] Acclimation medium: seed medium + vitamin B 12 + Gibberellic acid (vitamin B 12 The concentrations are 0g / L, 2g / L, 4g / L, and 6g / L respectively; the gibberellin concentrations are 0mg / L, 1mg / L, 2mg / L, 3mg / L, 4mg / L, 5mg / L, 6mg / L, 7mg / L, 8mg / L, 9mg / L, and 10mg / L respectively);

[0076] Fermentation medium: fermentation medium + gibberellin (the concentrations of gibberellin are 1 mg / L, 2 mg / L, 3 mg / L, 4 mg / L, 5 mg / L, 6 mg / L, 7 mg / L, 8 mg / L, 9 mg / L, and 10 mg / L respectively);

[0077] Fermentation medium: 80g / L glucose, 2g / L KH 2 PO 4 , 5g / L(NH 4 ) 2 SO 4 , 2g / L malic acid, 12g / L monosodium glutamate, 12g / L yeast extract powder, 6g / L corn syrup powder, 35g / L sea salt;

[0078] During the acclimation process, biomass was determined by:

[0079] Take 1 mL of fermentation broth in a centrifuge tube and centrifuge it at 8000 rpm for 5 min to separate the solid and liquid. Remove the supernatant and wash the precipitate once with 2 wt% sodium chloride solution and then with distilled water. Then put the precipitate in an oven and dry it at 100 °C for 6 hours to constant weight. The weight of the precipitate is the biomass.

[0080] The specific domestication process is as follows:

[0081] (1) Inoculating 50 mL of seed culture medium with a starting strain of Schizochytrium (deposit number: CCTCCNO.M209059) at an inoculum volume of 2%, culturing for 24 h at an initial pH of 7, a rotation speed of 170 rpm, and a temperature of 28° C., to obtain a Schizochytrium seed solution;

[0082] (2) Inoculate the Schizochytrium seed solution in step (1) with vitamin B 12 In a liquid acclimation medium with a concentration of 6 g / L and a gibberellin concentration of 0 mg / L, the culture was carried out for 24 hours at an initial pH of 7, a rotation speed of 200 rpm, and a temperature of 30°C to obtain a first-generation acclimation seed solution of Schizochytrium;

[0083] (3) Reduce the amount of vitamin B in the liquid acclimatization medium by 2 g / L 12At the same time, the concentration of gibberellin (vitamin B 12 The concentrations of the acclimated schizochytrium in step (2) are 4 g / L, 2 g / L, 0 g / L, 0 g / L, 0 g / L, 0 g / L, 0 g / L, 0 g / L, 0 g / L, and 0 g / L, respectively, and the concentrations of the gibberellins are 1 mg / L, 2 mg / L, 3 mg / L, 4 mg / L, 5 mg / L, 6 mg / L, 7 mg / L, 8 mg / L, 9 mg / L, and 10 mg / L, respectively, and the schizochytrium in step (2) is continuously acclimated and cultured in the liquid acclimation medium until normal growth is obtained to obtain a gibberellin-acclimated first-generation schizochytrium bacterial liquid;

[0084] (4) Repeat the cultivation and domestication according to the method of step (3), the domestication time is 3 days, 5 days, 10 days, 15 days, and 20 days, and the number of domestication passages is 3 generations, 5 generations, 10 generations, 15 generations, and 20 generations, until the domesticated strain can have a gibberellin content of 1 mg / L, 2 mg / L, 3 mg / L, 4 mg / L, 5 mg / L, 6 mg / L, 7 mg / L, 8 mg / L, 9 mg / L, and 10 mg / L respectively and does not contain vitamin B 12 The strains grew normally in the liquid acclimation medium. Among them, the Schizochytrium acclimated for 20 generations grew the best, with the highest cell biomass of 20 g / L. This strain was counted as Schizochytrium acclimated strain I.

[0085] The polyunsaturated fatty acid fermentation performance of Schizochytrium domesticated strain I was investigated. Schizochytrium domesticated strain I was inoculated into the fermentation medium with an inoculation amount of 15% by volume, an initial pH of 6, a rotation speed of 450 rpm, a temperature of 28° C., and a time of 120 h.

