L-sorbose-resistant associated bacterium Y31 and application thereof in fermentation of 2-keto-L-gulonic acid

Abstract

The invention relates to the technical field of microbial breeding and fermentation engineering, in particular to an L-sorbose-resistant associated bacterium Y31 and application thereof in fermentation of 2-keto-L-gulonic acid. The companion fungus Y31 is classified and named as bacillus endophyticus, and the preservation registration number of the companion fungus Y31 is CCTCC (China Center For Type Culture Collection) NO: M20251995. The strain can normally grow in a culture medium with high L-sorbose content, and can significantly improve the yield of 2-keto-L-gulonic acid (2-KLG) when mixed with acid-producing bacteria for fermentation, shorten the fermentation period and improve the production efficiency of the vitamin C precursor 2-keto-L-gulonic acid.

Description

Technical Field

[0001] This application relates to the field of microbial breeding and fermentation engineering technology, and in particular to a strain of L-sorbitol-tolerant commensal bacterium Y31 and its application in 2-keto-L-gulonic acid fermentation. Background Technology

[0002] Vitamin C (L-ascorbic acid) is an essential nutrient for the human body. Its industrial production mainly employs a two-step fermentation method, in which the second step of fermentation is carried out by common ketogenic gulonic acid bacteria (L-ascorbic acid bacteria). Ketogulonicigenium vulgare Small bacteria) and associated bacteria (such as Bacillus The process is completed jointly by the bacteria (including large bacteria). Although the symbiotic bacteria do not directly produce acid, they can provide the necessary symbiotic substances for the growth of acid-producing bacteria, significantly increasing the yield of 2-keto-L-gulonic acid (2-KLG).

[0003] However, the associated bacteria have poor tolerance to high concentrations of L-sorbose, limiting the increase in fermentation substrate concentration and thus affecting acid production efficiency. Existing technologies often employ methods such as ultraviolet light and chemical mutagenesis to improve strains, but these methods suffer from low mutation rates and poor stability. Non-thermal plasma (NTP) mutagenesis technology offers advantages such as high mutation rates, safe operation, and stable progeny, making it suitable for microbial breeding. Summary of the Invention

[0004] This application provides an L-sorbose-tolerant commensal bacterium and its application in the fermentation of 2-keto-L-gulonic acid, in order to solve the problem of poor tolerance of commensal bacteria to L-sorbose in related technologies.

[0005] In the first aspect, an L-sorbose-tolerant symbiotic bacterium Y31 is provided, which is classified as Bacillus endophyticus and was deposited at the China Center for Type Culture Collection on September 10, 2025, with the accession number CCTCCNO: M20251995.

[0006] Secondly, the application of the L-sorbitol-tolerant commensal bacterium Y31, as described above, in the fermentation of 2-keto-L-gulonic acid is provided.

[0007] Preferably, the application includes the following steps: L-sorbose-tolerant symbiotic bacteria Y31 and Ketogulonicigenium vulgare L4 were mixed and fermented in a mixed culture medium comprising: 8-15% L-sorbose, 0.8-1.6% urea, 1.1-1.9% corn steep liquor, 0.3-1.1% CaCO3, 0.005-0.025% MgSO4, with the remainder being water.

[0008] Preferably, the mixed fermentation conditions are that the liquid volume is 4.0-12.0%, the initial pH is 6.4-7.6, the temperature is 25-33 DEG C, and the inoculation amount is 5-25%.

[0009] The beneficial effects brought by the technical scheme provided in the application include: The application provides a L-sorbose-tolerant accompanying strain Y31 and application of the strain in 2-keto-L-gulonic acid fermentation. Bacillus endophyticus B0 is mutagenized, and a mutant strain Y31 tolerating high-concentration L-sorbose is screened. The strain can grow normally in a 15% L-sorbose culture medium, can significantly improve the 2-KLG yield when mixed fermentation is performed with an acid-producing strain L4, shortens the fermentation period, has good genetic stability, and can improve the production efficiency of a vitamin C precursor 2-keto-L-gulonic acid. BRIEF DESCRIPTION OF DRAWINGS

[0010] In order to more clearly illustrate the technical scheme in the embodiments of the application, the drawings needed in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the application, and other drawings can be obtained by those skilled in the art without creative labor.

