1. A bacillus subtilis and bacillus natto subspecies BNA-128/001/B128 strain is preserved in China center for type culture collection with the preservation number of CCTCC NO. M2020479.

2. A preparation method of natto fermented beverage is characterized by comprising the following steps:

preparing soybean paste: cooking and crushing soaked soybeans, and adding water and stirring uniformly to obtain soybean paste; the mass ratio of the soybeans to the water is 1:1-1: 20;

enzymolysis: adding complex enzyme into the soybean paste, and performing enzymolysis at 20-50 deg.C to obtain enzymolysis compound; the compound enzyme accounts for 0.1-5 wt% of the mass of the soybean paste; the complex enzyme comprises pectinase, neutral protease and cellulase; preparation of single cell solution: diluting the enzymolysis compound with water to obtain a soybean single cell solution;

primary fermentation: inoculating seed liquid of bacillus subtilis and bacillus natto subspecies strains into a soybean single cell solution, and performing primary fermentation in an aerobic environment to obtain a primary fermentation mixture; fermenting at 20-45 deg.C for 2-8 days while controlling pH to 5.5-8.5;

seasoning: adding a flavoring agent into the primary fermentation mixture for flavoring to obtain a flavoring mixture;

and (3) secondary fermentation: performing secondary fermentation on the flavoring mixture in oxygen-free environment to obtain fermented natto beverage, wherein the fermentation temperature is 4-20 deg.C, the fermentation time is 2-8 days, and the pH during fermentation is controlled at 5.5-8.5.

3. The method for preparing natto fermented drink according to claim 2, wherein in the step of preparing the mashed soybeans, the soybeans are soaked for 1-4 days and cooked in a high-pressure cooking manner.

4. The method for preparing natto fermented beverage according to claim 2, wherein in the enzymolysis step, the enzymolysis process is stirred at a stirring speed of 100-150 r/min; the compound enzyme contains 1-10 wt% of pectinase, 1-5 wt% of neutral protease and 1-5 wt% of cellulase.

5. According to claim4, the preparation method of the natto fermented drink is characterized in that in the enzymolysis step, the enzyme activity of pectinase is 5 multiplied by 10⁵u/g, enzyme activity of neutral protease is 10 multiplied by 10⁵u/g and the enzyme activity of the cellulase is 10 multiplied by 10⁵u/g。

6. The method for preparing fermented natto beverage according to claim 2, wherein in the primary fermentation step, the bacillus subtilis natto subspecies strain is bacillus subtilis natto subspecies BNA-128/001/B128 strain secreting nattokinase, the strain is preserved in the chinese type culture collection with the preservation number of CCTCC No. m 2020479.

7. The method for preparing fermented natto drink according to claim 2, wherein in the primary fermentation step, the bacteria content in the seed liquid is 5x10⁸cfu/ml, and the inoculation amount of the seed solution is 2-7 wt% of soybean single cell solution.

8. The method for preparing natto fermented beverage according to claim 2, wherein in the primary fermentation step, stirring is performed during fermentation, the stirring rotation number is 100-800rpm, and the ventilation amount during fermentation is 1-4L/min.

9. The method of preparing the natto fermented beverage according to claim 2, wherein the flavoring agent includes sugar.

10. A fermented natto drink characterized by being produced by the production method according to any one of claims 2 to 9.

Background

Natto is well known by people with unique taste and numerous health care functions (dissolving thrombus, reducing blood fat, removing carcinogens in vivo, regulating intestinal flora, relaxing bowel, and the like). For example, Chinese patent application 200810228955.7 discloses a Bacillus subtilis natto subspecies for producing natto kinase and application thereof; chinese patent application 201510588951.X discloses a microwave-resistant bacillus subtilis and application thereof in preparation of nattokinase. However, traditional natto is a solid food with strong smell. There are three existing natto products: traditional natto, natto composite beverage and natto kinase health care products. Traditional natto is popular in japan, but is rarely provided by restaurants due to excessive odor; the existing three natto products have the following defects respectively:

