A strain producing 1-deoxynojirimycin and its application

Abstract

The invention relates to a 1-deoxynojirimycin producing strain and application thereof, and relates to the field of microbial fermentation. Specifically, an alpha-glucosidase inhibition test is used to screen out a DNJ-producing strain Bacillus amyloliquefaciens S1 from 50 potential strains in a laboratory strain library; a fermentation medium and fermentation conditions with the highest DNJ yield are obtained through a single factor experiment; finally, a fermentation supernatant of the S1 strain has a good blood sugar lowering effect and an effect of improving the extraction rate of DNJ in leaves.

Description

Technical Field

[0001] The invention belongs to the field of microbial fermentation, and specifically relates to a 1-deoxynojirimycin producing strain and application thereof. Background Art

[0002] 1-Deoxynojirimycin (DNJ) is a natural polyhydroxypiperidine belonging to the class of iminosugar alkaloids. [1-2] Its molecular formula is C6H 13 NO4, molecular weight 163.2, chemical structure as shown in formula I:

[0003]

[0004] Formula I.

[0005] DNJ molecules have multiple hydroxyl groups and are easily soluble in water. Under acidic conditions, they react with hydrochloric acid to form hydrochloride, further increasing their solubility in water. DNJ is similar to glucose in molecular formula and structure. The complex formed by combining with the active center of α-glucosidase is more stable than other sugars. When the two coexist, the chance of the enzyme combining with other sugars is reduced, so it has strong α-glucosidase inhibitory activity. After entering the digestive tract, DNJ competes with other sugars for the active site of α-glucosidase, hindering the digestion and absorption of sugar substances by the digestive tract, and interfering with the synthesis of oligosaccharide chains by glycoproteins, forming the basis for its biological activities such as lowering blood sugar levels and antiviral.

[0006] The main sources of DNJ include plant sources such as mulberry leaves, animal sources such as insects that eat mulberry leaves, and microbial sources such as Streptomyces.

[0007] The earliest microorganisms found to produce DNJ were Streptomyces and Bacillus. In 1967, Inouye, Frommer and others successively obtained DNJ from Streptomyces ( S. roseochromogenes ), Bacillus ( Bacillus DNJ was discovered in 1984. Subsequently, the biosynthetic pathway of DNJ in microorganisms was also elucidated.

[26] et al. studied the DNJ fermentation process of Bacillus amyloliquefaciens DSM704 and found that DNJ was produced as a secondary metabolite and could not be used as a nitrogen or carbon source by the strain. Ye Jingjing et al. isolated and screened microorganisms from mulberry trees and isolated two DNJ-producing bacteria, Micrococcus luteus and Stenotrophomonas maltophilia. These studies enriched the microbial sources of DNJ and provided scientific basis and technical support for the production of DNJ through microbial fermentation.

[0008] Traditional methods of obtaining DNJ, including extraction from animals, plants, and microorganisms, often encounter the problems of cumbersome extraction procedures and low output efficiency. In view of these challenges, scientists began to seek chemical synthesis methods to efficiently mass-produce DNJ. The chemical synthesis of DNJ involves multiple steps, such as subjecting sugar substances such as D-glucose to amination, dephosphorylation, oxidation, and isomerization, and finally synthesizing the desired compound. The synthesis strategies can be roughly summarized into three types: one is the synthesis method starting with L-sorbose or D-glucose; the second is the synthesis technology using non-L-sorbose or non-D-glucose; and the third is the synthesis pathway that combines chemical synthesis and biological transformation methods. In 1967, researchers such as Paulsen set a precedent for synthesizing DNJ using sorbose as a raw material. In 1980, researchers began to explore innovative methods that combine microbial transformation and chemical synthesis to prepare DNJ. This method simplifies the complex steps in traditional chemical synthesis, such as the addition and removal of protecting groups, and effectively shortens the synthesis time. At the same time, this synthesis method has the advantages of mild conditions, high selectivity, and easy product purification. It meets the standards of economic benefits and environmental protection, and responds to the needs of contemporary society for green chemistry and sustainable development. Therefore, it has significant development potential and market prospects. Summary of the invention

[0009] In view of this, the main purpose of the present invention is to provide a microbial strain that can efficiently ferment and produce 1-deoxynojirimycin, and apply it to medical and agricultural production.

