Candida globosa LSH 221, application thereof and method for producing sophorolipid

Abstract

The invention provides candida utilis LSH 221, application thereof and a method for producing sophorolipid. The candida utilis LSH 221 is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC 34437. The candida globosa LSH 221 has high sophorolipid preparation capacity, significantly improves the sophorolipid yield, has excellent genetic stability, and can maintain high sophorolipid production capacity in continuous subculture.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biotechnology, and particularly relates to a Starmerella bombicola LSH 221 and application thereof, and a method for producing sophorolipid. BACKGROUND

[0002] Sophorolipid can be used as a glycolipid biosurfactant. Due to its unique molecular structure and environmental friendly characteristics, sophorolipid has shown a broad application prospect in the fields of oil exploitation, environmental remediation, medicine, daily chemical, food industry and agriculture. In addition, sophorolipid also shows significant antibacterial, anti-inflammatory and anticancer activities, such as inducing cancer cell apoptosis pathway or inhibiting pathogenic microorganism biofilm formation, which provides a scientific basis for its application in the field of medicine.

[0003] However, the traditional sophorolipid production technology faces multiple bottlenecks. First, the synthesis efficiency of wild strains is limited, second, in industrial production, the strain genetic stability, fermentation process amplification effect and product batch consistency problems are faced. The limitations of traditional sophorolipid production technology also exist in the environmental and cost pressure. Although the traditional chemical surfactant is low in price, it has ecological toxicity and is difficult to degrade, while the natural properties of sophorolipid are in line with the green manufacturing trend, but its production cost is still higher than that of chemical products, and it is urgent to realize cost reduction and efficiency increase through strain optimization.

[0004] In summary, developing sophorolipid production strains with high yield, stability and easy industrial amplification not only can meet the demand for high-performance biosurfactants in multiple fields, but also is a key breakthrough point to promote the industrialization of synthetic biology technology and help achieve the goal of carbon neutralization. SUMMARY

[0005] The present application provides a Starmerella bombicola LSH 221 and application thereof, and a method for producing sophorolipid. The Starmerella bombicola LSH 221 has high sophorolipid preparation capacity, significantly improves the sophorolipid yield, and has excellent genetic stability characteristics, and can maintain high sophorolipid production capacity in continuous subculture.

[0006] According to one aspect of the present application, a Starmerella bombicola LSH 221 is provided. The Starmerella bombicola LSH 221 is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC 34437.

[0007] The *Candida globosa* strain LSH 221 provided in this solution is a novel and highly efficient sophorolipid synthesizing strain with high sophorolipid production capacity, significantly improving sophorolipid yield and enhancing the biosynthetic pathway of lactone-type sophorolipids. High sophorolipid yield directly reduces the cost of raw materials, energy consumption, and fermentation time per unit product, making sophorolipids more price-competitive with chemically synthesized surfactants and accelerating their substitution in bulk markets such as detergents and oil extraction. Furthermore, the genetic stability of *Candida globosa* LSH 221 allows it to maintain high sophorolipid production capacity during continuous subculturing. The stable expression characteristics of *Candida globosa* LSH 221 reduce batch production fluctuations, ensuring product quality uniformity and meeting the stringent requirements for raw material consistency in high-end fields such as pharmaceuticals and cosmetics, providing a reliable guarantee for industrial continuous fermentation.

[0008] According to another aspect of the present invention, the use of the above-mentioned Candida albicans LSH 221 in the preparation of sophorolipids is provided.

[0009] According to another aspect of the present invention, a method for producing sophorolipids is provided, comprising the following operations: fermentation with Candida albicans LSH 221, which is deposited at the China General Microbiological Culture Collection Center with accession number CGMCC 34437.

