A method for improving the ability of Aureobasidium pullulans strain to produce heavy oil by fermentation

Abstract

The present invention discloses a method for improving the ability of a budding short-stalked mold strain to produce heavy oil by fermentation, comprising the following steps: S1, strain activation; S2, seed liquid culture; S3, preparation of fermentation medium; S4, fermentation culture; S5, heavy oil extraction. The present invention ensures the oxygen content in the medium by adding an oxygen-releasing body to the fermentation medium, so that the budding short-stalked mold has sufficient oxygen during the fermentation process, and at the same time, n-hexane is added as an oxygen carrier, and Tween 60 is added as a surfactant to improve the dispersibility of n-hexane in the fermentation medium and improve the oxygen transfer efficiency; and the composite carbon source added to the fermentation medium, monosaccharide glucose, xylose and polysaccharide inulin are compounded to better provide a variety of nutrients for strain growth, and the various component raw materials in the fermentation medium cooperate with each other to jointly promote the growth and metabolism of the budding short-stalked mold, thereby improving the ability of the budding short-stalked mold strain to produce heavy oil.

Description

Technical Field

[0001] The invention belongs to the field of fermentation engineering and biotechnology, and in particular relates to a method for improving the ability of a budding Aureobasidium pullulans strain to produce heavy oil by fermentation. Background Art

[0002] Budding Agromyces, also known as budding thallus, budding side thallus, and short-stalked mold, belongs to the genus Agromyces of the Ascomycetes. In nature, it often lives with yeasts. It has similar morphological or physiological characteristics to yeasts and is a "yeast-like fungus". Budding Agromyces is widely distributed in nature, mainly on the surface of plant materials (flowers, leaves, fruits, bark, etc.) or in the soil. It also exists on the surface of rotten fruits and can be isolated from deep-sea microorganisms and limestone. Its colony grows fast. In the early stage of colony growth, it is moist and smooth yeast-like, and in the later stage of growth, it becomes black leather-like. The life cycle of budding Agromyces is complex. During its growth, there are usually five cell forms: thick-walled spores, yeast-like, mycelium, swollen spores and budding spores. The cell morphology composition usually varies greatly due to different strains, fermentation conditions and culture media.

[0003] Studies in recent years have shown that fermentation of different Aureobasidium pullulans can produce a series of substances including extracellular polysaccharides, extracellular enzymes, melanin, β-polymalic acid, siderophore, L-sorbitol, etc., many of which have been purified and identified; at the same time, Aureobasidium pullulans can be used as a fermentation bacterium for single-cell protein due to its special growth characteristics.

[0004] Compared with the production of conventional animal and plant oils, microbial oils have fast microbial growth, high bacterial density, and can be artificially fermented and cultured industrially. The growth environment of the strain can be artificially controlled and has high safety performance. Unlike other oils, heavy oil (liamocin) is an active polyol ester produced by budding Aureobasidium under specific culture conditions. It consists of a polyol head group (monomannitol head group, arabinol head group, etc.) and some O-acyl groups acylated with polyester tails and polyester tails containing 1 to 5 3,5-dihydroxydecanoate groups. The heavy oil produced by budding Aureobasidium is a fat that can be directly secreted outside the cell. Since the density of heavy oil is greater than that of water, after the fermentation is completed, compared with most single-cell oils synthesized intracellularly, its extraction process not only saves the process of cell wall breaking, but also breaks through the restriction of biomass on oil yield in the fermentation results. The heavy oil can be completely precipitated at the bottom of the reactor by centrifugation, and then extracted by organic solvent. This method greatly reduces the cost required for the extraction and esterification reaction of traditional oils and facilitates the separation and large-scale preparation of long-chain normal alkanes. The special structure of polyol lipids gives them great application potential. In medical treatment, they have anti-proliferative effects on a variety of human tumor cells. For example, the growth of lung cancer cells, leukemia cells and cervical cancer cells is inhibited, while normal cells are not affected. In the chemical industry, they can be used as surfactants, biodiesel, biofuels, etc.

[0005] At present, the heavy oil yield of budding short-stalked mold is not high, and the research on heavy oil is still in the preliminary stage. There are very few studies at the molecular level, so its synthesis mechanism is still unclear. Studies have shown that the yield of known heavy oil-producing strains is limited by the type of carbon source, and the heavy oil yield of different carbon sources varies greatly. Some scholars have optimized the fermentation of budding short-stalked mold. The heavy oil yield has increased when glucose is used as the carbon source, but the content of polyol lipids is not high. Chinese patent application No. 201910978113.1 discloses a high-yield heavy oil budding short-stalked mold strain and its construction method and use, which belongs to the field of genetic engineering. The present invention significantly improves the heavy oil yield of budding short-stalked mold bred by knocking out the pullulan synthase gene and overexpressing the ACL gene. After 7 days of fermentation with xylose as the carbon source, the heavy oil yield of the recombinant strain reached 19.4372g / L, and the heavy oil yield of the parent strain was 10.0325g / L. The recombinant strain increased by 93.74% compared with the parent strain. This patent significantly improves the heavy oil yield of the selected Aureobasidium pullulans by overexpressing the ACL gene and knocking out (replacing) the pullulan synthase gene PUL. However, the method of encoding genes is relatively limited, has high requirements for strains, and is not suitable for industrial production. Summary of the invention

