Method for producing sophorolipid through fermentation

Abstract

The invention relates to a method for producing sophorolipid by fermentation, which comprises the following steps: inoculating a sophorolipid producing strain into a fermentation medium for fermentation, and supplementing hydrogen peroxide into a fermentation system in the initial stage of product synthesis, the supplementing amount of the hydrogen peroxide being 0.5-10% of the mass of the fermentation system when the hydrogen peroxide is added according to a 30% hydrogen peroxide solution; and continuously fermenting in the presence of hydrogen peroxide until the fermentation is finished. From the perspective of metabolic flux directional regulation, secondary metabolism of microorganisms is regulated by adding hydrogen peroxide with a certain concentration, and the enzyme activity of a key enzyme monooxygenase (CYP450) in a hydrophobic carbon source metabolic pathway in a sophorolipid synthesis process of thalli is induced and improved, so that the conversion rate of the hydrophobic carbon source and the fermentation yield of sophorolipid are improved; the invention solves the technical problems of low utilization efficiency of high-cost hydrophobic carbon source, low yield of sophorolipid, high production cost and the like in the existing fermentation production process of sophorolipid.

Description

Technical Field

[0001] The present invention relates to the technical field of biosurfactants produced by microbial fermentation, and particularly to a method for producing sophorolipids by fermentation. Background Art

[0002] Sophorolipids are fermented from Candida albicans and are currently the most widely studied biosurfactants. Sophorolipids have solubilizing, emulsifying, antibacterial and anti-inflammatory, wetting, and surface tension reduction functions. They are also non-toxic, biodegradable, and environmentally friendly. They are widely used in cosmetics, toiletries, medicine, environmental remediation, and other fields. However, the large-scale production of sophorolipids still faces the following problems: (1) High production costs. The fermentation substrates of Candida albicans include hydrophilic carbon sources (glucose) and hydrophobic carbon sources (oils and fats). The cost of these carbon sources accounts for a large proportion of the entire production process. However, due to the low efficiency of carbon source utilization and insufficient regulation of secondary metabolism, the yield of sophorolipids is low and the production cost is high. (2) There are bottlenecks in fermentation. The fermentation process of hydrophobic carbon sources has problems such as poor mass transfer, low substrate utilization, imbalance in metabolic flow distribution, and serious accumulation of by-products. In addition, in the later stage of fermentation, as the concentration of sophorolipids in the fermentation broth increases, the viscosity of the fermentation broth becomes increasingly higher, the DO in the fermentation broth is insufficient, which inhibits the activity of the bacteria and limits the conversion rate.

[0003] Hydrogen peroxide is a common oxidant that damages bacterial cell membranes, proteins, and DNA by generating reactive oxygen species (ROS). It is widely used for disinfection of medical devices and the environment. Most aerobic bacteria are inhibited in the presence of >0.1% H2O2, and anaerobic bacteria are more easily inactivated. Most bacterial fermentations rely on conventional parameters such as pH, temperature, and dissolved oxygen for regulation. The addition of hydrogen peroxide is generally avoided due to the risk of oxidative stress. Therefore, its use as a fermentation control method in industrial microbial fermentation is uncommon. Chinese patent application CN119081888A, "Bombus Candida and its application in preparing a fermentation broth of sophorolipids with low surface tension and low interfacial tension," discloses a bumblebee Candida (Candida bombicoia), deposited as CGMCC No. 31393. This strain has the ability to produce sophorolipids with low surface tension and low interfacial tension. The sophorolipid fermentation broth prepared by this method has the characteristics of low surface tension and low interfacial tension. According to testing, the surface tension of the sophorolipid fermentation broth is about 25mN / m and the interfacial tension is about 3mN / m. It can be widely used in the fields of improving wettability, removing oil stains, stabilizing emulsions, increasing residual oil recovery, and improving condensate fluidity. The solution discloses adding hydrogen peroxide at a mass concentration of 27%-30% after 40 hours of fermentation, with the amount added being 0.005%-0.008% of the mass of the fermentation medium. The solution does not clearly define the purpose of adding hydrogen peroxide. However, when applied to low-tension scenarios such as oil extraction and pesticide emulsification, the industry consensus is that carboxylic acid-type sophorolipids perform better in such applications. Therefore, it is speculated that the solution is to add hydrogen peroxide in the late fermentation period (40h) to oxidize the double bonds or primary alcohol groups in the sophorolipid molecules and increase the proportion of carboxylic acid-type sophorolipids (relative to lactone type). Carboxylic acid-type sophorolipids are more hydrophilic, easier to form micelles, have lower surface tension and interfacial tension, and are more suitable for improving the recovery rate of residual oil and improving the fluidity of condensate oil.