[0010] Preferably, the fermentation process includes the following steps: *Candida globosa* LSH 221 is mixed with a fermentation medium to obtain a fermentation broth. The fermentation medium includes oleic acid at a concentration of 50-70 g / L, glucose at a concentration of 50-70 g / L, and yeast extract at a concentration of 2-4 g / L. Based on the sophorolipid production method using *Candida globosa* LSH 221, the synthesis efficiency and product quality are simultaneously improved through synergistic optimization of strain characteristics and fermentation medium composition. By adjusting the content of oleic acid and glucose, the synergistic effect of carbon and nitrogen sources significantly improves the synthesis efficiency of sophorolipids. High-concentration oleic acid, as the main carbon source, directly provides the precursor for sophorolipid synthesis, while glucose and yeast extract indirectly enhance metabolic activity by promoting cell proliferation. This optimizes the fermentation environment of *Candida globosa* LSH 221, thereby directionally increasing the yield of lactone-type sophorolipids.

[0011] Preferably, the fermentation medium also includes potassium dihydrogen phosphate (KH₂PO₄), disodium hydrogen phosphate (Na₂HPO₄), and magnesium sulfate (MgSO₄). By adding these salts to the fermentation medium, the ion concentration and buffer system are optimized, further enhancing the synthesis capacity of sophorolipids. Specifically, in a fermentation medium system with high oleic acid concentration, phosphates are used to maintain the pH stability of the fermentation broth and avoid the accumulation of acidic byproducts, while MgSO₄... 2+ It can improve substrate conversion efficiency.

[0012] Preferably, the fermentation medium comprises KH2PO4 at a concentration of 0.5-1.5 g / L, Na2HPO4·12H2O at a concentration of 0.5-1.5 g / L, and MgSO4·7H2O at a concentration of 0.2-1.8 g / L.

[0013] Preferably, the amount of added LSH 221 is 1-3%. Based on the fermentation medium system with high oleic acid concentration, the inoculation amount of LSH 221 falls within the above range, which can balance the growth of the bacterial body and product synthesis. If the inoculation amount is too low, the fermentation period will be longer, and if the inoculation amount is too high, it will be easy to inhibit sophorolipid accumulation due to nutrient competition. When the inoculation amount of LSH 221 falls within the above range, LSH 221 is in the exponential growth phase in the fermentation medium, and the biomass and sophorolipid yield are greatly better than the preferred ratio, which can also reduce the residual glucose content in the fermentation broth and improve the substrate utilization rate.

[0014] Preferably, during the fermentation process, the fermentation temperature is 25-35°C, and the aeration amount is 1.3-1.5 m 3 / h. Based on the physicochemical properties of LSH 221, the fermentation temperature and aeration amount are adjusted to meet the above range, and the physical parameter control of the fermentation conditions is realized to maximize the sophorolipid production efficiency, and the specific fermentation temperature and aeration amount make LSH 221 have high activity.

[0015] Preferably, during the fermentation process, oleic acid is supplemented to the fermentation medium. The strategy of supplementing oleic acid during the fermentation process effectively prolongs the high-yield period and maintains product specificity. This design effectively reduces metabolic diversion caused by substrate depletion by continuously supplying oleic acid, while improving the efficiency of sophorolipid production.

[0016] Preferably, during the fermentation process, the fermentation time is at least 168 hours.

[0017] Preferably, during the fermentation process, when the fermentation is 96 hours, oleic acid is supplemented to the fermentation medium.

[0018] Preferably, during the fermentation process, when the fermentation is 144 hours, oleic acid is supplemented to the fermentation medium.

[0019] According to another aspect of the present application, a method for subculturing the above-mentioned LSH 221 is provided, which comprises the following steps: inoculating LSH 221 into a seed medium for culture, and the seed medium comprises glucose and proteose peptone in a mass ratio of 0.9-1.1:0.9-1.1. Based on the physicochemical properties of LSH 221, the composition of the seed medium is adjusted to optimize and ensure the genetic stability of the strain.

[0020] Preferably, the seed culture medium comprises glucose at a concentration of 15-25 g / L, and peptone at a concentration of 15-25 g / L.

[0021] Preferably, the seed culture medium further comprises yeast powder.