[0006] In view of the shortcomings of the prior art, the object of the present invention is to provide a method for improving the ability of a budding Aureobasidium strain to produce heavy oil by fermentation. The method promotes better metabolism and proliferation of budding Aureobasidium by preparing a specific fermentation medium and using specific carbon sources and nitrogen sources, thereby improving its ability to produce heavy oil.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] A method for improving the ability of a budding Aureobasidium pullulans strain to produce heavy oil by fermentation, comprising the following steps:

[0009] S1. Activation of bacterial strains: inoculating Aureobasidium pullulans into an activation medium and culturing at a constant temperature to obtain activated bacterial strains;

[0010] S2, seed liquid culture: inoculating the activated bacteria in step S1 into a seed liquid culture medium, performing seed culture, and obtaining a seed liquid;

[0011] S3. Preparation of fermentation medium: according to percentage, 8-10% of complex carbon source, 2-4% of nitrogen source, 5-7% of oxygen-releasing body, 2-4% of n-hexane, 0.05-0.1% of K2HPO4, 0.05-0.1% of MgSO4, 0.1-0.15% of Tween 60, and the balance of water are mixed evenly and sterilized at 110-120° C. for 15-20 min to obtain fermentation medium;

[0012] S4, fermentation culture: inoculating the seed liquid in step S2 into the fermentation medium in step S3, and fermenting and culturing, and obtaining a fermentation liquid after the fermentation is completed;

[0013] S5. Heavy oil extraction: The fermentation liquid in step S4 was centrifuged to discard the supernatant, an equal volume of ethyl acetate was added and mixed thoroughly, and the mixture was centrifuged at 7000 r / min for 10 min to obtain an organic layer containing heavy oil, which was transferred to a new graduated tube. The operation was repeated three times, and the obtained organic layer solution was rotary evaporated to remove the solvent to obtain heavy oil, which was weighed to calculate the yield.

[0014] Preferably, the constant temperature culture conditions in step S1 are cultured in a shaker at 26-28°C and 150-200 r / min for 2-3 days; the composition of the activation culture medium is: in percentage, 1-3% glucose, 1-3% peptone, 0.5-2% yeast extract, 1-4% agar, and the balance is water, and sterilized at 110-130°C for 20-40 min.

[0015] More preferably, the constant temperature culture conditions in step S1 are incubated at 27-28°C in a shaker at 150-180 r / min for 2-3 days; the activation culture medium is composed of: 2-3% glucose, 2-3% peptone, 0.5-1% yeast extract, 2-4% agar, and the balance is water, and is sterilized at 120-130°C for 30-40 min.

[0016] Preferably, the composition of the seed liquid culture medium in step S2 is: in percentage, 1-3% glucose, 1-3% peptone, 0.5-2% yeast extract, 0.5-1% K2HPO4, 0.1-0.3% MgSO4, and the balance is water, and is sterilized at 110-130°C for 20-40min.

[0017] More preferably, the composition of the seed liquid culture medium in step S2 is: in percentage, 2-3% glucose, 1-2% peptone, 1-2% yeast extract, 0.5-0.8% K2HPO4, 0.2-0.3% MgSO4, and the balance is water, and is sterilized at 120-130°C for 20-30min.

[0018] Preferably, the inoculation amount of the activated bacteria in step S2 is 2-4%; the seed culture conditions are in a shaker at 27-29° C. and 150-200 r / min for 2-3 days.

[0019] More preferably, the inoculation amount of the activated bacteria in step S2 is 3-4%; the seed culture conditions are in a shaker at 28-29° C. and 170-200 r / min for 2-3 days.

[0020] Preferably, the preparation method of the oxygen-releasing body in step S3 is as follows:

[0021] The sepiolite is added into a hydrochloric acid solution for heat treatment, and after the treatment is completed, it is filtered, washed and dried to obtain a pretreated sepiolite; the pretreated sepiolite is then added into deionized water, and then 3-[3-(cholesterolaminopropyl)dimethylamino]-1-propanesulfonic acid is added for constant temperature reaction, and after the reaction is completed, it is filtered and dried to obtain a modified sepiolite; the modified sepiolite is added into a calcium chloride solution, and after stirring evenly, a suspension is obtained, and then concentrated ammonia water is added to the suspension, and stirring is continued. After the stirring is completed, a hydrogen peroxide solution is added dropwise, and the temperature is controlled to rapidly stir the reaction, and after the reaction is completed, it is filtered, washed and dried to obtain the oxygen-releasing body.

[0022] Preferably, the mass fraction of the hydrochloric acid solution is 5-10%, the heat treatment temperature is 70-80°C, and the time is 2-3h; the mass ratio of the pretreated sepiolite and 3-[3-(cholesterylaminopropyl)dimethylamino]-1-propanesulfonic acid is 10:0.2-0.5, the temperature of the isothermal reaction is 6070°C, and the time is 4-6h; the mass fraction of the calcium chloride solution is 10-15%, the mass fraction of the concentrated ammonia water is 25-28%, the concentration of the hydrogen peroxide is 25-30%, the mass ratio of the modified sepiolite, calcium chloride solution, concentrated ammonia water, and hydrogen peroxide solution is 10:150-200:60-70:80-90, the temperature of the rapid stirring reaction is 0-5°C, the time is 8-10h, and the stirring speed is 400-500r / min.

