CN114958932B - Application of regulating factor in improving polyunsaturated fatty acid content of oleaginous microorganism and preparation method of microbial oil - Google Patents

Application of regulating factor in improving polyunsaturated fatty acid content of oleaginous microorganism and preparation method of microbial oil Download PDF

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CN114958932B
CN114958932B CN202210621417.4A CN202210621417A CN114958932B CN 114958932 B CN114958932 B CN 114958932B CN 202210621417 A CN202210621417 A CN 202210621417A CN 114958932 B CN114958932 B CN 114958932B
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黄和
刘孟珍
郭东升
陈子雷
任波
金素星
杜远航
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Nanjing Normal University
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Abstract

The invention relates to the field of fermentation, and discloses application of a regulating factor in improving polyunsaturated fatty acid content of oleaginous microorganisms and a preparation method of microbial oil. The method for increasing the polyunsaturated fatty acid content of oleaginous microorganisms comprises: inoculating oleaginous microorganisms into a culture medium containing a regulating factor for culturing; the regulatory factor comprises graphene oxide. The preparation method of the microbial oil comprises the following steps: culturing oleaginous microorganisms by the method to obtain a culture solution, breaking the wall of the culture solution, and extracting. The method provided by the invention can improve the content of polyunsaturated fatty acid in oleaginous microorganisms, and is simple, efficient and low in cost.

Description

Application of regulating factor in improving polyunsaturated fatty acid content of oleaginous microorganism and preparation method of microbial oil
Technical Field
The invention relates to the field of fermentation, in particular to application of a regulating factor in improving polyunsaturated fatty acid content of oleaginous microorganisms and a preparation method of microbial oil.
Background
Polyunsaturated fatty acid contains more than two double bonds, has special biological activity, has wide functions in biological systems, and plays an important role in stabilizing cell membrane functions, regulating gene expression, maintaining cytokine and lipoprotein balance, resisting cardiovascular diseases, promoting growth and development and the like. Polyunsaturated fatty acids mainly comprise the n-3, n-6 and n-9 series of fatty acids, of which the biological activity of the n-6 and n-3 series of fatty acids is more important.
Currently, the main source of polyunsaturated fatty acids is fish oil, the quality of which depends on the type of fish, the capturing season, the capturing region and the like, and the fatty acid content of various chain lengths and saturation in fish oil can directly influence the efficacy of the fish oil. In addition, fish oil has heavy iso-taste and high purification cost, and greatly hinders the development of large-scale production and commercial application of polyunsaturated fatty acids.
It was found that fish accumulate polyunsaturated fatty acids in the body by ingestion of marine algae microorganisms rich in polyunsaturated fatty acids, so that marine microorganisms are the true producer of polyunsaturated fatty acids. The microorganism classes that have been reported to contain polyunsaturated fatty acids include: algae, marine bacteria and fungi. The culture of algae is influenced by factors such as illumination, long culture period and the like, and the production of polyunsaturated fatty acid by using the algae has certain difficulty, so the algae is always in the primary stage of research; the yield of polyunsaturated fatty acid produced by bacteria is not high, the report is below 100mg/L at present, and the development potential is limited; the most studied marine oleaginous fungi have the characteristics of high grease content, easy culture, high biomass and the like at present, and the marine oleaginous fungi are widely applied to the fields of food, medical treatment, health care and the like for producing polyunsaturated fatty acids, such as EPA, DHA and the like, and the demands of people are gradually increased, but the level of synthesizing other polyunsaturated fatty acids is still lower.
Disclosure of Invention
The invention aims to overcome the problems in the prior art and provide an application of a regulating factor in improving the polyunsaturated fatty acid content of oleaginous microorganisms and a preparation method of microbial oil.
In a first aspect the present invention provides the use of a regulatory factor comprising graphene oxide for increasing the polyunsaturated fatty acid content of oleaginous microorganisms.
Preferably, the regulatory factor further comprises sulforaphane.
In a second aspect, the invention provides a method for increasing the polyunsaturated fatty acid content of an oleaginous microorganism, the method comprising: inoculating oleaginous microorganisms into a culture medium containing a regulating factor for culturing; the regulatory factor comprises graphene oxide.
Preferably, the regulatory factor further comprises sulforaphane.
Preferably, the graphene oxide is added in an amount of 1-5mg and the sulforaphane is added in an amount of 0.5-1mg relative to 1L of the culture medium.
Preferably, the culturing process comprises: inoculating oleaginous microorganisms into a first culture medium for subculture to obtain a seed solution, and inoculating the seed solution into a second culture medium for fermentation culture in an inoculum size of 0.5-10% by volume, wherein the graphene oxide is added into the first culture medium and/or the second culture medium, and the sulforaphane is added into the first culture medium and/or the second culture medium.
Preferably, the graphene oxide is added in the first medium, and the sulforaphane is added in the second medium.
Preferably, the addition time of the sulforaphane is 20-30h after fermentation begins.
Preferably, the subculture is carried out for 5 to 20 passages, and the subculture time is 22 to 26 hours per passage.
Preferably, the first medium contains: 30-50g/L glucose 、1-5g/L KH2PO4、10-14g/L Na2SO4、2-5g/L MgSO4、1-3g/L KCl、2-5g/L(NH4)2SO4、0.06-1g/L CaCl2、0.1-1g/L K2SO4、8-12g/L monosodium glutamate, 9-15g/L yeast powder; the second medium contains: 50-70g/L glucose 、1-5g/L KH2PO4、10-14g/L Na2SO4、2-5g/L MgSO4、1-3g/L KCl、2-5g/L(NH4)2SO4、0.06-1g/L CaCl2、0.1-1g/L K2SO4、1-5g/L malic acid, 8-12g/L monosodium glutamate, 9-15g/L yeast extract, 3-7g/L corn steep liquor dry powder and 0.5-1g/L metal ion solution, wherein the metal ion solution contains Zn 2+ and Cu 2+, and the mass ratio of Zn 2+ to Cu 2+ is 1:0.5-1.
