Bacillus subtilis WD01 and its application
By screening and identifying the WD01 strain of Bacillus subtilis, the problems of low DNJ yield and single function of natto product were solved, efficient production of DNJ and innovative natto food functions were achieved, and the competitiveness of the domestic natto industry was enhanced.
Patent Information
- Application Number
- CN202410836212.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-06-26
- Publication Date
- 2025-04-04
AI Technical Summary
In the existing diabetes treatment drugs, 1-deoxynojirimycin (DNJ) has low yield and complex process, making it difficult to produce on a large scale, and domestic natto products have a single function, making it difficult to compete with Japanese natto products.
By screening and identifying the Bacillus subtilis WD01 strain, this strain efficiently produces DNJ in fermented black beans, and Natto food with high yield of DNJ is prepared through soybean fermentation, innovating the functions of Natto food.
It has achieved high yield of DNJ, improved the functional diversity of natto food, broken through the limitations of the traditional natto industry, and has high commercial value.
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Abstract
Description
Technical Field
[0001] The invention relates to the technical field of microorganisms, and in particular to Bacillus subtilis WD01 and applications thereof. Background Art
[0002] As diabetes becomes increasingly globalized and younger, the treatment of diabetes has become a thorny issue in the medical community. Although there are various types of drugs for treating diabetes, long-term use generally has certain side effects. 1-Deoxynojirimycin (DNJ) and its derivatives, as highly effective α-glucosidase inhibitors, have been widely used in the development of drugs for treating diabetes. Currently, DNJ used in medicine is mainly extracted from mulberry leaves, but the content of DNJ in mulberry leaves is low. In addition, the process of artificial chemical synthesis of DNJ is complex, with low returns and high costs, so it is very difficult to obtain DNJ on a large scale. Therefore, how to increase the production of DNJ has become a research hotspot for scientific researchers.
[0003] Natto refers to a fermented food made from selected soybeans fermented by Bacillus subtilis natto. It has a unique flavor and viscosity, is rich in probiotics, a variety of nutrients and functional ingredients, and is particularly concerned about nattokinase as a thrombolytic active ingredient. It has important application value in the prevention and treatment of cardiovascular and cerebrovascular diseases, and is crucial to alleviating the health problems of the elderly in my country. Japan is the first country in the world to produce natto functional foods, and is at the forefront of the world in the field of research and development and application of natto fermented foods. The quality of domestic natto products is uneven and the functions are single. Especially in the face of the impact of Japanese natto product brands, the development is very slow. How domestic natto can gain a competitive advantage over Japanese natto products, stand out in the fierce competition, and revitalize the domestic natto industry is worthy of in-depth discussion. Some Bacillus subtilis have been reported to synthesize DNJ. The selection of Bacillus subtilis with high DNJ production for the production of natto food, the preparation of new natto food with hypoglycemic function, and the enrichment of new functions of natto food are expected to break through the problems existing in the traditional domestic natto industry. Summary of the invention
[0004] The purpose of the present invention is to provide a Bacillus subtilis WD01 and application thereof in order to solve the above problems.
[0005] In order to achieve the above object, the present invention adopts the following technical solutions:
[0006] A method for screening Bacillus subtilis WD01 strains comprises the following steps:
[0007] A DNJ-producing strain was screened out from fermented black beans, with a suitable growth temperature of 30-40°C and a suitable pH of 6.5-7.5. The strain was identified as Bacillus subtilis through morphological, physiological and biochemical identification and 16S rRNA gene molecular identification, and was named WD01 (preservation number CCTCC M 2022909).