[0086] After fermentation, the oil is collected by subcritical fluid extraction. The specific steps are as follows:

[0087] (1) According to the volume of the fermentation medium, 4 g / L of cell wall breaking enzyme was added relative to 1 L of the fermentation medium, and then the pH of the fermentation medium was adjusted to 12 with 1 mol / L NaOH solution. Cell wall breaking was carried out at a speed of 150 rpm, a temperature of 60°C, and a time of 8 h. During the cell wall breaking process, 0.1 mL of the solution was taken and placed under a microscope to observe whether the degree of cell enzymolysis was complete, to ensure that the cells were completely enzymolyzed to obtain the feed liquid.

[0088] (2) extracting the feed liquid obtained in step (1) by subcritical fluid extraction, wherein the extraction solvent is propane, and propane is formed into a subcritical fluid under the conditions of a temperature of 25° C. and a pressure of 0.5 MPa, and is introduced into an extraction device to mix with the feed liquid in reverse, and the oil is extracted repeatedly until the extract is colorless, and the extract is introduced into an evaporation device to reduce the pressure and concentrate the solvent to obtain extracted oil;

[0089] (3) The extracted oil in step (2) is placed in an oven and dried at 40° C. for 2 hours, and the oil mass is weighed.

[0090] The mass proportions of polyunsaturated fatty acids (PUFAs), eicosatetraenoic acid (ARA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in the above oils were determined, and the results are shown in Table 1.

[0091] Table 1

[0092]

[0093]

[0094] Example 2

[0095] Seed culture medium: 60g / L glucose, 5g / L KH 2 PO 4 、14g / L Na 2 SO 4 、5g / L MgSO 4 , 3g / L KCl, 5g / L (NH 4 ) 2 SO 4 , 1g / L CaCl 2 , 1g / LK 2 SO 4 , 10g / L monosodium glutamate, 12g / L yeast extract powder;

[0096] Acclimation medium 1: seed medium + vitamin B 12 (Vitamin B 12 The concentrations are 0g / L, 2g / L, 4g / L, and 6g / L respectively);

[0097] Acclimation medium 2: seed medium + gibberellin (the gibberellin concentrations were 0 mg / L, 1 mg / L, 2 mg / L, 3 mg / L, 4 mg / L, 5 mg / L, 6 mg / L, 7 mg / L, 8 mg / L, 9 mg / L, and 10 mg / L respectively);

[0098] Fermentation medium: fermentation medium + gibberellin (the concentrations of gibberellin are 1 mg / L, 2 mg / L, 3 mg / L, 4 mg / L, 5 mg / L, 6 mg / L, 7 mg / L, 8 mg / L, 9 mg / L, and 10 mg / L respectively);

[0099] Fermentation medium: 80g / L glucose, 2g / L KH 2 PO 4 , 5g / L(NH 4 ) 2 SO4 , 2g / L malic acid, 12g / L monosodium glutamate, 12g / L yeast extract powder, 6g / L corn syrup powder, 35g / L sea salt;

[0100] During the acclimation process, biomass was determined by:

[0101] Take 1 mL of fermentation broth in a centrifuge tube and centrifuge it at 8000 rpm for 5 min to separate the solid and liquid. Remove the supernatant and wash the precipitate once with 2 wt% sodium chloride solution and then with distilled water. Then put the precipitate in an oven and dry it at 100 °C for 6 hours to constant weight. The weight of the precipitate is the biomass.

[0102] The specific domestication process is as follows:

[0103] (1) Inoculating 50 mL of seed culture medium with a starting strain of Schizochytrium (deposit number: CCTCCNO.M209059) at an inoculum volume of 2%, culturing for 24 h at an initial pH of 7, a rotation speed of 170 rpm, and a temperature of 28° C., to obtain a Schizochytrium seed solution;

[0104] (2) Inoculate the Schizochytrium seed solution in step (1) with vitamin B 12 The concentration of 6g / L liquid acclimation medium was cultured for 24h at an initial pH of 7, a rotation speed of 200rpm, and a temperature of 30℃ to obtain vitamin B12 from Schizochytrium. 12 First generation domesticated seed solution;

[0105] (3) Reduce the amount of vitamin B in the liquid acclimatization medium by 2 g / L 12 Concentration (vitamin B 12 The concentrations of the Schizochytrium vitamin B in step (2) are 4 g / L, 2 g / L, and 0 g / L, respectively. 12 The first generation acclimated seed liquid is continuously acclimated and cultured in the liquid acclimation medium until the Schizochytrium grows normally to form the seed liquid after the Schizochytrium subculture;

