A method for preparing sophorolipids by one-step conversion

Abstract

A one-step conversion method for preparing sophorolipids belongs to the technical field of sophorolipid synthesis. The method uses corn stalks as raw materials, pre-treated with alkali, and then adding citric acid buffer, complex enzyme, concentrated fermentation liquid and a mutant strain of Candida albicans for fermentation. The mutant strain of Candida albicans is Starmerella bombicola U1-4, its deposit number is CCTCC NO: M 2025692. In the present invention, corn straw is pretreated with NaOH, and cellulase, β-glucosidase, xylanase and mutant strain U1-4 are used for one-step fermentation. Since the mutant strain U1-4 has excellent tolerance to phenolic inhibitors, the straw system pretreated with NaOH is suitable for the growth and fermentation of the mutant bacteria U1-4 in the one-step process, reducing the inhibition of the inhibitors on the growth and fermentation of microorganisms, and can also use phenolic inhibitors as a carbon source for growth and fermentation, thereby increasing the yield of sophorolipids, and the yield reaches 76.46 g / L.

Description

Technical Field

[0001] The present invention relates to the technical field of sophorolipid synthesis, and particularly to a method for preparing sophorolipids by a one-step conversion method. Background Art

[0002] Sophorolipids (SLs) are a class of glycolipid biosurfactants produced by yeast metabolism and are currently the most productive. They are mainly produced by yeast using hydrophilic and hydrophobic carbon sources for fermentation. [2][3][4] Due to its good surface activity, biodegradability and biocompatibility, it has been used in a small range of fields such as food, medicine, oil extraction, and environmental protection. [7][8] However, the high production cost of SLs has limited their widespread application. Exploring inexpensive fermentation substrates to replace the conventional hydrophilic carbon sources (glucose), hydrophobic carbon sources (plant oil), and nitrogen sources (yeast powder) is an effective way to reduce production costs. Furthermore, long fermentation cycles and low SLs yields pose challenges, necessitating further research into inexpensive alternative substrates for SLs fermentation production.

[0003] The high cost of glucose determines the production cost of SLs, necessitating the need for inexpensive, hydrophilic carbon sources to replace glucose. Corn stover is a significant agricultural waste stream. Rich in cellulose and hemicellulose, these polysaccharides decompose into intermediate sugars, which can then become a viable substrate for biofuel production. However, due to its dense structure and the interlinked carbon chains of lignin, cellulose, and hemicellulose, the lignocellulosic structure presents a difficult-to-break spatial structure, making it difficult for fermentation microorganisms to utilize it, and thus reducing the efficiency of enzymatic hydrolysis. Therefore, pretreatment of corn stover is essential.

[0004] In the prior art, the main methods used in the biorefining process of sophora fermentation biosurfactants using lignocellulose as raw materials are the step-by-step saccharification and fermentation method and the synchronous saccharification and fermentation method. The step-by-step saccharification and fermentation method includes the steps of straw pretreatment, cellulose saccharification, saccharification liquid detoxification and sophora fermentation biosurfactants. It is necessary to prepare the reaction liquid and fermentation liquid multiple times and recycle them. The relatively cumbersome production steps make the equipment use more and the operating cost higher, which restricts its industrial application to a certain extent. The synchronous saccharification and fermentation method includes straw pretreatment, solid-liquid separation, and the addition of cellulase and microorganisms to the solid for synchronous saccharification and fermentation. In this process, the liquid components in the pretreatment process are removed, resulting in a large amount of waste of carbohydrate components, and the microorganisms are required to have a certain degree of inhibitor tolerance, which leads to a low straw conversion rate. Compared with the synchronous saccharification and fermentation method, the one-step conversion method is to carry out the pretreatment, saccharification and fermentation of corn straw to produce sophora fermentation biosurfactants in the same space at the same time, without the need to prepare the feed liquid, solid-liquid separation, neutralization and detoxification in batches, so as to achieve a "one-step conversion" from substrate straw to product sophora fermentation biosurfactants. However, due to the low enzymatic hydrolysis efficiency of cellulase at the optimal fermentation temperature, and the large amount of inhibitors generated during the pretreatment process are stored in the slurry without detoxification, the inhibitory effect is stronger than that of the simultaneous saccharification and fermentation method, which results in most microorganisms being unable to grow and metabolize in this system. The above problems seriously affect the enzymatic hydrolysis and microbial growth and metabolism process. Summary of the Invention

[0005] Based on the above problems, the present invention aims to provide a Starmerella bombicola The mutant strain U1-4 of Candida albicans with high tolerance to inhibitors was obtained by mutagenesis of ATCC22214.

