A method for the biosynthesis of 1-deoxynojirimycin

Abstract

This invention belongs to the field of biotechnology, specifically disclosing a method for the biosynthesis of 1-deoxynojirimycin. The method includes the following steps: inoculating *Paenibacillus bovis* CGMCC No. 8333 into a skim milk starch medium for fermentation to obtain a fermentation broth containing 1-deoxynojirimycin. This preparation method, for the first time, utilizes *Paenibacillus bovis* CGMCC No. 8333 to ferment a skim milk starch medium to complete the biosynthesis of 1-deoxynojirimycin, revealing a new application for *Paenibacillus bovis* CGMCC No. 8333 in synthesizing 1-deoxynojirimycin through fermentation of skim milk starch medium, thus broadening the sources of 1-deoxynojirimycin. Furthermore, the simple composition and naturally derived culture medium result in higher safety for the synthesized 1-deoxynojirimycin.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for the biosynthesis of 1-deoxynojirimycin. Background Technology

[0002] With the rapid development of my country's economy, dietary structure and lifestyle are undergoing tremendous changes. Coupled with the accelerating aging of the population, the prevalence of diabetes is showing a rapid increasing trend year by year. Based on different pathogenesis mechanisms, diabetes is divided into type 1 diabetes (insulin-dependent) and type 2 diabetes (non-insulin-dependent), the latter accounting for more than 85% of all diabetes cases. To date, drugs for treating type 2 diabetes are mainly classified according to their treatment mechanisms into: insulin secretagogues, insulin sensitizers, and alpha-glucosidase inhibitors (α-GI). Because α-GI has the advantages of mild and long-lasting action, few or no toxic side effects, it has gained increasing favor from researchers both domestically and internationally.

[0003] 1-Deoxynojirimycin is an early-discovered α-glucosidase inhibitor of piperidine alkaloids. Its main natural source is plants, particularly mulberry leaves and roots, where it is most abundant. However, the relatively long growth cycle of plants results in high time costs for its preparation. Therefore, finding a rapid and efficient method for preparing 1-deoxynojirimycin, and completely resolving the issue of its source, is a primary problem urgently needing to be solved by those skilled in the art. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a method for the biosynthesis of 1-deoxynojirimycin. The 1-deoxynojirimycin is a known compound with the following structural formula: .

[0005] Specifically, the biosynthesis method of this 1-deoxynojirimycin includes the following steps: ... (The text abruptly ends here, likely due to an incomplete sentence or missing information.) Paenibacillus bovis CGMCC No. 8333 was fermented in the culture medium.

[0006] Preferably, the culture medium is a skim milk starch culture medium.

[0007] Preferably, the skim milk starch culture medium comprises skim milk powder, starch and water, wherein the skim milk powder accounts for 0.5-4.5% of the mass of the skim milk starch culture medium, the starch accounts for 0.5-4.5% of the mass of the skim milk starch culture medium, and the remainder is water.

[0008] Preferably, the step further includes inoculating the culture medium with Bacillus bovis CGMCC No. 8333; more preferably, the inoculation amount is 1.6 x 10⁻⁶.6 ~8x10 6 cfu / mL.

[0009] Preferably, the fermentation is carried out by shaking culture at a shaking speed of 100~300 rpm.

[0010] Preferably, the fermentation temperature is 25℃~35℃.

[0011] Preferably, the fermentation time is 12~48 h.

[0012] Furthermore, the content of 1-deoxynojirimycin in the fermentation broth at the end of fermentation was >0.5 mg / mL.

[0013] The present invention also provides a method for preparing 1-deoxynojirimycin or a pharmaceutical salt thereof, wherein the preparation comprises synthesizing 1-deoxynojirimycin using the biosynthetic method of 1-deoxynojirimycin described above.

[0014] The 1-deoxynojirimycin or its salt described in this invention may be one or a mixture of its various isomers.