[0008] A method for screening Bacillus subtilis WD01 strains comprises the following steps:
[0009] The skim milk plate method was used for initial screening, and then a new α-glucosidase inhibitor screening model was established by replacing the traditional pNPG with maltose as the substrate and glucose as the detection index. Take 1g of the crushed test sample in a sterile PA bottle, add 10mL of sterile water, shake on a shaker for 30min, heat at 80℃ in a water bath for 20min, let it stand and cool to room temperature, absorb the supernatant and dilute it on a skim milk plate (LB solid plate containing 1% skim milk powder), culture at 37℃ for 24h, pick a single colony with a larger transparent circle, transfer it to the LB solid plate after separation by streaking, and store it in a 4℃ refrigerator after culture. After strain activation, transfer it to soybeans and culture at 37℃ for 36-48h. Using maltose as the substrate and the fermentation supernatant as the inhibitor, the glucose content is detected after hydrolysis by α-glucosidase, and the inhibition rate of the fermentation broth on α-glucosidase is calculated, and the strain with a high inhibition rate of the fermentation supernatant on α-glucosidase is screened out. The reaction formula is as follows:
[0010]
[0011] The strain with the highest inhibition rate of α-glucosidase and the lowest odor was selected, and the strain was identified according to the prescribed method. The 16S rRNA gene sequence of the Bacillus subtilis strain provided by the present invention has 1484 bases, and the 16S rRNA gene sequence is shown in SEQ ID NO.1:
[0012]
[0013] The measured 16S rRNA sequence showed that strain WD01 belonged to Bacillus subtilis. Based on the morphological identification, physiological and biochemical identification and 16S rRNA gene molecular identification results, the strain was identified as Bacillus subtilis. The highest inhibitory activity of the fermentation supernatant of this strain on α-glucosidase was 84.2% (the fermentation broth was diluted 3000 times), which was 9 times higher than the inhibitory activity of the high-yield DNJ strain HZ-12 preserved in this experiment on α-glucosidase.
[0014] A Bacillus subtilis WD01 and its application, and a method for preparing natto food, comprising the following steps:
[0015] (1) The Bacillus subtilis WD01 strain of claim 1 was inoculated into LB liquid culture medium and cultured at 37°C for 12 hours, then placed in a sterile tube and frozen at -70°C for storage;
[0016] (2) preparing seed solution;
[0017] (3) preparing a bean fermentation substrate, i.e., soybean;
[0018] (4) Fermentation culture: Add the seed liquid to the bean fermentation substrate at an inoculation rate of 3%-5% (v / w), mix well, and culture statically at 37° C. for 36-48 hours to obtain natto food.
[0019] A Bacillus subtilis WD01 and its application, a seed liquid preparation method comprises the following steps:
[0020] (1) Streak the frozen bacterial strain on LB solid medium and culture at 37°C for 12 h;
[0021] (2) Transfer the activated strains to the seed solution and culture at 37°C and 180 rpm for 8-11 hours;
[0022] (3) According to the method for preparing natto food of the present invention, the method for preparing a bean fermentation substrate comprises the following steps:
[0023] 1) Selection: Remove discolored, moldy, and insect-infested beans, remove debris, and wash with water;
[0024] 2) Soaking: Add 5 times volume (v / w) of hot water and soak for 2h;
[0025] 3) Draining: Make the initial moisture content of the beans 50%-60%;
[0026] 4) Steaming: Steaming the whole beans or the crushed beans at 115° C. under high temperature and high pressure for 30 minutes to obtain a bean fermentation substrate.
[0027] A Bacillus subtilis WD01 and its application, a method for preparing a DNJ-rich meal replacement powder, comprising the following steps:
[0028] The natto food is dried at 100°C and then crushed with a grinder to obtain fermentation dry powder, which is added as a nutritional enhancer.
[0029] The invention separates Bacillus subtilis with high DNJ production from traditional fermented black beans, uses soybean as a fermentation substrate, obtains a high-production process of DNJ, prepares a novel natto food with high DNJ content, and innovates the function of traditional natto food. DETAILED DESCRIPTION
[0030] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments.
[0031] Example 1 Strain Screening
[0032] Add sterile water to the fermented black bean curd product, heat at 80℃ for 20min, take the supernatant and dilute it to the screening medium plate (peptone 10g / L, yeast powder 5g / L, sodium chloride 10g / L, agar powder 15g / L, skim milk powder 10g / L, pH 7.2), culture at 37℃ for 12-24h, pick the single colony with a larger transparent circle, amplify the nattokinase gene by PCR reaction, and first screen out the Bacillus subtilis that meets the conditions. The total DNA of the strain was extracted by a bacterial genome extraction kit, and the PCR amplification was performed using the nattokinase gene open reading frame specific primers aprNF (CCGTGAGAAGCAAAAAATTGTGGATCA) and aprNR (ATTTATTGTGCAGCTGCTTGTACGTTG). The PCR program was: 95℃ pre-denaturation for 5min; 95℃ denaturation for 30s, 55℃ annealing for 30s, 72℃ extension for 1min, 30 cycles; 72℃ extension for 10min. The Bacillus subtilis that can produce a clearing zone was selected.