[0010] Specifically, the first aspect of the present invention is to provide a strain of Bacillus amyloliquefaciens S1 that can produce 1-deoxynojirimycin, the deposit date of which is March 26, 2024, and the classification name is Bacillus amyloliquefaciens Bacillus amyloliquefaciens , deposited in the General Microbiology Center of China Microorganism Culture Collection (CGMCC), the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.30156.

[0011] The second aspect of the present invention is to provide a bacterial agent, which includes the strain described in the first aspect.

[0012] The third aspect of the present invention is to provide a microbial fermentation product, wherein the microbial fermentation product is the strain described in the first aspect or the bacterial agent fermentation supernatant described in the second aspect.

[0013] The fourth aspect of the present invention provides a method for preparing 1-deoxynojirimycin using the strain described in the first aspect or the bacterial agent described in the second aspect, wherein the method comprises fermenting and culturing the strain described in the first aspect or the bacterial agent described in the second aspect to separate and obtain 1-deoxynojirimycin.

[0014] Furthermore, in the fermentation culture, the culture medium contains 3-5% sorbitol, 3-5% soy peptone, 0.2-0.8 g / L FeSO4, and 0.5-1 g / L MnSO4.

[0015] Furthermore, in the fermentation culture, the culture conditions are: culture medium pH 6-7, rotation speed 70-90 rpm, culture temperature 35-39°C, seed age 8-12h, and fermentation 48-96h.

[0016] The fifth aspect of the present invention is the use of the strain described in the first aspect, the bacterial agent described in the second aspect, the fermentation product described in the third aspect, and the method described in the fourth aspect in the preparation of a therapeutic hypoglycemic preparation.

[0017] The sixth aspect of the present invention is the use of the strain described in the first aspect, the bacterial agent described in the second aspect, the fermentation product described in the third aspect, and the method described in the fourth aspect in preparing a preparation for improving the extraction rate of DNJ in plant leaves; further, the plant leaves are mulberry leaves; further, the specific steps of the application are:

[0018] 1) fermenting and culturing the strain using the method described in the fourth aspect of the present invention, and obtaining a supernatant by centrifugation;

[0019] 2) dilute the supernatant obtained in step 1) to 5-15% and spray on the leaves;

[0020] Wherein, the centrifugation is performed at 5000-8000 rpm for 10-20 min.

[0021] The beneficial effects achieved by the present invention include:

[0022] 1) A total of 8 strains with high α-glucosidase inhibition rates were screened out using the α-glucosidase inhibition experiment, and their inhibition rates were all greater than 40%. A high-yield Bacillus amyloliquefaciens S1 was obtained through repeated screening using HPLC-UV;

[0023] 2) The most effective formula obtained by optimizing the culture medium is: 4% sorbitol, 4% soy peptone, 0.5g / L FeSO4, 0.8g / L MnSO4. The fermentation experiment with the optimized formula obtained a DNJ yield of 264.18±4.36mg / L by Bacillus amyloliquefaciens S1;

[0024] 3) The best fermentation conditions obtained by optimizing the culture medium conditions were: culture medium pH 6.5, rotation speed 80rpm, culture temperature 37℃, seed age 10h, and fermentation 72h. The fermentation DNJ yield under the optimized culture medium formula and culture conditions was 317.21±5.32mg / L;

[0025] 4) The fermentation supernatant was used to gavage mice and the changes in blood glucose over time were detected. Compared with other control groups, the fermentation supernatant significantly reduced the postprandial blood glucose level of mice. Compared with the blank control, the fermentation supernatant ultimately reduced the blood glucose area under the curve (AUC) by 15.3%;

[0026] 5) After extracting the fermentation supernatant, it was diluted to 10% spraying concentration to treat mulberry leaves, which could reduce the cost of use and increase the DNJ extraction rate of mulberry leaves by 4.87%. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 A simplified synthetic pathway for microbial production of DNJ;

[0028] Figure 2 is the α-glucosidase inhibition rate of fermentation supernatants of different strains;

[0029] Figure 3 The results of DNJ liquid phase test: A is the standard; B is the fermentation supernatant;