[0015] The preparation method in the above technical solution is the first to use *Bacillus bovis* (…). Paenibacillus bovis The CGMCC No. 8333 method for the biosynthesis of 1-deoxynojirimycin via fermentation of skim milk starch medium has been successfully implemented. This discloses a new application for Bacillus bovis CGMCC No. 8333 in synthesizing 1-deoxynojirimycin via fermentation of skim milk starch medium, broadening the sources of 1-deoxynojirimycin. Furthermore, the simple composition and naturally derived culture medium enhance the safety of the synthesized 1-deoxynojirimycin for use. Attached Figure Description

[0016] Figure 1 This is a UHPLC-Q-TOF MS secondary mirror-matched image of 1-deoxynojirimycin. Detailed Implementation

[0017] To more clearly illustrate the technical solution of the present invention, the following will further describe the technical solution of the present invention in conjunction with specific embodiments: In one specific embodiment, a method for the biosynthesis of 1-deoxynojirimycin is provided, comprising the following steps: ... (The text abruptly ends here, likely due to an incomplete sentence or missing information.) Paenibacillus bovis CGMCC No. 8333 was inoculated into skim milk starch medium for fermentation, and a fermentation broth containing 1-deoxynojirimycin was obtained.

[0018] The preparation method in the above technical solution is the first to use *Bacillus bovis* (…). Paenibacillus bovisThe method described in CGMCC No. 8333 for the biosynthesis of 1-deoxynojirimycin via fermentation of skim milk starch medium reveals a new application for *Bacillus bovis* (CGMCC No. 8333) in synthesizing 1-deoxynojirimycin via fermentation of skim milk starch medium, broadening the sources of 1-deoxynojirimycin. Furthermore, the simple composition and naturally derived medium enhance the safety of the synthesized 1-deoxynojirimycin for use.

[0019] In addition, the fermentation medium used in biosynthesis is widely available, inexpensive, and natural and safe, avoiding the use of chemically synthesized medium; and the fermentation strain uses a single strain of Bacillus bovis. The combination of the above-mentioned raw materials and strains is conducive to the standardization of the synthesis process and cost control for large-scale industrial production.

[0020] Preferably, the inoculum size of Bacillus bovis CGMCC No. 8333 is 1.6 x 10⁻⁶. 6 ~8x10 6 cfu / mL; preferably 3.2 x 10⁻⁶. 6 ~6.4x10 6 cfu / mL, more preferably 4.8 x 10⁻⁶ 6 cfu / mL.

[0021] Preferably, the skim milk starch culture medium comprises skim milk powder, starch, and water, wherein the skim milk powder accounts for 0.5-4.5% of the mass percentage of the skim milk starch culture medium, the starch accounts for 0.5-4.5% of the mass percentage of the skim milk starch culture medium, and the remainder is water. The preparation method may include the following steps: adding skim milk powder and starch to distilled water, mixing thoroughly, sterilizing at 95-125°C for 5-20 minutes, and cooling to obtain the final product.

[0022] Preferably, the fermentation method is shaking culture, with a shaking speed of 100~300 rpm; more preferably, 150~250 rpm; and even more preferably, 200 rpm.

[0023] Preferably, the fermentation temperature is 25℃~35℃; more preferably, it is 28℃~32℃; and even more preferably, it is 30℃.

[0024] Preferably, the fermentation time is 12-48 h; more preferably, it is 18-36 h; and even more preferably, it is 24 h.

[0025] As can be seen from Comparative Example 1, outside the range of optimal fermentation parameters, the efficiency of *Bacillus bovis* CGMCC No. 8333 in synthesizing 1-deoxynojirimycin in skim milk starch medium significantly decreased. Within the optimal range, the inoculum size, shaking speed, fermentation temperature, and time interacted, resulting in a stronger ability of *Bacillus bovis* CGMCC No. 8333 to synthesize 1-deoxynojirimycin.

[0026] When fermentation was terminated, the content of 1-deoxynojirimycin in the fermentation broth was >0.5 mg / mL.

[0027] The present invention also provides 1-deoxynojirimycin obtained by the above-described biosynthetic method.

