Method for preparing high-activity nattokinase by taking beans as raw materials
By incorporating bacterial seed culture into seed culture medium and fermentation medium, high-active nattokinase is prepared, which solves the problem of insufficient enzyme activity of nattokinase in the prior art, and achieves high-activity, short fermentation cycle and low-cost product preparation , suitable for promotion and application.
Patent Information
- Application Number
- CN202411464141.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-21
- Publication Date
- 2024-12-17
AI Technical Summary
The prior art is difficult to improve the enzyme activity and production efficiency of unit products of nattokinase, which limits its application in thrombolytic drugs.
The bacterial seeds are inserted into seed culture medium to prepare seed activation solution, and then it is added to the fermentation medium for fermentation and culture. Finally, high-active nattokinase is obtained through leaching and centrifugation, and specific medium components and conditions such as trypsin, Beef paste, NaCl, yeast extract and aspartic acid, etc., ensure high enzyme activity and simple process.
The preparation of highly active nattokinase is achieved, the enzyme activity is improved, the fermentation cycle is shortened, the product is rich in nutrients, and the process cost is low, which ensures product quality and quality and broadens market application prospects.
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of nattokinase, in particular to a method for preparing high-activity nattokinase by taking beans as raw materials. Background Art
[0002] Nattokinase is extracted from food natto or produced by natto bacteria. It is a protein with a molecular weight far smaller than UK, SK, and tPA, and can be absorbed by the intestine. The in vitro and in vivo thrombolytic properties of nattokinase have also been determined through experiments. At the same time, it was found that the in vivo thrombolytic activity of nattokinase is four times that of plasmin. It acts rapidly and lasts for a long time in the body. It can also activate tPA in the body, so that it gently and continuously improves the fibrinolytic activity of the blood.
[0003] Since nattokinase has advantages that the thrombolytic drugs currently in use on the market do not have, improving the enzyme activity and production efficiency of the unit product of nattokinase is of great significance to the promotion and application of nattokinase.
[0004] Based on this, the present invention provides a method for preparing highly active nattokinase using beans as raw materials to solve the above-mentioned technical problems! Summary of the invention
[0005] The object of the present invention is to provide a method for preparing high-activity nattokinase using beans as raw materials. The method comprises the following steps: inoculating the strain into a seed culture medium, preparing a seed activation solution, then inoculating the seed activation solution into a fermentation culture medium, fermenting and culturing, and finally extracting and centrifuging the fermented product to obtain high-activity nattokinase. The high-activity nattokinase prepared by the present invention has high enzyme activity, short fermentation cycle, richer product nutrition, simple process and low cost, and at the same time, effectively guarantees its quality.
[0006] To achieve the above object, the present invention provides the following technical solutions:
[0007] The present invention provides a method for preparing high-activity nattokinase using beans as raw materials, comprising the following steps:
[0008] S1, inoculating the bacterial strain into the seed culture medium to prepare the seed activation solution;
[0009] S2, connecting the seed activation liquid to the fermentation medium for fermentation culture;
[0010] S3. Extract and centrifuge the fermented product to obtain highly active nattokinase.
[0011] The present invention is further configured as follows: in step S1, the preparation process of the seed activation solution is as follows:
[0012] Take a loopful of bacteria and inoculate it into 10-12 mL of seed culture medium, and culture it at 36-37°C and 170-180 rpm for 12 hours.
[0013] After the shaking culture is completed, transfer to 50-52 mL of seed culture medium and continue shaking culture at 36-37°C and 170-180 r / min for 12 h to obtain seed activation solution.
[0014] The present invention is further configured as follows: the bacterial species is selected from any one of Bacillus natto BNCC185324 and Bacillus licheniformis BNCC132622.
[0015] The present invention is further configured as follows: the seed culture medium comprises the following concentration components:
[0016] Tryptone 12-28 g / L, beef extract 3-5 g / L, NaCl 2-5 g / L, yeast extract 5-8 g / L, aspartic acid 0.1-0.3 g / L, pH 7.0-7.5.
