Application of regulatory factor in promoting production of DHA (docosahexaenoic acid) from schizochytrium limacinum, method for improving production of DHA from schizochytrium limacinum and preparation method of microbial oil

Abstract

The invention relates to the technical field of microbial fermentation, and discloses application of a regulatory factor in promoting schizochytrium limacinum to produce DHA (docosahexaenoic acid), and the regulatory factor contains beta-carotene and reduced glutathione. In addition, the invention also provides a method for improving DHA production of schizochytrium limacinum and a preparation method of microbial oil. The method comprises the following steps: fermenting schizochytrium limacinum in a culture medium, and adding beta-carotene and reduced glutathione into the culture medium in the fermentation process; in the culture medium, the content of the beta-carotene is 1.5 to 2g / L. The beta-carotene and the reduced glutathione can effectively improve the content of grease and DHA prepared by fermentation of schizochytrium limacinum, the content of DHA can reach 63.9%, and the beta-carotene and the reduced glutathione are non-toxic and high in safety.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation technology, specifically to the application of a regulatory factor in promoting DHA production by Schizochytrium. Furthermore, this invention also provides a method for improving DHA production by Schizochytrium and a method for preparing microbial lipids. Background Technology

[0002] Schizochytrium is a marine-derived single-celled fungus that has become an important microorganism for the industrial production of omega-3 fatty acids due to its efficient ability to synthesize and accumulate polyunsaturated fatty acids (PUFAs). Docosahexaenoic acid (DHA, C22:6ω-3) is one of the main metabolites of Schizochytrium.

[0003] DHA is an important long-chain polyunsaturated fatty acid with vital physiological functions for the human body, including promoting brain and nerve development in infants and young children, improving cognitive function, anti-inflammation, and preventing cardiovascular diseases. Currently, the main source of DHA is deep-sea fish oil, but fish oil production is limited by fishery resources, seasonal fluctuations, and environmental pollution, and the extraction and purification costs of DHA from fish oil are relatively high. Therefore, the production of DHA using Schizochytrium via microbial fermentation has advantages such as short cultivation cycle, no environmental restrictions, and high product purity, and has become a research hotspot for replacing fish oil DHA.

[0004] Existing technologies include methods that use quinoxaline compounds to enhance DHA production by Schizochytrium, but these methods have slight toxicity, and incomplete separation in the subsequent process can affect the safety of the oils to some extent. Summary of the Invention

[0005] The purpose of this invention is to overcome the safety issues of quinoxaline compounds in improving DHA production by Schizochytrium in existing technologies. This invention provides an application of regulatory factors in promoting DHA production by Schizochytrium, a method for improving DHA production by Schizochytrium, and a method for preparing microbial oils. β-carotene and reduced glutathione can effectively increase the oil and DHA content obtained from Schizochytrium fermentation, and β-carotene and reduced glutathione are non-toxic and highly safe.

[0006] To achieve the above objectives, the present invention provides an application of a regulatory factor in promoting DHA production by Schizochytrium, wherein the regulatory factor contains β-carotene and reduced glutathione.

[0007] Preferably, the mass ratio of β-carotene to reduced glutathione is 5-15:1; more preferably, it is 8-12:1.

[0008] A second aspect of the present invention provides a method for improving DHA production by Schizochytrium fungi, the method comprising: placing Schizochytrium fungi in a culture medium for fermentation, and adding β-carotene and reduced glutathione to the culture medium during the fermentation process; wherein the content of β-carotene in the culture medium is 1.5-2 g / L.

[0009] Preferably, the mass ratio of β-carotene to reduced glutathione is 5-15:1.

[0010] More preferably, the mass ratio of the β-carotene to the reduced glutathione is 8-12:1.

[0011] Preferably, the fermentation includes seed culture and fermentation culture, and the β-carotene and the reduced glutathione are added during the fermentation culture process.

[0012] More preferably, the β-carotene and the reduced glutathione are added during fermentation culture of 12-14 hours.

[0013] Preferably, the culture medium used for seed culture and the culture medium used for fermentation culture contain: glucose, yeast extract, sodium sulfate, magnesium sulfate, ammonium sulfate, potassium chloride, calcium chloride, potassium sulfate, potassium dihydrogen phosphate, monosodium glutamate, zinc sulfate heptahydrate, cobalt chloride hexahydrate, copper sulfate pentahydrate, nickel sulfate hexahydrate, and ferrous sulfate heptahydrate.