[0023] More preferably, the mass fraction of the hydrochloric acid solution is 8-10%, the heat treatment temperature is 75-80°C, and the time is 2-2.5h; the mass ratio of the pretreated sepiolite and 3-[3-(cholesterylaminopropyl)dimethylamino]-1-propanesulfonic acid is 10:0.3-0.5, the temperature of the isothermal reaction is 65-70°C, and the time is 4-5h; the mass fraction of the calcium chloride solution is 10-15%, the mass fraction of the concentrated ammonia water is 25-28%, the concentration of the hydrogen peroxide is 25-30%, the mass ratio of the modified sepiolite, calcium chloride solution, concentrated ammonia water, and hydrogen peroxide solution is 10:170-200:65-70:85-90, the temperature of the rapid stirring reaction is 0-3°C, the time is 9-10h, and the stirring speed is 450-500r / min.

[0024] Preferably, the composite carbon source in step S3 is a mixture of glucose, inulin and xylose in a mass ratio of 10:4-7:2-3.

[0025] More preferably, the composite carbon source in step S3 is a mixture of glucose, inulin and xylose in a mass ratio of 10:5-6:2.5-3.

[0026] Preferably, the nitrogen source in step S3 is one or more of corn protein, soybean meal, and yeast extract.

[0027] More preferably, the nitrogen source in step S3 is zein.

[0028] Preferably, the inoculation amount of the seed liquid in step S4 is 5-8%; the fermentation temperature is 28-30° C., the fermentation time is 7-10 days, and the rotation speed is 180-240 r / min.

[0029] More preferably, the inoculation amount of the seed liquid in step S4 is 6-8%; the fermentation temperature is 29-30° C., the fermentation time is 8-10 days, and the rotation speed is 200-240 r / min.

[0030] Preferably, the centrifugal speed in step S5 is 6000-8000 r / min, and the time is 10-15 min.

[0031] More preferably, the centrifugal speed in step S5 is 7000-8000 r / min, and the time is 10-15 min.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] (1) The method for improving the ability of a budding Aureobasidium strain to produce heavy oil by fermentation provided by the present invention comprises the following steps: adding an oxygen-releasing body to a fermentation medium to ensure the oxygen content in the medium, so that the budding Aureobasidium strain has sufficient oxygen during the fermentation process; adding n-hexane as an oxygen carrier and Tween 60 as a surfactant to improve the dispersibility of n-hexane in the fermentation medium and the oxygen transfer efficiency; and adding a composite carbon source to the fermentation medium to compound monosaccharides glucose and xylose with polysaccharide inulin to better provide a variety of nutrients for the growth of the strain; and the various component raw materials in the fermentation medium cooperate with each other to jointly promote the growth and metabolism of the budding Aureobasidium strain, thereby improving the ability of the budding Aureobasidium strain to produce heavy oil.

[0034] (2) The method provided by the present invention for improving the ability of a budding Aureobasidium strain to produce heavy oil by fermentation, wherein the oxygen-releasing body added has sepiolite as the basic skeleton, and sepiolite is a porous magnesium silicate in a fibrous form, has a high surface area and a developed internal pore structure, and has good adsorption performance. The sepiolite is first subjected to acid-heat modification to enlarge its micropore diameter and increase its specific surface area, thereby increasing its internal acid sites, which is beneficial to the subsequent reaction; then, 3-[3-(cholesterolaminopropyl)dimethylamino]-1-propanesulfonic acid is reacted with the pretreated sepiolite to introduce 3-[3-(cholesterolaminopropyl)dimethylamino]-1-propanesulfonic acid into the surface and internal micropores of the sepiolite, and 3-[3-(cholesterolaminopropyl)dimethylamino]-1-propanesulfonic acid is introduced into the surface and internal micropores of the sepiolite. As an amphoteric surfactant, it can not only make the sepiolite more evenly dispersed and allow the sepiolite to load more calcium peroxide in subsequent reactions, but also change the fluid properties of the culture medium, enhance the mass transfer of metabolites produced by A. budding B. septempunctata, and secrete heavy oil into the fermentation liquid, thereby relieving product inhibition and increasing the yield of heavy oil; finally, the modified sepiolite is added to a calcium chloride solution, reacted with concentrated ammonia water and hydrogen peroxide, and calcium peroxide is generated in situ. Compared with directly adding calcium peroxide, the in-situ generated calcium peroxide adheres to the surface and pores of the modified sepiolite with a complex structure, and can release oxygen continuously and stably; and the generated calcium hydroxide neutralizes the acidic sites inside the sepiolite, ensuring the stability of the pH of the culture, thereby promoting the metabolism of A. budding B. septempunctata and increasing the yield of heavy oil. DETAILED DESCRIPTION

[0035] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] Biological preservation information: The budding Aureobasidium melanogenum described in the present invention was deposited in the China Center for Type Culture Collection (CCTCC) on November 8, 2022, with a preservation number: CCTCC NO: M 20221742, and a classification name of Aureobasidium melanogenum XYN202201.

[0037] The sepiolite was purchased from Shijiazhuang Chenjin Mineral Products Co., Ltd., with a mesh size of 600 meshes.