[0106] (4) inoculating the seed liquid of Schizochytrium subcultured in step (3) into a liquid acclimation medium with a gibberellin concentration of 1 mg / L, with an inoculation amount of 2% by volume, and culturing for 24 hours at an initial pH of 7, a rotation speed of 150 rpm, and a temperature of 25° C. to obtain a first generation acclimation seed liquid of Schizochytrium gibberellin;

[0107] (5) increasing the concentration of gibberellins in the liquid acclimation medium by a gradient of 1 mg / L (the gibberellins concentrations are 2 mg / L, 3 mg / L, 4 mg / L, 5 mg / L, 6 mg / L, 7 mg / L, 8 mg / L, 9 mg / L, and 10 mg / L, respectively), and continuing to acclimate and culture the Schizochytrium in step (4) in the liquid acclimation medium until normal growth is obtained to obtain a first generation of Schizochytrium acclimated with gibberellins;

[0108] (6) Repeat the cultivation and domestication according to the method of step (4) and step (5), the domestication time is 3 days, 5 days, 10 days, 15 days, and 20 days, and the number of domestication passages is 3 generations, 5 generations, 10 generations, 15 generations, and 20 generations, until the domesticated strain can produce gibberellins at contents of 1 mg / L, 2 mg / L, 3 mg / L, 4 mg / L, 5 mg / L, 6 mg / L, 7 mg / L, 8 mg / L, 9 mg / L, and 10 mg / L, respectively, and does not contain vitamin B 12 The strains grew normally in the liquid acclimation medium. Among them, the Schizochytrium acclimated for 20 generations grew the best, with the highest cell biomass of 18 g / L. This strain was counted as Schizochytrium acclimated strain II.

[0109] The polyunsaturated fatty acid fermentation performance of the domesticated Schizochytrium strain II obtained by the domestication was investigated. The domesticated Schizochytrium strain II was inoculated into the fermentation medium with an inoculation amount of 15% by volume, an initial pH of 6, a rotation speed of 450 rpm, a temperature of 28° C., and a time of 120 h.

[0110] After fermentation, the oil is collected by subcritical fluid extraction. The specific steps are as follows:

[0111] (1) According to the volume of the fermentation medium, 4 g / L of cell wall breaking enzyme was added relative to 1 L of the fermentation medium, and then the pH of the fermentation medium was adjusted to 12 with 1 mol / L NaOH solution. Cell wall breaking was carried out at a speed of 150 rpm, a temperature of 60°C, and a time of 8 h. During the cell wall breaking process, 0.1 mL of the solution was taken and placed under a microscope to observe whether the degree of cell enzymolysis was complete, to ensure that the cells were completely enzymolyzed to obtain the feed liquid.

[0112] (2) extracting the feed liquid obtained in step (1) by subcritical fluid extraction, wherein the extraction solvent is propane, and propane is formed into a subcritical fluid under the conditions of a temperature of 25° C. and a pressure of 0.5 MPa, and is introduced into an extraction device to mix with the feed liquid in reverse, and the oil is extracted repeatedly until the extract is colorless, and the extract is introduced into an evaporation device to reduce the pressure and concentrate the solvent to obtain extracted oil;

[0113] (3) The extracted oil in step (2) is placed in an oven and dried at 40° C. for 2 hours, and the oil mass is weighed.

[0114] The mass proportions of polyunsaturated fatty acids (PUFAs), eicosatetraenoic acid (ARA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in the above oils were determined, and the results are shown in Table 2.

[0115] Table 2

[0116]

[0117]

[0118] Example 3

[0119] Seed culture medium: 50g / L glucose, 4g / L KH 2 PO 4 、12g / L Na 2 SO 4 、3g / L MgSO 4 , 2g / L KCl, 4g / L (NH 4 ) 2 SO 4 、0.5g / L CaCl 2 , 0.5g / LK 2 SO 4 , 12g / L monosodium glutamate, 15g / L yeast extract powder;

[0120] Acclimation medium 1: seed medium + vitamin B 12 (Vitamin B 12 The concentrations were 0g / L, 3g / L, and 6g / L respectively);

[0121] Acclimation medium 2: seed medium + gibberellin (gibberellin concentrations were 0 mg / L, 2 mg / L, 4 mg / L, 6 mg / L, 8 mg / L, and 10 mg / L);