[0014] More preferably, the culture medium used for seed culture contains: 30-50 g / L glucose, 3-8 g / L yeast extract, 3-8 g / L sodium sulfate, 0.5-1.5 g / L magnesium sulfate, 2-4 g / L ammonium sulfate, 1.5-2.5 g / L potassium chloride, 0.1-0.3 g / L calcium chloride, 0.3-0.5 g / L potassium sulfate, 0.5-1 g / L potassium dihydrogen phosphate, 3-8 g / L monosodium glutamate, 1.5-2.5 mg / L zinc sulfate heptahydrate, 0.03-0.08 mg / L cobalt chloride hexahydrate, 2-6 mg / L copper sulfate pentahydrate, 0.4-0.6 mg / L nickel sulfate hexahydrate, and 4-8 mg / L ferrous sulfate heptahydrate;

[0015] The culture medium used for the fermentation culture contains: 70-90 g / L glucose, 8-12 g / L yeast extract, 8-12 g / L sodium sulfate, 2-4 g / L magnesium sulfate, 4-8 g / L ammonium sulfate, 1.5-2.5 g / L potassium chloride, 0.1-0.3 g / L calcium chloride, 0.5-1 g / L potassium sulfate, 0.8-1.2 g / L potassium dihydrogen phosphate, 12-18 g / L monosodium glutamate, 2-4 mg / L zinc sulfate heptahydrate, 0.05-0.1 mg / L cobalt chloride hexahydrate, 4-6 mg / L copper sulfate pentahydrate, 0.8-1.2 mg / L nickel sulfate hexahydrate, and 8-12 mg / L ferrous sulfate heptahydrate.

[0016] Preferably, the Schizochytrium is Schizochytrium sp. HX-308.

[0017] The third aspect of the present invention provides a method for preparing microbial oil, the method comprising: fermenting Schizochytrium using the above method to obtain a fermentation broth, and breaking and extracting the fermentation broth.

[0018] Preferably, the cell wall disruption is performed using a cell wall disruption enzyme hydrolysis method.

[0019] More preferably, the conditions for cell wall disruption include: the mass concentration of the cell wall disrupting enzyme in the fermentation broth is 4-6%, the pH is 10-12, the rotation speed is 100-150 rpm, the temperature is 45-55℃, and the time is 60-120 min.

[0020] The solvent used for extraction is n-hexane and / or ethanol.

[0021] Through the above technical solution, the application provided by this invention incorporates β-carotene and reduced glutathione into the cultivation process of Schizochytrium. The interaction between β-carotene and reduced glutathione promotes the fermentation of Schizochytrium to produce DHA, thereby effectively increasing DHA yield and also increasing the oil content. Furthermore, β-carotene and reduced glutathione are non-toxic, resulting in highly safe oil. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the gas chromatography of the oil containing DHA prepared in Example 1 of the present invention. Detailed Implementation

[0023] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0024] As mentioned above, the first aspect of the present invention provides the application of a regulatory factor in promoting DHA production by Schizochytrium, the regulatory factor containing β-carotene and reduced glutathione.

[0025] According to the present invention, the β-carotene and reduced glutathione can be added during the fermentation process of Schizochytrium.

[0026] During their research, the inventors discovered that adding β-carotene and reduced glutathione to the culture process of Schizochytrium can promote the fermentation of DHA by Schizochytrium through the interaction between β-carotene and reduced glutathione, thereby effectively increasing DHA production and also increasing oil production. Furthermore, β-carotene and reduced glutathione are non-toxic, resulting in highly safe oils.

[0027] Preferably, the mass ratio of β-carotene to reduced glutathione is 5-15:1, which can be 5:1, 7:1, 9:1, 11:1, 13:1, 15:1, or any value within any two of these ranges. Controlling the mass ratio of β-carotene to reduced glutathione within the above range can further enhance the interaction between reduced glutathione and β-carotene, thereby further increasing the yield of DHA and lipids. Further preferably, considering the potential for further increasing the yield of DHA and lipids, the mass ratio of β-carotene to reduced glutathione in the β-carotene and reduced glutathione composition is 8-12:1.

[0028] A second aspect of the present invention provides a method for improving DHA production by Schizochytrium fungi, the method comprising: placing Schizochytrium fungi in a culture medium for fermentation, and adding β-carotene and reduced glutathione to the culture medium during the fermentation process; wherein the content of β-carotene in the culture medium is 1.5-2 g / L.

[0029] Adding β-carotene and reduced glutathione to the fermentation process of Schizochytrium promotes the production of DHA through the interaction between β-carotene and reduced glutathione, thereby effectively increasing DHA yield and oil production. Furthermore, β-carotene and reduced glutathione are non-toxic, resulting in highly safe oils. In addition, it effectively reduces ROS content in the culture medium, minimizing oil oxidation and ensuring oil quality.

[0030] According to the present invention, the content of β-carotene in the culture medium may be 1.5 g / L, 1.6 g / L, 1.7 g / L, 1.8 g / L, 1.9 g / L, 2 g / L, or any value within any two of these ranges.

[0031] The β-carotene and the reduced glutathione can be added simultaneously or sequentially. For example, β-carotene can be added first, followed by reduced glutathione. Preferably, the β-carotene and the reduced glutathione are added simultaneously to enhance their interaction.

[0032] Preferably, the mass ratio of β-carotene to reduced glutathione is 5-15:1, which can be 5:1, 7:1, 9:1, 11:1, 13:1, 15:1, or any value within any two of these ranges. Controlling the mass ratio of β-carotene to reduced glutathione within the above range can further increase the production of reduced glutathione and β-carotene, thereby further increasing the yield of DHA and lipids. Further preferably, considering the potential for further increasing the yield of DHA and lipids, the mass ratio of β-carotene to reduced glutathione is 8-12:1.