[0004] Therefore, no relevant literature reports on increasing the yield of sophorolipids by adding higher concentrations of hydrogen peroxide to the fermentation broth of Candida bumblebee have been retrieved so far. Summary of the Invention

[0005] (1) Technical issues to be solved

[0006] In view of the above-mentioned shortcomings of the prior art, the present invention provides a method for fermenting and producing sophorolipids. The method starts from the perspective of directional regulation of metabolic flow, and regulates the secondary metabolism of microorganisms by adding a certain concentration of hydrogen peroxide to the fermentation broth, thereby inducing and improving the enzyme activity of cytochrome monooxygenase (CYP450), a key enzyme in the hydrophobic carbon source metabolic pathway of the bacteria during the synthesis of sophorolipids, thereby improving the utilization rate of the hydrophobic carbon source and the yield of sophorolipids; solving the technical problems of high cost, low utilization efficiency of hydrophobic carbon source, low yield of sophorolipids, and high production cost in the existing sophorolipid fermentation production process.

[0007] Furthermore, low concentration of Tween 80 was added in combination with hydrogen peroxide induction. Through the synergistic effect of hydrogen peroxide and Tween 80, the mass transfer efficiency of hydrophobic substrates was increased, the permeability of bacterial cell membranes was increased to reduce metabolite inhibition caused by intracellular metabolite content, the viscosity of the fermentation broth was reduced, the dissolved oxygen was improved, the bacterial activity and enzyme activity were increased, and the yield of sophorolipids was increased.

[0008] (2) Technical solution

[0009] The technical solutions of the present invention are as follows:

[0010] A method for fermenting and producing sophorolipids, comprising: inoculating a seed liquid of a sophorolipid-producing strain into a fermentation medium for fermentation; in the early stage of product synthesis, adding hydrogen peroxide to the fermentation system, wherein the amount of hydrogen peroxide added is calculated as 30% hydrogen peroxide solution and accounts for 0.5%-10% of the mass of the fermentation system at the time of addition; and continuing fermentation in the presence of hydrogen peroxide until the fermentation is completed.

[0011] According to a preferred embodiment of the present invention, the sophorolipid-producing strain is Starmerella bombicola.

[0012] According to a preferred embodiment of the present invention, the preparation method of the seed liquid of the sophorolipid-producing strain is as follows: the sophorolipid-producing strain glycerol tube is inoculated into the YPD seed culture medium, and the shaking culture conditions are: temperature 20-30 ° C, speed 100-300 rpm, and culture for 20-48h; preferably, temperature 25-28 ° C, speed 200-300 rpm, and culture for 28-32h. Preferably, the sophorolipid-producing strain seed liquid OD 600 20-60.

[0013] According to a preferred embodiment of the present invention, the fermentation medium contains a hydrophilic carbon source, a hydrophobic carbon source, a nitrogen source and an inorganic salt; the hydrophilic carbon source includes but is not limited to any one of glucose, fructose, sucrose, molasses and lactose, or a combination of two or more thereof; the hydrophobic carbon source includes but is not limited to any one of vegetable oils, waste oils and fats, synthetic or semi-synthetic carbon sources, and novel natural carbon sources, or a combination of two or more thereof; the nitrogen source includes but is not limited to any one of peptone, yeast extract, corn flour, fish meal, ammonium sulfate, ammonium sulfate, ammonium nitrate and urea, or a combination of two or more thereof; the inorganic salt is any one of phosphate, sodium salt, calcium salt and magnesium salt, or a combination of two or more thereof.

[0014] Vegetable oils include but are not limited to soybean oil, corn oil, cottonseed oil, camellia oil, sunflower oil, palm oil, palm kernel oil, rapeseed oil, vegetable oil oleic acid, etc.; waste oils include but are not limited to catering waste oil (UCO) that has been pretreated by degumming and deacidification; synthetic or semi-synthetic carbon sources include but are not limited to C 12 -C18 Normal alkanes (such as n-hexadecane), fatty acid methyl esters (FAME); new natural carbon sources include but are not limited to microalgae oil, lignin-derived aromatics, such as lignin depolymerization products, vanillin, etc., which can be used to synthesize new sophorolipids containing benzene ring structures.

