Natto red yeast rice herb bean flour and preparation method thereof

Abstract

The invention relates to the technical field of microbial fermentation engineering, and particularly discloses natto red yeast rice herb bean flour and a preparation method thereof. The preparation method comprises the following steps: respectively activating specifically preserved bacillus natto and monascus to obtain secondary seed solutions; inoculating the two seed solutions into a solid-state fermentation culture medium for co-fermentation; and inactivating, drying and crushing the fermentation product to obtain a finished product. The solid-state fermentation culture medium is prepared by compounding a bean matrix (soybeans, black beans or chickpeas) and herb tea components (roses, lotus leaves, chrysanthemums, mulberry leaves, haws, cassia seeds and the like). Through the synergistic effect of the bacillus natto and the monascus, nattokinase and monacolin K are efficiently enriched in a single fermentation system at the same time, conversion of soy isoflavone to a high-bioavailability aglycone type is remarkably promoted, and the obtained product has multiple health-care functions of thrombolysis, lipid reduction, efficient absorption and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microbial fermentation engineering, and particularly relates to a preparation method of a natto Monascus flower grass bean powder. BACKGROUND

[0002] Soybean is a recognized healthy food resource, which is rich in high-quality protein and various bioactive substances. Among them, soy isoflavones have attracted much attention due to their potential in preventing cardiovascular diseases, osteoporosis and regulating endocrine. However, about 97% of isoflavones in natural soybean exist in the form of glycosides (such as daidzin and genistin). Such molecules have large polarity and low bioavailability, and can only be effectively absorbed by the human body after being hydrolyzed by intestinal microorganisms into aglycone (such as daidzein and genistein). The conversion process is inefficient and unstable, which seriously restricts the physiological function of soy isoflavones.

[0003] To break this bottleneck, microbial fermentation technology is considered as an effective solution. Among various fermentation technologies, mixed solid-state fermentation shows unique advantages. This technology can secrete more comprehensive enzyme systems by utilizing the complementarity and synergy of two or more microorganisms, making the enzyme reaction network more perfect, so as to simultaneously generate multiple high-value bioactive substances and achieve the multiplication of the nutritional and functional value of fermentation products. When this technology is applied to a complex substrate composed of beans and tea ingredients (such as lotus leaf, hawthorn, and cassia seed), the multiplication potential of its value is more significant.

[0004] Among the functional microorganisms, the combination of Bacillus natto and Monascus spp. is particularly attractive. Bacillus natto is a recognized safe strain, which can secrete a powerful protease system during fermentation and efficiently synthesize nattokinase with direct thrombolytic function. The enzyme has clear application value in preventing and improving cardiovascular thrombotic diseases. Monascus is a traditional medicinal fungus in China, which can ferment to produce monacolin K (lovastatin), a classic hydroxymethylglutaryl coenzyme A reductase competitive inhibitor, which can effectively block the endogenous synthesis pathway of cholesterol, thereby exerting the effect of lowering blood lipids.

[0005] Studies have shown that Bacillus natto and Monascus can promote the glycoside- aglycone conversion of soy isoflavones by using the β-glucosidase produced by themselves when they are fermented alone. However, the existing technology is mostly focused on the fermentation application of a single strain or is limited to physical mixing. Therefore, the present application generates an innovative idea: if the two are co-fermented in cooperation and a natural plant ingredient with specific health efficacy is introduced as part of the fermentation substrate, not only can the soy isoflavones be more thoroughly and efficiently converted into the aglycone type with high bioavailability through the superposition and synergistic effect of the enzyme systems, but also the two cardiovascular and cerebrovascular health functional factors of nattokinase and monacolin K can be simultaneously enriched in a single fermentation system. The final product will integrate soy isoflavone, nattokinase, monacolin K and natural plant active ingredients, forming a multi-target and synergistically effective health effect, thus creating an innovative product with multiple functions of "thrombolytic, lipid-lowering and high absorption".

[0006] The present application carries out systematic co-fermentation culture of Bacillus natto and Monascus and applies it to a specific compound substrate of beans and natural plants, so as to simultaneously achieve efficient conversion of isoflavones, high production of nattokinase and enrichment of monacolin K, and form a functional synergistic integrated technology with active ingredients of herbal tea. At present, it is still blank. Therefore, the present application aims to provide a brand-new process scheme to solve the deficiencies of the prior art and develop a high-value fermentation product with clear health guidance. SUMMARY

[0007] The present application aims to provide a natto-Monascus-herbal bean powder and a preparation method thereof. Bacillus natto and Monascus are activated respectively and then inoculated into a solid-state fermentation medium containing a bean substrate and herbal tea for co-fermentation to obtain the natto-Monascus-herbal bean powder. Through the synergistic effect of Bacillus natto and Monascus, nattokinase and monacolin K are simultaneously and efficiently enriched in a single fermentation system, and the conversion of soy isoflavones to the aglycone type with high bioavailability is significantly promoted. The obtained product has multiple health functions of thrombolytic, lipid-lowering and high absorption.

[0008] To achieve the above-mentioned object, the present application adopts the following technical scheme: The first object of the present application is to provide a preparation method of a natto-Monascus-herbal bean powder, which comprises: inoculating an activation solution of Bacillus natto and an activation solution of Monascus into a solid-state fermentation medium for solid-state fermentation to obtain the natto-Monascus-herbal bean powder, wherein, The solid-state fermentation medium contains a bean substrate and herbal tea, the bean substrate includes soybeans and / or black beans, and the herbal tea includes any one or more of rose, lotus leaf, chrysanthemum, mulberry leaf, hawthorn and cassia seed.

