CN101214263B - Method for preparing freeze-dried powder containing bacillus natto and nattokinase - Google Patents
Method for preparing freeze-dried powder containing bacillus natto and nattokinase Download PDFInfo
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- CN101214263B CN101214263B CN2007101646321A CN200710164632A CN101214263B CN 101214263 B CN101214263 B CN 101214263B CN 2007101646321 A CN2007101646321 A CN 2007101646321A CN 200710164632 A CN200710164632 A CN 200710164632A CN 101214263 B CN101214263 B CN 101214263B
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Abstract
The present invention relates to freeze-dried powder containing bacillus natto and nattokinase and a preparation method thereof, the process of which comprises that: firstly, culture medium is confected and sterilized, wherein, the culture medium components with the weight percentage are 8 percent to 15 percent of soybean flour, 1 percent to 5 percent of sucrose, 0.4 percent to 0.6 percent of NaCland the water with the rest quantity, the pH value of which is 6.8 to 7.2; secondly, the bacillus natto is inoculated, and the inoculation quantity is 2 percent to 8 percent; thirdly, the bacillus natto after the inoculation is cultured and fermented for 20 to 28 hours under the temperature of 36 DEG C to 38 DEG C; fourthly, fermentation liquid is frozen and dried in vacuum, the glycerin with 1 percent to 7 percent of the weight of the fermentation liquid is used protecting agent to be added into the fermentation liquid, which is frozen under the temperature of subzero 18 DEG C in advance tobe frozen and dried in vacuum for 12 to 20 hours and then is resolved under the temperature lower than 30 DEG C. The method has low cost and is in favor of the industrial production. Under the precondition of maintaining the nattokinase activity, the freeze-dried process also holds other nutrient component in the bacillus natto and the fermentation liquid, which reduces the nattokinase purification cost.
Description
Technical field
The present invention relates to a kind of preparation method that contains the lyophilized powder of Bacillus natto and nattokinase.
Background technology
According to data, Japanese digestive tract disease, thrombosis and thrombosis angiemphraxis disease incidence probability are very low, and this eats natto every days with them very big contact.Natto is a Semen sojae atricolor through soaking, anhydrate, after the process such as steaming and decocting, inserting Bacillus natto and ferment and form.Find that after deliberation natto has various health-care, this mainly has relation with following 3 aspects:
1. Bacillus natto has broad-spectrum antibacterial action, and gram positive bacteria, gram negative bacteria, yeast and mycete are all had certain antagonism, and unusual Hansenula yeast is had strong antibacterial action.Antibiotics that Bacillus natto can produce such as bacitracin, polymyxin, 2, dipicolimic acid 2 has the effects of pathogenic bacterium such as suppressing staphylococcus aureus, Salmonella, typhoid fever bacterium, dysentery bacillus and escherichia coli;
2. Semen sojae atricolor produces materials such as VK, genistein, thick antioxidant extract after Bacillus natto fermentation, to blood fat reducing, anticancer, prevent and treat osteoporosis etc. bigger effect arranged;
3. the nattokinase that produces after Bacillus natto ferments is thrombus effectively, also have blood viscosity lowering in addition, suppress platelet and solidify blood fat reducing, cholesterol reducing, improve the blood circulation situation, keep different physiological roles such as the normal morphology of hemocyte and function.
Because the natto that conventional art is produced, the room temperature following shelf-life of state is very short, and the maintenance that the activity of nattokinase can not be permanent, has limited its exploitation as health food greatly and has been worth.Because natto has wire drawing, special features such as abnormal smells from the patient do not have market in China again.In addition, the now natto and the nattokinase product of exploitation, or have only the nattokinase composition, complex process, cost costliness, or make after directly pulverizing drying with natto, but the exploitation of the Bacillus natto that plays antibacterial action is relatively ignored.
Go up to analyze according to this as can be seen, the key of exploitation natto and nattokinase goods be seek that technology is simple, productive rate is high, have the fermented soybean product concurrently, the new product of nattokinase and Bacillus natto health care.
Summary of the invention
Main purpose of the present invention is in order to overcome above-mentioned deficiency, a kind of preparation method that contains the lyophilized powder of Bacillus natto and nattokinase to be provided.This preparation method can keep the activity of back nattokinase of fermenting as far as possible, and keeps Bacillus natto viable bacteria and nutrient substance in the fermentation liquid simultaneously, and its health-care effect can be developed to greatest extent; And can the long period keep its functional component, can be made into capsule, be convenient to take.