[0015] According to a preferred embodiment of the present invention, the method further comprises: adding Tween 80 to the fermentation system when the bacterial growth ends and the product synthesis begins, wherein the amount of Tween 80 added accounts for 0.05%-0.3% of the mass of the fermentation system at the time of addition (i.e., the real-time mass percentage concentration); and the fermentation system continues to ferment in the presence of Tween 80 and hydrogen peroxide until the fermentation ends.

[0016] According to a preferred embodiment of the present invention, Tween 80 is added to the fermentation system at 24-64 hours of fermentation, preferably 32-48 hours of fermentation; the amount of Tween 80 added is 0.05-0.1% of the mass of the fermentation system at the time of addition; and Tween 80 is added to the fermentation system by a one-time addition, a feeding addition, or a combination of a one-time addition and a feeding addition.

[0017] According to a preferred embodiment of the present invention, hydrogen peroxide is added to the fermentation system at 48-144 hours of fermentation, preferably 63-96 hours of fermentation. Based on the hydrogen peroxide being a 30% hydrogen peroxide solution, the amount of hydrogen peroxide added is 1.0-5.0% (preferably 1.5-3%) of the mass of the fermentation system at the time of addition. The hydrogen peroxide can be added to the fermentation system by a single injection, a feeding method, or a combination of both.

[0018] In the present invention, Tween 80 or hydrogen peroxide is preferably added during the fermentation period when bacterial growth ends and product synthesis begins. If these exogenous factors are added too early, the proliferation and growth of the bacteria will be inhibited. If they are added too late, the metabolic flow of the bacteria has been established, and the effect of metabolic flow regulation becomes poor.

[0019] 30% hydrogen peroxide solution is the most common commercially available concentration. Other concentrations of hydrogen peroxide can also be used, but the dosage needs to be calculated and adjusted accordingly based on the dosage limits specified in this disclosure. For example, if a 33% hydrogen peroxide solution is used, the total dosage should be appropriately reduced; if a 29% hydrogen peroxide solution is used, the total dosage should be appropriately increased.

[0020] According to a preferred embodiment of the present invention, the fermentation process is carried out in a shake flask or in a fermenter;

[0021] Shake flask culture conditions: The seed liquid of the sophorolipid-producing strain was inoculated into a shake flask filled with fermentation medium and fermented at a temperature of 20-30°C and a rotation speed of 100-300 rpm for 96-168 hours;

[0022] Fermentation tank culture conditions: at 20-30°C, when the pH of the fermentation liquid naturally drops to <3.5, adjust with alkali or ammonia to maintain pH = 3.5 ± 0.1; stir at 300-1000 rpm, control DO ≥ 20%, and the total fermentation time is 168-400 hours; preferably, control DO ≥ 50%, and the fermentation time is 256-300 hours.

[0023] According to a preferred embodiment of the present invention, before starting fermentation, a basal medium and a feed medium are prepared, wherein the total concentration of the carbon source (calculated as element C) in the basal medium is in the range of 0 to 8.5%, wherein the concentration of the hydrophilic carbon source is in the range of 0 to 5.5%, the concentration of the hydrophobic carbon source is in the range of 0 to 3%, and the total concentration of the nitrogen source (calculated as element nitrogen) is in the range of 0.01 to 0.16%;

[0024] During the fermentation process, when the mass concentration of the hydrophilic carbon source in the basal culture medium is 0.5% or less, the hydrophilic carbon source is dynamically supplemented by a one-time addition or a feeding addition method until its mass concentration is maintained at 1% to 3%; when the mass concentration of the hydrophobic carbon source in the basal culture medium is 0.5% or less, the hydrophobic carbon source is supplemented by a one-time addition, a feeding addition or a combination of the two until its mass concentration is maintained at 1% to 3%.

[0025] When the carbon source in the basal culture medium is 0, it means that the basal culture medium does not contain a carbon source, and the feed medium is added from the beginning of fermentation and monitored in real time. If the carbon source concentration in the fermentation liquid is lower than the preset value, the feeding rate is increased; if it is higher than the preset value, the feeding rate is reduced or the feeding is suspended.