[0030] Figure 4 is the DNJ standard curve;

[0031] Figure 5 The results of strain rescreening;

[0032] Figure 6 Morphological observation of Bacillus amyloliquefaciens S1: A is the colony picture, B is the microscopic examination result;

[0033] Figure 7 is the phylogenetic tree of Bacillus amyloliquefaciens S1;

[0034] Figure 8 The growth curve of Bacillus amyloliquefaciens S1 (A) and the fermentation curve in LB medium (B);

[0035] Fig. 9 This is the heat map of drug resistance of Bacillus amyloliquefaciens S1;

[0036] Fig.10 Bubble diagram for KEGG enrichment analysis;

[0037] Fig.11 Results of nitrogen source screening and dosage for fermentation: A is the type of nitrogen source, B is the dosage of nitrogen source;

[0038] Fig.12 The results of carbon source screening and dosage for fermentation: A is the type of nitrogen source, B is the dosage of nitrogen source;

[0039] Fig.13 The selection and dosage of inorganic salts in the culture medium: A is MgSO4, B is ZnSO4, C is FeSO4, and D is MnSO4;

[0040] Fig.14 Experimental results for setting fermentation conditions: A is temperature, B is rotation speed, C is medium pH, D is seed age, E is inoculum size, and F is fermentation days;

[0041] Fig.15 The experimental results of the inhibition of blood glucose by the fermentation supernatant of Bacillus subtilis S1: A is the sucrose tolerance curve of mice, and B is the area under the blood glucose curve of each experimental group;

[0042] Fig.16 These are the results of the spraying experiment with diluted solution of Bacillus amyloliquefaciens S1. DETAILED DESCRIPTION

[0043] Example 1 Screening and identification of GNJ-producing strains

[0044] (1) Search for potential DNJ-producing bacteria in KEGG

[0045] KEGG was searched separately gutB1 , ykt , gabT1 Click the Taxonomy option in the GENES region to get the distribution of the orthologous genes corresponding to the gene in different species. According to the retrieved results and the strains preserved in the laboratory, determine the strains with the potential to produce DNJ. The results are as follows Figure 1 shown.

[0046] (2) Take out the strain stored at -80℃, inoculate it into LB broth or MRS liquid culture medium at a 2% inoculum, vortex for 10 seconds, and place the inoculated liquid culture medium in a 37℃ incubator for 12 hours. When subculturing, inoculate it into the subculture tube at a 2% inoculum, and other conditions remain the same. Take the third-generation tube for subsequent experiments.

[0047] Searching for DNJ synthesis gene related information in KEGG, we get gutB1 , ykt , gabT1 The KO numbers are: K00004, K01092, and K00823.

[0048] Our laboratory has preserved many strains of lactic acid bacteria and Bacillus. We used the KO database in KEGG to search for gutB1 , ykt , gabT1 The results are shown in Table 1.

[0049] Table 1 Bacterial genera containing orthologous genes of DNJ synthesis gene cluster

[0050]

[0051] Table Notes: Only some species of Lactobacillus, such as Lactobacillus fermentum and Lactobacillus plantarum, have orthologous genes of the DNJ synthesis gene cluster.

[0052] The search results showed that some strains of Lactobacillus, such as Lactobacillus fermentum and Lactobacillus plantarum, and almost all strains of Bacillus contained all the orthologous genes of the DNJ synthesis gene cluster. Combined with the laboratory preserved strains, 15 strains of Lactobacillus fermentum, 23 strains of Lactobacillus plantarum, and 12 strains of Bacillus (including Bacillus atrophaeus, Bacillus amyloliquefaciens, Bacillus coagulans, etc.) were selected for preliminary screening.

[0053] (3) Add 150 μL of 0.1 M phosphate buffer (pH 6.8), 20 μL of fermentation supernatant (the fermentation broth was centrifuged at 15000 r / min for 5 min and the supernatant was taken), 50 μL of α-glucosidase solution (25 mg / mL), and 50 μL of PNPG (0.9133 mg / mL) to a 96-well ELISA plate. Mix well and react in a 37°C incubator for 45 min. Add 50 μL of sodium carbonate solution (71.39 mg / mL) to terminate the reaction and measure the absorbance (405 nm) with an ELISA reader.