Preferably, the conditions of the subculture and the fermentation culture include: the initial pH is 5-6, the rotating speed is 150-200rpm, and the temperature is 25-30 ℃.
Preferably, the oleaginous microorganism is selected from at least one of schizochytrium, diatom, nannochloropsis and mortierella alpina, preferably schizochytrium.
The third aspect of the invention provides a preparation method of microbial oil, which comprises culturing by adopting the method of the second aspect to obtain a culture solution, breaking the wall of the culture solution, and extracting.
Preferably, the wall breaking is performed by a wall breaking enzymatic hydrolysis method.
Preferably, the conditions for breaking the wall include: the dosage of the wall breaking enzyme is 3-6g/L, the pH is 10-12, the rotating speed is 150-200rpm, the temperature is 25-30 ℃ and the time is 4-6h.
Preferably, the extraction solvent is n-hexane and/or ethanol.
Through the technical scheme, the invention has the beneficial effects that: according to the invention, graphene oxide is used as a regulating factor for increasing the polyunsaturated fatty acid content of oleaginous microorganisms for the first time, so that the polyunsaturated fatty acid content, in particular the content of docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA) and eicosatetraenoic acid (ARA), in grease produced by oleaginous microorganisms can be effectively increased, and a new thought is provided for increasing the polyunsaturated fatty acid content of oleaginous microorganisms.
Moreover, graphene oxide and sulforaphane are adopted as regulating factors, when the regulating factors are applied to the culture process of oleaginous microorganisms, the contents of docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA) and eicosatetraenoic acid (ARA) in the grease can be further improved while the total polyunsaturated fatty acid (PUFA) content in the grease is obviously improved, and the method is simple to operate, low in production cost and remarkable in development value.
Drawings
FIG. 1 shows the growth of cells in seed culture solutions obtained at different passage times in examples 1 to 3, 9, 10 and comparative example 1.
Detailed Description
The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.
In a first aspect the present invention provides the use of a regulatory factor comprising graphene oxide for increasing the polyunsaturated fatty acid content of oleaginous microorganisms.
The inventor of the invention discovers in the research process that the addition of graphene oxide can effectively improve the content of polyunsaturated fatty acid in grease produced by oleaginous microorganisms. In addition, the graphene oxide can promote the absorption of oleaginous microorganisms to nutrient substances, and improve the utilization rate of nutrients in the culture medium.
According to the present invention, preferably, the regulatory factor further contains sulforaphane. The inventors have found that in this preferred embodiment, the formation of saturated fatty acids during the growth of oleaginous microorganisms is allowed to be suppressed, thereby further increasing the polyunsaturated fatty acid content of the oils and fats produced by oleaginous microorganisms.
In a second aspect, the invention provides a method for increasing the polyunsaturated fatty acid content of an oleaginous microorganism, the method comprising: inoculating oleaginous microorganisms into a culture medium containing a regulating factor for culturing; the regulatory factor comprises graphene oxide.
The inventor of the invention discovers in the research process that the oleaginous microorganism is inoculated into a culture medium containing a regulating factor for culture, so that the oleaginous microorganism can promote the oleaginous microorganism to absorb nutrient substances, and the utilization rate of nutrition in the culture medium is improved, thereby further improving the content of polyunsaturated fatty acids in grease produced by oleaginous microorganism.
According to the present invention, preferably, the regulatory factor further contains sulforaphane. The inventors have found that in this preferred embodiment, the formation of saturated fatty acids during the growth of oleaginous microorganisms is allowed to be suppressed, thereby further increasing the polyunsaturated fatty acid content of the oils and fats produced by oleaginous microorganisms.
According to the present invention, the content of the regulatory factor in the medium is not particularly limited as long as the regulatory effect can be exerted, and the synthesis of polyunsaturated fatty acids during the growth of oleaginous microorganisms can be enhanced. In order to further increase the polyunsaturated fatty acid content after the culture of oleaginous microorganisms, preferably, the graphene oxide is added in an amount of 1 to 5mg, specifically 1mg, 2mg, 3mg, 4mg, 5mg or any value in the range consisting of any two values of the above, relative to 1L of the culture medium; the addition amount of the sulforaphane is 0.5mg, 0.6mg, 0.7mg, 0.8mg, 0.9mg, 1mg or any value in a range formed by any two values.
The method of culturing oleaginous microorganisms is not particularly limited as long as the oleaginous microorganisms can be proliferated in large amounts by the method of culturing. Preferably, the culturing process comprises: inoculating oleaginous microorganisms into a first culture medium for subculture to obtain a seed solution, and inoculating the seed solution into a second culture medium for fermentation culture in an inoculum size of 0.5-10% by volume, wherein the graphene oxide is added into the first culture medium and/or the second culture medium, and the sulforaphane is added into the first culture medium and/or the second culture medium.
In view of further increasing the polyunsaturated fatty acid content in the grease produced by the oleaginous microorganism, it is preferable that the graphene oxide is added in the first medium and the sulforaphane is added in the second medium.
According to the invention, preferably, the addition time of the sulforaphane is 20-30h after the fermentation starts. In this preferred embodiment, the formation of saturated fatty acids is inhibited during the growth of oleaginous microorganisms, thereby further increasing the polyunsaturated fatty acid content of the oils and fats produced by oleaginous microorganisms.
According to the present invention, preferably, the number of passages of the subculture is 5 to 20 passages, and the time for each passage is 22 to 26 hours. More preferably, the incubation time is 24 hours per generation. The inventors have found that in this preferred embodiment it is advantageous to increase the polyunsaturated fatty acid content of the grease produced by the oleaginous microorganisms.
In the present invention, before the oleaginous microorganism is inoculated to the first culture medium, an activation treatment is required, and specifically, the activation treatment may be to inoculate the oleaginous microorganism to the activation culture medium for activation culture, thereby obtaining an activation culture solution.
