Bacillus subtilis fermentation method and freeze-dried powder of natto prepared thereby

Abstract

The application provides a solid-state fermentation method, which comprises primary fermentation and secondary fermentation; in the primary fermentation, a culture medium containing beans is treated by using Bacillus subtilis BS-14 to obtain a culture; in the secondary fermentation, the culture and seed liquid of a nattokinase-producing strain are added into the beans to perform solid-state fermentation. The fermentation method provided by the application can effectively convert the nutrients in the solid-state culture into substances absorbed and utilized by Bacillus subtilis, thereby significantly improving the enzyme activity concentration of nattokinase. Furthermore, phospholipid substances (phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol) and fat can be further decomposed, which can not only further improve the spoilage odor of the freeze-dried powder, but also prolong the shelf life. Furthermore, active peptides with antibacterial activity against pathogenic microorganisms can be further produced, so that the quality safety of the freeze-dried powder is improved.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation technology, specifically to a Bacillus subtilis fermentation method and the natto freeze-dried powder prepared therefrom. Background Technology

[0002] Natto is made from soybeans through biological fermentation. As a traditional fermented soybean food, it has a long history of consumption, and its safety is widely recognized. Natto freeze-dried powder is obtained by vacuum freeze-drying the fermentation products, which maximizes the preservation of its nutritional and active substances. Therefore, natto is usually sold in freeze-dried powder form. Nattokinase is the most important active substance in freeze-dried natto powder. Nattokinase can effectively dissolve blood clots, acting directly or indirectly. Compared with other thrombolytic drugs, it has a longer half-life, faster onset of action, and is more easily absorbed by the body. It has excellent efficacy in preventing and treating thrombotic diseases and can significantly alleviate the symptoms and signs of ischemic stroke.

[0003] Currently, industrial production of natto powder generally uses Bacillus subtilis as the production strain, employing soybean oil extraction byproducts such as soybeans, soybean meal, and soybean cake powder as carriers and nitrogen sources for solid-state fermentation, followed by freeze-drying to obtain freeze-dried natto powder. However, after soybean products are fermented into natto, the large molecular proteins and oils are not degraded. The plant proteins are not optimized for microbial utilization, making them difficult for the human intestine to absorb and utilize. The presence of oils results in poor natto powder properties, making it prone to discoloration, spoilage, acidification, and even a strong beany odor. Furthermore, some heat-resistant anti-nutritional factors, such as phytic acid, soybean antigens, and some oligosaccharides, are not consumed by microorganisms, leading to poor absorption and utilization by the human body, and even some toxic reactions. Moreover, due to the solid-state fermentation method, microorganisms have difficulty utilizing solid cultures, resulting in a significant difference in fermentation potency compared to liquid fermentation. Summary of the Invention

[0004] To improve the nutrient utilization of solid culture medium by Bacillus subtilis and promote the production of nattokinase, this invention provides a solid fermentation method, including primary fermentation and secondary fermentation.

[0005] In a single fermentation process, a culture medium containing legumes is treated with a complex enzyme to obtain a culture.

[0006] In the secondary fermentation, the culture and the seed liquid of the nattokinase-producing strain are added to the beans for solid-state fermentation.

[0007] The complex enzyme includes at least neutral protease, alkaline protease, lipase, α-amylase, phytase, and glucanase.

[0008] The nattokinase-producing strain is Bacillus subtilis, or Bacillus subtilis MQ013-26.

[0009] The present invention also provides a solid-state fermentation method, including primary fermentation and secondary fermentation;

[0010] In the primary fermentation, Bacillus subtilis BS-14 was used to treat the culture medium containing legumes to obtain the culture.

[0011] In the secondary fermentation, the culture and the seed liquid of the nattokinase-producing strain are added to the beans for solid-state fermentation.

[0012] The nattokinase-producing strain is Bacillus subtilis, or Bacillus subtilis MQ013-26.

[0013] Preferably, the Bacillus subtilis BS-14 and / or the Bacillus subtilis MQ013-26 are added in the form of seed liquid.

[0014] Preferably, during secondary fermentation, phospholipase D, β-glucanase, and α-galactosidase are optionally added.

