Mannosylerythritol lipid produced by utilizing fermentation of aphid yeast XM01 strain as well as fermentation method and application of mannosylerythritol lipid

Abstract

The invention discloses mannosylerythritol lipid produced by fermentation of an aphid yeast XM01 strain as well as a fermentation method and application of the mannosylerythritol lipid, and belongs to the technical field of microorganisms. The invention provides a fermentation method using the aphid torulopsis XM01 strain, through compounding and screening of a carbon source, a secondary metabolism integration pathway of sugar alcohol is effectively activated, meanwhile, synthesis of mannosylerythritol lipid MELs is effectively promoted, and the yield reaches up to 82.21 g / L; the invention further provides the MELs prepared by using the safflower oil as the first carbon source, the C10 content in a fatty acid side chain of the MELs accounts for 80% or above, and the MELs is good in emulsifying property, has antioxidant and anti-inflammatory capacities, can effectively inhibit candida albicans and promote cell proliferation, and has reference significance in application of preparing biological preparations in cosmetics and drugs by using the MELs; according to the MELs prepared by the invention, the technical problems of emulsification control and metabolic balance in the amplification process are solved by decoupling thallus growth and a product anabolism path through a time sequence.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of microbiology, and particularly relates to a mannose erythritol lipid produced by fermentation of aphid pseudococcus XM01 strain and a fermentation method and application thereof. BACKGROUND

[0002] Biological surfactants can be divided into two categories according to their source: chemical synthesis and biological synthesis, both of which have hydrophobic and hydrophilic amphiphilic properties.

[0003] The most studied lipopeptide biological surfactant is produced by Bacillus subtilis, but its solubility in water is very poor, and it is only soluble at high pH (8-8.5), which is a disadvantage in cosmetic or pharmaceutical formulations. Phospholipids are a class of zwitterionic substances, usually derived from plant and animal cells, and are used as emulsifiers in the food industry. Compared with chemical surfactants, biological surfactants do not rely on petrochemical raw materials, and can reduce the consumption of non-renewable resources.

[0004] Mannose erythritol lipid (MELs) is composed of hydrophilic and hydrophobic parts, with mannose erythritol as the hydrophilic part and fatty acid chains and acetyl groups as the hydrophobic part. The structure of MELs is composed of a hydrophilic mannose erythritol head and a hydrophobic fatty acid chain tail, and its unique amphiphilic nature allows it to spontaneously form various self-assembly structures such as vesicles, micelles and liquid crystals in solution. These self-assembly behaviors not only depend on the molecular structure of MELs, but are also significantly influenced by environmental conditions such as pH, temperature and ionic strength.

[0005] The self-assembly behavior and emulsifying performance of MELs are closely related to their molecular structure and environmental conditions. By adjusting the degree of acetylation of MELs, the properties of fatty acid chains and environmental parameters, the self-assembly ability and emulsifying efficiency of MELs can be significantly improved. Therefore, it is of great significance to further explore the self-assembly behavior of MELs in complex systems and their interaction with other functional components to expand their application potential in the fields of food, medicine and materials science. SUMMARY

[0006] The purpose of the present application is to provide a mannose erythritol lipid produced by fermentation of aphid pseudococcus XM01 strain and a fermentation method and application thereof.

[0007] To achieve the above-mentioned purpose of the application, the following technical solutions are adopted:

[0008] The present application provides a fermentation method using aphid pseudococcus XM01 strain, which specifically comprises the following steps:

[0009] (1) Aphid Pseudozyma XM01 strain is inoculated into YPD solid culture medium for constant temperature culture to form a colony;

[0010] (2) The colony is inoculated into seed culture medium for shaking culture to obtain a seed liquid;

[0011] (3) The seed liquid is inoculated into fermentation culture medium for fermentation to obtain a fermentation liquid;

[0012] (4) The fermentation liquid is added into ethyl acetate, mixed and centrifuged to obtain a crude extract containing oil; the crude extract is added into methanol, centrifuged and evaporated to obtain mannose erythritol lipid MELs.

[0013] Further, the aphid Pseudozyma XM01 strain has a classification name of Pseudozyma aphidis XM01, and a preservation number of CCTCC NO: M2021517.

[0014] Further, the carbon source of the fermentation culture medium comprises a first carbon source, and the first carbon source is at least one of soybean oil, macadamia nut seed oil, safflower seed oil, evening primrose oil, wheat germ oil, camellia seed oil, sunflower seed oil, grape seed oil, flaxseed oil, rapeseed oil and olive oil; the concentration of the first carbon source is 2%-15% (w / v).

[0015] Further, when the first carbon source is safflower seed oil, the concentration of the safflower seed oil is 4%-12% (w / v), and the yield of the mannose erythritol lipid is 50-80 g / L.

[0016] Further, the concentration of the safflower seed oil is 8%, and the yield of the mannose erythritol lipid is 77.78±1.36 g / L.

[0017] Further, the carbon source further comprises a second carbon source, and the second carbon source is at least one of glucose, fructose, mannose, L-arabinose, xylose, sorbitol, erythritol, mannitol, L-arabitol and xylitol; the concentration of the second carbon source is 1%-6% (w / v).

[0018] Further, the second carbon source is preferably glucose, erythritol, mannitol, L-arabitol, fructose and mannose, and the concentration of the second carbon source is 2%-4% (w / v).

[0019] Further, the nitrogen source of the fermentation culture medium is sodium nitrate, and the concentration of the sodium nitrate is 0.05%-0.3% (w / v).

[0020] Further, the optimal components and concentrations of the carbon source and nitrogen source of the fermentation medium are as follows: the first carbon source is safflower seed oil, the concentration is 8% (w / v); the second carbon source is glucose, the concentration is 2% (w / v); and the nitrogen source is sodium nitrate, the concentration is 0.2% (w / v).

[0021] Further, the yield of the prepared mannose erythritol lipid MELs after optimization of the components of the fermentation medium is >82 g / L.

[0022] Further, the yield of the prepared mannose erythritol lipid MELs after optimization of the components of the fermentation medium is 82.21 ± 0.67 g / L.

[0023] The application further provides the mannose erythritol lipid obtained by the preparation method, wherein the content of C10 in the fatty acid side chain of the mannose erythritol lipid is ≥80%, and the critical micelle concentration of the mannose erythritol lipid is 4-5.5 mg / L.

[0024] Further, the critical micelle concentration of the mannose erythritol lipid is 5.34 mg / L.

[0025] Further, the concentration of the mannose erythritol lipid is 20 mg / mL, and the DPPH free radical scavenging activity is 20%-30%.

[0026] Further, the emulsification value of the mannose erythritol lipid to n-hexane is >60%.

[0027] The application further provides the use of the mannose erythritol lipid in the preparation of cosmetics and / or drugs of antioxidant and / or anti-inflammatory biological agents.

[0028] Further, the inhibition concentration of the mannose erythritol lipid to TNF-α is 10-100 μg / mL.