[0028] The following examples further illustrate the specific implementation methods described above, but do not limit the invention to the scope of these examples. Experimental methods in the following examples, unless otherwise specified, were performed according to conventional methods and conditions, or as per the product instructions. Unless otherwise stated, all reagents used in the examples are analytical grade reagents purchased from Sinopharm Group. Other experimental instruments, reagents, and bacterial strains, unless otherwise specified, can be purchased directly through commercial channels. Example 1

[0029] 1. Materials and Methods (a) Preparation of seed (fermentation strain): Lyophilized powder of *Bacillus bovis* CGMCC No. 8333 (see Chinese Patent Publication No. CN 103740618A for the source of this strain) was dissolved in a small amount of sterile distilled water. A loopful was used to streak a single colony onto TYC solid medium (purchased from OXOID Co., UK). The culture was aerobically incubated at 30°C for 48 h. A single colony was then picked up using an inoculation loop and placed into 10 mL of TYC liquid medium (purchased from OXOID Co., UK). The colony was evenly dispersed in the liquid medium using a vortex mixer. The culture was incubated at 30°C and 180 rpm for 24 h. The culture was then inoculated at a 2% (v / v) inoculation rate onto TYC liquid medium (purchased from OXOID Co., UK). After incubation at 30°C and 180 rpm for 24 h, the culture was cultured to 15,000 ml. Centrifuge at rpm for 10 minutes, discard the supernatant, wash the bacterial cells twice with sterile distilled water, and then resuspend them in the original culture volume of sterile distilled water to obtain the seed culture for fermentation. The bacterial concentration of the seed culture is 1.6 x 10⁻⁶. 8 cfu / mL.

[0030] (b) Preparation of skim milk starch culture medium: Mix 1% skim milk powder, 4% starch and distilled water thoroughly, sterilize at 125°C for 5 min, and cool to room temperature to obtain the skim milk starch culture medium of the required concentration.

[0031] (c) Structural identification of 1-deoxynojirimycin in fermentation samples: Ultra-high performance liquid chromatography-tandem time-of-flight mass spectrometry (UHPLC-Q-TOF MS).

[0032] Preparation of the test sample: Add pre-cooled methanol / acetonitrile / water solution (2:2:1, v / v) to an appropriate amount of fermentation sample, vortex mix, sonicate at low temperature for 30 min, let stand at -20℃ for 10 min, centrifuge at 14,000 g at 4℃ for 20 min, and collect the supernatant to dry under vacuum. For mass spectrometry analysis, add 100 μL of acetonitrile aqueous solution (acetonitrile:water = 1:1, v / v) to reconstitute, vortex, centrifuge at 14,000 g at 4℃ for 15 min, and collect the supernatant to obtain the test sample.

[0033] Chromatographic conditions: Agilent 1290 Infinity LC system (Agilent Technologies, USA), ACQUITY UPLC BEH Amide HILIC column (Waters, USA), injection volume 2 μL, column temperature 25℃, flow rate 0.5 mL / min, mobile phase A: water + 25 mM ammonium acetate + 25 mM ammonia, mobile phase B: acetonitrile. The gradient elution program is shown in Table 1. Table 1 Gradient elution program for liquid chromatography 0.0 5 % 95 % 0.5 0.5 5 % 95 % 0.5 7.0 35 % 65 % 0.5 8.0 60 % 40 % 0.5 9.0 60 % 40 % 0.5 9.1 5 % 95 % 0.5 12.0 5 % 95 % 0.5 Mass spectrometry conditions: Mass spectrometer: AB Triple TOF 6600 (SCIEX Corporation, USA); Detection method: Electrospray ionization (ESI) positive and negative ion modes; ESI source settings: Nebulizer gas, auxiliary heating gas 1 (Gas1): 60, auxiliary heating gas 2 (Gas2): 60, curtain gas (CUR): 30 psi, ion source temperature: 600℃, spray voltage (ISVF): ±5500 V (positive and negative modes), primary mass-to-charge ratio detection range: 60-1000 Da, secondary daughter ion mass-to-charge ratio detection range: 25-1000 Da, primary mass spectrum scan cumulative time: 0.20 s / spectra, secondary mass spectrum scan cumulative time: 0.05 s / spectra, secondary mass spectrometry was acquired using data-dependent acquisition mode (IDA) with peak intensity screening mode, declustering voltage (DP): ±60 V (positive and negative modes), collision energy: 35±15 eV, IDA... The settings are as follows: Dynamic exclusion range of isotope ions: 4 Da, 10 fragment spectra are collected per scan.