According to the present invention, preferably, the oleaginous microorganism is inoculated in an amount of 0.5 to 2% by volume in the activation medium upon the activation culture; if the microorganism strain stored in glycerol tubes is inoculated, the strain in each glycerol tube is inoculated into 100mL of the activated medium. The parameters such as temperature, pH, rotation speed, time and the like used for the activation culture may be conventional settings in the art. Preferably, the conditions of the activation culture include: the initial pH is 5.5-7, the rotating speed is 150-200rpm, the temperature is 25-30 ℃ and the time is 20-24h. The inventors have found that in this preferred embodiment, it is advantageous to promote the growth of oleaginous microorganisms and to increase the biomass of the fermentation.
According to the present invention, the activation medium, the first medium and the second medium each independently contain a carbon source, a nitrogen source, inorganic salt ions and trace elements, which may be any of the corresponding species for microbial fermentation. In order to be able to further increase the polyunsaturated fatty acid content of the oils and fats produced by oleaginous microorganisms, it is preferred that the activation medium and the first medium each independently contain: 30-50g/L glucose 、1-5g/L KH2PO4、10-14g/L Na2SO4、2-5g/L MgSO4、1-3g/L KCl、2-5g/L(NH4)2SO4、0.06-1g/L CaCl2、0.1-1g/L K2SO4、8-12g/L monosodium glutamate and 9-15g/L yeast powder. The second medium contains: 50-70g/L glucose 、1-5g/L KH2PO4、10-14g/L Na2SO4、2-5g/L MgSO4、1-3g/L KCl、2-5g/L(NH4)2SO4、0.06-1g/L CaCl2、0.1-1g/L K2SO4、1-5g/L malic acid, 8-12g/L monosodium glutamate, 9-15g/L yeast extract, 3-7g/L corn steep liquor dry powder and 0.5-1g/L metal ion solution, wherein the metal ion solution contains Zn 2+ and Cu 2 +, and the mass ratio of Zn 2+ to Cu 2+ is 1:0.5-1. Specifically, the zinc source providing Zn 2+ may be any zinc salt, such as zinc sulfate, zinc chloride, zinc nitrate, etc., and the copper source providing Cu 2+ may be any copper salt, such as copper sulfate, copper chloride, copper nitrate, etc. In view of further increasing the polyunsaturated fatty acid content in the grease produced by the oleaginous microorganism, it is preferable that the zinc source providing Zn 2+ is zinc sulfate and the copper source providing Cu 2+ is copper sulfate. Wherein, the inoculation amount of the first culture medium is generally 0.5-2% by volume, and if the microorganism strain stored in the glycerol tube is used for inoculation, the strain in each glycerol tube is inoculated into 100mL of the first culture medium.
Parameters such as temperature, pH, rotation speed, time and the like adopted for subculture and fermentation culture in the invention can be conventional settings in the field. Preferably, the conditions of the subculture and the fermentation culture include: the initial pH is 5-6, the rotating speed is 150-200rpm, and the temperature is 25-30 ℃. It was found that subculturing and fermentation culturing under the above conditions can further increase the polyunsaturated fatty acid content in the oils and fats produced by oleaginous microorganisms.
According to the present invention, the oleaginous microorganism may be any one capable of producing microbial oils by fermentation, preferably, the oleaginous microorganism is selected from at least one of schizochytrium, diatom, nannochloropsis and mortierella alpina. The schizochytrium is preferable in terms of further increasing the polyunsaturated fatty acid content in the grease produced by the oleaginous microorganism.
The third aspect of the present invention provides a method for producing microbial oil, comprising: culturing oleaginous microorganisms by the method of the second aspect to obtain a culture solution, and breaking the wall and extracting the culture solution.
According to the invention, the cell wall breaking of the oleaginous microorganism can be performed in a conventional manner in the art, preferably by a wall breaking enzymatic hydrolysis method, so as to improve the wall breaking efficiency and reduce the damage to metabolites in the microbial cells.
Preferably, the conditions for breaking the wall include: the dosage of the wall breaking enzyme is 3-6g/L, and can be specifically 3g/L, 4g/L, 5g/L, 6g/L or any value in the range formed by any two values; the pH is 10-12, and can be specifically 10, 11, 12 or any value in a range formed by any two values; the rotation speed is 150-200rpm, and can be specifically 150rpm, 160rpm, 170rpm, 180rpm, 190rpm, 200rpm or any value in a range formed by any two values; the temperature is 25-30deg.C, specifically 25 deg.C, 26 deg.C, 27 deg.C, 28 deg.C, 29 deg.C, 30 deg.C or any value in the range of any two values; the time is 4-6h, and specifically can be 4h, 5h, 6h or any value in a range formed by any two values.
According to the present invention, in order to enhance the extraction efficiency of the microbial oil, it is preferable that the extraction solvent is n-hexane and/or ethanol, preferably n-hexane and ethanol, to extract the oil with n-hexane while disrupting the microbial cells with ethanol. The extracted oil contains polyunsaturated fatty acids such as oleic acid, palmitic acid, linoleic acid, eicosatetraenoic acid, eicosapentaenoic acid, and docosahexaenoic acid.
