Preparation method and application of vitamin K2 and tea polyphenol reinforced fermented bean flour

Abstract

The invention relates to the field of functional food preparation by a fermentation method, and discloses a preparation method and application of vitamin K2 and tea polyphenol reinforced fermented bean flour. The method comprises the following steps: activating bacillus natto serving as a starting strain into primary seeds, culturing the primary seeds in a seed culture medium at 37 DEG C for 12 hours according to an inoculum size of 1% to realize secondary seed culture, then culturing the secondary seeds in a fermentation culture medium containing bean flour and herb tea raw materials according to an inoculum size of 2%, culturing the secondary seeds at 40 DEG C for 6 days to obtain fermentation liquor, and filtering the fermentation liquor to obtain the finished product. And finally, adding a carrier into the fermentation liquid, and performing low-temperature freeze drying or spray drying. Different bean flour and herb tea raw materials are added into a fermentation culture medium, synchronous enrichment of vitamin K2 and tea polyphenol is achieved, and the obtained fermented bean flour is suitable for functional food, brewing drinks, nutrient enrichment ingredients and the like. The technological process is simple, parameters are clear, and amplification and industrial application are facilitated.

Description

Technical Field

[0001] This invention relates to the field of functional food preparation by fermentation, specifically to a method and application for preparing fermented soybean powder fortified with vitamin K2 and tea polyphenols. Background Technology

[0002] Fermented soybean flour is a functionalized plant protein powder obtained by inoculating soybeans and their graded raw materials into a substrate with selected functional microorganisms, such as Bacillus natto, under controlled conditions for solid or semi-solid fermentation. The fermentation is then terminated, and the product is dried and pulverized. Compared to unfermented soybean flour, its protein is partially hydrolyzed into soluble peptides and free amino acids, isoflavones are converted from glycosides to aglycones, and anti-nutritional factors such as phytic acid, trypsin inhibitors, and oligosaccharides are reduced. Simultaneously, the beany odor is weakened, a characteristic fermented flavor is formed, and beneficial metabolites such as vitamin K2 can be produced along with the microbial strain. Therefore, it has application value in meal replacements, baking, ready-to-drink solid beverages, special diets, and pet nutrition. However, current fermented soybean flour preparation methods still suffer from problems such as large batch-to-batch variations in microbial stability, difficulty in quantitatively controlling key flavor and functional indicators, insufficient thermal / oxygen stability of effective components, poor bitterness and dispersibility, and high costs associated with controlling the risks of microorganisms and fungal toxins in the final product. These issues restrict large-scale and high-value-added applications.

[0003] Vitamin K2 belongs to the fat-soluble vitamin K family and is structurally a menaquinone (MK-n) compound with a polyisoprene side chain. Vitamin K was first proposed in the 1930s during research on avian clotting factors and was subsequently named "Koagulations-Vitamin (K)". Subsequent research revealed that, in addition to vitamin K1 derived from leafy green plants, fermented foods and specific microbial communities can synthesize a type of vitamin K2 with a longer side chain structure, which was isolated and its structure confirmed in the 1940s. Compared to vitamin K1, which has a shorter half-life and primarily participates in the clotting process, vitamin K2 circulates longer in the body, effectively activating osteocalcin and matrix Gla protein (MGP), promoting calcium deposition in bones and inhibiting vascular calcification, thus having significant implications for bone metabolism and cardiovascular health. In recent years, numerous studies have further revealed the application potential of vitamin K2 in improving osteoporosis, reducing fracture risk, regulating blood lipids, delaying arteriosclerosis, and its potential antioxidant and anti-inflammatory effects. As a result, demand for vitamin K2 has grown rapidly in the fields of nutritional supplements, functional foods, and clinical adjuvant therapy.

[0004] Tea polyphenols are a class of natural phenolic compounds found in the highest concentration and with significant activity in tea. They mainly include catechins such as epigallocatechin gallate (EGCG), epicatechin (EC), epicatechin gallate (ECG), and epigallocatechin (EGC), as well as various components such as flavonoids, flavonols, and phenolic acids. Tea polyphenols were first isolated and identified from green tea in the early 20th century. With the development of separation, purification, and analytical techniques, their structural characteristics, physiological functions, and application value have been systematically studied. Numerous studies have shown that tea polyphenols possess physicochemical properties such as free radical scavenging, lipid oxidation inhibition, and metal ion chelation. They can be used as natural antioxidants, antibacterial agents, or quality stabilizers in the food, health product, and daily chemical industries. Therefore, tea polyphenols can also be added to fermented soybean flour systems to enhance the product's antioxidant stability and antibacterial ability, slow down oil oxidation and color deterioration, and synergistically improve flavor and nutrition under certain conditions. Summary of the Invention

[0005] Purpose of the invention: In order to overcome the shortcomings of the existing technology, the present invention provides a method for preparing and applying fermented soybean powder fortified with vitamin K2 and tea polyphenols. By adding different soybean powders and herbal tea raw materials to the fermentation culture medium and inoculating with Bacillus natto for fermentation culture, the simultaneous enrichment of vitamin K2 and tea polyphenols is achieved. The resulting fermented soybean powder is suitable for functional foods, instant beverages and nutritional fortification ingredients.