[0054] The top 8 strains with the highest α-glucosidase inhibition rate were selected for subsequent rescreening.

[0055] The α-glucosidase inhibition test was used to detect the fermentation supernatants of 50 strains to be screened. The results are as follows Figure 2 As shown. Overall, the α-glucosidase inhibition rate of Bacillus is higher than that of Lactobacillus, which may be because Bacillus has stronger metabolic ability and can produce more secondary metabolites. The α-glucosidase inhibition rate of strain 410-3 was the highest, at 57.89±5.22%. Eight strains with an α-glucosidase inhibition rate of more than 40% were selected for liquid phase detection (strain information is shown in Table 2), and strains with higher DNJ production were further screened.

[0056] Table 2 Information of 8 strains selected in the initial screening

[0057]

[0058] (4) Detection of DNJ content in fermentation broth by HPLC-UV

[0059] High performance liquid chromatography (HPLC-UV) was used to determine the DNJ content in the fermentation broth. The detection method was slightly modified based on the national standard GB / T40642-2021 "Detection of 1-deoxynojirimycin in mulberry leaf extract: High performance liquid chromatography" and the literature method of Ye Jingjing et al. The detailed steps are as follows:

[0060] a. Prepare DNJ standard solution

[0061] Weigh 1000 mg of DNJ standard in a 1L volumetric flask, add 0.01 mol / L dilute hydrochloric acid to make up to volume, and prepare a mother solution with a concentration of 1000 mg / L. Dilute the mother solution with dilute hydrochloric acid to obtain a series of DNJ standard solutions with concentration gradients such as 1 mg / L, 5 mg / L, 10 mg / L, 20 mg / L, 50 mg / L, and 100 mg / L.

[0062] b. Treatment of fermentation broth

[0063] Pipette 1 ml of fermentation liquid into a 1.5 ml centrifuge tube, centrifuge at 15000 r / min for 5 min in a low-temperature high-speed centrifuge, take out the supernatant, dilute it seven times and set aside.

[0064] c. DNJ derivatization reaction

[0065] Take 100 μL of fermentation supernatant or DNJ standard solution in a 1.5 mL centrifuge tube, add 125 μL of 0.4 mol / L potassium borate buffer (pH 8.5), 250 μL of 5 mmol derivatization reagent FMOC-C1 (dissolved in acetonitrile), mix for 5 seconds, react in a 25°C constant temperature water bath for 25 minutes, add 1 mol / L glycine to react for 20 minutes to neutralize the remaining FMOC-C1. Then add 75 μL of 0.1% acetic acid solution, dilute to 1000 μL with water, filter with a 0.22 μm pore size microporous filter membrane, bottle and test on the machine.

[0066] d. High performance liquid chromatography detection method

[0067] The chromatographic column was Shimadzu ShimNex CS C18 (specification: 5μm, 4.6x150mm), the detector was UVD ultraviolet detector, the detection wavelength was 254 nm, the mobile phase was acetonitrile-0.1% acetic acid (36:64), the flow rate was 1.0mL / min, and the injection volume was 10μL; the detection results of the standard and fermentation supernatant are shown in Figure 3 As shown: The DNJ derivative peak time of the DNJ standard is 9.92min ( Figure 3 A), the DNJ derivative peak time of the fermentation supernatant was 9.87min ( Figure 3 B), the two are basically the same.

[0068] e. Draw the DNJ standard curve

[0069] A series of DNJ standard solutions were tested, and the DNJ standard curve was drawn with the DNJ concentration as the horizontal axis and the peak area as the vertical axis.

[0070] The DNJ standard curve was drawn with DNJ concentration as the horizontal axis and the peak area as the vertical axis. Figure 4 As shown, the calculated standard curve regression equation is: y = 3400.2x + 34076, and the correlation coefficient R² = 0.9997, which has a good linear relationship.

[0071] (5) Rescreening of DNJ-producing bacteria

[0072] The 8 strains obtained in the initial screening were tested by high performance liquid chromatography to select the strain with the highest yield. The results are as follows Figure 5 As shown in the figure, Bacillus amyloliquefaciens S1 had the highest DNJ yield in the fermentation supernatant, which was 1.10±0.07 mg / L. Bacillus amyloliquefaciens S1 was selected for subsequent fermentation optimization experiments.