[0006] Another object of the present invention is to provide a one-step method for preparing sophorolipids based on the above mutant strain.

[0007] The object of the present invention is achieved through the following technical solutions:

[0008] A one-step conversion method for preparing sophorolipids is characterized in that: corn straw is used as raw material, a pretreatment mixture is obtained by alkali pretreatment, and then a citric acid buffer, a complex enzyme, a concentrated fermentation broth and a mutant strain of Candida albicans are added to the pretreatment mixture for fermentation.

[0009] Further, the mutant strain is Starmerella bombicola The strain obtained by sequentially subjecting ATCC 22214 to ARTP mutagenesis and UV mutagenesis is classified as Candida albicans ( Starmerella bombicola )U1-4, deposited in China Center for Type Culture Collection (CCTCC), the deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, the deposit number is CCTCC NO: M 2025692, and the deposit date is April 3, 2025.

[0010] Furthermore, the alkaline pretreatment is to soak the corn straw in a NaOH aqueous solution with a mass concentration of 0.25-1.0% for 12-36 hours.

[0011] Further preferably, the concentration of the NaOH aqueous solution is 1%, and the immersion time is 24 h.

[0012] Furthermore, the solid-liquid ratio of the corn straw to the NaOH aqueous solution is 1:8-12, preferably, the solid-liquid ratio is 1:10.

[0013] Furthermore, the complex enzyme is composed of cellulase, β-glucosidase and xylanase in an enzyme activity ratio of 1:1 to 2:3.

[0014] Further preferably, the complex enzyme is composed of cellulase, β-glucosidase and xylanase in an enzyme activity ratio of 1:1:3.

[0015] Furthermore, during the fermentation process, the temperature was maintained at 30-35° C. and the fermentation reaction was carried out at 150-180 rpm for 7 days.

[0016] More preferably, during the fermentation process, the temperature is maintained at 32° C. and the fermentation reaction is carried out at 160 rpm for 7 days.

[0017] Further, the ARTP mutagenesis is to Starmerella bombicola Inoculate cells in YPD seed medium and then dilute the cell suspension cultured to the logarithmic growth phase with 5% glycerol-normal saline to an OD value between 0.6 and 0.8. Place a 1 cm diameter copper disc for mutagenesis in a sterilized empty culture dish. Add 10 μL of the freshly prepared cell suspension to the disc and expose to ARTP radiation for 0, 40, 60, 80, 100, 120, 140, and 160 s. Set the instrument parameters as follows:

[0018] The distance was approximately 2 mm, 120 W (Pin), 20 W (Pout), 2 mm (D), and a helium flow rate of 10 sLm. After treatment, sterile tweezers were used to place the copper disc into a 1.5 mL centrifuge tube containing 1 mL of sterile water. The tube was shaken for 2 minutes to fully dissolve the cell suspension attached to the copper disc in sterile water. The sample was then diluted 10-5 times and plated for culture. The tube was then incubated at 30°C for 4 days.

[0019] Furthermore, the UV mutagenesis method involves inoculating a mutant strain with increased yield after ARTP mutagenesis into YPD medium, and diluting the cell suspension cultured to the logarithmic growth phase with 5% glycerol-normal saline to an OD value between 0.6 and 0.8. 5 mL of the cultured cell suspension and a sterile magnetic rotor are added to a sterile culture dish, which is then placed under a 254 nm UV mutagenesis lamp at a treatment distance of 25 cm for 0, 40, 50, 70, 90, 100, 110, and 180 seconds. After treatment, the sample is diluted 10-5 times and plated for culture. The culture is then incubated in the dark at a constant temperature of 30°C for 4 days.

[0020] In the traditional step-by-step preparation of sophorolipids, the pretreated straw needs to undergo multiple steps such as enzymatic hydrolysis to prepare saccharification liquid, detoxification of the saccharification liquid, and fermentation to produce sophorolipids. Although the yield of sophorolipids is considerable, the steps are complicated and waste of sugar sources is caused, resulting in a significant increase in production costs. Therefore, we do not perform solid-liquid separation after pretreatment, directly add cellulase and fermentation strains, and synthesize sophorolipids through a one-step method.