For achieving the above object, technical solution of the present invention is as follows: a kind of preparation method that contains the lyophilized powder of Bacillus natto and nattokinase is characterized in that its process comprises:
A. culture medium preparation and sterilization, wherein, its mass percent composition of culture medium is an analysis for soybean powder 8%～15%, sucrose 1%～5%, NaCl 0.4%～0.6%, and surplus is a water, and pH value is 6.8～7.2;
B. Bacillus natto inoculation, the Bacillus natto inoculum concentration is 2%～8%;
C. fermentation, inoculation back cultivation and fermentation 20～28h under 36～38 ℃ of temperature;
D. fermentation liquid vacuum lyophilization: get fermentation liquid, the glycerol that adds 1%～7% fermentation liquid quality is cooked protective agent, after-18 ℃ of following temperature pre-freezes, and vacuum lyophilization 12～20h, parsing-desiccation below 30 ℃ promptly gets lyophilized powder again.
Semen Glycines obtains analysis for soybean powder by pulverizing, and analysis for soybean powder is crossed 200 mesh sieves, is preferably to be equal to or less than 200 purpose analysis for soybean powder, is convenient to inoculation.
The preparation method that contains the lyophilized powder of Bacillus natto and nattokinase of the present invention, its mass percent composition of its described culture medium is preferably analysis for soybean powder 10%, sucrose 2%, NaCl 0.5%, and surplus is a water, and pH value is 7.0; Described Bacillus natto inoculum concentration is preferably 5%; Be preferably cultivation and fermentation 24h under 37 ℃ of temperature after the Bacillus natto inoculation in the sweat.
Different being that the present invention and nattokinase lyophilized powder are in the past made: carry out the fermentation of Bacillus natto as culture medium with common materials such as analysis for soybean powder, sucrose, NaCl, with low cost, be beneficial to commercial production, freezing dry process is keeping under the natto kinase activity prerequisite, taken into account the reservation of other nutritional labeling in Bacillus natto and the fermentation liquid, and simplified processing step, reduced the natto kinase purifying cost.Lyophilized powder can be made into capsule, not only makes active substance keep the activity of long period, and can cover special odor, is convenient to consumer and accepts.
The specific embodiment
Below by embodiment the present invention program is done detailed description.
1. culture medium is prepared and sterilization steps embodiment
The preparation culture medium, wherein, its mass percent composition of culture medium is an analysis for soybean powder 8%～15%, sucrose 1%～5%, NaCl 0.5%, and surplus is a water, and pH value is 7.0.Each concrete content is shown in Table 1 (content of only having listed analysis for soybean powder and sucrose in the table), and Semen Glycines obtains analysis for soybean powder by pulverizing, and analysis for soybean powder is crossed 200 mesh sieves.To medium sterilization, insert Bacillus natto by inoculum concentration 5%, at 37 ℃ of bottom fermentation 24h.Get fermentation liquid, the glycerol that adds 5% fermentation liquid quality is cooked protective agent, after-19 ℃ of pre-freezes, and vacuum lyophilization 16h, 25 ℃ of parsing-desiccations promptly get lyophilized powder again.(above percentage ratio is mass percent.) nattokinase fibrin plate method mensuration, the Bacillus natto number is counted with blood counting chamber, the results are shown in Table 1.
Select common beef-protein medium, its mass percent composition is: peptone 1%, Carnis Bovis seu Bubali cream 0.3%, NaCl 0.5%, agar 2%, and surplus is a water, pH value is 6.8-7.2, all the other operation preparation processes are tested its Bacillus natto number and natto kinase activity with embodiment 1 behind the fermented frozen, the result also lists in table 1.