[0011] Figure 1 Effects of different mutagenic voltages provided in the application on lethality; Figure 2 Effects of different mutagenic times provided in the application on lethality; Figure 3 A schematic diagram of effects of different L-sorbose concentrations provided in the application on mutagenized accompanying strains; Figure 4 A schematic diagram of effects of different L-sorbose concentrations provided in the application on mutagenized accompanying strains; Figure 5 A schematic diagram of L-sorbose tolerance of accompanying strains (mutagenized strain B0 and mutagenized strain Y31) provided in the application; Figure 6 Growth curves of B0 and Y31 in culture media with different L-sorbose concentrations provided in the application; Figure 7 Colony morphology and gram staining (10x100 times) of B0 and Y31 provided in the application; Figure 8 A broken line graph of 2-KLG production capacity of B0 and Y31 in 8% L-sorbose mixed fermentation culture medium provided in the application; Figure 9The B0 and Y31 provided in the present application in the mixed fermentation medium with different concentrations of L-sorbose, the yield of 2-KLG is shown in the schematic diagram; Figure 10 The L-sorbose concentration provided in the present application has an effect on the production of 2-KLG by Y31, which is shown in the schematic diagram; Figure 11 The urea concentration provided in the present application has an effect on the production of 2-KLG by Y31, which is shown in the schematic diagram; Figure 12 The corn steep liquor concentration provided in the present application has an effect on the production of 2-KLG by Y31, which is shown in the schematic diagram; Figure 13 The CaCO3 concentration provided in the present application has an effect on the production of 2-KLG by Y31, which is shown in the schematic diagram; Figure 14 The MgSO4 concentration provided in the present application has an effect on the production of 2-KLG by Y31, which is shown in the schematic diagram; Figure 15 The response surface and contour plot of the interaction of each factor (L-sorbose, urea and corn steep liquor) on the acid production is shown in the schematic diagram provided in the present application; Figure 16 The liquid volume has an effect on the production of 2-KLG in the mixed fermentation of Y31 and L4, which is shown in the schematic diagram provided in the present application; Figure 17 The inoculum has an effect on the production of 2-KLG in the mixed fermentation of Y31 and L4, which is shown in the schematic diagram provided in the present application; Figure 18 The initial pH has an effect on the production of 2-KLG in the mixed fermentation of Y31 and L4, which is shown in the schematic diagram provided in the present application; Figure 19 The temperature has an effect on the production of 2-KLG in the mixed fermentation of Y31 and L4, which is shown in the schematic diagram provided in the present application; Figure 20 The response surface and contour plot of the interaction of each factor (liquid volume, initial pH and temperature) on the acid production is shown in the schematic diagram provided in the present application; Figure 21 The stability of Y31 in the continuous passage for the production of 2-KLG is shown in the schematic diagram provided in the present application. DETAILED DESCRIPTION

[0012] In order to make the purpose, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0013] Reference is made toFigures 1-21 As shown, the present application provides a L-sorbose-tolerant companion bacteria Y31 and its application in 2-keto-L-gulonic acid fermentation.

[0014] Some of the materials and reagents used in the following examples are as follows: Ketogulonicigenium vulgare L4 (commonly known as acid-producing bacteria, small bacteria), hereinafter referred to as L4. Bacillus endophyticus B0 (commonly known as companion bacteria, large bacteria), hereinafter referred to as B0.

[0015] Yeast extract powder, beef extract, urea, magnesium sulfate, iodine, potassium iodide, ferrous sulfate, Guangdong Huanke Biological Technology Co., Ltd.; agar, L-sorbose, calcium carbonate, potassium dihydrogen phosphate, hydrochloric acid, concentrated sulfuric acid, National Pharmaceutical Group Chemical Reagent Co., Ltd.; soluble starch, glucose, protein peptone, Tianjin Huihang Chemical Technology Co., Ltd.

[0016] Some of the culture media used and the preparation solutions are as follows (in the culture medium, % is the concentration of each component in water): (1) Seed culture medium (%): glucose 0.2, L-sorbose 2.0, urea 0.1, corn syrup 0.5, calcium carbonate 0.2, pH 6.7-7.0. 121℃, 20min sterilization.

[0017] (2) Solid separation culture medium (%): yeast extract 0.3, beef extract 0.3, corn syrup 0.3, protein peptone 1.0, potassium dihydrogen phosphate 0.1, magnesium sulfate 0.02, urea 0.1, calcium carbonate 0.1, L-sorbose 2.0, agar 2.3, 10ppm ferrous sulfate 0.1mL / L, pH 6.7-7.0. 121℃, 20min sterilization.

[0018] (3) Mixed bacteria fermentation medium (%): L-sorbose 8.0~15.0, corn syrup 1.1~1.9, calcium carbonate 0.3~1.1, potassium dihydrogen phosphate 0.1, magnesium sulfate 0.005~0.025, urea 0.8~1.6, 10ppm ferrous sulfate 0.1mL / L, pH 6.7-7.0 (sorbitol and urea are sterilized separately, and other culture medium components are added after being dissolved and pH adjusted). 121℃, 20min sterilization.

[0019] (5) 0.1% starch: 1g starch is added to water and boiled with stirring until dissolved, and then made up to 100mL.

[0020] It should be noted that the above culture medium is only part of the culture medium used in the examples, and some of the components (such as L-sorbose) therein can be adjusted according to actual needs.

[0021] 2-Keto-L-gulonic acid was obtained by mixing L-sorbitol-tolerant commensal bacteria Y31 and Ketogulonicigenium vulgare L4 in a mixed culture fermentation medium.

