CN112998206A - Preparation method of bacillus natto fermented soybean curd residue, product and application - Google Patents
Preparation method of bacillus natto fermented soybean curd residue, product and application Download PDFInfo
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Abstract
The invention discloses a preparation method of bacillus natto fermented soybean curd residue, a product and application. The invention prepares the natto bacterium fermented soybean curd residue by inoculating the natto bacterium leavening agent into the cooked soybean curd residue, fermenting, culturing and then freezing and drying. The invention develops and produces a new product by taking the soybean curb residue as a raw material, provides a functional series of foods with health, safety, nutrition and health care for people, improves the utilization rate and the added value of the soybean curb residue and prolongs the industrial chain of soybean processing.
Description
Technical Field
The invention relates to the field of fermented food processing, in particular to a preparation method of bacillus natto fermented soybean curd residue, a product and application.
Background
The bean curd residue is a main byproduct in the production of bean products such as bean curd, dried bean curd, bean curd skin and the like, and accounts for about 15-20% of the total dried bean curd. The bean curd residue is rich in dietary fiber, protein, mineral, vitamins, etc., and has high nutritive value. The bean curd residue also contains various functional components including isoflavone, total saponin, dietary fiber and the like, the edible bean curd residue can reduce the content of cholesterol in blood, reduce the consumption of insulin of a diabetic patient and is beneficial to preventing intestinal cancer, hypertension, diabetes and other diseases, and the bean curd residue is regarded as a new dietary fiber source. In China, which is a major country for producing and consuming bean products, the annual production amount of wet soybean curd residues is more than 2000 million tons based on 2 tons of wet soybean residues produced by processing 0.5 tons of dry soybeans. At present, only a small part of bean curd residues are used as auxiliary materials for food production, such as battercake, bean dreg egg cake, ball, steamed bread, bean dreg cake, bean dreg biscuit and the like, but the bean curd residues have rough mouthfeel and have bean smell and other unpleasant odors, so the application of the bean curd residues in food processing is limited, most of the bean curd residues are used as feed, the water content is high, the bean curd residues are extremely easy to decay, the environment is polluted, and the resource is greatly wasted. Therefore, the production of food which is easy to be accepted by consumers by utilizing bean curd residue resources is an urgent problem to be solved by bean product manufacturers.
Bacillus subtilis natto is a strain used for producing natto food. The natto prepared by fermenting the soybeans with the bacillus natto has very high nutritional value, contains cellulose, vitamins, saponin, total flavonoids and some trace elements which are higher than those of the steamed soybeans, contains 6 times of vitamin B2 of the steamed soybeans, contains 22 mineral elements, 19 amino acids, multiple vitamins such as folic acid, tocopherol, nattokinase, vitamin K2 and the like, and contains a large amount of unsaturated fatty acids and the like. Meanwhile, natto has various health care functions, including anti-tumor, bacteriostasis, blood pressure reduction and antioxidation. The fibrinolysis effect of the nattokinase determined by the fibrin plate method is obvious, and the thrombolysis effect can inhibit the formation of thrombus when being applied to in vivo tests of healthy people. Reports on the preparation of food series by fermenting soybean curb residue with bacillus natto are not seen at present.
Disclosure of Invention
The invention aims to provide a preparation method of bacillus natto fermented soybean curd residue, a product and application. The bean curd residue is used as a raw material to develop and produce a new product, and a functional series of foods with health, safety, nutrition and health care are provided for people. Improving the utilization rate and the added value of the soybean curd residue and prolonging the industrial chain of soybean processing.
In order to achieve the purpose, the invention provides the following technical scheme:
one of the technical schemes of the invention is as follows: a preparation method of Bacillus natto fermented bean curd residue comprises the following steps:
inoculating the bacillus natto starter to the steamed soybean curd residue, fermenting, culturing, and freeze-drying to obtain the bacillus natto fermented soybean curd residue.
Preferably, the specific steps of the bacillus natto fermented bean curd residue comprise: selecting a bean curd residue raw material with the water content of 73-93%, cooking for 20-40 min, and inoculating a bacillus natto fermenting agent into the cooked bean curd residue; uniformly stirring, flattening, and fermenting and culturing for 24-44 h; freeze drying after fermentation culture to obtain Bacillus natto fermented bean curd residue;
the volume-to-mass ratio of the bacillus natto starter to the steamed bean curd residues is 2-8 mL:100 g;
the thickness of the flattening is 1-5 cm;
the content of Bacillus natto in the Bacillus natto starter is 1 × 105～109CUF/mL。
More preferably, the water content of the soybean curd residue raw material is 83%; the cooking time is 30min, and the volume-to-mass ratio of the bacillus natto starter to the cooked bean curd residue is 6mL:100 g; the thickness of the flattening is 3 cm; the time of fermentation culture is 36 h.
Preferably, the cooking condition is 0.07-0.13 MPa and 115-125 ℃.
Preferably, the method for controlling the water content of the soybean curd refuse comprises the following steps: adding soybean milk when the water content of the bean curd residue is too low, and removing excessive water with filter cloth or centrifuge when the water content of the bean curd residue is too high.
