Microbial oil as well as preparation method and application thereof

Abstract

The invention relates to the field of microorganisms, and discloses microbial oil as well as a preparation method and application thereof. The weight ratio of DHA to DPA in the microbial oil is greater than or equal to 4; the proportion of the long-chain polyunsaturated fatty acid on the Sn-2 site of the triglyceride is greater than or equal to 30wt%; the proportion of DHA in the Sn-2 position of the triglyceride is > = 30% by weight. The preparation method comprises the steps of fermentation culture, separation and extraction. The fermentation culture process comprises the following steps: inoculating the oleaginous microorganisms into a fermentation culture medium added with ascorbate and benzoic acid para-position derivatives, and carrying out fermentation culture. The microbial oil can effectively increase the content of DHA at the Sn-2 site, so that the DHA absorption rate of a human body can be effectively increased.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and in particular to a microbial oil and fat and a preparation method and application thereof. Background Art

[0002] Docosahexaenoic acid (DHA) is an important long-chain polyunsaturated fatty acid (PUFA) that has vital physiological functions for the human body. The human body cannot synthesize it and must be ingested through diet. During the digestion and absorption process, triglyceride-type DHA needs to be broken down by pancreatic lipase in the digestive tract, and pancreatic lipase is a digestive enzyme that specifically hydrolyzes the Sn-1 and Sn-3 positions of triglycerides. The Sn-1 and Sn-3 positions of triglycerides will be hydrolyzed to form free fatty acids; the fatty acids at the Sn-2 position of triglycerides will form monoglycerides at the Sn-2 position. Sn-2 DHA monoglyceride can directly enter the blood through the intestinal mucosa, and combine with phospholipids in the body to form phospholipid structured lipids (PLS, which we often call phospholipid-type DHA), thereby entering the brain, so a higher proportion of sn-2 DHA means a higher absorption rate.

[0003] As one of the main sources of DHA, microbial oils and fats have been widely used in infant foods and health foods as people's health awareness increases. Their nutritional effects are increasingly recognized and accepted by the public, and the absorption rate of DHA in microbial oils and fats is also increasingly concerned by the public. More than 90% of the fatty acids in microbial oils and fats are in the form of triglyceride structures, and the current microbial-derived DHA oils and fats are obtained by fermentation of microorganisms such as Oukenbergia, Schizochytrium, Thraustochytriales, Crypthecodinium, and yeast. The distribution ratio of the obtained triglyceride-type DHA on the glycerol skeleton is that the content of Sn-2 is much lower than that of Sn-1 and Sn-3. A large amount of Sn-1 and Sn-3 DHA forms soap salts and is lost during human digestion, which limits the health effects of microbial oils and fats. Summary of the invention

[0004] The purpose of the present invention is to overcome the problems in the prior art that the Sn-2 DHA content in microbial oils is low and the ratio of DHA to DPA is unbalanced, which affects the absorption rate of DHA by the human body. A microbial oil and a preparation method and application thereof are provided. The microbial oil can effectively increase the Sn-2 DHA content and the weight ratio of DHA to DPA, thereby effectively increasing the absorption rate of DHA by the human body.

[0005] The first aspect of the present invention provides a microbial oil, in which the weight ratio of DHA to DPA is ≥4; the proportion of long-chain polyunsaturated fatty acids at the Sn-2 position of triglycerides is ≥30 weight%, and the proportion of DHA at the sn-2 position of triglycerides is ≥30 weight%.

[0006] The second aspect of the present invention provides a method for preparing microbial oil, which comprises the following steps: fermentation culture, separation and extraction; the fermentation culture process comprises: inoculating the oil-producing microorganism into a fermentation medium added with ascorbate and a para-derivative of benzoic acid for fermentation culture.

[0007] Preferably, the ascorbate is selected from at least one of sodium ascorbate, magnesium ascorbate, calcium ascorbate and zinc ascorbate.

[0008] Preferably, the para-derivative of benzoic acid is p-aminobenzoic acid and / or folic acid.

[0009] Preferably, relative to 1 L of the fermentation medium, the added amount of the ascorbate is 2-50 g, and the added amount of the para-benzoic acid derivative is 50-500 mg.

[0010] More preferably, relative to 1 L of the fermentation medium, the added amount of the ascorbate is 5-10 g, and the added amount of the para-benzoic acid derivative is 100-200 mg.

[0011] Preferably, the weight ratio of the ascorbate to the para-benzoic acid derivative is 40-60:1.

[0012] Preferably, the pH value is maintained at 5-7.5 by adding a pH adjuster during the fermentation culture.

[0013] Preferably, the pH adjuster is a small molecule organic acid, more preferably citric acid and / or malic acid.

[0014] Preferably, the fermentation culture includes a first stage fermentation and a second stage fermentation, and the conditions of the first stage fermentation include: pH 6-7.5, dissolved oxygen content 2-55%, temperature 25-30°C, and time 40-60h; the conditions of the second stage fermentation include: pH 5.5-7, dissolved oxygen content 10-70%, temperature 18-28°C, and time 48-60h.