[0022] The specific strain cultivation method is as follows: (1) Preparation of separation plates: Activate the bacterial culture in cryovials stored at -80℃ to liquid culture medium. After shaking and incubating at 29℃ for 1-2 days, if the associated bacteria and acid-producing bacteria appear normal under a microscope, the bacteria can be isolated. Spread the activated bacterial culture onto solid isolation medium plates and incubate at 29℃ for 4-5 days. Then store in a 4℃ refrigerator for later use.

[0023] (2) Preparation of bacterial suspensions of acid-producing bacteria and associated bacteria: Associated bacteria: Select a loopful of a single colony, place it in 2 mL of sterile water, shake well, and prepare an associated bacteria suspension.

[0024] Acid-producing bacteria: Select a number of single-colony acid-producing bacteria, place them in 3 mL of sterile water, shake well, and the concentration of acid-producing bacteria is determined by the milky white color of the bacterial suspension.

[0025] Acid-producing bacteria and associated bacteria mixed suspension: Pick 1-2 loops of associated bacteria suspension and mix them with acid-producing bacteria suspension.

[0026] (3) Preparation of seed solution: Take 4 mL of the mixed bacterial suspension of acid-producing bacteria and associated bacteria into a 250 mL Erlenmeyer flask with a volume of 20 mL, and incubate at a constant temperature of 29 °C with shaking for 20-24 h. When the acid content of the inoculum reaches 5 g / L or more, it is considered a qualified inoculum and can be inoculated into the mixed bacterial fermentation medium.

[0027] (4) Isolation of acid-producing bacteria and associated bacteria: Take the two-step seed culture medium, dilute it appropriately, spread it on isolation plates, and incubate at 29℃ for 4-5 days to grow single colonies of acid-producing bacteria and associated bacteria. Pick and preserve the single colony slants of acid-producing bacteria and associated bacteria separately.

[0028] Example 1: Mutagenesis and screening of L-sorbitol-resistant symbiotic bacteria Y31 Mutagenesis of Bacillus endophyticus B0 (B0) bacterial culture was induced using an atmospheric pressure dielectric barrier discharge plasma jet (APPJ) mutagenesis system with a voltage of 12000 V and a treatment time of 110 s. Place the bacterial culture container onto the coated quartz plate of the mutagen to induce mutagenesis. Adjust the sample stage knob to set the processing time and voltage, and begin sample processing. Store the mutagenized bacterial culture in a sterilized EP tube.

[0029] Lethality rate calculation: the above mutagenized bacteria were diluted at different gradients and plated on solid separation medium and cultured at 29°C for 48h, and then the plate count was performed. The control group was the dilution of the bacteria without mutagenesis treatment. The lethality rate was calculated according to the following formula:

[0030] Different plasma mutagenesis voltages had a significant effect on the lethality rate of the original strain B0, and the results are shown in Figure 1 and Figure 2 . Referring to Figure 1 , when the voltage was 1000V, the lethality rate was 48.95%±4.67%, when the voltage was 11000V, the lethality rate was 76.30%±2.22%, when the voltage was 12000V, the lethality rate was 94.61%±0.44%, when the voltage was 13000V, the lethality rate was 98.16%±0.96%, and when the voltage was 14000V, the lethality rate was 98.63%±0.72%. By variance analysis, it was found that mutagenesis at a voltage higher than 12000V could significantly improve the lethality rate (P<0.05). However, when the voltage increased from 12000V to 13000V and 14000V, the lethality rate did not change significantly (P>0.05).

[0031] Referring to Figure 2 , different plasma mutagenesis times had a significant effect on the lethality rate of the strain B0. When the time was 90s, the lethality rate was 84.37%±0.45%, when the time was 110s, the lethality rate was 94.61%±0.44%, when the time was 130s, the lethality rate was 96.41%±0.18%, when the time was 150s, the lethality rate was 97.98%±0.29%, and when the time was 170s, the lethality rate was 98.98%±0.50%. By variance analysis, it was found that there were significant differences between each mutagenesis time group (P<0.05). The study believed that the lethality rate of mutagenesis was best when it was 90~95%. Therefore, the following examples selected 110s as the best mutagenesis time for the strain and selected 12000V as the best mutagenesis voltage.

[0032] Screening: the mutagenized bacteria were diluted at an appropriate concentration and plated on a screening medium (solid separation medium containing 15% L-sorbitol) and cultured at 29°C for 48h, and then the strains cultured on the solid separation medium were inoculated into a mixed bacteria fermentation medium. The accompanying bacteria that could normally grow in the mixed bacteria fermentation medium with 15% L-sorbitol were selected as high-sugar-tolerant strains.