[0009] Optionally, in some embodiments of the present application, the bean substrate is soybean.

[0010] Optionally, in some embodiments of the present application, the legume matrix is black beans.

[0011] Optionally, in some embodiments of the present application, the herbal tea is rose, lotus leaf, chrysanthemum, mulberry leaf, hawthorn, cassia seed.

[0012] Further optionally, in some embodiments of the present application, the herbal tea is rose, lotus leaf, chrysanthemum, mulberry leaf, hawthorn, cassia seed, which are mixed in a mass ratio of 1:1:(0.8~0.4):(0.8~0.4):(0.8~0.4):(0.8~0.4), and further optionally 1:1:0.8:0.6:0.4:0.4.

[0013] Optionally, in some embodiments of the present application, the inoculation amount ratio of the activated liquid of Bacillus natto and the activated liquid of Monascus is 2:1~1:2.

[0014] Further optionally, in some embodiments of the present application, the inoculation amount ratio of the activated liquid of Bacillus natto and the activated liquid of Monascus is 2:(1~2), and more further 2:1.

[0015] Optionally, in some embodiments of the present application, the inoculation amount of the activated liquid of Bacillus natto and the activated liquid of Monascus is 10~15%.

[0016] Optionally, in some embodiments of the present application, the solid-state fermentation medium contains 90~120 g of the crushed mixture of legume matrix and herbal tea powder.

[0017] Optionally, in some embodiments of the present application, the mass ratio of the legume and the herbal tea is 1:(0.1~0.4), preferably 1:(0.1~0.3), further preferably 1:(0.2~0.3), and more preferably 1:0.2.

[0018] Optionally, in some embodiments of the present application, the configuration method of the solid-state fermentation medium comprises: taking 90~120 g of the crushed mixture of legume matrix and herbal tea powder, wherein the ratio of the legume matrix and the herbal tea is 1:(0.1~0.3), 2~4 g of sucrose, 0.2~0.4 g of magnesium sulfate, 1~2 g of dipotassium hydrogen phosphate, 0.2~0.4 g of calcium chloride, and 20~40 g of water are added and mixed uniformly, and then sterilized to obtain the solid-state fermentation medium.

[0019] Optionally, in some embodiments of the present application, the solid-state fermentation condition is that the culture temperature is 30±2℃, the aeration amount is 0.2~0.4 m 3 / min, and after standing culture for 48±2 h, the culture temperature is changed to 36±2℃ for standing culture for 60±2 h to obtain the natto Monascus flower and grass bean fermentation product. In the early stage, the fermentation temperature of 30±2℃ is beneficial to the growth of Monascus; and in the later stage, the culture temperature of 36±2℃ is beneficial to the growth of Bacillus natto. The two-stage culture temperature mode is adopted to ensure the growth of the two strains.

[0020] Optionally, in some embodiments of the present application, the fermentation product after solid-state fermentation is subjected to high-temperature inactivation, drying and crushing treatment to obtain natto Monascus flower and grass bean powder.

[0021] Further optionally, in some embodiments of the present application, the high-temperature inactivation temperature is 75±5℃, the drying temperature is 55±5℃, the drying is performed until the water content is 3-8%, and after cooling to room temperature, the crushing treatment is performed to obtain natto Monascus flower and grass bean powder.

[0022] Optionally, in some embodiments of the present application, a preparation method of natto Monascus flower and grass bean powder includes the following specific process: S1, respectively activating and culturing Bacillus natto and Monascus to obtain Bacillus natto secondary seed liquid and Monascus secondary seed liquid; S2, mixing and crushing the bean matrix and flower and grass tea, and adding other fermentation components to obtain a solid-state fermentation medium; S3, inoculating the activated and cultured Bacillus natto secondary seed liquid and Monascus secondary seed liquid into the solid-state fermentation medium in a ratio of 2:1-1:2 at an inoculation amount of 10-15% for fermentation to obtain a fermentation product; S4, subjecting the fermentation product to high-temperature inactivation, drying and crushing treatment to obtain natto Monascus flower and grass bean powder.

[0023] Optionally, in some embodiments of the present application, the Bacillus natto is Bacillus natto ND-1-A27, the strain name is ND-1-A27, the classification name is Bacillus subtilis natto, the preservation date is March 14, 2018, the preservation unit is China Center for Type Culture Collection, the preservation address is Wuhan University in Wuhan, China, and the preservation number is CCTCC NO: M2018131.

[0024] Optionally, in some embodiments of the present application, the Monascus is purchased from Ningbo Mingzhou Biotechnology Co., Ltd. (Monascus BMZ147769, original number: WM-12.3), and the preservation number is CCIC 41601.

[0025] Optionally, in some embodiments of the present application, in step S1, the activation method of Bacillus natto comprises: taking Bacillus natto from a strain cryopreservation tube, inoculating in an activation medium at a volume ratio of 1:10-1:25, and culturing at 32-37°C for 20-26 h to obtain a first-level seed liquid, inoculating the first-level seed liquid in the activation medium at a volume ratio of 1:10-1:25, and culturing at 32-37°C for 20-26 h to obtain a second-level seed liquid.

