Application of Bacillus licheniformis WX-02 in promoting DNJ production by Bacillus amyloliquefaciens HZ-12 in soybean solid fermentation
By using mixed fermentation of Bacillus licheniformis WX-02 and Bacillus amyloliquefaciens HZ-12 and optimizing culture medium and conditions, the problem of low DNJ yield in microbial fermentation was solved, and efficient DNJ production was achieved.
Patent Information
- Application Number
- CN202510111606.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-23
- Publication Date
- 2025-09-30
- Estimated Expiration
- 2045-01-23
AI Technical Summary
The existing microbial fermentation synthesis of 1-deoxynojirimycin (DNJ) has the problems of low yield and long fermentation time.
Bacillus licheniformis WX-02 and Bacillus amyloliquefaciens HZ-12 were mixed and inoculated into soybean solid fermentation medium, and the inoculation ratio and culture conditions, including fermentation time, temperature and medium composition, were optimized for mixed fermentation.
The yield of DNJ was significantly increased to 512 mg/kg, which was 35.45% higher than that of inoculating Bacillus amyloliquefaciens HZ-12 alone. The process is simple and easy to scale up for production.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of fermentation engineering, and particularly relates to the application of Bacillus licheniformis WX-02 in promoting the production of DNJ by Bacillus amyloliquefaciens HZ-12 soybean solid fermentation. Background Art
[0002] 1-Deoxynojirimycin (DNJ for short) is a polyhydroxypiperidine alkaloid with a similar structure to many sugar substances. It can bind to or near the active center of α-glucosidase and interfere with the interaction between the substrate and the enzyme. Therefore, DNJ has strong α-glucosidase inhibitory activity and is a natural hypoglycemic active ingredient. In addition, a large number of studies have shown that DNJ has multiple pharmacological functions and has great potential in the treatment of many non-infectious metabolic diseases. DNJ can regulate sugar metabolism, lipid and fat metabolism, and can also reduce the risk of metabolic diseases related to abnormal cholesterol regulation, showing multiple functions such as treating diabetes, anti-cancer, and antiviral.
[0003] Microbial fermentation has the characteristics of rapid growth, simple nutritional requirements, and easy cultivation. Currently, Bacillus is reported to be the main microorganism that produces 1-deoxynojirimycin. For example, Bai Dou et al. reported that a black variant of Bacillus subtilis could produce 1-DNJ after mutagenesis, but its yield was only 20.7 mg / L (Bai Dou, Long Ling, Zhu Naishuo. Mutagenesis breeding of a black variant of Bacillus subtilis producing 1-deoxynojirimycin (DNJ) [J]. Journal of Fudan University (Natural Science Edition), 2016, 55(01): 104-111). Currently, the methods for synthesizing 1-deoxynojirimycin through microbial fermentation generally have problems such as low 1-deoxynojirimycin yield and long fermentation time. Therefore, it is of great significance to establish a high-yield 1-deoxynojirimycin fermentation method and process. Summary of the Invention
[0004] The present invention aims to provide an application of Bacillus licheniformis WX-02 in promoting the production of DNJ by Bacillus amyloliquefaciens HZ-12 in soybean solid fermentation.
[0005] In order to achieve the above object, the present invention adopts the following technical measures:
[0006] The protection scope of the present invention includes:
[0007] Application of Bacillus licheniformis WX-02 in promoting DNJ production from soybean solid fermentation by Bacillus amyloliquefaciens HZ-12;
[0008] In the above application, preferably, the application process is to simultaneously inoculate Bacillus licheniformis WX-02 and Bacillus amyloliquefaciens HZ-12 into soybean solid fermentation medium for mixed fermentation;
[0009] In the above application, preferably, the ratio of the inoculum amount: the effective bacterial concentration ratio of Bacillus licheniformis WX-02 to Bacillus amyloliquefaciens HZ-12 is 1:0.5-1:2, and more preferably the ratio is 1:1;
[0010] In the above application, preferably, the soybean solid fermentation medium comprises: 50-70 g soybeans / 250 mL soybean solid fermentation medium, 20-40 g lactose / kg soybeans, and 5-15 g malt extract / kg soybeans, wherein the soybeans are soaked and filtered; the water content of the medium is 120-150%, and the pH is natural;
[0011] In the above application, preferably, the fermentation conditions are: fermentation time 24-72h, inoculation amount of Bacillus amyloliquefaciens HZ-12 5-20% (v / v), and culture temperature 28-40°C.
[0012] In the above application, preferably, the fermentation conditions are: fermentation time 60h, inoculation amount of Bacillus amyloliquefaciens HZ-12 8%, and culture temperature 37°C.