[0015] Preferably, oxygen uptake is monitored during primary fermentation; and / or, oxygen uptake is monitored during secondary fermentation.

[0016] Preferably, during primary fermentation, the oxygen uptake rate is monitored, and when the oxygen uptake rate exceeds 250 mol / m³, the oxygen uptake rate is determined to be higher than 250 mol / m³. 3 / h, increase air volume; and / or, in secondary fermentation, monitor oxygen uptake rate, when the oxygen uptake rate is higher than 350 mol / m³ 3 / h, increase airflow.

[0017] Preferably, during primary fermentation, the oxygen uptake rate is monitored, and when the oxygen uptake rate exceeds 250 mol / m³, the oxygen uptake rate is determined to be higher than 250 mol / m³. 3 / h, increase the air volume, up to a maximum of 30L / min; and / or, during secondary fermentation, monitor the oxygen uptake rate, when the oxygen uptake rate exceeds 350mol / m 3 / h, increase air volume, up to 350L / min.

[0018] Preferably, during the first fermentation, when the CO2 release rate begins to decrease, the fermentation is extended for another 4-8 hours to end the first fermentation.

[0019] Preferably, during the secondary fermentation, when the CO2 release rate begins to decrease, the fermentation is extended for another 6-8 hours to end the secondary fermentation.

[0020] The present invention also provides a fermentation product obtained by any of the fermentation methods described herein.

[0021] The present invention also provides a natto freeze-dried powder, which is prepared from the fermentation product.

[0022] The Bacillus subtilis BS-14 provided by this invention is deposited at the China Center for Type Culture Collection, located at Wuhan University, Wuhan, China, on October 24, 2024, with accession number CCTCCNO:M20242248.

[0023] In the fermentation method described above, the following parameters can be further selected.

[0024] Preferably, fermentation is carried out in a solid culture medium, which includes legumes, a carbon source, inorganic salts, and water.

[0025] Preferably, the fermentation temperature is not higher than 45℃, or is 30~45℃, or is 30~42℃.

[0026] Preferably, the humidity is controlled at 70-80 HR.

[0027] Preferably, in the primary fermentation, the seed culture of the strain is added to a culture medium containing soybean meal, maltodextrin, dipotassium hydrogen phosphate, and sodium chloride.

[0028] Preferably, during the primary fermentation process, in the solid-state fermentation stage, the air volume, temperature, and humidity are controlled to maintain the solid-state fermentation temperature between 27°C and 36°C.

[0029] Preferably, during the primary fermentation, the air volume is controlled at 10L / min, the temperature at 33℃, and the humidity at 70-80%.

[0030] Preferably, the primary fermentation process contains 9 kg of soybean meal, 1000 g of maltodextrin, 30 g of dipotassium hydrogen phosphate, 15 g of sodium chloride, and 10 kg of water.

[0031] Preferably, 200 ml of BS-14 seed liquid is added during the first fermentation.

[0032] Preferably, the strain producing nattokinase in the secondary fermentation is strain MQ013-26.

[0033] Preferably, 40 kg of beans are added during the secondary fermentation.

[0034] Preferably, during the secondary fermentation, the MQ013-26 seed liquid is added by spraying.

[0035] Preferably, during the secondary fermentation, 1L of MQ013-26 seed liquid is added.

[0036] Preferably, during the secondary fermentation, in the solid-state fermentation stage, the air volume, temperature, and humidity are controlled to maintain the solid-state fermentation temperature between 30℃ and 42℃.

[0037] Preferably, during the secondary fermentation, in the solid-state fermentation stage, the air volume is controlled at 100L / min, the temperature at 37℃, and the humidity at 60-70%.

[0038] Preferably, phospholipase, β-glucanase, and α-galactosidase are added during the secondary fermentation.

[0039] Preferably, during the secondary fermentation, phospholipase D 20 U / g, β-glucanase 30 U / g, and α-galactosidase 30 U / g are added.

[0040] Preferably, the legume used in the secondary fermentation is soybean.

[0041] Preferably, during the secondary fermentation, the soybeans are crushed to a mesh size of 5-10.