[0026] Optionally, in some embodiments of the present application, the configuration method of the activation medium of Bacillus natto comprises: uniformly mixing 8-12 g / L of proteose peptone, 5-10 g / L of beef extract, 1-3 g / L of sodium chloride, 10-15 g / L of glucose, and 3-7 g / L of yeast extract, and sterilizing at 115°C for 15 min to obtain the activation medium of Bacillus natto.

[0027] Optionally, in some embodiments of the present application, the activation method of Monascus comprises: taking Monascus WM-12.3 from a strain cryopreservation tube, inoculating in an activation medium at a volume ratio of 1:10-1:25, and culturing at 28-32°C for 20-26 h to obtain a first-level seed liquid, inoculating the first-level seed liquid in the activation medium at a volume ratio of 1:10-1:25, and culturing at 28-32°C for 20-26 h to obtain a second-level seed liquid.

[0028] Optionally, in some embodiments of the present application, the configuration method of the activation medium of Monascus WM-12.3 comprises: uniformly mixing 8-12 g / L of proteose peptone, 25-40 g / L of glucose, 1-3 g / L of sodium nitrate, 70-90 g / L of glycerol, 0.3-1.5 g / L of potassium phosphate dibasic, 0.3-1.5 g / L of magnesium sulfate, and 10-20 g / L of soybean meal powder, and adjusting the pH value to 4-4.5, and sterilizing at 115°C for 15 min to obtain the activation medium of Monascus.

[0029] Optionally, in some embodiments of the present application, in step S2, the configuration method of the solid-state fermentation medium comprises: taking 90-120 g of a crushed mixture of legume matrix and herbal tea powder, wherein the ratio of the legume matrix to the herbal tea is 1:0.1-1:0.3, adding 2-4 g of sucrose, 0.2-0.4 g of magnesium sulfate, 1-2 g of potassium phosphate dibasic, and 0.2-0.4 g of calcium chloride, and adding 20-40 g of water, and uniformly mixing to obtain the solid-state fermentation medium.

[0030] Optionally, in some embodiments of the present application, the legume matrix comprises soybeans and / or black beans.

[0031] Optionally, in some embodiments of the present application, the herbal tea includes any one or more of rose, lotus leaf, chrysanthemum, mulberry leaf, hawthorn, cassia seed.

[0032] Optionally, in some embodiments of the present application, in step S2, the conditions for solid-state fermentation are as follows: the culture temperature is 30±2℃, the ventilation amount is 0.2 m 3 / min, and after static culture for 48±2 h, the culture temperature is changed to 36±2℃ for static culture for 60±2 h to obtain the fermented product of the natto red koji herbal soybean.

[0033] Optionally, in some embodiments of the present application, in step S4, the temperature for high-temperature inactivation is 75±5℃, the drying temperature is 55±5℃, the drying is performed until the water content is 3-8%, and after cooling to room temperature, the product is crushed to obtain the natto red koji herbal soybean powder.

[0034] A second object of the present application is to provide a natto red koji herbal soybean powder prepared by the above method.

[0035] Optionally, in some embodiments of the present application, in the natto red koji herbal soybean powder, the natto kinase activity is not less than 760 U / g, the monacolin K content is not less than 0.40 mg / g, and the aglycone type soybean isoflavone content is not less than 0.80 mg / g.

[0036] Further optionally, in some embodiments of the present application, in the natto red koji herbal soybean powder, the natto kinase activity is 760-830 U / g, the monacolin K content is 0.40-0.51 mg / g, and the aglycone type soybean isoflavone content is 0.80-1.50 mg / g.

[0037] A third object of the present application is to provide a use of the natto red koji herbal soybean powder in the preparation of a drug for dissolving thrombus or reducing blood lipid.

[0038] The present application has the following advantages and positive effects: (1) The present application uses a specific ratio (2:1-1:2) of Bacillus natto and Monascus to be compounded and two-stage temperature control fermentation, which synergistically and efficiently enriches natto kinase, monacolin K (lovastatin) and other functional factors in one system, and significantly promotes the conversion of soybean isoflavone to high-bioavailability aglycone type.

[0039] (2) The present application first scientifically compounds a legume matrix and various herbal tea components (such as lotus leaf, hawthorn, cassia seed, etc.) into a solid-state fermentation medium, which not only provides rich nutrition for the growth of the bacterial cells, but also makes the microbial metabolites and the natural active ingredients of plants mutually beneficial, and together endow the product with the synergistic effects of reducing fat, dissolving thrombus and reducing lipid, etc. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 is a flowchart of the present application; Figure 2 is a graph showing the effect of fermentation time on the activity of nattokinase, the content of monacolin K and the content of aglycone type soybean isoflavone; Figure 3 is a graph showing the effect of fermentation temperature on the activity of nattokinase, the content of monacolin K and the content of aglycone type soybean isoflavone; Figure 4 is a graph showing the effect of the total mass of the mixture of the legume matrix and the crushed herbal tea on the activity of nattokinase, the content of monacolin K and the content of aglycone type soybean isoflavone; Figure 5 is a graph showing the effect of the mass ratio of the legume and the herbal tea on the activity of nattokinase, the content of monacolin K and the content of aglycone type soybean isoflavone; Figure 6 is a graph showing the effect of the inoculation ratio of the strain on the activity of nattokinase, the content of monacolin K and the content of aglycone type soybean isoflavone; Figure 7 is a graph showing the effect of different legume matrices on the activity of nattokinase, the content of monacolin K and the content of aglycone type soybean isoflavone. DETAILED DESCRIPTION

[0041] In order to more clearly illustrate the present application, the present application will be further described below in conjunction with examples. Those skilled in the art should understand that the specific description below is illustrative rather than limiting, and should not limit the protection scope of the present application.

