Strain Bacillus natto.SWS-001 and application thereof in preparation of nattokinase active soybean meal product

Abstract

The invention discloses a strain Bacillus natto.SWS-001 and application thereof.The active strain Bacillus natto.SWS-001 is used for producing a soybean meal feed additive through a solid state fermentation technology, a fermented soybean meal product with high nattokinase activity is prepared, in the prepared soybean meal feed additive, the nattokinase activity is larger than or equal to 3500 U / g, the bacillus natto content is larger than or equal to 2.0 * 10 < 8 > cfu / g, and the crude protein content is larger than or equal to 28%; the water content is less than or equal to 9.0% w / w. The bacillus natto fermented soybean meal product is produced from raw materials such as liquid submerged fermentation culture seed liquid, solid fermented soybean meal and bran, and the bacillus natto fermented soybean meal product is used for being added into feed main materials or auxiliary materials and can effectively improve the feed absorption rate of the livestock breeding industry.

Description

Technical Field

[0001] The invention relates to the technical field of biological fermentation, in particular to a soybean meal solid fermentation process suitable for high nattokinase activity and application thereof. Background Art

[0002] Soybean meal is one of the important protein raw materials for livestock and aquatic feed, with a protein content of about 80%-90%. Soybean protein is insoluble in water and contains anti-nutritional factors such as phytic acid and trypsin inhibitors. Fermenting soybean meal with Bacillus subtilis can effectively improve the absorption rate of protein and reduce anti-nutritional factors. Therefore, soybean meal fermented with Bacillus subtilis is a commonly used additive in feed, and the fermentation level is mainly based on the number of Bacillus subtilis and protein content.

[0003] Bacillus subtilis is a general term for a genus of bacteria. The protease it secretes is mainly alkaline protease, which can decompose soy protein into small peptides, which is beneficial for animal absorption. Bacillus natto belongs to the subgenus of Bacillus subtilis. Its biological characteristic is the production of nattokinase. The activity of this enzyme is positively correlated with the stringy viscosity of the fermented soybeans. Previous feeding studies on animals such as broilers and pigs found that the fermentation products of Bacillus natto have the effect of improving absorption rate and reducing feed-to-meat ratio, effectively reducing the stress response of young poultry, and improving immunity. Its effect is better than the Bacillus subtilis fermentation agent sold on the market. In addition, the bacterial count of Bacillus natto fermentation agent is only 1-1.5 billion / g, which is much lower than the 10-20 billion / g of Bacillus subtilis fermentation agent. Through the study of the biological characteristics of the strain, it was found that the nattokinase activity of Bacillus natto is about 5-10 times higher than that of ordinary Bacillus subtilis. At the same time, under the same culture conditions, the number of its strain reproduction is much lower than that of ordinary Bacillus subtilis, so its nattokinase activity is a characteristic influencing factor in exerting biological effects.

[0004] Based on the above technical status, it can be seen that developing a process for preparing nattokinase-active soybean meal products based on active strains has important practical significance. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a strain-based Bacillus natto. The use of SWS-001 in preparing nattokinase active soybean meal products.

[0006] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows.

[0007] strain Bacillus natto. SWS-001 is used for preparing nattokinase active soybean meal products.

[0008] As a preferred technical solution of the present invention, using active strains Bacillus natto.SWS-001 produces soybean meal feed additives through solid-state fermentation process to prepare fermented soybean meal products with high nattokinase activity.

[0009] As a preferred technical solution of the present invention, the nattokinase activity in the prepared soybean meal feed additive is ≥3500U / g.

[0010] As a preferred technical solution of the present invention, the nattokinase activity in the prepared soybean meal feed additive is ≥3500U / g, and the content of Bacillus subtilis (natto) is ≥2.0×10 8 cfu / g.

[0011] As a preferred technical solution of the present invention, the nattokinase activity in the prepared soybean meal feed additive is ≥3500U / g, and the content of Bacillus subtilis (natto) is ≥2.0×10 8 cfu / g, and crude protein content ≥28%; As a preferred technical solution of the present invention, the moisture content of the prepared soybean meal feed additive is ≤9.0% w / w.