The comparison of table 1. natto kinase activity and Bacillus natto number
| Culture medium | Natto kinase activity (IU/mL) | The Bacillus natto number (individual/mL) |
| Beef-protein medium | 867 | 4.10×10 9 |
| Analysis for soybean powder 8%, sucrose 1% | 976 | 4.00×10 9 |
| Analysis for soybean powder 8%, sucrose 2% | 976 | 3.85×10 9 |
| Analysis for soybean powder 8%, sucrose 5% | 921 | 3.73×10 9 |
| Analysis for soybean powder 10%, sucrose 1% | 991 | 3.71×10 9 |
| Analysis for soybean powder 10%, sucrose 2% | 1080 | 3.90×10 9 |
| Culture medium | Natto kinase activity (IU/mL) | The Bacillus natto number (individual/mL) |
| Analysis for soybean powder 10%, sucrose 5% | 1036 | 4.11×10 9 |
| Analysis for soybean powder 15%, sucrose 1% | 976 | 3.77×10 9 |
| Analysis for soybean powder 15%, sucrose 2% | 932 | 3.65×10 9 |
| Analysis for soybean powder 15%, sucrose 5% | 924 | 3.54×10 9 |
The result as can be seen by experiment, at medium component is analysis for soybean powder 8%～15%, sucrose 1%～5%, NaCl 0.5%, and surplus is a water, and pH value is under 7.0 conditions, natto kinase activity and Bacillus natto number do not have obvious variation, all be higher than the resulting natto kinase activity unit of Comparative Examples, but optimum condition is an analysis for soybean powder 10%, sucrose 2%.
2. Bacillus natto inoculation step embodiment
Be mixed with and be divided into analysis for soybean powder (Semen Glycines was pulverized 200 mesh sieves) 10%, sucrose 2%, NaCl 0.5%, the culture medium of pH 6.8-7.2, the inoculum concentration with 2%, 5%, 8% inserts Bacillus natto, at 37 ℃ of bottom fermentation 24h.Get fermentation liquid, the glycerol that adds 5% fermentation liquid weight is cooked protective agent, after-18 ℃ of following temperature pre-freezes, and vacuum lyophilization 16h, parsing-desiccation below 30 ℃ promptly gets lyophilized powder again.Nattokinase is measured with the fibrin plate method, and the Bacillus natto number is counted with blood counting chamber, the results are shown in Table 2.
The comparison of table 2. natto kinase activity and Bacillus natto number
| Inoculum concentration | Natto kinase activity (IU/mL) | The Bacillus natto number (individual/mL) |
| 2％ | 976 | 3.75×10 9 |
| 5％ | 1080 | 3.90×10 9 |
| 8％ | 991 | 3.84×10 9 |
By the result as can be seen, inoculum concentration does not have much influences to natto kinase activity and Bacillus natto number in the scope of 2-8%, and comparatively speaking, 5% inoculum concentration is preferred.
3. fermentation liquid vacuum lyophilization step embodiment
Add in the comparative preparation process and glycerol adding do not prepare the natto kinase activity and the Bacillus natto survival rate of lyophilized powder: be mixed with and be divided into analysis for soybean powder (Semen Glycines was pulverized 200 mesh sieves) 10%, sucrose 2%, NaCl 0.5%, the culture medium of pH 6.8-7.2, inoculum concentration with 5% inserts Bacillus natto, at 37 ℃ of bottom fermentation 24h.Get fermentation liquid, the glycerol of a part of 5% fermentation liquid weight is cooked protective agent, and another part not glycerol adding is cooked protective agent, after-18 ℃ of following temperature pre-freezes, and vacuum lyophilization 16h, parsing-desiccation below 30 ℃ promptly gets lyophilized powder again.Nattokinase is measured with the fibrin plate method, and the Bacillus natto number is counted with blood counting chamber, the results are shown in Table 3.
The interpolation of table 3. glycerol (protective agent) is to the influence of natto kinase activity and Bacillus natto survival rate
| Processing method | The lyophilizing of common fermentation liquid | Add 5% glycerol fermented broth lyophilizing |
| Natto kinase activity (IU/g dry powder) | 8000 | 5000 |
| The enzyme response rate (%) | 27.2 | 33.8 |
| Fermentation liquid gets dry powder heavy (g/100mL fermentation liquid) | 12.67 | 15.33 |
| Viable count (individual/g dry powder) | 4.19×10 9 | 7.53×10 9 |
| Survival rate (%) | 13.6 | 29.6 |
Stability experiment
Can keep enzyme to live and the Bacillus natto viable count at normal temperatures and pressures for understanding contains Bacillus natto nattokinase lyophilized powder, the present invention be contained the lyophilized powder that the preparation method of the lyophilized powder of Bacillus natto and nattokinase makes carried out stable mensuration, the results are shown in Table 4:
Table 4. contains Bacillus natto nattokinase lyophilized powder stability and measures
By experiment the result as can be seen, the enzyme work of nattokinase has descended 2.56% after 12 months, the Bacillus natto viable count has descended 14.5%, therefore, lyophilized powder can keep the long period at normal temperatures and pressures.