According to a particularly preferred embodiment of the present invention, there is provided a method for producing microbial oil, comprising:
(1) Inoculating schizochytrium strains into an activation culture medium in an inoculum size of 0.5-2 vol%, and performing activation culture for 20-24 hours under the conditions that the initial pH is 5.5-7, the rotating speed is 150-200rpm and the temperature is 25-30 ℃ to obtain an activation culture solution;
(2) Inoculating the activated culture solution obtained in the step (1) into a first culture medium with an inoculum size of 0.5-2 vol%, and subculturing for 5-20 generations (24 h per generation of culture time) under the conditions of an initial pH of 5-6, a rotating speed of 150-200rpm and a temperature of 25-30 ℃ to obtain a seed culture solution;
(3) Inoculating the seed culture solution obtained in the step (2) into a second culture medium with the inoculum size of 0.5-10 vol%, and fermenting and culturing for 110-130h under the conditions of initial pH of 5-6, rotating speed of 150-200rpm and temperature of 25-30 ℃ to obtain a fermentation culture solution;
(4) Adding wall breaking enzyme into the fermentation culture solution obtained in the step (3) at an adding amount of 3-6g/L, carrying out enzymolysis for 4-6h under the conditions of pH of 10-12, rotating speed of 150-200rpm and temperature of 25-30 ℃ to obtain wall breaking liquid, and mixing the wall breaking liquid with ethanol and n-hexane at a ratio of 1:1:1, mixing and extracting to obtain an n-hexane phase, and removing the n-hexane from the n-hexane phase by rotary evaporation to obtain microbial oil;
wherein, graphene oxide is added in the first culture medium, and the addition amount of the graphene oxide is 1-5mg/L; the sulforaphane is added into the second culture medium, the addition amount of the sulforaphane is 0.5-1mg/L, and the addition time of the sulforaphane is 20-30h after fermentation begins;
The activation medium contains: 30-50g/L glucose 、1-5g/L KH2PO4、10-14g/L Na2SO4、2-5g/L MgSO4、1-3g/L KCl、2-5g/L(NH4)2SO4、0.06-1g/L CaCl2、0.1-1g/L K2SO4、8-12g/L monosodium glutamate, 9-15g/L yeast powder;
The first medium contains: 30-50g/L glucose 、1-5g/L KH2PO4、10-14g/L Na2SO4、2-5g/L MgSO4、1-3g/L KCl、2-5g/L(NH4)2SO4、0.06-1g/L CaCl2、0.1-1g/L K2SO4、8-12g/L monosodium glutamate, 9-15g/L yeast powder;
The second medium contains: 50-70g/L glucose 、1-5g/L KH2PO4、10-14g/L Na2SO4、2-5g/L MgSO4、1-3g/L KCl、2-5g/L(NH4)2SO4、0.06-1g/L CaCl2、0.1-1g/L K2SO4、1-5g/L malic acid, 8-12g/L monosodium glutamate, 9-15g/L yeast extract, 3-7g/L corn steep liquor dry powder and 0.5-1g/L metal ion solution, wherein the metal ion solution contains Zn 2+ and Cu 2+, and the mass ratio of Zn 2+ to Cu 2+ is 1:0.5-1; the oleaginous microorganism is selected from at least one of schizochytrium, diatom, nannochloropsis and mortierella alpina.
The microbial oil prepared by the method provided by the preferred embodiment has higher polyunsaturated fatty acid content.
The present invention will be described in detail by examples.
In the following examples and comparative examples, schizochytrium sp.HX-308, which was isolated and screened from coastal areas autonomously by the laboratory, was deposited at China center for type culture Collection (CCTCC for short), with a deposit number of CCTCCM 209059, and was described in the patent application publication No. CN 106947706A.
Graphene oxide is purchased from Shanghai Yuan Yes Biotechnology Co., ltd, model S25045; the sulforaphane is purchased from Shanghai Jiding biotechnology limited company and is of the model L-MD782; other materials and reagents are commercially available.
The biomass measuring method comprises the following steps: 5mL of the culture solution is taken in a weighed centrifuge tube, the supernatant is removed by centrifugation, and the culture solution is dried to constant weight in an oven, so that weighing calculation is performed.
The measurement method of the lipid content comprises the following steps: and (5) drying the obtained microbial oil in an oven at 65 ℃ to constant weight, and weighing.
The fatty acid component of schizochytrium is obtained by:
(1) Methyl esterification of fats and oils: adding 1mL of KOH-methanol solution into a20 mL volumetric flask, adding 50 mu L of grease into the volumetric flask, uniformly mixing, adding 2mL of KOH-methanol solution into the volumetric flask, uniformly mixing, carrying out water bath at 65 ℃ for 17min, and cooling to room temperature; adding 2ml of boron trifluoride-diethyl etherate (boron trifluoride: diethyl etherate=3:7), mixing well, and then carrying out water bath at 65 ℃ for 7min; adding 2mL of saturated KCl solution, shaking, adding 3mL of n-hexane (chromatographic grade), standing and layering;
(2) Removing impurities from the layered upper layer liquid by microporous membrane, preparing sample, and detecting fatty acid component by gas chromatography of Shimadzu DB-23. The gas chromatography test method comprises the following steps: referring to Chen Lizhu, chromatographic conditions of a detection method for detecting docosahexaenoic acid in a method for detecting docosahexaenoic acid by fermentation production of schizochytrium in fed-batch culture are as follows: capillary chromatography columns (60 m x 0.32nm x 15 um) were selected. The temperature programming is adopted: the initial temperature was 200℃for 2min, then raised to 240℃at 10℃per min and maintained for 40min. Column pressure was 200kPa, sample inlet temperature was 250℃and detector temperature was 280 ℃.