[0029] Further, the concentration of the mannose erythritol lipid is 0.01%-0.0005%, and the cell survival rate is all greater than 70%, the mannose erythritol lipid does not have cytotoxicity, and effectively promotes the proliferation of L929 cells.

[0030] The application further provides the use of the mannose erythritol lipid in the preparation of biological agents for inhibiting Candida albicans.

[0031] Further, the bacteriostatic concentration of the mannose erythritol lipid to Candida albicans is 15 mg / mL-25 mg / mL.

[0032] Further, the minimum bacteriostatic concentration of the mannose erythritol lipid to Candida albicans is 15.6 mg / mL.

[0033] Further, the mannose erythritol lipid has a bacteriostatic concentration of 20 mg / mL for Candida albicans, and a bacteriostatic ring diameter of 13.2 ± 0.4 mm.

[0034] Compared with the prior art, the present application has the following advantages and beneficial effects

[0035] 1. The present application effectively activates the secondary metabolic integration pathway of sugar alcohol by compounding and screening of carbon sources, realizes the customization of the hydrophilic head of the fermentation product, and experimentally verifies that the compounding of safflower seed oil and glucose promotes the synthesis of MELs and the yield breaks through 80 g / L.

[0036] 2. The MELs prepared by the fermentation method optimized by the present application have high yield, and the C10 content in the fatty acid side chain of MELs accounts for 80%; experimentally verified that the prepared MELs have good emulsifying performance, antioxidant and anti-inflammatory capacity, and effectively inhibit Candida albicans and promote cell proliferation.

[0037] 3. The present application experimentally verifies that the minimum bacteriostatic concentration of MELs on Candida albicans is 15.6 mg / mL, and the critical micelle concentration of MELs produced by fermentation with safflower seed oil as carbon source is 5.34 mg / L, indicating that MELs have surface activity, can reduce surface tension, and promote micelle formation. The concentration of 5.34 mg / L of MELs has important reference significance in the application fields of drug delivery and cosmetics. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 is a sugar metabolism pathway prediction diagram drawn based on M. aphidis XM01 genome sequencing;

[0039] Figure 2 is the influence of the first carbon source on the yield of MELs;

[0040] Figure 3 is the GC-MS result of the MELs fatty acid side chain composition corresponding to 11 kinds of first carbon sources;

[0041] Figure 4 is a data chart of the MELs yield, biomass and intracellular oil data of MELs fermented in the soybean oil fermentation medium added with different types of second carbon sources;

[0042] Figure 5 is the nitrogen source and carbon source optimization result of the fermentation medium; (A) is the nitrogen source type optimization of the fermentation medium; (B) is the nitrogen source concentration optimization of the fermentation medium; (C) is the first carbon source (safflower seed oil) concentration optimization of the fermentation medium; (D) is the second carbon source (glucose) concentration optimization of the fermentation medium;

[0043] Figure 6MELs antioxidant capacity determination results;

[0044] Figure 7 MELs emulsification capacity determination; (A) is the emulsion appearance chart: three groups of test tubes from left to right are soybean oil, liquid paraffin and n-hexane, and each group of test tubes from left to right is MELs, Tween 80, SDS; (B) is the MELs emulsification data chart;

[0045] Figure 8 Critical micelle concentration of MELs with safflower seed oil as carbon source;

[0046] Figure 9 Inhibition of MELs on bacteria and fungi;

[0047] Figure 10 Annexin V-FITC and PI staining results of MELs on Candida albicans, (A) is the staining result without MELs treatment, (B) is the staining result of Candida albicans treated with MELs;

[0048] Figure 11 Minimum inhibitory concentration determination results of MELs on Candida albicans;

[0049] Figure 12 Anti-inflammatory capacity of MELs with safflower seed oil as carbon source;

[0050] Figure 13 Cytotoxicity of MELs with safflower seed oil as carbon source;

[0051] Figure 14 Cell proliferation promoting results of MELs with safflower seed oil as carbon source;

[0052] Figure 15 MELs biomass, yield, intracellular oil content, and residual glucose and safflower seed oil changes during fermentation of XM01 strain with safflower seed oil as first carbon source and glucose as second carbon source in a 10-liter fermentation tank. DETAILED DESCRIPTION

[0053] The technical solutions of the present application will be further described in detail below in combination with the drawings and specific embodiments, but the scope of protection claimed by the present application is not limited to the scope expressed in the examples. The experimental methods not specified in the following embodiments are usually carried out according to the conventional conditions, or according to the conditions suggested by the manufacturers, and the materials not specified in detail are all commercially available materials.

[0054] The aphid Pseudozyma aphidis XM01 strain is screened from the mangrove in Hainan in the early stage by the laboratory, and the Pseudozyma aphidis XM01 strain producing mannose erythritol lipid (MEL) is preserved in China Center for Type Culture Collection, and the preservation number of the Pseudozyma aphidis XM01 is CCTCC NO: M2021517, and the preservation date is May 12, 2021.

[0055] The first carbon source used in the application includes soybean oil, macadamia nut seed oil, safflower seed oil, evening primrose oil, wheat germ oil, camellia seed oil, sunflower seed oil, grape seed oil, flaxseed oil, rapeseed oil, olive oil, white pool flower seed oil, glycerol, lecithin, phosphatidylglycerol and yolk oil.

[0056] The second carbon source used in the application includes glucose, fructose, mannose, L-arabinose, xylose, sorbitol, erythritol, mannitol, L-arabitol and xylitol.

[0057] The concentration of the medium component in the application is based on the volume of the whole medium, wherein w / v represents the mass-volume ratio, the mass unit is g, and the volume is ml.

[0058] Example 1: Genome sequencing KEGG map analysis of XM01 strain

[0059] 1. Illumina next-generation sequencing method is used for whole genome sequencing of XM01 strain, according to the whole genome sequencing results of XM01 strain, KEGG database (https: / / www.kegg.jp / kegg / pathway.html) is used to classify genes according to the involved pathways, including glycolysis pathway, pentose phosphate pathway, fructose and mannose pathway, etc., and the metabolism and synthesis pathway of the alcohol head of mannose erythritol lipid is drawn.

[0060] 2. Predict the glycol metabolism pathway combined with the KEGG map results of genome sequencing

[0061] GDP-D-mannose and erythritol are key precursor substances for the biosynthesis of MELs, and the intracellular accumulation level directly affects the yield of glycolipids. Based on the KEGG metabolic pathway annotation system of whole genome sequencing of XM01 strain, the regulation of glucose, fructose, mannose, arabinose, xylose, ribose, sorbose, xylitol, sorbitol, erythritol, mannitol and arabinol on the synthesis of GDP-D-mannose is analyzed, and the results are shown in Figure 1 The addition of glucose, fructose, mannose, xylitol and ribose can significantly promote the synthesis of GDP-D-mannose, which provides a key metabolic regulation strategy for optimizing glycolipid biosynthesis.