[0026] As a preferred embodiment of the present invention, the inorganic salt composition of the basal culture medium is: magnesium sulfate 0.8-1.2 g / kg, sodium dihydrogen phosphate 0.8-1.2 g / kg, and dipotassium hydrogen phosphate 0.4-0.6 g / kg.

[0027] (3) Beneficial effects

[0028] 1. The present invention adds a high concentration of hydrogen peroxide to the fermentation broth of sophorolipids at the end of fermentation bacterial growth and the early stage of product synthesis, and uses hydrogen peroxide to induce the activity of monooxygenase (CYP450), a key enzyme for sophorolipid synthesis in the bacteria, to increase the enzyme activity, thereby promoting the conversion of more hydrophobic carbon sources into sophorolipids. At the same time, hydrogen peroxide oxidative stress inhibits the fatty acid β-oxidation pathway, thereby increasing the yield of sophorolipids, reducing the production cost of sophorolipids, and shortening the fermentation time when the same conversion rate is achieved.

[0029] 2. Further, in a preferred embodiment of the present invention, when the bacterial growth ends and the product synthesis begins, a low concentration of Tween 80 is added to the fermentation broth of the sophorolipid. On the one hand, the hydrophobic carbon source is emulsified, which makes the hydrophobic carbon source easier to be taken up and utilized by the bacteria. On the other hand, after the hydrophobic carbon source is emulsified, it is beneficial to the uniform dissolved oxygen inside the fermentation broth, thereby ensuring that the cells maintain high activity after a period of fermentation; in addition, Tween 80 can increase the fluidity of the cell membrane, so that the metabolites accumulated inside can be discharged to the extracellular space and promote H2O2 / O2 transmembrane transport into the cell, reducing the inhibition of intracellular metabolites on the bacteria and their enzymes, and is more conducive to making the intracellular H2O2 / O2 reach a concentration that activates the monooxygenase CYP450, thereby improving the conversion rate of the hydrophobic carbon source and the yield of sophorolipids.

[0030] 3. According to the experimental results, after adding hydrogen peroxide, the maximum yield of sophorolipids in a 500mL shake flask can reach 30.20g / kg, which is 11.38-31.70% higher than that of the control without adding hydrogen peroxide. The 5L tank was added with hydrogen peroxide by one-time addition and flow addition, and the sophorolipid yields reached 302.08g / kg and 308.16g / kg, respectively. Compared with the control without adding hydrogen peroxide, the yield increased by 32.29% and 34.95%, respectively. The enzyme activity of monooxygenase (CYP450) increased by 11.18% and 12.28%, respectively. The conversion rates based on vegetable oil were 1.30 and 1.31, respectively, which were 31.31% and 32.32% higher than those in Comparative Example 2, respectively.

[0031] Furthermore, under the synergistic effect of Tween 80 and hydrogen peroxide, the yield of sophora biosurfactant in the 5L tank was increased by 48.07% and 44.98% respectively compared with the comparative example 2, the enzyme activity of monooxygenase (CYP450) was increased by 19.08% and 20.18% respectively compared with the comparative example 2, and the conversion rate of glycolipid based on oil was increased by 41.41% and 42.42% respectively compared with the comparative example 2.

[0032] In summary, the present invention significantly improves the conversion rate of oil and fat and the yield of sophorolipids, which lays the foundation for process amplification and industrialization. DETAILED DESCRIPTION

[0033] In order to better explain the present invention and facilitate understanding, the present invention is described in detail below through specific implementation methods.