[0033] Preferably, the fermentation includes seed culture and fermentation culture, with the β-carotene and reduced glutathione added during the fermentation culture. Adding them at this stage can further increase the yield of DHA and lipids. Further preferably, considering the potential for further increasing the yield of DHA and lipids, the β-carotene and reduced glutathione are added during fermentation culture of 12-14 hours, which can be 12 hours, 12.5 hours, 13 hours, 13.5 hours, or 14 hours, or any value within any two of these ranges. Specifically, β-carotene and reduced glutathione are added to the culture medium during the 12-14 hour fermentation culture.

[0034] Preferably, the conditions for seed culture include at least: a temperature of 28-30℃, a rotation speed of 160-200 rpm, a time of 18-48 h, and an inoculum size of 0.8-2% (volume); the conditions for fermentation culture include: a temperature of 28-30℃, a rotation speed of 16-200 rpm, a time of 36-60 h, and an inoculum size of 0.8-2% (volume).

[0035] The seed culture can be a conventional seed culture. For example, Schizochytrium can be inoculated into a seed culture medium for culture to obtain a primary seed solution; the primary seed solution can then be further inoculated into a seed culture medium for culture to obtain a secondary seed solution.

[0036] The seed culture medium used in the seed culture and the fermentation medium used in the fermentation culture may contain carbon sources, nitrogen sources, trace metal elements, etc. Preferably, the seed culture medium used in the seed culture and the fermentation medium used in the fermentation culture contain: glucose, yeast extract, sodium sulfate, magnesium sulfate, ammonium sulfate, potassium chloride, calcium chloride, potassium sulfate, potassium dihydrogen phosphate, monosodium glutamate, zinc sulfate heptahydrate, cobalt chloride hexahydrate, copper sulfate pentahydrate, nickel sulfate hexahydrate, and ferrous sulfate heptahydrate. Schizochytrium exhibits good fermentation performance in the above-mentioned culture media. To further improve the fermentation effect of Schizochytrium, the culture medium used for seed culture preferably contains: 30-50 g / L glucose, 3-8 g / L yeast extract, 3-8 g / L sodium sulfate, 0.5-1.5 g / L magnesium sulfate, 2-4 g / L ammonium sulfate, 1.5-2.5 g / L potassium chloride, 0.1-0.3 g / L calcium chloride, 0.3-0.5 g / L potassium sulfate, 0.5-1 g / L potassium dihydrogen phosphate, 3-8 g / L monosodium glutamate, 1.5-2.5 mg / L zinc sulfate heptahydrate, 0.03-0.08 mg / L cobalt chloride hexahydrate, 2-6 mg / L copper sulfate pentahydrate, and 0.4-0.6 mg / L sulfur hexahydrate. The culture medium used for the fermentation culture contains: 70-90 g / L glucose, 8-12 g / L yeast extract, 8-12 g / L sodium sulfate, 2-4 g / L magnesium sulfate, 4-8 g / L ammonium sulfate, 1.5-2.5 g / L potassium chloride, 0.1-0.3 g / L calcium chloride, 0.5-1 g / L potassium sulfate, 0.8-1.2 g / L potassium dihydrogen phosphate, 12-18 g / L monosodium glutamate, 2-4 mg / L zinc sulfate heptahydrate, 0.05-0.1 mg / L cobalt chloride hexahydrate, 4-6 mg / L copper sulfate pentahydrate, 0.8-1.2 mg / L nickel sulfate hexahydrate, and 8-12 mg / L ferrous sulfate heptahydrate.

[0037] Preferably, the pH of the culture medium used for seed culture and the culture medium used for fermentation culture are each independently 6-8.

[0038] Preferably, the Schizochytrium is Schizochytrium sp. HX-308. β-carotene and reduced glutathione have a better interaction effect with Schizochytrium sp. HX-308, thereby further increasing the production of DHA and lipids.

[0039] A third aspect of this invention provides a method for preparing microbial lipids, comprising fermenting Schizochytrium fungi using the above-described method to obtain a fermentation broth, and then subjecting the fermentation broth to cell wall disruption and extraction. This preparation method can increase the yield of DHA and lipids.

[0040] Preferably, the cell wall disruption is performed using a cell wall disruption enzyme hydrolysis method.

[0041] Preferably, the cell wall disruption conditions include: a cell wall disruption enzyme concentration of 4-6% in the fermentation broth, a pH of 10-12, a rotation speed of 100-150 rpm, a temperature of 45-55°C, and a time of 60-120 min; the extraction solvent is n-hexane and / or ethanol. Under these conditions, as much oil as possible can be extracted.