the traditional natto has overlarge smell, and can affect other people when being eaten in public places, so the natto is not sold in restaurants basically, and only can be eaten at home. The natto capsules are the main products of the natto kinase health care products in the market, and the natto capsules are prepared by fermenting, concentrating and drying the natto bacteria, so the natto kinase health care products are expensive in price and bad in taste. The natto composite beverage is prepared by compounding and seasoning freeze-dried natto ground pulp, and the way can cause the nutrition of natto to be greatly lost and the natto flavor to be lost; meanwhile, the mechanical grinding mode causes the loss of the viable bacteria of the bacillus subtilis natto to be more than half, and the content of the nattokinase is relatively reduced. For example, Chinese patent application 201510452976.7 discloses a blended health milk drink containing nattokinase, which is prepared from soybean milk, milk and bacillus subtilis natto seed solution, and has low nattokinase content.

Disclosure of Invention

Therefore, the drink which has mild smell and soft taste and contains high nattokinase is needed to be provided for the public to drink daily.

In order to achieve the object of the present invention, the first aspect of the present invention provides a bacillus natto strain, which is a bacillus subtilis subspecies natto BNA-128/001/B128 (strain name/strain number/symbol), and the latin classification name is: bacillus subtilis natto, wherein the strain is preserved in China center for type culture Collection and is addressed to Wuhan university in Wuhan, China; the preservation number is CCTCC NO. M2020479; the preservation date is 2020, 9 months and 9 days.

The bacillus subtilis and bacillus natto subspecies BNA-128/001/B128 bacterial strain has the characteristics of high activity and low odor, and the enzyme activity of the secreted nattokinase reaches 1500U/ml.

The invention provides a preparation method of natto fermented beverage, which comprises the following steps: preparing soybean paste: cooking and crushing soaked soybeans, and adding water and stirring uniformly to obtain soybean paste; the mass ratio of the soybeans to the water is 1:1-1: 20;

enzymolysis: adding complex enzyme into the soybean paste, and performing enzymolysis at 20-50 deg.C to obtain enzymolysis compound; the compound enzyme accounts for 0.1-5 wt% of the mass of the soybean paste; the complex enzyme comprises pectinase, neutral protease and cellulase; preparation of single cell solution: diluting the enzymolysis compound with water to obtain a soybean single cell solution; primary fermentation: inoculating the seed solution of the bacillus subtilis and bacillus natto subspecies strain into the soybean single cell solution, and performing primary fermentation in an aerobic environment to obtain a primary fermentation mixture; fermenting at 20-45 deg.C for 2-8 days while controlling pH to 5.5-8.5;

seasoning: adding a flavoring agent into the primary fermentation mixture for flavoring to obtain a flavoring mixture;

and (3) secondary fermentation: performing secondary fermentation on the flavoring mixture in oxygen-free environment to obtain fermented natto beverage, wherein the fermentation temperature is 4-20 deg.C, the fermentation time is 2-8 days, and the pH during fermentation is controlled at 5.5-8.5.

The inventor finds in research that soybean is rich in pectin among intercellular spaces, which is a complex polysaccharide widely present in higher plants, and is usually combined with lignin, cellulose, hemicellulose and the like in plant tissues to form different textures and forms of the plant tissues. In the enzymolysis step, the pectin is subjected to enzymolysis by adopting the compound enzyme, so that the connection among the soybean cells can be broken, and the soybean single cells which are not mutually bonded are obtained. Meanwhile, the decomposition of the polysaccharide is also beneficial to the mass utilization and the rapid propagation of the bacillus natto in the primary fermentation step.

In the primary fermentation step, the bacillus natto self-propagates in a large amount in an aerobic fermentation environment at the temperature of 20-45 ℃. In the secondary fermentation step, in an anaerobic environment, the bacillus natto enters a strain stabilization period, the strain growth is slowed down at low temperature under anaerobic condition, the strain is more inclined to produce fermentation products, including nattokinase, vitamin K, isoflavone, amino acid and the like, so that the taste is more mellow; meanwhile, the low temperature inhibits the further propagation of the strains, the fermented smell of the natto is well covered, and the natto tea is suitable for daily drinking.

The invention adopts soybean single cells and seed liquid of bacillus natto (bacillus subtilis bacillus natto subspecies strains) for fermentation, increases the contact area of bacillus natto and soybean cells, and improves the fermentation utilization rate. Fermentation produces nattokinase and other nutrient substances (vitamin K, isoflavone and amino acid), and the flavor of natto is also kept.