[0122] Fermentation medium: fermentation medium + gibberellin (the concentrations of gibberellin are 1 mg / L, 2 mg / L, 3 mg / L, 4 mg / L, 5 mg / L, 6 mg / L, 7 mg / L, 8 mg / L, 9 mg / L, and 10 mg / L respectively);

[0123] Fermentation medium: 90g / L glucose, 1g / L KH 2 PO 4 , 4g / L(NH 4 ) 2 SO 4 , 1g / L malic acid, 10g / L monosodium glutamate, 10g / L yeast extract powder, 5g / L corn syrup powder, 32g / L sea salt;

[0124] Biomass was determined by:

[0125] Take 1 mL of fermentation broth in a centrifuge tube and centrifuge it at 1000 rpm for 6 min to separate the solid and liquid. Remove the supernatant and wash the precipitate once with 3 wt% sodium chloride solution and then with distilled water. Then put the precipitate in an oven and dry it at 110 ° C for 5 hours to constant weight. The weight of the precipitate is the biomass.

[0126] The specific domestication process is as follows:

[0127] (1) Inoculating 50 mL of seed culture medium with a starting strain of Schizochytrium (deposit number: CCTCCNO.M209059) at an inoculation amount of 0.5% by volume, culturing for 30 h at an initial pH of 6, a rotation speed of 200 rpm, and a temperature of 30° C. to obtain a Schizochytrium seed solution;

[0128] (2) Inoculate the Schizochytrium seed solution in step (1) with vitamin B 12 Vitamin B1 was obtained by culturing the culture medium with a concentration of 6 g / L at an initial pH of 6, a rotation speed of 200 rpm, and a temperature of 30 °C for 30 h. 12 First generation domesticated seed solution;

[0129] (3) Reduce the amount of vitamin B in the liquid acclimatization medium by 3 g / L 12 Concentration (vitamin B 12 The concentrations of the Schizochytrium vitamin B in step (2) are 3 g / L and 0 g / L, respectively. 12 The first generation acclimated seed liquid is continuously acclimated and cultured in the liquid acclimation medium until the Schizochytrium grows normally to form the seed liquid after the Schizochytrium subculture;

[0130] (4) inoculating the seed liquid of Schizochytrium subcultured in step (3) into a liquid acclimation medium with a gibberellin concentration of 0 mg / L, with an inoculation amount of 0.5% by volume, and culturing for 24 hours at an initial pH of 6, a rotation speed of 200 rpm, and a temperature of 30° C. to obtain a first generation acclimation seed liquid of Schizochytrium gibberellin;

[0131] (5) increasing the concentration of gibberellins in the liquid acclimation medium by a gradient of 2 mg / L (the gibberellins concentrations are 2 mg / L, 4 mg / L, 6 mg / L, 8 mg / L, and 10 mg / L, respectively), and continuing to acclimate and culture the Schizochytrium in step (4) in the liquid acclimation medium until normal growth is obtained to obtain a first generation of Schizochytrium acclimated with gibberellins;

[0132] (6) Repeat the cultivation and domestication according to the method of step (4) and step (5), the domestication time is 3 days, 5 days, 10 days, 15 days, and 20 days, and the number of domestication passages is 3 generations, 5 generations, 10 generations, 15 generations, and 20 generations, until the domesticated strain can produce gibberellins at contents of 1 mg / L, 2 mg / L, 3 mg / L, 4 mg / L, 5 mg / L, 6 mg / L, 7 mg / L, 8 mg / L, 9 mg / L, and 10 mg / L, respectively, and does not contain vitamin B 12 The strains grew normally in the liquid acclimation medium. Among them, the Schizochytrium acclimated for 20 generations grew the best, with the highest cell biomass of 21 g / L. This strain was counted as Schizochytrium acclimated strain III.

[0133] The polyunsaturated fatty acid fermentation performance of Schizochytrium domesticated strain III was investigated. Schizochytrium domesticated strain III was inoculated into the fermentation medium, the inoculation amount was 10 volume %, the initial pH was 7, the rotation speed was 500 rpm, the temperature was 30° C., and the time was 150 h.