[0042] The microbial combination used in the present application includes Bacillus subtilis ND-1-A27, which is obtained by independent screening.

[0043] The natto Bacillus ND-1-A27 has the strain name of ND-1-A27, the classification name of Bacillus subtilis natto, the preservation date of March 14, 2018, the preservation unit of China Center for Type Culture Collection, the preservation address of Wuhan University in Wuhan, China, and the preservation number of CCTCC NO: M2018131. Morphological observation, physiological and biochemical identification, and 16s rDNA strain identification analysis are performed on the strain. The colony is grayish white, nearly round, dry on the surface, opaque, wrinkled, not shiny, with a rough edge in the form of a split leaf, and the central part is darker than the edge part.

[0044] Monascus purpureus, preserved in China Center of Industrial Culture Collection (CCIC), and purchased from Ningbo Mingzhou Biotechnology Co., Ltd. (Monascus BMZ147769, original number: WM-12.3), with the preservation number of CCIC 41601.

[0045] The legume substrate in the solid-state fermentation medium is soybean and / or black soybean, and the pretreatment of the legume substrate is to soak whole soybeans for 6-8 hours and then drain the water.

[0046] The flower and tea ingredient in the solid-state fermentation medium is any one or more of rose, lotus leaf, chrysanthemum, mulberry leaf, hawthorn, and cassia seed, and the pretreatment of the flower and tea ingredient includes removing yellow and withered leaves, washing with tap water, draining the water, cutting into segments, and mixing uniformly.

[0047] Example 1 A kind of natto red mold flower and tea bean powder and a preparation method thereof, the present embodiment is to ferment soybeans and natural plant substrates by mixed fermentation of Bacillus natto and Monascus, including the following steps: S1, Bacillus natto ND-1-A27 strain activation method: Bacillus natto ND-1-A27 is taken from the strain cryopreservation tube, inoculated in the activation medium at a volume ratio of 1:20, incubated at 37°C for 24h, and then the first-stage seed liquid is obtained. The first-stage seed liquid is inoculated in the activation medium at a volume ratio of 1:20, incubated at 37°C for 24h, and then the second-stage seed liquid is obtained. The activation medium for Bacillus natto ND-1-A27 is prepared by mixing 10 g / L of proteose peptone, 5 g / L of beef extract, 3 g / L of sodium chloride, 15 g / L of glucose, and 5 g / L of yeast extract, and then sterilizing at 115°C for 15 min.

[0048] S2, Monascus WM-12.3 strain activation method: Monascus WM-12.3 is taken from the strain cryopreservation tube, inoculated in the activation medium at a volume ratio of 1:20, incubated at 30°C for 24h, and then the first-stage seed liquid is obtained. The first-stage seed liquid is inoculated in the activation medium at a volume ratio of 1:20, incubated at 30°C for 24h, and then the second-stage seed liquid is obtained. The configuration method of the activated culture medium of Monascus WM-12.3 is as follows: 10 g / L of proteose peptone, 30 g / L of glucose, 2 g / L of sodium nitrate, 70 g / L of glycerol, 1 g / L of potassium phosphate dibasic, 1 g / L of magnesium sulfate, and 15 g / L of soybean meal powder are uniformly mixed, and the pH value is adjusted to 4.5, and then sterilized at 115 ℃ for 15 min to obtain the activated culture medium of Monascus.

[0049] S3, the activated culture medium of Bacillus natto ND-1-A27 and Monascus WM-12.3 is inoculated into the solid-state fermentation medium at a ratio of 1:2 and a inoculation amount of 10%, and then fermented; the solid-state fermentation conditions are as follows: the culture temperature is 30 ℃, the ventilation amount is 0.2 m 3 / min, and after static culture for 48 h, the culture temperature is changed to 36 ℃ for static culture for 60 h to obtain the Bacillus natto Monascus flower and grass yellow bean fermentation product; S4, the solid-state culture medium is obtained by mixing and crushing the legume matrix and the flower and grass tea, and adding other fermentation components; The configuration method of the solid-state fermentation medium is as follows: 110 g of the crushed mixture of yellow beans and flower and grass tea is obtained, wherein the ratio of yellow beans to flower and grass tea is 1:0.2, the flower and grass tea is selected from rose, lotus leaf, chrysanthemum, mulberry leaf, hawthorn, and cassia seed, and the ratio of each component is 1:1:0.8:0.6:0.4:0.4; 2 g of sucrose, 0.2 g of magnesium sulfate, 1 g of potassium phosphate dibasic, and 0.2 g of calcium chloride are added, and then 20 g of water is added to obtain the solid-state fermentation medium which is sterilized at 121 ℃ for 20 min.

[0050] S5, the fermentation product is inactivated at a high temperature of 75 ℃, dried at a temperature of 55 ℃ until the water content is 5%, cooled to room temperature, and then crushed to obtain the Bacillus natto Monascus flower and grass yellow bean powder.

[0051] Example 2 In this example, the preparation process of the Bacillus natto Monascus flower and grass bean powder is optimized through single-factor experiments and orthogonal experiments.