[0006] To achieve the above objectives, the present invention provides the following technical solution: The first objective of this invention is to provide a method for preparing fermented soybean powder fortified with vitamin K2 and tea polyphenols. The method comprises: inoculating an activated solution of Bacillus natto into a fermentation medium, fermenting and culturing at 35-40°C and 120-140 r / min for 6 days to obtain fermented soybean powder containing vitamin K2 and tea polyphenols. The fermentation medium contains 20-30 g of soybean powder and 6-30 g of herbal tea per 1 L. The soybean flour includes soybeans, black beans, and chickpeas. The herbal tea includes peppermint, and also includes chamomile and / or roselle.

[0007] Optionally, in some embodiments of the present invention, the inoculation amount of the Bacillus natto activation solution is 2%.

[0008] Optionally, in some embodiments of the present invention, the Bacillus natto is Bacillus natto ND-1-A27, deposited at the China Center for Type Culture Collection, with the number CCTCC NO:M 2018131.

[0009] Optionally, in some embodiments of the present invention, the fermentation medium comprises, per 1 L: 20-30 g soybean flour, 6-30 g herbal tea, 20-30 g glycerol, 15-30 g yeast extract, 0.06-0.1 g K2HPO4, and 0.06-0.1 g MgSO4; with a pH of 6-8.

[0010] Optionally, in some embodiments of the present invention, the soybean flour further includes broad beans.

[0011] Optionally, in some embodiments of the present invention, the herbal tea further includes chrysanthemum.

[0012] Optionally, in some embodiments of the present invention, the fermentation medium contains 15%-30% w / w carrier.

[0013] Further optionally, in some embodiments of the present invention, the carrier includes, but is not limited to, any one or more of the following: maltodextrin, fructooligosaccharides, inulin, galactooligosaccharides, xylooligosaccharides, isomaltooligosaccharides, mannitol, maltitol, resistant dextrin, and sucrose.

[0014] A second objective of this invention is to provide a fermented soybean powder fortified with vitamin K2 and tea polyphenols, prepared according to any of the methods described above.

[0015] Optionally, in some embodiments of the present invention, the fermented soybean powder contains not less than 160 mg / kg of vitamin K2 and not less than 240 mg / kg of tea polyphenols.

[0016] Further optional, in some embodiments of the present invention, the fermented soybean powder contains not less than 170 mg / Kg of vitamin K2 and not less than 250 mg / Kg of tea polyphenols.

[0017] A third objective of this invention is to provide a food or medicine comprising fermented soybean flour fortified with vitamin K2 and tea polyphenols as described above.

[0018] A fourth objective of this invention is to provide the use of the food or medicine described above in the preparation of treatments or preventative treatments of bone metabolic or cardiovascular diseases.

[0019] Compared with existing technologies, this invention provides a method for preparing and applying fermented soybean powder fortified with vitamin K2 and tea polyphenols, which has the following beneficial effects: 1. Dual-function nutritional fortification: By introducing vitamin K2 and tea polyphenol-rich components into the fermentation system, the synergistic enrichment of fat-soluble and polyphenolic active substances in the plant protein carrier is achieved, significantly enhancing the product's antioxidant and bone metabolism regulation potential.

[0020] 2. Microbial metabolic regulation and optimization: Using a single-strain fermentation system of Bacillus natto, the pH and carbon-nitrogen ratio are controlled to balance the K2 production efficiency and polyphenol stability, effectively improving the bioavailability of vitamin K2 and the retention rate of tea polyphenols. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the process of this invention.

[0022] Figure 2 This refers to the vitamin K2 and tea polyphenol content determined by fermenting soybeans with various herbal teas. The various herbal teas include chrysanthemum, peppermint, chamomile, and roselle.

[0023] Figure 3 This is a determination of vitamin K2 and tea polyphenol content based on the fermentation of black beans and various herbal teas. The various herbal teas include chrysanthemum, peppermint, chamomile, and roselle.

[0024] Figure 4 This refers to the vitamin K2 and tea polyphenol content determined by fermenting broad beans with various herbal teas. The various herbal teas include chrysanthemum, peppermint, chamomile, and roselle.