[0022] Preferably, the seed culture medium comprises yeast powder at a concentration of 5-15 g / L.

[0023] Preferably, the seed culture medium comprises a liquid seed culture medium and a solid seed culture medium.

[0024] Preferably, the solid seed culture medium further comprises agar.

[0025] Preferably, the inoculation amount of Starmerella bombicola LSH 221 is 2%. By controlling the inoculation amount of Starmerella bombicola LSH 221 in the process of subculture, metabolic differences caused by random inoculation are avoided, process repeatability is improved, the fluctuation range of sophorolipid yield among offspring is reduced, and a uniform and stable seed liquid source is provided for large-scale production. DETAILED DESCRIPTION

[0026] In order for those skilled in the art to better understand the technical solutions in the present application, the technical solutions of the present application will be described clearly and completely below in combination with the drawings of the embodiments and examples of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor should fall within the scope of protection of the present application.

[0027] The experimental reagents and instruments designed in the examples and comparative examples of the present application are all common reagents and instruments unless otherwise specified, and can be obtained from commercial channels. In the examples and comparative examples, the experimental methods used are all conventional methods unless otherwise specified, and the raw materials used in parallel experiments are the same batch of raw materials unless otherwise specified.

[0028] The raw materials used in the examples are described as follows, but are not limited to these materials: Reagents: Physiological saline: 0.9 g of sodium chloride is dissolved in 100 ml of distilled water, and sterilized at 121℃ for 20 min.

[0029] 5% glycerol-physiological saline solution: prepare a 10% glycerol aqueous solution, then add an equal amount of physiological saline, stir uniformly, and sterilize at 121℃ for 20 min.

[0030] Instruments: Electronic balance, ultrasonic cleaner, vertical pressure steam sterilizer, constant temperature incubation oscillator, ion beam biological engineering device (LF-50C), double person single surface purification workbench, frozen high-speed centrifuge, biochemical incubator, ultraviolet spectrophotometer, high performance liquid chromatograph, surface tension instrument, enzyme marker, constant temperature water bath, SBA-40C biological sensor.

[0031] Example 1 Preparation of Starmerella bombicola LSH 221 Step one: preparation of culture medium Seed culture medium (YEPD) includes liquid seed culture medium and solid seed culture medium.

[0032] The liquid seed culture medium includes glucose with a concentration of 20.0 g / L, proteose peptone with a concentration of 20.0 g / L, and yeast powder with a concentration of 10.0 g / L, wherein the yeast powder provides the nitrogen source required for the growth of the strain. The preparation method of the liquid seed culture medium includes mixing the raw materials for preparing the liquid seed culture medium, sterilizing in a 121℃ environment for 20 minutes, and preparing the liquid seed culture medium.

[0033] The solid seed culture medium includes glucose with a concentration of 20.0 g / L, proteose peptone with a concentration of 20.0 g / L, yeast powder with a concentration of 10.0 g / L, and agar with a concentration of 20.0 g / L. The preparation method of the liquid seed culture medium includes mixing the raw materials for preparing the solid seed culture medium, sterilizing in a 121℃ environment for 20 minutes, pouring an appropriate amount of the solid seed culture medium into a culture dish while it is hot, and allowing it to solidify, thereby preparing the solid seed culture medium.

[0034] The fermentation culture medium includes oleic acid with a volume fraction of 60%, glucose with a concentration of 60.0 g / L, yeast powder with a concentration of 3.0 g / L, KH2PO4 with a concentration of 1.0 g / L, Na2HPO4·12H2O with a concentration of 1.0 g / L, and MgSO4·7H2O with a concentration of 0.5 g / L.

[0035] Step two: preparation of Starmerella bombicola LSH 221 Starmerella bombicola CGMCC 1576 strain as the starting strain, Starmerella bombicola LSH 221 was prepared.