Example 1
(1) The formula of the activation medium is as follows: glucose 40g/L、KH2PO43g/L、Na2SO412g/L、MgSO43g/L、KCl 2g/L、(NH4)2SO44g/L、CaCl20.5g/L、K2SO40.5g/L、 monosodium glutamate 10g/L, yeast powder 12g/L, and sterilizing at 121deg.C for 20 min; the formula of the first culture medium is as follows: glucose 40g/L、KH2PO43g/L、Na2SO412g/L、MgSO43g/L、KCl 2g/L、(NH4)2SO44g/L、CaCl20.5g/L、K2SO40.5g/L、 monosodium glutamate 10g/L, yeast powder 12g/L, and sterilizing at 121deg.C for 20 min; the formula of the second culture medium is as follows: glucose 60g/L、KH2PO43g/L、Na2SO412g/L、MgSO43g/L、KCl 2g/L、(NH4)2SO44g/L、CaCl20.5g/L、K2SO40.5g/L、 malic acid 3g/L, monosodium glutamate 10g/L, yeast extract 12g/L, corn steep liquor dry powder 5g/L and metal ion solution 0.5-1g/L (the mass ratio of zinc ions in ZnSO 4 to copper ions in CuSO 4 is 1:0.725), and sterilizing at 121 ℃ for 20min for later use;
(2) Inoculating schizochytrium strains into an activation culture medium in an inoculum size of 1 vol%, and performing activation culture for 24 hours under the conditions that the initial pH is 6.5, the rotating speed is 170rpm and the temperature is 28 ℃ to obtain an activation culture solution;
(3) Inoculating the activated culture solution obtained in the step (2) to a first culture medium with an inoculum size of 1 vol%, adding graphene oxide to a content of 3mg/L, and performing subculture for 20 generations (24 hours per subculture time) under the conditions that the initial pH is 5.5, the rotating speed is 170rpm and the temperature is 28 ℃ to obtain a seed culture solution;
(4) Inoculating seed culture solution obtained in the step (3) for 15 generations into a second culture medium with the inoculum size of 10 vol%, fermenting and culturing at the initial pH of 5.5 and the rotating speed of 170rpm and the temperature of 28 ℃ for 24 hours, adding sulforaphane to the culture solution to enable the content of the sulforaphane to be 0.7mg/L, and culturing for 96 hours to obtain fermentation culture solution;
(5) Adding wall breaking enzyme into the fermentation broth in an adding amount of 4g/L, carrying out enzymolysis for 5 hours under the conditions of pH 11, rotating speed of 180rpm and temperature of 28 ℃ to obtain wall breaking liquid, and mixing the wall breaking liquid with ethanol and n-hexane in a volume ratio of 1:1:1, mixing and extracting to obtain an n-hexane phase, and removing the n-hexane from the n-hexane phase by rotary evaporation to obtain the microbial oil.
Performing biomass measurement on the seed culture solution obtained in the step (3), wherein the result is shown in figure 1; and (3) carrying out biomass measurement on the fermentation culture solution obtained in the step (4), calculating the oil content of the microbial oil obtained in the step (5), and measuring the polyunsaturated fatty acid content in the microbial oil, wherein the result is shown in Table 1.
Example 2
(1) The formula of the activation medium is as follows: glucose 30g/L、KH2PO45g/L、Na2SO410g/L、MgSO42g/L、KCl 3g/L、(NH4)2SO42g/L、CaCl20.06g/L、K2SO40.1g/L、 monosodium glutamate 8g/L and yeast powder 9g/L, sterilizing at 121deg.C for 20 min; the formula of the first culture medium is as follows: glucose 30g/L、KH2PO45g/L、Na2SO410g/L、MgSO42g/L、KCl 3g/L、(NH4)2SO42g/L、CaCl20.06g/L、K2SO40.1g/L、 monosodium glutamate 8g/L and yeast powder 9g/L, sterilizing at 121deg.C for 20 min; the formula of the second culture medium is as follows: glucose 50g/L、KH2PO41g/L、Na2SO414g/L、MgSO45g/L、KCl 1g/L、(NH4)2SO42g/L、CaCl20.06g/L、K2SO40.1g/L、 malic acid 1g/L, monosodium glutamate 8g/L, yeast extract 9g/L, corn steep liquor dry powder 3g/L and metal ion solution 0.5g/L (the mass ratio of zinc ions in ZnSO 4 to copper ions in CuSO 4 is 1:1), and sterilizing at 121deg.C for 20min for use;
(2) Inoculating schizochytrium strains into an activation culture medium in an inoculum size of 0.5 vol%, and performing activation culture for 20 hours under the conditions that the initial pH is 5.5, the rotating speed is 150rpm and the temperature is 25 ℃ to obtain an activation culture solution;
(3) Inoculating the activated culture solution obtained in the step (2) to a first culture medium with an inoculum size of 0.5 vol%, adding graphene oxide to make the content of the graphene oxide be 1mg/L, and carrying out subculture for 20 generations (24 hours per subculture time) under the conditions of an initial pH value of 5, a rotating speed of 150rpm and a temperature of 25 ℃ to obtain a seed culture solution;
(4) Inoculating seed culture solution obtained in the step (3) for 15 generations into a second culture medium with the inoculum size of 0.5 vol%, fermenting and culturing at the initial pH of 5, the rotating speed of 150rpm and the temperature of 25 ℃ for 20 hours, adding sulforaphane to the culture solution to enable the content of the sulforaphane to be 0.5mg/L, and culturing for 90 hours to obtain fermentation culture solution;
(5) Adding wall breaking enzyme into the fermentation broth in an adding amount of 3g/L, carrying out enzymolysis for 4 hours under the conditions of pH of 10, rotating speed of 150rpm and temperature of 25 ℃ to obtain wall breaking liquid, and mixing the wall breaking liquid with ethanol and n-hexane in a volume ratio of 1:1:1, mixing and extracting to obtain an n-hexane phase, and removing the n-hexane from the n-hexane phase by rotary evaporation to obtain the microbial oil.
Performing biomass measurement on the seed culture solution obtained in the step (3), wherein the result is shown in figure 1; and (3) carrying out biomass measurement on the fermentation culture solution obtained in the step (4), calculating the oil content of the microbial oil obtained in the step (5), and measuring the polyunsaturated fatty acid content in the microbial oil, wherein the result is shown in Table 1.