[0015] Preferably, the conditions of the second stage fermentation also include: reducing the dissolved oxygen content to 2-30% within 10-30 hours and maintaining it until the end of the fermentation.

[0016] Preferably, the method further comprises: inoculating the oil-producing microorganism into a seed culture medium for culture, and then transferring the oil-producing microorganism into the fermentation culture medium for fermentation culture.

[0017] Preferably, the seed culture conditions include: pH 5.5-7, rotation speed 150-200 rpm, temperature 16-30°C, and time 20-24 h.

[0018] Preferably, the oil-producing microorganism is selected from at least one of Schizochytrium, Thraustochytrium, Ukenella and Cryptodinium, more preferably Schizochytrium.

[0019] Preferably, the separation and extraction process comprises: subjecting the fermentation broth obtained by the fermentation culture to enzymatic cell wall breaking treatment or mechanical cell wall breaking treatment and then centrifugal extraction.

[0020] Preferably, the enzyme used in the enzymatic cell wall breaking treatment is selected from at least one of protease, pectinase, cellulase and mannanase; and the mechanical cell wall breaking treatment uses a homogenization method to break the cell wall.

[0021] Preferably, the method further comprises: refining the oil obtained by separation and extraction.

[0022] Preferably, the oil refining process includes: dewaxing, degumming, washing, deodorization and winterization.

[0023] The third aspect of the present invention provides the use of the microbial oil described in the first aspect and / or the microbial oil obtained by the method described in the second aspect in infant formula food, health food and health food.

[0024] Through the above technical solution, the beneficial effects of the present invention are:

[0025] The microbial oil provided by the present invention can improve the absorption rate of DHA in the microbial oil by the human body by controlling the weight ratio of DHA to DPA in the microbial oil to be ≥4, the proportion of long-chain polyunsaturated fatty acids at the Sn-2 position of triglycerides to be ≥30% by weight, and the proportion of DHA at the Sn-2 position of triglycerides to be ≥30% by weight.

[0026] The preparation method of the microbial oil provided by the present invention can effectively increase the weight ratio of DHA to DPA and the content of DHA in the microbial oil by using ascorbate and a para-derivative of benzoic acid for the fermentation culture of oil-producing microorganisms, and can also increase the proportion of long-chain polyunsaturated fatty acids at the Sn-2 position of triglycerides, thereby significantly improving the absorption rate of DHA in the microbial oil by the human body. DETAILED DESCRIPTION

[0027] 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.

[0028] The first aspect of the present invention provides a microbial oil, in which the weight ratio of DHA to DPA is ≥4; the proportion of long-chain polyunsaturated fatty acids at the Sn-2 position of triglycerides is ≥30 weight %; and the proportion of DHA at the Sn-2 position of triglycerides is ≥30 weight %.

[0029] In the present invention, long-chain polyunsaturated fatty acids refer to straight-chain fatty acids containing 18 to 22 carbon atoms, and these fatty acid molecules contain two or more double bonds. Exemplarily, the long-chain polyunsaturated fatty acids can be eicosapentaenoic acid, docosapentaenoic acid, docosahexaenoic acid, etc.

[0030] During the research process, the inventors of the present invention unexpectedly discovered that by controlling the weight ratio of DHA to DPA in microbial oil to ≥4, the proportion of long-chain polyunsaturated fatty acids at the Sn-2 position of triglycerides to ≥30% by weight, and the proportion of DHA at the Sn-2 position of triglycerides to ≥30% by weight, the human body's absorption rate of DHA in microbial oil can be improved.

[0031] The second aspect of the present invention provides a method for preparing the microbial oil described in the first aspect, which method comprises the following steps: fermentation culture, separation and extraction; the fermentation culture process comprises: inoculating the oil-producing microorganisms into a fermentation medium added with ascorbate and a para-derivative of benzoic acid for fermentation culture.

[0032] The inventors of the present invention discovered during the research process that the preparation method can effectively increase the weight ratio of DHA to DPA and the DHA content in the microbial oil by using ascorbate and para-derivatives of benzoic acid to ferment and produce microbial oils with oil-producing microorganisms, and can also increase the proportion of long-chain polyunsaturated fatty acids at the Sn-2 position of triglycerides, thereby improving the human body's absorption rate of DHA in the microbial oil.

[0033] In the present invention, there is no particular limitation on the type of the ascorbate, and it can be any ascorbate that can be dissolved in the culture medium. Preferably, the ascorbate is selected from at least one of sodium ascorbate, magnesium ascorbate, calcium ascorbate and zinc ascorbate. The inventors have found that under this preferred embodiment, the content of Sn-2 DHA in the microbial oil formed by the oil-producing microorganism can be effectively increased.

[0034] According to the present invention, preferably, the para-benzoic acid derivative is p-aminobenzoic acid and / or folic acid. The inventors have found that under this preferred embodiment, the content of DHA at the Sn-2 position in the microbial oil formed by the oil-producing microorganism can be effectively increased.