[0034] Data analysis: The raw data in Wiff format was converted into .mzXML format using ProteoWizard, and then analyzed using XCMS software. The data was then matched with standard substances in the database to determine the metabolites.

[0035] (d) Quantitative detection of 1-deoxynojirimycin in fermentation samples: liquid chromatography-tandem mass spectrometry (HPLC-MS / MS).

[0036] Preparation of the test sample: Centrifuge the fermentation broth (15,000 g, 15 min) and take the supernatant. Add three times the volume of anhydrous ethanol to the supernatant, centrifuge again (15,000 g, 15 min) and take the supernatant. Remove the ethanol by rotary evaporation, freeze dry, and dissolve in the original volume of deionized water of the fermentation broth to obtain the test sample.

[0037] Preparation of standard samples: 1-Deoxynojirimycin was prepared into standard samples with 1, 2, 5, 10, 20, 50, 100, 200, 500 and 1000 ppb concentration gradients using deionized water.

[0038] Chromatographic conditions: Column: Acquity uplc HSS T3 1.8 μm 2.1×100 mm, injection volume: 4 μL, column temperature: 35℃, flow rate: 0.25 mL / min, mobile phase: A is water + 0.1% formic acid, B is 0.1% acetonitrile, gradient elution program is shown in Table 2.

[0039] Table 2 Gradient elution program for liquid chromatography 0.0 98.0 2.0 0.25 1.0 98.0 2.0 0.25 9.0 50.0 50.0 0.25 12.0 2.0 98.0 0.25 13.5 2.0 98.0 0.25 14.0 98.0 2.0 0.25 20.0 98.0 2.0 0.25 Mass spectrometry conditions: ionization mode: electrospray ionization (ESI); positive ion mode; scanning mode: Full-MS-ddMS 2; spray voltage: 3,500 V; temperature: 350 °C; sheath gas flow rate: 30 psi; sweep gas flow rate: 0 psi; aux gas flow rate: 10 psi.

[0040] Data analysis: The above-mentioned standard samples of 1-deoxynojirimycin were sequentially loaded into a liquid chromatography-tandem mass spectrometry system, and a standard curve of the relationship between the peak area and concentration of the standard samples was obtained by fitting. Then, the sample to be tested was loaded, and its concentration was calculated based on the peak area of ​​1-deoxynojirimycin.

[0041] 2. Biosynthesis of 1-deoxynojirimycin Bacillus bovis CGMCC No. 8333 seed was aseptically inoculated at an inoculum of 3% (v / v) into a skim milk starch medium containing 1% (w / w) skim milk powder and 4% (w / w) starch, and cultured at 30°C and 200 rpm for 24 h to obtain a fermentation broth containing 1-deoxynojirimycin.

[0042] 3. Structural identification and quantitative determination of 1-deoxynojirimycin in fermentation samples After analyzing the fermentation samples using the UHPLC-Q-TOF MS method described above, combined with XCMS software analysis, it was found that the substance with the code M164T75 in the metabolites completely matched the mass spectrometric characteristic signal of the 1-deoxynojirimycin standard in the database. Therefore, this substance was identified as 1-deoxynojirimycin. The results are as follows. Figure 1 As shown in the figure. The fermentation sample was analyzed by the above HPLC-MS / MS method, and the content of 1-deoxynojirimycin was determined to be 0.62 mg / mL. Example 2

[0043] 1. Materials and Methods (a) Preparation of seeds (fermentation strains): Same as in Example 1.

[0044] (b) Preparation of skim milk starch culture medium: 0.5% skim milk powder, 4.5% starch and distilled water by mass percentage were thoroughly mixed, sterilized at 95°C for 20 min and cooled to room temperature to obtain the skim milk starch culture medium of the required concentration.