[0036] (1) Preparation of bacterial suspension Take 1 mL containing the starting strain of liquid seed stored in 4 ℃ environment, inoculate the liquid seed in the liquid seed culture medium for activation, centrifuge at 180 rpm, and cultivate at 30 ℃ for 10 hours, so that the starting strain reaches the logarithmic growth phase. Take a proper amount of activated starting strain, wash twice with 5% glycerol-saline solution to obtain a bacterial suspension. Take 5% glycerol-saline solution as a blank control, measure the OD value (λ=600 nm) of the bacterial suspension, and dilute the bacterial suspension with 5% glycerol-saline solution to control the OD value of the bacterial suspension between 0.6 and 0.8.

[0037] (2) Low-energy ion beam implantation mutagenesis S1. Mutagenesis preliminary experiment Ion beam implantation was carried out in the Key Laboratory of Ion Beam Bioengineering of the Institute of Plasma Physics, Chinese Academy of Sciences. Take 100 μL of bacterial suspension and spread it on a 90 mm flat dish, blow for about 30 min on a clean bench, and then dry and perform ion implantation. Natural and vacuum controls were set. Implantation parameters: energy 15 ke V N + , target chamber vacuum degree about 10 -3 Pa, implantation dose 769, 1538, 2308, 3077, 3846, 4615, 5385, 6154 units, i.e. 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0×1015 ions / cm 2 , two dishes as a group, one for the experimental group and one for the vacuum control group, to exclude the influence of vacuum on cell survival rate. After implantation, immediately elute the bacterial film with 1.0 mL of saline and place it in a 2.0 ml EP tube. A total of 16 samples were obtained.

[0038] S2. Draw survival rate curve The eluted bacterial film of the experimental and control groups was diluted with saline to 10 3 times, and 100 μL of each sample was spread on the solid seed culture medium, with 3 parallel samples for each dilution. After 30 ℃ cultivation for 4 days, colony counting was performed, and the average value of 3 parallel samples was taken. The relative survival rate was obtained by comparing each dose experimental group with the corresponding vacuum control group. According to the N + implantation dose and relative survival rate, curve fitting was performed to obtain the survival rate curve of the strain after ion implantation. According to the survival rate curve, the implantation dose with a survival rate in the "saddle part" was selected for large-scale mutagenesis experiment.

[0039] S3. Culture of mutagenized strains Take 100 μL mutagenic elution of bacteria on solid seed medium for coating, randomly select single colonies on solid seed medium containing mutant strains after constant temperature culture at 30°C for 2 days, respectively store on agar slant formed by solid seed medium, then carry out primary screening and rescreening on mutant strains. Among them, the mutant strains include Trichococcus sp. LSH 221.

[0040] (3) Screening to obtain Trichococcus sp. LSH 221 S1. Primary screening: Use a sterile toothpick to pick single colonies of the starting strain and the mutant strain on the solid seed medium into a 96-deep-well plate (seed plate) (400ul of seed medium per well), transfer to the culture well and shake gently several times, discard the toothpick, repeat in turn until the selection is completed, and set two negative control wells without adding bacteria at the same time. Cover with a sandwich cover plate and fix on a shaker, 30°C, 180 r / min, culture for 10h.

[0041] Remove 50µL of bacteria and transfer to the corresponding 48-deep-well plate (malonic acid stress seed plate) for culture (1mL of seed medium per well), and culture at 30°C, 180r / min for 10h. Use a 96-well transparent enzyme-coated plate to take the cultured seed liquid and measure the OD600 value. Select the seed liquid with high OD600 value from the 48-deep-well plate for deep-well plate rescreening of mutant strains, and store the 48 seed plates in a 4°C refrigerator for future use.

[0042] S2. Rescreening: Inoculate the 48-deep-well plate (seed plate) liquid seed into a 24-deep-well plate (fermentation plate) at an inoculation amount of 2% (v / v) for culture (1.2 mL of fermentation medium per well), set 3 parallel controls for each strain, and culture at 30°C, 180r / min for 7 days. Select mutant strains that produce sophorolipids for shake flask rescreening.