[0035] According to the present invention, in order to further increase the Sn-2 DHA content in microbial oils and fats, preferably, relative to 1L of the fermentation medium, the added amount of the ascorbate is 2-50g, specifically 2g, 10g, 20g, 30g, 40g, 50g, or any value between the aforementioned values; the added amount of the para-derivative of benzoic acid is 50-500mg, specifically 50mg, 100mg, 150mg, 200mg, 250mg, 300mg, 350mg, 400mg, 450mg, 500mg, or any value between the aforementioned values.

[0036] According to the present invention, in order to further increase the Sn-2 DHA content in microbial oils and fats, preferably, relative to 1L of the fermentation medium, the added amount of the ascorbate is 5-10g, specifically 5g, 6g, 7g, 8g, 9g, 10g, or any value between the aforementioned values; the added amount of the para-derivative of benzoic acid is 100-200mg, specifically 100mg, 150mg, 200mg, or any value between the aforementioned values.

[0037] According to the present invention, preferably, the weight ratio of the ascorbate to the para-benzoic acid derivative is 40-60:1, specifically 40:1, 50:1, 60:1, or any value between the aforementioned values. The inventors have found that under this preferred embodiment, it is beneficial to enhance the effect of ascorbate and para-benzoic acid derivatives in promoting the synthesis of Sn-2 DHA in microbial oils produced by oil-producing microorganisms.

[0038] In the present invention, the fermentation medium contains a carbon source, a nitrogen source, an inorganic salt ion and a trace element, and the carbon source, the nitrogen source, the inorganic salt ion and the trace element can be any one of the corresponding substance types used for microbial fermentation; illustratively, the carbon source can be glucose, the nitrogen source can be yeast extract and / or corn steep liquor powder, the inorganic salt ion can be any one or more of sodium salt, potassium salt, magnesium salt, calcium salt, ammonium salt and phosphate, and the trace element can be Mn 2+ 、Co 2+ 、Ni 2+ and Fe 2+Any one or more of. In order to further increase the content of Sn-2 DHA in microbial oils and fats, preferably, the fermentation medium contains glucose, KH2PO4, Na2SO4, MgSO4, KCl, (NH4)2SO4, NaCl, CaCl2, sodium glutamate, yeast powder and corn steep liquor powder. Further preferably, the fermentation medium contains: 60-100g / L glucose, 0.5-5g / LKH2PO4, 4-15g / L Na2SO4, 1.5-6g / LMgSO4, 0.1-1g / L KCl, 1-5g / L(NH4)2SO4, 4-10g / L NaCl, 0.01-0.2g / L CaCl2, 5-25g / L sodium glutamate, 1-5g / L yeast powder and 0.5-10 / L corn steep liquor powder. Wherein, the inoculation amount of the fermentation medium is generally 0.5-10% by volume.

[0039] According to the present invention, preferably, during the fermentation culture, the pH value is maintained at 5-7.5 by adjusting the pH regulator, specifically 5, 5.5, 6, 6.5, 7, 7.5, or any value between the above two values. The inventors have found that under this preferred embodiment, it is beneficial to promote the growth of oil-producing microorganisms and increase the fermentation biomass.

[0040] According to the present invention, there is no particular limitation on the selection of pH regulators, as long as the pH regulation can be completed. Preferably, the pH regulator is a small molecule organic acid, more preferably citric acid and / or malic acid. Specifically, the pH regulator is citric acid and malic acid. There is no limitation on the dosage ratio of the citric acid to the malic acid in the pH regulator, and it can be a conventional choice in the art. In order to further promote the growth of oil-producing microorganisms, preferably, the dosage ratio of citric acid to malic acid in the pH regulator is 1:1-5, specifically 1:1, 1:2, 1:3, 1:4, 1:5, or any value between the aforementioned values.

[0041] According to the present invention, in order to further increase the Sn-2 DHA content in microbial oils and fats, the pH and dissolved oxygen content of the fermentation culture are controlled in stages during the fermentation culture stage. Preferably, the fermentation culture includes a first stage fermentation and a second stage fermentation, and the conditions of the first stage fermentation include: pH is 6-7.5, specifically 6, 6.5, 7, 7.5, or any value between the two values; dissolved oxygen content is 2-55%, specifically 2%, 20%, 40%, 55%, or any value between the two values; temperature is 25-30°C, specifically 25°C, 27°C, 30°C, or any value between the two values; time is 40-60h, specifically 40h, 50h, 60h, or the above any value between the two values; the conditions for the second stage fermentation include: pH 5.5-7, specifically 5.5, 6, 6.5, 7, or any value between the aforementioned two values; dissolved oxygen content is 10-70%, specifically 10%, 30%, 50%, 70%, or any value between the aforementioned two values; temperature is 18-28°C, specifically 18°C, 22°C, 24°C, 28°C, or any value between the aforementioned two values; time is 48-60h, specifically 48h, 54h, 60h, or any value between the aforementioned two values.