[0025] Figure 5 This is a determination of vitamin K2 and tea polyphenol content based on the fermentation of red beans and various herbal teas. The various herbal teas include chrysanthemum, peppermint, chamomile, and roselle.

[0026] Figure 6 This is a determination of vitamin K2 and tea polyphenol content based on the fermentation of chickpeas and various herbal teas. The various herbal teas include chrysanthemum, peppermint, chamomile, and roselle.

[0027] Figure 7 This refers to the vitamin K2 content determined by fermentation of various beans, either alone or in combination. These beans include soybeans, black beans, broad beans, adzuki beans, and chickpeas.

[0028] Figure 8 This refers to the polyphenol content of various herbal teas, determined by fermentation alone or in combination. These herbal teas include chrysanthemum, peppermint, chamomile, and roselle.

[0029] Figure 9 This is a fermentation assay of the optimal combination of legumes and herbal teas, measuring vitamin K2 and tea polyphenol content. The optimal legumes are soybeans, black beans, and chickpeas; the optimal herbal teas are peppermint and chamomile, and peppermint and roselle. Detailed Implementation

[0030] To more clearly illustrate the present invention, the following description, in conjunction with embodiments, further clarifies the invention. Those skilled in the art should understand that the specific descriptions below are illustrative rather than restrictive, and should not be construed as limiting the scope of protection of the present invention.

[0031] Example 1: Activation of microbial strains and preparation of seed culture Using Bacillus natto ND-1-A27 as the starting strain, which is deposited at the China Center for Type Culture Collection (CCTCC) under the number CCTCC NO:M 2018131, an appropriate amount of the strain was aspirated from a glycerol tube stored at -80℃ and streaked onto LB agar plates. The plates were incubated at 37℃ for 12 h. Suitable single colonies were selected and transferred to flasks containing 10 mL of LB liquid medium. The flasks were then incubated at 37℃ and 220 r / min for 12 h. Finally, the culture was inoculated into LB medium at a volume fraction of 1% and incubated at 37℃ and 220 r / min for 12 h to achieve primary seed culture.

[0032] The LB medium for activating the strain consisted of 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, and 20 g / L agar added to the solid LB medium.

[0033] Primary seeds were inoculated into the seed culture medium at a volume fraction of 1%, and cultured at 37℃ and 220 r / min for 12 h to achieve secondary seed culture.

[0034] The seed culture medium consisted of 18 g / L glucose, 20 g / L yeast extract, 8 g / L corn steep liquor, and 13 g / L soybean meal.

[0035] Example 2 In this embodiment, soybeans were selected as the raw material for soybean flour, and chrysanthemum, mint, chamomile, or roselle were selected as the raw materials for herbal tea, respectively, in order to study the effect of adding soybeans and different herbal tea raw materials to the fermentation medium on the fermentation effect.

[0036] A method for preparing fermented soybean powder fortified with vitamin K2 and tea polyphenols, comprising the following steps: Soybeans undergo the following multiple treatment steps before being added to the culture medium: A1. Cleaning and Impurity Removal: Remove dust, impurities, and broken beans; control moisture content to ≤13% of the original bean moisture content. A2. Soaking and cooking: Material-to-water ratio 1:2 (w / w), 20℃ for 10 h; then steam at 121℃ for 20 min until there is no hard center; A3. Drying, pulverizing, and sieving: Dry with hot air at 60°C until moisture content ≤8%; pulverize to 100 mesh; A4. Pre-fermentation treatment: Before preparation, dry powder of raw materials is heated at 85°C for 15 minutes. In this embodiment, the herbal teas are made from chrysanthemum, mint, chamomile, or roselle, and are treated through the following steps before being added to the culture medium: B1. Raw material preparation: Remove impurities and select intact flower / leaf parts; dry with hot air at 60°C until moisture content ≤8%; B2. Grinding and sieving: Most herbal teas are ground to 100 mesh; roselle (calyx) can be ground to 120 mesh. B3. Pre-fermentation treatment: The finished culture medium is uniformly sterilized by moist heat at 115°C for 10 min; The seed culture solution was inoculated into the fermentation medium at a 2% inoculation rate and cultured at 40℃ and 120 r / min for 6 days.

[0037] The fermentation medium consisted of: 25 g / L soybean, 18 g / L herbal tea, 25 g / L glycerol, 22.5 g / L yeast extract, 0.08 g / L K2HPO4, and 0.08 g / L MgSO4; pH 7.

[0038] Add the carrier to the fermentation medium to a concentration of 18%, and then freeze-dry or spray-dry at low temperature to obtain fermented soybean powder rich in vitamin K2 and tea polyphenols.