[0017] The present invention is further configured as follows: in step S2, the seed activation liquid is connected to the fermentation medium, and the fermentation culture process is as follows:
[0018] Add an appropriate amount of seed activation solution to the fermentation medium at a mass ratio of 0.05 to 0.08:1;
[0019] Then culture at 36-37°C for 48-49h.
[0020] The present invention is further configured as follows: the preparation process of the fermentation medium is as follows:
[0021] Place an appropriate amount of the composite fermentation material in water at a solid-liquid ratio of 0.22-0.32 g / mL, and soak in a water bath at 50°C for 100-120 min;
[0022] After soaking, place at 110-115℃ and sterilize for 30-34 minutes.
[0023] The present invention is further configured as follows: the composite fermentation material is formed by mixing cyperus oleifera meal and Xanthoceras sorbifolia meal in a mass ratio of 1:0.3-0.4.
[0024] The present invention is further configured as follows: in step S3, the extraction and centrifugation process is as follows:
[0025] Place an appropriate amount of the product in distilled water at a mass ratio of 1:5.2-10.2 and extract for 120-200 minutes;
[0026] Then centrifuge at 10000-12000 r / min for 10-15 min.
[0027] Compared with the prior art, the present invention has the following beneficial effects:
[0028] The invention provides a method for obtaining high-activity nattokinase by inoculating bacteria into a seed culture medium, preparing a seed activation liquid, then inoculating the seed activation liquid into a fermentation culture medium, fermenting and culturing, and finally extracting and centrifuging the fermented product, wherein the seed culture medium comprises the following concentration components: 12-28 g / L of tryptone, 3-5 g / L of beef extract, 2-5 g / L of NaCl, 5-8 g / L of yeast extract, 0.1-0.3 g / L of aspartic acid, pH 7.0-7.5, and the fermentation culture medium is obtained by soaking and sterilizing an appropriate amount of composite fermentation material in water, wherein the composite fermentation material is mixed with serrata meal and Xanthoceras sorbifolia meal in a mass ratio of 1:0.3-0.4. The high-activity nattokinase prepared by the invention has high enzyme activity, short fermentation cycle, richer product nutrition, simple process and low cost, and at the same time, effectively guarantees its quality. The method for preparing high-activity nattokinase using beans as raw materials provided by the invention has a broader market prospect and is more suitable for promotion. DETAILED DESCRIPTION
[0029] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0030] Example 1
[0031] The present embodiment provides a method for preparing highly active nattokinase using beans as raw materials, comprising the following steps:
[0032] S1. Inoculate the bacterial strain into the seed culture medium to prepare the seed activation solution.
[0033] The preparation process of the seed activation solution is as follows:
[0034] Take a loopful of bacteria and inoculate it into 10 mL of seed culture medium. Incubate it at 36°C and 170 rpm for 12 h.
[0035] After the shaking culture was completed, the culture was transferred to 50 mL of seed culture medium and continued to be shaken for 12 h at 36°C and 170 r / min to obtain a seed activation solution.
[0036] Furthermore, the bacterial strain selected is Bacillus natto BNCC185324.
[0037] The seed culture medium includes the following concentration components:
[0038] Tryptone 12g / L, beef extract 3g / L, NaCl 2g / L, yeast extract 5g / L, aspartic acid 0.1g / L, pH 7.0.
[0039] In this embodiment, it should be noted that Bacillus natto BNCC185324 was purchased from Beina Chuanglian Biotechnology Co., Ltd., and yeast extract was purchased from Jinan Weiduofeng Biotechnology Co., Ltd.
[0040] S2, connecting the seed activation liquid to the fermentation medium for fermentation culture.
[0041] The seed activation liquid is connected to the fermentation medium, and the fermentation culture process is as follows:
[0042] Add an appropriate amount of seed activation solution to the fermentation medium at a mass ratio of 0.05:1;
[0043] Then, the cells were cultured at 36°C for 48 h.