[0021] There are multiple problems in the process of preparing sophora biosurfactants using a one-step method. Specifically, the saccharification liquid cannot be detoxified, the presence of inhibitors produced during pretreatment will lead to the inhibition of microorganisms during the fermentation process, and because the reaction temperature exceeds 30°C, the activity of the fermentation bacteria is inhibited and sophora biosurfactants are not produced. At this temperature, the efficiency of cellulose enzymatic hydrolysis is low. The above problems seriously affect the enzymatic hydrolysis and microbial growth and metabolism processes in the synchronous process, resulting in low straw utilization and low sophora biosurfactant production.

[0022] In the present invention, NaOH is used to pretreat the straw, the types and concentrations of inhibitors generated in the pretreated straw are adjusted, and then the strain obtained by mutation is synchronously treated with a composite enzyme composed of cellulase, β-glucosidase and xylanase, so that saccharification and fermentation to produce sophorolipids are carried out in one step. In this process, the main inhibitors generated in the straw system after NaOH pretreatment are phenolic aldehydes, and the mutant strain U1-4 has a high tolerance to such substances, which is conducive to the growth and fermentation of the strain. At the same time, the mutant strain U1-4 has a significantly reduced content of phenolic inhibitors after fermentation in the system. It is speculated that in the mixed system of NaOH-pretreated straw, the mutant strain U1-4 can further utilize phenolic substances as a carbon source for growth and fermentation. In this system, since the strain utilizes the phenolic inhibitors in the system, the inhibitory effect of the inhibitor on the fermentation production of SLs by the strain is low, thereby improving the efficiency of sophorolipid production.

[0023] Most specifically, a method for preparing sophorolipids by a one-step conversion method is characterized by comprising the following steps:

[0024] Corn straw was immersed in a 0.25-1.0% NaOH aqueous solution for 12-36 h and sterilized at 121°C for 60 min to obtain a pretreatment mixture with a solid-liquid ratio of corn straw to NaOH aqueous solution of 1:8-12.

[0025] Without solid-liquid separation, the pH of the pretreatment mixture was directly adjusted to 3.0, and a citric acid buffer solution with a concentration of 50mM and a pH of 3.5 was added and mixed, and then a composite enzyme, concentrated fermentation broth and mutant strain were added, and the mixture was reacted at 32°C and 160rpm for 7 days. The composite enzyme was composed of cellulase, β-glucosidase and xylanase in an enzyme activity ratio of 1:1:3, wherein the amount of cellulase added was 30 FPU / L, and the mutant strain was used. Starmerella bombicola ATCC 22214 was obtained by sequentially subjecting it to ARTP mutagenesis and UV mutagenesis. The inoculum size was 3-6%. The concentrated fermentation broth was composed of 2% oleic acid, 0.3% yeast powder, 0.10% KH2PO4, 0.10% Na2HPO4·12H2O, and 0.05% MgSO4·7H2O. The volume ratio of the liquid system, citric acid buffer, and concentrated fermentation broth was 50:2.5:1.

[0026] The present invention has the following technical effects:

[0027] In the present invention, corn straw is pretreated with NaOH to generate inhibitors of specific types and concentrations, and a composite enzyme consisting of cellulase, β-glucosidase and xylanase and a mutant strain U1-4 are used for one-step fermentation. Since the mutant strain U1-4 has excellent tolerance to phenolic inhibitors, the straw system pretreated with NaOH is suitable for the growth and fermentation of the mutant strain U1-4 during the one-step process, reducing the inhibition of the inhibitors on the growth and fermentation of microorganisms. In addition, the phenolic inhibitors can be used as a carbon source for growth and fermentation, thereby increasing the yield of sophorolipids to 76.46 g / L. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 : Effects of different organic acid inhibitors on the SLs fermentation and survival abilities of the original strain and mutant strains.

[0029] Figure 2 : Effects of different phenolic inhibitors on the SLs fermentation and survival abilities of the original strain and mutant strains.

[0030] Figure 3 : Fourier transform infrared (FTIR) spectra of corn stover before and after NaOH and KOH pretreatment.

[0031] Figure 4 : Effects of different concentrations of NaOH and KOH pretreatment on sugar production after enzymatic hydrolysis of corn straw.

[0032] Figure 5 : Sugar production efficiency of corn straw enzymatic hydrolysis treated with 1.00% NaOH for different soaking times.

[0033] Figure 6 : Changes of glucose, xylose and sophorolipid contents over time during the one-step conversion of corn straw to sophorolipids by pretreatment with different concentrations of NaOH and KOH (A1-A4: NaOH concentrations of 0.25%, 0.50%, 0.75% and 1.00%, respectively; B1-B4: KOH concentrations of 0.25%, 0.50%, 0.75% and 1.00%, respectively).