[0042] The beneficial effects of this invention are as follows:

[0043] 1) The fermentation method provided by the present invention can effectively convert nutrients in solid culture into substances that can be absorbed and utilized by Bacillus subtilis, thereby significantly increasing the concentration of nattokinase enzyme activity.

[0044] 2) In addition, the microenvironment created by the fermentation of strain BS-14 used in this invention is conducive to promoting the fermentation of MQ013-26, thereby further increasing the concentration of nattokinase activity.

[0045] 3) The fermentation method provided by the present invention can further decompose phospholipids (phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol) and fats, which can not only further improve the putrid odor of freeze-dried powder, but also extend its shelf life.

[0046] 4) The fermentation method provided by the present invention can further produce active peptides that resist pathogenic microorganisms, which have significant antibacterial activity, thereby improving the quality and safety of freeze-dried powder.

[0047] 5) The fermentation method provided by the present invention can further decompose phospholipids and fats by monitoring OUR and CER.

[0048] 6) The fermentation method provided by this invention can further increase the concentration of nattokinase by monitoring OUR and CER and adjusting the fermentation parameters, and the improvement effect is outstanding. Detailed Implementation

[0049] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0050] Unless otherwise specified, the materials and reagents used in the following examples are all commercially available products that can be purchased on the market.

[0051] The present invention will be further described below through embodiments, but these descriptions are not intended to further limit the scope of the invention. Those skilled in the art should understand that equivalent substitutions or corresponding improvements made to the present invention still fall within the protection scope of the present invention.

[0052] As an example, the Bacillus subtilis MQ013-26 mentioned below is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2023432. This strain has been published in Chinese patent CN202311096027.0.

[0053] Bacillus subtilis BS-14, deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, on October 24, 2024, with accession number CCTCC NO: M 20242248.

[0054] In the following text, the nattokinase detection method was performed according to the operation procedure of the ultraviolet spectrophotometry section of the Guangdong Provincial Food Safety Local Standard (DBS44 / 013-2019).

[0055] Method for detecting neutral protease activity: GB / T 23527-2009 Protease preparations - Folin method;

[0056] Alkaline protease activity detection method: GB / T 23527-2009 Protease preparations - Fully automated biochemical analysis method;

[0057] Lipase activity detection method: GB / T 23535-2009 Lipase preparations;

[0058] Phytase activity detection method: GB / T 18634-2009 Determination of phytase activity in feed - spectrophotometric method;

[0059] Dextranase activity assay method: QB / T 4481-2013 β-glucanase preparations;

[0060] α-Amylase activity detection method: GB 8275-2009 Food additives α-amylase preparations.

[0061] Example 1

[0062] Bacillus subtilis MQ013-26, which was preserved, was used to prepare agar plates on sterile LB agar plates. The agar plates were then scraped and placed in several shake flasks (500 ml) containing sterile LB liquid culture medium, and cultured for 12 h to obtain seed culture.

[0063] 1) Primary fermentation

[0064] Take 9 kg of small-grain soybean meal, 1000 g of maltodextrin, 30 g of dipotassium hydrogen phosphate, 15 g of sodium chloride, and 10 kg of drinking water. Mix well and sterilize at high temperature for 30 min. Add 200 ml of MQ013-26 seed culture, neutral protease (960 U / g), alkaline protease (565.8 U / g), lipase (253.1 U / g), α-amylase (1474.2 U / g), phytase (274 U / g), and dextranase (104 U / g). Mix well and spread evenly on a solid-state fermentation bed. Control the air volume at 10 L / min, the temperature at 33℃, and the humidity at 70-80%. Turn the material over for 5 min every 2 hours using an automatic turner. Maintain the solid-state fermentation temperature at 27℃~36℃. Stop fermentation when the solid culture medium becomes slightly sticky and stringy, thus obtaining the bacterial residue.