[0038] A method for improving the ability of a budding Aureobasidium pullulans strain to produce heavy oil by fermentation, comprising the following steps:

[0039] S1. Activation of strains: inoculating Aureobasidium shortbacillus into an activation medium, culturing in a shaker at 26-28° C. at 150-200 r / min for 2-3 days to obtain an activated strain; wherein the activation medium is composed of, by percentage, 1-3% glucose, 1-3% peptone, 0.5-2% yeast extract, 1-4% agar, and the balance is water, and sterilizing at 110-130° C. for 20-40 min;

[0040] S2, seed liquid culture: inoculate the activated bacteria in step S1 into the seed liquid culture medium at an inoculum rate of 2-4%, and culture in a shaker at 27-29°C at 150-200r / min for 2-3 days to obtain seed liquid; wherein the composition of the seed liquid culture medium is: by percentage, 1-3% glucose, 1-3% peptone, 0.5-2% yeast extract, 0.5-1% K2HPO4, 0.1-0.3% MgSO4, and the balance is water, and sterilize at 110-130°C for 20-40min;

[0041] S3. Preparation of fermentation medium: according to percentage, 8-10% of complex carbon source, 2-4% of nitrogen source, 5-7% of oxygen-releasing body, 2-4% of n-hexane, 0.05-0.1% of K2HPO4, 0.05-0.1% of MgSO4, 0.1-0.15% of Tween 60, and the balance of water are mixed evenly and sterilized at 110-120° C. for 15-20 min to obtain fermentation medium;

[0042] S4, fermentation culture: the seed liquid in step S2 is inoculated into the fermentation medium in step S3 at an inoculum amount of 5-8%, and fermented and cultured in a shaking incubator at 28-30° C. at 180-240 r / min for 7-10 days, and a fermentation liquid is obtained after the fermentation is completed;

[0043] S5. Extraction of heavy oil: centrifuge the fermentation broth in step S4 at 6000-8000 r / min for 10-15 min, discard the supernatant, add an equal volume of ethyl acetate and mix thoroughly, centrifuge at 7000 r / min for 10 min to obtain an organic layer containing heavy oil; transfer the organic layer to a new graduated tube, repeat the above operation three times, and rotary evaporate the obtained organic layer at 75° C. to remove the solvent to obtain heavy oil, and weigh it to calculate the yield.

[0044] Wherein, the composite carbon source in step S3 is a mixture of glucose, inulin and xylose in a mass ratio of 10:4-7:2-3; the nitrogen source is one or more of corn protein, soybean meal and yeast extract;

[0045] The preparation method of the oxygen-releasing body is as follows:

[0046] 50g of sepiolite was added to 800mL of 5-10% hydrochloric acid solution, stirred and immersed at 70-80°C for 2-3h, filtered, washed and dried after treatment to obtain pretreated sepiolite; then 10g of pretreated sepiolite was added to 150mL of deionized water, followed by 0.2-0.5g 3-[3-(cholesterol aminopropyl) dimethylamino]-1-propanesulfonic acid, react at a constant temperature of 60-70°C for 4-6h, filter and dry after the reaction is completed to obtain modified sepiolite; add 10g of modified sepiolite to 150-200g of calcium chloride solution with a mass fraction of 10-15%, stir evenly to obtain a suspension, then add 60-70g of concentrated ammonia water with a mass fraction of 25-28% to the suspension, continue stirring, and after the stirring is completed, drop 80-90g of hydrogen peroxide solution with a mass fraction of 25-30%, control the temperature at 0-5°C, stir at a speed of 400-500r / min for 8-10h, filter, wash and dry after the reaction is completed to obtain the oxygen-releasing body.

[0047] The present invention will be further described below through specific examples.

[0048] Example 1

[0049] A method for improving the ability of a budding Aureobasidium pullulans strain to produce heavy oil by fermentation, comprising the following steps:

[0050] S1. Activation of strains: inoculating Aureobasidium bracteata into an activation medium, culturing in a shaker at 27° C. and 180 r / min for 2 days to obtain an activated strain; wherein the activation medium is composed of: 2% glucose, 2% peptone, 1% yeast extract, 2% agar, and the balance is water, sterilized at 121° C. for 30 min;

[0051] S2, seed liquid culture: inoculate the activated bacteria in step S1 into the seed liquid culture medium at an inoculum rate of 3%, and culture in a shaker at 28°C and 180r / min for 3 days to obtain seed liquid; wherein the composition of the seed liquid culture medium is: in percentage, 2% glucose, 2% peptone, 1% yeast extract, 0.8% K2HPO4, 0.2% MgSO4, and the balance is water, and sterilize at 121°C for 30min;

[0052] S3. Preparation of fermentation medium: 9% of composite carbon source, 3% of corn protein, 6% of oxygen-releasing body, 3% of n-hexane, 0.08% of K2HPO4, 0.08% of MgSO4, 0.13% of Tween 60, and the balance of water are mixed evenly and sterilized at 115° C. for 20 min to obtain fermentation medium;

[0053] S4, fermentation culture: the seed solution in step S2 is inoculated into the fermentation medium in step S3 at an inoculum amount of 7%, and fermented and cultured in a shaking incubator at 29° C. at 210 r / min for 9 days to obtain a fermentation broth after the fermentation is completed;

[0054] S5. Heavy oil extraction: The fermentation liquid in step S4 was centrifuged at 7000 r / min for 15 min, the supernatant was discarded, an equal volume of ethyl acetate was added and mixed thoroughly, and the mixture was centrifuged at 7000 r / min for 10 min to obtain an organic layer containing heavy oil; the organic layer was transferred to a new graduated tube, and the above operation was repeated three times, and the obtained organic layer was subjected to rotary evaporation at 75° C. to remove the solvent to obtain heavy oil, and the yield was calculated by weighing.