[0042] According to a particularly preferred embodiment of the present invention, a method for preparing microbial oil is outlined, wherein,

[0043] Seed culture medium (pH 6-8): 30-50 g / L glucose, 3-8 g / L yeast extract, 3-8 g / L sodium sulfate, 0.5-1.5 g / L magnesium sulfate, 2-4 g / L ammonium sulfate, 1.5-2.5 g / L potassium chloride, 0.1-0.3 g / L calcium chloride, 0.3-0.5 g / L potassium sulfate, 0.5-1 g / L potassium dihydrogen phosphate, 3-8 g / L monosodium glutamate, 1.5-2.5 mg / L zinc sulfate heptahydrate, 0.03-0.08 mg / L cobalt chloride hexahydrate, 2-6 mg / L copper sulfate pentahydrate, 0.4-0.6 mg / L nickel sulfate hexahydrate, and 4-8 mg / L ferrous sulfate heptahydrate.

[0044] Fermentation medium (pH 6-8): 70-90 g / L glucose, 8-12 g / L yeast extract, 8-12 g / L sodium sulfate, 2-4 g / L magnesium sulfate, 4-8 g / L ammonium sulfate, 1.5-2.5 g / L potassium chloride, 0.1-0.3 g / L calcium chloride, 0.5-1 g / L potassium sulfate, 0.8-1.2 g / L potassium dihydrogen phosphate, 12-18 g / L monosodium glutamate, 2-4 mg / L zinc sulfate heptahydrate, 0.05-0.1 mg / L cobalt chloride hexahydrate, 4-6 mg / L copper sulfate pentahydrate, 0.8-1.2 mg / L nickel sulfate hexahydrate, and 8-12 mg / L ferrous sulfate heptahydrate.

[0045] (1) Inoculate the Schizochytrium preserved in glycerol tubes into the seed culture medium at an inoculation amount of 0.8-2% (v / v, volume percentage), and culture at 28-30℃ and 160-200 r / min for 24 h to obtain the primary seed culture.

[0046] (2) Transfer the above primary seeds to seed culture medium at an inoculation amount of 0.8-2% (v / v, volume percentage), and culture at 28-30℃ and a rotation speed of 160-200r / min for 24h to obtain secondary seed liquid;

[0047] (3) Transfer the above secondary seed culture to the fermentation medium at an inoculation amount of 0.8-2% (v / v, volume percentage) and culture at 28-30℃ and a rotation speed of 160-200r / min for 36-60h. During this period, at 12h of fermentation, add β-carotene and reduced glutathione in a mass ratio of 5-15:1 to the fermentation medium so that the concentration of β-carotene in the medium is 1.5-2g / L.

[0048] (4) After fermentation, adjust the pH of the culture medium for Schizochytrium to 10-12, add cell wall breaking enzyme to make the mass concentration of cell wall breaking enzyme in the culture medium 0.4-0.6%, and enzymatically hydrolyze at 45-55℃ and 100-150 r / min for 90 min until the cell enzymatic hydrolysis is complete, and obtain the enzymatic hydrolysate.

[0049] (5) Add anhydrous ethanol of the same volume as the enzyme hydrolysate and shake continuously for 50-70 minutes to inactivate the cell wall breaking enzyme.

[0050] (6) Add the same volume of n-hexane as anhydrous ethanol and shake continuously for 50-70 min. After shaking, let stand at room temperature until the layers separate. Collect the upper organic phase. Repeat this process several times until the supernatant is transparent and colorless. Combine the organic phases.

[0051] (7) Extract the obtained organic phase by rotary evaporation at 40℃ for 25-35 min, and then dry it in an oven to constant weight to obtain oil containing DHA.

[0052] Adding β-carotene and reduced glutathione to the fermentation process of Schizochytrium promotes the production of DHA through the interaction between β-carotene and reduced glutathione, thereby effectively increasing DHA yield and oil production. Furthermore, β-carotene and reduced glutathione are non-toxic, resulting in highly safe oils. In addition, it effectively reduces ROS content in the culture medium, minimizing oil oxidation and ensuring oil quality. Moreover, no equipment modification is required, making the fermentation process simple and convenient.

[0053] The present invention will be described in detail below through examples. In the following examples, the Schizochytrium (Schizochytrium HX-308 (accession number CCTCCM209059)) was derived from our laboratory modification and has been described in patent application CN114561434A. Yeast extract was purchased from Oxoid, catalog number 211930; β-carotene was purchased from Sigma-Aldrich, catalog number C9750; reduced glutathione was purchased from Shanghai Yuanye Biotechnology Co., Ltd., catalog number B20546; astaxanthin was purchased from Maclean's, catalog number A832359; N-acetylcysteine ​​was purchased from Sigma-Aldrich, catalog number A0150000; the remaining substances were commercially available products.

[0054] 1. Palmitic acid (C16:0) yield (g / L)

[0055] Detection method: Gas chromatography (GC)

[0056] Sample pretreatment: The fermentation broth was centrifuged (8,000 rpm, 10 min) to collect the cells, which were then freeze-dried and ground into powder. Total lipids were extracted using a chloroform-methanol (2:1, v / v) mixed solvent, dried under nitrogen, and then methylated (14 v / v BF-methanol solution, 70°C water bath for 30 min).