[0012] As a preferred technical solution of the present invention, the active strain Bacillus natto. The main biological characteristics of SWS-001 include: Gram staining positive G+, rod-shaped cells, spores oval or columnar, Gram staining positive, catalase positive, VP reaction positive, nitrate reduction positive, D-xylose fermentation positive, D-glucose fermentation positive, gelatin liquefaction positive, starch hydrolysis positive, and colony morphology milky white opaque round; its preservation information is: preservation date January 23, 2019, preservation number CGMCC NO: 17226.

[0013] As a preferred technical solution of the present invention, using Bacillus natto. The SWS-001 microbial strain performs solid fermentation on soybean meal and bran raw materials, and a fermented soybean meal feed additive with high nattokinase activity is prepared through a post-processing process.

[0014] As a preferred technical solution of the present invention, the following implementation steps are included: C. Solid fermentation of soybean meal and bran raw materials: C-1. The formula of culture medium is: by weight, soybean meal 1-1.5, bran 2-3, inorganic salt KH2PO4 0.01-0.03, K2HPO4·3H2O 0.04-0.06, MgSO4·7H2O 0.005-0.02; C-2. Ratio of culture medium to bacterial liquid and water content: After mixing the above raw materials, add the fermentation liquid of the microbial strain at an amount of 8%-20% w / w of the solid material. Add water to make the solid-liquid ratio 3:1-3.3:1 w / w, adjust the pH to 7.5-8.0, the temperature of the fermentation workshop is 33-37℃, the humidity is 85%-95%, and the total fermentation time is 48-72h.

[0015] As a preferred technical solution of the present invention, it specifically includes the following implementation steps: A. Preparation of bacterial slant and bacterial activation: A-1. Preparation of culture medium: prepare PB culture medium according to the following formula, in weight proportion: beef extract 0.3-0.8, yeast powder 0.5-1.8, disodium hydrogen phosphate 0.3-0.8, sodium dihydrogen phosphate 0.3-0.8, water 100, agar 1-3; heat and dissolve the slant culture medium, adjust the pH to 6.0-6.2, and then divide it into eggplant bottles or test tubes, put them into a sterilizer for wet heat sterilization, and make slant after cooling for use; A-2. Activation and expansion of strains: Use an inoculation loop to pick up the bacterial moss from the original strain slant under sterile conditions, draw a line on the slant of a test tube or eggplant bottle, and then culture it in a 30±1℃ constant temperature box for 24 hours and store it for later use; B. Bacterial culture: B-1. Preparation of seed culture medium, which includes, by weight, 0.3-0.8 of beef extract, 0.5-1.8 of yeast powder, 0.3-0.8 of disodium hydrogen phosphate, 0.3-0.8 of sodium dihydrogen phosphate, and 100 parts of water. Adjust the pH to 7.0-7.8, and sterilize for use. B-2. Inoculation and culture: Take the eggplant bottle slope or test tube slope, inoculate the bacterial moss into the seed solution with an inoculation loop, and culture in a constant temperature shaker at 30±3℃, 250 rpm for 6 hours; after the seed solution passes the microscopic examination, store it at 4℃ for later use; B-3. ​​Expansion culture: The expansion culture medium includes 2-4 parts by weight of soybean powder, 0.3-1 parts by weight of glucose, 0.3-0.8 parts by weight of disodium hydrogen phosphate, 0.3-0.8 parts by weight of sodium dihydrogen phosphate, 0.3-0.8 parts by weight of water, and the pH is adjusted to 7.0-7.8. After sterilization, the temperature is lowered. The seed liquid is inoculated into the expansion culture medium with an inoculation amount of 2%-5%, 30±3℃, 200 rpm, and a ventilation rate of 1:1.5-1.8 (V gas / V liquid / min). The culture is carried out for 8-16 hours and the culture is ready for use after passing the microscopic examination. C. Solid fermentation of soybean meal and bran raw materials: C-1. The culture medium formula is: soybean meal, bran, inorganic salts KH2PO4, K2HPO4·3H2O, MgSO4·7H2O; the specific formula is, by weight: soybean meal 1, bran 2, KH2PO4 0.02%, K2HPO4·3H2O 0.052%, MgSO4·7H2O0.01%, pH 7.5 before sterilization; C-2. Ratio of culture medium to bacterial liquid and water content: After mixing the above raw materials, add the fermentation liquid of microbial strains at an amount of 8%-20% w / w of the solid material, add water to make the solid-liquid ratio 3:1-3.3:1 w / w, adjust the pH to 7.5-8.0, the temperature of the fermentation workshop is 33-37℃, the humidity is 85%-95%, and the total fermentation time is 48-72h; D. Drying process of solid fermentation feed additives: Adopt fluidized bed drying and crushing, inlet temperature 120℃, outlet temperature 60℃, time 20min, crushing mesh 40, and obtain yellow-brown powder with special fermentation taste, which is solid fermentation feed additive product; E. Conduct quality inspection on the fermented soybean meal product with high nattokinase activity, and the inspection indicators shall at least include crude protein content, nattokinase enzyme activity, and the number of live bacteria of Bacillus natto.