[0062] According to Figure 1 It can be known that through KEGG metabolic map analysis, the glycolysis pathway and the pentose phosphate pathway of the XM01 strain are closely related to the mannose metabolic pathway. The metabolism of glucose and fructose mainly generates pyruvate and acetyl-CoA through the glycolysis pathway, and at the same time, NADPH and pentose phosphate intermediates are provided through the pentose phosphate pathway, both of which provide energy and precursor substances for the synthesis of GDP-D-mannose. When glucose is added, the strain accumulates 6-phosphofructose and 3-phosphoglyceraldehyde through glycolysis, thereby promoting the activity of key enzymes in the mannose metabolic pathway, so as to improve the yield of GDP-D-mannose. The metabolism of xylitol and ribose plays a unique role through the pentose phosphate pathway. After xylitol is catalyzed by dehydrogenase to generate xylulose-5-phosphate, it enters the pentose phosphate pathway to generate ribulose-5-phosphate, and is finally converted into 5-phosphoribose, thereby enhancing the cross node of the pentose phosphate pathway and the mannose metabolism, and promoting the synthesis of GDP-D-mannose.

[0063] Example 2: Exploring the influence of the type of first carbon source in the fermentation medium on MELs

[0064] 1. Influence of the type of first carbon source on the yield of MELs

[0065] YPD solid medium: 2% (w / v) glucose, 2% (w / v) peptone, 1% (w / v) yeast extract, 2% (w / v) agar, dissolved in distilled water, sterilized at 115°C for 30 min.

[0066] Seed medium: 0.1% (w / v) ammonium nitrate, 0.1% (w / v) yeast powder, 0.03% (w / v) potassium dihydrogen phosphate, 3% (w / v) glucose. Dissolved in distilled water, sterilized at 115°C for 30 min.

[0067] The following are the operation steps for preparing MELs by fermentation:

[0068] (1) The XM01 strain was inoculated on the surface of the YPD solid medium for streak isolation, and placed in a 28°C constant temperature incubator for inverted culture for 48 h.

[0069] (2) After the formation of colonies, a typical single colony was selected and transferred to a test tube containing 5 mL of seed medium, and cultured at 28°C and 180 rpm for 48 h.

[0070] (3) Then, 50 mL of fermentation medium was inoculated at a 10% inoculation amount, and fermented for 7 days under the same temperature (28°C) and rotation speed (180 rpm) conditions to obtain the fermentation broth.

[0071] The fermentation medium formula is: 0.2% (w / v) sodium nitrate, 0.1% (w / v) yeast powder, 0.02% (w / v), potassium dihydrogen phosphate, 0.02% (w / v) magnesium sulfate heptahydrate, 8% (w / v) first carbon source. Dissolved in distilled water, sterilized at 115 ℃ for 30 min.

[0072] The first carbon source is respectively set as soybean oil, macadamia nut seed oil, safflower seed oil, evening primrose oil, wheat germ oil, camellia seed oil, sunflower seed oil, grape seed oil, flaxseed oil, rapeseed oil, olive oil, white pool flower seed oil, glycerol, lecithin, phosphatidylglycerol, and egg yolk oil.

[0073] (4) 45 mL of the above obtained 16 kinds of fermentation broth was respectively added with 15 mL of ethyl acetate, and after fully oscillating and mixing, it was transferred to a 50 mL centrifuge tube. Strong mixing was performed using a vortex oscillator to ensure that the MELs were fully extracted into the organic phase. Then, centrifugation was performed at 8000 rpm for 5 min, and the upper organic phase was collected and transferred to a flat-bottom flask. Ethyl acetate was removed by a rotary evaporator to obtain a crude extract containing oil. The crude extract was dissolved in 15 mL of methanol, and after vortex mixing, centrifugation was performed at 5000 rpm for 5 min. The methanol phase was collected, and rotary evaporation was performed again to finally obtain MELs. The product weight was accurately weighed and the yield was calculated for yield analysis.

[0074] In combination Figure 2 As described above, the 11 kinds of first carbon sources screened can significantly improve the yield of MELs, with the lowest MELs yield being 58.34±1.22 g / L (flaxseed oil) and the highest yield being 86.11±2.78 g / L (macadamia nut seed oil). At the same time, the yield corresponding to safflower seed oil as the first carbon source is 77.78 g / L. It can be seen that the addition of the first carbon source significantly improves the yield of MELs, and the corresponding first carbon source is soybean oil, macadamia nut seed oil, safflower seed oil, evening primrose oil, wheat germ oil, camellia seed oil, sunflower seed oil, grape seed oil, flaxseed oil, rapeseed oil, and olive oil.

[0075] 2. Effect of first carbon source type on the fatty acid side chain composition of MELs

[0076] The composition and length of the fatty acid side chain have important influence on the physicochemical properties of MELs (such as surface activity, emulsifying performance, and cytotoxicity). GC-MS was used to determine the fatty acid side chain composition and proportion of MELs produced by the above 11 kinds of first carbon sources.

[0077] (1) 100 mg of MELs was weighed into a 10 mL vial, 1 mL of chloroform was added, and after vortex oscillation, 2.5 mL of 2% sulfuric acid-methanol solution was added to the mixed solution. After sealing, it was reacted in an oven at 80℃ for 2.5 h, and the reaction was frequently oscillated to ensure complete reaction.

[0078] (2) Take out the vial and cool to room temperature, add 1 mL saturated sodium chloride solution, mix well.

[0079] (3) Add 1 mL of n-hexane, shake and mix well, stand until the sample is layered, take the upper n-hexane phase (containing fatty acid methyl ester), filter through an organic filter membrane into a chromatographic sample vial, and use for GC-MS analysis.

[0080] (4) Chromatographic conditions: DB-5MS capillary column, non-split injection, injection volume 1 μL, carrier gas high-purity helium, flow rate 1 mL / min. The temperature program of the column is optimized as follows: equilibrate at 50°C for 1 min, then heat at a rate of 8°C / min to 180°C, hold for 5 min, then heat at a rate of 30°C / min to 280°C, hold for 20 min. Mass spectrometry conditions: mass scan range 35-550 amu. The injector and interface temperature is maintained at 280°C, and the ion source temperature is maintained at 230°C.

[0081] The experimental results are shown in Table 1. Figure 3 It has been shown that alkyl chain length of C10 or longer has a good repair effect on damaged skin cells, and when the first carbon source is safflower seed oil, it contains a large amount of unsaturated fatty acid linolenic acid. The C10 content in the MELs fatty acid side chain produced by fermentation is more than 80%, and unsaturated fatty acids such as C10:1 and C10:2 also have good antioxidant effects.

[0082] Example 3: Exploring the effect of the second carbon source in the fermentation medium on the yield of MELs synthesized by the strain, biomass and intracellular oil content

[0083] Based on the sugar metabolism prediction pathway generated by genome sequencing in Example 1, and based on the fermentation conditions of the soybean oil fermentation medium, monosaccharides or sugar alcohols involved in the pathway are added to the fermentation medium to explore the changes in MELs synthesis.

[0084] The carbon sources are divided into first carbon sources and second carbon sources, and the first carbon source is set as 8% (w / v) soybean oil as the control group, and different second carbon sources are added to prepare MELs for analysis.