[0013] Compared with the prior art, the present invention has the following advantages:
[0014] The production process of the present invention is simple and easy to scale. The inoculum ratio of Bacillus licheniformis WX-02 to Bacillus amyloliquefaciens HZ-12 is 1:1, significantly increasing DNJ yields under different culture medium compositions and fermentation conditions. The process of the present invention is highly stable, facilitating scale-up. Solid-state fermentation of spore bacilli using the specific culture medium formulation and culture conditions provided by the present invention demonstrated a DNJ yield of 512 mg / kg, a 35.45% increase compared to inoculation with a single Bacillus amyloliquefaciens HZ-12 strain. BRIEF DESCRIPTION OF THE DRAWINGS
[0015] Figure 1 Effect of adding Bacillus licheniformis WX-02 strain on DNJ yield.
[0016] Figure 2 Effect of adding different proportions of Bacillus licheniformis WX-02 strain on DNJ production.
[0017] Figure 3 Effect of adding Bacillus licheniformis DW2 strain on DNJ production.
[0018] Figure 4 Effect of adding Bacillus subtilis and lactic acid bacteria on DNJ yield. DETAILED DESCRIPTION
[0019] The following will further illustrate the fermentation process for high-yield DNJ by mixed fermentation of Bacillus licheniformis WX-02 (CN115058400A) and DW2 with Bacillus amyloliquefaciens HZ-12 (CN112226437A) provided by the present invention in conjunction with specific implementation cases. The embodiments described below are exemplary and are only used to explain the present invention and are not to be construed as limiting the present invention. The experimental methods in the following examples, unless otherwise specified, are conventional methods. The experimental materials used in the following examples, unless otherwise specified, were purchased from the market.
[0020] The detection and quantification method of DNJ in this embodiment is as follows: after the fermentation is completed, 3 volumes of distilled water are added, shaken at 230 rpm (2h), the mixed fermentation liquid is centrifuged at 12000 rpm for 5 min, and diluted 600 times for measurement. Prepare a 96-well plate, add 120 μl of PBS buffer, add 30 ul of fermentation liquid, add 50 μl of α-glucosidase, incubate at 37°C for 15 min, add 50 μl of PNPG, and measure the initial value (OD) with a microplate reader. 405 ), then placed in 37 ° C for 45 min, added 50ul of Na2CO3 solution to terminate the reaction, and the final value (OD 405 ), α-glucosidase inhibition rate = 1-measured average value / level average value; DNJ production = 0.0298×e 4.3698×抑制率 × dilution factor.
[0021] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or materials described, unless otherwise specified, are all from commercial channels.
[0022] Example 1:
[0023] Application of Bacillus licheniformis WX-02 in promoting DNJ production from soybean solid fermentation by Bacillus amyloliquefaciens HZ-12:
[0024] 1) Seed culture medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl; pH 7.0-7.2, 121°C, sterilize for 20 min.
[0025] 2) Preparation of seed culture of Bacillus amyloliquefaciens HZ-12 and Bacillus licheniformis WX-02: Bacillus amyloliquefaciens HZ-12 and Bacillus licheniformis WX-02 were removed from a -80°C ultra-low temperature freezer, inoculated onto LB solid plates, and cultured overnight at 37°C. One loopful of bacteria was inoculated into the seed culture medium using an inoculating loop. The culture temperature was 37°C, the liquid volume in a 250-mL Erlenmeyer flask was 50 mL, the shaker speed was 230 rpm / min, and the culture time was 10 h.
[0026] 3) Soybean pretreatment: Weigh an appropriate amount of soybeans, add clean water, soak for 12 hours, and drain the water.
[0027] 4) Preparation of solid fermentation medium: Weigh 60 g of soaked soybeans, add 40 g / kg of soybean lactose and 10 g / kg of soybean malt extract, add water to make the moisture content 130%, and sterilize at 121° C. for 20 min.
[0028] 5) The Bacillus amyloliquefaciens pre-cultured in step 2) was inoculated into the solid fermentation medium at a volume ratio of 8%, and then inoculated with Bacillus licheniformis WX-02 so that the effective bacterial concentration ratio of Bacillus amyloliquefaciens HZ-12 and Bacillus licheniformis WX-02 was 1:1; Bacillus amyloliquefaciens and Bacillus licheniformis were inoculated into the solid fermentation medium alone as a control, and the culture was placed at a constant temperature of 37° C. for DNJ solid fermentation for 60 h.
[0029] After the experiment, the content of DNJ was measured. Figure 1 As shown, the best results were achieved by adding Bacillus licheniformis WX-02 and Bacillus amyloliquefaciens HZ-12 to the mixed fermentation, with the highest DNJ yield reaching 512 mg / kg, a 35.4% increase compared to the 378 mg / kg produced by fermentation with Bacillus amyloliquefaciens HZ-12 alone. Bacillus licheniformis WX-02 cannot synthesize DNJ on its own.
[0030] Example 2:
[0031] Application of different inoculation amounts of Bacillus licheniformis WX-02 in promoting DNJ production by Bacillus amyloliquefaciens HZ-12 in soybean solid fermentation:
[0032] The difference between this embodiment and embodiment 1 is that the ratios of the effective bacterial concentrations of Bacillus amyloliquefaciens and Bacillus licheniformis are 1:1, 2:1, and 1:2, respectively. The remaining steps are the same as those in embodiment 1.