[0042] According to the present invention, in order to further increase the Sn-2 DHA content in microbial oils, preferably, the conditions of the second stage fermentation also include: within 10-30 hours, the dissolved oxygen content is reduced to 2-30% and maintained until the end of fermentation.

[0043] In the present invention, the method for controlling the dissolved oxygen content of the culture medium may be a conventional method in the art, for example, the dissolved oxygen content of the fermentation culture medium may be controlled by adjusting the air flow rate and / or the stirring speed.

[0044] According to the present invention, in order to obtain more fermentation products, preferably, the method comprises: inoculating the oil-producing microorganism into a seed culture medium for culture, and then transferring the oil-producing microorganism into the fermentation culture medium for fermentation culture.

[0045] According to the present invention, the seed culture medium contains a carbon source, a nitrogen source, an inorganic salt ion and a trace element, wherein the carbon source, the nitrogen source, the inorganic salt ion and the trace element can be any one of the corresponding substance types used for microbial fermentation; illustratively, the carbon source can be glucose, the nitrogen source can be yeast extract and / or corn steep liquor powder, the inorganic salt ion can be any one or more of sodium salt, potassium salt, magnesium salt, calcium salt, ammonium salt and phosphate, and the trace element can be Mn 2+ 、Co 2+ 、Ni 2+ and Fe 2+In order to further increase the content of Sn-2 DHA in microbial oils and fats, preferably, the seed culture medium contains glucose, KH2PO4, Na2SO4, MgSO4, KCl, (NH4)2SO4, sodium glutamate and yeast powder, and more preferably, the seed culture medium contains: 40-80g / L glucose, 0.5-5g / L KH2PO4, 5-45g / L Na2SO4, 1.5-6g / L MgSO4, 0.5-2g / L KCl, 1-8g / L (NH4)2SO4, 5-25g / L sodium glutamate and 2-10g / L yeast powder. Wherein, the inoculation amount of the seed culture medium is generally 0.5-2% by volume. If the microbial strains preserved in glycerol tubes are inoculated, the strains in each glycerol tube are inoculated into 100mL of the seed culture medium.

[0046] In the present invention, the parameters such as temperature, pH, rotation speed, time of seed culture can be conventional settings in the art. Preferably, the conditions of seed culture include: pH is 5.5-7, specifically 5.5, 6, 6.5, 7, or any value between the above two values; the rotation speed is 150-200rpm, specifically 150rpm, 170rpm, 200rpm, or any value between the above two values; the temperature is 16-30℃, specifically 16℃, 20℃, 25℃, 30℃, or any value between the above two values; the time is 20-24h, specifically 20h, 22h, 24h, or any value between the above two values. The inventors found that under this preferred specific embodiment, it is conducive to promoting the growth of oil-producing microorganisms and increasing the fermentation biomass.

[0047] According to the present invention, the oil-producing microorganism can be any microorganism that can produce microbial oil by fermentation. Preferably, the oil-producing microorganism is selected from at least one of Schizochytrium, diatom, Nannochloropsis and Mortierella alpina. In order to further increase the content of Sn-2 DHA in the oil produced by the oil-producing microorganism, Schizochytrium is more preferred.

[0048] According to the present invention, in order to further increase the content of effective ingredients in microbial oils and fats, preferably, the separation and extraction process comprises: subjecting the fermentation culture fluid obtained by the fermentation culture to enzymatic or mechanical cell wall breaking treatment and then centrifugal extraction.

[0049] According to the present invention, in order to further improve the separation and extraction effect of microbial oils, preferably, the enzyme used in the enzymatic cell wall breaking treatment is selected from at least one of protease, pectinase, cellulase and mannanase; and the mechanical cell wall breaking treatment adopts homogenization method for cell wall breaking.

[0050] For example, when enzymatic cell wall breaking treatment is used, the extraction method of microbial oil can be centrifugal separation. Specifically, a cell wall breaking enzyme is added to the culture solution at an addition amount of 0.2-5 g / L, and the cell wall breaking solution is obtained by enzymatic hydrolysis for 3-6 hours under the conditions of pH 10-12, rotation speed 150-200 rpm, and temperature 25-30°C. The cell wall breaking solution is placed in a centrifuge and centrifuged at a rotation speed of 8000 rpm to obtain microbial oil.

[0051] According to the present invention, the oil obtained by separation and extraction can be directly applied to the product, or the oil obtained by separation and extraction can be refined before being applied to the product. In order to further increase the content of effective ingredients in the microbial oil, preferably, the method also includes: refining the oil obtained by separation and extraction.

[0052] According to the present invention, in order to further increase the content of effective ingredients in microbial oils, the oil refining process includes: dewaxing, degumming, washing, deodorization, and winterization. The methods of dewaxing, degumming, washing, deodorization, and winterization can all be conventionally selected in the art and will not be described in detail here.