[0034] Figure 7 : Changes of glucose, xylose and sophorolipid contents with pH during the one-step conversion fermentation process for preparing sophorolipids. DETAILED DESCRIPTION

[0035] The present invention is described in detail below through examples. It is necessary to point out that the following examples are only used to further illustrate the present invention and are not to be construed as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above-mentioned contents of the present invention.

[0036] The corn stalks used in the present invention were harvested at Jilin Agricultural University, and the cellulase, β-glucosidase, pectinase, and xylanase used were purchased from Novozymes Biotechnology Co., Ltd. (Tianjin, China), Beijing Solebold Technology Co., Ltd. (Beijing, China), Sigma-Aldrich Co., Ltd. (USA), and Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China), respectively.

[0037] The mutant strain used in the present invention is Candida albicans ( Starmerella bombicola ) ATCC 22214 was mutagenized to obtain the Candida albicans ( Starmerella bombicola ) ATCC 22214 was purchased from Beinan Biotechnology Co., Ltd. (the corresponding Beinan number is BNCC340735).

[0038] Example 1

[0039] The steps for obtaining the mutant strain U1-4 are as follows:

[0040] Each culture medium:

[0041] (1) YPD medium: 2% peptone, 1% yeast extract, 2% glucose (solid medium containing 2% agar), sterilized at 115°C for 30 min. The inoculum size is 1% of the seed solution and cultured at 30°C, 160 rpm for 16 h.

[0042] (2) Conventional fermentation medium: glucose 6%, oleic acid 6%, yeast extract 0.3%, KH2PO4 0.10%, Na2HPO4·12H2O 0.10%, MgSO4·7H2O 0.05%, sterilized at 115°C for 30 min. The medium pH was 3.0, the inoculum size was 4%, and the culture was carried out at 30°C and 160 rpm for 7 days.

[0043] (3) Fermentation medium with inhibitors: Different concentrations of formic acid, acetic acid, glycolic acid, vanillin and syringaldehyde were added to the ordinary fermentation medium and cultured at 30°C and 160 rpm for 7 days.

[0044] The specific mutagenesis steps of the mutant strain are as follows:

[0045] (1) ARTP mutagenesis: The original strain was inoculated into YPD seed medium. The cell suspension cultured to the logarithmic growth phase was then diluted with 5% glycerol-normal saline to an OD value between 0.6 and 0.8. A 1 cm diameter copper sheet for mutagenesis was placed in a sterilized empty culture dish. 10 μL of the freshly prepared cell suspension was added to the copper sheet and exposed to ARTP radiation for 0, 40, 60, 80, 100, 120, 140, and 160 s. The instrument parameters were set as follows:

[0046] The distance was approximately 2 mm, 120 W (Pin), 20 W (Pout), 2 mm (D), and a helium flow rate of 10 sLm. After treatment, sterile tweezers were used to place the copper disc into a 1.5 mL centrifuge tube containing 1 mL of sterile water. The tube was shaken for 2 minutes to fully dissolve the cell suspension attached to the copper disc in sterile water. The sample was then diluted 10-5 times and plated for culture. The tube was then incubated at 30°C for 4 days.

[0047] (2) UV mutagenesis: The mutant strain with increased yield after ARTP mutagenesis was inoculated into YPD medium. The cell suspension cultured to the logarithmic growth phase was diluted with 5% glycerol-normal saline to an OD value of 0.6-0.8. 5 mL of the cultured cell suspension and a sterile magnetic rotor were added to a sterile culture dish and placed under a 254 nm UV mutagenesis lamp at a treatment distance of 25 cm. The irradiation treatments were performed for 0, 40, 50, 70, 90, 100, 110, and 180 s, respectively. After treatment, the sample was diluted 10-5 times and then spread for culture. The culture was kept in the dark at 30°C for 4 days.

[0048] The inhibitory effects of different organic acid inhibitors on the growth, survival and fermentation production of SLs of the original strain ATCC22214 and the mutant strain U1-4 were tested. Figure 1As shown (a: formic acid, b: acetic acid, c: glycolic acid), it can be seen that, under the action of formic acid, as the concentration increases, the growth activity of the original strain and mutant strain U1-4 is inhibited, and the inhibition rate of fermentation SLs production gradually increases. However, the inhibition of mutant strain U1-4 decreases significantly. When the formic acid concentration exceeds 0.8 g / L, the inhibition of the original strain and mutant strain U1-4 is comparable. Under the action of acetic acid, as the concentration increases, the growth activity and inhibition rate of fermentation SLs production of the original strain and mutant strain U1-4 are basically the same when the acetic acid concentration is below 3 g / L. However, at a concentration of 5 g / L, the inhibition of mutant strain U1-4 is significantly higher than that of the original strain, and then lower than that of the original strain. Under the action of glycolic acid, as the glycolic acid concentration increases, the inhibition of mutant strain U1-4 is significantly higher than that of the original strain. Overall, mutant strain U1-4 does not have improved resistance to toxicity from organic acid inhibitors compared to the original strain.