[0055] Wherein, the composite carbon source in step S3 is a mixture of glucose, inulin and xylose in a mass ratio of 10:7:3;

[0056] The preparation method of the oxygen-releasing body is as follows:

[0057] 50g of sepiolite was added to 800mL of 8% hydrochloric acid solution, stirred and immersed at 75°C for 2.5h, filtered, washed and dried after the treatment was completed to obtain pretreated sepiolite; then 10g of the pretreated sepiolite was added to 150mL of deionized water, followed by adding 0.4g of 3-[3-(cholesterolaminopropyl)dimethylamino]-1-propanesulfonic acid, and reacted at a constant temperature of 65°C for 5h. After the reaction was completed, filtered and dried to obtain modified sepiolite; 10g of the modified sepiolite was added to 180g of 13% calcium chloride solution, stirred evenly to obtain a suspension, then 65g of 25% concentrated ammonia water was added to the suspension, and stirring was continued. After the stirring was completed, 85g of 30% hydrogen peroxide solution was added dropwise, the temperature was controlled at 3°C, and the reaction was stirred at a speed of 450r / min for 9h. After the reaction was completed, filtered, washed and dried to obtain the oxygen-releasing body.

[0058] Example 2

[0059] A method for improving the ability of a budding Aureobasidium pullulans strain to produce heavy oil by fermentation, comprising the following steps:

[0060] S1. Activation of strains: inoculate Aureobasidium bracteata into an activation medium, and culture in a shaker at 27° C. and 170 r / min for 2 days to obtain an activated strain; wherein the activation medium is composed of: 2% glucose, 2% peptone, 1.5% yeast extract, 3% agar, and the balance is water, and sterilize at 121° C. for 30 min;

[0061] S2, seed liquid culture: inoculate the activated bacteria in step S1 into the seed liquid culture medium at an inoculum rate of 3%, and culture in a shaker at 28.5°C and 170r / min for 3 days to obtain seed liquid; wherein the composition of the seed liquid culture medium is: in percentage, 2% glucose, 2% peptone, 1.5% yeast extract, 0.7% K2HPO4, 0.2% MgSO4, and the balance is water, and sterilize at 121°C for 30min;

[0062] S3. Preparation of fermentation medium: according to percentage, 9% of composite carbon source, 2-4% of soybean meal, 6% of oxygen-releasing body, 3% of n-hexane, 0.08% of K2HPO4, 0.07% of MgSO4, 0.12% of Tween 60, and the balance of water are mixed evenly and sterilized at 115° C. for 15 min to obtain fermentation medium;

[0063] S4, fermentation culture: the seed solution in step S2 is inoculated into the fermentation medium in step S3 at a 6% inoculum amount, and fermented and cultured in a shaking incubator at 29.5° C. at 210 r / min for 8 days, and a fermentation liquid is obtained after the fermentation is completed;

[0064] S5. Extraction of heavy oil: The fermentation liquid in step S4 was centrifuged at 7000 r / min for 10 min, the supernatant was discarded, an equal volume of ethyl acetate was added and mixed thoroughly, and the mixture was centrifuged at 7000 r / min for 10 min to obtain an organic layer containing heavy oil; the organic layer was transferred to a new graduated tube, and the above operation was repeated three times, and the obtained organic layer was subjected to rotary evaporation at 75° C. to remove the solvent to obtain heavy oil, and the yield was calculated by weighing.

[0065] Wherein, the composite carbon source in step S3 is a mixture of glucose, inulin and xylose in a mass ratio of 10:5:2.5;

[0066] The preparation method of the oxygen-releasing body is as follows:

[0067] 50g of sepiolite was added to 800mL of 8% hydrochloric acid solution, stirred and immersed at 75°C for 2.5h, filtered, washed and dried after the treatment was completed to obtain pretreated sepiolite; then 10g of the pretreated sepiolite was added to 150mL of deionized water, followed by adding 0.3g of 3-[3-(cholesterolaminopropyl)dimethylamino]-1-propanesulfonic acid, and reacted at a constant temperature of 65°C for 5h. After the reaction was completed, filtered and dried to obtain modified sepiolite; 10g of the modified sepiolite was added to 180g of 10% calcium chloride solution, stirred evenly to obtain a suspension, then 65g of 28% concentrated ammonia water was added to the suspension, and stirring was continued. After the stirring was completed, 85g of 30% hydrogen peroxide solution was added dropwise, the temperature was controlled at 4°C, and the reaction was stirred at a speed of 450r / min for 9h. After the reaction was completed, filtered, washed and dried to obtain the oxygen-releasing body.