[0044] Further, the preparation process of the fermentation medium is as follows:
[0045] Place an appropriate amount of the composite fermentation material in water at a solid-liquid ratio of 0.22 g / mL and soak it in a water bath at 50°C for 100 min;
[0046] After soaking, place it at 110℃ and sterilize it for 30 minutes.
[0047] Furthermore, the composite fermentation feed is prepared by mixing sesame seed meal and Xanthoceras sorbifolia meal in a mass ratio of 1:0.3.
[0048] S3. Extract and centrifuge the fermented product to obtain highly active nattokinase.
[0049] Among them, the process of leaching and centrifugation is as follows:
[0050] An appropriate amount of the product was placed in distilled water at a mass ratio of 1:5.2 and extracted for 120 min;
[0051] Then centrifuge at 10000 r / min for 10 min.
[0052] Example 2
[0053] The present embodiment provides a method for preparing highly active nattokinase using beans as raw materials, comprising the following steps:
[0054] S1. Inoculate the bacterial strain into the seed culture medium to prepare the seed activation solution.
[0055] The preparation process of the seed activation solution is as follows:
[0056] Take a loopful of bacteria and inoculate it into 11 mL of seed culture medium. Shake and culture it at 36°C and 172 rpm for 12 h.
[0057] After the shaking culture was completed, the culture medium was transferred to 51 mL of seed culture medium and continued to be shaken for 12 h at 37°C and 172 r / min to obtain a seed activation solution.
[0058] Furthermore, the bacterial strain selected is Bacillus natto BNCC185324.
[0059] The seed culture medium includes the following concentration components:
[0060] Tryptone 13g / L, beef extract 4g / L, NaCl 3g / L, yeast extract 6g / L, aspartic acid 0.2g / L, pH 7.1.
[0061] In this embodiment, it should be noted that Bacillus natto BNCC185324 was purchased from Beina Chuanglian Biotechnology Co., Ltd., and yeast extract was purchased from Jinan Weiduofeng Biotechnology Co., Ltd.
[0062] S2, connecting the seed activation liquid to the fermentation medium for fermentation culture.
[0063] The seed activation liquid is connected to the fermentation medium, and the fermentation culture process is as follows:
[0064] Add an appropriate amount of seed activation solution to the fermentation medium at a mass ratio of 0.05:1;
[0065] Then, the cells were cultured at 36°C for 48 h.
[0066] Further, the preparation process of the fermentation medium is as follows:
[0067] Place an appropriate amount of the composite fermentation material in water at a solid-liquid ratio of 0.24 g / mL and soak it in a water bath at 50°C for 105 min;
[0068] After soaking, sterilize at 111°C for 31 minutes.
[0069] Furthermore, the composite fermentation feed is prepared by mixing sesame seed meal and Xanthoceras sorbifolia meal in a mass ratio of 1:0.3.
[0070] S3. Extract and centrifuge the fermented product to obtain highly active nattokinase.
[0071] Among them, the process of leaching and centrifugation is as follows:
[0072] An appropriate amount of the product was placed in distilled water at a mass ratio of 1:5.3 and extracted for 130 min;
[0073] Then centrifuge at 11000 r / min for 11 min.
[0074] Example 3
[0075] The present embodiment provides a method for preparing highly active nattokinase using beans as raw materials, comprising the following steps:
[0076] S1. Inoculate the bacterial strain into the seed culture medium to prepare the seed activation solution.
[0077] The preparation process of the seed activation solution is as follows:
[0078] Take a loopful of bacteria and inoculate it into 11 mL of seed culture medium. Incubate it at 37°C and 175 rpm for 12 h.
[0079] After the shaking culture was completed, the cells were transferred to 51 mL of seed culture medium and continued to be shaken at 37°C and 175 r / min for 12 h to obtain a seed activation solution.
[0080] Furthermore, the bacterial strain selected is Bacillus natto BNCC185324.