[0034] The present invention utilizes the addition of low concentrations of Tween 80 and hydrogen peroxide to the fermentation broth of sophorolipids at the end of fermentation bacterial growth and the early stage of product synthesis to achieve directional regulation of metabolic flow, thereby promoting the conversion of more high-cost hydrophobic carbon sources into sophorolipids, increasing the yield of sophorolipids, reducing the production cost of sophorolipids, and shortening the fermentation time when the same conversion rate is achieved. Tween 80 and hydrogen peroxide play the following synergistic regulatory mechanism in the metabolic regulation of fermentation production of sophorolipids: ① Tween 80 promotes the transmembrane transport of hydrogen peroxide by increasing cell membrane fluidity, making the intracellular H2O2 concentration gradient more conducive to activating the monooxygenase CYP450. Hydrogen peroxide oxidative stress inhibits the fatty acid β-oxidation pathway, while the emulsification effect of Tween 80 forces the carbon source to preferentially enter the sophorolipid synthesis pathway (reducing acetyl-CoA shunt). ② The oil droplets emulsified with Tween 80 (particle size <5 μm) expose more hydrophobic chain end groups, and hydrogen peroxide can selectively oxidize these sites to generate hydroxy fatty acids (direct precursors). Tween 80 reduces the surface tension of the fermentation broth (the viscosity of the fermentation broth will also increase as the metabolites increase), and cooperates with hydrogen peroxide to decompose and produce oxygen, thereby increasing the dissolved oxygen efficiency by 40%. ③ Tween 80 dissolves membrane lipids, while hydrogen peroxide induces cells to synthesize stress proteins (such as heat shock protein HSP70), forming a controllable "leak-repair" balance and continuously promoting product secretion. Hydrogen peroxide activates the oxidative stress pathway (SOD / catalase), and Tween 80 stabilizes the free radical intermediate and prolongs the signal duration. In addition, by adjusting the Tween 80 / H2O2 ratio, the degree of acetylation of sophorolipids can be controlled, such as adjusting the molar ratio of acidic type: lactone type from 1:1 to 3:1. Tween 80 and hydrogen peroxide accelerate the production of sophorolipids and increase the yield of sophorolipids through the coupling of multiple synergistic regulatory mechanisms.

[0035] The following is a detailed description of the preferred embodiments and comparative examples of the present invention.

[0036] It should be noted that in the following examples, the 30% hydrogen peroxide solution was purchased from Sichuan Jinshan Pharmaceutical Co., Ltd. Tween 80 was purchased from Xilong Chemical and is commercially available. The bumblebee-derived Candida species is classified as Starmerella bombicola.

[0037] In the following examples, the amount of 30% hydrogen peroxide added refers to the percentage of the mass of the 30% hydrogen peroxide solution added to the fermentation system to the mass of the fermentation system at the time of addition (i.e., the real-time mass percentage concentration), and the amount of Tween 80 added refers to the percentage of the mass of Tween 80 added to the fermentation system to the mass of the fermentation system at the time of addition.

[0038] In the following examples and comparative examples, the residual oil content was detected by weighing after n-hexane extraction, the residual sugar was determined by a bioanalyzer, and the sophorolipid yield was detected by the sulfuric acid-anthrone method.

[0039] In the following examples and comparative examples, the method for determining the monooxygenase CYP450 enzyme activity of the bacteria is as follows:

[0040] (1) Preparation of crude enzyme:

[0041] Take 3 mL of fermentation broth by rapid sampling, centrifuge at 4000 rpm for 5 min at 4°C, discard the supernatant, then add an equal volume of 20 mmol / L Tris-HCl buffer at pH 8.0 and wash the bacteria twice. After washing twice, resuspend the bacteria in buffer and then ultrasonically disrupt (this step must be performed on ice). After completion, centrifuge at 4°C and 12000 rpm for 10 min to obtain crude enzyme solution.

[0042] (2) Drawing a standard curve: Monooxygenase CYP450 enzyme activity was determined using an enzyme-linked immunosorbent assay kit with a detection range of 7.5 U / L-120 U / L. For the preparation of a series of standards with different concentrations (120 U / L, 60 U / L, 30 U / L, 15 U / L, 7.5 U / L), the specific measurement steps, and precautions, please refer to the instructions for the Monooxygenase CYP450 Enzyme Activity Assay Kit.

[0043] (3) Determination of enzyme activity: First, add 40 μL of diluent to the sample well to be tested, then take 10 μL of the treated sample diluted to an appropriate multiple and shake gently. The remaining steps are the same as the determination method of the standard. Finally, the absorbance value A is measured at 450 nm. 450 , the measured absorbance value A 450 Substitute the calculated value into the standard curve and multiply it by the dilution factor to obtain the monooxygenase CYP450 enzyme concentration in the sample (U / L).

[0044] Examples 1-7

[0045] A glycerol tube of Starmerella bombicola ATCC 22214 was placed in a 500 ml shake flask containing 150 g of liquid YPD seed medium and cultured at 25°C, 200 rpm, for 28 h to obtain the OD value. 600 The seed solution was 28% H₂O₂; then, at a 5% (m / m) inoculum, the solution was inoculated into a 500 mL shake flask containing 150 g of fermentation medium and cultured at 25°C, 200 rpm, for 156 h. The time points and real-time mass percentage concentrations of 30% H₂O₂ added to the fermentation system in Examples 1-7, as well as the total fermentation time, are all shown in Table 2.