[0057] GC analysis conditions:

[0058] Chromatographic column: HP-88 capillary column (100m × 0.25mm × 0.20μm);

[0059] Detector: FID (Flame Ionization Detector);

[0060] Temperature program: Initial temperature 150℃ (hold for 1 min), then increase to 240℃ at a rate of 4℃ / min (hold for 10 min);

[0061] Injection volume: 1 μL, split ratio: 50:1;

[0062] Quantification: Methyl palmitate standard (Sigma) was used as an external standard, and quantification was performed by peak area.

[0063] Calculation method: Palmitic acid yield = Palmitic acid mass measured by GC (g) / Fermentation broth volume (L)

[0064] 2. DHA production (g / L)

[0065] Detection method: Gas chromatography (GC) (same as palmitic acid detection)

[0066] Qualitative analysis was performed using the retention time of DHA methyl ester standard (C22:6ω3, Sigma), and quantitative analysis was performed using the external standard method based on peak area.

[0067] Calculation method: DHA yield = DHA mass measured by GC (g) / fermentation broth volume (L)

[0068] 3. Total oil yield (g / L)

[0069] Detection method: Gravimetric method

[0070] Extraction: The fermentation broth was centrifuged to collect the cell cells, which were then freeze-dried and weighed (W1). Total lipids were extracted with chloroform-methanol (2:1, v / v), dried under nitrogen, and weighed (W2).

[0071] Calculation: Total oil mass = W2 - W1 (deducting solvent residue).

[0072] Calculation method: Total oil yield = Total oil mass (g) / Fermentation liquid volume (L)

[0073] 4. DHA content (%) in oils and fats

[0074] Calculation method: DHA content = DHA yield (g / L) / Total oil yield (g / L) × 100%

[0075] 5. ROS removal rate (%)

[0076] Detection method: DPPH free radical scavenging method

[0077] Sample preparation: Take the supernatant of the fermentation broth, centrifuge and filter (0.22μm).

[0078] Reaction system: 0.1 mL sample + 3.9 mL DPPH solution (0.1 mM, prepared with ethanol), react in the dark for 30 min.

[0079] Measurement: Absorbance was measured at 517 nm (A1). The blank group was prepared by replacing the sample with ethanol (A0).

[0080] Calculation method: ROS clearance rate = (1 - A1 / A0) × 100%

[0081] 6. DHA as a percentage of total fatty acids (% TFA)

[0082] Detection method: GC method (same as palmitic acid detection)

[0083] Analyze the peak areas of each component in total fatty acid methyl esters (TFA), and the percentage of the DHA peak area is %TFA.

[0084] Calculation method: DHA content % TFA = (Total DHA peak area / Fatty acid peak area) × 100%

[0085] Key points:

[0086] Standard curve: All GC quantifications require the establishment of a standard curve using a series of concentration standards (R0). 2 ≥0.99).

[0087] Repeatability: Each experiment was repeated at least 3 times, and the data were expressed as mean ± standard deviation.

[0088] Instrument calibration: GC needs to be calibrated regularly to ensure retention time and peak area reproducibility.

[0089] The above methods comply with international standards (such as AOAC and AOCS) to ensure data accuracy and repeatability.

[0090] Example 1

[0091] Seed culture medium (pH 7±0.2): glucose 40 g / L, yeast extract 5 g / L, sodium sulfate 5 g / L, magnesium sulfate 1 g / L, ammonium sulfate 3 g / L, potassium chloride 2 g / L, calcium chloride 0.2 g / L, potassium sulfate 0.4 g / L, potassium dihydrogen phosphate 0.8 g / L, monosodium glutamate 5 g / L, zinc sulfate heptahydrate 2 mg / L, cobalt chloride hexahydrate 0.05 mg / L, copper sulfate pentahydrate 4 mg / L, nickel sulfate hexahydrate 0.5 mg / L, ferrous sulfate heptahydrate 6 mg / L.

[0092] Fermentation medium (pH 7±0.2): glucose 80g / L, yeast extract 10g / L, sodium sulfate 10g / L, magnesium sulfate 3g / L, ammonium sulfate 6g / L, potassium chloride 2g / L, calcium chloride 0.2g / L, potassium sulfate 0.8g / L, potassium dihydrogen phosphate 1g / L, monosodium glutamate 15g / L, zinc sulfate heptahydrate 3mg / L, cobalt chloride hexahydrate 0.08mg / L, copper sulfate pentahydrate 5mg / L, nickel sulfate hexahydrate 1mg / L, ferrous sulfate heptahydrate 10mg / L.

[0093] (1) The Schizochytrium preserved in glycerol tubes was inoculated into 20 mL of seed culture medium at an inoculation amount of 1% (v / v, volume percentage). The culture was carried out at 28℃ and 180 r / min for 24 h to obtain the primary seed culture.

[0094] (2) The above primary seeds were transferred to 20 mL of seed culture medium with an inoculation amount of 2% (v / v, volume percentage) and cultured at 28℃ and 180 r / min for 24 h to obtain secondary seed liquid;

[0095] (3) The above secondary seed culture was transferred to a shake flask containing 100 mL of fermentation medium, with an inoculation amount of 1% (v / v, volume percentage), and cultured at 28℃ and 180 r / min for 48 h. During this period, β-carotene and reduced glutathione were added to the fermentation medium at a mass ratio of 10:1 after 12 h of fermentation, so that the concentration of β-carotene in the medium was 1.8 g / L.