[0016] The beneficial effect of adopting the above technical scheme is that the present invention abandons the simple use of the Bacillus subtilis fermentation process with the number of strains as an indicator, and the liquid deep fermentation culture seed liquid, solid fermentation soybean meal and bran and other raw materials, produce the natto Bacillus fermentation soybean meal product, the nattokinase activity of which reaches more than 4000U / g, which is used for the main material or auxiliary material of feed, and can effectively improve the feed absorption rate of animal husbandry. The detailed beneficial effects of the present invention and the relevant detailed experimental research data can be further referred to in the following embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a microscopic examination of the test tube slant strain.

[0018] Figure 2 This is a graph of the 6-hour culture of bacterial culture solution in a shake flask.

[0019] Figure 3 16-hour chart of seed solution in a triangular flask.

[0020] Figure 4 It is a standard curve drawn for testing the activity of nattokinase.

[0021] The deposited strain is classified as Bacillus subtilis subspecies natto, and its Latin name is Bacillus natto , named Bacillus natto.SWS-001The depository is the General Microbiology Center of China Microbiological Culture Collection Administration, the deposit address is Beijing, China, the deposit date is January 23, 2019, and the deposit number is CGMCC NO: 17226. DETAILED DESCRIPTION

[0022] The following examples illustrate the present invention in detail. Various raw materials and equipment used in the present invention are conventional commercial products, which can be directly obtained by market purchase. In the description of the following embodiments, specific details such as specific system structures, technologies, etc. are proposed for illustration rather than limitation, so as to thoroughly understand the embodiments of the present application. However, it should be clear to those skilled in the art that the present application can also be implemented in other embodiments without these specific details. In other cases, detailed descriptions of well-known systems, devices, circuits and methods are omitted to prevent unnecessary details from hindering the description of the present application. It should be understood that when used in the present application specification and the attached claims, the term "including" indicates the presence of the described features, wholes, steps, operations, elements and / or components, but does not exclude the presence or addition of one or more other features, wholes, steps, operations, elements, components and / or their collections. It should also be understood that the term "and / or" used in the present application specification and the attached claims refers to any combination of one or more of the items listed in association and all possible combinations, and includes these combinations. As used in the specification of the present application and the appended claims, the term "if" can be interpreted as "when..." or "once" or "in response to determination" or "in response to detection" according to the context. Similarly, the phrase "if it is determined" or "if [the described condition or event] is detected" can be interpreted as meaning "once it is determined" or "in response to determination" or "once [the described condition or event] is detected" or "in response to detection [the described condition or event]" according to the context. In addition, in the description of the specification of the present application and the appended claims, the terms "first", "second", "third", etc. are only used to distinguish the description and cannot be understood as indicating or implying relative importance. The reference "one embodiment" or "some embodiments" described in the specification of the present application means that one or more embodiments of the present application include specific features, structures or characteristics described in conjunction with the embodiment. Thus, the sentences "in one embodiment", "in some embodiments", "in some other embodiments", "in some other embodiments", etc. that appear in different places in the specification are not necessarily all referring to the same embodiment, but mean "one or more but not all embodiments", unless otherwise specifically emphasized in other ways. The terms "include", "comprising", "having" and variations thereof all mean "including but not limited to", unless specifically emphasized otherwise.