[0065] 2) Secondary fermentation

[0066] Take 40 kg of small soybeans and grind them to a mesh size of 5-10. Add 40 kg of drinking water for high-temperature sterilization. After cooling, mix in 20 kg of the bacterial residue obtained from the primary fermentation. Spread the mixture evenly on a solid-state fermentation bed to a thickness of 3-5 cm. Add 1 L of the prepared MQ013-26 seed liquid by spraying. Control the airflow at 100 L / min, the temperature at 37℃, and the humidity at 60-70%. Turn the material for 5 minutes every 2 hours, maintaining the solid-state fermentation temperature at 30℃~42℃. The secondary fermentation is completed after 36 hours. The nattokinase activity concentration of the wet-weight bacterial residue was found to be 1925 FU / g, with a moisture content of 46%.

[0067] 3) Natto powder is dried and ground into powder.

[0068] The bacterial residue obtained from the secondary fermentation is dried at 50-60℃ with forced air until the moisture content is 20-30%, then transferred to a vacuum freeze-drying oven and freeze-dried for 72 hours. The resulting natto freeze-dried powder is then prepared and sealed for drying and storage.

[0069] The concentration of nattokinase in the freeze-dried natto powder was measured to be 2317 FU / g, and the water content was 3.9%.

[0070] Example 2

[0071] The procedure of Example 1 was repeated. During the secondary fermentation, phospholipase D (enzyme activity concentration 20 U / g), β-glucanase (enzyme activity concentration 30 U / g), and α-galactosidase (enzyme activity concentration 30 U / g) were added. The secondary fermentation was completed after 36 hours. The nattokinase enzyme activity concentration of the wet-weight bacterial residue was measured to be 2059 FU / g, and the moisture content was 49%.

[0072] Natto freeze-dried powder was obtained according to Example 1. The natto kinase activity concentration was measured to be 2562 FU / g, and the water content was 4.1%.

[0073] In this embodiment, the addition of phospholipase D can reduce the phospholipid content in the fermentation product, thereby improving the taste of the freeze-dried powder. β-glucanase and α-galactosidase can reduce the content of oligosaccharides, thereby promoting the absorption of nutrients in the freeze-dried powder by the human body and reducing the probability of toxic reactions.

[0074] Compared with Example 1, it can be found that the addition of these three enzymes did not reduce the activity concentration of nattokinase.

[0075] Comparative Example 1

[0076] According to Example 1, the difference is that only a second fermentation is carried out, without a first fermentation process.

[0077] Take 40 kg of small soybeans and grind them to a mesh size of 5-10. Add 40 kg of drinking water for high-temperature sterilization. After cooling, spread the mixture evenly on a solid-state fermentation bed to a thickness of 3-5 cm. Inoculate with 1 L of MQ013-26 seed liquid by spraying. Control the airflow at 100 L / min, the temperature at 37℃, and the humidity at 60-70%. Turn the material for 5 minutes every 2 hours, maintaining the solid-state fermentation temperature at 30℃~42℃. Fermentation is completed after 36 hours. The nattokinase activity concentration of the wet-weight bacterial residue was found to be 765 FU / g, with a moisture content of 47%.

[0078] Natto freeze-dried powder was obtained according to the freeze-drying process in Example 1. The natto kinase activity concentration was measured to be 864 FU / g, and the water content was 4.5%.

[0079] In addition, two fermentations were carried out in accordance with the method of Example 1, but without the addition of compound enzymes (neutral protease, alkaline protease, lipase, α-amylase, phytase, glucanase). The concentration of nattokinase activity in the bacterial residue and natto freeze-dried powder obtained after fermentation was not significantly different from the results of the two fermentations alone.

[0080] This shows that strain MQ013-26 cannot effectively decompose and transform nutrients in solid culture, which leads to limited growth and fermentation processes, and also limits the production of nattokinase.

[0081] Example 3

[0082] Bacillus subtilis BS-14, which had been preserved, was used to prepare agar plates on sterile LB agar plates. The agar plates were then scraped off and placed in several shake flasks (500 ml) containing sterile LB liquid culture medium. The plates were incubated for 12 h to obtain Bacillus subtilis BS-14 seed culture.

[0083] Seed culture of Bacillus subtilis MQ013-26 was prepared according to Example 1.

