Application of bacillus licheniformis WX-02 in promoting production of DNJ by soybean solid fermentation of bacillus amyloliquefaciens HZ-12

Abstract

The invention belongs to the technical field of fermentation engineering, and discloses an application of bacillus licheniformis WX-02 in promoting soybean solid fermentation of bacillus amyloliquefaciens HZ-12 to produce DNJ. And inoculating the bacillus licheniformis into a DNJ solid fermentation system of the bacillus amyloliquefaciens HZ-12 according to an inoculation ratio of (1: 0.5)-(1: 2). Under a mixed bacterium culture system, the yield of DNJ is higher than that of DNJ inoculated with a single bacillus amyloliquefaciens HZ-12 strain. Under the optimal condition, the yield of the DNJ reaches 512 mg / kg, and compared with inoculation of a single bacillus amyloliquefaciens HZ-12 strain, the yield of the DNJ is increased by 35.45%. The process disclosed by the invention has the advantages of cheap and easily available raw materials, simple culture medium composition, high DNJ yield and the like, and has wide application prospects of DNJ-rich soybean food for preventing and improving diabetes and cardiovascular diseases.

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 Bacillus amyloliquefaciens HZ-12 to produce DNJ by solid fermentation of soybean. Background Art

[0002] 1-Deoxynojirimycin (DNJ for short) is a polyhydroxypiperidine alkaloid with similar structures 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 blood sugar-lowering 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 the treatment of diabetes, anti-cancer, and anti-virus.

[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, Baidou 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 (Baidou, Long Ling, Zhu Naishuo. Study on the 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). At present, the method of synthesizing 1-deoxynojirimycin by microbial fermentation generally has the problems of 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 object of the present invention is to provide an application of Bacillus licheniformis WX-02 in promoting the production of DNJ by soybean solid fermentation of Bacillus amyloliquefaciens HZ-12.

[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 by Bacillus amyloliquefaciens HZ-12 in soybean solid fermentation;

[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 inoculation amount: the ratio of the effective bacterial concentration of Bacillus licheniformis WX-02 to Bacillus amyloliquefaciens HZ-12 is 1:0.5-1:2, and the more preferred ratio is 1:1;

[0010] In the above application, preferably, the soybean solid fermentation medium comprises: 50-70g / 250mL soybean solid fermentation medium, 20-40g / kg lactose, 5-15g / kg malt extract, wherein the soybean is 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 convenient to promote. The inoculation ratio of Bacillus licheniformis WX-02 and Bacillus amyloliquefaciens HZ-12 is 1:1, and the DNJ yield is significantly increased under different culture medium components and fermentation conditions. The process of the present invention has the characteristics of high stability and is convenient for large-scale production. The spore bacilli were verified by soybean solid fermentation using the specific culture medium formula and culture conditions provided by the present invention, and the DNJ yield reached 512 mg / kg. Compared with the inoculation of a single Bacillus amyloliquefaciens HZ-12 strain, the DNJ yield was increased by 35.45%. 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 yield.

[0017] Figure 3 Effect of adding Bacillus licheniformis DW2 strain on DNJ yield.

[0018] Figure 4 Effect of adding Bacillus subtilis and lactic acid bacteria on DNJ yield. DETAILED DESCRIPTION

[0019] 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 will be further described below in conjunction with specific implementation cases. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limitations of the present invention. The experimental methods in the following embodiments, unless otherwise specified, are conventional methods. The experimental materials used in the following embodiments, 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 405 ), then placed at 37°C for 45 min, 50 μl of Na2CO3 solution was added 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.

[0022] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or materials, unless otherwise specified, are all from commercial channels.

[0023] Embodiment 1:

[0024] Application of Bacillus licheniformis WX-02 in promoting DNJ production by soybean solid fermentation of Bacillus amyloliquefaciens HZ-12:

[0025] 1) Seed culture medium configuration: 10 g / L peptone, 5 g / L yeast extract powder, 10 g / L NaCl; pH 7.0-7.2, 121°C, sterilization for 20 min.

[0026] 2) Obtaining seed solution of Bacillus amyloliquefaciens HZ-12 and Bacillus licheniformis WX-02: Take out the strains of Bacillus amyloliquefaciens HZ-12 and Bacillus licheniformis WX-02 from a -80°C ultra-low temperature freezer, inoculate them on an LB solid plate, and culture them at 37°C overnight. Use an inoculation loop to inoculate one loop of bacteria into the seed culture medium. The culture temperature is 37°C, the liquid volume in a 250mL Erlenmeyer flask is 50mL, the shaking speed is 230rpm / min, and the culture time is 10h.