[0033] DNJ detection is performed by glucose oxidation method, the specific operation is as follows:
[0034] Blank group: 2g / L maltose 500μL + 550μL HAC-NaAC buffer (0.2M pH4.5) + 150μL 0.002g / mL α-glucosidase solution, incubate in a water bath at 40°C for 30min, then immediately in a boiling water bath for 5min, and use a glucose detection kit to detect the Glu content before and after the reaction.
[0035] Experimental group: 2g / L maltose 500μL + 450μL HAC-NaAC buffer (0.2M pH4.5) + 100μL fermentation supernatant (or DNJ standard solution) diluted a certain number of times + 150μL 0.002g / ml α-glucosidase solution, water bath at 40℃ for 30min, immediately ice for 5min, and use a glucose detection kit to detect the Glu content before and after the reaction.
[0036] α-glucosidase inhibition rate = (A-B+C / 12) / (AD)×100%
[0037] Where: A: Glu value of the system after the blank group reaction
[0038] B: Glu value of the system after the reaction in the experimental group
[0039] C: Glu value after dilution of fermentation supernatant
[0040] D: Initial Glu value of blank group reaction system
[0041] Example 2 Strain Identification
[0042] Morphological and physiological and biochemical identification
[0043] Bacterial morphology and physiological and biochemical identification were carried out according to the Bergey's Manual of Bacterial Identification. When the isolated strains grew on LB solid medium, the edges of the colonies were rough and the surface of the bacterial lawn was wrinkled. The suitable growth temperature was 28-40℃ and the suitable pH was 6.5-7.5. Casein hydrolysis was positive, starch hydrolysis was positive, nitrate utilization was positive, citrate utilization was positive, and gelatin liquefaction test was positive. The bacteria were straight rod-shaped, 1.8-2.9×0.8-1.2μm, and Gram staining was positive.
[0044] 16S rRNA molecular identification
[0045] The total DNA of the strain was extracted by a bacterial genome extraction kit, and the 16S rRNA gene PCR amplification was performed using universal primers (27F: agagtttgatcctggctcag) and (1492R: ggttaccttgttacgactt). The PCR program was: 95℃ pre-denaturation for 5min; 95℃ denaturation for 45s, 55℃ annealing for 45s, 72℃ extension for 1.5min, 28 cycles; 72℃ extension for 10min. The sequencing results showed that the 16S rRNA gene sequence of the strain had 1484 bases, and the similarity with Bacillus subtilis ZJ-1 reached 99%. The phylogenetic tree was constructed by MEG.4 software, and the phylogenetic tree also showed that the strain belonged to Bacillus subtilis. Combining the 16SrRNA molecular identification with the previous morphological and physiological and biochemical identification results, the strain was identified as Bacillus subtilis of the genus Bacillus and named WD01.
[0046] Example 3 Soybean solid fermentation
[0047] 1. Use Bacillus subtilis WD01 as the starting strain.
[0048] 2. Activate the strain: Streak the refrigerated strain onto an LB plate and culture at 37°C for 12 hours.
[0049] 3. Seed liquid culture: transfer the seeds activated on LB solid plate to LB liquid medium and culture at 37°C, 180r / min for 8h;
[0050] 4. Preparation of soybean substrate:
[0051] (1) Soybean selection: Remove discolored, moldy, and worm-eaten soybeans, remove sand, gravel, soil, and other debris mixed with oil, and wash both sides with water.
[0052] (2) Soaking: Add 5 times the volume (v / w) of hot water and soak for 2 h.
[0053] (3) Draining: Drain the water on the surface of the soaked soybeans. The initial water content is 50%-60%.
[0054] (4) Steaming: Steam at 115°C for 30 min and cool to room temperature.