[0084] 1) Primary fermentation

[0085] Take 9 kg of small-grain soybean meal, 1000 g of maltodextrin, 30 g of dipotassium hydrogen phosphate, 15 g of sodium chloride, and 10 kg of drinking water. Mix well and sterilize at high temperature for 30 minutes. Add 200 ml of BS-14 seed liquid and mix well. Spread the mixture evenly on a solid-state fermentation bed. Control the airflow at 10 L / min, the temperature at 33℃, and the humidity at 70-80%. Turn the mixture for 5 minutes every 2 hours using an automatic turner to maintain the solid-state fermentation temperature between 27℃ and 36℃. Stop fermentation when the solid culture medium becomes slightly sticky and stringy, thus obtaining the bacterial residue.

[0086] 2) Secondary fermentation

[0087] Take 40 kg of small soybeans and grind them to a mesh size of 5-10. Add 40 kg of drinking water for high-temperature sterilization. After cooling, mix in 20 kg of the above-mentioned BS-14 solid-state fermentation residue and spread it evenly on the solid-state fermentation bed to a thickness of 3-5 cm. Add 1 L of MQ013-26 seed liquid by spraying. Control the air volume at 100 L / min, the temperature at 37℃, and the humidity at 60-70%. Turn the material for 5 minutes every 2 hours, maintaining the solid-state fermentation temperature at 30℃~42℃. The secondary fermentation is completed after 36 hours. The nattokinase activity concentration of the wet-weight residue was found to be 2803 FU / g, and the moisture content was 51%.

[0088] Natto freeze-dried powder was obtained according to the freeze-drying process in Example 1. The nattokinase enzyme activity concentration was measured to be 3365 FU / g, and the water content was 5.2%.

[0089] By comparing Examples 1, 2 and 3, it can be seen that the strain BS-14 screened by the applicant can also convert nutrients in solid culture into substances that can be absorbed and utilized by Bacillus subtilis. In addition, the applicant speculates that the microenvironment produced by the fermentation of strain BS-14 is also conducive to promoting the fermentation of MQ013-26, thus resulting in a further increase in the concentration of nattokinase activity.

[0090] Example 4

[0091] Repeat the operation of Example 3. During the secondary fermentation, inoculate with phospholipase D 20 U / g, β-glucanase 30 U / g, and α-galactosidase 30 U / g. The nattokinase activity concentration of the wet-weight bacterial residue was measured to be 2752 FU / g, and the water content was 51%.

[0092] Natto freeze-dried powder was obtained according to the freeze-drying process in Example 1. The nattokinase activity concentration was measured to be 3415 FU / g, and the water content was 4.0%.

[0093] Example 5

[0094] Seed cultures of strains BS-14 and MQ013-26 were prepared as described above.

[0095] 1) Primary fermentation

[0096] Take 9 kg of small-grain soybean meal, 1000 g of maltodextrin, 30 g of dipotassium hydrogen phosphate, 15 g of sodium chloride, and 10 kg of drinking water. Mix well and sterilize at high temperature for 30 min. Add 200 ml of BS-14 seed liquid and mix well. Spread evenly on a solid-state fermentation bed, control the air volume at 10 L / min, the temperature at 33℃, and the humidity at 70-80%. Turn the material over for 5 min every 2 hours using an automatic turner to maintain the solid-state fermentation temperature at 27℃~36℃. When the oxygen uptake (OUR) is higher than 250 mol / m³... 3 Increase the air volume to a maximum of 30L / min. When the CO2 release rate (CER) begins to decrease from its maximum value, extend the fermentation time by 4-8 hours to end the first fermentation.

[0097] 2) Secondary fermentation

[0098] Take 40 kg of small soybeans and grind them to a mesh size of 5-10. Add 40 kg of drinking water for high-temperature sterilization. After cooling, mix in 20 kg of the above-mentioned BS-14 solid-state fermentation residue and spread it evenly on the solid-state fermentation bed to a thickness of 3-5 cm. Add 1 L of MQ013-26 seed liquid by spraying. Inoculate with phospholipase D 20 U / g, β-glucanase 30 U / g, and α-galactosidase 30 U / g. Control the air volume at 100 L / min, the temperature at 37℃, and the humidity at 60-70%. Turn the material for 5 minutes every 2 hours to maintain the solid-state fermentation temperature at 30℃~42℃. When the OUR is higher than 350 mol / m³... 3 Increase the airflow to a maximum of 350 L / min. When the CER begins to decrease, extend the fermentation time by another 6-8 hours to end the secondary fermentation. The nattokinase activity concentration in the wet-weight bacterial residue was measured to be 3755 FU / g, with a moisture content of 47%.