[0053] The third aspect of the present invention provides the use of the microbial oil described in the first aspect and / or the microbial oil obtained by the method described in the second aspect in infant formula, health products and health foods.

[0054] According to a particularly preferred embodiment of the present invention, a method for preparing microbial oil is provided, the method comprising:

[0055] (1) inoculating the strain of Schizochytrium into a seed culture medium at an inoculum amount of 0.5-2% by volume, and culturing for 20-24 hours at a pH of 5.5-7, a rotation speed of 150-200 rpm, and a temperature of 16-30° C. to obtain a seed solution;

[0056] (2) inoculating the seed solution obtained in step (1) into a fermentation medium supplemented with ascorbate and a para-derivative of benzoic acid at an inoculum amount of 0.5-10% by volume, first culturing at a pH of 6-7.5, a dissolved oxygen content of 2-55%, and a temperature of 25-30° C. for 40-60 h, and then culturing at a pH of 5.5-7, a dissolved oxygen content of 10-70%, and a temperature of 18-28° C., wherein the dissolved oxygen content is reduced to 2-30% within 5-30 h of culturing and maintained until the end of fermentation, thereby obtaining a fermentation culture solution;

[0057] (3) adding a cell wall-breaking enzyme in an amount of 2-4 g / L to the fermentation culture medium obtained in step (2), performing enzymolysis for 3-6 h at a pH of 10-12, a rotation speed of 150-200 rpm, and a temperature of 25-30° C. to obtain a cell wall-breaking liquid, mixing the cell wall-breaking liquid with n-hexane for extraction to obtain an aqueous phase, a n-hexane phase, and a solid, and removing the n-hexane from the n-hexane phase by rotary evaporation to obtain microbial oil;

[0058] Wherein, during the fermentation culture, the pH value is maintained at 5-7.5 by adding a pH regulator, and the pH regulator is citric acid and / or malic acid;

[0059] The seed culture medium contains: 40-80g / L glucose, 0.5-5g / L KH2PO4, 5-45g / L Na2SO4, 1.5-6g / LMgSO4, 0.5-2g / L KCl, 1-8g / L (NH4)2SO4, 5-25g / L sodium glutamate and 2-10g / L yeast powder; the fermentation medium contains: 60-100g / L glucose, 0.5-5g / L KH2PO4, 4-15g / L Na2SO4, 1.5-6g / L MgSO4, 0.1-1g / L KCl, 1-5g / L (NH4)2SO4, 4-10g / L NaCl, 0.01-0.2g / L CaCl2, 5-25g / L sodium glutamate, 1-5g / L yeast powder and 0.5-10g / L corn steep liquor powder; ascorbate is selected from at least one of sodium ascorbate, magnesium ascorbate, calcium ascorbate and zinc ascorbate; the para-benzoic acid derivative is para-aminobenzoic acid and / or folic acid; relative to 1L of fermentation medium, the added amount of ascorbate is 5-10g, the added amount of the para-benzoic acid derivative is 100-200mg, and the mass ratio of ascorbate to the para-benzoic acid derivative is 40-60:1.

[0060] In the microbial oil prepared by the method provided in the above preferred embodiment, the content of Sn-2 DHA is higher.

[0061] The present invention will be described in detail below through examples. In the following examples and comparative examples, Schizochytrium is a self-owned strain, which is currently deposited in Guangdong Provincial Microbiological Collection Center with a deposit number of GDMCC.NO 60733.

[0062] Sodium ascorbate was purchased from CSPC Pharmaceutical Group Co., Ltd. Other raw materials and reagents were purchased from commercial suppliers.

[0063] The biomass was measured by taking 50 mL of the culture solution into a weighed centrifuge tube, centrifuging to remove the supernatant, and drying in an oven to a constant weight.

[0064] The method for determining the content of DHA and fatty acid composition in microbial oils and fats adopts GB 5009.168-2016. The method for determining the proportion of fatty acids at the sn-2 position in microbial oils and fats adopts the method in CN202311180318.8.

[0065] Example 1

[0066] (1) The formula of the seed culture medium is as follows: 60 g / L glucose, 3 g / L KH2PO4, 35 g / L Na2SO4, 4 g / L MgSO4, 1 g / L KCl, 5 g / L (NH4)2SO4, 20 g / L sodium glutamate and 5 g / L yeast powder, which is sterilized at 121°C for 20 min before use; the formula of the fermentation culture medium is as follows: 5 g / L sodium ascorbate, 100 mg / L p-aminobenzoic acid, 80 g / L glucose, 3 g / L KH2PO4, 8 g / L Na2SO4, 4 g / L MgSO4, 0.5 g / L KCl, 3 g / L (NH4)2SO4, 7 g / L NaCl, 0.1 g / L CaCl2, 20 g / L sodium glutamate, 3 g / L yeast powder and 1.5 g / L corn steep liquor powder, which is sterilized at 121°C for 20 min before use;

[0067] (2) inoculating the Schizochytrium species into a seed culture medium at an inoculum amount of 1 volume %, and culturing for 24 hours at a pH of 6.5, a rotation speed of 170 rpm, and a temperature of 28° C. to obtain a seed solution;