[0049] The results of the inhibitory effects of different phenolic inhibitors on the growth, survival and fermentation SLs production of the original strain ATCC22214 and the mutant strain U1-4 are as follows: Figure 2 As shown in Figure 2, it can be seen that under the action of phenolic inhibitors, the mutant strain U1-4 has a significantly improved ability to resist phenolic inhibitor toxicity compared with the original strain.

[0050] The mutant strain U1-4 was biologically deposited and the classification name of the strain was Candida albicans ( Starmerella bombicola )U1-4, deposited in China Center for Type Culture Collection (CCTCC), the deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, the deposit number is CCTCC NO: M 2025692, and the deposit date is April 3, 2025.

[0051] Example 2

[0052] The one-step method for preparing sophorolipids comprises the following steps:

[0053] Corn straw was soaked in a 1.0% NaOH aqueous solution for 24 h and sterilized at 121°C for 60 min to obtain a pretreatment mixture. The solid-liquid ratio of corn straw to NaOH aqueous solution was 1:10.

[0054] Without solid-liquid separation, the pH of the pre-treated mixture was directly adjusted to 3.0, and 50 mM citric acid buffer with a pH of 3.0 was added and mixed, and then cellulase, concentrated fermentation broth and Candida albicans ( Starmerella bombicola) mutant strain, reacting at 32° C. and 160 rpm for 7 days, the cellulase added at a rate of 30 FPU / mL, the inoculum rate of the Torulopsis mutant strain was 4%, the concentrated fermentation broth was composed of 2% oleic acid, 0.3% yeast powder, 0.10% KH2PO4, 0.10% Na2HPO4·12H2O, and 0.05% MgSO4·7H2O, and the volume ratio of the liquid system, citric acid buffer, and concentrated fermentation broth was 50:2.5:1.

[0055] During the pretreatment process, different chemical pretreatments (HCl, H2SO4, NaOH, KOH) were tried. The types and contents of inhibitors produced in the straw system under different pretreatments were significantly different. Therefore, after different pretreatments, the concentrations of various inhibitors in the pretreated system were tested. The inhibitor components were analyzed by HPLC (Agilent 1260) equipped with a VWD detector (G7114A) and a SilGreen ODS C18 column.

[0056] Detection of organic acid inhibitors:

[0057] Formic acid and acetic acid were determined using potassium dihydrogen phosphate / methanol (2:8, v / v) as the mobile phase with isocratic elution, a detection wavelength of 210 nm, and a flow rate of 0.4 mL / min. The mobile phase and sample were filtered through a 0.22 μm filter.

[0058] Detection of furan inhibitors:

[0059] For the determination of furfural and 5-hydroxymethylfurfural, methanol and water were used in a ratio of 1:9 as the mobile phase, with isocratic elution, a detection wavelength of 280 nm, a flow rate of 1 mL / min, and the mobile phase and sample were filtered through a 0.22 μm filter.

[0060] Detection of phenolic inhibitors:

[0061] For the determination of syringaldehyde and vanillin contents, methanol and water were used in a ratio of 3:7 as the mobile phase, with isocratic elution, detection wavelength of 280 nm, flow rate of 1 mL / min, and the mobile phase and sample were filtered through a 0.22 μm filter.

[0062] Plotting the standard curve of each inhibitor:

[0063] Prepare a 2 mg / mL inhibitor stock solution based on the density of each inhibitor. This solution was serially diluted to yield three replicates of 0.2, 0.4, 0.6, 0.8, and 1.0 mg / mL standard solutions. Detection was performed at 280 nm. A standard curve was plotted with peak area on the vertical axis and inhibitor content on the horizontal axis, and a regression line was fitted.

[0064] The content of each inhibitor was calculated based on the standard curve. The results are shown in Table 1.