[0068] Example 3

[0069] A method for improving the ability of a budding Aureobasidium pullulans strain to produce heavy oil by fermentation, comprising the following steps:

[0070] S1. Activation of strains: inoculating Aureobasidium bracteata into an activation medium, culturing in a shaker at 26°C and 150 r / min for 3 days to obtain an activated strain; wherein the activation medium is composed of: 2% glucose, 2% peptone, 1% yeast extract, 2% agar, and the balance is water, sterilized at 121°C for 30 min;

[0071] S2, seed liquid culture: inoculate the activated bacteria in step S1 into the seed liquid culture medium at an inoculum rate of 2-4%, and culture in a shaker at 27-29°C at 150-200r / min for 2-3 days to obtain seed liquid; wherein the composition of the seed liquid culture medium is: in percentage, 2% glucose, 2% peptone, 1% yeast extract, 0.8% K2HPO4, 0.2% MgSO4, and the balance is water, and sterilize at 121°C for 30min;

[0072] S3. Preparation of fermentation medium: 8% of complex carbon source, 2% of yeast extract, 5% of oxygen-releasing body, 2% of n-hexane, 0.05% of K2HPO4, 0.05% of MgSO4, 0.1% of Tween 60, and the balance of water are mixed evenly and sterilized at 110° C. for 20 min to obtain fermentation medium;

[0073] S4, fermentation culture: the seed liquid in step S2 is inoculated into the fermentation medium in step S3 at a 5% inoculum amount, and fermented and cultured in a shaking incubator at 28° C. at 180 r / min for 7 days to obtain a fermentation liquid after the fermentation is completed;

[0074] S5. Extraction of heavy oil: The fermentation liquid in step S4 was centrifuged at 6000 r / min for 15 min, the supernatant was discarded, an equal volume of ethyl acetate was added and mixed thoroughly, and the mixture was centrifuged at 7000 r / min for 10 min to obtain an organic layer containing heavy oil; the organic layer was transferred to a new graduated tube, and the above operation was repeated three times. The obtained organic layer was subjected to rotary evaporation at 75° C. to remove the solvent to obtain heavy oil, and the yield was calculated by weighing.

[0075] Wherein, the composite carbon source in step S3 is a mixture of glucose, inulin and xylose in a mass ratio of 10:4:2;

[0076] The preparation method of the oxygen-releasing body is as follows:

[0077] 50g of sepiolite was added to 800mL of 5% hydrochloric acid solution, stirred and immersed at 70°C for 3h, filtered, washed and dried after the treatment was completed to obtain pretreated sepiolite; then 10g of the pretreated sepiolite was added to 150mL of deionized water, followed by adding 0.2g of 3-[3-(cholesterolaminopropyl)dimethylamino]-1-propanesulfonic acid, and reacted at a constant temperature of 60°C for 6h. After the reaction was completed, filtered and dried to obtain modified sepiolite; 10g of the modified sepiolite was added to 150g of 15% calcium chloride solution, stirred evenly to obtain a suspension, then 60g of 28% concentrated ammonia water was added to the suspension, and stirring was continued. After the stirring was completed, 80g of 30% hydrogen peroxide solution was added dropwise, the temperature was controlled at 0°C, and the reaction was stirred at a speed of 400r / min for 10h. After the reaction was completed, filtered, washed and dried to obtain the oxygen-releasing body.

[0078] Example 4

[0079] A method for improving the ability of a budding Aureobasidium pullulans strain to produce heavy oil by fermentation, comprising the following steps:

[0080] S1. Activation of strains: inoculating Aureobasidium bracteata into an activation medium, culturing in a shaker at 28°C and 200 r / min for 2 days to obtain an activated strain; wherein the activation medium is composed of: 2% glucose, 2% peptone, 1.5% yeast extract, 3% agar, and the balance is water, sterilized at 121°C for 30 min;

[0081] S2, seed liquid culture: inoculate the activated bacteria in step S1 into the seed liquid culture medium at a rate of 4%, and culture in a shaker at 29°C and 200 r / min for 2 days to obtain seed liquid; wherein the composition of the seed liquid culture medium is: by percentage, 2% glucose, 2% peptone, 1.5% yeast extract, 0.7% K2HPO4, 0.2% MgSO4, and the balance is water, and sterilize at 121°C for 30 minutes;

[0082] S3. Preparation of fermentation medium: according to percentage, 10% of composite carbon source, 4% of corn protein, 7% of oxygen-releasing body, 4% of n-hexane, 0.1% of K2HPO4, 0.1% of MgSO4, 0.15% of Tween 60, and the balance of water are mixed evenly and sterilized at 120° C. for 15 min to obtain fermentation medium;

[0083] S4, fermentation culture: the seed solution in step S2 is inoculated into the fermentation medium in step S3 at an inoculum amount of 8%, and fermented and cultured in a shaking incubator at 30° C. at 240 r / min for 10 days to obtain a fermentation liquid after the fermentation is completed;

[0084] S5. Heavy oil extraction: The fermentation liquid in step S4 was centrifuged at 8000 r / min for 10 min, the supernatant was discarded, an equal volume of ethyl acetate was added and mixed thoroughly, and the mixture was centrifuged at 7000 r / min for 10 min to obtain an organic layer containing heavy oil; the organic layer was transferred to a new graduated tube, and the above operation was repeated three times, and the obtained organic layer was subjected to rotary evaporation at 75° C. to remove the solvent to obtain heavy oil, and the yield was calculated by weighing.

[0085] Wherein, the composite carbon source in step S3 is a mixture of glucose, inulin and xylose in a mass ratio of 10:7:3;

[0086] The preparation method of the oxygen-releasing body is as follows:

[0087] 50g of sepiolite was added to 800mL of 10% hydrochloric acid solution, stirred and immersed at 80°C for 2h, filtered, washed and dried after the treatment was completed to obtain pretreated sepiolite; then 10g of the pretreated sepiolite was added to 150mL of deionized water, followed by adding 0.5g of 3-[3-(cholesterolaminopropyl)dimethylamino]-1-propanesulfonic acid, and reacted at 70°C for 4h. After the reaction was completed, filtered and dried to obtain modified sepiolite; 10g of the modified sepiolite was added to 200g of 10% calcium chloride solution, stirred evenly to obtain a suspension, then 70g of 25% concentrated ammonia water was added to the suspension, and stirring was continued. After the stirring was completed, 90g of 25% hydrogen peroxide solution was added dropwise, the temperature was controlled at 5°C, and the reaction was stirred at a speed of 500r / min for 8h. After the reaction was completed, filtered, washed and dried to obtain the oxygen-releasing body.