[0043] S3. Shake flask rescreening: Determine the seed liquid of mutant strains that can produce sophorolipids through deep-well plate rescreening, take the seed liquid from the 48-deep-well plate (seed plate), inoculate the seed liquid into a 250mL flask containing 50mL of liquid seed medium at an inoculation amount of 2%, and culture at 30°C, 180r / min for 10 days to obtain activated liquid seed.

[0044] Inoculate the activated seed liquid into a 250mL flask containing 50mL of fermentation medium at an inoculation amount of 2%, set 3 parallel controls, and culture at 30°C, 180r / min for 7 days.

[0045] The lactone sophorolipid and total sophorolipid content in the fermentation broth, biomass, residual glucose content, and the negative mutation rate and positive mutation rate of the strain were determined, and the Trichosporonoides globosa LSH 221 was screened. Among them, the definition of positive mutation and negative mutation is: compared with the starting strain, the yield of lactone sophorolipid, acid sophorolipid or total sophorolipid is increased by more than 10% which belongs to positive mutation, or the yield of lactone sophorolipid, acid sophorolipid or total sophorolipid is decreased by more than 10% which belongs to negative mutation. Positive mutation rate = sophorolipid yield increase rate, negative mutation rate = sophorolipid yield decrease rate.

[0046] (4) Determination items 1. Sophorolipid yield: Lactone sophorolipid yield: 0.5 mL of fermentation broth was added to 1 mL of ethyl acetate, mixed and stood for 5 min, centrifuged at 10000 rpm for 10 min, and the content of lactone sophorolipid in the ethyl acetate layer was determined by anthrone method.

[0047] Total sophorolipid yield: 0.5 mL of fermentation broth was added to 1 mL of ethanol, mixed and centrifuged at 10000 rpm for 10 min, and the total sugar concentration in the supernatant and the residual amount of glucose in the supernatant were determined by anthrone method, and then the total sophorolipid yield was calculated. Total sophorolipid yield = (total sugar concentration in supernatant - residual amount of glucose in supernatant) x (M 槐糖 / M 葡萄糖 ), wherein M 槐糖 is the molar mass of sophorose, and M 葡萄糖 is the molar mass of glucose.

[0048] 2. Biomass: According to the volume ratio, n-butanol: ethanol: chloroform = 10:10:1 was calculated to obtain the extraction liquid. 5 mL of fermentation broth was added to 5 mL of extraction liquid, mixed with a vortex mixer, centrifuged at 8000 rpm for 10 min, washed with distilled water for 2 times, dried to constant weight, and the weight was recorded as biomass.

[0049] 3. Residual glucose content: The fermentation broth was diluted with water to a measurable multiple, and the residual amount of glucose in the diluted fermentation broth was determined using a SBA-40C biosensor, and the residual amount of glucose in the undiluted fermentation broth was calculated.

[0050] (5) The test results are shown in Table 1.

[0051] Table 1. Test performance of Trichosporonoides globosa LSH 221 and starting strain

[0052] Note: The improvement rate refers to the improvement rate of the performance index measured by P. bombicola LSH 221 compared with the performance index measured by the starting strain. For example, the improvement rate of the production of lactone sophorolipids = (the production of lactone sophorolipids by P. bombicola LSH 221 - the production of lactone sophorolipids by the starting strain) / the production of lactone sophorolipids by the starting strain x 100%.

[0053] Result analysis: As can be seen from the test data shown in Table 1, P. bombicola LSH 221 has a significant performance advantage in sophorolipid production compared with the starting strain (Starmerella bombicola CGMCC 1576). In terms of sophorolipid synthesis capacity, the production of lactone sophorolipids by P. bombicola LSH 221 reached 32.61 ± 1.26 g / L, which was 61.49% higher than that of the starting strain (21.97 ± 2.64 g / L). Compared with the starting strain, the total sophorolipid yield obtained by using P. bombicola LSH 221 for fermentation increased by as much as 86.35%.