[0023] In general, the present invention uses the Bacillus subtilis (natto) strain SWS-001 (preservation number CGMCC NO: 17226) to develop a production process for producing soybean meal feed additives by solid-state fermentation. The experimental production process includes: activation of the original strain, seed liquid culture, liquid deep fermentation, solid fermentation, and quality inspection. The soybean meal feed additive prepared according to the production process of the present invention has very excellent biological activity indicators: ① The content of Bacillus subtilis (natto) ≥ 2.0×10 8 cfu / g; ② Nattokinase activity ≥3500U / g; ③ Crude protein content ≥28%; ④ Water content ≤9.0%. Detailed experimental research data are as follows.

[0024] Example 1: Strain Slant Production Operation a. Instruments, equipment and reagents Sterile room and clean workbench, autoclave, test tubes, eggplant bottles, refrigerator, constant temperature incubator, microscope, sodium hydroxide, hydrochloric acid, sodium chloride, beef extract, peptone and agar, etc.

[0025] b. Culture medium preparation Prepare the culture medium (PB culture medium) according to the following formula: prepare the PB culture medium according to the following formula: 0.5 beef extract, 1.0 yeast powder, 0.5 disodium hydrogen phosphate, 0.5 sodium dihydrogen phosphate, 2 agar, 100 water; heat and dissolve the slant culture medium, adjust the pH to 7.0-7.8, and then divide it into eggplant bottles or test tubes, put them into the autoclave for wet heat sterilization, and make slant after cooling for use; c. Activation and expansion of bacterial strains Use an inoculating loop to pick up the bacterial moss from the original strain slant under sterile conditions, streak it on the slant of the test tube or eggplant bottle, then culture it in a constant temperature box at 30±1℃ for 24 hours and store it for later use.

[0026] Example 2: Seed liquid culture method a. Instruments, equipment and reagents Sterile room and clean workbench, autoclave, refrigerator, constant temperature incubator, microscope, sodium hydroxide, hydrochloric acid, etc.

[0027] b. Preparation of seed liquid culture medium Prepare the seed culture medium as follows: 0.5% beef extract, 1.0% yeast powder, 0.5% disodium hydrogen phosphate, 0.5% sodium dihydrogen phosphate, adjust the pH to 7.0-7.8, sterilize and set aside; c. Inoculation and bacterial culture Take the eggplant bottle slant or test tube slant, inoculate the bacterial moss into the seed solution with an inoculation loop, and culture in a constant temperature shaker at 30±3℃, 250 rpm for 6 hours; after the seed solution passes the microscopic examination, store it at 4℃ for later use.

[0028] All seed liquids are combined, placed in a sterile stainless steel inoculation pot and stored in the refrigerator for later use. At the same time, a sterility check is performed using the streaking method. The seed liquid should not be stored for more than three days.

[0029] Example 3: Scale-up culture of Bacillus subtilis (Natto) SWS-001 The expansion medium is calculated by weight ratio of 2 soybean powder, 1 glucose, 0.5 disodium hydrogen phosphate, 0.5 sodium dihydrogen phosphate, and 100 water, totaling 300L. The pH is adjusted to 7.8 and the temperature is cooled after sterilization. The seed liquid is inoculated into the expansion medium with an inoculation amount of 2%, 33℃, 200 rpm, ventilation rate of 1:1.5 (V gas / V liquid / min), and culture for 12h.