Preferably, the preparation method of the bacillus natto starter comprises the following steps: taking soybeans soaked in water at the temperature of 30-40 ℃ for 10-14 h, mixing the soybeans and the water according to the mass ratio of 1: 4-5, treating the soybeans at the temperature of 115-125 ℃ under the pressure of 0.07-0.13 MPa for 15-25 min, cooling, filtering, adding 4-6% of white sugar and 0.01-0.03% of dipotassium hydrogen phosphate into filtrate, sterilizing to prepare a culture medium, inoculating the activated and expanded cultured bacillus natto into the culture medium, and culturing to obtain the bacillus natto starter.
Preferably, the step of freeze-drying comprises: the soybean curd residue after fermentation culture is pre-frozen at a temperature of between 45 ℃ below zero and 35 ℃ below zero for 1.5 to 2.5 hours, and then is dried in vacuum at a temperature of between 60 ℃ below zero and 50 ℃ below zero for 25 to 35 hours with a vacuum degree of 10 to 50 Pa.
The second technical scheme of the invention is as follows: provides the bacillus natto fermented bean curd residue prepared by the preparation method.
The third technical scheme of the invention is as follows: provides an application of the bacillus natto fermented soybean curd residue in food or health care products.
Preferably, the food is a crisp cake prepared from the bean curd residues fermented by the bacillus natto.
Preferably, the health-care product is one of powder, tablets or capsules prepared from the bean curd residues fermented by the bacillus natto.
The invention has the following beneficial technical effects:
the invention develops and produces a new product by taking the bean curd residue as a raw material, and provides a functional series of foods with health, safety, nutrition and health care for people. Improves the utilization rate and the added value of the soybean curd residue and prolongs the industrial chain of soybean processing. The food produced by fermenting the bean curd residue with the bacillus natto is rich in nattokinase, flavone and saponin; besides food, the product can also be used for preparing health products.
Drawings
FIG. 1 is a fibrin plate for measuring the nattokinase activity of the Bacillus natto fermented bean curd residue prepared in example 1 by fibrin plate method.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
Example 1
Preparing the natto bacterium fermented bean curd residue:
(1) the raw material requirements are as follows: selecting fresh bean curd residue which is just separated from bean curd and soybean product production lines, wherein the fresh bean curd residue is required to have no chemical or physical pollution, no insect and mouse damage, no mildew and no putrefaction;
(2) treating and proportioning bean curd residues: controlling the water content of the bean curd residue at 83%, adding appropriate amount of water when the water content is too low, stirring, and removing excessive water with filter cloth or centrifuge when the water content is too high;
(3) boiling and sterilizing raw materials: putting the bean curd residue meeting the requirement into a container, and then putting into high-pressure steam sterilization equipment to steam and boil for 30min at the temperature of 121 ℃ and under the pressure of 0.1 MPa;
(4) and (3) cooling: after the cooking is finished, taking out the bean curd residues and cooling to 40 ℃ for later use;
(5) preparation of slant strain (activation): putting 1.0g of beef extract, 2.0g of peptone, 0.2g of sodium chloride, 4g of agar and 200mL of distilled water into an enamel jar, heating on a heater, stirring with a glass rod to dissolve the beef extract, supplementing evaporated water after the beef extract is fully melted, subpackaging the beef extract into a clean test tube with a tampon while hot, and sterilizing at the temperature of 121 ℃ and 0.1MPa for 20 min. After sterilization, the test tube is placed on a 15-degree inclined plane while the test tube is hot, and the test tube is condensed with the opening facing upwards to prepare an inclined plane culture medium. Picking up a ring of bacterial lawn from the inclined surface of the bacillus natto by using an inoculating ring on a super clean bench, streaking the bacterial lawn onto a prepared inclined surface culture medium (beef extract peptone inclined surface culture medium), culturing the bacterial lawn in an incubator at 37 ℃ for 26 hours, taking out the bacterial lawn, and storing the bacterial lawn in a refrigerator at 4 ℃ for later use;
(6) preparation of stock seed liquid leaven (expanded culture): 1% of peptone, 0.5% of beef extract, 0.5% of sodium chloride, 100mL of water, sterilizing at 121 ℃ and 0.1MPa for 20min, and cooling to obtain a stock liquid culture medium for later use. Inoculating a loop of lawn fungus from the cultured slant strains into a stock solution culture medium, culturing in a shaking incubator at 37 deg.C and 150r/min for 26h, and preserving in a refrigerator at 4 deg.C for use;
(7) preparing a bacillus natto fermenting agent: cleaning soybeans with complete particles, free of impurities and mildew, putting the soybeans into tap water at 37 ℃, soaking for 12 hours, taking out the soaked soybeans, adding tap water in a ratio of 1:5, putting the soybeans into a sterilization pot, sterilizing at 121 ℃ and 0.1MPa for 20 minutes, cooling to room temperature, filtering with four layers of gauze, adding 5% of white sugar and 0.02% of dipotassium hydrogen phosphate into filtrate, dissolving the white sugar and the dipotassium hydrogen phosphate into the filtrate, filtering, putting the filtrate into a 100mL conical bottle, plugging with a cotton plug, adding damp-proof paper, tying the mouth to prevent dropping, and filling liquid in each bottle to be not more than 67 mL. Placing the conical flask into a sterilizing pot, sterilizing at 121 deg.C and 0.1MPa for 20min, and cooling. Inoculating the cultured stock seed liquid leaven into the culture medium for producing the liquid leaven according to the inoculation amount of 6 percent, culturing for 26 hours at 37 ℃ in an incubator, and diluting until the content of the bacillus natto is 1 multiplied by 108CUF/mL to become bacillus natto starter;
(8) inoculating and fermenting: inoculating the bacillus natto starter into the bean curd residue steamed and cooled to 40 ℃ for later use in the step (4) according to the inoculation amount of 6% vol/m, fully and uniformly stirring, spreading to the thickness of 3cm, and putting into an incubator for fermentation culture at 37 ℃ for 36 hours;
(9) vacuum freeze drying: after the fermentation culture is finished, transferring the fermented materials into a mold (the mold can be in different shapes such as square, round, polygonal and the like according to requirements, and the thickness is about 2 cm), pre-freezing the materials in a refrigerator at the temperature of-40 ℃ for 2h, and then putting the pre-frozen materials into a vacuum freeze dryer (the vacuum degree is 10Pa, the temperature of a cold trap is-54 ℃, the temperature of a clapboard is 20 ℃, and the operation time is 30h) for vacuum freeze drying to obtain the bacillus natto fermented bean curd residue.