[0073] (6) Morphological observation of Bacillus amyloliquefaciens S1

[0074] Streak the plate on LB nutrient agar, invert it and incubate it in a 37℃ incubator for 24 hours, then observe the colony morphology; pick the colonies for Gram staining and examine them under a high-power microscope. Figure 6 As shown. After streaking culture on the plate, white colonies grow, with a diameter of about 6-9 mm, milky white and opaque, showing bulges, roughness, dullness, and opacity ( Figure 6 A). After Gram staining, the strain appears rod-shaped under a high-power microscope and is a Gram-positive bacterium ( Figure 6 B).

[0075] (7) 16Sr RNA sequencing of Bacillus amyloliquefaciens

[0076] The bacterial genome was extracted using Tiangen Bio's DNJ genome extraction kit (DP305) and sent to Beijing Sangon Biotechnology for 16Sr RNA sequencing. The full length of the 16Sr RNA sequence of Bacillus amyloliquefaciens S1 was 1427 bp (SEQ ID NO: 1). After sequencing, the sequence was aligned on NCBI, and a phylogenetic tree based on 16Sr RNA was constructed using MEGA (V11.0). The results are as follows Figure 7 shown.

[0077] 16sRNA sequence:

[0078]

[0079] After sequence comparison, strain S1 was identified as Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ), the strain was deposited in the General Microbiology Center (CGMCC) of China Microorganism Culture Collection Administration on March 26, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.30156.

[0080] (8) Determination of growth curve and fermentation curve of Bacillus amyloliquefaciens S1

[0081] The third generation seeds were inoculated into LB medium at a 2% inoculum and cultured in a 37°C incubator. Samples were taken at 0, 2, 4, 6, 8, 9, 10, 11, 12, 13, 14, 16, and 18 h of culture, and the OD of the culture medium was measured using a multifunctional microplate reader. 600 , with time as the horizontal axis, OD 600 The growth curve is drawn with the value as the vertical axis.

[0082] The third generation seeds were inoculated into LB medium at 2% inoculation, and cultured in a 37°C incubator. Samples were taken every 4 hours and immediately placed in a refrigerator to terminate the fermentation. The DNJ content of the supernatant was detected. The fermentation curve was drawn with time as the horizontal axis and DNJ content as the vertical axis. The results are shown in Figure 8 It can be seen that Bacillus amyloliquefaciens S1 is in the hysteresis phase from 0 to 6 hours and grows relatively slowly ( Figure 8 A); 6-12h in the logarithmic growth phase, rapid growth, OD 600 The value changes rapidly; it enters the stable period after 14 hours, OD 600 Maintaining stability ( Figure 8 B).

[0083] Bacillus amyloliquefaciens S1 was inoculated into LB medium, and samples were taken every 4 hours to detect the DNJ content of the medium. Fig.11 The DNJ produced by strain S1 in LB medium accumulated over time, with 10h-20h being the fastest period of DNJ accumulation, and 44-48h DNJ production basically stopped accumulating, with the maximum production reaching 3.91±0.23mg / L.

[0084] (9) Whole genome sequencing

[0085] The genome of strain S1 was extracted using Tiangen Biotechnology’s DNA extraction kit (DP305) and sent to Beijing Biomaike Cloud Technology Co., Ltd. for whole-genome sequencing.

[0086] The number of coding genes (CDS) predicted by the whole genome sequence is 4241, and its genome is predicted to contain 11 rRNAs, 82 tRNAs, and 1 16sRNA. CARD analysis showed that it contains 229 drug resistance genes, and its drug resistance heat map is shown in the figure below: Fig. 9 shown.

[0087] KEGG enrichment analysis was performed on the genes of Bacillus amyloliquefaciens S1, such as Fig.10 As shown, Bacillus amyloliquefaciens S1 has 933 genes related to metabolism and has strong metabolic capacity, among which there are 307 genes related to carbohydrate metabolism and 378 genes related to amino acid metabolism, respectively.