[0088] Comparative Example 1

[0089] A method for improving the ability of a budding Aureobasidium pullulans strain to produce heavy oil by fermentation, comprising the following steps:

[0090] S1. Activation of strains: inoculating Aureobasidium bracteata into an activation medium, culturing in a shaker at 27° C. and 180 r / min for 2 days to obtain an activated strain; wherein the activation medium is composed of: 2% glucose, 2% peptone, 1% yeast extract, 2% agar, and the balance is water, sterilized at 121° C. for 30 min;

[0091] S2, seed liquid culture: inoculate the activated bacteria in step S1 into the seed liquid culture medium at an inoculum rate of 3%, and culture in a shaker at 28°C and 180r / min for 3 days to obtain seed liquid; wherein the composition of the seed liquid culture medium is: in percentage, 2% glucose, 2% peptone, 1% yeast extract, 0.8% K2HPO4, 0.2% MgSO4, and the balance is water, and sterilize at 121°C for 30min;

[0092] S3. Preparation of fermentation medium: 9% of composite carbon source, 3% of corn protein, 3% of n-hexane, 0.08% of K2HPO4, 0.08% of MgSO4, 0.13% of Tween 60, and the balance of water were mixed evenly and sterilized at 115° C. for 20 min to obtain a fermentation medium;

[0093] S4, fermentation culture: the seed solution in step S2 is inoculated into the fermentation medium in step S3 at an inoculum amount of 7%, and fermented and cultured in a shaking incubator at 29° C. at 210 r / min for 9 days to obtain a fermentation broth after the fermentation is completed;

[0094] S5. Heavy oil extraction: The fermentation liquid in step S4 was centrifuged at 7000 r / min for 15 min, the supernatant was discarded, an equal volume of ethyl acetate was added and mixed thoroughly, and the mixture was centrifuged at 7000 r / min for 10 min to obtain an organic layer containing heavy oil; the organic layer was transferred to a new graduated tube, and the above operation was repeated three times, and the obtained organic layer was subjected to rotary evaporation at 75° C. to remove the solvent to obtain heavy oil, and the yield was calculated by weighing.

[0095] Wherein, the composite carbon source in step S3 is a mixture of glucose, inulin and xylose in a mass ratio of 10:7:3.

[0096] Comparative Example 2

[0097] A method for improving the ability of a budding Aureobasidium pullulans strain to produce heavy oil by fermentation, comprising the following steps:

[0098] S1. Activation of strains: inoculating Aureobasidium bracteata into an activation medium, culturing in a shaker at 27° C. and 180 r / min for 2 days to obtain an activated strain; wherein the activation medium is composed of: 2% glucose, 2% peptone, 1% yeast extract, 2% agar, and the balance is water, sterilized at 121° C. for 30 min;

[0099] S2, seed liquid culture: inoculate the activated bacteria in step S1 into the seed liquid culture medium at an inoculum rate of 3%, and culture in a shaker at 28°C and 180r / min for 3 days to obtain seed liquid; wherein the composition of the seed liquid culture medium is: in percentage, 2% glucose, 2% peptone, 1% yeast extract, 0.8% K2HPO4, 0.2% MgSO4, and the balance is water, and sterilize at 121°C for 30min;

[0100] S3. Preparation of fermentation medium: 9% of composite carbon source, 3% of corn protein, 6% of oxygen-releasing body, 3% of n-hexane, 0.08% of K2HPO4, 0.08% of MgSO4, 0.13% of Tween 60, and the balance of water are mixed evenly and sterilized at 115° C. for 20 min to obtain fermentation medium;

[0101] S4, fermentation culture: the seed solution in step S2 is inoculated into the fermentation medium in step S3 at an inoculum amount of 7%, and fermented and cultured in a shaking incubator at 29° C. at 210 r / min for 9 days to obtain a fermentation broth after the fermentation is completed;

[0102] S5. Heavy oil extraction: The fermentation liquid in step S4 was centrifuged at 7000 r / min for 15 min, the supernatant was discarded, an equal volume of ethyl acetate was added and mixed thoroughly, and the mixture was centrifuged at 7000 r / min for 10 min to obtain an organic layer containing heavy oil; the organic layer was transferred to a new graduated tube, and the above operation was repeated three times, and the obtained organic layer was subjected to rotary evaporation at 75° C. to remove the solvent to obtain heavy oil, and the yield was calculated by weighing.

[0103] Wherein, the composite carbon source in step S3 is a mixture of glucose, inulin and xylose in a mass ratio of 10:7:3;

[0104] The preparation method of the oxygen-releasing body is as follows:

[0105] 50 g of sepiolite was added into 800 mL of 8% hydrochloric acid solution, and stirred and immersed at 75° C. for 2.5 h. After the treatment, the solution was filtered, washed and dried to obtain the pretreated sepiolite. 10 g of the pretreated sepiolite was evenly mixed with 5 g of calcium peroxide to obtain the oxygen-releasing body.