[0045] (c) Structural identification of 1-deoxynojirimycin in fermentation samples: same as in Example 1.

[0046] (d) Quantitative detection of 1-deoxynojirimycin in fermentation samples: Same as in Example 1.

[0047] 2. Biosynthesis of 1-deoxynojirimycin Bacillus bovis CGMCC No. 8333 seed was aseptically inoculated at an inoculum size of 5% (v / v) into a skim milk starch medium containing 0.5% (w / w) skim milk powder and 4.5% (w / w) starch, and cultured at 32°C and 300 rpm for 12 h to obtain a fermentation broth containing 1-deoxynojirimycin.

[0048] 3. Structural identification and quantitative determination of 1-deoxynojirimycin in fermentation samples The fermentation sample was analyzed using the UHPLC-Q-TOF MS method described above, combined with XCMS software. The results were the same as in Example 1. The fermentation sample was analyzed using the HPLC-MS / MS method described above, and the content of 1-deoxynojirimycin was determined to be 0.51 mg / mL. Example 3

[0049] 1. Materials and Methods (a) Preparation of seeds (fermentation strains): Same as in Example 1.

[0050] (b) Preparation of skim milk starch culture medium: 4.5% skim milk powder, 0.5% starch and distilled water by mass percentage were thoroughly mixed, sterilized at 100°C for 15 min and cooled to room temperature to obtain the skim milk starch culture medium of the required concentration.

[0051] (c) Structural identification of 1-deoxynojirimycin in fermentation samples: same as in Example 1.

[0052] (d) Quantitative detection of 1-deoxynojirimycin in fermentation samples: Same as in Example 1.

[0053] 2. Biosynthesis of 1-deoxynojirimycin Bacillus bovis CGMCC No. 8333 seed was aseptically inoculated at an inoculum of 1% (v / v) into a skim milk starch medium containing 4.5% (w / w) skim milk powder and 0.5% (w / w) starch, and cultured at 28°C and 100 rpm for 48 h to obtain a fermentation broth containing 1-deoxynojirimycin.

[0054] 3. Structural identification and quantitative determination of 1-deoxynojirimycin in fermentation samples The fermentation sample was analyzed using the UHPLC-Q-TOF MS method described above, combined with XCMS software. The results were the same as in Example 1. The fermentation sample was analyzed using the HPLC-MS / MS method described above, and the content of 1-deoxynojirimycin was determined to be 0.67 mg / mL. Example 4

[0055] 1. Materials and Methods (a) Preparation of seeds (fermentation strains): Same as in Example 1.

[0056] (b) Preparation of skim milk starch culture medium: 2.5% skim milk powder, 2.5% starch and distilled water were thoroughly mixed, sterilized at 120°C for 10 min and cooled to room temperature to obtain the skim milk starch culture medium of the required concentration.

[0057] (c) Structural identification of 1-deoxynojirimycin in fermentation samples: same as in Example 1.

[0058] (d) Quantitative detection of 1-deoxynojirimycin in fermentation samples: Same as in Example 1.

[0059] 2. Biosynthesis of 1-deoxynojirimycin Bacillus bovis CGMCC No. 8333 seed was aseptically inoculated at an inoculum size of 2% (v / v) into a skim milk starch medium containing 2.5% (w / w) skim milk powder and 2.5% (w / w) starch, and cultured at 25°C and 250 rpm for 36 h to obtain a fermentation broth containing 1-deoxynojirimycin.

[0060] 3. Structural identification and quantitative determination of 1-deoxynojirimycin in fermentation samples The fermentation sample was analyzed using the UHPLC-Q-TOF MS method described above, combined with XCMS software. The results were the same as in Example 1. The fermentation sample was analyzed using the HPLC-MS / MS method described above, and the content of 1-deoxynojirimycin was determined to be 0.58 mg / mL. Example 5

[0061] 1. Materials and Methods (a) Preparation of seeds (fermentation strains): Same as in Example 1.