[0054] In addition, it should be noted that biomass represents the dry weight of the bacterial cells. In the early stage of fermentation, the biomass gradually increases with time, and then remains basically unchanged. The bacterial cells grow and consume glucose, and the glucose content decreases with the increase of the bacterial cells. Therefore, the higher the biomass and the lower the glucose content, the faster the bacterial cells grow and reproduce. As can be seen from the data shown in Table 1, the biomass of P. bombicola LSH 221 is slightly lower than that of the starting strain, and the residual glucose content is higher, indicating that P. bombicola LSH 221 directs more carbon sources to sophorolipid synthesis rather than bacterial cell proliferation, verifying its high-efficiency metabolic reconstitution characteristics. This "low biomass, high product output" mode highlights the success of P. bombicola LSH 221 in strengthening specific metabolic pathways.

[0055] Example 2: Genetic stability analysis (1) Genetic stability analysis of P. bombicola LSH 221: Step one: subculture P. bombicola LSH 221 was inoculated in a 250 mL triangular flask containing 50 mL of liquid seed culture medium at an inoculation amount of 2%, and cultured at 30°C and 180 r / min for 10 hours (as the first generation of subculture). Then, an appropriate amount of seed liquid after the first generation of subculture was diluted with physiological saline to two concentration gradients of 100,000 times and 1,000,000 times, respectively, and 100 μL of the diluted liquid was spread on the solid seed culture medium and incubated at 30°C.

[0056] After single colonies were grown on solid seed medium, single colonies were collected and transferred to a 250 mL flask containing 50 mL of liquid seed medium and incubated at 30°C, 180 r / min for 10 hours (as the second generation of subculture).

[0057] The above process was repeated 5 times until the fifth subculture of Candida curvata LSH 221 was obtained.

[0058] Step two: fermentation The fifth subculture of Candida curvata LSH 221 was inoculated into fermentation medium at an inoculation amount of 2%, and incubated at 30°C, 180 r / min for 7 days.

[0059] (2) Genetic stability analysis of the starting strain: Step one: refer to the subculture operation steps of Candida curvata LSH 221, the difference is that the same concentration of the starting strain is used instead of Candida curvata LSH 221.

[0060] Step two: refer to the fermentation steps of Candida curvata LSH 221, the difference is that the same concentration of the starting strain is used instead of Candida curvata LSH 221.

[0061] (3) Test items 1. sophorolipid yield: Lactone-type sophorolipid yield: the test method of lactone-type sophorolipid yield in this example is strictly the same as the test method of lactone-type sophorolipid yield in Example 1.

[0062] Total sophorolipid yield: the test method of total sophorolipid yield in this example is strictly the same as the test method of total sophorolipid yield in Example 1.

[0063] 2. Biomass: the test method of biomass in this example is strictly the same as the test method of biomass in Example 1.

[0064] 3. Residual glucose content: the test method of residual glucose content in this example is strictly the same as the test method of residual glucose content in Example 1.

[0065] (4) Test results: the genetic stability test results of Candida curvata LSH 221 are shown in Table 2; the genetic stability test results of the starting strain are shown in Table 3.

[0066] Table 2. Genetic stability test results of Candida curvata LSH 221

[0067] Note: Genetic stability refers to the change of performance indicators measured by the fifth generation compared with the first generation strain, such as the retention rate of lactone sophorolipid yield = (lactone sophorolipid yield of the fifth generation strain / lactone sophorolipid yield of the first generation strain x 100%).

[0068] Table 3. Genetic stability test results of the starting strain

[0069] Note: Genetic stability refers to the change of performance indicators measured by the fifth generation compared with the first generation strain, such as the retention rate of lactone sophorolipid yield = (lactone sophorolipid yield of the fifth generation strain / lactone sophorolipid yield of the first generation strain x 100%).