[0099] Natto freeze-dried powder was obtained according to the freeze-drying process in Example 1. The nattokinase activity concentration was measured to be 4356 FU / g, and the water content was 4.3%.

[0100] Compared with Example 4, further adjusting the fermentation parameters by monitoring OUR and CER can further increase the concentration of nattokinase enzyme activity, and the improvement effect is significant.

[0101] Comparative Example 2

[0102] The procedure was performed according to Example 1, except that no compound enzyme was added and the MQ013-26 strain was replaced with the BS-14 strain for fermentation. All other operations remained unchanged. The secondary fermentation was completed after 36 hours. The nattokinase activity concentration in the wet-weight bacterial residue was measured to be 235 FU / g, with a moisture content of 51%.

[0103] Natto freeze-dried powder was obtained according to the freeze-drying process in Example 1. The nattokinase activity concentration was measured to be 336 FU / g, and the water content was 3.8%.

[0104] Therefore, although strain BS-14 can convert nutrients in solid cultures into substances that can be absorbed and utilized by Bacillus subtilis and improve the fermentation environment of the microorganisms, its own ability to produce nattokinase is not outstanding. Thus, the increased nattokinase activity concentration resulting from co-fermentation with MQ013-26 is mainly due to the improved culture environment, which in turn promotes the fermentation capacity of MQ013-26.

[0105] Comparative Example 3

[0106] The strain BS-14 used in Example 3 was replaced with a preserved vitamin K2-producing Bacillus subtilis strain for natto, deposited at the China General Microbiological Culture Collection Center (CGMCC NO. 21799). All other procedures remained unchanged, and the secondary fermentation was completed after 36 hours. The nattokinase activity concentration in the wet-weight fermented residue was measured to be 1159 FU / g, with a moisture content of 45%.

[0107] Natto freeze-dried powder was obtained according to the freeze-drying process in Example 1. The nattokinase enzyme activity concentration was measured to be 1475 FU / g, and the water content was 4.4%.

[0108] This shows that strains BS-14 and MQ013-26 can achieve the greatest synergistic effect, resulting in the highest concentration of nattokinase activity in the product.

[0109] Test Example 1

[0110] Protein content detection

[0111] The protein content in the freeze-dried powders prepared according to GB5009.5—2010 "National Food Safety Standard - Determination of Protein in Food" (Kjeldahl method) was obtained. The results are shown in the table below.

[0112] Protein content (%) Example 1 42.9 Example 2 44.5 Comparative Example 1 39.6 Example 3 43.5 Example 4 47.2 Example 5 48.9 Comparative Example 2 41.5 Comparative Example 3 42.2

[0113] Protein molecular weight distribution

[0114] Samples from Example 4 and Comparative Example 1 were sent to an external testing institution (Three Gorges Public Testing and Inspection Center) for SDS-PAGE protein gel electrophoresis. The results showed that a large amount of low-molecular-weight protein was present in Example 4, indicating that the method of the present invention can degrade and optimize large-molecular-weight proteins and antigenic proteins in soybeans, thereby helping to promote their absorption by the human body.

[0115] Test Example 2

[0116] Fat and phospholipid content

[0117] Method for detecting phospholipid content: GB5009.272-2016 National Food Safety Standard - Determination of phospholipids (PC PIPE) in food.

[0118] Oil content detection method: Detected according to GB 5009.6-2016 National Food Safety Standard - Determination of fat in food - Soxhlet extraction method.

[0119]

[0120]

[0121] Therefore, it is evident that, in addition to improving the solid-state fermentation environment and promoting the fermentation of strain MQ013-26, the BS-14 strain provided by this invention can further decompose phospholipids (phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol) and fats. This not only further improves the spoilage odor of the freeze-dried powder but also extends its shelf life. Moreover, during the fermentation process, monitoring OUR and CER can further enhance the ability of strain BS-14 to decompose phospholipids and fats.