Example 3
(1) The formula of the activation medium is as follows: glucose 50g/L、KH2PO41g/L、Na2SO414g/L、MgSO45g/L、KCl 1g/L、(NH4)2SO45g/L、CaCl21g/L、K2SO41g/L、 monosodium glutamate 12g/L, yeast powder 15g/L, and sterilizing at 121deg.C for 20 min; the formula of the first culture medium is as follows: glucose 50g/L、KH2PO41g/L、Na2SO414g/L、MgSO45g/L、KCl 1g/L、(NH4)2SO45g/L、CaCl21g/L、K2SO41g/L、 monosodium glutamate 12g/L, yeast powder 15g/L, and sterilizing at 121deg.C for 20 min; the formula of the second culture medium is as follows: glucose 70g/L、KH2PO45g/L、Na2SO410g/L、MgSO42g/L、KCl 3g/L、(NH4)2SO45g/L、CaCl21g/L、K2SO41g/L、 g/L malic acid, monosodium glutamate 12g/L, yeast extract 15g/L, corn steep liquor dry powder 7g/L and metal ion solution 1g/L (the mass ratio of zinc ions in ZnSO 4 to copper ions in CuSO 4 is 1:0.5), and sterilizing at 121deg.C for 20min for use;
(2) Inoculating schizochytrium strains into an activation culture medium in an inoculum size of 2 vol%, and performing activation culture for 24 hours under the conditions that the initial pH is 7, the rotating speed is 200rpm and the temperature is 30 ℃ to obtain an activation culture solution;
(3) Inoculating the activated culture solution obtained in the step (2) to a first culture medium with an inoculum size of 2 vol%, and then adding graphene oxide to enable the content of the graphene oxide to be 5mg/L, and carrying out subculture for 20 generations (24 hours per generation of culture time) under the conditions that the initial pH is 6, the rotating speed is 200rpm and the temperature is 30 ℃ to obtain a seed culture solution;
(4) Inoculating seed culture solution obtained in the step (3) for 15 generations into a second culture medium with an inoculum size of 5 vol%, fermenting and culturing at an initial pH of 6 and a rotating speed of 200rpm and a temperature of 30 ℃ for 30 hours, adding sulforaphane to make the content of the sulforaphane be 1mg/L, and culturing for 100 hours to obtain fermentation culture solution;
(5) Adding wall breaking enzyme into the fermentation broth in an adding amount of 6g/L, carrying out enzymolysis for 6 hours under the conditions of pH of 12, rotating speed of 200rpm and temperature of 30 ℃ to obtain wall breaking liquid, and mixing the wall breaking liquid with ethanol and n-hexane in a volume ratio of 1:1:1, mixing and extracting to obtain an n-hexane phase, and removing the n-hexane from the n-hexane phase by rotary evaporation to obtain the microbial oil.
Performing biomass measurement on the seed culture solution obtained in the step (3), wherein the result is shown in figure 1; and (3) carrying out biomass measurement on the fermentation culture solution obtained in the step (4), calculating the oil content of the microbial oil obtained in the step (5), and measuring the polyunsaturated fatty acid content in the microbial oil, wherein the result is shown in Table 1.
Example 4
A microbial oil was produced in the same manner as in example 1, except that in step (4), the addition amount of sulforaphane in the second medium was replaced with 0.5mg/L.
And (3) biomass measurement is carried out on the fermentation culture solution obtained in the step (4), the oil content calculation is carried out on the microbial oil obtained in the step (5), and the polyunsaturated fatty acid content in the microbial oil is measured, so that the results are shown in Table 1.
Example 5
A microbial oil was produced in the same manner as in example 1, except that in step (4), the addition amount of sulforaphane in the second medium was replaced with 0.6mg/L.
And (3) biomass measurement is carried out on the fermentation culture solution obtained in the step (4), the oil content calculation is carried out on the microbial oil obtained in the step (5), and the polyunsaturated fatty acid content in the microbial oil is measured, so that the results are shown in Table 1.
Example 6
A microbial oil was produced in the same manner as in example 1, except that in step (4), the addition amount of sulforaphane in the second medium was replaced with 0.8mg/L.
And (3) biomass measurement is carried out on the fermentation culture solution obtained in the step (4), the oil content calculation is carried out on the microbial oil obtained in the step (5), and the polyunsaturated fatty acid content in the microbial oil is measured, so that the results are shown in Table 1.
Example 7
A microbial oil was produced in the same manner as in example 1, except that in step (4), the addition amount of sulforaphane in the second medium was replaced with 0.9mg/L.
And (3) biomass measurement is carried out on the fermentation culture solution obtained in the step (4), the oil content calculation is carried out on the microbial oil obtained in the step (5), and the polyunsaturated fatty acid content in the microbial oil is measured, so that the results are shown in Table 1.
Example 8
A microbial oil was produced in the same manner as in example 1, except that in step (4), the addition amount of sulforaphane in the second medium was replaced with 1mg/L.
And (3) biomass measurement is carried out on the fermentation culture solution obtained in the step (4), the oil content calculation is carried out on the microbial oil obtained in the step (5), and the polyunsaturated fatty acid content in the microbial oil is measured, so that the results are shown in Table 1.
Example 9
A microbial oil was produced in the same manner as in example 1, except that in step (3), the amount of graphene oxide added to the first medium was replaced with 2mg/L.
Performing biomass measurement on the seed culture solution obtained in the step (3), wherein the result is shown in figure 1; and (3) biomass measurement is carried out on the fermentation culture solution obtained in the step (4), the oil content calculation is carried out on the microbial oil obtained in the step (5), and the polyunsaturated fatty acid content in the microbial oil is measured, so that the results are shown in Table 1.
Example 10
A microbial oil was produced in the same manner as in example 1, except that in step (3), the amount of graphene oxide added to the first medium was replaced with 4mg/L.
Performing biomass measurement on the seed culture solution obtained in the step (3), wherein the result is shown in figure 1; and (3) biomass measurement is carried out on the fermentation culture solution obtained in the step (4), the oil content calculation is carried out on the microbial oil obtained in the step (5), and the polyunsaturated fatty acid content in the microbial oil is measured, so that the results are shown in Table 1.
Example 11
Microbial oil was prepared as in example 1, except that in step (3), sulforaphane was added to the first medium to a content of 0.7mg/L; in the step (4), the sulforaphane is not added to the second medium.