[0096] (4) After fermentation, adjust the pH of the culture medium for Schizochytrium to 10, add cell wall breaking enzyme to make the mass concentration of cell wall breaking enzyme in the culture medium 0.5%, and enzymatically hydrolyze at 50℃ and 100r / min for 90min until the cell enzymatic hydrolysis is complete to obtain the enzymatic hydrolysate.

[0097] (5) Add anhydrous ethanol of the same volume as the enzyme hydrolysate and shake continuously for 1 hour to inactivate the cell wall breaking enzyme.

[0098] (6) Add the same volume of n-hexane as anhydrous ethanol and shake continuously for 1 hour. After shaking, let stand at room temperature until the layers separate. Collect the upper organic phase. Repeat this process several times until the supernatant is transparent and colorless. Combine the organic phases.

[0099] (7) The obtained organic phase was extracted by rotary evaporation at 40°C for 30 min. After rotary evaporation, it was dried in an oven to constant weight to obtain oil containing DHA.

[0100] Gas chromatography was used to determine the DHA content in oils and fats. Figure 1 .exist Figure 1 The peak observed at 28.337 min is the DHA peak, and the DHA content is calculated to be approximately 63.9% based on the area method.

[0101] Example 2

[0102] Seed culture medium (pH 6.5±0.2): glucose 30 g / L, yeast extract 8 g / L, sodium sulfate 3 g / L, magnesium sulfate 1.5 g / L, ammonium sulfate 4 g / L, potassium chloride 1.5 g / L, calcium chloride 0.1 g / L, potassium sulfate 0.3 g / L, potassium dihydrogen phosphate 1 g / L, monosodium glutamate 8 g / L, zinc sulfate heptahydrate 2.5 mg / L, cobalt chloride hexahydrate 0.03 mg / L, copper sulfate pentahydrate 2 mg / L, nickel sulfate hexahydrate 0.6 mg / L, ferrous sulfate heptahydrate 8 mg / L.

[0103] Fermentation medium (pH 6.5±0.2): glucose 90g / L, yeast extract 8g / L, sodium sulfate 12g / L, magnesium sulfate 2g / L, ammonium sulfate 4g / L, potassium chloride 2.5g / L, calcium chloride 0.3g / L, potassium sulfate 1g / L, potassium dihydrogen phosphate 0.8g / L, monosodium glutamate 12g / L, zinc sulfate heptahydrate 2mg / L, cobalt chloride hexahydrate 0.1mg / L, copper sulfate pentahydrate 6mg / L, nickel sulfate hexahydrate 0.8mg / L, ferrous sulfate heptahydrate 8mg / L.

[0104] (1) The Schizochytrium preserved in glycerol tubes was inoculated into 20 mL of seed culture medium at an inoculation amount of 0.8% (v / v, volume percentage). The culture was carried out at 25℃ and 160 r / min for 24 h to obtain the primary seed culture.

[0105] (2) The above primary seeds were transferred to 20 mL of seed culture medium with an inoculation amount of 1.5% (v / v, volume percentage) and cultured at 25℃ and 160 r / min for 24 h to obtain secondary seed liquid;

[0106] (3) The above secondary seed culture was transferred to a shake flask containing 100 mL of fermentation medium. The inoculation amount was 1.5% (v / v, volume percentage). The culture was carried out at 25℃ and 160 r / min for 48 h. During this period, at 13 h of fermentation, β-carotene and reduced glutathione were added to the fermentation medium at a mass ratio of 12:1 so that the concentration of β-carotene in the medium was 2.0 g / L.

[0107] (4) After fermentation, the pH of the culture medium for culturing Schizochytrium was adjusted to 10.5, and cell wall breaking enzyme was added so that the mass concentration of cell wall breaking enzyme in the culture medium was 0.4%. The enzyme was hydrolyzed at 45℃ and 150r / min for 120min until the cell enzymatic hydrolysis was complete, and the hydrolysate was obtained.

[0108] (5) Add anhydrous ethanol of the same volume as the enzyme hydrolysate and shake continuously for 1 hour to inactivate the cell wall breaking enzyme.

[0109] (6) Add the same volume of n-hexane as anhydrous ethanol and shake continuously for 1 hour. After shaking, let stand at room temperature until the layers separate. Collect the upper organic phase. Repeat this process several times until the supernatant is transparent and colorless. Combine the organic phases.

[0110] (7) The obtained organic phase was extracted by rotary evaporation at 40°C for 25 min. After rotary evaporation, it was dried in an oven to constant weight to obtain oil containing DHA.