[0081] The seed culture medium includes the following concentration components:
[0082] Tryptone 17g / L, beef extract 4g / L, NaCl 3g / L, yeast extract 6g / L, aspartic acid 0.2g / L, pH 7.2.
[0083] In this embodiment, it should be noted that Bacillus natto BNCC185324 was purchased from Beina Chuanglian Biotechnology Co., Ltd., and yeast extract was purchased from Jinan Weiduofeng Biotechnology Co., Ltd.
[0084] S2, connecting the seed activation liquid to the fermentation medium for fermentation culture.
[0085] The seed activation liquid is connected to the fermentation medium, and the fermentation culture process is as follows:
[0086] Add an appropriate amount of seed activation solution to the fermentation medium at a mass ratio of 0.06:1;
[0087] Then, the cells were cultured at 37°C for 49 h.
[0088] Further, the preparation process of the fermentation medium is as follows:
[0089] Place an appropriate amount of the composite fermentation material in water at a solid-liquid ratio of 0.27 g / mL and soak it in a water bath at 50°C for 110 minutes;
[0090] After soaking, sterilize at 112℃ for 32 minutes.
[0091] Furthermore, the composite fermented feed is prepared by mixing sesame seed meal and Xanthoceras sorbifolia meal in a mass ratio of 1:0.4.
[0092] S3. Extract and centrifuge the fermented product to obtain highly active nattokinase.
[0093] Among them, the process of leaching and centrifugation is as follows:
[0094] An appropriate amount of the product was placed in distilled water at a mass ratio of 1:7.2 and extracted for 160 min;
[0095] Then centrifuge at 11000 r / min for 12 min.
[0096] Example 4
[0097] The present embodiment provides a method for preparing highly active nattokinase using beans as raw materials, comprising the following steps:
[0098] S1. Inoculate the bacterial strain into the seed culture medium to prepare the seed activation solution.
[0099] The preparation process of the seed activation solution is as follows:
[0100] Take a loopful of bacteria and inoculate it into 11 mL of seed culture medium. Incubate it at 37°C and 178 rpm for 12 h.
[0101] After the shaking culture was completed, the cells were transferred to 51 mL of seed culture medium and continued to be shaken at 37°C and 178 rpm for 12 h to obtain a seed activation solution.
[0102] Furthermore, the bacterial strain selected is Bacillus natto BNCC185324.
[0103] The seed culture medium includes the following concentration components:
[0104] Tryptone 26g / L, beef extract 5g / L, NaCl 5g / L, yeast extract 7g / L, aspartic acid 0.3g / L, pH7.4.
[0105] In this embodiment, it should be noted that Bacillus natto BNCC185324 was purchased from Beina Chuanglian Biotechnology Co., Ltd., and yeast extract was purchased from Jinan Weiduofeng Biotechnology Co., Ltd.
[0106] S2, connecting the seed activation liquid to the fermentation medium for fermentation culture.
[0107] The seed activation liquid is connected to the fermentation medium, and the fermentation culture process is as follows:
[0108] Add an appropriate amount of seed activation solution to the fermentation medium at a mass ratio of 0.06:1;
[0109] Then, the cells were cultured at 37°C for 49 h.
[0110] Further, the preparation process of the fermentation medium is as follows:
[0111] Place an appropriate amount of the composite fermentation material in water at a solid-liquid ratio of 0.27 g / mL and soak it in a water bath at 50°C for 110 minutes;
[0112] After soaking, sterilize at 112℃ for 32 minutes.
[0113] Furthermore, the composite fermented feed is prepared by mixing sesame seed meal and Xanthoceras sorbifolia meal in a mass ratio of 1:0.4.
[0114] S3. Extract and centrifuge the fermented product to obtain highly active nattokinase.
[0115] Among them, the process of leaching and centrifugation is as follows:
[0116] An appropriate amount of the product was placed in distilled water at a mass ratio of 1:7.2 and extracted for 160 min;
[0117] Then centrifuge at 11000 r / min for 12 min.