[0052] (1) Single-factor experiment In this example, the fermentation time and temperature after temperature change, the total mass of the crushed mixture of yellow beans and flower and grass tea, the mass ratio of legume matrix to flower and grass tea, and the inoculation ratio of Bacillus natto to Monascus in step S3 of Example 1 are sequentially changed, and the effects of single factors on the activity of Bacillus natto kinase and the content of monacolin K are measured, and the specific grouping is as follows: The fermentation time after temperature change is 24, 36, 48, 60, 72, and 84 h, respectively; The fermentation temperature after temperature change is 27, 30, 33, 36, 39, and 42 ℃, respectively; The total mass of the mixture of soybean and herbal tea powder was 80, 90, 100, 110, 120, and 130 g, respectively; The mass ratio of legume to herbal tea was 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, and 1:0.6, respectively. The legume substrate was soybean. The herbal tea was a mixture of rose, lotus leaf, chrysanthemum, mulberry leaf, hawthorn, and cassia seed in a mass ratio of 1:1:0.8:0.6:0.4:0.4.

[0053] The inoculation ratio of Bacillus natto to Monascus was 3:1, 2:1, 1:1, 1:2, and 1:3, respectively. The determination results are shown in Table 2. Figures 2-6

[0054] Then, according to the single-factor experiment results, the fermentation time, fermentation temperature, total mass of the mixture of legume substrate and herbal tea powder, mass ratio of legume substrate to herbal tea, and inoculation ratio of Bacillus natto to Monascus were selected as the five factors with more significant influence for orthogonal experiment. The experimental design is shown in Table 1. Table 1: Arrangement of factors and levels (2) Orthogonal experiment The orthogonal experiment results are shown in Table 2. Table 2: Orthogonal experiment design and range analysis results Taking the activity of natto kinase as the index, through range R analysis, the primary and secondary order of the influence of each factor on the activity of natto kinase was: A (time) > B (temperature) > D (mass ratio of legume to herbal tea) > E (inoculation ratio of strains) > C (total mass). By comparing the average values of each factor level, the theoretical optimal process parameters were obtained as follows: A3B3D2E4C3, i.e., fermentation time 60 h, fermentation temperature 36 ℃, mass ratio of legume to herbal tea 1:0.2, inoculation ratio of strains 2:1, and total mass 110 g.

[0055] Taking the content of monacolin K as the index, through range R analysis, the primary and secondary order of the influence of each factor on the content of monacolin K was: D (mass ratio of legume to herbal tea) > A (time) > B (temperature) > E (inoculation ratio of strains) > C (total mass). The optimal process parameters were obtained as follows: D4A3B1E3C2, i.e., mass ratio of legume to herbal tea 1:0.4, fermentation time 60 h, fermentation temperature 30 ℃, inoculation ratio of strains 1:1, and total mass 100 g.

[0056] ​With the aglycone type soy isoflavone content as an index, through range R analysis, the primary and secondary order of the influence of each factor on the aglycone type soy isoflavone content is: D (bean and herbal tea mass ratio) > A (time) > B (temperature) > E (strain inoculation ratio) > C (total mass). Through comparison of the average of each factor, the theoretical optimal process parameters are obtained as: D4A3B3E4C4, i.e. fermentation time 60 h, fermentation temperature 36 ℃, bean and herbal tea mass ratio 1:0.4, strain inoculation ratio 2:1, and total mass 120 g.

[0057] With the nattokinase activity, monacolin K content and aglycone type soy isoflavone content in the fermentation product as indexes, the optimal process parameters obtained through orthogonal test are different. Considering the product function positioning (mainly thrombolytic function) and the main index nattokinase activity, the theoretical optimal combination A3B3D2E4C3 is determined as the final optimal process.

[0058] Since the selected optimal combination (A3B3D2E4C3) is not included in the 16 experiments that have been performed, a verification experiment is needed. Through three parallel verifications, the nattokinase activity under the condition is measured as 935.20 ± 12.45 U / g, the monacolin K content is 0.503 ± 0.015 mg / g, and the aglycone type soy isoflavone content is 1.28 ± 0.05 mg / g. The comprehensive effect is better than that of any experimental group in the orthogonal table. Therefore, A3B3D2E4C3 is finally determined as the optimal process condition, i.e. fermentation time 60 h, fermentation temperature 36 ℃, bean and herbal tea mass ratio 1:0.2, natto Bacillus and Monascus inoculation ratio 2:1, and total mass 110 g.

[0059] Example 3 This example is used to illustrate the influence of using different bean-based materials in the solid-state fermentation medium on the functional component content of the fermentation product and the aglycone type soy isoflavone content.

[0060] The difference between this example and the optimal combination of Example 2 is only that the bean-based material in the solid-state fermentation medium is chickpea. The remaining preparation steps and parameters are exactly the same as those of the optimal combination of Example 2.

[0061] A natto Monascus herbal bean powder and a preparation method thereof, this example is a mixed solid-state fermentation of natto Bacillus and Monascus on chickpea and natural plant substrate, including the following steps: S1, method for activating the natto Bacillus ND-1-A27 strain: The Bacillus natto ND-1-A27 is taken out from a strain cryopreservation tube, inoculated in an activated culture medium at a volume ratio of 1:20, and cultured at 37 DEG C for 24 hours to obtain a first-stage seed liquid; the first-stage seed liquid is inoculated in an activated culture medium at a volume ratio of 1:20, and cultured at 37 DEG C for 24 hours to obtain a second-stage seed liquid. The activated culture medium of the Bacillus natto ND-1-A27 is prepared by mixing 10 g / L of proteose peptone, 5 g / L of beef extract, 3 g / L of sodium chloride, 15 g / L of glucose and 5 g / L of yeast extract uniformly, sterilizing at 115 DEG C for 15 minutes to obtain the activated culture medium of the Bacillus natto.