[0068] (3) inoculating the seed liquid obtained in step (2) into the fermentation medium at an inoculum amount of 5% by volume, adjusting the pH using citric acid and malic acid as pH regulators, culturing for 48 hours under the conditions of pH 7.2, dissolved oxygen content of 50%, and temperature of 28° C., and then culturing under the conditions of pH 6.5 and temperature of 26° C., reducing the dissolved oxygen content to 20% within 20 hours of culturing and continuing fermentation for 36 hours to obtain a fermentation culture medium;

[0069] (4) adding a cell wall-breaking enzyme at an addition amount of 3 g / L to the fermentation culture medium, performing enzymatic hydrolysis for 4 h under the conditions of pH 11, rotation speed 180 rpm, and temperature 28° C. to obtain a cell wall-breaking liquid, mixing the cell wall-breaking liquid with ethanol and n-hexane at a volume ratio of 1:1:1 to extract a n-hexane phase, and removing n-hexane from the n-hexane phase by rotary evaporation to obtain microbial oil;

[0070] Among them, in the pH adjuster, the weight ratio of citric acid to malic acid is 1:1.

[0071] The microbial oil was measured and its component contents were shown in Table 1.

[0072] Table 1

[0073]

[0074]

[0075] Example 2

[0076] (1) The formula of the seed culture medium is as follows: 40 g / L glucose, 5 g / L KH2PO4, 45 g / L Na2SO4, 6 g / L MgSO4, 2 g / L KCl, 8 g / L (NH4)2SO4, 25 g / L sodium glutamate and 8 g / L yeast powder, which is sterilized at 121°C for 20 min before use; the formula of the fermentation culture medium is as follows: 8 g / L sodium ascorbate, 200 mg / L p-aminobenzoic acid, 60 g / L glucose, 5 g / L KH2PO4, 12 g / L Na2SO4, 6 g / L MgSO4, 1 g / L KCl, 5 g / L (NH4)2SO4, 10 g / L NaCl, 0.2 g / L CaCl2, 25 g / L sodium glutamate, 5 g / L yeast powder and 2.5 g / L corn steep liquor powder, which is sterilized at 121°C for 20 min before use;

[0077] (2) inoculating the Schizochytrium species into a seed culture medium at an inoculum amount of 0.5 volume %, and culturing for 20 h under the conditions of pH 7.0, a rotation speed of 150 rpm, and a temperature of 25° C. to obtain a seed solution;

[0078] (3) inoculating the seed liquid obtained in step (2) into the fermentation medium at an inoculum amount of 0.5% by volume, adjusting the pH using citric acid and malic acid as pH regulators, culturing for 24 hours under the conditions of pH 6.5, dissolved oxygen 45%, and temperature 28° C., and then culturing under the conditions of pH 5.5 and temperature 26° C., reducing the dissolved oxygen to 15% within 10 hours of culturing and continuing fermentation for 50 hours to obtain a fermentation culture medium;

[0079] (4) adding a cell wall-breaking enzyme at an addition amount of 2 g / L to the fermentation culture medium, performing enzymolysis for 3 h under the conditions of pH 12, rotation speed 200 rpm, and temperature 30° C. to obtain a cell wall-breaking liquid, mixing the cell wall-breaking liquid with ethanol and n-hexane at a volume ratio of 1:1:1 to extract a n-hexane phase, and removing n-hexane from the n-hexane phase by rotary evaporation to obtain microbial oil;

[0080] Among them, in the pH adjuster, the weight ratio of citric acid to malic acid is 1:4.

[0081] The microbial oil was measured and its component contents were shown in Table 2.

[0082] Table 2

[0083]

[0084]

[0085] Example 3

[0086] (1) The formula of the seed culture medium is as follows: glucose 80 g / L, KH2PO4 1 g / L, Na2SO4 25 g / L, MgSO4 2 g / L, KCl 0.5 g / L, (NH4)2SO4 2 g / L, sodium glutamate 15 g / L and yeast powder 2 g / L, which is sterilized at 121 °C for 20 min and then used. The formula of the fermentation culture medium is as follows: sodium ascorbate 10 g / L, p-aminobenzoic acid 150 mg / L, glucose 100 g / L, KH2PO4 1 g / L, Na2SO4 4 g / L, MgSO4 2 g / L, KCl 0.1 g / L, (NH4)2SO4 1 g / L, NaCl 4 g / L, CaCl2 0.01g / L, sodium glutamate 15g / L, yeast powder 1g / L and corn steep liquor powder 0.5g / L, sterilize at 121℃ for 20min and set aside;

[0087] (2) inoculating the Schizochytrium species into a seed culture medium at an inoculum amount of 2% by volume, and culturing for 24 hours at a pH of 5.5, a rotation speed of 200 rpm, and a temperature of 30° C. to obtain a seed solution;