[0027] 3) Soybean pretreatment: Weigh an appropriate amount of soybeans, add clean water, soak for 12 hours, and drain the water.

[0028] 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 water content 130%, sterilize at 121° C. for 20 min.

[0029] 5) The Bacillus amyloliquefaciens pre-cultured in step 2) was inoculated into the above-mentioned 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 separately inoculated into the above-mentioned solid fermentation medium as a control, and placed at a constant temperature of 37°C for DNJ solid fermentation, and the fermentation time was 60h.

[0030] After the experiment, the content of DNJ was measured. Figure 1 As shown, the best effect was achieved by adding Bacillus licheniformis WX-02 and Bacillus amyloliquefaciens HZ-12 for mixed fermentation, with the highest DNJ yield of 512 mg / kg, which was 35.4% higher than the 378 mg / kg produced by fermentation with Bacillus amyloliquefaciens HZ-12 alone. Bacillus licheniformis WX-02 cannot synthesize DNJ by itself.

[0031] Embodiment 2:

[0032] Application of different inoculation amounts of Bacillus licheniformis WX-02 in promoting soybean solid fermentation of Bacillus amyloliquefaciens HZ-12 to produce DNJ:

[0033] The difference between this embodiment and embodiment 1 is that the ratios of effective bacterial concentrations of Bacillus amyloliquefaciens and Bacillus licheniformis are 1:1, 2:1, and 1:2, respectively, and the remaining steps are the same as those in embodiment 1.

[0034] 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 as high as 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).

[0035] Embodiment 3:

[0036] Effect of mixed fermentation on DNJ production under different proportions of components in the same solid fermentation medium

[0037] 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, and the rest of the steps are the same as in embodiment 1.

[0038] Table 1 Composition of different culture media

[0039] 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

[0040] After the experiment, the content of DNJ was determined. The yield is shown in Table 2. According to the results, under different component ratios, the addition of Bacillus licheniformis WX-02 can increase the yield of DNJ synthesized by Bacillus amyloliquefaciens HZ-12. When 60g / 250mL soybean, 40g / kg lactose soybean, and 10g / kg malt extract soybean medium were added, the inoculation ratio of Bacillus amyloliquefaciens and Bacillus licheniformis was 1:1 for solid fermentation, and the DNJ yield increased the most, increasing by 35.45%.

[0041] Table 2

[0042]

[0043]

[0044] Embodiment 4:

[0045] Effect of Bacillus licheniformis DW2 on DNJ production by Bacillus amyloliquefaciens HZ-12 in soybean solid fermentation:

[0046] The difference between this embodiment and embodiment 1 is that Bacillus licheniformis WX-02 is replaced by Bacillus licheniformis DW2 (CN118995551A), and the remaining steps are the same as those in embodiment 1.

[0047] After the experiment, the content of DNJ 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. However, its promoting effect was not as good as that of Bacillus licheniformis WX-02.

[0048] Embodiment 5:

[0049] Effects of lactic acid bacteria or Bacillus subtilis on DNJ production by Bacillus amyloliquefaciens HZ-12 in soybean solid fermentation:

[0050] This embodiment is different from Embodiment 1 in that Bacillus licheniformis WX-02 is replaced by lactic acid bacteria (commercially purchased) or Bacillus subtilis 168 (CN113106095A), and the remaining steps are the same as those in Embodiment 1.

[0051] After the experiment, the content of DNJ was measured. Figure 4 As shown, 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 reduced the DNJ yield.

Claims

1. Application of Bacillus licheniformis WX-02 in promoting the production of DNJ by soybean solid fermentation of Bacillus amyloliquefaciens HZ-12.

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 2, wherein the ratio of the effective bacterial concentration of WX-02 Bacillus amyloliquefaciens HZ-12 to that of Bacillus licheniformis WX-02 is 1:0.5-1:

2.

4. The use according to claim 3, wherein the ratio of the effective bacterial concentration of Bacillus licheniformis WX-02 to that of Bacillus amyloliquefaciens HZ-12 is 1:

1.

5. The use according to claim 1, wherein the soybean solid fermentation medium 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 medium is 120-150%, and the pH is natural.

6. 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.

7. The use according to claim 6, wherein the fermentation conditions are: fermentation time 60 h, inoculation amount of Bacillus amyloliquefaciens HZ-12 8%, and culture temperature 37°C.

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