[0088] The DNJ synthesis gene cluster in Bacillus amyloliquefaciens S1 was analyzed by blast comparison on NCBI. The results are shown in Table 3:

[0089] Table 3 DNJ biosynthesis gene cluster in Bacillus amyloliquefaciens S1

[0090]

[0091] Example 2 Optimization of fermentation conditions for DNJ production by Bacillus amyloliquefaciens S1

[0092] (1) Screening of dominant nitrogen sources for DNJ production

[0093] The nitrogen sources in LB were replaced with peptone, yeast extract powder, beef extract powder, malt extract powder, soy peptone, casein, ammonium sulfate, etc., and other components remained unchanged to prepare different fermentation media. The third-generation seeds were inoculated with 2% into the fermentation medium, placed in a 37°C incubator for fermentation for 48 hours, and the DNJ content in the fermentation supernatant was detected. The nitrogen source with the highest DNJ production was selected, and its concentration was adjusted to 2%, 4%, 6%, 8%, and 10% to prepare the fermentation medium. After inoculation and fermentation for 48 hours, the DNJ content in the supernatant was detected.

[0094] The results are as follows Fig.11 As shown, the yield of Bacillus amyloliquefaciens S1 under different nitrogen sources varies greatly ( Fig.11 A), among which yeast extract and soy peptone are more suitable nitrogen sources, the DNJ yield of other nitrogen sources is low, basically below 10 mg / L. S1 has the highest DNJ yield in soy peptone, which is 36.03±2.36 mg / L. The DNJ yield of Bacillus amyloliquefaciens S1 fermentation increases first and then decreases with the increase of soy peptone concentration. When the soy peptone concentration is 4%, the maximum yield is 64.32±2.34 mg / L ( Fig.11 B).

[0095] (2) Screening of superior carbon sources for DNJ production

[0096] Add 2% lactose, glucose, fructose, sucrose, sorbitol, and glycerol to 4% soy peptone to prepare different fermentation media. Inoculate 2% of the third-generation seeds into the fermentation medium, place it in a 37°C incubator for fermentation for 48 hours, and detect the DNJ content in the fermentation supernatant. Select the carbon source with the highest DNJ yield, adjust its concentration to 2%, 4%, 6%, 8%, and 10% to prepare the fermentation medium, and detect the DNJ content in the supernatant after inoculation and fermentation for 48 hours.

[0097] The results are as follows Fig.12 As shown in Figure 2, the yield of Bacillus amyloliquefaciens S1 under different carbon sources varies greatly. Its DNJ yield is the highest when sorbose is used as the carbon source, which is 71.46±4.56 mg / L ( Fig.12 A), with the increase of carbon source concentration in fermentation broth, the DNJ production of Bacillus amyloliquefaciens first increased and then decreased, with the highest yield of 81.46±5.27 mg / L at 4% sorbitol ( Fig.12 B).

[0098] (3) Effects of different inorganic salts on DNJ production by strains

[0099] Four inorganic salts, MgSO4, ZnSO4, FeSO4, and MnSO4, were selected and added to 4% soy peptone and 4% sorbitol culture medium at 0, 0.2, 0.4, 0.6, 0.8, and 1.0 g / L, respectively, to prepare different fermentation mediums. After inoculation, the medium was placed in a 37°C incubator for fermentation for 48 hours, and the DNJ content in the fermentation supernatant was detected. Fig.13 It can be seen that under different concentrations of MgSO4 and ZnSO4 ( Fig.13 A. Fig.13 B), the DNJ production of strain S1 did not change much, indicating that it had little effect on the DNJ production of strain S1. Fig.13 C. Fig.13 D), the strain yield changed significantly, indicating that it had a greater impact on the DNJ yield of strain S1.

[0100] (4) Orthogonal experiment to optimize culture medium composition

[0101] The four factors of sorbitol, soy peptone, FeSO4 and MnSO4 that have a greater impact on DNJ production in the single factor experiment were selected, and a four-factor four-level orthogonal table was designed to obtain the optimal formula for DNJ production by Bacillus amyloliquefaciens S1 through orthogonal experiments. The experimental results are shown in Table 4.

[0102] Table 4 Orthogonal experiment for medium formulation optimization

[0103]

[0104] The orthogonal results were analyzed by range analysis, and the best medium formula was obtained as follows: 4% sorbitol, 4% soy peptone, 0.5g / L FeSO4, 0.8g / L MnSO4. The fermentation experiment with the optimized formula obtained the maximum yield of DNJ produced by Bacillus amyloliquefaciens S1 to be 264.18±4.36mg / L.