[0062] S2, the activation method of the Monascus WM-12.3 strain is as follows: The Monascus WM-12.3 is taken out from a strain cryopreservation tube, inoculated in an activated culture medium at a volume ratio of 1:20, and cultured at 30 DEG C for 24 hours to obtain a first-stage seed liquid; the first-stage seed liquid is inoculated in an activated culture medium at a volume ratio of 1:20, and cultured at 30 DEG C for 24 hours to obtain a second-stage seed liquid. The activated culture medium of the Monascus WM-12.3 is prepared by mixing 10 g / L of proteose peptone, 30 g / L of glucose, 2 g / L of sodium nitrate, 70 g / L of glycerol, 1 g / L of potassium phosphate dibasic, 1 g / L of magnesium sulfate, 15 g / L of soybean meal powder, adjusting the pH value to 4.5, and sterilizing at 115 DEG C for 15 minutes to obtain the activated culture medium of the Monascus.

[0063] S3, the activated culture medium of the Bacillus natto ND-1-A27 second-stage seed liquid and the Monascus WM-12.3 second-stage seed liquid are inoculated in a solid-state fermentation medium at a ratio of 2:1 and a 10% inoculation amount for fermentation; the solid-state fermentation conditions are as follows: the culture temperature is 30 DEG C, the aeration amount is 0.2 m 3 / min, and after being cultured at rest for 48 hours, the culture temperature is changed to 36 DEG C for culturing at rest for 60 hours to obtain the Bacillus natto and Monascus fermented product of the hawk's beak beans and herbal tea; S4, the solid-state culture medium is prepared by mixing and crushing the legume matrix and the herbal tea, and adding other fermentation components to obtain the solid-state fermentation medium. The solid-state fermentation medium is prepared by mixing and crushing 110 g of the hawk's beak beans and the herbal tea, wherein the ratio of the yellow beans to the herbal tea is 1:0.2, the herbal tea is selected from rose, lotus leaf, chrysanthemum, mulberry leaf, hawthorn and cassia seed, the ratio of the components is 1:1:0.8:0.6:0.4:0.4, 2 g of sucrose, 0.2 g of magnesium sulfate, 1 g of potassium phosphate dibasic, 0.2 g of calcium chloride, and 20 g of water are added, and then the mixture is sterilized at 121 DEG C for 20 minutes to obtain the solid-state fermentation medium.

[0064] S5. The fermentation product is subjected to high-temperature inactivation at a temperature of 75°C and a drying temperature of 55°C. After drying to a moisture content of 5%, it is cooled to room temperature and then pulverized to obtain natto red yeast rice flower chickpea powder.

[0065] The detection and evaluation of the product's active ingredients should refer to the methods in the experimental examples. Specific results are as follows: Figure 7 As shown.

[0066] This embodiment uses chickpeas as the legume substrate for solid-state fermentation, and co-fermentation is carried out under optimal process conditions. The results show that, compared with the soybean substrate (Example 2), the nattokinase activity (820.40 U / g) and monacolin K content (0.435 mg / g) in the chickpea substrate fermentation product are significantly lower. Simultaneously, its content of aglycone-type soy isoflavones (0.95 mg / g) is also at a low level. These results clearly indicate that the nutritional composition or physical structure of chickpeas may be unfavorable to the growth, metabolism, and synergistic effect of Bacillus natto and Monascus purpureus WM-12.3, resulting in unsatisfactory yields of the three core functional components. Therefore, chickpeas are not an ideal substrate for the process described in this patent.

[0067] Example 4 This example illustrates the effect of using different soybean-based materials in a solid-state fermentation medium on the content of functional components and aglycone-type soybean isoflavones in the fermentation product.

[0068] The only difference between this embodiment and the optimal combination in Example 2 is that the soybean-based material in the solid-state fermentation medium is black soybean. All other preparation steps and parameters are exactly the same as the optimal combination in Example 2.

[0069] A method for preparing natto red yeast rice powder and its preparation method is disclosed. This embodiment involves solid-state fermentation of black soybeans and natural plant substrates using a mixed culture of Bacillus natto and Monascus purpureus, including the following steps: Activation method of S1 Bacillus ND-1-A27 strain: Bacillus natto ND-1-A27 was taken out of the strain cryopreservation tube and inoculated into activation medium at a volume ratio of 1:20. It was then incubated at 37°C for 24 hours to obtain the primary seed culture. The primary seed culture was then inoculated into activation medium at a volume ratio of 1:20 and incubated at 37°C for 24 hours to obtain the secondary seed culture. The method for preparing the activation medium for Bacillus natto ND-1-A27 is as follows: mix 10 g / L peptone, 5 g / L beef extract, 3 g / L sodium chloride, 15 g / L glucose, and 5 g / L yeast extract evenly, and then sterilize at 115℃ for 15 min to obtain the Bacillus natto activation medium.