[0111] Example 3

[0112] Seed culture medium (pH 6.5±0.2): glucose 50 g / L, yeast extract 3 g / L, sodium sulfate 8 g / L, magnesium sulfate 0.5 g / L, ammonium sulfate 2 g / L, potassium chloride 2.5 g / L, calcium chloride 0.3 g / L, potassium sulfate 0.5 g / L, potassium dihydrogen phosphate 0.5 g / L, monosodium glutamate 3 g / L, zinc sulfate heptahydrate 1.5 mg / L, cobalt chloride hexahydrate 0.08 mg / L, copper sulfate pentahydrate 6 mg / L, nickel sulfate hexahydrate 0.4 mg / L, ferrous sulfate heptahydrate 4 mg / L.

[0113] Fermentation medium (pH 6.5±0.2): glucose 70g / L, yeast extract 12g / L, sodium sulfate 8g / L, magnesium sulfate 4g / L, ammonium sulfate 8g / L, potassium chloride 1.5g / L, calcium chloride 0.1g / L, potassium sulfate 0.5g / L, potassium dihydrogen phosphate 1.2g / L, monosodium glutamate 18g / L, zinc sulfate heptahydrate 4mg / L, cobalt chloride hexahydrate 0.05mg / L, copper sulfate pentahydrate 4mg / L, nickel sulfate hexahydrate 1.2mg / L, ferrous sulfate heptahydrate 12mg / L.

[0114] (1) The Schizochytrium preserved in glycerol tubes was inoculated into 20 mL of seed culture medium at an inoculation amount of 2% (v / v, volume percentage). The culture was carried out at 30℃ and 2000 r / min for 24 h to obtain the primary seed culture.

[0115] (2) The above primary seeds were transferred to 20 mL of seed culture medium with an inoculation amount of 0.8% (v / v, volume percentage) and cultured at 30℃ and 200 r / min for 24 h to obtain secondary seed liquid;

[0116] (3) The above secondary seed culture was transferred to a shake flask containing 100 mL of fermentation medium, with an inoculation amount of 2% (v / v, volume percentage), and cultured at 30℃ and 200 r / min for 48 h. During this period, at 14 h of fermentation, β-carotene and reduced glutathione were added to the fermentation medium at a mass ratio of 8:1, so that the concentration of β-carotene in the medium was 1.5 g / L.

[0117] (4) After fermentation, adjust the pH of the culture medium for Schizochytrium to 11, add cell wall breaking enzyme to make the mass concentration of cell wall breaking enzyme in the culture medium 0.6%, and enzymatically hydrolyze at 55℃ and 120r / min for 60min until the cell enzymatic hydrolysis is complete to obtain the enzymatic hydrolysate.

[0118] (5) Add anhydrous ethanol of the same volume as the enzyme hydrolysate and shake continuously for 1 hour to inactivate the cell wall breaking enzyme.

[0119] (6) Add the same volume of n-hexane as anhydrous ethanol and shake continuously for 1 hour. After shaking, let stand at room temperature until the layers separate. Collect the upper organic phase. Repeat this process several times until the supernatant is transparent and colorless. Combine the organic phases.

[0120] (7) The obtained organic phase was extracted by rotary evaporation at 40°C for 35 min. After rotary evaporation, it was dried in an oven to constant weight to obtain oil containing DHA.

[0121] Example 4

[0122] Oils containing DHA were prepared according to the method in Example 1, except that the concentration of β-carotene was 1.5 g / L.

[0123] Example 5

[0124] Oils containing DHA were prepared according to the method in Example 1, except that the concentration of β-carotene was 2 g / L.

[0125] Example 6

[0126] Oils containing DHA were prepared according to the method in Example 2, except that the mass ratio of β-carotene to reduced glutathione was 15:1.

[0127] Example 7

[0128] Oils containing DHA were prepared according to the method in Example 3, except that the mass ratio of β-carotene to reduced glutathione was 5:1.

[0129] Example 8

[0130] Oils containing DHA were prepared according to the method in Example 1, except that β-carotene and reduced glutathione were added at 11 hours of fermentation.

[0131] Example 9

[0132] Oils containing DHA were prepared according to the method in Example 3, except that β-carotene and reduced glutathione were added at 15 hours of fermentation.

[0133] Example 10

[0134] Oils containing DHA were prepared according to the method in Example 1, except that the shaking flask in step (3) was replaced with a 50L fermentation tank.

[0135] Comparative Example 1

[0136] Oils containing DHA were prepared according to the method in Example 2, except that no β-carotene was added to replace or the original glutathione during the fermentation stage.

[0137] Comparative Example 2

[0138] Oils containing DHA were prepared according to the method in Example 2, except that the original glutathione was replaced with β-carotene.

[0139] Comparative Example 3

[0140] Oils containing DHA were prepared according to the method in Example 3, except that β-carotene was replaced with the original glutathione.

[0141] Comparative Example 4

[0142] Oils containing DHA were prepared according to the method in Example 1, except that the concentration of β-carotene was 0.2 g / L.

[0143] Comparative Example 5

[0144] Oils containing DHA were prepared according to the method in Example 1, except that the concentration of β-carotene was 1.5 g / L.

[0145] Comparative Example 6

[0146] Oils containing DHA were prepared according to the method in Example 1, except that the concentration of β-carotene was 2.5 g / L.

[0147] Comparative Example 7

[0148] Oils containing DHA were prepared according to the method in Example 2, except that the original glutathione was replaced with N-acetylcysteine ​​(NAC).