Example 2
The processing technology of the natto fermented bean curd residue food comprises the following steps:
(1) the raw material requirements are as follows: selecting fresh bean curd residue which is just separated from bean curd and soybean product production lines, wherein the fresh bean curd residue is required to have no chemical or physical pollution, no insect and mouse damage, no mildew and no putrefaction;
(2) treating and proportioning bean curd residues: controlling the water content of the bean curd residue at 80%, adding appropriate amount of water when the water content is too low, stirring, and removing excessive water with filter cloth or centrifuge when the water content is too high;
(3) boiling and sterilizing raw materials: putting the bean curd residue meeting the requirement into a container, and then putting into high-pressure steam sterilization equipment to steam and boil for 25min at the temperature of 125 ℃ and under 0.13 MPa;
(4) and (3) cooling: after the cooking is finished, taking out the bean curd residues and cooling to 40 ℃ for later use;
(5) preparation of slant strain (activation): putting 1.0g of beef extract, 2.0g of peptone, 0.2g of sodium chloride, 4g of agar and 200mL of distilled water into an enamel jar, heating on a heater, stirring with a glass rod to dissolve the beef extract, supplementing evaporated water after the beef extract is fully melted, subpackaging the beef extract into a clean test tube with a tampon while hot, and sterilizing at the temperature of 121 ℃ and 0.1MPa for 20 min. After sterilization, the test tube is placed on a 15-degree inclined plane while the test tube is hot, and the test tube is condensed with the opening facing upwards to prepare an inclined plane culture medium. Picking up a ring of bacterial lawn from the inclined surface of the bacillus natto by using an inoculating ring on a super clean bench, streaking the bacterial lawn onto a prepared inclined surface culture medium (beef extract peptone inclined surface culture medium), culturing the bacterial lawn in an incubator at 37 ℃ for 26 hours, taking out the bacterial lawn, and storing the bacterial lawn in a refrigerator at 4 ℃ for later use;
(6) preparation of stock seed liquid leaven (expanded culture): 1% of peptone, 0.5% of beef extract, 0.5% of sodium chloride, 100mL of water, sterilizing at 121 ℃ and 0.1MPa for 20min, and cooling to obtain a stock liquid culture medium for later use. Inoculating a loop of lawn fungus from the cultured slant strains into a stock solution culture medium, culturing in a shaking incubator at 37 deg.C and 150r/min for 26h, and preserving in a refrigerator at 4 deg.C for use;
(7) preparing a bacillus natto fermenting agent: cleaning semen glycines with clear water, soaking in tap water of 35 deg.C for 13 hr, and taking out soaked semen glycinesAdding tap water into bean according to a ratio of 1:5, placing into a sterilizing pot, sterilizing at 115 deg.C and 0.07MPa for 25min, cooling to room temperature, filtering with four layers of gauze, adding 4% white sugar and 0.03% dipotassium hydrogen phosphate into the filtrate, dissolving, filtering, placing the filtrate into a 100mL conical bottle, plugging with a cotton plug, adding a damp-proof paper, tying, and preventing dropping, wherein the amount of liquid in each bottle is not more than 67 mL. Placing the conical flask into a sterilizing pot, sterilizing at 121 deg.C and 0.1MPa for 20min, and cooling. Inoculating the cultured stock seed liquid leaven into the culture medium for producing the liquid leaven according to the inoculation amount of 6 percent, culturing for 26 hours at 37 ℃ in an incubator, and diluting until the content of the bacillus natto is 1 multiplied by 108CUF/mL to become bacillus natto starter;
(8) inoculating and fermenting: inoculating the bacillus natto starter into the bean curd residue steamed and cooled to 40 ℃ for later use in the step (4) according to the inoculation amount of 8% vol/m, fully and uniformly stirring, spreading to the thickness of 2cm, and putting into an incubator for fermentation culture at 37 ℃ for 32 hours;
(9) vacuum freeze drying: after the fermentation culture is finished, transferring the materials after the fermentation culture into a mold, pre-freezing the materials in a refrigerator at the temperature of-35 ℃ for 2.5 hours, and then putting the materials in a vacuum freeze dryer (the vacuum degree is 30Pa, the temperature of a cold trap is-50 ℃, the temperature of a partition plate is 20 ℃, and the running time is 35 hours) for vacuum freeze drying;
(10) packaging: vacuum freeze-drying the dried material, immediately finishing and then vacuum packaging to prevent moisture absorption from affecting the quality, and taking the material as a crisp biscuit;
(11) and (4) finished product preservation: the packaged product can be preserved for 24 months under the condition of cool and dry room temperature, and can also be preserved for more than 24 months in a refrigerator at 4 ℃.