[0070] Activation method for S2 and Monascus purpureus strain WM-12.3: Remove Monascus purpureus WM-12.3 from the strain cryopreservation tube and inoculate it into activation medium at a volume ratio of 1:20. Incubate at 30°C for 24 hours to obtain the primary seed culture. Inoculate the primary seed culture into activation medium at a volume ratio of 1:20 and incubate at 30°C for 24 hours to obtain the secondary seed culture. The method for preparing the activation medium for Monascus purpureus WM-12.3 is as follows: mix 10 g / L peptone, 30 g / L glucose, 2 g / L sodium nitrate, 70 g / L glycerol, 1 g / L dipotassium hydrogen phosphate, 1 g / L magnesium sulfate, and 15 g / L soybean meal powder evenly, adjust the pH to 4.5, and sterilize at 115℃ for 15 min to obtain the activation medium for Monascus purpureus.

[0071] S3. The activated Bacillus natto ND-1-A27 secondary seed culture and Monascus purpureus WM-12.3 secondary seed culture were inoculated into solid-state fermentation medium at a ratio of 1:2 and an inoculum volume of 10% for fermentation. The solid-state fermentation conditions were: culture temperature of 30℃ and aeration rate of 0.2 m³ / h. 3 After static incubation for 48 hours, the incubation temperature was changed to 36℃ and static incubation was continued for 60 hours to obtain the fermented product of natto, red yeast rice, and black soybean. S4. Solid culture medium: The legume substrate is mixed and pulverized with herbal tea, and other fermentation ingredients are added to obtain a solid fermentation culture medium. The solid-state fermentation medium was prepared as follows: 110g of a mixture of black beans and herbal tea powder was taken, with the ratio of soybeans to herbal tea being 1:0.1. The herbal tea ingredients were selected from six kinds of herbal tea components: rose, lotus leaf, chrysanthemum, mulberry leaf, hawthorn, and cassia seed, with the ratio of each component being 1:1:0.8:0.6:0.4:0.4. 2g of sucrose, 0.2g of magnesium sulfate, 1g of dipotassium hydrogen phosphate, and 0.2g of calcium chloride were added. 20g of water was added and mixed evenly. The mixture was then sterilized at 121℃ for 20min to obtain the solid-state fermentation medium.

[0072] S5. The fermentation product is subjected to high-temperature inactivation at a temperature of 75°C and a drying temperature of 55°C. After drying to a moisture content of 5%, it is cooled to room temperature and then pulverized to obtain natto red yeast rice flower and black bean powder.

[0073] The detection and evaluation of the product's active ingredients should refer to the methods in the experimental examples. Specific results are as follows: Figure 7 As shown.

[0074] In this embodiment, black soybeans were used as the soybean substrate for solid-state fermentation, and co-fermentation was carried out under optimal process conditions. The results showed that the content of aglycone-type soy isoflavones in the fermentation product of the black soybean substrate (1.52 mg / g) was significantly higher than that of the soybean substrate, proving that black soybeans are extremely beneficial for microbial conversion into highly bioavailable aglycone-type isoflavones. However, its content of nattokinase activity (875.60 U / g) and monacolin K content (0.485 mg / g) were slightly lower than the optimal levels of the soybean substrate. This comparison indicates that black soybeans have unique value in specific application scenarios focused on isoflavone conversion; however, if the goal is to achieve the optimal combined output of nattokinase and monacolin K, soybeans remain the preferred substrate for a more balanced and efficient process.

[0075] Experimental Example: Detection and Evaluation of Active Ingredients in Products This experimental example aims to determine the content and function of key active ingredients in the fermented soybean flour products prepared in the above examples and comparative examples. All tests were conducted under the same conditions to ensure the reliability and comparability of the results. The specific test methods are as follows: The content of soybean isoflavones was determined by high performance liquid chromatography (HPLC). The specific method is as follows: Following GB / T 26625-2011 and other methods with slight modifications, the collected natto and red yeast rice flower powder samples were pulverized and passed through a 60-mesh sieve. 1 g of soybean powder from Examples 1-4 was accurately weighed into 25 ml centrifuge tubes, and 10 mL of 90% methanol was added as the extraction solvent. The mixture was ultrasonically extracted in a water bath at 60℃ for 1 h, followed by shaking extraction 2-3 times. After centrifugation at 10000 r / min for 15 min, 1 mL of the extract was filtered through a 0.45 μm microporous membrane for HPLC determination. The chromatographic column was an RPC18 column (5 μm, 250 mm × 4.6 mm). Mobile phase: A was 0.1% acetic acid solution, and B was 0.1% acetic acid-acetonitrile solution; mobile phase flow rate: 1 mL / min; UV detection wavelength: 260 nm; column temperature: 40℃ constant temperature; injection volume: 20 μL; elution time: 26 min. 20 μL of the standard working solution of soy isoflavones and the sample solutions of Examples 1-4 were respectively subjected to liquid chromatography for determination, and the content of soy isoflavones was calculated. The method for determining nattokinase activity (NK) is as follows: Accurately weigh 1.00 g of the pulverized and mixed solid-state fermentation samples from Examples 1-4, and place them in 50 mL centrifuge tubes. Add 25 mL of phosphate buffer (pH 7.4, 0.05 mol / L), and extract by shaking at 150 r / min for 60 min at 4℃. Centrifuge the extract at 10000 r / min for 15 min at 4℃, collect the supernatant, which is the crude nattokinase enzyme solution, and store it on ice for later use. Use a cannula to evenly punch 6 wells with a diameter of 2 mm on a prepared fibrin plate, and use a microsyringe to spot the sample into each well, injecting 8 mL into each well. Incubate at 37℃ for 18 h, and measure the two vertical diameters of the lysis zone. Substitute the logarithm of the product of the two diameters into the standard curve prepared with urokinase standard to calculate the enzyme activity of the sample. The method for determining Monacolin K is as follows: Accurately weigh 1.00 g of the pulverized and mixed solid-state fermentation samples from Examples 1-4, place them in 50 mL centrifuge tubes, add 10 mL of 75% (v / v) ethanol solution, shake thoroughly to mix, and ultrasonically extract for 1 h. Centrifuge at 8000 r / min for 10 min. Filter the supernatant through a 0.22 μm filter membrane and determine the MK content in the fermentation product using high-performance liquid chromatography (HPLC). Chromatographic conditions: Waters Sun Fire C18 column (5 μm, 4.6 mm × 150 mm); mobile phase: acetonitrile: 0.1% phosphoric acid aqueous solution = 55:45 (v / v); flow rate: 1 mL / min; column temperature: 30℃; injection volume: 20 μL; detection wavelength: 238 nm. Perform HPLC analysis on 20 μL of the Monacolin K standard working solution and the sample solutions from Examples 1-4, respectively, and calculate the Monacolin K content. Conclusion: This invention, through a specific combination of microbial strains, a compound culture medium of legumes and herbal teas, and an optimized co-fermentation process, efficiently and synergistically enriches three core functional components in one system, overcoming the shortcomings of existing technologies and possessing outstanding substantive features and significant progress.