[0033] After the experiment, the DNJ content was determined. Figure 2 As shown, according to the results, when the inoculation ratio of Bacillus licheniformis WX-02 to Bacillus amyloliquefaciens HZ-12 was 1:1, the DNJ yield was the highest at 512 mg / kg. When the inoculation ratio was 1:0.5 and 1:2, the solid fermentation DNJ yields were 441 and 421 mg / kg, respectively, which were higher than that of Bacillus amyloliquefaciens HZ-12 alone (378 mg / kg).
[0034] Example 3:
[0035] Effect of mixed fermentation on DNJ production under different proportions of components in the same solid fermentation medium
[0036] The difference between this embodiment and embodiment 1 is that the components of the solid fermentation medium are the same, but the proportions of the components are different. According to the proportions in Table 1, multiple culture media are prepared for DNJ solid fermentation. The rest of the steps are the same as in embodiment 1.
[0037] Table 1 Composition of different culture media
[0038] serial number Amount of soybeans g Malt extract (g / kg soybean) Lactose (g / kg soybean) 1 50 5 20 2 50 10 30 3 50 15 40 4 60 10 40 5 60 5 50 6 60 5 30 7 70 10 40 8 70 15 50 9 70 5 30
[0039] After the experiment, the DNJ content was measured. The yield is shown in Table 2. The results show that adding Bacillus licheniformis WX-02 increased the DNJ yield of Bacillus amyloliquefaciens HZ-12 at varying component ratios. The highest increase in DNJ yield, by 35.45%, was achieved when the inoculum ratio of Bacillus amyloliquefaciens to Bacillus licheniformis was 1:1 and the culture medium was supplemented with 60 g of soybeans per 250 mL, 40 g of lactose per kg of soybeans, and 10 g of malt extract per kg of soybeans.
[0040] Table 2
[0041]
[0042]
[0043] Example 4:
[0044] Effect of Bacillus licheniformis DW2 on DNJ production by Bacillus amyloliquefaciens HZ-12 in soybean solid fermentation:
[0045] This embodiment differs from embodiment 1 in that Bacillus licheniformis WX-02 is replaced by Bacillus licheniformis DW2 (CN118995551A), and the remaining steps are the same as those in embodiment 1.
[0046] After the experiment, the DNJ content was determined. Figure 3 As shown, when Bacillus licheniformis DW2 and Bacillus amyloliquefaciens HZ-12 were added for mixed fermentation, the maximum DNJ production was 486 mg / kg, which was 28.5% higher than the 378 mg / kg produced by fermentation with Bacillus amyloliquefaciens HZ-12 alone. Its promoting effect was not as good as that of Bacillus licheniformis WX-02.
[0047] Example 5:
[0048] Effects of lactic acid bacteria or Bacillus subtilis on DNJ production by Bacillus amyloliquefaciens HZ-12 in soybean solid fermentation:
[0049] This embodiment differs from Example 1 in that Bacillus licheniformis WX-02 is replaced by lactic acid bacteria (commercially available) or Bacillus subtilis 168 (CN113106095A), and the remaining steps are the same as those in Example 1.
[0050] After the experiment, the content of DNJ was measured. Figure 4 As shown in the results, the addition of Bacillus subtilis and lactic acid bacteria did not increase the DNJ yield of Bacillus amyloliquefaciens HZ-12, and the addition of lactic acid bacteria even decreased the DNJ yield.
Claims
1. Application of Bacillus licheniformis WX-02 in promoting the production of 1-deoxynojirimycin by Bacillus amyloliquefaciens HZ-12 in soybean solid fermentation, wherein the ratio of the effective bacterial concentration of Bacillus amyloliquefaciens HZ-12 to Bacillus licheniformis WX-02 is 1:0.5-1:
2.
2. The use according to claim 1, wherein the application process comprises simultaneously inoculating Bacillus licheniformis WX-02 and Bacillus amyloliquefaciens HZ-12 into soybean solid fermentation medium for mixed fermentation.
3. The use according to claim 1, wherein the ratio of the effective bacterial concentration of the Bacillus licheniformis WX-02 to the effective bacterial concentration of the Bacillus amyloliquefaciens HZ-12 is 1:
1.
4. The use according to claim 1, wherein the culture medium for soybean solid fermentation comprises: 50-70 g soybeans / 250 mL soybean solid fermentation medium, 20-40 g lactose / kg soybeans, 5-15 g malt extract / kg soybeans, wherein the soybeans are soaked and filtered; the water content of the culture medium is 120-150%, and the pH is natural.
5. The use according to claim 2, wherein the fermentation conditions are: fermentation time 24-72 hours, inoculation amount of Bacillus amyloliquefaciens HZ-12 5-20%, and culture temperature 28-40°C.
6. The use according to claim 5, wherein the fermentation conditions are: fermentation time 60 h, inoculation amount of Bacillus amyloliquefaciens HZ-12 8%, and culture temperature 37°C.