Example 3
The processing technology of the bacillus natto fermented bean curd residue health product comprises the following steps:
(1) the raw material requirements are as follows: selecting fresh bean curd residue which is just separated from bean curd and soybean product production lines, wherein the fresh bean curd residue is required to have no chemical or physical pollution, no insect and mouse damage, no mildew and no putrefaction;
(2) treating and proportioning bean curd residues: controlling the water content of the bean curd residue at 88%, adding appropriate amount of water when the water content is too low, stirring, and removing excessive water with filter cloth or centrifuge when the water content is too high;
(3) boiling and sterilizing raw materials: putting the bean curd residue meeting the requirements into a container, and then putting into high-pressure steam sterilization equipment to steam at the temperature of 115 ℃ for 35min under the pressure of 0.07 MPa;
(4) and (3) cooling: after the cooking is finished, taking out the bean curd residues and cooling to 40 ℃ for later use;
(5) preparation of slant strain (activation): putting 1.0g of beef extract, 2.0g of peptone, 0.2g of sodium chloride, 4g of agar and 200mL of distilled water into an enamel jar, heating on a heater, stirring with a glass rod to dissolve the beef extract, supplementing evaporated water after the beef extract is fully melted, subpackaging the beef extract into a clean test tube with a tampon while hot, and sterilizing at the temperature of 121 ℃ and 0.1MPa for 20 min. After sterilization, the test tube is placed on a 15-degree inclined plane while the test tube is hot, and the test tube is condensed with the opening facing upwards to prepare an inclined plane culture medium. Picking up a ring of bacterial lawn from the inclined surface of the bacillus natto by using an inoculating ring on a super clean bench, streaking the bacterial lawn onto a prepared inclined surface culture medium (beef extract peptone inclined surface culture medium), culturing the bacterial lawn in an incubator at 37 ℃ for 26 hours, taking out the bacterial lawn, and storing the bacterial lawn in a refrigerator at 4 ℃ for later use;
(6) preparation of stock seed liquid leaven (expanded culture): 1% of peptone, 0.5% of beef extract, 0.5% of sodium chloride, 100mL of water, sterilizing at 121 ℃ and 0.1MPa for 20min, and cooling to obtain a stock liquid culture medium for later use. Inoculating a loop of lawn fungus from the cultured slant strains into a stock solution culture medium, culturing in a shaking incubator at 37 deg.C and 150r/min for 26h, and preserving in a refrigerator at 4 deg.C for use;
(7) preparing a bacillus natto fermenting agent: cleaning soybeans with complete particles, free of impurities and mildew, putting the soybeans into tap water at 40 ℃ for soaking for 10 hours after being cleaned by clear water, taking out the soaked soybeans, adding tap water in a ratio of 1:4, putting the soybeans into a sterilization pot for sterilization at 125 ℃ and 0.13MPa for 15 minutes, cooling to room temperature, filtering by using four layers of gauze, adding 6% of white sugar and 0.01% of dipotassium hydrogen phosphate into filtrate, dissolving the white sugar and the dipotassium hydrogen phosphate into the filtrate, filtering, putting the filtrate into a 100mL conical bottle, plugging the conical bottle with a cotton plug, adding damp-proof paper, tying the opening to prevent the soybeans from falling off, wherein the amount of liquid filled in each bottle is not more than 67 mL. Sterilizing the conical flask in a sterilizing pot at 121 deg.C and 0.1MPa for 20min, taking out, and coolingAnd (5) standby. Inoculating the cultured stock seed liquid leaven into the culture medium for producing the liquid leaven according to the inoculation amount of 6 percent, culturing for 26 hours at 37 ℃ in an incubator, and diluting until the content of the bacillus natto is 1 multiplied by 107CUF/mL to become bacillus natto starter;
(8) inoculating and fermenting: inoculating the bacillus natto starter into the bean curd residue steamed and cooled to 40 ℃ for later use in the step (4) according to the inoculation amount of 4% vol/m, fully and uniformly stirring, spreading to 4cm in thickness, and putting into an incubator for fermentation culture at 37 ℃ for 40 hours;
(9) vacuum freeze drying: after the fermentation culture is finished, transferring the materials after the fermentation culture into a mold, pre-freezing the materials in a refrigerator at the temperature of-45 ℃ for 1.5h, and then putting the materials in a vacuum freeze dryer (the vacuum degree is 50Pa, the temperature of a cold trap is-60 ℃, the temperature of a partition plate is 20 ℃, and the running time is 25h) for vacuum freeze drying;
(10) packaging: directly crushing the material after vacuum freeze-drying to prepare powder, bagging, sealing and packaging;
(11) and (4) finished product preservation: the packaged product can be preserved for 24 months under the condition of cool and dry room temperature, and can also be preserved for more than 24 months in a refrigerator at 4 ℃.