[0106] The heavy oil yields in Examples 1-4 and Comparative Examples 1-2 were calculated, and the results are shown in Table 1:

[0107] Table 1

[0108] Heavy oil production (g / L) Example 1 52.37 Example 2 49.52 Example 3 47.89 Example 4 50.71 Comparative Example 1 40.26 Comparative Example 2 42.63

[0109] As can be seen from Table 1 above, the method for improving the ability of Aureobasidium pullulans strain to produce heavy oil by fermentation provided by the present invention can enable Aureobasidium pullulans to metabolize and proliferate better, thereby improving its ability to produce heavy oil, and has good application prospects.

[0110] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for improving the ability of Aureobasidium pullulans strain to produce heavy oil by fermentation, characterized in that: The following steps are involved: S1. Activation of bacterial strains: inoculating Aureobasidium pullulans into an activation medium and culturing at a constant temperature to obtain activated bacterial strains; S2, seed liquid culture: inoculating the activated bacteria in step S1 into a seed liquid culture medium, performing seed culture, and obtaining a seed liquid; S3. Preparation of fermentation medium: according to percentage, 8-10% of complex carbon source, 2-4% of nitrogen source, 5-7% of oxygen-releasing body, 2-4% of n-hexane, 0.05-0.1% of K2HPO4, 0.05-0.1% of MgSO4, 0.1-0.15% of Tween 60, and the balance of water are mixed evenly and sterilized at 110-120° C. for 15-20 min to obtain fermentation medium; S4, fermentation culture: inoculating the seed liquid in step S2 into the fermentation medium in step S3, and fermenting and culturing, and obtaining a fermentation liquid after the fermentation is completed; S5, heavy oil extraction: centrifuge the fermentation liquid in step S4, discard the supernatant, add an equal volume of ethyl acetate and mix thoroughly, centrifuge at 7000r / min for 10min, transfer the obtained organic layer containing heavy oil to a new graduated tube, repeat the operation three times, and rotary evaporate the obtained organic layer solution to remove the solvent to obtain heavy oil, and weigh it to calculate the yield; Wherein, the preparation method of the oxygen-releasing body in step S3 is as follows: The sepiolite is added to a hydrochloric acid solution for heat treatment, and after the treatment is completed, it is filtered, washed, and dried to obtain a pretreated sepiolite; then the pretreated sepiolite is added to deionized water, and then 3-[3-(cholesterylaminopropyl)dimethylamino]-1-propanesulfonic acid is added to react at a constant temperature, and after the reaction is completed, it is filtered and dried to obtain a modified sepiolite; The modified sepiolite is added to the calcium chloride solution, stirred evenly to obtain a suspension, then concentrated ammonia water is added to the suspension, and stirring is continued. After the stirring is completed, a hydrogen peroxide solution is added dropwise, the temperature is controlled to quickly stir the reaction, and after the reaction is completed, the oxygen-releasing body is filtered, washed, and dried to obtain the oxygen-releasing body; the mass fraction of the hydrochloric acid solution is 5-10%, the heat treatment temperature is 70-80°C, and the time is 2-3h; the mass ratio of the pretreated sepiolite and 3-[3-(cholesterylaminopropyl)dimethylamino]-1-propanesulfonic acid is 10:0.2-0 .5, the temperature of the isothermal reaction is 60-70°C, the time is 4-6h; the mass fraction of the calcium chloride solution is 10-15%, the mass fraction of the concentrated ammonia water is 25-28%, the concentration of the hydrogen peroxide is 25-30%, the mass ratio of the modified sepiolite, calcium chloride solution, concentrated ammonia water, and hydrogen peroxide solution is 10:150-200:60-70:80-90, the temperature of the rapid stirring reaction is 0-5°C, the time is 8-10h, and the stirring speed is 400-500r / min; In step S3, the composite carbon source is a mixture of glucose, inulin and xylose in a mass ratio of 10:4-7:2-3; the nitrogen source is one or more of corn protein, soybean meal and yeast extract.

2. The method according to claim 1, characterized in that The constant temperature culture conditions in step S1 are culturing in a shaker at 26-28° C. and 150-200 r / min for 2-3 days; the composition of the activation culture medium is: in percentage, 1-3% glucose, 1-3% peptone, 0.5-2% yeast extract, 1-4% agar, and the balance is water, and sterilized at 110-130° C. for 20-40 minutes.

3. The method according to claim 1, characterized in that The composition of the seed liquid culture medium in step S2 is: in percentage, 1-3% glucose, 1-3% peptone, 0.5-2% yeast extract, 0.5-1% K2HPO4, 0.1-0.3% MgSO4, and the balance is water, and it is sterilized at 110-130°C for 20-40min.

4. The method according to claim 1, characterized in that The inoculation amount of the activated bacteria in step S2 is 2-4%; the seed culture conditions are cultured in a shaker at 27-29° C. and 150-200 r / min for 2-3 days.

5. The method according to claim 1, characterized in that The inoculation amount of the seed liquid in step S4 is 5-8%; the fermentation temperature is 28-30° C., the fermentation time is 7-10 days, and the rotation speed is 180-240 r / min.

6. The method according to claim 1, characterized in that The centrifugal speed in step S5 is 6000-8000 r / min, and the time is 10-15 min.