[0118] Example 5
[0119] The present embodiment provides a method for preparing highly active nattokinase using beans as raw materials, comprising the following steps:
[0120] S1. Inoculate the bacterial strain into the seed culture medium to prepare the seed activation solution.
[0121] The preparation process of the seed activation solution is as follows:
[0122] Take a loopful of bacteria and inoculate it into 12 mL of seed culture medium. Incubate it at 37°C and 180 rpm for 12 h.
[0123] After the shaking culture was completed, the culture was transferred to 52 mL of seed culture medium and continued to be shaken for 12 h at 37°C and 180 r / min to obtain a seed activation solution.
[0124] Further, Bacillus licheniformis BNCC132622.
[0125] The seed culture medium includes the following concentration components:
[0126] Tryptone 28g / L, beef extract 5g / L, NaCl 5g / L, yeast extract 8g / L, aspartic acid 0.3g / L, pH7.5.
[0127] In this example, it should be noted that Bacillus licheniformis BNCC132622 was purchased from Beina Biotechnology Co., Ltd., and yeast extract was purchased from Jinan Weiduofeng Biotechnology Co., Ltd.
[0128] S2, connecting the seed activation liquid to the fermentation medium for fermentation culture.
[0129] The seed activation liquid is connected to the fermentation medium, and the fermentation culture process is as follows:
[0130] Add an appropriate amount of seed activation solution to the fermentation medium at a mass ratio of 0.08:1;
[0131] Then, the cells were cultured at 37°C for 49 h.
[0132] Further, the preparation process of the fermentation medium is as follows:
[0133] Place an appropriate amount of the composite fermentation material in water at a solid-liquid ratio of 0.32 g / mL and soak it in a water bath at 50°C for 120 min;
[0134] After soaking, sterilize at 115℃ for 34 minutes.
[0135] Furthermore, the composite fermented feed is prepared by mixing sesame seed meal and Xanthoceras sorbifolia meal in a mass ratio of 1:0.4.
[0136] S3. Extract and centrifuge the fermented product to obtain highly active nattokinase.
[0137] Among them, the process of leaching and centrifugation is as follows:
[0138] An appropriate amount of the product was placed in distilled water at a mass ratio of 1:10.2 and extracted for 200 min;
[0139] Then centrifuge at 12000 r / min for 15 min.
[0140] Performance test: The highly active nattokinase samples provided in Examples 1 to 5 are respectively labeled as Examples 1 to 5; and the relevant properties of the highly active nattokinase provided in Examples 1 to 5 are respectively tested as follows:
[0141] Enzyme activity test: The test method is agarose-fibrin plate method: prepare UK standard solutions with concentration gradients of 10U / mL, 20U / mL, 40U / mL, 60U / mL, 80U / mL, and 100U / mL, punch holes in the fibrin plate after solidification, add 10μL of UK solution of each concentration, set up 3 parallels, incubate at a constant temperature of 37℃ in a biochemical incubator for 18h, use a vernier caliper to measure the two vertical diameters of the transparent dissolution circle, take the average value to calculate its area S=πd 2 / 4, and draw a standard curve. After punching the fibrin plate, add 10 μL of crude enzyme solution, set up 3 parallels, incubate at 37℃ biochemical incubator for 18h, calculate the average area of transparent dissolution zone, and calculate the fibrinolytic activity of crude enzyme solution according to the UK standard curve equation.
[0142] The obtained test data are recorded in Table 1 below:
[0143] Table 1 Enzyme activity performance test results of each group
[0144] Group Activity (IU / g) Example 1 Group 37400 Example 2 Group 37100 Example 3 Group 37200 Example 4 group 37400 Example 5 group 37300
[0145] By comparing and analyzing the relevant data in Table 1, it can be known that the high-activity nattokinase prepared by the present invention has high enzyme activity, short fermentation cycle, richer product nutrition, simple process and low cost, and at the same time, effectively guarantees its quality. This shows that the method for preparing high-activity nattokinase using beans as raw materials provided by the present invention has a broader market prospect and is more suitable for promotion.