Example 4
The water content of the soybean curd refuse in the step (2) was controlled to 73% as compared with that in the example 1, and the other conditions were the same as those in the example 1.
Example 5
The water content of the soybean curd refuse in the step (2) was controlled to 93% as compared with that in the example 1, and the other conditions were the same as those in the example 1.
Example 6
The cooking time in step (3) was set to 20min as compared with example 1, and the other conditions were the same as in example 1.
Example 7
The cooking time in step (3) was set to 40min as compared with example 1, and the other conditions were the same as in example 1.
Example 8
The procedure of the step (8) was followed by inoculating the Bacillus natto starter at an inoculation amount of 2% vol/m as compared with that of example 1, and the other conditions were the same as those of example 1.
Example 9
The procedure of the step (8) was followed by inoculating the Bacillus natto starter at an inoculation amount of 8% vol/m as compared with that of example 1, and the other conditions were the same as those of example 1.
Example 10
The fermentation was carried out in step (8) at a thickness of 1cm as compared with example 1, and the other conditions were the same as in example 1.
Example 11
The fermentation was carried out in the step (8) with a thickness of 5cm as compared with example 1, and the other conditions were the same as in example 1.
Example 12
The fermentation time in step (8) was 24 hours as compared with example 1, and the other conditions were the same as in example 1.
Example 13
The fermentation time in step (8) was 44 hours as compared with example 1, and the other conditions were the same as in example 1.
Comparative example 1
Compared with the example 1, the drying process in the step (9) adopts 50 ℃ drying.
Test example 1
The enzyme activity of nattokinase in the fermented soybean curd refuse of Bacillus natto obtained in examples 1 and 4 to 13 was measured by Folin's phenol method, and the results are shown in Table 1.
TABLE 1
| Numbering | Nattokinase enzyme activity (U/g) |
| Example 1 | 459.8 |
| Example 4 | 135.6 |
| Example 5 | 367.3 |
| Example 6 | 407.6 |
| Example 7 | 398.2 |
| Example 8 | 306.3 |
| Example 9 | 356.9 |
| Example 10 | 380.9 |
| Example 11 | 405.1 |
| Example 12 | 368.3 |
| Example 13 | 410.6 |
As can be seen from Table 1, the enzyme activity of nattokinase of example 4 is lower than that of example 1 because the moisture content of the soybean curd residue is too small, the soybean curd residue becomes dry due to the evaporation of moisture at the late stage of fermentation, the growth of Bacillus natto is inhibited, and the enzyme activity is not high; the soybean curd residue obtained in the example 5 has too high water content, so that the soybean curd residue has poor air permeability, external oxygen hardly enters the inside of the soybean curd residue, the growth of the bacillus natto is slow, and meanwhile, a liquid film is formed on the surface of the soybean curd residue, so that the growth of the bacillus natto in the soybean curd residue is influenced, and even the bacillus natto is infected with mixed bacteria; the enzyme activity of the nattokinase in the embodiment 4 is the lowest in all the embodiments, which shows that the influence of the water content on the nattokinase produced by the bacillus natto is the largest;
the enzyme activity of the natto kinase of example 6 is lower than that of example 1 because the steaming time is insufficient, the soybean curd residue is not matured enough to affect the fermentation of the natto bacillus, while the steaming of example 7 for a long time can cause the loss of the nutrient components of the soybean curd residue, and the gaps of the soybean curd residue become tight, so that the transmission rate of oxygen among the soybean curd residue particles is reduced, which is not beneficial to the enzyme production of the natto bacillus;
the nattokinase activity of the examples 8-9 is lower than that of the example 1 because when the inoculation amount is too low, because the nattokinase generated by the bacillus natto is too little, and when the inoculation amount is too large, the water content of the soybean curd residue is increased, the air permeability of the soybean curd residue is influenced, the normal metabolic activity of the bacillus natto is not facilitated, and the nattokinase generated by the bacillus natto is influenced;
the enzyme activities of the nattokinase of the embodiments 10 to 11 are all lower than that of the embodiment 1, because the natto bacteria draw oxygen from air for respiration in solid state fermentation, the thickness of the soybean curd residue can directly influence the transmission of the oxygen in the soybean curd residue, the thickness of the soybean curd residue is too small, the amount of nutrient substrates available for the natto bacteria in a certain time in the growth process is reduced, the activity of the produced nattokinase is lower, and when the thickness of the soybean curd residue is too large, the exchange between the inside of the soybean curd residue and the outside air is not facilitated, and the enzyme production of the natto bacteria is influenced;
the nattokinase activity of examples 12-13 is lower than that of example 1 because the fermentation time is too short, the bacillus natto does not grow sufficiently, the activity of the produced nattokinase is not maximum, when the fermentation time is too long, the growth of the bacillus natto shifts from a peak to a stable period until the bacillus natto dies, the bacterial bodies are autolyzed, and the enzyme activity of the nattokinase is gradually reduced.