[0033] The mutant strains on solid isolation medium were inoculated into mixed fermentation media containing 8% and 15% L-sorbose, respectively, and their OD values ​​were measured after 12 h of cultivation. Strains with OD values ​​significantly different from those of B0 (P<0.05) were considered mutants tolerant to high concentrations of L-sorbose. The results are as follows: Figure 3 As shown, the OD values ​​of A3 and Y31 in 8% L-sorbitol medium were significantly higher than those of B0 (P<0.05). Figure 3 a and Figure 3 c). Strains A5, B11, B17, B18, B19, B20, and C35 showed significantly lower levels of B0 in 8% L-sorbitol medium (P<0.05). Figure 3 a, Figure 3 b and Figure 3 c). In high-concentration (15%) L-sorbose medium, only Y31 had a significantly higher OD value than the original strain (P<0.05). Figure 3 c).

[0034] Further screening was conducted on mutant strains (Y31, A3, A5, and C35) that showed significant L-sorbose tolerance compared to the original strain. Figure 4 As shown, the OD values ​​of Y31 in 8% and 15% L-sorbate media were significantly higher than those of B0 (P<0.05). While strain A3 showed significant growth in 8% L-sorbate medium (P<0.05), it exhibited poor tolerance to 15% L-sorbate medium (P>0.05). A5 and C35 showed significantly lower growth rates than the original strain in 8% L-sorbate medium (P<0.05), indicating no application potential. Therefore, Y31 demonstrated stronger L-sorbate tolerance, and was selected as the application material for subsequent examples.

[0035] It should be noted that, Figure 3 and Figure 4 In the diagram, CK represents the blank culture medium control group, and B0 represents the pre-mutation strain. Uppercase letters indicate the analysis of variance between groups using 8% L-sorbose culture medium, and lowercase letters indicate the analysis of variance between groups using 15% L-sorbose culture medium. In subsequent examples, B0 will refer to the pre-mutation strain.

[0036] Example 2: Physiological and biochemical identification of L-sorbitol-resistant commensal bacterium Y31 According to the Manual of Systematic Bacteriology, partial physiological and biochemical tests were performed on Y31 and B0 (glucose acid and gas production test, beta-galactose test, citrate test, gelatin liquefaction test, starch hydrolysis test, hydrogen sulfide test, sucrose test, maltose test, nitrate test and lactose test). The results are shown in Table 1. The characteristics of Y31 were basically the same as those of B0. Both Y31 and B0 were positive in glucose, beta-galactose, citrate and gelatin liquefaction tests. The strains could not hydrolyze starch, and were negative in hydrogen sulfide, sucrose, maltose, nitrate and lactose tests, indicating that the mutation did not change the basic metabolic type.

[0037] Table 1. Physiological and biochemical identification

[0038] In Table 1, + is positive and - is negative.

[0039] In addition, the gene sequence of Y31 (SEQ ID No. 1) is as follows:

[0040] Example 3 Evaluation of L-sorbose tolerance of L-sorbose-resistant companion bacteria Y31 For the tolerance of Y31 to L-sorbose, Y31 was cultured at L-sorbose concentrations of 5%, 8%, 11%, 14%, and 17%, respectively, and the OD value after 12 hours of culture was used to evaluate the tolerance of Y31 to L-sorbose, with the results shown in Table 1. Figure 5 The OD value of B0 decreased with the increase of L-sorbose concentration, and the OD value of Y31 increased first and then decreased with the increase of L-sorbose concentration. Different companion bacteria have different degrees of tolerance to L-sorbose. B0 can tolerate 11% L-sorbose concentration, and Y31 can grow normally in a 14% L-sorbose culture medium, which is significantly higher than the L-sorbose tolerance of B0 (P<0.05).

[0041] Example 4 Growth characteristics of L-sorbose-resistant companion bacteria Y31 The preserved strains were inoculated into seed culture medium for activation and cultured at 29°C for 24 h. The OD value was measured by an enzyme marker, and the OD value was measured every 2 h. The blank mixed bacteria fermentation medium (without inoculation of strains) was used as a control, and the measurement was continuously performed for 24 h. According to the measured results, the growth curve of the strains was drawn. 650 nm, and the OD value was measured every 2 h. The blank mixed bacteria fermentation medium (without inoculation of strains) was used as a control, and the measurement was continuously performed for 24 h. According to the measured results, the growth curve of the strains was drawn.

[0042] This example studies the growth rules of the companion bacteria before and after mutagenesis by drawing the growth curve, with the results shown in Table 2. Figure 6 The growth curves of Y31 and B0 in the mixed bacteria fermentation medium with different L-sorbose concentrations were measured, and the OD value was measured every 2 h. In the L-sorbose culture medium with a concentration of 8%, the first 4 h of Y31 and B0 were the lag phase. The period from 4 h to 10 h was the logarithmic growth phase of B0, and the period from 4 h to 14 h was the logarithmic growth phase of Y31. The period from 10 h to 16 h was the stationary phase of B0, and the period from 14 h to 18 h was the stationary phase of Y31. After 16 h of culture, B0 entered the decline phase, and after 18 h, Y31 entered the decline phase. In the L-sorbose culture medium with a concentration of 15%, the first 4 h of Y31 was the lag phase, the period from 4 h to 10 h was the logarithmic growth phase of B0, the period from 10 h to 14 h was the stationary phase of B0, and after 14 h of culture, Y31 entered the decline phase.