And (3) biomass measurement is carried out on the fermentation culture solution obtained in the step (4), the oil content calculation is carried out on the microbial oil obtained in the step (5), and the polyunsaturated fatty acid content in the microbial oil is measured, so that the results are shown in Table 1.
Example 12
A microbial oil was prepared in the same manner as in example 1, except that in step (3), graphene oxide in the first medium was replaced with sulforaphane and the amount of sulforaphane added was 0.7mg/L; in the step (4), the sulforaphane in the second culture medium is replaced by graphene oxide, and the addition amount of the graphene oxide is 3mg/L.
And (3) biomass measurement is carried out on the fermentation culture solution obtained in the step (4), the oil content calculation is carried out on the microbial oil obtained in the step (5), and the polyunsaturated fatty acid content in the microbial oil is measured, so that the results are shown in Table 1.
Example 13
A microbial oil was produced in the same manner as in example 1, except that in step (3), graphene oxide was not added to the first medium, and step (4) was replaced with:
(4) Inoculating seed culture solution obtained in the step (3) for 15 generations into a second culture medium with the inoculum size of 10 vol%, fermenting and culturing under the conditions that the initial pH is 5.5 and the rotating speed is 170rpm, adding graphene oxide and sulforaphane after culturing for 24 hours, enabling the content of the graphene oxide to be 3mg/L and the content of the sulforaphane to be 0.7mg/L, and culturing for 96 hours to obtain fermentation culture solution.
And (3) biomass measurement is carried out on the fermentation culture solution obtained in the step (4), the oil content calculation is carried out on the microbial oil obtained in the step (5), and the polyunsaturated fatty acid content in the microbial oil is measured, so that the results are shown in Table 1.
Example 14
A microbial oil was produced in the same manner as in example 1, except that in step (3), the amount of graphene oxide added to the first medium was replaced with 0.5mg/L.
And (3) biomass measurement is carried out on the fermentation culture solution obtained in the step (4), the oil content calculation is carried out on the microbial oil obtained in the step (5), and the polyunsaturated fatty acid content in the microbial oil is measured, so that the results are shown in Table 1.
Example 15
A microbial oil was produced in the same manner as in example 1, except that in step (4), the addition amount of sulforaphane in the second medium was replaced with 2mg/L.
And (3) biomass measurement is carried out on the fermentation culture solution obtained in the step (4), the oil content calculation is carried out on the microbial oil obtained in the step (5), and the polyunsaturated fatty acid content in the microbial oil is measured, so that the results are shown in Table 1.
Example 16
A microbial oil was produced in the same manner as in example 1, except that in step (3), the amount of graphene oxide added in the first medium was replaced with 0.5mg/L; in the step (4), the addition amount of the sulforaphane in the second culture medium is replaced by 2mg/L.
And (3) biomass measurement is carried out on the fermentation culture solution obtained in the step (4), the oil content calculation is carried out on the microbial oil obtained in the step (5), and the polyunsaturated fatty acid content in the microbial oil is measured, so that the results are shown in Table 1.
Example 17
A microbial oil was prepared as in example 1, except that step (4) was replaced with:
(4) Inoculating the seed culture solution obtained in the step (3) for 15 generations into a second culture medium with the inoculation amount of 10 vol%, fermenting and culturing at the initial pH of 5.5, the rotating speed of 170rpm and the temperature of 28 ℃ for 10 hours, adding sulforaphane to the culture solution to enable the content of the sulforaphane to be 0.7mg/L, and culturing for 96 hours to obtain a fermentation culture solution.
And (3) biomass measurement is carried out on the fermentation culture solution obtained in the step (4), the oil content calculation is carried out on the microbial oil obtained in the step (5), and the polyunsaturated fatty acid content in the microbial oil is measured, so that the results are shown in Table 1.
Example 18
A microbial oil was prepared as in example 1, except that in step (4), sulforaphane was not added to the second medium.
And (3) biomass measurement is carried out on the fermentation culture solution obtained in the step (4), the oil content calculation is carried out on the microbial oil obtained in the step (5), and the polyunsaturated fatty acid content in the microbial oil is measured, so that the results are shown in Table 1.
Comparative example 1
A microbial oil was prepared according to the method of example 1, except that in step (3), graphene oxide was not added to the first medium; in the step (4), the sulforaphane is not added to the second culture medium.
Performing biomass measurement on the seed culture solution obtained in the step (3), wherein the result is shown in figure 1; and (3) carrying out biomass measurement on the fermentation culture solution obtained in the step (4), calculating the oil content of the microbial oil obtained in the step (5), and measuring the polyunsaturated fatty acid content in the microbial oil, wherein the result is shown in Table 1.
Comparative example 2
A microbial oil was prepared according to the method of example 1, except that in step (3), graphene oxide was not added to the first medium; in the step (4), the addition amount of the sulforaphane in the second culture medium is replaced by 0.5mg/L.
And (3) biomass measurement is carried out on the fermentation culture solution obtained in the step (4), the oil content calculation is carried out on the microbial oil obtained in the step (5), and the polyunsaturated fatty acid content in the microbial oil is measured, so that the results are shown in Table 1.
Comparative example 3
A microbial oil was prepared according to the method of example 1, except that in step (3), graphene oxide was not added to the first medium; in the step (4), the addition amount of the sulforaphane in the second culture medium is replaced by 0.6mg/L.
And (3) biomass measurement is carried out on the fermentation culture solution obtained in the step (4), the oil content calculation is carried out on the microbial oil obtained in the step (5), and the polyunsaturated fatty acid content in the microbial oil is measured, so that the results are shown in Table 1.
Comparative example 4
A microbial oil was prepared according to the method of example 1, except that in step (3), graphene oxide was not added to the first medium; in the step (4), the addition amount of the sulforaphane in the second culture medium is replaced by 0.7mg/L.
And (3) biomass measurement is carried out on the fermentation culture solution obtained in the step (4), the oil content calculation is carried out on the microbial oil obtained in the step (5), and the polyunsaturated fatty acid content in the microbial oil is measured, so that the results are shown in Table 1.