[0065] Table 1:

[0066]

[0067] Among them, the content of formic acid, acetic acid and furan inhibitors in the acid pretreatment group was much higher than that in the alkali pretreatment group. In the alkali pretreatment group, the content of organic acid and phenolic inhibitors in the NaOH group was significantly higher than that in the KOH group, and the content of furan inhibitors was lower than that in the KOH group. In the one-step preparation of sophorolipids, since no solid-liquid separation, detoxification and other processes were performed after the pretreatment of the straw, the inhibitors generated by the pretreatment were all present in the liquid. The inhibitors generated during the acid pretreatment seriously affected the cellulase activity and subsequent microbial activity. It can be seen that organic acid pretreatment is not suitable for the one-step method. Therefore, the alkali pretreatment group was considered for the one-step preparation of sophorolipids.

[0068] Fourier transform infrared spectroscopy (FT-IR) analysis was performed on the corn straw pretreated with NaOH and KOH, respectively, and the untreated corn straw (CK) to study the changes in their chemical structures. The results are shown in Figure 3. Figure 3 It can be seen from the figure that the infrared absorption peaks of pretreated and untreated corn straw are basically the same, but the intensity of some characteristic peaks has changed significantly, and some characteristic peaks have disappeared in the spectrum. -1 The absorption peak at is caused by the ring vibration in cellulose and sugars such as β-saccharides, galactose, mannose, arabinose and polysaccharides. Figure 3 It can be seen that the pretreated straw is 1274.26 cm thicker than the CK group. -1 The C-O-C antisymmetric vibration peak at wave number 1607.89 cm represents the ether bond between phenylpropane monomers in lignin. -1 The absorption peak of the benzene ring in the pretreated straw decreased compared with that in the CK group. The peak of the untreated corn straw was more obvious. The intensity of the peak of the straw pretreated with NaOH and KOH was significantly weakened. All of the above indicated that the lignin was degraded to a certain extent. -1 The absorption peak of the corn straw before and after pretreatment is the ester group in the hemicellulose, which is related to the carbon-oxygen ratio stretching of this component. The absorption peak of the pretreated straw is lower than that of the CK group, indicating that the pretreatment group has better removed the hemicellulose in the corn straw. The infrared absorption peak of the corn straw before and after pretreatment is at 3433.69cm -1 There are similar broad absorption peaks at the 400 nm and 400 nm, which indicates that the alkali-heat pretreatment has no adverse effect on the configuration of cellulose.

[0069] Different concentrations of KOH and NaOH were used for pretreatment, and then the same cellulase was used for enzymatic saccharification. The glucose content in the supernatant after the reaction was determined using a glucose (GLU) kit (glucose oxidase method GOD), and the xylose content in the supernatant after the reaction was determined using a D-xylose kit. The results are shown in Figure 2. Figure 4 As shown in the data, with the increase of alkali concentration, the reducing sugar content in the enzymatic hydrolyzate increased accordingly. When the concentration reached 1.0%, the mass concentrations of glucose and xylose reached the highest, reaching 10.79 g / L and 12.27 g / L in the NaOH group and 9.32 g / L and 9.59 g / L in the KOH group, respectively. Compared with the KOH pretreatment, the glucose and xylose yields in the saccharification liquid obtained by enzymatic hydrolysis of the straw after NaOH pretreatment were higher than those in the KOH treatment group.

[0070] The sugar production of corn straw after enzymatic hydrolysis with different soaking times under 1.00% NaOH is shown in the figure. Figure 5 As shown in the figure, glucose content increases with increasing soaking time, reaching its highest level at 10.87 g / L after 24 hours of soaking. Xylose content peaked at 15.52 g / L after 12 hours of soaking. However, reducing sugar content decreased with further soaking time. This is because, as soaking time increases, corn straw produces byproducts in an alkaline environment that inhibit subsequent fermentation, thereby affecting reducing sugar content. Therefore, a soaking time of 24 hours is preferred during pretreatment.

[0071] According to Example 2, a one-step conversion method was carried out to produce sophorolipids. The fermentation broth was sampled at regular intervals and the following indicators were mainly measured: residual glucose content, residual xylose content, and sophorolipid yield. The results are as follows Figure 6 As shown in the figure (A1~A4 are NaOH concentrations of 0.25%, 0.50%, 0.75% and 1.00%, respectively; B1~B4 are KOH concentrations of 0.25%, 0.50%, 0.75% and 1.00%, respectively), it can be seen that the content of sophorolipids produced by one-step conversion of corn straw pretreated with NaOH as a carbon source is higher. Among them, the sophorolipid content produced by one-step conversion after pretreatment with 1% NaOH concentration is the highest, reaching 52.31 g / L on the 4th day of fermentation, while the sophorolipid yields under one-step conversion after pretreatment with other concentrations of NaOH are all lower than those under pretreatment with 1% NaOH, indicating that pretreatment with 1% NaOH concentration is more conducive to the synthesis of sophorolipids. And from the overall detection, it can be seen that the first 3 days of one-step conversion are mainly the enzymatic hydrolysis period of corn straw. At the same time, the strain continuously uses glucose and a small amount of xylose for self-reproduction and fermentation to produce sophorolipids. As the reaction continues, the glucose is almost consumed on the 7th day, and the product yield decreases during the overall conversion process. It may be that the bacteria consume the product as a carbon source to promote growth.