[0085] (1) Cultivation method: XM01 strain was inoculated on the surface of YPD solid medium for streak isolation, and placed in a 28°C constant temperature incubator for inverted culture for 48 h. After the formation of colonies, typical single colonies were selected and transferred to a test tube containing 5 mL of seed culture medium, and cultured at 28°C, 180 rpm for 48 h. Subsequently, 10% inoculation was performed into 50 mL of soybean oil fermentation medium, and the fermentation culture was carried out at the same temperature (28°C) and speed (180 rpm) for 7 days to obtain the fermentation broth.

[0086] YPD solid medium: 2% (w / v) glucose, 2% (w / v) peptone, 1% (w / v) yeast extract, 2% (w / v) agar, dissolved in distilled water, sterilized at 115°C for 30 min.

[0087] Seed medium: 0.1% (w / v) ammonium nitrate, 0.1% (w / v) yeast powder, 0.03% (w / v) potassium dihydrogen phosphate, 3% (w / v) glucose. Dissolved in distilled water, sterilized at 115°C for 30 min.

[0088] Soybean oil fermentation medium: 0.2% (w / v) sodium nitrate, 0.1% (w / v) yeast powder, 0.02% (w / v) potassium dihydrogen phosphate, 0.02% (w / v) magnesium sulfate heptahydrate, add carbon source. Dissolved in distilled water, sterilized at 115°C for 30 min.

[0089] The second carbon source of the soybean fermentation medium is added as follows:

[0090] Table 1: Different second carbon source addition amount

[0091]

[0092] (2) Add 15 mL of ethyl acetate to each of the 11 fermentation broths obtained above, mix thoroughly by shaking, and then transfer to a 50 mL centrifuge tube. Use a vortex shaker to mix vigorously to ensure that the MELs are fully extracted into the organic phase. Then centrifuge at 8000 rpm for 5 min, collect the upper organic phase and transfer to a flat-bottom flask, remove the ethyl acetate by rotary evaporation to obtain the crude extract containing oil. Dissolve the crude extract in 15 mL of methanol, mix well by vortexing, and then centrifuge at 5000 rpm for 5 min. Collect the methanol phase and perform rotary evaporation again to obtain the MELs. Accurately weigh the product and calculate the yield.

[0093] (3) Dry the biomass obtained by centrifuging the fermentation broth in an 80°C oven, weigh it, and calculate the biomass.

[0094] (4) Add 5 mL of concentrated hydrochloric acid and 5 mL of water to the centrifuge tube containing the dry biomass, seal it and place it in an 80°C oven for 6 h. After cooling, add 7 mL of 60% methanol solution and 7 mL of chloroform, mix well by vortexing, centrifuge at 8000 rpm for 10 min, and take the lower chloroform phase. Dry at 80°C to obtain the intracellular oil, and weigh it to calculate the content.

[0095] The experimental results are as follows: Figure 4The experimental results show that, after adding monosaccharides or sugar alcohols, the MELs yield is higher than that of the control group (only soybean oil as carbon source), among which the promoting effect of mannose and fructose is the most significant, and the biomass accumulation also increases to different degrees. The biomass of the glucose group is the highest, and the MELs yield of the glucose group is 76.77±8.38, which is 12.4% higher than that of the control group, indicating that the addition of monosaccharides or sugar alcohols not only provides additional glycosyl donors for glycolipid synthesis, but also promotes bacterial growth by strengthening central carbon metabolism (such as glycolysis and pentose phosphate pathway). In addition, the intracellular oil content analysis shows that, after adding monosaccharides or sugar alcohols, the intracellular oil content of most samples decreases, indicating that the exogenous sugar substances improve the carbon source conversion efficiency to MELs by adjusting the metabolic flow direction of fatty acid beta-oxidation and glycolipid synthesis. It can be known from this embodiment that the addition of the second carbon source significantly improves the MELs yield, and the biomass of the glucose group is the highest, which provides an optimization idea for the later experiment with safflower oil as the first carbon source, and selecting glucose as the second carbon source is also the best choice for designing the experiment.

[0096] Example 4: Effect of carbon source amount, nitrogen source type and amount on MELs

[0097] It can be known from Example 2 that safflower oil contains a large amount of unsaturated fatty acid linolenic acid, and the content of C10 in the fatty acid side chain of the MELs produced by fermentation is more than 80%; it can be known from Example 3 that the biomass of the glucose group as the second carbon source is the highest, indicating that the addition of monosaccharides not only provides glycosyl donors for glycolipid synthesis, but also promotes bacterial growth by strengthening central carbon metabolic pathways such as glycolysis and pentose phosphate. Therefore, in this embodiment, safflower oil is selected as the first carbon source and glucose is selected as the second carbon source in the fermentation medium. The effects of carbon source amount, nitrogen source type and amount in the fermentation medium on MELs yield, biomass and intracellular oil are explored.

[0098] (1) Cultivation method: XM01 strain was inoculated on the surface of YPD solid medium for streak isolation, and placed in a 28°C constant temperature incubator for inverted culture for 48 h. After the formation of colonies, typical single colonies were selected and transferred to a test tube containing 5 mL seed culture medium, and cultured at 28°C and 180 rpm for 48 h. Subsequently, 10% inoculation amount was inoculated into 50 mL fermentation medium, and the fermentation culture was carried out under the same temperature (28°C) and rotation speed (180 rpm) conditions for 7 days to obtain the fermentation broth.

[0099] The YPD solid medium is 2% (w / v) glucose, 2% (w / v) peptone, 1% (w / v) yeast extract, 2% (w / v) agar, dissolved in distilled water, and sterilized at 115°C for 30 min.

[0100] Seed culture medium: 0.1% (w / v) ammonium nitrate, 0.1% (w / v) yeast extract, 0.03% (w / v) potassium dihydrogen phosphate, 3% (w / v) glucose. Dissolve in distilled water and sterilize at 115°C for 30 min.

[0101] A single-factor optimization experiment was conducted on the fermentation medium. The methods for determining the MELs yield, biomass, and intracellular oil content during the fermentation medium optimization process were the same as those described in steps (2), (3), and (4) of Example 3. The fermentation medium optimization process was as follows: (1) Optimization of nitrogen source types

[0102] The fermentation medium formula was as follows: 0.2% (w / v) sodium nitrate / potassium nitrate / ammonium nitrate / ammonium sulfate as nitrogen sources, 0.1% (w / v) yeast extract, 0.02% (w / v) potassium dihydrogen phosphate, 0.02% (w / v) magnesium sulfate heptahydrate, and 8% safflower oil. The medium was dissolved in distilled water and sterilized at 115°C for 30 min.

[0103] (2) Optimization of nitrogen source concentration:

[0104] The fermentation medium formula was: 0.5 g / L, 1 g / L, 2 g / L, 3 g / L, and 4 g / L sodium nitrate, 0.1% (w / v) yeast extract, 0.02% (w / v) potassium dihydrogen phosphate, 0.02% (w / v) magnesium sulfate heptahydrate, and 8% safflower oil. The medium was dissolved in distilled water and sterilized at 115°C for 30 min.