Further, the bacillus natto seed liquid adopts shake bacteria to culture the bacillus natto strain to logarithmic phase; the efficiency of the first fermentation is improved.

Further, in the preparation step of the soybean paste, the soybean is soaked for 1-4 days and cooked in a high-pressure cooking mode.

Further, in the enzymolysis step, the enzymolysis process is stirred at the stirring speed of 100-; the compound enzyme contains 1-10 wt% of pectinase, 1-5 wt% of neutral protease and 1-5 wt% of cellulase.

Further, in the enzymolysis step, the enzyme activity of the pectinase is 5 multiplied by 10⁵u/g, enzyme activity of neutral protease is 10 multiplied by 10⁵u/g and the enzyme activity of the cellulase is 10 multiplied by 10⁵u/g。

Further, in the single cell solution preparation step, the enzymolysis solution is diluted by adding water until the quality of the soybean paste is reached, and then the soybean single cell solution is obtained by sterilizing at the temperature of 100 ℃ and 130 ℃.

Further, in the primary fermentation step, the bacillus subtilis and bacillus natto subspecies strain is bacillus subtilis and bacillus natto subspecies BNA-128/001/B128 strain, and the strain is preserved in China center for type culture collection with the preservation number of CCTCC NO. M2020479.

Further, in the first fermentation step, the bacteria content in the seed liquid is 5 multiplied by 10⁸cfu/ml, and the inoculation amount of the seed solution is 2-7 wt% of soybean single cell solution.

Further, in the first fermentation step, stirring is carried out in the fermentation process, the stirring rotation number is 100-800rpm, and the ventilation volume in the fermentation is 1-4L/min.

Further, the flavoring agent comprises sugar. The addition of sugar can regulate taste, provide C source, inhibit reproduction of Bacillus natto, and convert Bacillus natto into secretion fermentation product.

Further, the flavoring agent comprises white granulated sugar, sodium cyclamate, essence and sodium benzoate.

The third aspect of the invention provides a natto fermented beverage which is prepared by the preparation method of the second aspect of the invention.

Different from the prior art, the technical scheme at least comprises the following beneficial effects: the Bacillus natto BNA-128/001/B128 has high nattokinase yield and low odor, and can be used for preparing fermented natto drinks. The invention adopts the seed liquid of the soybean single cell and the bacillus natto for fermentation, increases the contact area of the bacillus natto and the soybean cell, and improves the fermentation utilization rate by adopting the liquid fermentation process. Fermentation produces nattokinase and other nutrient substances (vitamin K, isoflavone and amino acid), and the flavor of natto is also kept.

Drawings

FIG. 1 is a micrograph a of soybean cells in the supernatant of an enzymatic complex;

FIG. 2 is a micrograph b of soybean cells in the supernatant of the enzymatic complex.

Detailed Description

To explain technical contents, structural features, and objects and effects of the technical solutions in detail, the following detailed description is given with reference to the accompanying drawings in conjunction with the embodiments.

Example 1 screening of Bacillus natto (Bacillus subtilis Bacillus natto strain BNA-128) and preparation of culture Medium

(1) Primary screening of culture medium: peptone 1-5%, sodium chloride 0.1-1%, beef extract 0.3-1%, casein 1-5%, and solid agar 1-4%.

(2) Re-screening the culture medium: white sugar 0.1-0.5%, bacteriological peptone 0.1-0.5%, pH7.2

(3) Fibrin flat plate

(4) Slant culture medium: peptone 0.5-2.5%, beef extract powder 0.2-2%, sodium chloride 0.5-2.5%, agar 1-5%, and pH7.3

2. Operation of

(1) Primary screening: soaking 1-2g soybean fermented by peasant family in small amount of sterile normal saline, shaking for 30-60min, diluting the soaking solution by 10 times gradient method, spreading 0.1-1ml of the above diluted solution in a primary sieve culture medium, culturing at 30-37 deg.C for 24-48h, and observing the result. 5 single colonies with round, leafy edges, wrinkled surfaces and large circles of hydrolysis were picked and streaked until pure.