[0088] (3) inoculating the seed liquid obtained in step (2) into the fermentation medium at an inoculum amount of 10% by volume, adjusting the pH using citric acid and malic acid as pH regulators, culturing for 60 hours under the conditions of pH 7.2, dissolved oxygen content of 55%, and temperature of 28° C., and then culturing under the conditions of pH 5.5, initial dissolved oxygen content of 55%, and temperature of 26° C., reducing the dissolved oxygen content to 30% within 30 hours of culturing and continuing fermentation for 24 hours to obtain a fermentation culture medium;

[0089] (4) adding a cell wall-breaking enzyme at an addition amount of 4 g / L to the fermentation broth, performing enzymolysis for 6 h under the conditions of pH 10, rotation speed 150 rpm, and temperature 25° C. to obtain a cell wall-breaking liquid, mixing the cell wall-breaking liquid with ethanol and n-hexane at a volume ratio of 1:1:1 to obtain a n-hexane phase, and removing n-hexane from the n-hexane phase by rotary evaporation to obtain microbial oil;

[0090] Among them, in the pH adjuster, the weight ratio of citric acid to malic acid is 1:3.

[0091] The microbial oil was measured and its component contents were shown in Table 3.

[0092] Table 3

[0093]

[0094] Example 4

[0095] The microbial oil was prepared according to the method of Example 1, except that in step (1), p-aminobenzoic acid was replaced by folic acid.

[0096] The microbial oil was measured and its component contents were shown in Table 4.

[0097] Table 4

[0098]

[0099] Example 5

[0100] The microbial oil was prepared according to the method of Example 1, except that in step (1), p-aminobenzoic acid was replaced by p-aminobenzoic acid and folic acid, wherein the content of p-aminobenzoic acid was 50 mg / L, and the content of folic acid was 50 mg / L.

[0101] The microbial oil was measured and its component contents were shown in Table 5.

[0102] Table 5

[0103]

[0104] Example 6

[0105] The microbial oil was prepared according to the method of Example 1, except that in step (1), sodium ascorbate was replaced by magnesium ascorbate.

[0106] The microbial oil was measured and its component contents were shown in Table 6.

[0107] Table 6

[0108]

[0109] Example 7

[0110] Microbial oil was prepared according to the method of Example 1, except that in step (1), sodium ascorbate was replaced by calcium ascorbate and zinc ascorbate, wherein the content of calcium ascorbate was 2.5 g / L and the content of zinc ascorbate was 2.5 g / L.

[0111] The microbial oil was measured and its component contents were shown in Table 7.

[0112] Table 7

[0113]

[0114] Example 8

[0115] The microbial oil was prepared according to the method of Example 1, except that in step (1), the amount of sodium ascorbate added was 2 g / L.

[0116] The microbial oil was measured and its component contents were shown in Table 8.

[0117] Table 8

[0118]

[0119] Example 9

[0120] The microbial oil was prepared according to the method of Example 1, except that in step (1), the amount of p-aminobenzoic acid added was 50 mg / L.

[0121] The microbial oil was measured and its component contents were shown in Table 9.

[0122] Table 9

[0123]

[0124] Example 10

[0125] The microbial oil was prepared according to the method of Example 1, except that in step (1), the amount of sodium ascorbate added was 2 g / L and the amount of p-aminobenzoic acid added was 50 mg / L.

[0126] The microbial oil was measured and its component contents were shown in Table 10.

[0127] Table 10

[0128]

[0129]

[0130] Embodiment 11

[0131] Microbial oil was prepared according to the method of Example 1, except that in step (1), the amount of sodium ascorbate added was 5 g / L and the amount of p-aminobenzoic acid added was 200 mg / L.

[0132] The microbial oil was measured and its component contents were shown in Table 11.

[0133] Table 11

[0134]

[0135] Example 12

[0136] The microbial oil was prepared according to the method of Example 1, except that step (3) was replaced by:

[0137] (3) inoculating the seed solution obtained in step (2) into a fermentation medium at an inoculum amount of 5% by volume, and fermenting and culturing for 96 hours at a pH of 7.2, a dissolved oxygen content of 50%, and a temperature of 28° C. to obtain a fermentation culture medium;

[0138] The microbial oil was measured and its component contents were shown in Table 12.

[0139] Table 12

[0140]

[0141] Comparative Example 1

[0142] The microbial oil was prepared according to the method of Example 1, except that in step (1), p-aminobenzoic acid was not added to the fermentation medium.

[0143] The microbial oil was measured and its component contents were shown in Table 13.

[0144] Table 13

[0145]

[0146] Comparative Example 2

[0147] The microbial oil was prepared according to the method of Example 1, except that in step (1), sodium ascorbate was not added to the fermentation medium.

[0148] The microbial oil was measured and its component contents were shown in Table 14.

[0149] Table 14

[0150]

[0151] Comparative Example 3

[0152] The microbial oil was prepared according to the method of Example 1, except that in step (1), p-aminobenzoic acid and sodium ascorbate were not added to the fermentation medium.