[0134] After fermentation, the oil is collected by subcritical fluid extraction. The specific steps are as follows:

[0135] (1) According to the volume of the fermentation medium, 3 g / L of cell wall breaking enzyme was added relative to 1 L of the fermentation medium, and then the pH of the fermentation medium was adjusted to 11 with a 2 mol / L NaOH solution. Cell wall breaking was performed at a speed of 135 rpm, a temperature of 55° C., and a time of 7 h. During the cell wall breaking process, 0.2 mL of the solution was taken and placed under a microscope to observe whether the degree of cell enzymolysis was complete, to ensure that the cells were completely enzymolyzed to obtain the feed liquid.

[0136] (2) extracting the feed liquid obtained in step (1) by subcritical fluid extraction, wherein the extraction solvent is butane, and butane is formed into a subcritical fluid under the conditions of a temperature of 30° C. and a pressure of 0.4 MPa, and then introduced into an extraction device to mix with the feed liquid in reverse, and the oil is repeatedly extracted until the extract is colorless, and the extract is introduced into an evaporation device to reduce the pressure and concentrate the solvent to obtain extracted oil;

[0137] (3) The extracted oil in step (2) is placed in an oven and dried at 45° C. for 1.5 hours, and the oil mass is weighed.

[0138] The mass proportions of polyunsaturated fatty acids (PUFAs), eicosatetraenoic acid (ARA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in the above oils were determined, and the results are shown in Table 3.

[0139] Table 3

[0140]

[0141]

[0142] Example 4

[0143] Seed culture medium: 30g / L glucose, 1g / L KH 2 PO 4 、10g / L Na 2 SO 4 、2g / L MgSO 4 , 1g / L KCl, 3g / L (NH 4 ) 2 SO 4 , 0.06g / L CaCl 2 , 0.1g / LK 2 SO 4 , 8g / L monosodium glutamate, 9g / L yeast extract powder;

[0144] Acclimation medium 1: seed medium + vitamin B 12 (Vitamin B 12 The concentrations were 0g / L, 3g / L, and 6g / L respectively);

[0145] Acclimation medium 2: seed medium + gibberellin (the gibberellin concentrations were 1 mg / L, 3 mg / L, 5 mg / L, 7 mg / L, 9 mg / L, and 10 mg / L respectively);

[0146] Fermentation medium: fermentation medium + gibberellin (the concentrations of gibberellin are 1 mg / L, 2 mg / L, 3 mg / L, 4 mg / L, 5 mg / L, 6 mg / L, 7 mg / L, 8 mg / L, 9 mg / L, and 10 mg / L respectively);

[0147] Fermentation medium: 70g / L glucose, 0.5g / L KH 2 PO 4 、3g / L(NH 4 ) 2 SO 4 , 0.5g / L malic acid, 8g / L monosodium glutamate, 8g / L yeast extract powder, 4g / L corn syrup powder, 30g / L sea salt;

[0148] During the acclimation process, biomass was determined by:

[0149] Take 1 mL of fermentation broth in a centrifuge tube and centrifuge it at 8000 rpm for 5 min to separate the solid and liquid. Remove the supernatant and wash the precipitate once with 2 wt% sodium chloride solution and then with distilled water. Then put the precipitate in an oven and dry it at 100 °C for 6 hours to constant weight. The weight of the precipitate is the biomass.

[0150] The specific domestication process is as follows:

[0151] (1) Inoculating 50 mL of seed culture medium with a starting strain of Schizochytrium (deposit number: CCTCCNO.M209059) at an inoculation amount of 1% by volume, culturing for 25 h at an initial pH of 6.5, a rotation speed of 150 rpm, and a temperature of 25° C., to obtain a Schizochytrium seed solution;

[0152] (2) Inoculate the Schizochytrium seed solution in step (1) with vitamin B 12 The concentration of 6g / L liquid acclimation medium was cultured at an initial pH of 6.5, a rotation speed of 150rpm, and a temperature of 25°C to obtain vitamin B12 from Schizochytrium. 12 First generation domesticated seed solution;

[0153] (3) Reduce the amount of vitamin B in the liquid acclimatization medium by 3 g / L 12 Concentration (vitamin B 12 The concentrations of the Schizochytrium vitamin B in step (2) are 3 g / L and 0 g / L, respectively. 12 The first generation acclimated seed liquid is continuously acclimated and cultured in the liquid acclimation medium until the Schizochytrium grows normally to form the seed liquid after the Schizochytrium subculture;