[0105] (3) Optimization of the concentration of safflower oil as the first carbon source

[0106] The fermentation medium formula is: 0.2% (w / v) sodium nitrate, 0.1% (w / v) yeast extract, 0.02% (w / v) potassium dihydrogen phosphate, 0.02% (w / v) magnesium sulfate heptahydrate, and 4%, 6%, 8%, 10%, and 12% safflower oil, respectively. Dissolve in distilled water and sterilize at 115°C for 30 minutes.

[0107] (4) Optimization of glucose concentration as the second carbon source

[0108] The fermentation medium formula was: 0.2% (w / v) sodium nitrate, 0.1% (w / v) yeast extract, 0.02% (w / v) potassium dihydrogen phosphate, 0.02% (w / v) magnesium sulfate heptahydrate, 8% (w / v) safflower oil, and 0%, 1%, 2%, 3%, and 4% glucose. The solution was dissolved in distilled water and sterilized at 115°C for 30 min.

[0109] The MELs yield obtained by the above single factor experiment was combined with Figure 5 Analyze as shown.

[0110] like Figure 5(A) As shown, first, compared the nitrogen source effect of potassium nitrate and sodium nitrate on nitrogen source selection, it was found that the yield of MELs was 73.91 ± 1.56 g / L when sodium nitrate was used as the nitrogen source, which was slightly higher than that of potassium nitrate 72.35 ± 1.02 g / L, and the cost was lower, so sodium nitrate was selected as the best nitrogen source.

[0111] As Figure 5 (B) As shown, then the sodium nitrate concentration was optimized, and the optimal concentration of sodium nitrate was determined to be 2 g / L through gradient experiments, at which the yield of MELs reached a peak of 77.5 ± 1.33 g / L.

[0112] As Figure 5 (C) As shown, the amount of carbon source safflower oil was analyzed, and when the concentration of safflower oil was increased to 8%, the yield of MELs tended to be saturated, and the yield of MELs was 77.78 ± 1.36 g / L, and there was no significant gain in subsequent concentration increase, so 8% was selected as the optimal carbon source concentration.

[0113] Combining Example 3, it was found that glucose could significantly improve the yield of MELs, so glucose was selected as the second carbon source for optimization, and the concentration was further optimized. As Figure 5 (D) As shown, when the concentration of glucose was 2%, the yield of MELs reached the optimal level of 82.21 ± 0.67 g / L, which was about 4.62% higher than that of the single carbon source (safflower oil) system.

[0114] Through the above multi-factor optimization, the yield of MELs was finally improved to 82.21 ± 0.67 g / L under the condition of shake flask fermentation, which was 11.31% higher than that of the initial medium, laying an important foundation for large-scale production.

[0115] Combining Examples 2, 3 and 4, the optimization of carbon source and nitrogen source of the fermentation medium can be known, and the optimal component and concentration of the carbon source in the optimized fermentation medium are safflower oil 8% and glucose 2%, and the optimal component and amount of the nitrogen source are sodium nitrate 0.2%. The present application first applies safflower oil and glucose in combination to MELs fermentation, breaks through the yield bottleneck through the synergistic effect of carbon sources, and at the same time considers economy and sustainability, which provides key technical support for the industrialization application of MELs in the field of cosmetics.

[0116] From the above, according to the combination of the first carbon source and the second carbon source, the optimal method for improving the fermentation product MELs was optimized, and combining Example 2, it can be known that when the first carbon source is safflower oil, it contains a large amount of unsaturated fatty acid linolenic acid, and the C10 content in the fatty acid side chain of the fermented MELs is more than 80%, and the unsaturated fatty acids C10:1 and C10:2 also have good antioxidant effect, then, the biochemical indicators of MELs were explored by using safflower oil alone as the first carbon source.

[0117] MELs described in the following examples 5-12 are prepared by fermentation of a fermentation medium with only 8% safflower oil as carbon source.

[0118] Example 5: Determination of antioxidant capacity of MELs

[0119] (1) Accurately weigh 2.0 mg of DPHH powder, dissolve in 50 mL of absolute ethanol to prepare a working solution of 0.1 mM. After the solution is prepared, transfer it to a brown reagent bottle, store at 4°C in the dark, and the shelf life is not more than 7 days.

[0120] (2) Prepare 10 mg / mL, 20 mg / mL, 30 mg / mL glycolipid solutions:

[0121] As (MELs produced by safflower oil in experimental group I): 10 mg / mL, 20 mg / mL, 30 mg / mL + DPPH alcohol solution (0.1 mM); (MELs produced by olive oil in experimental group II): 10 mg / mL, 20 mg / mL, 30 mg / mL + DPPH methanol solution (0.1 mM).

[0122] Ab (MELs produced by safflower oil in blank group I): 10 mg / mL, 20 mg / mL, 30 mg / mL + absolute ethanol (0.1 mM); (MELs produced by olive oil in blank group II): 10 mg / mL, 20 mg / mL, 30 mg / mL + absolute ethanol (0.1 mM).

[0123] Ac (control group): DPPH alcohol solution + methanol

[0124] Background: methanol + ethanol

[0125] (3) Add 100 μL of Ⅰ, Ⅱ glycolipid (10 mg / mL, 20 mg / mL, 30 mg / mL) in 96-well plates, respectively, with 100 μL DPPH alcohol solution (0.1 mM) / 100 μL absolute ethanol, mix well for 20 min. All measurements are made on triplicate samples. Take 100 μL supernatant to measure absorbance OD 517 .

[0126] Glycolipid scavenging efficiency formula for DPPH free radicals:

[0127]

[0128] Bioactivity is an important parameter for screening functional cosmetic raw materials. The fatty acid side chain of MELs produced from safflower seed oil contains a large amount of unsaturated fatty acids, which should have good antioxidant properties. The antioxidant capacity of MELs produced from safflower seed oil was determined as shown in Figure 6 The experimental results show that the MELs produced from safflower seed oil have a concentration-dependent DPPH radical scavenging activity, and the MELs at 20 mg / mL have a DPPH radical scavenging activity of 22.4%. This shows that MELs can neutralize free radicals and have antioxidant and anti-aging effects on the skin.

[0129] Example 6: Investigation of the emulsifying properties of MELs

[0130] The emulsifying capacity of MELs on soybean oil, liquid paraffin, and n-hexane was determined, with Tween 80 and sodium dodecyl sulfate (SDS) as controls. MELs were diluted to a concentration of 20 mg / L, and 3 ml of MELs were mixed with 3 ml of soybean oil, 3 ml of liquid paraffin, and 3 ml of n-hexane, respectively. The mixture was vortexed for 2 min and allowed to stand at room temperature for 24 h. The emulsion layer height was measured and compared with the total liquid layer height, with Tween 80 and SDS used as experimental controls.