Test example 2
The number of viable bacteria in the fermented soybean curd refuse of Bacillus natto obtained in example 1 was measured by dilution coating method, and the measurement result was 8.46X 108CUF/g。
Test example 3
The enzyme activity of nattokinase in the fermented soybean curd residue of Bacillus natto prepared in example 1 was measured by the fiber plate method, and 3g of the fermented soybean curd residue of Bacillus natto prepared in example 1 was added to 90mL of sterile water, stirred with a magnetic stirrer for 5min, and then allowed to stand for 12 h. Lightly placing four Oxford cups on a flat plate respectively, adding 10 mu L of the uniformly mixed natto fermentation liquor into the small holes by using a liquid transfer gun for two Oxford cups, and marking; and adding 20 mu L of the uniformly mixed natto fermentation liquor into the small holes of the other two oxford cups by using a liquid transfer gun, and marking. The fibrin plate was then incubated in a 37 ℃ incubator for 18h, and after removal, the diameter of the transparent circle in the fibrin plate was measured with a vernier caliper and the area of the transparent circle was calculated, and the results are shown in FIG. 1. The size of the transparent circle was measured with a vernier caliper, and it was found that the transparent circles to which 10. mu.L of the fermentation broth was added to the Oxford cup were 20.6 mm. times.20.6 mm, 21.1 mm. times.21.1 mm, respectively, and the transparent circles to which 20. mu.L of the fermentation broth was added to the Oxford cup were 23.8 mm. times.23.7 mm, respectively; 23.9mm, the transparent ring is not only present, but also is very large, which indicates that the bean curd residue fermented by the optimal fermentation process contains a lot of nattokinase, so that a fibrin plate which is large enough can be dissolved in a specified time.
Test example 4
The fermented soybean curd refuse of Bacillus natto obtained in example 1 was added with 0.01% by mass of sodium hydroxide solution at a ratio of 1:30(g: mL), stirred with a magnetic stirrer for 5min to completely disperse the freeze-dried product, and then subjected to water bath at 50 ℃ for 30 min. Then the mixture is centrifuged in a centrifuge at the rotating speed of 5000r/min for 30 min. Discarding supernatant, washing precipitate with distilled water to neutrality, and oven drying at 60 deg.C to constant weight to obtain dietary fiber. The content of dietary fiber in the fermented soybean curd refuse of Bacillus natto prepared in example 1 was 7450 mg/g.
The characteristics of the dietary fiber in the fermented soybean curd refuse of Bacillus natto prepared in example 1 were measured:
(1) water binding capacity
Accurately weighing 0.5000g of dietary fiber powder in a 100mL beaker by using an analytical balance, slowly adding 35mL of distilled water, stirring for 2 hours by using a magnetic stirrer, centrifuging for 30 minutes at 6000r/min, removing the weight of supernatant fluid, and weighing the water holding capacity by using the following calculation formula:
water binding capacity (WHC) (g/g) ═ wet mass-dry mass)/sample mass;
the water retention of the dietary fiber was found to be 8.6188 g/g.
(2) Expansibility of expansion
Accurately weighing 0.5000g dietary fiber powder in an analytical balance, reading dry dietary fiber volume (V)0) Adding 10mL distilled water, shaking, standing at room temperature for 24 hr, and reading volume (V) of dietary fiber after swelling1) The expansion calculation formula is as follows:
expansibility (SC) (mL/g) ═ V1－V0) (ii)/sample mass;
the dietary fiber expansibility was measured to be 8.48 mL/g.
(3) Combined with hydraulic power
Accurately weighing 0.5000g of dry dietary fiber powder as m by using an analytical balance0Placing in a 50mL centrifuge tube, adding 50mL distilled water, centrifuging at 6000r/min for 1h, removing supernatant, standing for 1h, weighing the sample and the centrifuge tube, and recording as m1. Then the precipitate is dried in an oven, and the mass of the sample and the centrifuge tube is weighed and recorded as m2。m1And m2The difference value is the mass of the dietary fiber combined water, and the combined hydraulic calculation formula is as follows:
combined water power (CWC) (g/g) ═ m1－m2)/m0；
The dietary fiber bound hydraulic power was found to be 8.4244 g/g.
(4) Oil retention property
Accurately weighing 0.5g of dietary fiber powder in a 100mL beaker by using an analytical balance, adding 35mL of rapeseed oil, stirring for 2h by using a magnetic stirrer, centrifuging for 30min at 6000r/min, removing the weight of supernatant fluid, and calculating the oil retention property according to the following formula:
oil retention (OHC) (g/g) ═ wet mass-dry mass)/sample mass;
the dietary fiber was measured to maintain an oiliness of 2.2212 g/g.
Test example 5
The contents of total flavonoids (rutin was used as a control) and total saponins (oleanolic acid was used as a control) in the fermented soybean curd residue of Bacillus natto obtained in example 1 and comparative example 1 were measured according to a standard curve method, and the results are shown in Table 2.