[0055] 5. Fermentation culture: Add 3% inoculum into soybeans and mix well. Culture statically at 37°C for 48 hours to obtain soybean natto food.
[0056] 6. DNJ yield analysis: 4 volumes (v / w) of water were added to the fermented soybeans, and the mixture was extracted with water at 180 rpm for 1 hour. The bacteria were removed by centrifugation at 10,000 rpm for 10 minutes. The supernatant was the DNJ water extract. The DNJ analysis method described in Example 1 was used to detect and analyze the α-glucosidase inhibitory activity of the fermented supernatant in the fermented soybeans, which reached 74.1% (the fermentation broth was diluted 3,000 times).
[0057] Example 4 Soybean solid fermentation
[0058] 1. Use frozen Bacillus subtilis WD01 as the original strain;
[0059] 2. Activate the strains: streak the strains onto LB plates and culture at 37°C for 12 hours.
[0060] 3. Seed liquid culture:
[0061] The seeds activated by LB solid culture were transferred to LB liquid culture medium and cultured at 37°C and 180 rpm for 12 h.
[0062] 4. Preparation of soybean substrate:
[0063] (1) Soybean selection: Remove discolored, moldy, and worm-eaten soybeans, remove sand, gravel, soil, and other debris mixed with oil, and wash both sides with water.
[0064] (2) Soaking: Add 5 times the volume (v / w) of hot water and soak for 2 h.
[0065] (3) Draining: Drain the water from the soaked soybeans. The initial water content is 50%-60%.
[0066] (4) Steaming: Steam at 115°C for 30 min and cool to room temperature.
[0067] 5. Fermentation culture: Add 3% inoculum into soybeans and mix well. Culture statically at 37°C for 48 hours to obtain soybean natto food.
[0068] 6. DNJ yield analysis: 4 volumes (v / w) of water were added to the fermented soybeans, and the mixture was extracted with water at 180 rpm for 1 hour. The cells were removed by centrifugation at 10,000 rpm for 10 minutes, and the supernatant was the DNJ water extract. The DNJ analysis method described in Example 1 was used to detect and analyze the α-glucosidase inhibitory activity of the fermented supernatant in the fermented soybeans, which reached 84.2% (the fermentation broth was diluted 3,000 times).
[0069] Example 5 Soybean solid fermentation
[0070] 1. Use frozen Bacillus subtilis WD01 as the original strain;
[0071] 2. Activate the strains: streak the strains onto LB plates and culture at 37°C for 12 hours.
[0072] 3. Seed liquid culture:
[0073] The seeds activated by LB solid culture were transferred to LB liquid culture medium and cultured at 37°C and 180 rpm for 12 h.
[0074] 4. Preparation of soybean substrate:
[0075] (1) Soybean selection: Remove discolored, moldy, and worm-eaten soybeans, remove sand, gravel, soil, and other debris mixed with oil, and wash both sides with water.
[0076] (2) Soaking: Add 5 times the volume (v / w) of hot water and soak for 2 h.
[0077] (3) Draining: Drain the water on the surface of the soaked soybeans. The initial water content is 50%-60%.
[0078] (4) Steaming: Steam at 115°C for 30 min and cool to room temperature.
[0079] 5. Fermentation culture: Add 3% inoculum into soybeans and mix well. Culture statically at 37°C for 48 hours to obtain soybean natto food.
[0080] 6. DNJ yield analysis: The fermented soybeans were dried at 100°C, crushed with a crusher, and extracted with 4 times the volume (v / w) of water at 180r / min for 1h. The dregs were removed by centrifugation at 10000rpm for 10min. The supernatant was the DNJ water extract. The DNJ analysis method described in Example 1 was used to detect and analyze the inhibitory activity of the extract supernatant on α-glucosidase, reaching 44.4% (the fermentation broth was diluted 3000 times).
[0081] Example 6 Soybean solid fermentation
[0082] 1. Use frozen Bacillus subtilis WD01 as the original strain.
[0083] 2. Activate the strains: streak the strains onto LB plates and culture at 37°C for 12 hours.
[0084] 3. Seed liquid culture:
[0085] The seeds activated by LB solid culture medium were transferred to LB liquid culture medium and cultured at 37°C and 180 r / min for 12 h.