[0131] Emulsification index EI 24 Calculation formula:

[0132] ,

[0133] The experimental results are shown in Figure 7 As shown in Figure 7 (A), the emulsion was initially uniform and milky white with moderate flowability. After standing for 24 hours, the water phase was separated at the bottom, but the remaining emulsion remained stable without flocculation. As shown in Figure 7 (B), the emulsifying effect of MELs on soybean oil and liquid paraffin was inferior to that of Tween 80 and SDS, with an emulsification value of about 52.6%. The emulsifying capacity of MELs on n-hexane was significantly better than that of Tween 80 and SDS, with an emulsification value of more than 60%.

[0134] Example 7: Determination of the critical micelle concentration CMC value of MELs

[0135] To evaluate the surface activity of MELs produced by fermentation of safflower seed oil as the only carbon source, pyrene was used as a fluorescent probe to determine the critical micelle concentration (cmc).

[0136] (1) Pyrene stock solution preparation: 12.54 mg pyrene was accurately weighed and dissolved in acetone under light protection, and then diluted to 5 mL to obtain the mother liquor. 50 μL of the mother liquor was diluted to 5 mL with acetone to prepare the working stock solution, which was stored at room temperature under light protection for future use.

[0137] (2) 100 μL of the pyrene stock solution was taken and added to a 15 mL vial, which was left to stand at room temperature overnight to allow the acetone to evaporate completely. The glycolipid sample was prepared into a micellar aqueous solution, and a concentration gradient series of 0.5-100 mg / L was prepared by dilution. Ice water bath ultrasonic treatment was performed for 15 min (working 2 s / intermittent 2 s), followed by light protection cooling and standing overnight.

[0138] (3) The fluorescence intensity of the sample was measured using a TECAN microplate reader. The standard curve was plotted with the fluorescence intensity ratio at 373 nm and 383 nm as the vertical coordinate and Log C (mg / L) as the horizontal coordinate. The critical micelle concentration of different glycolipid samples was determined by the inflection point of the curve.

[0139] As shown in Figure 8 , the critical micelle concentration of MELs produced by safflower seed oil as carbon source fermentation was 5.34 mg / L, indicating that MELs had surface activity, could reduce surface tension, and promote micelle formation. The higher the CMC value, the lower the stability of the micelle in the solution, and the MELs concentration of 5.34 mg / L had important reference significance in the application of drug delivery, cosmetics, etc.

[0140] Example 8: Oxford cup method antibacterial experiment

[0141] MELs had significant inhibitory effect on a variety of bacteria and fungi, and its mechanism involved destroying cell membrane structure, interfering with biofilm formation, and inducing oxidative stress, etc. This experiment determined the inhibitory effect of MELs on Staphylococcus aureus, Listeria monocytogenes in Gram-positive bacteria, Escherichia coli, Salmonella in Gram-negative bacteria, and Candida albicans in fungi.

[0142] (1) The MELs sample was dissolved in DMSO to prepare a stock solution of 20 mg / mL, which was stored at 4°C for future use.

[0143] (2) Escherichia coli, Listeria monocytogenes, Staphylococcus aureus, and Salmonella were cultured in 5 mL of LB liquid medium at 37°C with 180 rpm shaking, and 10 μL (1 × 10 5Spores / mL) were spread evenly on the surface of LB solid medium, and after the surface dried, four oxford cups were placed by sterile operation. 100 μΙ_ DMSO was used as a blank control, and 100 μΙ_ of the prepared stock solution was injected into the other three cups, which were incubated at 37 °C overnight. The diameters of the inhibition zones were measured to determine the antibacterial activity at 37 °C.

[0144] (3) A single colony of activated white Candida was picked from the plate and cultured in 5 mL YPD liquid medium at 28 °C and 180 rpm overnight. 10 μΙ_ of spore suspension (1 x 10 5 Spores / mL) were spread evenly on the surface of YPD solid medium, and after the surface dried, four oxford cups were placed by sterile operation. 100 μΙ_ DMSO was used as a blank control, and 100 μΙ_ of the prepared stock solution was injected into the other three cups, which were incubated at 28 °C for 24 h. The diameters of the inhibition zones were measured to determine the antibacterial activity at 28 °C.

[0145] LB liquid medium: 1% (w / v) tryptone, 0.5% (w / v) yeast extract, 1% (w / v) sodium chloride, dissolved in distilled water, sterilized at 115 °C for 30 min.

[0146] LB solid medium: 1% (w / v) tryptone, 0.5% (w / v) yeast extract, 1% (w / v) sodium chloride, 2% (w / v) agar, dissolved in distilled water, sterilized at 115 °C for 30 min.

[0147] YPD liquid medium: 2% (w / v) glucose, 2% (w / v) tryptone, 1% (w / v) yeast extract, dissolved in distilled water, sterilized at 115 °C for 30 min.

[0148] The experimental results are shown in Table 1. Figure 9 MELs had no inhibitory effect on bacteria at 20 mg / mL, but showed inhibition on white Candida. The diameter of the inhibition zone of MELs synthesized by red safflower seed oil as a carbon source was 13.2 ± 0.4 mm.

[0149] Example 9: Annexin V-FITC / PI double staining of MELs-treated white Candida

[0150] To determine whether the inhibitory effect of MELs on C. albicans is due to apoptosis or necrosis, an Annexin V-FITC Apoptosis Detection Kit was used. Propidium iodide (PI) can selectively label cells with damaged membrane integrity, showing red fluorescence under a fluorescence microscope. Annexin V-FITC can specifically bind to phosphatidylserine on the surface of apoptotic cells, showing green fluorescence. According to the staining results, different cell states can be distinguished: normal cells show no fluorescence signal; early apoptotic cells show only green fluorescence; late apoptotic cells show both red and green fluorescence; and necrotic cells show strong red fluorescence due to complete loss of membrane integrity, accompanied by green fluorescence.

[0151] C. albicans single colonies on the plate were inoculated in 5 mL of YPD liquid medium (as in Example 11) and cultured at 28°C and 180 rpm for 8-10 h. MELs stock solution was added to the bacterial solution to a final concentration of 20 mg / mL, and an equal amount of DMSO was added to the bacterial solution as a control group. Both groups were cultured at 28°C and 180 rpm for 24 h. The specific steps were performed according to the instructions of the Biyun Tian Apoptosis Kit. After the final preparation, the samples were observed under a fluorescence microscope.

[0152] The experimental results are shown in Figure 10 As shown in the results, the C. albicans bacterial solution with added MELs showed green fluorescence with Annexin V-FITC and red fluorescence after staining with PI, indicating that the addition of MELs caused apoptosis of C. albicans, thereby exhibiting antibacterial effects.

[0153] Example 10: Determination of the minimum inhibitory concentration by MTT method

[0154] MTT colorimetric method is a classic technique for quantitatively evaluating cell survival and growth based on mitochondrial enzyme activity. The core mechanism is that succinate dehydrogenase in metabolically active cells can catalyze the reduction of tetrazolium salt MTT to insoluble blue-purple formazan crystals, while inactive cells lose this reduction ability.