[0043] Example 5 Cell and colony morphology of L-sorbose-resistant companion bacteria Y31 The cell and colony morphology of the two companion bacteria (B0 and Y31) before and after mutagenesis were observed. The morphological characteristics of the two strains before and after mutagenesis were smooth surface, round protrusions, white opaque strains, and no obvious changes, as shown in Table 3. Figure 7

[0044] It should be noted that, Figure 7 ​(a) indicates Gram staining of B0. Figure 7 (b) shows the colony morphology of plate B0. Figure 7 (c) indicates Gram staining of Y31. Figure 7 (d) indicates the plate colony morphology of Y31.

[0045] Example 6: Application of mixed-culture fermentation Two strains (B0 and Y31) before and after mutagenesis were mixed with acid-producing bacterium L4 for co-fermentation to evaluate their 2-KLG production performance. Figure 8 As shown, in the mixed fermentation medium with 8% L-sorbose, strain Y31 stopped producing 2-KLG at 48 h, with a yield of 55.38 mg / mL. strain B0 stopped producing 2-KLG at 50 h, with a yield of 54.10 mg / mL. Y31 reached the fermentation endpoint 2 h earlier than B0, indicating a faster fermentation rate. This demonstrates that Y31 has a stronger acid-producing capacity, a faster fermentation rate, and a higher acid production.

[0046] Example 7 Effect of different concentrations of L-sorbose on the production of 2-KLG by mixed fermentation of Y31 and L4 To determine the optimal L-sorbose concentration for mixed-culture fermentation of the mutagenized strains, inoculum solutions of the two strains, before and after mutagenesis, were prepared and inoculated into mixed-culture fermentation media with different concentrations of L-sorbose. Specifically, inoculum solutions of the two strains were prepared separately, and 15% of the qualified inoculum solutions of the two strains were inoculated into mixed-culture fermentation media with L-sorbose concentrations of 8%, 10%, 12%, 14%, and 16%, respectively, and cultured at 29°C and 180 rpm / min for 48 h in a shaker.

[0047] like Figure 9 As shown, the yield of 2-KLG produced by B0 decreased with increasing L-sorbate concentration. This indicates that B0 is intolerant to L-sorbate, and the higher the L-sorbate concentration, the lower the acid production. The mutant strain Y31 produced the highest amount of 2-KLG (57.43 mg / mL) in the 10% L-sorbate mixed fermentation medium, significantly higher than other concentrations (P<0.05), and significantly higher than the B0 mixed fermentation (P<0.05). Subsequently, with further increases in L-sorbate concentration, the acid production decreased significantly (P<0.05), indicating that 10% L-sorbate concentration is the optimal concentration for 2-KLG production by Y31 mixed fermentation, representing a 2% improvement over the starting strain.

[0048] It should be noted that, Figure 9 In the chart, uppercase letters represent B0 in the analysis of variance among the L-sorbitol concentration groups, and lowercase letters represent Y31 in the analysis of variance among the L-sorbitol concentration groups.

[0049] Furthermore, the cultivation conditions were optimized.

[0050] For mixed fermentation medium, the optimization process includes single factor test, Plackett-Burman test (PB), steepest ascent test and response surface analysis.

[0051] Single factor test: Based on the initial mixed fermentation medium, the effects of the concentrations of main nutrients (L-sorbose, urea, corn steep liquor, calcium carbonate and magnesium sulfate) in the mixed fermentation medium on the fermentation of the strains were investigated with the yield of 2-KLG as the main index. Plackett-Burman test (PB): PB test was designed by using Design-expert, and the test with N=12 was created. Five factors, including the concentrations of L-sorbose (A, %), urea (B, %), corn steep liquor (C, %), MgSO4 (D, %) and CaCO3 (E, %), were investigated. The effect of each factor on the yield of 2-KLG was investigated with the yield of 2-KLG as the response value. Steepest ascent test: After the PB test, the three factors with the most significant effects were screened. The gradient direction presented by the change of the effect value was used as the clear climbing direction. By considering the specific size of the positive and negative effect values of each factor, the step size of each factor was determined, and the steepest ascent test was carried out. Response surface analysis: 3-factor 3-level response surface test was carried out by using Design-expert software. During the test, the yield of 2-KLG was used as the response value index. Through the analysis of the test data under different factor level combinations, the optimal conditions were determined to realize the optimization control and improvement of the fermentation process.