[0046] During the fermentation process, samples were taken to determine the residual sugar, residual oil, and sophorolipid contents, and the monooxygenase CYP450 enzyme activity was determined 12 h after the addition of H2O2. The composition of the fermentation medium is shown in Table 1.

[0047] Table 1: Fermentation medium composition

[0048]

[0049] Comparative Example 1

[0050] The only difference between this comparative example and Example 1 is that H2O2 was not added to the fermentation liquid during the fermentation process, and the other conditions remained the same as those in Example 1.

[0051] Table 2: H2O2 addition conditions, sophorolipid production and CYP450 enzyme activity in Examples 1-7 and Comparative Example 1

[0052]

[0053] According to the measured results in Table 1, after adding hydrogen peroxide, the maximum yield of sophorolipids in a 500 mL shake flask can reach 30.20 g / kg. Compared with Comparative Example 1 without adding hydrogen peroxide, the sophorolipid yields of Examples 1-7 were increased by 11.38-31.70%, respectively. In addition, the CYP450 enzyme activity detected in Examples 1-7 was also significantly improved compared to Comparative Example 1. From the shake flask culture results in Table 1, when the amount of hydrogen peroxide added was 3.00%, the yield of sophorolipids was the highest, which was better than the cases where the amount of hydrogen peroxide added was 1.50%, 4.50% and 6.00%.

[0054] Examples 8-9

[0055] In Example 8-9, a fermentation experiment was carried out in which Candida bombicola ATCC22214 was inoculated in a 5 L fermenter to prepare sophorolipids. The fermentation method was as follows:

[0056] (1) A glycerol tube of Starmerella bombicola ATCC 22214 was placed in a 500 ml shake flask containing 150 g of liquid YPD seed medium. The flask was cultured at 25°C, 200 rpm, and 28 h to obtain the OD value. 600 It is 28% seed liquid.

[0057] (2) The seed solution was inoculated at a concentration of 5% (m / m) into a 5 L fermenter containing 2.5 kg of fermentation medium. Fermentation was carried out at 28°C. When the pH of the fermentation solution naturally dropped to <3.5, ammonia water was automatically added to maintain the pH of the fermentation solution at 3.5 ± 0.1. The stirring speed was 300-800 rpm. When the DO was lower than 20%, the speed was adjusted to adjust the DO to ≥ 20%. The total fermentation period was 302.5 h. At 72 h of fermentation, 3.00% of 30% H2O2 (see Table 3) was added to the fermentation system by a one-time or fed-batch method.

[0058] Before starting fermentation, a basal culture medium and a feed culture medium were prepared. The carbon source in the basal culture medium had a total concentration of 9 wt% calculated as element C, of which the hydrophilic carbon source concentration was 6 wt% and the hydrophobic carbon source concentration was 3 wt%; the nitrogen source had a total concentration of 0.085 wt% calculated as element nitrogen; magnesium sulfate was 1.0 g / kg, sodium dihydrogen phosphate was 1.0 g / kg, and potassium dihydrogen phosphate was 0.5 g / kg.

[0059] During the fermentation process, the carbon source in the fermentation system is monitored in real time. When the concentration of the hydrophilic carbon source in the basal culture medium drops to 0.5% or below, the hydrophilic carbon source is dynamically supplemented to a concentration within the range of 1-3wt% by one-time addition, feeding, or a combination of the two. When the mass concentration of the hydrophobic carbon source in the basal culture medium drops to 0.5% or below, the hydrophobic carbon source is supplemented to a concentration within the range of 1-3wt% by one-time addition, feeding, or a combination of the two.

[0060] The above composition of the fermentation medium and the control level of the carbon source during the fermentation process are specific examples actually used in Examples 8-11 and Comparative Examples 2-4, but they are not intended to limit the present invention. In different embodiments, the operator can adjust the concentration of each component in the basal medium and the control level of the carbon source during the fermentation process as needed, thereby regulating the yield and speed of sophorolipids.

[0061] Comparative Example 2

[0062] Referring to Table 3, the only difference between this comparative example and Example 8 is that no hydrogen peroxide solution was added to the fermentation broth in any manner during the fermentation process; the other conditions remained the same as those in Example 8.