[0155] Under standard culture conditions, the amount of formazan crystals generated is positively linearly correlated with the density of active cell populations, providing a quantitative basis for cell proliferation / toxicity detection. MELs solutions at 4.0, 8.0, 12.0, 16.0, 20.0, 24.0, and 28.0 mg / mL were prepared, and the minimum inhibitory concentration of MELs on C. albicans was determined according to the instructions of the Biyun Tian MTT Kit.

[0156] The results of determining the minimum inhibitory concentration of MELs produced by safflower seed oil are shown in Figure 11 As shown in the results, the minimum inhibitory concentration of MELs produced by safflower seed oil was 15.6 mg / mL.

[0157] Example 11: Anti-inflammatory ability assay

[0158] The activity of RAW 264.7 macrophages after MELs treatment was determined according to the MTT method, and the appropriate concentration was selected for TNF-α inhibition ability assay according to the cytotoxicity determination results. The entire experimental procedure was strictly carried out according to the operation specification of the ELISA kit to ensure the reliability of the experimental data.

[0159] RAW 264.7 cells (1.0 x 10 5 Cells / mL, 200 microliters) were inoculated into a 96-well cell culture plate and incubated in a humidified incubator at 37°C, 5% CO2 for 4 hours. Then, the cells were exposed to 200 microliters of LPS (1.0 microgram / milliliter) for 6 hours. After removing LPS, 200 microliters of the sample to be tested was added to each well. After 6 hours, 100.0 microliters of supernatant was collected and centrifuged at 4°C, 2000 rpm for 20 minutes. The content of TNF-α was determined using an ELISA kit. All measurements were repeated three times (n = 3).

[0160] Results: As Figure 12 The extracellular product MELs with safflower seed oil as carbon source had a concentration-dependent inhibitory effect on TNF-α, and had an anti-inflammatory effect at a concentration range of 10-100 μg / mL.

[0161] Example 12: Cytotoxicity and cell proliferation experiments

[0162] 1. Cell toxicity assay using MTT method:

[0163] (1) Cells in good growth state were inoculated in a 96-well culture plate at a density of 5 x 10 4 -1 x 10 5 cells per well and incubated at 37°C, 5% CO2 overnight.

[0164] (2) Discard the culture solution and add different concentrations of samples, with 5 replicates for each concentration. The cell control group was added with culture medium and incubated for 24 h.

[0165] (3) Discard the culture medium, wash twice with PBS, add 100 μL MTT (1.0 g·L -1 solution, and incubate at 37°C, 5% CO2 for 4 h. Discard the liquid, add 150 μL DMSO, and incubate at 37°C for 10 min. Measure the absorbance value of each well at 490 nm wavelength.

[0166] Calculation formula:

[0167] ,

[0168] V (%) - cell survival rate, %;

[0169] OD sample - absorbance of the reaction system containing the sample to be tested;

[0170] OD blank control - absorbance of the empty plate without any substance;

[0171] OD cell control - absorbance without the reaction system of the sample to be tested.

[0172] Combined Figure 13 It can be seen that the extracellular products MELs with safflower seed oil as carbon source have no cytotoxicity at a concentration range of 0.01%-0.0005%, and the cell survival rate is greater than 70%.

[0173] 2. Cell proliferation promoting experiment method:

[0174] (1) L929 mouse fibroblasts in good growth state were inoculated in a 96-well culture plate at a density of 2x10 4 / mL per well, 100 μL was inoculated per well, and incubated at 37℃ in a 5% CO2 environment overnight.

[0175] (2) Discard the culture solution, add different concentrations of samples, dilute with DMEM complete culture solution, set 6 parallels for each concentration, add DMEM complete culture solution to the cell control group, add complete culture solution containing 20 ng / mL transforming growth factor β1 (TGF-β1) to the positive control group, 100 μL per well, and incubate for 48-72 h.

[0176] (3) Aspirate the culture medium, wash twice with PBS, add 100 μL MTT (1.0 g·L -1 ) solution, incubate at 37℃ in a 5% CO2 environment for 4 h, discard the liquid, add 150 μL DMSO, and incubate at 37℃ for 10 min, then measure the absorbance value of each well at 490 nm wavelength.

[0177] Calculation formula:

[0178] ,

[0179] In the formula:

[0180] V (%) - cell survival rate, %;

[0181] OD sample - absorbance of the reaction system containing the sample to be tested;

[0182] OD blank control - absorbance of the empty plate without any substance;

[0183] OD cell control - absorbance without the reaction system of the sample to be tested.

[0184] In combination Figure 14 It can be seen that the fermentation product MELs prepared by safflower seed oil as the first carbon source has obvious effect on promoting the proliferation of L929 cells in the concentration range of 0.0001%-0.005%, and the cell survival rate is higher when the concentration is 0.0005%.

[0185] Example 13: fed-batch fermentation in a 10-liter fermenter

[0186] Firstly, the XM01 strain was coated on YPD solid medium, and the YPD solid medium was prepared by dissolving 2% (w / v) glucose, 2% (w / v) peptone, 1% (w / v) yeast extract, 2% (w / v) agar in distilled water and sterilizing at 115°C for 30 min. It was cultured at 28°C for 2 days, then inoculated into seed medium for 48 h, and then inoculated into a total volume of 7 liters of fermentation medium.

[0187] The fermentation medium was prepared by dissolving 0.2% (w / v) sodium nitrate, 0.1% (w / v) yeast powder, 0.02% (w / v) potassium dihydrogen phosphate, 0.02% (w / v) magnesium sulfate heptahydrate, 8% (w / v) safflower seed oil, and 2% (w / v) glucose in distilled water, and sterilizing at 115°C for 30 min. However, this fermentation medium can easily cause emulsification in the tank during fermentation. At the same time, it significantly inhibits the growth of the bacterial cells and the production of MELs.

[0188] Three-stage fermentation process: the fermentation medium was prepared by dissolving 2g / L sodium nitrate, 0.1% (w / v) yeast powder, 0.02% (w / v) potassium dihydrogen phosphate, 0.02% (w / v) magnesium sulfate heptahydrate, 120g / L safflower seed oil, and 2% glucose in a total volume of 7 liters; wherein the safflower seed oil was added in batches, 35g / L at 0h, 25g / L at 36h, and 15g / L at 60h, 72h, 84h, and 96h. In the actual operation process, the safflower seed oil was added at 15-25g / L each time, and the safflower seed oil content was close to consistent after each addition according to the results of the previous oil consumption. And in the early stage of 0-36h, the aeration was 720 L / h, and the stirring speed was 300 r / min; in the middle stage of 36-96h, the aeration and stirring were reduced to 150 L / h and 180 r / min respectively, and the carbon source was precisely added to maintain the balance of C / N ratio; in the later stage of 96-216h, the limiting feeding strategy was adopted, and the aeration rate was maintained at 100 L / h, and the stirring rate was maintained at 120 r / min.