TABLE 2
| Numbering | Total Flavonoids content (mg/g) | Total saponins content (mg/g) |
| Example 1 | 9.77 | 10.63 |
| Comparative example 1 | 8.05 | 10.13 |
Test example 6
The DPPH.radical scavenging ability of the fermented soybean curd refuse of Bacillus natto obtained in example 1 and comparative example 1 was measured, and the results are shown in Table 3.
TABLE 3 DPPH & radical scavenging Capacity
Test example 7
Treating with different water contents:
100g of untreated wet soybean curd residue with a water content of 83% was weighed, and spread on a plate to be dried, and weighed once at intervals so that the water content of the soybean curd residue was controlled to be 53%, 63%, and 73%, and distilled water was added to the wet soybean curd residue so that the water content of the soybean curd residue was 93%. Adding bean curd residue with water content of 53%, 63%, 73%, 83%, 93% into a hexagonal bottle, sterilizing at 121 deg.C under 0.1MPa for 20min, cooling to room temperature, inoculating 4% bacterial liquid, culturing in an incubator at 37 deg.C for 36h, and aging in a refrigerator at 4 deg.C for 14 h. The fermented soybean curd residues with water content of 53%, 63%, 73%, 83%, 93% are A1, A2, A3, A4, and A5. A1, A2, A3, A4 and A5 were used to see whether or not the increase or decrease of water had an effect on the fermentation of the strain. After the fermentation is finished, the bacterial count is measured by using a dilution coating method as a flat plate and the enzyme activity of the nattokinase is measured by using a forskolin phenol method.
After the bean curd residue bacillus natto is fermented, observing and measuring the enzyme activity of the nattokinase, finding that the bean curd residue with the water content of 53 percent is basically not long after the bean curd residue is added with the nattokinase, and measuring that the enzyme activity of the nattokinase is 21.28U/g, which is particularly small, because the water content is too low and the bean curd residue is too dry, the nattokinase cannot normally grow and propagate; adding Bacillus natto into bean curd residue with water content of 63%, observing with naked eyes after fermentation, slightly picking up bean curd residue with glass rod, and finding no wiredrawing; adding bacillus natto into the soybean curd residue with the water content of 73%, observing partial bacterial colonies on the surface of the soybean curd residue by naked eyes after fermentation is finished, slightly picking up the soybean curd residue by using a glass rod, and finding that no wiredrawing exists; adding Bacillus natto into bean curd residue with water content of 83%, after fermentation, smelling bean curd residue, and observing thallus Porphyrae on the surface of bean curd residue with naked eye, slightly picking up bean curd residue with glass rod, and measuring the enzyme activity of natto kinase to be 247.76U/g; the soybean curb residue with the water content of 93 percent is added with the bacillus natto, after the fermentation is finished, the soybean curb residue is very tight, the water content can be obviously sensed to be very high, white bacterial colonies exist on the surface of the soybean curb residue, the enzyme activity of the nattokinase is measured to be 228.38U/g, and the enzyme activity is found to be small.
Test example 8
Treatment with protein addition:
accurately weighing 200g of wet soybean curd residue, drying until the water content is 53%, 63% and 73%, and adding appropriate amount of soybean milk to make the water content of soybean curd residue 83% again. Meanwhile, soybean milk is added on the basis of untreated soybean curd residue with the water content of 83 percent, so that the water content of the soybean curd residue is 93 percent. Adding bean curd residue with water content of 53%, 63%, 73%, 83%, 93% into a hexagonal bottle, sterilizing at 121 deg.C under 0.1MPa for 20min, inoculating 4% bacterial solution, culturing in an incubator at 37 deg.C for 36h, and aging in a refrigerator at 4 deg.C for 14 h. The fermented soybean curd residues with water content of 53%, 63%, 73%, 83% and 93% are B1, B2, B3, B4 and B5. The reason for the treatment of increasing protein is that the protein content of the soybean curd residue is much lower than that of soybean, and the natto bacteria can be well fermented by supplementing protein.
After the fermentation of the soybean curd residue and the Bacillus natto, the soybean curd residue containing 83% of water and the soybean curd residue containing 93% of water were found to be well fermented, while the soybean curd residue containing soybean milk after drying was found to be slightly inferior in fermentation effect, and the soybean curd residue was gently picked up with a glass rod, and the soybean curd residue B4 and B5 were found to have a 4-5 cm-long stringy, and the enzyme activity of nattokinase was measured, and it was found that B1 was 76.38U/g, B2 was 100.70U/g, B3 was 121.98U/g, B4 was 249.28U/g, and B5 was 233.70U/g. B1, B2, B3 and B4 are compared, the water content of the soybean milk is 83 percent, the difference is that B1, B2 and B3 are dried firstly and then added with the soybean milk, the addition amount of the soybean milk is B1 > B2 > B3, and the comparison of B1, B2, B3 and B4 can deduce that: the soybean milk added in the fermentation process of the bean curd residue bacillus natto is not beneficial to the fermentation of the bean curd residue bacillus natto; the structure of the bean curd refuse is destroyed during the drying process of the bean curd refuse, and although the soybean milk is supplemented later, the destruction caused during the drying process is irreversible. B4 and B5 are different in that the B5 is added with soybean milk, and other treatments are the same, so that the enzyme activity of B5 is lower than that of B4, and the reason is that the water content is too high, so that the air permeability inside the soybean curd residue is poor, and meanwhile, heat cannot be rapidly discharged, so that the propagation of strains is influenced. In conclusion, the drying treatment of the soybean curb residue is not favorable for the fermentation of the soybean curb residue bacillus natto.