[0072] After the fermentation was completed, the inhibitor content in the system was tested again, and the results are shown in Table 2.

[0073] Table 2:

[0074]

[0075] Through detection, it was found that the content of organic acid and furan inhibitors in the alkali pretreatment group did not change significantly, while the phenolic inhibitors were significantly reduced. This shows that the mutant strain U1-4 effectively utilized the phenolic inhibitors as a carbon source for fermentation during the one-step preparation of sophorolipids. In the NaOH pretreatment group, the content of phenolic substances was higher, which provided more carbon sources for the one-step sugar production and fermentation synthesis of sophorolipids by the mutant strain U1-4. It can be seen from the table that the content of phenolic inhibitors in the NaOH group decreased more significantly. It can be seen that the mutant strain U1-4 is not only more resistant to the toxic effects of phenolic inhibitors, but also inferred from the above results that although phenolic inhibitors have a certain inhibitory effect on the growth and metabolism of the mutant strain U1-4, in the composite system containing multiple inhibitors obtained by NaOH pretreatment, the mutant strain U1-4 can effectively utilize such inhibitors as a carbon source for fermentation, thereby making its synthesis of sophorolipids more efficient.

[0076] Example 3

[0077] The one-step method for preparing sophorolipids comprises the following steps:

[0078] Corn straw was soaked in a 1.0% NaOH aqueous solution for 24 h and sterilized at 121°C for 60 min to obtain a pretreatment mixture. The solid-liquid ratio of corn straw to NaOH aqueous solution was 1:10.

[0079] Without solid-liquid separation, the pH of the pre-treated mixture was directly adjusted to 2.0-5.0, and then 50 mM citric acid buffer with a pH of 3.0 was added and mixed, and then the complex enzyme, concentrated fermentation broth and Candida albicans ( Starmerella bombicola ) mutant strain, reacting at 32° C. and 160 rpm for 7 days, wherein the complex enzyme comprises cellulase, β-glucosidase, and xylanase in an enzyme activity ratio of 1:1:3, wherein the cellulase is added at a rate of 30 FPU / mL, the inoculum size of the Torulopsis mutant strain is 4%, the concentrated fermentation broth comprises 2% oleic acid, 0.3% yeast powder, 0.10% KH2PO4, 0.10% Na2HPO4·12H2O, and 0.05% MgSO4·7H2O, and the volume ratio of the liquid system, citric acid buffer, and concentrated fermentation broth is 50:2.5:1.

[0080] According to the one-step method in Example 3, the dynamic changes of glucose and xylose in the system and the yield of sophorolipids were as follows: Figure 7 It can be seen that at a pH of 3.0, the yield of sophorolipids was the highest, reaching 76.46 g / L.

[0081] About the optimization of complex enzymes:

[0082] Since the fermentation temperature of the mutant strain U1-4 is about 30°C, and the enzymatic activity of cellulase at this temperature is only 19.42%, other enzymes need to be combined with cellulase to improve the efficiency of enzymatic hydrolysis and sugar production. During the experiment, β-glucosidase, pectinase, and xylanase were used in different combinations with cellulase. The optimal reaction temperatures of the four enzymes were: cellulase 55°C; β-glucosidase 55°C; pectinase 45°C; and xylanase 30°C. After pretreatment with 1.0% NaOH, the enzymes and mutant strain U1-4 were added simultaneously for fermentation and enzymatic hydrolysis to test the effects of different complex enzymes on the final sophorolipid yield (the enzyme addition amounts involved in each group were: cellulase 30 FPU / mL, β-glucosidase 30 U / mL, pectinase 30 U / mL, and xylanase 90 U / mL). The anthrone reagent reaction method is a sugar determination method that can be used to determine the content of free monosaccharides and polysaccharides of various structures. After the reaction, they all show a blue-green color and have a maximum light absorption at a wavelength of 620 nm. The structure of sophorolipids contains sophorose formed by two molecules of glucose linked together. Therefore, the total sugar concentration can be obtained based on the glucose standard curve. The total sugar content in the supernatant is determined using the anthrone-sulfuric acid sugar determination method. According to the ratio between the molecular weights of sophorolipids and glucose, that is, 1g of glucose is equivalent to 1.91g of sophorolipids, the total sophorolipid content in the sample is calculated.