[0062] (b) Preparation of skim milk starch culture medium: 3% skim milk powder, 2% starch and distilled water by mass percentage are thoroughly mixed, sterilized at 110°C for 12 min, and cooled to room temperature to obtain the skim milk starch culture medium of the required concentration.

[0063] (c) Structural identification of 1-deoxynojirimycin in fermentation samples: same as in Example 1.

[0064] (d) Quantitative detection of 1-deoxynojirimycin in fermentation samples: Same as in Example 1.

[0065] 2. Biosynthesis of 1-deoxynojirimycin Bacillus bovis CGMCC No. 8333 seed was aseptically inoculated at an inoculum size of 4% (v / v) into a skim milk starch medium containing 3% (w / w) skim milk powder and 2% (w / w) starch, and cultured at 35°C and 150 rpm for 18 h to obtain a fermentation broth containing 1-deoxynojirimycin.

[0066] 3. Structural identification and quantitative determination of 1-deoxynojirimycin in fermentation samples The fermentation sample was analyzed using the UHPLC-Q-TOF MS method described above, combined with XCMS software. The results were the same as in Example 1. The fermentation sample was analyzed using the HPLC-MS / MS method described above, and the content of 1-deoxynojirimycin was determined to be 0.69 mg / mL.

[0067] Comparative Example 1 The inoculum size, culture temperature, fermentation time, and fermentation oscillation speed in Example 1 were adjusted one by one to obtain the following set of fermentation broths prepared by different methods. The 1-deoxynojirimycin was quantitatively determined in each group of fermentation broths, and the results are shown in Table 3.

[0068] Table 3. Content of 1-deoxynojirimycin in fermentation broth prepared by different methods Inoculation amount (volume percentage) 0.1 3 3 3 Fermentation temperature (°C) 30 10 30 30 Oscillation speed (rpm) 200 200 50 200 Fermentation time (h) 24 24 24 6 1-Deoxynojirimycin content (mg / mL) 0.06 0.03 0.17 0.02 As shown in Table 3, when the inoculum amount, fermentation temperature, fermentation time, and fermentation oscillation speed in the biosynthesis method of 1-deoxynojirimycin are adjusted to outside the preferred range, Bacillus bovis CGMCC No. 8333 can still synthesize 1-deoxynojirimycin, but its yield is significantly reduced.

[0069] Comparative Example 2 Referring to the method described in Example 1, the results of comparing *Bacillus bovis* CGMCC No. 8333 and *Lactobacillus casei* were analyzed. L. casei ATCC 393 (purchased from ATCC), Lactobacillus bulgaricus ( L. bulgaricus LB340 (supplied by Danisco), Streptococcus thermophilus ( S. thermophilus The content of 1-deoxynojirimycin in the fermentation broth prepared with ST-BODY-3 (provided by Chr. Hansen) is determined by the following procedure: 1. Materials and Methods (a) Preparation of seeds (fermentation strains): Preparation of seeds of Bacillus bovis CGMCC No. 8333: Same as in Example 1.

[0070] Preparation of Lactobacillus casei and Lactobacillus bulgaricus seeds: Lyophilized Lactobacillus casei ATCC 393 and Lactobacillus bulgaricus LB340 powders were dissolved in a small amount of sterile distilled water. One loopful of each was streaked onto MRS solid medium (purchased from Merck Co., Germany). After anaerobic incubation at 37°C for 24 h, a single colony was picked up and placed into 1 mL of MRS liquid (purchased from Merck Co., Germany). The colony was evenly dispersed in the liquid medium using a vortex mixer. After anaerobic incubation at 37°C for 24 h, the colony was inoculated into 50 mL of MRS liquid at a 2% (v / v) inoculation rate. After incubation at 37°C for 24 h, the culture was centrifuged at 9,000 rpm for 10 minutes, the supernatant was discarded, and the cells were washed twice with sterile distilled water and then resuspended in the original culture volume of sterile distilled water to obtain the corresponding fermentation seeds.