[0063] Examples 10-11

[0064] Referring to Table 3, Examples 10-11 were fermentation experiments in a 5 L fermentor inoculated with Starmerella bombicola ATCC 22214 to prepare sophorolipids. The only difference between Examples 10-11 and Example 8 is that not only hydrogen peroxide was added during the fermentation process, but also Tween 80 was added to the fermentation system at a concentration of 0.10 wt% or 0.05 wt% in a one-time or feeding manner when the fermentation was carried out for 32 h or 38 h. The other conditions remained the same as in Example 8.

[0065] Comparative Example 3

[0066] As shown in Table 3, this comparative example is based on Example 10, retaining the addition of 0.10% Tween 80 to the fermentation system. However, no hydrogen peroxide solution was added during the entire fermentation process. All other conditions remained the same as in Example 10.

[0067] Comparative Example 4

[0068] Referring to Table 3, the method of adding hydrogen peroxide in this comparative example was adjusted as follows: 0.008% of 30% hydrogen peroxide was added at one time after 72 hours of fermentation, and the other conditions remained the same as those in Example 8.

[0069] Table 3: Addition conditions of 4H2O2, glycolipid production and CYP450 enzyme activity of Examples 8-11 and Comparative Example 2-4

[0070]

[0071] As shown in Table 3, Examples 8, 9, and Comparative Example 2, hydrogen peroxide was added in a 5L fermentor by both one-time addition and flow addition, and the sophorolipid yields reached 302.08 g / kg and 308.16 g / kg, respectively. Compared with the control without hydrogen peroxide, the yields increased by 32.29% and 34.95%, respectively, and the monooxygenase (CYP450) enzyme activities increased by 11.18% and 12.28%, respectively; the conversion rates of vegetable oil in Examples 8-9 were 1.30 and 1.31, respectively, which were 31.31% and 32.32% higher than those in Comparative Example 2, respectively. In Comparative Example 4, after adding 0.008% of 30% H2O2 at 72 h of fermentation, the CYP450 enzyme activity did not increase compared with Comparative Example 2, and the sophorolipid yield hardly increased.

[0072] It can be seen from Examples 10-11 and Comparative Example 2 in Table 3 that under the synergistic effect of Tween 80 and hydrogen peroxide, the yield of sophorolipids was increased by 48.07% and 44.98% respectively compared with Comparative Example 2, the enzyme activity of monooxygenase (CYP450) was increased by 19.08% and 20.18% respectively compared with the comparative example, and the conversion rate of glycolipids based on oil and fat was increased by 41.41% and 42.42% respectively compared with Comparative Example 2.

[0073] As can be seen from Examples 10-11 in Table 3 compared with Example 9, under the synergistic effect of Tween 80 and hydrogen peroxide, the yield of sophorolipids was further increased by 9.72% and 7.43% respectively compared with Example 9 (only hydrogen peroxide was added) (the enzyme activity of CYP450 enzymes was increased by 6.05% and 7.03% respectively compared with the control example, and the conversion rate of glycolipids based on oil and fat was increased by 6.87% and 7.63% respectively compared with Example 9). This shows that the addition of hydrogen peroxide and Tween 80 to the fermentation system can further effectively improve the conversion rate of the hydrophobic carbon source and the yield of sophorolipids. In Comparative Example 3, only 0.10% of Tween 80 was added, and the sophorolipid yield and CYP450 enzyme activity were not significantly improved. Therefore, the addition of low-concentration Tween 80 alone has almost no promoting effect on the yield of sophorolipids.

[0074] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or make equivalent replacements for some or all of the technical features therein. These modifications or replacements, or when the technical features in the above embodiments do not conflict with each other, can be combined in the manner described in the embodiments, and these modifications, replacements or combinations do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for producing sophorolipids by fermentation, characterized in that: It includes: The seed liquid of the sophorolipid-producing strain is inoculated into the fermentation medium for fermentation. In the early stage of product synthesis, hydrogen peroxide is added to the fermentation system. The amount of hydrogen peroxide added is calculated as 30% hydrogen peroxide solution, accounting for 0.5%-10% of the mass of the fermentation system at the time of addition; fermentation is continued in the presence of hydrogen peroxide until the end of fermentation.