Test example 9
Treatment for increasing sugar and protein:
5g of the soybean curd refuse dried in an oven at 60 ℃ to a constant weight was weighed, and 13.8mL of soybean milk was added to ensure that the soybean curd refuse contained sufficient protein, designated as C1. 5g of bean curd residue dried in an oven at 60 ℃ to a constant weight is weighed, 5g of white sugar is added, and then 13.8mL of distilled water is added to ensure that the bean curd residue contains enough saccharides, which is marked as C2. Weighing 5g of bean curd residue dried in an oven at 60 ℃ to constant weight, adding 5g of white sugar, and then adding 13.8mL of soybean milk to ensure that the bean curd residue contains enough protein and saccharide, and marking as C3. 20 soaked soybeans were weighed and recorded as C4. Adding into a hexagonal bottle, sterilizing at 121 deg.C and 0.1MPa for 20min, cooling to room temperature, inoculating 4% bacterial solution, culturing in 37 deg.C incubator for 36h, and aging in 4 deg.C refrigerator for 12 h. C1 has the same moisture content as C2, with the difference that C1 adds protein, C2 adds white sugar; both C2 and C3 are added with white sugar, and the difference is that C3 is added with soybean milk; c1 compared to C3, the difference was that C3 was supplemented with white sugar; the purpose of C4 is to verify whether the strain is normal or not, and whether the strain is contaminated by other bacteria or not.
C4 is fermented well, white wrinkles are formed on the soybeans, and the soybeans are lightly picked up by a glass rod to form a 10-20cm long wire; simulating the main components of the soybean through C1, C2 and C3, and finding that the fermentation effects of C1, C2 and C3 are poor, no obvious white film exists, no wiredrawing exists, and the soybean milk or the white sugar possibly has no influence on the fermentation of the soybean curd residue; the drying of the soybean curd residue destroys the structure between the soybean curd residue molecules, and the destruction is irreversible, and the excessive water content can be caused by directly adding soybean milk to the fresh soybean curd residue with the water content of 83%.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Claims (10)
1. A preparation method of bacillus natto fermented soybean curd residues is characterized by comprising the following steps:
inoculating the bacillus natto starter to the steamed soybean curd residue, fermenting, culturing, and freeze-drying to obtain the bacillus natto fermented soybean curd residue.
2. The method for preparing the Bacillus natto fermented soybean curd residue according to claim 1, comprising the steps of:
selecting a bean curd residue raw material with the water content of 73-93%, cooking for 20-40 min, and inoculating a bacillus natto fermenting agent into the cooked bean curd residue; uniformly stirring, flattening, and fermenting and culturing for 24-44 h; freeze drying after fermentation culture to obtain Bacillus natto fermented bean curd residue;
the volume-to-mass ratio of the bacillus natto starter to the steamed bean curd residues is 2-8 mL:100 g;
the thickness of the flattening is 1-5 cm;
the content of Bacillus natto in the Bacillus natto starter is 1 × 105～109CUF/mL。
3. The method for preparing Bacillus natto-fermented soybean curd refuse according to claim 2, wherein the soybean curd refuse raw material has a water content of 83%; the cooking time is 30min, and the volume-to-mass ratio of the bacillus natto starter to the cooked bean curd residue is 6mL:100 g; the thickness of the flattening is 3 cm; the time of fermentation culture is 36 h.
4. The method for preparing the bacillus natto fermented soybean curd residue according to claim 1, wherein the cooking conditions are 0.07 to 0.13MPa, 115 to 125 ℃.
5. The method for preparing Bacillus natto fermented soybean curd residue according to claim 1, wherein the Bacillus natto starter is prepared by the steps of: taking soybeans soaked in water at the temperature of 30-40 ℃ for 10-14 h, mixing the soybeans and the water according to the mass ratio of 1: 4-5, treating the soybeans at the temperature of 115-125 ℃ under the pressure of 0.07-0.13 MPa for 15-25 min, cooling, filtering, adding 4-6% of white sugar and 0.01-0.03% of dipotassium hydrogen phosphate into filtrate, sterilizing to prepare a culture medium, inoculating activated and expanded cultured bacillus natto into the culture medium, and culturing to obtain the bacillus natto starter.
6. The method for preparing Bacillus natto fermented soybean curd residue according to claim 1, wherein the step of freeze-drying comprises: the soybean curd residue after fermentation culture is pre-frozen at a temperature of between 45 ℃ below zero and 35 ℃ below zero for 1.5 to 2.5 hours, and then is dried in vacuum at a temperature of between 60 ℃ below zero and 50 ℃ below zero for 25 to 35 hours with a vacuum degree of 10 to 50 Pa.
7. A Bacillus natto fermented soybean curd residue obtained by the method according to any one of claims 1 to 6.
8. The use of the fermented soybean curd residue with Bacillus natto according to claim 7 in food or health food.
9. Use according to claim 8, wherein the food product is a pastry.
10. The use of claim 8, wherein the nutraceutical is one of a powder, a tablet or a capsule.
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