[0189] Based on the previous experimental observations, we found that MELs products themselves have excellent emulsification properties, which can easily cause in-tank emulsification during fermentation. At the same time, excessive oil in the fermentation broth can significantly inhibit the proliferation activity of cells in the early growth stage. Through systematic optimization of the culture parameters of a 10-liter fermenter, we finally developed a three-stage fermentation process to effectively solve the above technical problems, which divides the complete fermentation process into three stages: (1) the early stage of continuous growth and reproduction of the strain (2) the middle stage of MELs synthesis and carbon source supplementation (3) the end of the feeding, and a large amount of MELs synthesis. During the fermentation process, samples were taken every 12 h to measure glucose residual amount, MELs yield, intracellular oil content, biomass, and safflower seed oil residual amount.

[0190] Glucose residual amount: glucose residual amount was determined using a glucose detection kit;

[0191] MELs yield, intracellular oil content, biomass, and safflower seed oil residual amount determination method:

[0192] (1) Take 45 mL of fermentation broth and add 15 mL of ethyl acetate, mix thoroughly, and then transfer to a 50 mL centrifuge tube. Use a vortex mixer to mix intensively to ensure that the MELs are fully extracted into the organic phase. Then centrifuge at 8000 rpm for 5 min, collect the upper organic phase, and transfer it to a flat-bottom flask. Remove the ethyl acetate by rotary evaporation to obtain the crude oil-containing extract.

[0193] (2) Dissolve the crude extract in 15 mL of methanol, mix well with a vortex mixer, and centrifuge at 5000 rpm for 5 min. Collect the methanol phase and perform rotary evaporation again to obtain the MELs crude product.

[0194] (3) Dry the bacterial cells obtained by centrifugation of the fermentation broth in an 80°C oven, weigh, and calculate the biomass.

[0195] (4) Add 5 mL of concentrated hydrochloric acid and 5 mL of water to the centrifuge tube containing the bacterial cell dry weight, seal, and place in an 80°C oven for 6 h. After cooling, add 7 mL of 60% methanol solution and 7 mL of chloroform, vortex mix, centrifuge at 8000 rpm for 10 min, and take the lower chloroform phase. Dry at 80°C to obtain the intracellular oil, and weigh to calculate the intracellular oil content.

[0196] (5) Safflower seed oil residual amount: the safflower seed oil residual amount obtained by subtracting the MELs crude product from the oil-containing crude extract.

[0197] In the process of fermentation scale-up, the inherent strong emulsification characteristics of MELs can cause the emulsification of fermentation system, and the excessive lipid accumulation can significantly inhibit the early growth of the cells. Based on the repeated experiments in a 10-L scale fermenter, a new three-stage fermentation strategy was developed: the initial cell proliferation period (0-36 h) focused on promoting the growth metabolism of the cells, with a ventilation rate of 720 L / h and a stirring rate of 300 r / min, and the glucose was basically consumed at this stage; the product induction period (36-96 h) reduced the ventilation and stirring to maintain the balance of C / N ratio by accurately adding carbon source, which not only relieved the metabolic pressure but also promoted the synthesis of MELs; the product enrichment period (96-216 h) adopted a restrictive feeding strategy, with a ventilation rate of 100 L / h and a stirring rate of 120 r / min, to promote the directional accumulation of secondary metabolites.

[0198] In combination Figure 15 As shown in the fermentation kinetics data, the final yield of MELs reached 92.56±0.83 g / L when the system reached the process endpoint at 216 h, which was 13.44% higher than that of the shake flask fermentation, and the biomass level was maintained at 20.06 g / L, and the intracellular lipid content was stable at 64.73±1.05%. The segmented fermentation strategy decoupled the cell growth and product synthesis metabolic pathways by time sequence, effectively solving the key technical bottlenecks of emulsion control and metabolic balance in the scale-up process.

[0199] The above examples are only used to illustrate the technical solutions of the present application, but not to limit it; although the present application has been described in detail with reference to the foregoing examples, for those skilled in the art, the technical solutions recorded in the foregoing examples can be modified, or some technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions claimed by the present application.

Claims

1. A fermentation method using the aphid yeast strain XM01, characterized in that: The specific steps include: (1) Inoculate the aphid yeast strain XM01 into YPD solid medium and culture at constant temperature to form colonies; (2) Inoculate the bacterial colony into the seed culture medium and culture it on a shaking platform to obtain the seed solution; (3) inoculating the seed liquid into a fermentation medium and fermenting to obtain a fermentation liquid; (4) Add ethyl acetate to the fermentation broth, mix, and centrifuge to obtain a crude extract containing oil; add methanol to the crude extract, centrifuge, and evaporate to obtain mannitol erythritol lipids (MELs).

2. The fermentation method using the Aphid Yeast XM01 strain according to claim 1, characterized in that: The classification name of the aphid yeast Pseudozyma XM01 strain is Pseudozyma aphidis XM01, and its preservation number is CCTCC NO: M2021517.

3. The fermentation method using the Aphid Yeast XM01 strain according to claim 1, characterized in that: The carbon source of the fermentation medium in step (3) includes a first carbon source, which is selected from at least one of soybean oil, macadamia seed oil, safflower seed oil, evening primrose oil, wheat germ oil, camellia seed oil, sunflower seed oil, grape seed oil, linseed oil, rapeseed oil, and olive oil; and the concentration of the first carbon source is 2%-15%.

4. A fermentation method using the Aphid Yeast XM01 strain according to claim 3, characterized in that: The carbon source also includes a second carbon source, which is selected from at least one of glucose, fructose, mannose, L-arabinose, xylose, sorbitol, erythritol, mannitol, L-arabinol, and xylitol; the concentration of the second carbon source is 1%-6%.

5. The method for fermenting using the Aphid Yeast XM01 strain according to claim 1, characterized in that: The nitrogen source of the fermentation medium is sodium nitrate, and the concentration of the sodium nitrate is 0.05%-0.3%.

6. The fermentation method using the Aphid Yeast XM01 strain according to any one of claims 1 to 5, characterized in that: The optimal components and concentrations of the carbon source and nitrogen source of the fermentation medium are: the first carbon source is safflower seed oil, with a concentration of 8%; the second carbon source is glucose, with a concentration of 2%; and the nitrogen source is sodium nitrate, with a concentration of 0.2%.

7. The mannitol erythritol lipid obtained by the preparation method according to any one of claims 1 to 3, characterized in that: The C10 content in the fatty acid side chain of the mannitol erythritol lipid is ≥80%, and the critical micelle concentration of the mannitol erythritol lipid is 4-5.5 mg / L.

8. Use of the mannitol-erythritol lipid according to claim 7 in the preparation of antioxidant and / or anti-inflammatory cosmetics and / or medicines.

9. Use of the mannitol-erythritol lipid according to claim 7 in the preparation of a biological preparation for inhibiting Candida albicans.

10. The use according to claim 9, characterized in that The antibacterial concentration of the mannitol erythritol lipid in inhibiting Candida albicans is 15 mg / mL-25 mg / mL.

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