[0076] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing natto red yeast rice flower and bean powder, characterized in that, Both the activated solutions of Bacillus natto and Monascus purpureus were inoculated into a solid-state fermentation medium for solid-state fermentation to obtain natto red yeast rice flour. The solid fermentation culture medium contains a legume substrate and herbal tea. The legume substrate includes soybeans and / or black beans, and the herbal tea includes any one or more of rose, lotus leaf, chrysanthemum, mulberry leaf, hawthorn, and cassia seed.

2. The method for preparing natto red yeast rice flower and bean powder according to claim 1, characterized in that: The inoculation ratio of the Bacillus natto activation solution to the Monascus purpureus activation solution is 2:1 to 1:2, preferably 2:(1 to 2), and more preferably 2:1; The inoculation amount of the Bacillus natto activation solution and the Monascus purpureus activation solution is 10-15%.

3. The method for preparing natto red yeast rice flower and bean powder according to claim 1, characterized in that: The solid fermentation culture medium contains 90-120g of a mixture of legume substrate and powdered herbal tea, preferably 110g. The mass ratio of beans to herbal tea is 1:(0.1~0.4), preferably 1:(0.1~0.3), more preferably 1:(0.2~0.3), and even more preferably 1:0.2; The herbal tea is prepared by mixing rose, lotus leaf, chrysanthemum, mulberry leaf, hawthorn and cassia seed in a mass ratio of 1:1:(0.8~0.4):(0.8~0.4):(0.8~0.4):(0.8~0.4), preferably 1:1:0.8:0.6:0.4:0.

4.

4. The method for preparing natto red yeast rice flower and bean powder according to claim 1, characterized in that: The Bacillus subtilis described is Bacillus subtilis ND-1-A27, strain name: ND-1-A27, classified as Bacillus subtilis natto, deposited on March 14, 2018, deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number: CCTCC NO: M2018131.

5. The method for preparing natto red yeast rice flower and bean powder according to claim 1, characterized in that: The method for preparing the solid-state fermentation medium includes: taking 90-120g of a mixture of pulverized legume substrate and herbal tea, wherein the ratio of legume substrate to herbal tea is 1:0.1-1:0.3, 2-4g of sucrose, 0.2-0.4g of magnesium sulfate, 1-2g of dipotassium hydrogen phosphate, 0.2-0.4g of calcium chloride, adding 20-40g of water, mixing evenly, and then sterilizing to obtain the solid-state fermentation medium.

6. The method for preparing natto red yeast rice flower and bean powder according to claim 1, characterized in that: The solid-state fermentation conditions were as follows: a culture temperature of 30±2℃ and an aeration rate of 0.2~0.4 m³ / s. 3 After incubating at a constant temperature of 36±2℃ for 48±2 h, the fermentation product of natto red yeast rice flowers and grass beans was obtained.

7. The method for preparing natto red yeast rice flower and bean powder according to claim 1, characterized in that: The fermentation products after solid-state fermentation are subjected to high-temperature inactivation, drying, and pulverization to obtain natto red yeast rice flower and bean powder. Preferably, the high-temperature inactivation temperature is 75±5℃, the drying temperature is 55±5℃, the drying is carried out until the moisture content is 3-8%, and after cooling to room temperature, it is pulverized to obtain natto red yeast rice flower bean powder.

8. A natto red yeast rice flower powder, prepared by the method described in any one of claims 1-7.

9. The natto red yeast rice flower and bean powder according to claim 8, characterized in that: The nattokinase activity is not less than 760 U / g, the monacolin K content is not less than 0.40 mg / g, and the content of aglycone-type soy isoflavones is not less than 0.80 mg / g.

10. The use of natto red yeast rice flower powder as described in claim 8 or 9 in the preparation of thrombolytic or lipid-lowering drugs.

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