[0154] (4) inoculating the seed liquid of Schizochytrium subcultured in step (3) into a liquid acclimation medium with a gibberellin concentration of 1 mg / L, with an inoculation amount of 1% by volume, and culturing for 24 hours at an initial pH of 6.5, a rotation speed of 150 rpm, and a temperature of 25° C. to obtain a first generation acclimation seed liquid of Schizochytrium gibberellin;

[0155] (5) increasing the concentration of gibberellins in the liquid acclimation medium by a gradient of 2 mg / L (the gibberellins concentrations are 3 mg / L, 5 mg / L, 7 mg / L, 9 mg / L, and 10 mg / L, respectively), and continuing to acclimate and culture the Schizochytrium in step (4) in the liquid acclimation medium until normal growth is obtained to obtain a first generation of Schizochytrium acclimated with gibberellins;

[0156] (6) Repeat the cultivation and domestication according to the method of step (4) and step (5), the domestication time is 3 days, 5 days, 10 days, 15 days, and 20 days, and the number of domestication passages is 3 generations, 5 generations, 10 generations, 15 generations, and 20 generations, until the domesticated strain can produce gibberellins at contents of 1 mg / L, 2 mg / L, 3 mg / L, 4 mg / L, 5 mg / L, 6 mg / L, 7 mg / L, 8 mg / L, 9 mg / L, and 10 mg / L, respectively, and does not contain vitamin B 12 The strain grew normally in the liquid acclimation medium with a maximum cell biomass of 22 g / L. The strain was counted as Schizochytrium acclimation strain IV.

[0157] The polyunsaturated fatty acid fermentation performance of Schizochytrium domesticated strain IV was investigated. Schizochytrium domesticated strain IV was inoculated into the fermentation medium, the inoculation amount was 5% by volume, the initial pH was 6.5, the rotation speed was 350 rpm, the temperature was 25° C., and the time was 100 h.

[0158] After fermentation, the oil is collected by subcritical fluid extraction. The specific steps are as follows:

[0159] (1) According to the volume of the fermentation medium, 2 g / L of cell wall-breaking enzyme was added relative to 1 L of the fermentation medium, and then the pH of the fermentation medium was adjusted to 10 with a 1.5 mol / L NaOH solution. Cell wall breaking was performed at a speed of 120 rpm, a temperature of 50°C, and a time of 6 h. During the cell wall breaking process, 0.5 mL of the solution was taken and placed under a microscope to observe whether the degree of cell enzymolysis was complete, to ensure that the cells were completely enzymolyzed to obtain the feed liquid.

[0160] (2) extracting the feed liquid obtained in step (1) by subcritical fluid extraction, wherein the extraction solvent is dimethyl ether, and dimethyl ether is formed into a subcritical fluid under the conditions of a temperature of 30° C. and a pressure of 0.6 MPa, and introduced into an extraction device to mix with the feed liquid in reverse, and the oil is repeatedly extracted until the extract is colorless, and the extract is introduced into an evaporation device to reduce the pressure and concentrate the solvent to obtain extracted oil;

[0161] (3) The extracted oil in step (2) is placed in an oven and dried at 50° C. for 1 hour, and the oil mass is weighed.

[0162] The mass proportions of polyunsaturated fatty acids (PUFAs), eicosatetraenoic acid (ARA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in the above oils were determined, and the results are shown in Table 4.

[0163] Table 4

[0164]

[0165]

[0166] Example 5

[0167] The method of Example 2 was followed, except that gibberellin was first used for acclimatization, followed by vitamin B 12 Acclimation. Fermentation was performed to produce oil according to the fermentation conditions of Example 2, and the mass proportions of polyunsaturated fatty acids (PUFAs), eicosatetraenoic acid (ARA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in the oil were determined. The determination results are shown in Table 5.

[0168] Table 5

[0169]

[0170]

[0171] Example 6

[0172] The method of Example 2 was followed, except that vitamin B 12 After the domestication, no gibberellin domestication is performed. According to the fermentation conditions of Example 2, the fermentation is carried out to produce oil, and the mass proportions of polyunsaturated fatty acids (PUFAs), eicosatetraenoic acid (ARA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in the oil are determined. The results are shown in Table 6.