[0070] Result analysis: From the test data shown in Table 2, through the genetic stability test of five generations of continuous subculture, the Candida bombicola LSH 221 showed excellent industrial applicability. The total sophorolipid yield (68.13 ± 2.46 g / L) and lactone yield (35.48 ± 1.23 g / L) of the fifth generation of Candida bombicola LSH 221 were 109.25% and 108.80% respectively compared with the first generation of Candida bombicola LSH 221, indicating that its high-yield trait can be stably inherited. This characteristic provides a core guarantee for industrial continuous fermentation, which can greatly reduce the risk of strain degradation in the production process. From the test data shown in Table 3, the total sophorolipid and lactone yield retention rate of the starting strain after five generations of subculture was only 100.52% and 99.00%.

[0071] Example 3 Large-scale fermentation (1) Large-scale fermentation In a 100 L fermenter containing 80 L fermentation medium, Candida bombicola LSH 221 was added at an amount of 2%. The fermentation was carried out at a fermentation temperature of 30℃, a rotation speed of 37.1 HZ, and a ventilation volume of 1.3~1.5 m 3 / h for 168 hours. At 96 hours of fermentation and 144 hours of fermentation, oleic acid was added to the fermenter, and the volume of added oleic acid was 2% of the initial fermentation liquid. During the entire fermentation period, samples were collected at different times to determine the content of lactone sophorolipid, total sophorolipid, biomass, residual glucose, and pH value.

[0072] (2) Test items 1. Sophorolipid yield: The test method of lactone sophorolipid yield in this embodiment is strictly the same as the test method of lactone sophorolipid yield in Example 1.

[0073] Total sophorolipid yield: The testing method of total sophorolipid yield in this example is strictly consistent with the testing method of total sophorolipid yield in Example 1.

[0074] 2, Biomass: The testing method of biomass in this example is strictly consistent with the testing method of biomass in Example 1.

[0075] 3, Residual glucose content: The testing method of residual glucose content in this example is strictly consistent with the testing method of residual glucose content in Example 1.

[0076] (4) Test results: The large-scale fermentation test results of Starmerella bombicola LSH 221 are shown in Table 4.

[0077] Table 4. Large-scale fermentation test results of Starmerella bombicola LSH 221

[0078] Result analysis: In the scale-up experiment of 100 L fermenter, Starmerella bombicola LSH 221 showed strong process adaptability, with total sophorolipid yield of 224.31 ± 8.02 g / L and lactonic sophorolipid yield of 116.39 ± 6.48 g / L, confirming that Starmerella bombicola LSH 221 still maintains high-efficiency substrate conversion ability under large-scale culture. This result breaks through the yield bottleneck of traditional sophorolipid fermentation, and the proportion of lactonic in the product is stable, laying a solid foundation for industrial mass production.

[0079] Example 4 Fermentation condition optimization Experimental group 1 In this experimental group, Starmerella bombicola LSH 221 was used for fermentation, and the specific activation and fermentation operations in this experimental group were strictly consistent with those in Example 1. In addition, the raw materials, seed medium and fermentation medium used in this experimental group were strictly consistent with those used in Example 1.

[0080] In this example, Starmerella bombicola LSH 221 was used for fermentation with fermentation conditions as variables. The specific conditions of product numbers and variables are shown in Table 5. In this experimental group, except for the variables shown in Table 5, the other raw material ratios and preparation methods were strictly consistent with those in experimental group 1.

[0081] Table 5. Specific conditions of product numbers and corresponding variables in Example 4

[0082] Table 6. Test results of each product in Example 4

[0083] Results analysis: The above experimental groups 1-13 are divided into three groups according to the variable type, wherein the variables between experimental groups 1-5 are the content of oleic acid in the fermentation medium, which is divided into one group; the variables between experimental groups 1, 6-9 are the fermentation time, which is divided into one group; the variables between experimental groups 1, 10-13 are the inoculum size, which is divided into one group.