[0030] Example 4: Solid fermentation of soybean meal Take soybean meal: bran = 1:2, a total of 3 tons, add KH2PO4 0.02%, K2HPO4·3H2O 0.05%, MgSO4·7H2O0.01% and mix evenly, access the fermentation liquid of the expanded culture, the addition amount is 10% of the solid material, add water to make the solid-liquid ratio 3.5:1, adjust the pH to 8.0, the temperature of the fermentation workshop is 30°C, the humidity is 85%-95%, and the total fermentation time is 56h.

[0031] The fluidized bed is used for drying and crushing, with an inlet temperature of 120°C, an outlet temperature of 60°C, and a mesh size of 40, to obtain a yellow-brown powder with a special fermented taste, which is a solid fermented feed additive product.

[0032] Example 5: Fermentation test results of solid fermentation feed additive The following are the results of the fermentation test of the four batches of Example 1, the main indicators of the solid fermentation feed additive

[0033] It can be seen that after using Bacillus subtilis (natto) solid fermentation of soybean meal feed additives for multiple batches, it can be found that the liquid deep fermentation and solid fermentation processes of strain SWS-001 meet the production requirements of the feed additive, the equipment is conventional, and the process design is appropriate; the differences in indicators between batches are small, meeting the requirements of enterprise standards.

[0034] In the above embodiments, the description of each embodiment has its own emphasis. For the part that is not described or recorded in detail in a certain embodiment, please refer to the relevant description of other embodiments. The above embodiments are only used to illustrate the technical solution of the present invention, not to limit it; although the present invention is described in detail with reference to the above embodiments, ordinary technicians in this field should understand that they can still modify the technical solutions recorded in the above embodiments, or replace some of the technical features therein with equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention, and should be included in the protection scope of the present invention.

Claims

1. Strains Bacillus natto. SWS-001 is used for preparing nattokinase active soybean meal products.

2. The use according to claim 1, characterized in that: Using active strains Bacillus natto. SWS-001 produces soybean meal feed additives through solid-state fermentation process to prepare fermented soybean meal products with high nattokinase activity.

3. The use according to claim 2, characterized in that: The nattokinase activity in the prepared soybean meal feed additive is ≥3500U / g.

4. The use according to claim 2, characterized in that: The nattokinase activity in the prepared soybean meal feed additive is ≥3500U / g, and the active strain content is ≥2.0×10 8 cfu / g.

5. The use according to claim 2, characterized in that: The nattokinase activity in the prepared soybean meal feed additive is ≥3500U / g, and the active strain content is ≥2.0×10 8 cfu / g, and crude protein content ≥28%, w / w.

6. The use according to any one of claims 1 or 2 or 3 or 4 or 5, characterized in that: The moisture content of the prepared soybean meal feed additive is ≤9.0% w / w.

7. The use according to any one of claims 1 or 2 or 3 or 4 or 5, characterized in that: The active strain Bacillus natto. The main biological characteristics of SWS-001 include: Gram staining positive G+, rod-shaped cells, spores oval or columnar, Gram staining positive, catalase positive, VP reaction positive, nitrate reduction positive, D-xylose fermentation positive, D-glucose fermentation positive, gelatin liquefaction positive, starch hydrolysis positive, and colony morphology milky white opaque round; its preservation information is: preservation date January 23, 2019, preservation number CGMCC NO: 17226.

8. The use according to any one of claims 1 or 2 or 3 or 4 or 5, characterized in that: use Bacillus natto. The SWS-001 microbial strain performs solid fermentation on soybean meal and bran raw materials, and a fermented soybean meal feed additive with high nattokinase activity is prepared through a post-processing process.

9. The use according to claim 1, characterized in that: The implementation steps include: C. Solid fermentation of soybean meal and bran raw materials: C-1. The formula of culture medium is: by weight, soybean meal 1-1.5, bran 2-3, inorganic salt KH2PO4 0.01-0.03, K2HPO4·3H2O 0.04-0.06, MgSO4·7H2O 0.005-0.02; C-2. Ratio of culture medium to bacterial liquid and water content: After mixing the above raw materials, inoculate the microbial strains Bacillus natto. SWS-001 fermentation liquid, the addition amount is 8%-20% w / w of the solid material, water is added to make the solid-liquid ratio 3:1-3.3:1 w / w, the pH is adjusted to 7.5-8.0, the temperature of the fermentation workshop is 33-37℃, the humidity is 85%-95%, and the total fermentation time is 48-72h.