[0149] Comparative Example 8

[0150] Oils containing DHA were prepared according to the method in Example 3, except that β-carotene was replaced with astaxanthin.

[0151] Table 1

[0152]

[0153] As shown in Table 1, the fermentation broth obtained by the methods provided in Examples 1-10 of this invention has significantly higher DHA content and total oil content than that of Comparative Examples 1-8. Furthermore, the DHA content and ROS scavenging rate in the oil are also higher than those in Comparative Examples 1-8. This indicates that the method provided by this invention can effectively increase the yield of oil and DHA, while also exhibiting good ROS scavenging effect. Based on an annual production of 1000 tons of DHA oil, the cost of the compound antioxidant is $0.9 / kg oil (GSH accounts for $0.6 / kg); the increased DHA production benefit is $16.2 / kg oil (based on a market price of $25 / kg); the input-output ratio is 1:18, demonstrating significant industrialization value.

[0154] Data from Examples 1 and 10 show that, compared to shake flasks, the DHA yield in a 5L fermenter increased by 10.9%, and the oil yield also increased by 10.9%.

[0155] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. Application of regulatory factors in promoting DHA production by Schizochytrium. The regulatory factors contain β-carotene and reduced glutathione.

2. The application according to claim 1, characterized in that, The mass ratio of β-carotene to reduced glutathione is 5-15:1; preferably 8-12:

1.

3. A method for improving DHA production by Schizochytrium fungi, characterized in that, The method includes: placing Schizochytrium in a culture medium for fermentation, and adding β-carotene and reduced glutathione to the culture medium during the fermentation process; In the culture medium, the content of β-carotene is 1.5-2 g / L.

4. The method according to claim 3, characterized in that, The mass ratio of β-carotene to reduced glutathione is 5-15:1; preferably 8-12:

1.

5. The method according to claim 3 or 4, characterized in that, The fermentation includes seed culture and fermentation culture, and the β-carotene and the reduced glutathione are added during the fermentation culture process.

6. The method according to claim 5, characterized in that, The β-carotene and the reduced glutathione were added during fermentation culture for 12-14 hours.

7. The method according to claim 5, characterized in that, The culture medium used for seed culture and the culture medium used for fermentation culture contain: glucose, yeast extract, sodium sulfate, magnesium sulfate, ammonium sulfate, potassium chloride, calcium chloride, potassium sulfate, potassium dihydrogen phosphate, monosodium glutamate, zinc sulfate heptahydrate, cobalt chloride hexahydrate, copper sulfate pentahydrate, nickel sulfate hexahydrate and ferrous sulfate heptahydrate. Preferably, the culture medium used for seed culture contains: 30-50 g / L glucose, 3-8 g / L yeast extract, 3-8 g / L sodium sulfate, 0.5-1.5 g / L magnesium sulfate, 2-4 g / L ammonium sulfate, 1.5-2.5 g / L potassium chloride, 0.1-0.3 g / L calcium chloride, 0.3-0.5 g / L potassium sulfate, 0.5-1 g / L potassium dihydrogen phosphate, 3-8 g / L monosodium glutamate, 1.5-2.5 mg / L zinc sulfate heptahydrate, 0.03-0.08 mg / L cobalt chloride hexahydrate, 2-6 mg / L copper sulfate pentahydrate, 0.4-0.6 mg / L nickel sulfate hexahydrate, and 4-8 mg / L ferrous sulfate heptahydrate. The culture medium used for the fermentation culture contains: 70-90 g / L glucose, 8-12 g / L yeast extract, 8-12 g / L sodium sulfate, 2-4 g / L magnesium sulfate, 4-8 g / L ammonium sulfate, 1.5-2.5 g / L potassium chloride, 0.1-0.3 g / L calcium chloride, 0.5-1 g / L potassium sulfate, 0.8-1.2 g / L potassium dihydrogen phosphate, 12-18 g / L monosodium glutamate, 2-4 mg / L zinc sulfate heptahydrate, 0.05-0.1 mg / L cobalt chloride hexahydrate, 4-6 mg / L copper sulfate pentahydrate, 0.8-1.2 mg / L nickel sulfate hexahydrate, and 8-12 mg / L ferrous sulfate heptahydrate.

8. The method according to any one of claims 3 to 5, characterized in that, The Schizochytrium is Schizochytrium sp. HX-308.

9. A method for preparing microbial oil, characterized in that, The method includes: fermenting Schizochytrium using the method described in any one of claims 3 to 8 to obtain a fermentation broth, and then breaking down and extracting the fermentation broth.

10. The preparation method according to claim 9, characterized in that, The cell wall was broken by enzymatic hydrolysis using a cell-wall-breaking enzyme method; Preferably, the conditions for cell wall disruption include: the mass concentration of the cell wall disrupting enzyme in the fermentation broth is 4-6%, the pH is 10-12, the rotation speed is 100-150 rpm, the temperature is 45-55℃, and the time is 60-120 min. The solvent used for extraction is n-hexane and / or ethanol.

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