[0105] (5) Effect of culture temperature on DNJ yield

[0106] Using the optimized culture medium, DNJ fermentation experiments were carried out at 28°C, 31°C, 34°C, 37°C, 40°C, and 43°C, and the DNJ content in the fermentation supernatant was detected.

[0107] Fig.14 A shows that with the increase of temperature, the DNJ production of strain S1 first increases and then decreases, and 37℃ is the optimal temperature for its DNJ production.

[0108] (6) Effect of culture speed on DNJ yield

[0109] The optimized culture medium was used to perform DNJ fermentation experiments at rotation speeds of 0, 40, 80, 120, and 160 r / min, and the DNJ content in the fermentation supernatant was detected.

[0110] Depend on Fig.14 B shows that with the increase of rotation speed, the DNJ production first increases and then decreases, and the optimal rotation speed for strain S1 to produce DNJ is 80rpm.

[0111] (7) Effect of culture medium pH on DNJ production

[0112] Using the optimized culture medium, the pH of the culture medium was adjusted to 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0, and the DNJ content in the supernatant was detected after fermentation.

[0113] from Fig.14 C shows that with the increase of pH in the culture medium, the DNJ production of strain S1 first increases and then decreases, and the optimal pH is 6.5.

[0114] (8) Effect of seed age on DNJ yield

[0115] Seed tubes with seed ages of 6h, 8h, 10h, 12h, 14h, and 16h were inoculated into the optimized culture medium, and the DNJ content in the supernatant was detected after fermentation.

[0116] Depend on Fig.14 D It can be seen that when the seed culture time is too long or too short, the DNJ content in the culture medium will be degraded, and the optimal seed age is 10h.

[0117] (9) Effect of inoculum amount on DNJ yield

[0118] The inoculation amount was set to 1%, 2%, 3%, 4%, and 5%, and inoculated into the optimized culture medium. After fermentation, the DNJ content in the supernatant was detected. Fig.14 E, there was no significant change in the DNJ content in the culture medium under different inoculation sizes, indicating that the inoculation size had little effect on the DNJ production of strain S1.

[0119] (10) Fermentation process of Bacillus amyloliquefaciens S1

[0120] Under the optimized culture medium and culture conditions, Bacillus amyloliquefaciens S1 was fermented, and the DNJ content was detected on the 1st, 2nd, 3rd, 4th, 5th and 6th day of fermentation. Fig.14 As shown in F, it can be seen that the production of DNJ reached the maximum after 3 days of fermentation and remained basically stable, and the maximum DNJ production was 317.21±5.32 mg / L.

[0121] In summary, the most effective formula obtained by optimizing the culture medium is: 4% sorbitol, 4% soy peptone, 0.5g / L FeSO4, 0.8g / L MnSO4. The fermentation experiment with the optimized formula obtained a DNJ yield of 264.18±4.36mg / L by Bacillus amyloliquefaciens S1.

[0122] The optimal fermentation conditions obtained by optimizing the culture medium conditions were: culture medium pH 6.5, rotation speed 80rpm, culture temperature 37℃, seed age 10h, and fermentation 72h. The fermentation DNJ yield under the optimized culture medium formula and culture conditions was 317.21±5.32mg / L. The final yield was 81.3 times higher than that of the initial LB culture medium.

[0123] Example 3 Application of fermentation supernatant of Bacillus amyloliquefaciens S1

[0124] (1) Using the results of Example 2, the optimal culture medium formula and fermentation conditions were used to prepare a fermentation broth for 3 days. After centrifugation at 6000 rpm for 15 min, the fermentation supernatant was collected for later use.

[0125] (2) Sucrose tolerance test in mice

[0126] 12 mice were divided into four groups, and the amount of sucrose gavage was calculated according to 3g / kg body weight. The gavage sucrose solution was mixed with 100uL water, sterile culture medium, fermentation supernatant of Bacillus amyloliquefaciens S1, and 5mg / kg acarbose, and water was added to the mixture to finally prepare 200uL gavage solution. After gavage of mice, the blood glucose values ​​at 0, 30min, 60min, 90min, and 12min were measured, and the blood glucose concentration was plotted over time, and the area under the curve was calculated.