[0086] 4. Preparation of soybean substrate:
[0087] (1) Soybean selection: Remove discolored, moldy, and worm-eaten soybeans, remove sand, gravel, soil, and other debris mixed with oil, and wash both sides with water.
[0088] (2) Soaking: Add 5 times the volume (v / w) of hot water and soak for 2 h.
[0089] (3) Draining: Drain the water from the soaked soybeans. The initial water content is 50%-60%.
[0090] (4) Steaming: Steam at 115°C for 30 min and cool to room temperature.
[0091] 5. Fermentation culture: Add 3% inoculum into soybeans and mix well. Culture statically at 37°C for 48 hours to obtain soybean natto food.
[0092] 6. DNJ yield analysis: The fermented soybeans were dried at 55°C, crushed with a crusher, and extracted with 4 times the volume (v / w) of water at 180r / min for 1h. The dregs were removed by centrifugation at 10000rpm for 10min. The supernatant was the DNJ water extract. The DNJ analysis method described in Example 1 was used to detect and analyze the inhibitory activity of the extract supernatant on α-glucosidase, reaching 49.7% (the supernatant was diluted 3000 times).
[0093] Name of depository: China Center for Type Culture Collection
[0094] Address of depository: Wuhan University, China
[0095] Date of deposit: June 17, 2022
[0096] Deposit number: CCTCC NO:M2022909
[0097] Classification name: Bacillus subtilis WD01
[0098] Bacillus subtilis WDO1
[0099] <110> Wuhan Junan Biotechnology Co., Ltd.
[0100] <120> Bacillus subtilis WD01 and its application
[0101] <160> 1
[0102] <210> 1
[0103] <211> 1484
[0104] <212> DNA
[0105] <213> Bacillus subtilis WD01
[0106] <400> 1
[0107]
Claims
1. A method for screening Bacillus subtilis WD01 strain, characterized in that: The following steps are involved: A DNJ-producing strain was screened out from fermented black beans, with a suitable growth temperature of 30-40°C and a suitable pH of 6.5-7.
5. The strain was identified as Bacillus subtilis through morphological, physiological and biochemical identification and 16S rRNA gene molecular identification, and was named WD01 (preservation number CCTCC M 2022909).
2. The method for screening Bacillus subtilis WD01 strain according to claim 1, comprising the following steps: The skim milk plate method was used for initial screening, and then a new α-glucosidase inhibitor screening model was established by replacing the traditional pNPG with maltose as the substrate and glucose as the detection index. Take 1g of the crushed test sample in a sterile PA bottle, add 10mL of sterile water, shake on a shaker for 30min, heat at 80℃ in a water bath for 20min, let it stand and cool to room temperature, absorb the supernatant and dilute it on a skim milk plate (LB solid plate containing 1% skim milk powder), culture at 37℃ for 24h, pick out the single colony with a larger transparent circle, transfer it to the LB solid plate after streaking separation, and store it in a 4℃ refrigerator after culture. After strain activation, transfer it to soybeans and culture at 37℃ for 48h. Using maltose as the substrate and the fermentation supernatant as the inhibitor, the glucose content is detected after hydrolysis by α-glucosidase, and the inhibition rate of the fermentation broth on α-glucosidase is calculated, and the strain with a high inhibition rate of the fermentation supernatant on α-glucosidase is screened out. The reaction formula is as follows: The strain with the highest inhibition rate of α-glucosidase and the lowest odor was selected, and the strain was identified according to the prescribed method. The 16S rRNA gene sequence of the Bacillus subtilis strain provided by the present invention has 1484 bases, and the 16S rRNA gene sequence is shown in SEQ ID NO.1: atgcctgcgggtgctatacatgcagtcgagcggacagatgggagcttgctccctgatgttagcggcggacgggtgagtaacacgtgggtaacctgcctgtaagactgggataactccgggaaaccggggctaataccggatggttgtttgaaccgcatggttcaaacataaaaggtggcttcggctaccacttacagatggacccgcggcgcattagctagttggtgaggtaacggctcaccaaggcgacgatgcgtagccgacctgagagggtgatcggccacactgggactgagacacggcccagactcctacgggaggcagcagtagggaatcttccgcaatggacgaaagtctgacggagcaacgccgcgtgagtgatgaaggttttcggatcgtaaagctctgttgttagggaagaacaagtaccgttcgaatagggcggtaccttgacggtacctaaccagaaagccacggctaactacgtgccagcagccgcggtaatacgtaggtggcaagcgttgtccggaattattgggcgtaaagggctcgcaggcggtttcttaagtctgatgtgaaagcccccggctcaaccggggagggtcattggaaactggggaacttgagtgcagaagaggagagtggaattccacgtgtagcggtgaaatgcgtagagatgtggaggaacaccagtggcgaaggcgactctctggtctgtaactgacgctgaggagcgaaagcgtggggagcgaacaggattagataccctggtagtccacgccgtaaacgatgagtgctaagtgttagggggtttccgccccttagtgctgcagctaacgcattaagcactccgcctggggagtacggtcgcaagactgaaactcaaaggaattgacgggggcccgcacaagcggtggagcatgtggtttaattcgaagcaacgcgaagaaccttaccaggtcttgacatcctctgacaatcctagagataggacgtccccttcgggggcagagtgacaggtggtgcatggttgtcgtcagctcgtgtcgtgagatgttgggttaagtcccgcaacgagcgcaacccttgatcttagttgccagcattcagttgggcactctaaggtgactgccggtgacaaaccggaggaaggtggggatgacgtcaaatcatcatgccccttatgacctgggctacacacgtgctacaatggacagaacaaagggcagcgaaaccgcgaggttaagccaatcccacaaatctgttctcagttcggatcgcagtctgcaactcgactgcgtgaagctggaatcgctagtaatcgcggatcagcatgccgcggtgaatacgttcccgggccttgtacacaccgcccgtcacaccacgagagtttgtaacacccgaagtcggtgaggtaaccttttaggagccagccgccgaaggtgggacagatgattggggtgaagtcgtaacaagagcctgcc The measured 16S rRNA sequence showed that strain WD01 belonged to Bacillus subtilis. Based on the morphological identification, physiological and biochemical identification and 16S rRNA gene molecular identification results, the strain was identified as Bacillus subtilis. The highest inhibitory activity of the fermentation supernatant of this strain on α-glucosidase was 84.2% (the fermentation broth was diluted 3000 times), which was 9 times higher than the inhibitory activity of the high-yield DNJ strain HZ-12 preserved in this laboratory on α-glucosidase.
3. The Bacillus subtilis WD01 and its use according to claim 2, characterized in that: The method for preparing natto food comprises the following steps: 1) The Bacillus subtilis WD01 strain of claim 1 was inoculated into LB liquid culture medium and cultured at 37° C. for 12 h, then placed in a sterile tube and stored frozen at -70° C.; 2) preparing seed solution; 3) preparing a bean fermentation substrate, i.e. soybean; 4) Fermentation culture: Add the seed liquid to the bean fermentation substrate at an inoculation rate of 3%-5% (v / w), mix well, and culture statically at 37° C. for 36-48 hours to obtain natto food.
4. The Bacillus subtilis WD01 and use thereof according to claim 3, characterized in that: The seed solution preparation method comprises the following steps: (1) Streak the frozen bacterial strain on LB solid medium and culture at 37°C for 12 h; (2) Transfer the activated strains to the seed solution and culture at 37°C and 180 rpm for 8-11 hours; (3) According to the method for preparing natto food of the present invention, the method for preparing a bean fermentation substrate comprises the following steps: (1) Selection: Remove discolored, moldy, and insect-infested beans, remove foreign matter, and wash with water; (2) Soaking: Add 5 times the volume (v / w) of hot water and soak for 2 h; (3) draining the beans so that the initial moisture content is 50%-60%; (4) Steaming: Steam the whole beans or the crushed beans at 115°C for 30 minutes under high temperature and high pressure to obtain a bean fermentation substrate.
5. The Bacillus subtilis WD01 and use thereof according to claim 3, characterized in that: A method for preparing a DNJ-rich meal replacement powder comprises the following steps: The natto food is dried at 100°C and then crushed with a grinder to obtain fermentation dry powder, which is added as a nutritional enhancer.