Comparative example 5
A microbial oil was prepared according to the method of example 1, except that in step (3), graphene oxide was not added to the first medium; in the step (4), the addition amount of the sulforaphane in the second culture medium is replaced by 0.8mg/L.
And (3) biomass measurement is carried out on the fermentation culture solution obtained in the step (4), the oil content calculation is carried out on the microbial oil obtained in the step (5), and the polyunsaturated fatty acid content in the microbial oil is measured, so that the results are shown in Table 1.
Comparative example 6
A microbial oil was prepared according to the method of example 1, except that in step (3), graphene oxide was not added to the first medium; in the step (4), the addition amount of the sulforaphane in the second culture medium is replaced by 0.9mg/L.
And (3) biomass measurement is carried out on the fermentation culture solution obtained in the step (4), the oil content calculation is carried out on the microbial oil obtained in the step (5), and the polyunsaturated fatty acid content in the microbial oil is measured, so that the results are shown in Table 1.
Comparative example 7
A microbial oil was prepared according to the method of example 1, except that in step (3), graphene oxide was not added to the first medium; in the step (4), the addition amount of the sulforaphane in the second culture medium is replaced by 1mg/L.
And (3) biomass measurement is carried out on the fermentation culture solution obtained in the step (4), the oil content calculation is carried out on the microbial oil obtained in the step (5), and the polyunsaturated fatty acid content in the microbial oil is measured, so that the results are shown in Table 1.
Comparative example 8
A microbial oil was prepared as in example 1, except that graphene oxide was replaced with graphene in step (3).
And (3) biomass measurement is carried out on the fermentation culture solution obtained in the step (4), the oil content calculation is carried out on the microbial oil obtained in the step (5), and the polyunsaturated fatty acid content in the microbial oil is measured, so that the results are shown in Table 1.
TABLE 1
As can be seen from FIG. 1, the seed culture solutions obtained in the passages 15 were optimal for cell growth when the seed activation culture solutions in examples 1 to 3, 9, 10 and comparative example 1 were subjected to the passage culture for different passages.
As can be seen from the results in table 1, the polyunsaturated fatty acid (PUFA) content in the microbial oils and fats obtained by the microbial cultivation in examples 1 to 18 using the method provided by the present invention is significantly improved compared with comparative examples 1 to 8, which indicates that the regulatory factor in the present invention can significantly improve the polyunsaturated fatty acid content of oleaginous microorganisms. Moreover, compared with comparative example 1, the EPA content in the microbial oil prepared in example 1 was increased by 668%, and the ARA content was increased by 2500%.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, a number of simple variants of the technical solution of the invention are possible, including combinations of the individual technical features in any other suitable way, which simple variants and combinations should likewise be regarded as being disclosed by the invention, all falling within the scope of protection of the invention.

Claims (13)

1. The application of graphene oxide and sulforaphane in improving the production of ARA and EPA by schizochytrium.
2. A method for increasing the amount of ARA and EPA produced by schizochytrium, comprising: inoculating schizochytrium into a culture medium containing graphene oxide and sulforaphane for culturing.
3. The method according to claim 2, wherein the graphene oxide is added in an amount of 1 to 5mg and the sulforaphane is added in an amount of 0.5 to 1mg relative to 1L of the medium.
4. A method according to claim 3, wherein the culturing comprises: inoculating schizochytrium into a first culture medium for subculture to obtain a seed solution, and inoculating the seed solution into a second culture medium for fermentation culture in an inoculum size of 0.5-10 vol%, wherein the graphene oxide is added into the first culture medium and/or the second culture medium, and the sulforaphane is added into the first culture medium and/or the second culture medium.
5. The method of claim 4, wherein the graphene oxide is added in the first medium and the sulforaphane is added in the second medium.
6. The method of claim 5, wherein the time for adding the sulforaphane is 20-30 hours after the start of fermentation.
7. The method according to claim 6, wherein the subculture is carried out for a number of passages of 5 to 20 passages and a time period of 22 to 26 hours per passage.
8. The method of claim 6, wherein the first medium comprises: 30-50g/L glucose 、1-5g/L KH2PO4、10-14g/L Na2SO4、2-5g/L MgSO4、1-3g/L KCl、2-5g/L (NH4)2SO4、0.06-1g/L CaCl2、0.1-1g/L K2SO4、8-12g/L monosodium glutamate, 9-15g/L yeast powder;
The second medium contains: 50-70g/L glucose 、1-5g/L KH2PO4、10-14g/L Na2SO4、2-5g/L MgSO4、1-3g/L KCl、2-5g/L (NH4)2SO4、0.06-1g/L CaCl2、0.1-1g/L K2SO4、1-5g/L malic acid, 8-12g/L monosodium glutamate, 9-15g/L yeast extract, 3-7 g/L corn steep liquor dry powder and 0.5-1g/L metal ion solution, wherein the metal ion solution contains Zn 2+ and Cu 2+, and the mass ratio of Zn 2+ to Cu 2+ is 1:0.5-1.
9. The method of claim 8, wherein the conditions of the subculture and the fermentation culture comprise: the initial pH is 5-6, the rotating speed is 150-200rpm, and the temperature is 25-30 ℃.
10. A process for the preparation of ARA and/or EPA, the process comprising: culturing schizochytrium by the method of any one of claims 2 to 9 to obtain a culture solution, and breaking and extracting the wall of the culture solution.
11. The method according to claim 10, wherein the breaking of the wall is performed by enzymatic hydrolysis of the wall by a wall breaking enzyme.
12. The method of claim 11, wherein the wall breaking conditions comprise: the dosage of the wall breaking enzyme is 3-6g/L, the pH is 10-12, the rotating speed is 150-200rpm, the temperature is 25-30 ℃ and the time is 4-6h.
13. The method according to claim 12, wherein the extraction solvent is n-hexane and/or ethanol.
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