[0122] Test Example 5

[0123] Antimicrobial active peptides have antibacterial effects

[0124] 1g of lyophilized natto powder was reconstituted with 10ml of physiological saline, mixed thoroughly, and centrifuged at 14000rpm / min for 10min. The supernatant was then passed through a 0.22um membrane and kept for later use. The cultured Staphylococcus aureus and Escherichia coli were inoculated into 100ml of culture medium using 100ul glycerol seed tubes and cultured at 36℃ and 220rpm / min for 24h. 200ul of the cultured Staphylococcus aureus and Escherichia coli stock solutions were spread onto air-dried culture medium plates, and four holes were punched evenly on the plates using a 4mm punch. 200ul of the supernatant was added to each hole, and the plates were cultured for 24h. The inhibition zones were counted using calipers.

[0125]

[0126]

[0127] Therefore, it can be seen that, in addition to improving and promoting the fermentation of strain MQ013-26, the strain BS-14 provided by this invention can also further produce active peptides that resist pathogenic microorganisms, exhibiting significant antibacterial activity, thereby improving the quality and safety of freeze-dried powder.

Claims

1. A solid-state fermentation method, characterized in that: Includes primary fermentation and secondary fermentation; In a single fermentation process, a culture medium containing legumes is treated with a complex enzyme to obtain a culture. In the secondary fermentation, the culture and the seed liquid of the nattokinase-producing strain are added to the beans for solid-state fermentation. The complex enzyme includes at least neutral protease, alkaline protease, lipase, α-amylase, phytase, and glucanase. The nattokinase-producing strain is Bacillus subtilis, or Bacillus subtilis MQ013-26.

2. A solid-state fermentation method, characterized in that: Includes primary fermentation and secondary fermentation; In the primary fermentation, Bacillus subtilis BS-14 was used to treat the culture medium containing legumes to obtain the culture. In the secondary fermentation, the culture and the seed liquid of the nattokinase-producing strain are added to the beans for solid-state fermentation. The nattokinase-producing strain is Bacillus subtilis or Bacillus subtilis MQ013-26. The Bacillus subtilis BS-14 is deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, on October 24, 2024, with accession number CCTCC NO:M 20242248.

3. The fermentation method according to claim 2, characterized in that: During secondary fermentation, phospholipase D, β-glucanase, and α-galactosidase may be added optionally.

4. The fermentation method according to claim 2, characterized in that: During primary fermentation, oxygen uptake rate is monitored; And / or, during secondary fermentation, monitor oxygen uptake.

5. The fermentation method according to claim 4, characterized in that: During primary fermentation, the oxygen uptake rate was monitored; when the oxygen uptake rate exceeded 250 mol / m³, [further action was taken]. 3 / h, increase air volume; And / or, during secondary fermentation, monitor oxygen uptake; when the oxygen uptake rate exceeds 350 mol / m³, [further action is taken]. 3 / h, increase airflow.

6. The fermentation method according to claim 5, characterized in that: During primary fermentation, the oxygen uptake rate was monitored; when the oxygen uptake rate exceeded 250 mol / m³, [further action was taken]. 3 / h, increasing the air volume, up to a maximum of 30L / min; And / or, during secondary fermentation, monitor oxygen uptake; when the oxygen uptake rate exceeds 350 mol / m³, [further action is taken]. 3 / h, increase air volume, up to 350L / min.

7. The fermentation method according to claim 5, characterized in that: In the first fermentation, when the CO2 release rate begins to decrease, the fermentation is extended for another 4-8 hours to end the first fermentation.

8. The fermentation method according to claim 5, characterized in that: During the secondary fermentation, when the CO2 release rate begins to decrease, the fermentation is extended for another 6-8 hours to end the secondary fermentation.

9. A fermentation product obtained by the fermentation method according to any one of claims 1 to 8.

10. A freeze-dried natto powder, prepared from the fermentation product of claim 9.

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