[0146] The present invention obtains high-activity nattokinase by inoculating the strain into a seed culture medium, preparing a seed activation liquid, then inoculating the seed activation liquid into a fermentation culture medium, fermenting and culturing, and finally extracting and centrifuging the fermented product, wherein the seed culture medium includes the following concentration components: tryptone 12-28 g / L, beef extract 3-5 g / L, NaCl 2-5 g / L, yeast extract 5-8 g / L, aspartic acid 0.1-0.3 g / L, pH 7.0-7.5, and the fermentation culture medium is obtained by soaking and sterilizing an appropriate amount of composite fermentation material in water, and the composite fermentation material is mixed with cyperus sorbifolia meal and Xanthoceras sorbifolia meal in a mass ratio of 1:0.3-0.4. The high-activity nattokinase prepared by the present invention has high enzyme activity, short fermentation cycle, richer product nutrition, simple process and low cost, and at the same time, effectively guarantees its quality.
[0147] In the description of this specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.
[0148] The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to the specific implementation methods described. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and use the present invention well. The present invention is limited only by the claims and their full scope and equivalents.
Claims
1. A method for preparing highly active nattokinase using beans as raw materials, characterized in that: The following steps are involved: S1, inoculating the bacterial strain into the seed culture medium to prepare the seed activation solution; S2, connecting the seed activation liquid to the fermentation medium for fermentation culture; S3. Extract and centrifuge the fermented product to obtain highly active nattokinase.
2. A method for preparing highly active nattokinase using beans as raw materials according to claim 1, characterized in that, In step S1, the preparation process of the seed activation solution is as follows: Take a loopful of bacteria and inoculate it into 10-12 mL of seed culture medium, and culture it at 36-37°C and 170-180 rpm for 12 hours. After the shaking culture is completed, transfer to 50-52 mL of seed culture medium and continue shaking culture at 36-37°C and 170-180 r / min for 12 h to obtain seed activation solution.
3. A method for preparing highly active nattokinase using beans as raw materials according to claim 2, characterized in that, The bacterial strain is selected from any one of Bacillus natto BNCC185324 and Bacillus licheniformis BNCC132622.
4. A method for preparing highly active nattokinase using beans as raw materials according to claim 2, characterized in that, The seed culture medium includes the following concentration components: Tryptone 12-28 g / L, beef extract 3-5 g / L, NaCl 2-5 g / L, yeast extract 5-8 g / L, aspartic acid 0.1-0.3 g / L, pH 7.0-7.
5.
5. A method for preparing highly active nattokinase using beans as raw materials according to claim 1, characterized in that, In step S2, the seed activation solution is introduced into the fermentation medium, and the fermentation culture process is as follows: Add an appropriate amount of seed activation solution to the fermentation medium at a mass ratio of 0.05 to 0.08:1; Then culture at 36-37°C for 48-49h.
6. A method for preparing highly active nattokinase using beans as raw materials according to claim 5, characterized in that: The preparation process of the fermentation medium is as follows: Place an appropriate amount of the composite fermentation material in water at a solid-liquid ratio of 0.22-0.32 g / mL, and soak in a water bath at 50°C for 100-120 minutes; After soaking, place it at 110-115℃ and sterilize it for 30-34 minutes.
7. A method for preparing highly active nattokinase using beans as raw materials according to claim 6, characterized in that: The composite fermentation material is prepared by mixing cyperus oleifera meal and Xanthoceras sorbifolia meal in a mass ratio of 1:0.3-0.
4.
8. A method for preparing highly active nattokinase using beans as raw materials according to claim 1, characterized in that: In step S3, the process of extraction and centrifugation is as follows: Place an appropriate amount of the product in distilled water at a mass ratio of 1:5.2-10.2 and extract for 120-200 minutes; Then centrifuge at 10000-12000 r / min for 10-15 min.