Above embodiment is to the explanation of patent and further explains, rather than limitation of the present invention, and in spirit of the present invention and rights protection scope, any modification of being made all falls into protection scope of the present invention.
Claims (5)
1. preparation method that contains the lyophilized powder of Bacillus natto and nattokinase is characterized in that its process comprises:
A. culture medium preparation and sterilization, wherein, its mass percent composition of culture medium is an analysis for soybean powder 8%～15%, sucrose 1%～5%, NaCl 0.4%～0.6%, and surplus is a water, and pH value is 6.8～7.2;
B. Bacillus natto inoculation, the Bacillus natto inoculum concentration is 2%～8%;
C. fermentation, inoculation back cultivation and fermentation 20～28h under 36～38 ℃ of temperature;
D. fermentation liquid vacuum lyophilization: get fermentation liquid, the glycerol that adds 1%～7% fermentation liquid quality is cooked protective agent, after-18 ℃ of following temperature pre-freezes, and vacuum lyophilization 12～20h, parsing-desiccation below 30 ℃ promptly gets lyophilized powder again.
2. the preparation method that contains the lyophilized powder of Bacillus natto and nattokinase according to claim 1 is characterized in that described analysis for soybean powder particle diameter is equal to or less than 200 orders.
3. the preparation method that contains the lyophilized powder of Bacillus natto and nattokinase according to claim 1 and 2 is characterized in that its mass percent composition of described culture medium is an analysis for soybean powder 10%, sucrose 2%, and NaCl 0.5%, and surplus is a water, and pH value is 7.0.
4. the preparation method that contains the lyophilized powder of Bacillus natto and nattokinase according to claim 3 is characterized in that described Bacillus natto inoculum concentration is 5%.
5. the preparation method that contains the lyophilized powder of Bacillus natto and nattokinase according to claim 4 is characterized in that Bacillus natto inoculation back cultivation and fermentation 24h under 37 ℃ of temperature in the sweat.
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| CN101816395B (en) * | 2009-11-12 | 2013-03-13 | 天津市百奥生物技术有限公司 | Process for producing natto powder |
| CN102669581A (en) * | 2011-03-17 | 2012-09-19 | 潘峰 | Preparation method of bacillus natto freeze-dried powder for making natto at home |
| CN103238829B (en) * | 2012-02-13 | 2015-08-19 | 中国科学院过程工程研究所 | A kind of nattokinase enteric capsule and preparation method thereof |
| CN102787091A (en) * | 2012-08-31 | 2012-11-21 | 韶关市颐林泉生物科技有限公司 | Industrial production method of bacillus natto |
| CN103141837A (en) * | 2013-02-28 | 2013-06-12 | 东北林业大学 | Method for fermenting pigeonpea seed by immobilizing bacillus natto |
| CN104560790A (en) * | 2014-12-15 | 2015-04-29 | 北京农学院 | Functional lactobacillus plantarum and preparation method of compound bacterial powder of functional lactobacillus plantarum |
| CN105918576B (en) * | 2016-04-21 | 2019-11-26 | 广州市佰沃生物科技有限公司 | Fermented type coffee bean and preparation method thereof |
| CN106420659B (en) * | 2016-10-27 | 2019-05-21 | 北京浩奇科技有限责任公司 | A kind of bean-containing capsule and preparation method thereof |
| CN108048441B (en) * | 2017-12-10 | 2021-03-16 | 河北省微生物研究所 | Preparation method of hexokinase freeze-dried powder |
| CN108514079B (en) * | 2018-04-28 | 2021-10-29 | 山东润泽制药有限公司 | Preparation method of natto freeze-dried powder |
| CN109182312A (en) * | 2018-09-25 | 2019-01-11 | 武汉黄家医圈生物医药有限公司 | A kind of method and application thereof of composite microbial Tetramune high efficient expression |
| CN109645344A (en) * | 2019-01-25 | 2019-04-19 | 纽斯葆广赛（广东）生物科技股份有限公司 | A kind of preparation method of the freeze dry powder of fermented soybean of high-content Nattokinase |
| CN109706135A (en) * | 2019-03-06 | 2019-05-03 | 武汉轻工大学 | A kind of Nattokinase liquid state fermentation culture medium and the fermentation process for producing Nattokinase |
| CN111088196B (en) * | 2020-01-18 | 2022-04-22 | 河北省科学院生物研究所 | Strain SWS-001 for preparing high-activity nattokinase and application thereof |
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