[0071] Preparation of Streptococcus thermophilus seed: The lyophilized powder of Streptococcus thermophilus ST-BODY-3 was dissolved in a small amount of sterile distilled water. A loopful was used to streak a single colony onto M17 solid medium (purchased from Merck Co., Germany). After anaerobic incubation at 40°C for 24 h, the colony was removed and picked up with an inoculation loop and placed into 1 mL of M17 liquid (purchased from Merck Co., Germany). The colony was evenly dispersed in the liquid medium using a vortex mixer and anaerobic incubated at 40°C for 24 h. The colony was then inoculated into 50 mL of M17 liquid at a 2% (v / v) inoculation rate and incubated at 40°C for 24 h. After incubation, the culture was centrifuged at 9,000 rpm for 10 minutes, the supernatant was discarded, and the cells were washed twice with sterile distilled water and then resuspended in the original culture volume of sterile distilled water to obtain the seed for fermentation.

[0072] (b) Preparation of skim milk starch culture medium: Same as in Example 1.

[0073] (c) Quantitative detection of 1-deoxynojirimycin in fermentation samples: Same as in Example 1.

[0074] 2. Fermentation in skim milk starch medium Each strain was aseptically inoculated at a 3% (v / v) inoculation rate into a skim milk starch medium containing 1% (w / w) skim milk powder and 4% (w / w) starch, and cultured separately (Lactobacillus bulgaricus and Lactobacillus casei anaerobic culture at 37°C, Streptococcus thermophilus anaerobic culture at 40°C, and Bacillus bovis var. bovis anaerobic culture at 30°C and 200 rpm shaking culture) for 7 h to obtain the corresponding fermentation broth.

[0075] 3. Quantitative determination of 1-deoxynojirimycin in fermentation samples The fermentation samples were analyzed using the above HPLC-MS / MS method, and the results are shown in Table 4: Table 4. Content of 1-deoxynojirimycin in fermentation broth prepared from different strains 1-Deoxynojirimycin content (mg / mL) 0.62 - - - As shown in Table 4, except for Bacillus bovis CGMCC No. 8333, 1-deoxynojirimycin could not be detected in the fermentation broth of other conventional fermentation strains, indicating that other conventional fermentation bacteria do not have the ability to produce 1-deoxynojirimycin by fermenting skim milk starch medium.

[0076] The above provides a detailed description of the biosynthesis method for 1-deoxynojirimycin provided by this invention. Specific examples have been used to illustrate the principles and implementation methods of this invention. The descriptions of the above embodiments are merely for the purpose of helping to understand the method and core ideas of this invention. It should be noted that those skilled in the art can make various improvements and modifications to this invention without departing from its principles, and these improvements and modifications also fall within the protection scope of the claims of this invention.

Claims

1. A method for the biosynthesis of 1-deoxynojirimycin, characterized in that, Includes the following steps: Bacillus bovis ( Paenibacillus bovis CGMCC No. 8333 was fermented in a culture medium to obtain a fermentation broth containing 1-deoxynojirimycin; the culture medium was a skim milk starch medium, which included skim milk powder, starch and water, with skim milk powder accounting for 0.5~4.5% of the mass of the skim milk starch medium, starch accounting for 0.5~4.5% of the mass of the skim milk starch medium, and the remainder being water.

2. The method for biosynthesizing 1-deoxynojirimycin according to claim 1, characterized in that, The step also comprises inoculating the Bacillus bovis CGMCC No. 8333 into the culture medium at an inoculation amount of 1.6x10 6 8x10 6 cfu / mL.

3. The method for biosynthesizing 1-deoxynojirimycin according to claim 1, characterized in that, The fermentation is carried out by shaking culture at a shaking speed of 100-300 rpm.

4. The method for biosynthesizing 1-deoxynojirimycin according to claim 1, characterized in that, The fermentation temperature is 25℃~35℃.

5. The method for biosynthesizing 1-deoxynojirimycin according to claim 1, characterized in that, The fermentation time is 12-48 hours.

6. The method for biosynthesizing 1-deoxynojirimycin according to claim 1, characterized in that, When fermentation was terminated, the content of 1-deoxynojirimycin in the fermentation broth was >0.5 mg / mL.

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