[0173] Table 6

[0174]

[0175] Example 7

[0176] The same domestication method as in Example 2 was used, except that only gibberellin domestication was performed. Fermentation was performed to produce oil under the fermentation conditions of Example 2, and the mass proportions of polyunsaturated fatty acids (PUFAs), eicosatetraenoic acid (ARA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in the oil were determined. The results are shown in Table 7.

[0177] Table 7

[0178]

[0179] It can be seen from the results of Tables 1 to 7 that, using Examples 1 to 7 of the domestication method of the present invention, after gibberellin and vitamin B 12 The domesticated strain produces a higher yield of oil than the original strain, and the content of unsaturated fatty acids in the oil is also increased, especially the DHA content in the polyunsaturated fatty acids, and has a significantly better fermentation effect.

[0180] The present invention uses gibberellin and vitamin B 12 The induction of domestication of oil-producing microorganisms can achieve the directional domestication of oil-producing microorganisms with high polyunsaturated fatty acid production. It has a strong target-oriented nature, simple domestication method, high efficiency, and the domesticated strains of oil-producing microorganisms have fast growth rate and higher polyunsaturated fatty acid production. The oil extraction method is simple to operate, with high extraction quality, and is suitable for large-scale production.

[0181] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A method for domesticating oil-producing microorganisms, characterized in that: The method comprises: using the oil-producing microorganism to produce 12 The culture medium was used for acclimatization.

2. The method according to claim 1, characterized in that The oil-producing microorganism is at least one selected from Schizochytrium, Saccharomyces lipolytica and Mortierella alpina, preferably Schizochytrium.

3. The method according to claim 1, characterized in that The domestication process comprises: using the gibberellin and vitamin B 12 domesticating the oil-producing microorganism using a domestication medium; Preferably, the addition amount of the gibberellin is 0-10g / L; Preferably, the vitamin B 12 The addition amount is 0-6g / L.

4. The method according to claim 3, characterized in that During the acclimation process, the concentration of the gibberellin in the acclimation medium is increased in a gradient of 1-3 mg / L each time, and the vitamin B 12 The concentration was decreased in a gradient of 2-3 g / L each time; Preferably, the acclimation time is 5-20 days; Preferably, the number of generations of domestication is 3-20 generations.

5. The method according to any one of claims 1 to 4, characterized in that: The acclimation culture medium also contains: 30-60 g / L glucose, 1-5 g / L KH2PO4, 10-14 g / L Na2SO4, 2-5 g / LMgSO4, 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 powder.

6. The method according to claim 5, characterized in that The conditions of each subculture include: inoculation volume of 0.5-2% by volume, initial pH of 6-7, rotation speed of 150-200 rpm, temperature of 25-30° C., and time of 20-30 h.

7. Use of the domesticated strain of oil-producing microorganisms obtained by domestication according to the method of any one of claims 1 to 6 in the production of polyunsaturated fatty acids.

8. A method for producing polyunsaturated fatty acids, characterized in that: The method comprises the following steps: fermenting the domesticated strain of oil-producing microorganisms obtained by domestication according to the method according to any one of claims 1 to 6.

9. The method according to claim 8, characterized in that The fermentation process comprises: culturing the domesticated strain of the oil-producing microorganism in a fermentation medium containing gibberellins; Preferably, the content of gibberellin in the fermentation medium is 1-10 mg / L; Preferably, the fermentation medium further 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; Preferably, the culture conditions include: inoculation volume of 5-15% by volume, initial pH of 6-7, rotation speed of 350-500 rpm, temperature of 25-30° C., and time of 100-150 h.

10. The method according to claim 8 or 9, characterized in that: The method further comprises: breaking the cell wall of the fermented liquid obtained by the fermentation with a cell wall breaking enzyme, mixing the fermented liquid with an extraction solvent, and extracting the polyunsaturated fatty acids by a subcritical fluid extraction method; Preferably, the wall-breaking enzyme is selected from at least one of wall-lytic enzyme, snail enzyme and alkaline protease; Preferably, the amount of the cell wall-breaking enzyme is 2-4 g relative to 1 L of the fermentation medium; Preferably, the conditions for enzymatic hydrolysis of the cell wall-breaking enzyme include: pH 10-12, rotation speed 120-150rpm, temperature 50-60°C, and time 6-8h; Preferably, the extraction solvent is selected from at least one of propane, butane and dimethyl ether.

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