(2) Re-screening: the obtained bacteria cultured in the primary screening solid state are inoculated to a secondary screening culture medium according to the inoculation rate of 1-5 percent, the mixture is subjected to shake flask fermentation culture at the temperature of 30-37 ℃ and at the speed of 100-.

(3) Slant culture preservation: selecting a ring from a primary screening solid culture medium corresponding to the selected nattokinase high-yield strain, inoculating the selected primary screening solid culture medium to a solid slant culture medium, and culturing at the temperature of 30-37 ℃ for 24-48h to obtain the high-yield strain, namely a bacillus subtilis and bacillus natto subspecies BNA-128 strain.

Example 2 preparation of fermented natto drink

The method comprises the following steps:

preparing soybean paste: soaking semen glycines for 1-4 days, cooking and pulverizing by high pressure steaming, adding water, and stirring to obtain semen glycines paste; the mass ratio of the soybeans to the water is 1:1-1: 20;

enzymolysis: adding complex enzyme into the soybean paste, stirring for 2-8h at 20-50 ℃ and 100-; the compound enzyme accounts for 0.1-5 wt% of the mass of the soybean paste; the compound enzyme contains 1-10 wt% of pectase and 5 × 10 of enzyme activity⁵u/g, the content of neutral protease is 1-5 wt%, and the enzyme activity is 10 multiplied by 10⁵u/g, cellulase content of 1-5 wt%, and enzyme activity of 10 × 10⁵u/g。

Preparation of single cell solution: diluting the enzymolysis compound with water until the quality of the soybean paste is reached, and sterilizing at the temperature of 100-;

wherein, the microscopic pictures a and b of the soybean cells in the supernatant of the enzymolysis complex are shown in figures 1 and 2, and it can be seen from the figures that the pectinase between the cells is enzymolyzed, and the soybean cells are basically separated and are not adhered to each other. Primary fermentation: inoculating seed solution of Bacillus subtilis and Bacillus natto subspecies strain (BNA-128/001/B128, preservation number of CCTCC NO. M2020479) cultured to logarithmic phase into soybean single cell solution, wherein the bacterial content in the seed solution is 5 × 10⁸cfu/ml; performing primary fermentation in an aerobic environment to obtain a primary fermentation mixture; fermenting at 20-45 deg.C for 2-8 days while controlling pH to 5.5-8.5; stirring is carried out in the fermentation process, the stirring revolution is 100-800rpm, and the ventilation volume in the fermentation process is 1-4L/min. Seasoning: adding a flavoring agent into the primary fermentation mixture for flavoring to obtain a flavoring mixture; in the flavoring agent, white sugar content is 1-4 wt%, sodium cyclamate is 0.01-0.07 wt%, essence (milk flavor, rose flavor, grape flavor, etc.) is 0.01-0.1 wt%, and sodium benzoate is 0.01-0.05 wt%.

And (3) secondary fermentation: performing secondary fermentation on the flavoring mixture in oxygen-free environment to obtain fermented natto beverage, wherein the fermentation temperature is 4-20 deg.C, the fermentation time is 2-8 days, and the pH during fermentation is controlled at 5.5-8.5.

Example 2Detecting the nutrient substances of the natto fermented beverage prepared in the step (1). Tests show that the fermented beverage prepared in example 2 has the average value of enzyme activity of the nattokinase of 1500U/ml and the bacteria content of 1.5x10⁹cfu/ml; isoflavone content of 0.2% -0.8%, and vitamin K content of 2.1 × 10³g/ml.

It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or terminal that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or terminal. Without further limitation, an element defined by the phrases "comprising … …" or "comprising … …" does not exclude the presence of additional elements in a process, method, article, or terminal that comprises the element. Further, herein, "greater than," "less than," "more than," and the like are understood to exclude the present numbers; the terms "above", "below", "within" and the like are to be understood as including the number.

It should be noted that, although the above embodiments have been described herein, the invention is not limited thereto. Therefore, based on the innovative concepts of the present invention, the technical solutions of the present invention can be directly or indirectly applied to other related technical fields by making changes and modifications to the embodiments described herein, or by using equivalent structures or equivalent processes performed in the content of the present specification and the attached drawings, which are included in the scope of the present invention.