[0083] The formula for calculating sophorolipid yield is as follows: Sophorolipid content (g / L) = (total sugar concentration - residual sugar concentration) × 1.91. The maximum sophorolipid yield during the entire fermentation cycle (7 days) was calculated.

[0084] The formula for calculating cellulose conversion rate is as follows:

[0085]

[0086] The results are shown in Table 3.

[0087] Table 3:

[0088]

[0089] It can be seen that in the one-step preparation of sophorolipids, the glucose yield in the system with different complex enzymes for enzymatic hydrolysis is significantly different, and the efficiency of the final mutant strain U1-4 in fermenting sophorolipids is also significantly different. The content of sophorolipid produced by the mutant strain U1-4 in the single cellulase enzymatic hydrolysis system is 50.42 g / L. After compounding different enzymes on this basis, the sophorolipid yield has changed significantly, among which the sophorolipid yield in the cellulase + β-glucosidase system reached 55.76 g / L, while the sophorolipid yield in the cellulase + xylanase group was slightly lower than that of single cellulose. On this basis, further compounding, among which the mutant strain U1-4 had the highest efficiency in fermenting sophorolipids in the complex enzyme system consisting of cellulase + β-glucosidase + xylanase, reaching 76.46 g / L. After further adding pectinase on this basis, the glucose yield did not change much, but the sophorolipid yield decreased significantly. It can be seen that in the one-step preparation of sophorolipids, enzymatic sugar production and fermentation synthesis of sophorolipids are carried out simultaneously, and the difference in dynamic sugar production by different complex enzymes also has a significant effect on the efficiency of fermentation synthesis of sophorolipids by the mutant strain U1-4.

Claims

1. A method for preparing sophorolipids by a one-step conversion process, characterized in that: The method uses corn straw as raw material, obtains a pretreatment mixture by alkali pretreatment, and then adds citric acid buffer, complex enzyme, concentrated fermentation liquid and Candida albicans ( Starmerella bombicola ) mutant strain for fermentation, the alkaline pretreatment is to add corn straw into a NaOH aqueous solution with a mass concentration of 0.25-1.0% for soaking for 12-36 hours, the solid-liquid ratio of corn straw to NaOH aqueous solution is 1:8-12, the complex enzyme is cellulase, β-glucosidase and xylanase according to the enzyme activity ratio of 1:1-2:3, during the fermentation process, the temperature is maintained at 30-35 ° C, and the fermentation reaction is carried out at 150-180 rpm for 4-7 days, the mutant strain is used Starmerella bombicola The strain obtained by sequentially subjecting ATCC 22214 to ARTP mutagenesis and UV mutagenesis is classified as Candida albicans ( Starmerella bombicola )U1-4, deposited in China Center for Type Culture Collection (CCTCC), the deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, the deposit number is CCTCC NO: M2025692, and the deposit date is April 3, 2025.

2. The one-step method for preparing sophorolipids according to claim 1, wherein The steps include: Corn straw was immersed in a 0.25-1.0% NaOH aqueous solution for 12-36 h and sterilized at 121°C for 60 min to obtain a pretreatment mixture with a solid-liquid ratio of corn straw to NaOH aqueous solution of 1:8-12. Without performing solid-liquid separation, the pH of the pretreatment mixed liquid was directly adjusted to 3.0, a citric acid buffer with a concentration of 50 mM and a pH of 3.5 was added to form a liquid system, a composite enzyme, a concentrated fermentation broth and a mutant strain U1-4 were added, and the mixture was reacted at 32° C. and 160 rpm for 7 days, wherein the composite enzyme was composed of cellulase, β-glucosidase and xylanase in an enzyme activity ratio of 1:1:3, wherein the cellulase was added in an amount of 30 FPU / L, the inoculation amount of the mutant strain U1-4 was 3-6%, the concentrated fermentation broth was composed of 2% oleic acid, 0.3% yeast powder, 0.10% KH2PO4, 0.10% Na2HPO4·12H2O, and 0.05% MgSO4·7H2O, and the volume ratio of the liquid system, the citric acid buffer and the concentrated fermentation broth was 50:2.5:1.

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