[0052] Single factor test: The addition amount of L-sorbose had a significant effect on the acid production (P<0.05). When the initial concentration of L-sorbose was 10%, the acid production was the highest (see Table 1, 57.43 mg / mL). Therefore, the concentration of 10% L-sorbose was selected as the optimal substrate concentration. Different concentrations of L-sorbose affected the size of the acid production. When the concentration of L-sorbose was too high, the growth of the associated bacteria was inhibited. Figure 10 When the urea concentration was 1%, the acid production reached the highest value (see Table 2, 58.20 mg / mL). When the urea concentration was lower than 1%, the reaction did not reach the endpoint. When the urea concentration was higher than 1%, it would inhibit the growth of the bacteria. Therefore, 1% was selected as the optimal urea concentration. There was a significant correlation between the urea concentration in the medium and the acid production (P<0.05). The yield of 2-KLG increased first and then decreased with the increase of the urea concentration.

[0053] Figure 11

[0054] ​​There was a significant correlation between corn steep liquor concentration and acid production (P<0.05). The highest 2-KLG production was observed when the corn steep liquor concentration was 1.7% (see [reference needed]). Figure 12 (59.48 mg / mL). Corn steep liquor serves as a major nutrient in mixed-culture fermentation media, providing not only the carbon and nitrogen sources required for bacterial fermentation, but also essential amino acids and bioactive substances.

[0055] The acid production is highest when the CaCO3 concentration is 0.5% (see [reference]). Figure 13 (60.51 mg / mL). When the CaCO3 concentration is too low, it is difficult to effectively neutralize the produced acid, and an overly acidic culture medium is not conducive to the growth of associated bacteria. Conversely, when the CaCO3 concentration is too high, it inhibits bacterial growth, interferes with bacterial physiological activities, leads to reduced acid production, and affects the overall fermentation effect. Therefore, the optimal CaCO3 concentration is 0.5%.

[0056] The highest acid production was observed in mixed-culture fermentation when the MgSO4 concentration was 0.010% (see [reference]). Figure 14 (61.02 mg / mL). High concentrations of MgSO4 activate 2-KLG reductase, prompting further reduction of 2-KLG to L-iduronic acid. This conversion not only directly leads to a decrease in acid production but also results in the generation of more byproducts during metabolism.

[0057] Plackett-Burman experiment: Based on the results of the single-factor experiments above, L-sorbose, urea, corn steep liquor, CaCO3 and MgSO4 were selected as Plackett-Burman experimental factors for the experiment. The specific coding and design are shown in Table 2, and the experimental design and results are shown in Table 3.

[0058] Table 2 Plackett-Burman design factor levels and coding values

[0059] Table 3 Plackett-Burman Experimental Design and Results

[0060] The analysis results are shown in Table 4. The data in Table 4 shows that the p-value of the model is 0.0007, proving that the model has a good fit. From the correlation coefficient, R... 2 The value is 0.9525, indicating a good correlation between the model and the actual data. 2adj is 0.9130, meaning that 91.30% of the experimental data can be reasonably explained, fully embodying the effectiveness of the model. Generally, the smaller the coefficient of variation (CV), the higher the reliability and accuracy of the test. The CV in this test was 2.38%, further confirming the stability and accuracy of the test results. When the precision (Adeq Precision) is greater than 4.0, the model is considered reasonable, and the precision value in this test is 15.4502.

[0061] Through multiple regression analysis method, the regression equation model is obtained: Y = 53.46 + 3.90A + 1.47B + 1.58C - 0.03D - 0.42E Table 4 Plackett-Burman test significance analysis

[0062] Steep climb test: Considering the workload and production cost, the concentration of L-sorbose is 1%, urea is 0.04%, and corn syrup is 0.04%. The test design and results are shown in Table 5. The acid production of treatment 3 reaches the maximum value. Therefore, treatment 3 is used as the center point of the response surface test for research.

[0063] Table 5 Steepest ascent test design and results

[0064] In Table 5, Δ represents a step unit.

[0065] Box-Behnken response surface test: According to the above steepest ascent test, treatment 3 is used as the center point for Box-Behnken test. A, B, and C are used as independent variables, and the specific coding is shown in Table 6. According to the Box-Behnken design, a 3-factor 3-level test is established, and the acid production is used as the response value. The test design and results are shown in Table 7.

[0066] Table 6 Box-Behnken design factor levels and coding values

[0067] Table 7 Box-Behnken test design and results

[0068] The polynomial equation is: Y = 57.43 + 3.09A + 1.84B + 2.66C - 0.48AB + 0.64AC + 0.25BC - 6.11A 2 -0.66B2 -1.33C 2 , R 2 =0.9700, which indicated that the model fitted the actual situation well. The analysis of variance of response surface experiment is shown in Table 8.