[0153] The microbial oil was measured and its component contents were shown in Table 15.

[0154] Table 15

[0155]

[0156] Comparative Example 4

[0157] The microbial oil was prepared according to the method of Example 1, except that p-aminobenzoic acid and sodium ascorbate were not added to the fermentation medium, and step (3) was replaced by:

[0158] (3) The seed liquid obtained in step (2) is inoculated into the fermentation medium at an inoculum amount of 5 volume %, and fermented for 96 hours under the conditions of pH 7.2, dissolved oxygen content of 50%, and temperature of 28° C. to obtain a fermentation culture medium.

[0159] The microbial oil was measured and its component contents were shown in Table 16.

[0160] Table 16

[0161]

[0162] It can be seen from the results in Tables 1 to 16 that, compared with Comparative Examples 1 to 4, the weight ratio of DHA to DPA, the content of Sn-2 DHA and the proportion of the microbial oils prepared by the preparation method provided by the present invention in Examples 1 to 12 have been significantly improved, indicating that adding ascorbate and para-derivatives of benzoic acid into the culture medium for biological fermentation can promote the absorption of nutrients in the culture medium by oil-producing microorganisms, thereby improving the utilization rate of nutrients in the culture medium, and can also increase the content of Sn-2 DHA in the oils produced by oil-producing microorganisms.

[0163] 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 microbial oil, characterized in that: The weight ratio of DHA to DPA in the microbial oil is ≥4; the proportion of long-chain polyunsaturated fatty acids at the Sn-2 position of triglycerides is ≥30 weight %; and the proportion of DHA at the Sn-2 position of triglycerides is ≥30 weight %.

2. A method for preparing microbial oil, characterized in that: The method comprises the following steps: fermentation culture and separation and extraction; the fermentation culture process comprises: inoculating the oil-producing microorganism into a fermentation medium added with ascorbate and a para-derivative of benzoic acid for fermentation culture.

3. The preparation method according to claim 2, characterized in that: The ascorbate is selected from at least one of sodium ascorbate, magnesium ascorbate, calcium ascorbate and zinc ascorbate; Preferably, the para-derivative of benzoic acid is p-aminobenzoic acid and / or folic acid.

4. The preparation method according to claim 3, characterized in that: Relative to 1L of the fermentation medium, the amount of ascorbate added is 2-50g, and the amount of the para-benzoic acid derivative added is 50-500mg; Preferably, relative to 1 L of the fermentation medium, the amount of ascorbate added is 5-10 g, and the amount of the para-benzoic acid derivative added is 100-200 mg; Preferably, the weight ratio of the ascorbate to the para-benzoic acid derivative is 40-60:

1.

5. The preparation method according to any one of claims 2 to 4, characterized in that: During the fermentation culture, the pH value is maintained at 5-7.5 by adding a pH regulator; Preferably, the pH regulator is a small molecule organic acid, more preferably citric acid and / or malic acid; Preferably, the fermentation culture includes a first stage fermentation and a second stage fermentation, wherein the conditions of the first stage fermentation include: pH 6-7.5, dissolved oxygen 2-55%, temperature 25-30°C, and time 40-60h; the conditions of the second stage fermentation include: pH 5.5-7, dissolved oxygen 10-70%, temperature 18-28°C, and time 48-60h; Preferably, the conditions of the first stage fermentation also include: within 5-30 hours, the dissolved oxygen content is reduced to 2-30% and maintained until the end of the fermentation.

6. The preparation method according to any one of claims 2 to 4, characterized in that: The method further comprises: inoculating the oil-producing microorganism into a seed culture medium for culture, and then transferring the oil-producing microorganism into the fermentation culture medium for fermentation culture; Preferably, the seed culture conditions include: pH 5.5-7, rotation speed 150-200 rpm, temperature 16-30°C, and time 20-24 h.

7. The preparation method according to any one of claims 2 to 4, characterized in that: The oil-producing microorganism is at least one selected from Schizochytrium, Crypthecodinium cohnii, Chytridium wukenii, diatoms, Nannochloropsis and Mortierella alpina, preferably Schizochytrium.

8. The preparation method according to any one of claims 2 to 4, characterized in that: The separation and extraction process comprises: subjecting the fermentation culture fluid obtained by the fermentation culture to enzymatic cell wall breaking treatment or mechanical cell wall breaking treatment and then centrifugal extraction; Preferably, the enzyme used in the enzymatic cell wall breaking treatment is selected from at least one of protease, pectinase, cellulase and mannanase; and the mechanical cell wall breaking treatment uses a homogenization method to break the cell wall.

9. The preparation method according to any one of claims 2 to 4, characterized in that: The method further comprises: refining the oil obtained by separation and extraction; Preferably, the oil refining process includes: dewaxing, degumming, washing, deodorization and winterization.

10. Use of the microbial oil according to claim 1 and / or the microbial oil obtained by the preparation method according to any one of claims 2 to 9 in infant formula food, health food and health food.

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