[0127] Depend on Fig.15It can be seen that there was no significant difference in the postprandial blood glucose level of mice between the pure water group and the sterile fermentation liquid group. The fermentation supernatant of strain S1 significantly reduced the postprandial blood glucose level of mice compared with other control groups, and its effect of reducing blood glucose level was slightly worse than that of the positive control (5 mg / kg acarbose) ( Fig.15 A). Compared with the blank control group (pure water), the fermentation supernatant ultimately reduced the blood glucose area under the curve (AUC, Fig.15 B).

[0128] (3) Experiment on spraying diluted solution of Bacillus amyloliquefaciens S1

[0129] The supernatant of Bacillus amyloliquefaciens S1 was diluted 10 times in a gradient to prepare 10%, 1%, and 0.1% diluted bacterial solutions, which were sprayed on the surface of mulberry leaves. The spraying amount was calculated as 1 ml / 10 g. After spraying, the mulberry leaves were placed outdoors to dry. The treated mulberry leaves were placed in an oven and dried at 120 ° C for 6 hours. The dried mulberry leaves were ground into powder using a grinder for later use. The extraction experiment was carried out under the optimized conditions to detect the DNJ content in the extract.

[0130] Depend on Fig.16 It can be seen that the spraying concentration (fermentation supernatant concentration) of 10% and 100% can significantly increase the DNJ content in the extract. When applying in agriculture, the cost should be comprehensively considered. Using a 10% spraying concentration to treat mulberry leaves can reduce the cost of use and increase the DNJ extraction rate of mulberry leaves by 4.87%.

[0131] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A strain of Bacillus amyloliquefaciens producing 1-deoxynojirimycin ( Bacillus amyloliquefaciens ) S1, It is characterized in that The strain was deposited in the General Microbiology Center (CGMCC) of China Microorganism Culture Collection Administration on March 26, 2024, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.30156.

2. A bacterial agent, It is characterized in that The bacterial agent comprises the strain according to claim 1.

3. A microbial fermentation product, It is characterized in that The microbial fermentation product is the supernatant obtained by centrifugation after fermentation of the strain according to claim 1 or the bacterial agent according to claim 2, and the fermentation medium used in the fermentation is: 3~5% sorbitol, 3~5% soy peptone, 0.2~0.8g / L FeSO 4 and 0.5~1g / L MnSO 4 ; The culture conditions of the fermentation culture are: medium pH 6-7, rotation speed 70-90 rpm, culture temperature 35-39°C, seed age 8-12h, fermentation 48-96h; The fermentation solution was prepared on day 3 of fermentation and the fermentation supernatant was obtained after centrifugation at 6000 rpm for 15 min.

4. A method for preparing a fermentation product using the strain according to claim 1 or the bacterial agent according to claim 2, It is characterized in that The method comprises fermenting and culturing the strain according to claim 1 or the bacterial agent according to claim 2, and separating and obtaining a fermentation supernatant; the fermentation medium used in the fermentation and culturing comprises 3-5% sorbitol, 3-5% soy peptone, 0.2-0.8 g / L FeSO 4 and 0.5~1g / L MnSO 4 ; The culture conditions of the fermentation culture are: medium pH 6-7, rotation speed 70-90 rpm, culture temperature 35-39°C, seed age 8-12h, fermentation 48-96h; The fermentation solution was prepared on day 3 of fermentation and the fermentation supernatant was obtained after centrifugation at 6000 rpm for 15 min.

5. Use of the strain according to claim 1, the bacterial agent according to claim 2, the fermentation product according to claim 3, or the method according to claim 4 in the preparation of a hypoglycemic preparation.

6. Use of the strain according to claim 1, the bacterial agent according to claim 2, the fermentation product according to claim 3, or the method according to claim 4 in improving the extraction rate of DNJ in mulberry leaves; It is characterized in that The specific steps are: 1) obtaining a supernatant by using the fermentation product of claim 3 or the method of claim 4; 2) Dilute the supernatant obtained in step 1) to 5-15% and spray on the leaves.

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