[0069] Table 8 Analysis of variance of Box-Behnken quadratic model

[0070] According to the regression equation, the response surface graph and the contour graph under the interaction of each factor were drawn (Fig. 1, Fig. 2, Fig. 3 and Fig. 4), which could reflect the influence of L-sorbose, urea and corn syrup on the production of 2-KLG. Figure 15

[0071] Using Design-Expert 13 to predict the optimal conditions of mixed fermentation medium, the optimal conditions were L-sorbose 10.277%, urea 0.91106% and corn syrup 1.61912%. Under these conditions, the acid production was 60.623 mg / mL.

[0072] For the fermentation conditions, the optimization process included single factor experiment, Plackett-Burman experiment (PB), steepest ascent experiment and response surface analysis.

[0073] Among them, the single factor experiment: taking the production of 2-KLG as the main index, on the basis of the initial mixed fermentation medium, the influence of fermentation conditions (liquid loading, inoculum size, initial pH and temperature) on strain fermentation was explored.

[0074] Plackett-Burman experiment: using Design-expert to design PB experiment, creating an experiment with N=12, investigating 5 factors of liquid loading (A, %), inoculum size (B, %), initial pH (C) and temperature (D, ℃), and taking the production of 2-KLG as the response value to investigate the influence of each factor on the production of 2-KLG.

[0075] The process of steepest ascent experiment and response surface analysis refers to the optimization process of mixed fermentation medium.

[0076] Single factor experiment: Y31 and L4 are both aerobic bacteria, and their growth and metabolism are oxygen-consuming processes. When the liquid loading is 8%, the acid production reaches the highest value (see Fig. 5, 57.17 mg / mL). When the liquid loading is too small, the nutrients in the medium are difficult to meet the needs of the bacteria. When the liquid loading is too large, the oxygen in the flask is insufficient, which inhibits the metabolic activity of the bacteria and hinders the metabolic process. Figure 16

[0077] ​​Inoculum size plays an important role in acid production during mixed fermentation. To ensure the acid production rate of mixed fermentation, it is particularly important to control the inoculum size. The inoculum size and acid production have a significant correlation (P < 0.05). With the increase of inoculum size, the acid production first increases and then decreases. When the inoculum size is 15%, the 2-KLG production is the highest (see Table 1, 57.43 mg / mL). Figure 17

[0078] When the initial pH is 6.7, the acid production is the highest (see Table 2, 59.35 mg / mL). When the initial pH is too high, the alkaline condition inhibits the growth, physiological activity and metabolic function of the bacteria, which is not conducive to the acid production of the bacterial system. When the initial pH is too low, the environment is acidic, and the acidic substances produced during the mixed fermentation cannot be effectively neutralized, which will continuously reduce the pH of the fermentation broth. The over-acid environment is not conducive to the growth and metabolic activity of the associated bacteria. Figure 18 Temperature has a significant effect on acid production (P < 0.05), and the acid production is the best at 29°C (see Table 3, 59.74 mg / mL). When the environmental temperature is low, the physiological activity of the associated bacteria is inhibited, and their growth rate slows down. When the environmental temperature is too high, it will inhibit the growth of the acid-producing bacteria, hinder the normal metabolism and reproduction of the acid-producing bacteria. Considering the different needs of the associated bacteria and the acid-producing bacteria for temperature, the application preferentially selects 29°C as the best culture temperature.

[0079] Figure 19 Plackett-Burman test: Based on the test results of the single-factor fermentation conditions, the liquid loading, initial pH and temperature were selected as the key factors of the Plackett-Burman test. The specific coding and design scheme of each factor are shown in Table 9, and the test design and results are shown in Table 10.

[0080] Table 9 Plackett-Burman design factor level and coding value

[0081]

[0082] Table 10 Plackett-Burman test design and results

[0083] From the analysis data in Table 11, the P value of the model is 0.0007, indicating that the model fitting degree is good. R 2 is 0.9163, representing good correlation. R 2 ​​​adj = 0.8685, meaning 86.85% of the experimental data can be explained by the regression model. The CV in this experiment was 2.32%, reflecting the reliability of the Plackett-Burman experiment. In addition, an Adeq Precision value greater than 4.0 is considered reasonable, and the value in this experiment was 12.8463.

[0084] Table 11 Plackett-Burman experiment significance analysis

[0085] The regression equation was obtained by fitting the data by multiple regression: Y = 53.44 + 1.20A - 0.58B - 1.60C + 2.35D Steep climb experiment: The temperature was 1°C, the liquid loading was 0.5%, and the initial pH was 0.1. The experimental design and results are shown in Table 12. From the results, the acid production of treatment 3 reached the maximum value. Therefore, treatment 3 was used as the center point of the response surface experiment for subsequent experiments.

[0086] Table 12 Steepest ascent experiment design and results

[0087] In Table 12, Δ represents a step unit.

[0088] Box-Behnken response surface method experiment: According to the results of the steepest ascent experiment in 3.5.3, the liquid loading, initial pH, and temperature were used as independent variables, and the specific coding is shown in Table 13. A 3-factor 3-level experiment was designed with 2-KLG production as the response value. The experimental design and results are shown in Table 14.