2. The method according to claim 1, characterized in that The sophorolipid-producing strain is Starmerella bombicola.

3. The method according to claim 1, characterized in that The preparation method of the seed liquid of the sophorolipid-producing strain is as follows: inoculating a glycerol tube of the sophorolipid-producing strain into a YPD seed culture medium, and culturing under shaking conditions: temperature 20-30° C., rotation speed 100-300 rpm, and culturing for 20-48 hours.

4. The method according to claim 1, wherein The fermentation medium comprises a hydrophilic carbon source, a hydrophobic carbon source, a nitrogen source and an inorganic salt; The hydrophilic carbon source is not limited to any one or a combination of two or more of glucose, fructose, sucrose, molasses and lactose; The hydrophobic carbon source includes but is not limited to any one or a combination of two or more of plant oils, waste oils, synthetic or semi-synthetic carbon sources, and new natural carbon sources; The nitrogen source includes but is not limited to any one or a combination of two or more of peptone, yeast extract, corn meal, fish meal, ammonium sulfate, ammonium sulfate, ammonium nitrate and urea; The inorganic salt is any one of phosphate, sodium salt, calcium salt and magnesium salt, or a combination of two or more thereof.

5. The method according to claim 4, characterized in that The vegetable oil is at least one of soybean oil, corn oil, cottonseed oil, camellia oil, sunflower oil, palm oil, palm kernel oil, rapeseed oil, and vegetable oil oleic acid; the waste oil is catering waste oil that has been pretreated by degumming and deacidification; the synthetic or semi-synthetic carbon source is C 12 -C 18 The novel natural carbon source is microalgae oil or lignin-derived aromatics.

6. The method according to claim 1, characterized in that Also includes: When the bacterial growth ends and the product synthesis begins, Tween 80 is added to the fermentation system, and the amount of Tween 80 added accounts for 0.05%-0.3% of the mass of the fermentation system at the time of addition; the fermentation system continues to ferment in the presence of Tween 80 and hydrogen peroxide until the fermentation is completed.

7. The method according to claim 6, characterized in that The timing for adding Tween 80 to the fermentation system is: when the fermentation is in progress for 24-64 hours; the amount of Tween 80 added is 0.05-0.1% of the mass of the fermentation system at the time of addition; the method of adding Tween 80 is a one-time addition, a feeding addition, or a combination of a one-time addition and a feeding addition.

8. The method according to claim 1, characterized in that The timing for adding hydrogen peroxide to the fermentation system is: when the fermentation has been going on for 48-144 hours; based on the hydrogen peroxide being a 30% hydrogen peroxide solution, the amount of hydrogen peroxide added is 1.0-5% of the mass of the fermentation system at the time of addition; the hydrogen peroxide is added in a one-time addition, a feeding method, or a combination of one-time addition and feeding method.

9. The method according to claim 1, characterized in that The fermentation process is carried out in a shake flask or in a fermenter; Shake flask culture conditions: The seed liquid of the sophorolipid-producing strain was inoculated into a shake flask filled with fermentation medium and fermented at a temperature of 20-30°C and a rotation speed of 100-300 rpm for 96-168 hours; Fermentation tank culture conditions: at 20-30°C, when the pH of the fermentation liquid naturally drops to <3.5, adjust with alkali or ammonia to maintain pH = 3.5 ± 0.1; stir at 300-1000 rpm, maintain DO ≥ 20%, and the total fermentation time is 168-400 hours.

10. The method according to claim 1, characterized in that Before starting fermentation, a basal medium and a feed medium are prepared. The carbon source in the basal medium, calculated as element C, has a total concentration range of 0 to 8.5%, wherein the concentration of the hydrophilic carbon source is 0 to 5.5%, the concentration of the hydrophobic carbon source is 0 to 3%, and the total concentration of the nitrogen source, calculated as element nitrogen, ranges from 0.01 to 0.16%. During the fermentation process, when the mass concentration of the hydrophilic carbon source in the basal culture medium is 0.5% or less, the hydrophilic carbon source is dynamically supplemented by a one-time addition or a feeding addition method until its mass concentration is maintained at 1% to 3%; when the mass concentration of the hydrophobic carbon source in the basal culture medium is 0.5% or less, the hydrophobic carbon source is supplemented by a one-time addition, a feeding addition or a combination of the two until its mass concentration is maintained at 1% to 3%.

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