[0084] In the group formed by experimental groups 1-5, it can be found that with the increase of the content of oleic acid in the fermentation medium, the yield of sophorolipids shows a trend of first increasing and then decreasing, and adding more oleic acid has an adverse effect on the production of sophorolipids. High concentration of fatty acids can inhibit the production of NADPH, leading to the decrease of hydroxy fatty acid synthesis, thereby inhibiting the production of sophorolipids. Compared with the yield of sophorolipids in experimental groups 2 and 5, the yield of sophorolipids in experimental groups 1, 3-4 is higher. That is, when using Malassezia globosa LSH 221 to produce sophorolipids, using 50-70 g / L of oleic acid content in the fermentation medium can obtain higher yield of sophorolipids.

[0085] In the group formed by experimental groups 1, 6-9, it can be found that with the increase of fermentation time, the yield of sophorolipids shows a trend of first increasing and then stabilizing. The yield of sophorolipids is 62.36 g / L when fermented for 7 days, and the highest yield of sophorolipids is 64.97 g / L when fermented for 9 days. The yield of sophorolipids increases slightly during 7-9 days, and considering the time cost, 7 days is chosen as the optimal fermentation time. That is, when using Malassezia globosa LSH 221 to produce sophorolipids, using 7 days of fermentation time can obtain higher yield of sophorolipids.

[0086] In the group formed by experimental groups 1, 10-13, it can be found that with the increase of inoculum size, the yield of sophorolipids shows a trend of first increasing and then decreasing. When the inoculum size is too large, it will consume the nutrients in the medium, leading to insufficient nutrients in the subsequent sophorolipid fermentation process and reducing product accumulation; when the inoculum size is too small, it will lead to low production efficiency and prolonged fermentation period. Compared with the yield of sophorolipids in experimental groups 10 and 13, the yield of sophorolipids in experimental groups 1, 11-12 is higher, that is, when using Malassezia globosa LSH 221 to produce sophorolipids, using 1-3% of the inoculum size can obtain higher yield of sophorolipids.

[0087] The above examples are only used to illustrate the technical solutions of the present application and not to limit the protection scope of the present application. Although the present application is described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present application.

Claims

1. A type of Candida albicans LSH 221, characterized in that, The *Candida globosa* LSH 221 is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC 34437.

2. The application of Candida albicans LSH 221 as described in claim 1 in the preparation of sophorolipids.

3. A method for producing sophorolipids, characterized in that, The following operations are included, using Candida albicans LSH 221 for fermentation, which is deposited at the China General Microbiological Culture Collection Center with accession number CGMCC 34437.

4. The method for producing sophorolipids as described in claim 3, characterized in that, The fermentation process includes the following operations: the Candida albicans LSH 221 is mixed with a fermentation medium to obtain a fermentation broth, wherein the fermentation medium includes oleic acid at a concentration of 50-70 g / L, glucose at a concentration of 50-70 g / L, and yeast extract at a concentration of 2-4 g / L.

5. The method for producing sophorolipids as described in claim 4, characterized in that, The fermentation medium also includes potassium dihydrogen phosphate, disodium hydrogen phosphate, and magnesium sulfate.

6. The method for producing sophorolipids as described in claim 4, characterized in that, The amount of *Candida globosa* LSH 221 added is 1-3%.

7. The method for producing sophorolipids as described in claim 4, characterized in that, Fermentation time is 6 to 8 days.

8. The method for producing sophorolipids as described in claim 4, characterized in that, During the fermentation process, the fermentation temperature is 25~35℃, and the aeration rate is 1.3~1.5 m³ / s. 3 / h.

9. The method for producing sophorolipids as described in claim 4, characterized in that, During the fermentation process, oleic acid is added to the fermentation medium.

10. A method for subculturing Candida albicans LSH 221 as described in claim 1, characterized in that, The procedure includes the following steps: inoculating the *Candida globosa* LSH 221 into a seed culture medium for cultivation, wherein the seed culture medium comprises glucose and peptone in a mass ratio of 0.9-1.1:0.9-1.1.

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