[0089] Table 13 Box-Behnken design factor levels and coding values

[0090] Table 14 Box-Behnken experiment design and results

[0091] The Box-Behnken experiment data was fitted by quadratic multiple regression to obtain the polynomial equation: Y = 59.65 + 1.19A - 1.41C + 2.28D + 0.25AC - 0.45AB + 0.38CD - 2.23A 2 -1.02C 2 -0.31D 2 .

[0092] R 2 0.9456, the experimental results can be analyzed by the equation, and the results of variance analysis of the response surface experiment are shown in Table 15.

[0093] Table 15 Variance analysis of the quadratic model of the Box-Behnken experiment

[0094] As can be seen from Table 15, the model has significance (P < 0.05), R 2 = 0.9456, R 2 adj= 0.8477, indicating that the model can explain 84.77% of the change of the response value. As can be seen from the results of Table 15, the first-order terms A, C, D and the second-order terms A 2 have a significant effect on the acid production (P < 0.05). The degree of influence of each factor on the acid production is ranked as follows: D > C > A.

[0095] According to the equation, the response surface graph and the contour graph of the interaction of each factor (see Figure 20 ) can be drawn, which can reflect the influence of the liquid loading, the initial pH and the temperature on the production of 2-KLG.

[0096] The Design-Expert 13 software is used to optimize the medium conditions, and the optimal conditions are obtained as follows: the liquid loading is 7.11%, the initial pH is 6.856, and the temperature is 29.928℃. Under the above conditions, the acid production is 65.657 mg / mL.

[0097] Example 8 Verification of the optimal culture conditions of mixed bacteria fermentation In view of the feasibility of the actual operation, the optimal parameters are adjusted as follows: the liquid loading is 7.1%, the initial pH is 6.8~6.9, and the temperature is 29~30℃. Using the above parameters for fermentation, the acid production is 64.86±0.48 mg / mL, which is close to the predicted value. It is proved that the response surface method is reliable for the optimization result and has practical value.

[0098] Example 9 Stability of L-sorbose-resistant companion bacteria Y31 in producing 2-KLG The passage stability of the mutagenized strain Y31 is detected to ensure the quality of the mixed bacteria fermentation system. The Y31 is continuously passed on the solid separation medium for 5 times, and the companion bacteria Y31 and the acid-producing bacteria L4 of each generation are made into qualified seed liquid, which is inoculated into the mixed bacteria fermentation medium, and the production of 2-KLG of each generation of companion bacteria Y31 and acid-producing bacteria L4 is measured as shown in Figure 21 . The mixed bacteria fermentation system of the mutagenized strain has good stability, and the acid production is 63.46 mg / mL, which indicates that Y31 has good industrial application potential.

[0099] In summary, the application provides the L-sorbitol-resistant accompanying bacteria Y31 and its application in 2-keto-L-gulonic acid fermentation. The preferred mutagenesis condition of Y31 is 12000V voltage and 110s time, under which the lethality rate is 94.61%, and the mutant strain Y31 is screened under the condition. Y31 has improved tolerance to L-sorbitol and can grow normally in a 15% L-sorbitol fermentation medium. Meanwhile, the acid production performance of Y31 strain is significantly improved, and the conversion rate is faster, which is 2h faster than that of B0. The carbon source utilization is consistent with that of B0. In addition, the strain Y31 obtained after mutagenesis is continuously passed for 5 times, and after mixed fermentation, the average acid production is 63.46mg / mL, indicating that the bacterial system has good stability.

[0100] The above description is merely a specific implementation of the application, which enables those skilled in the art to understand or implement the application. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the application. Therefore, the application will not be limited to these embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A L-sorbose tolerant companion Y31, characterized in that: The classification name is Bacillus endophyticus , and was preserved in China Center for Type Culture Collection on September 10, 2025, with a preservation registration number of CCTCC NO: M20251995.

2. The application of the L-sorbose-tolerant accompanying bacteria Y31 in the fermentation of 2-keto-L-gulonic acid according to claim 1.

3. Use according to claim 2, wherein the compound is ###0002### The application comprises the following steps: After mixed fermentation of L-sorbose-tolerant accompanying bacteria Y31 and Ketogulonicigenium vulgare L4 in a mixed fermentation medium, 2-keto-L-gulonic acid was obtained. The mixed bacterial fermentation medium comprises 8-15% of L-sorbose, 0.8-1.6% of urea, 1.1-1.9% of corn syrup, 0.3-1.1% of CaCO3, 0.005-0.025% of MgSO4, and the rest is water.

4. The application according to claim 3, characterized in that: The mixed fermentation conditions are as follows: liquid loading amount of 4.0-12.0%, initial pH of 6.4-7.6, temperature of 25-33℃, and inoculum amount of 5-25%.

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