10. The use according to claim 9, characterized in that: The specific implementation steps include the following: A. Preparation of bacterial slant and bacterial activation: A-1. Preparation of culture medium: prepare PB culture medium according to the following formula, in weight proportion: beef extract 0.3-0.8, yeast powder 0.5-1.8, disodium hydrogen phosphate 0.3-0.8, sodium dihydrogen phosphate 0.3-0.8, water 100, agar 1-3; heat and dissolve the slant culture medium, adjust the pH to 6.0-6.2, and then divide it into eggplant bottles or test tubes, put them into a sterilizer for wet heat sterilization, and make slant after cooling for use; A-2. Activation and expansion of strains: Use an inoculation loop to pick up the bacterial moss from the original strain slant under sterile conditions, draw a line on the slant of a test tube or eggplant bottle, and then culture it in a 30±1℃ constant temperature box for 24 hours and store it for later use; B. Bacterial culture: B-1. Preparation of seed culture medium, which includes, by weight, 0.3-0.8 of beef extract, 0.5-1.8 of yeast powder, 0.3-0.8 of disodium hydrogen phosphate, 0.3-0.8 of sodium dihydrogen phosphate, and 100 parts of water. Adjust the pH to 7.0-7.8, and sterilize for use. B-2. Inoculation and culture: Take the eggplant bottle slope or test tube slope, inoculate the bacterial moss into the seed solution with an inoculation loop, and culture in a constant temperature shaker at 30±3℃, 250 rpm for 6 hours; after the seed solution passes the microscopic examination, store it at 4℃ for later use; B-3. ​​Expansion culture: The expansion culture medium includes 2-4 parts by weight of soybean powder, 0.3-1 parts by weight of glucose, 0.3-0.8 parts by weight of disodium hydrogen phosphate, 0.3-0.8 parts by weight of sodium dihydrogen phosphate, 100 parts by weight of water, and the pH is adjusted to 7.0-7.

8. After sterilization, the temperature is lowered. The seed liquid is inoculated into the expansion culture medium with an inoculation amount of 2%-5%. The temperature is maintained at 30±3℃, 200 rmp, and the ventilation volume is 1:1.5-1.8; V gas / V liquid / min. The culture is carried out for 8-16 hours and the culture medium is ready for use after passing the microscopic examination. C. Solid fermentation of soybean meal and bran raw materials: C-1. The culture medium formula is: soybean meal, bran, inorganic salts KH2PO4, K2HPO4·3H2O, MgSO4·7H2O; the specific formula is, by weight: soybean meal 1, bran 2, KH2PO4 0.02%, K2HPO4·3H2O 0.052%, MgSO4·7H2O0.01%, pH 7.5 before sterilization; C-2. Ratio of culture medium to bacterial solution and water content: After mixing the above raw materials, inoculate the microbial strains Bacillus natto. SWS-001 fermentation liquid, the addition amount is 8%-20% w / w of the solid material, water is added to make the solid-liquid ratio 3:1-3.3:1 w / w, pH is adjusted to 7.5-8.0, the temperature of the fermentation workshop is 33-37℃, the humidity is 85%-95%, and the total fermentation time is 48-72h; D. Drying process of solid fermentation feed additives: Adopt fluidized bed drying and crushing, inlet temperature 120℃, outlet temperature 60℃, time 20min, crushing mesh 40, and obtain yellow-brown powder with special fermentation taste, which is solid fermentation feed additive product; E. Conduct quality inspection on the fermented soybean meal product with high nattokinase activity, and the inspection indicators shall at least include crude protein content, nattokinase enzyme activity, and the number of live bacteria of Bacillus natto.

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