[0039] Total vitamin K2 and tea polyphenols are the bioactive components of fermented soybean flour; the higher the content, the better the fermentation effect and the higher the quality of the soybean flour. Results are as follows... Figure 2 As shown, the vitamin K2 obtained by fermenting soybeans and mint was 112.6 mg / Kg, and the tea polyphenols were 203.6 mg / Kg.

[0040] The carrier added to the fermentation medium in the fourth step is 10% maltodextrin and 8% galactooligosaccharides.

[0041] Vitamin K2 assay method In this invention, 1 g of fermentation sample was dissolved in 1 mL of deionized water, and then an equal volume of organic extractant was added for extraction. The extractant was prepared by mixing isopropanol and n-hexane in a 1:2 (v / v) ratio. After mixing, the mixture was vigorously shaken for 3 min to ensure that the fat-soluble vitamin K2 in the sample was fully dissolved in the organic phase. Then, the mixture was centrifuged at 8000 rpm for 1 min to separate the upper organic phase. This step was repeated three times, and the supernatant was collected after each centrifugation. The supernatants were combined to obtain the organic extract of the fermentation product. The extract was transferred to a 2 mL centrifuge tube, allowed to stand and air dry, and then ready for quantitative analysis. The quantitative analysis of the fermentation product was performed using high-performance liquid chromatography (HPLC). The chromatographic conditions were set as follows: mobile phase: 100% methanol; detection wavelength: 270 nm; flow rate: 1 mL / min; column temperature: 40±10℃; retention time: approximately 40 min. Based on the above steps and a standard curve, the vitamin K2 content in the fermentation sample was quantitatively analyzed.

[0042] Methods for determining tea polyphenols In this invention, 10 g of fermentation sample was weighed and added to 1000 mL of boiling pure water. Extraction was carried out at boiling temperature for 5–10 min to fully dissolve soluble polyphenols in the sample. After extraction, the mixture was cooled to room temperature to obtain a crude extract containing tea polyphenols. The extracted tea solution was then filtered through a 30,000 Da ultrafiltration membrane to remove large molecular impurities and interfering substances such as proteins. The resulting filtrate was directly fed into a D301 macroporous adsorption resin column for adsorption and enrichment, followed by elution with 85% ethanol to obtain the tea polyphenol eluent. Then, 2 mL of the tea polyphenol eluent was added to 4 mL of pure water and 5 mL of ferrous tartrate solution, mixed thoroughly, and brought to a final volume of 25 mL with pH 7.5 phosphate buffer to obtain the test solution. The ferrous tartrate solution was prepared by dissolving 0.1 g of ferrous sulfate and 0.5 g of potassium sodium tartrate in pure water and bringing the volume to 100 mL. This ratio stabilizes iron ions and allows them to complex with tea polyphenols to form a blue-green complex. Using phosphate buffer as a blank reference, the absorbance A1 of the blank solution and the absorbance A2 of the sample solution were measured at 540 nm using a 10 mm cuvette. Based on the principle that the color intensity of the colorimetric reaction is directly proportional to the concentration of tea polyphenols, the content of tea polyphenols in the sample was calculated using the following formula: Where X is the content of tea polyphenols in the sample (mg / L), K is the dilution factor, and V is the volume of test solution taken during the test (mL).

[0043] Example 3 In this embodiment, black soybeans were selected as the raw material for soybean flour, and chrysanthemum, mint, chamomile, or roselle were selected as the raw materials for herbal tea, respectively, in order to study the effect of adding black soybeans and different herbal tea raw materials to the fermentation medium on the fermentation effect.

[0044] A method for preparing fermented soybean powder fortified with vitamin K2 and tea polyphenols, comprising the following steps: (1) Before being added to the culture medium, black beans undergo the following multiple treatment steps: A1. Cleaning and Impurity Removal: Remove dust, impurities, and broken beans; control moisture content to ≤13% of the original bean moisture content. A2. Soaking and cooking: Material-to-water ratio 1:2 (w / w), 20℃ for 10 h; then steam at 121℃ for 20 min until there is no hard center; A3. Drying, pulverizing, and sieving: Dry with hot air at 60°C until moisture content ≤8%; pulverize to 100 mesh; A4. Pre-fermentation treatment: Before preparation, dry powder of raw materials is heated at 85°C for 15 minutes. (2) The herbal teas in this embodiment are chrysanthemum, mint, chamomile or roselle, and are treated with the following steps before being added to the culture medium: B1. Raw material preparation: Remove impurities and select intact flower / leaf parts; dry with hot air at 60°C until moisture content ≤8%; B2. Grinding and sieving: Most herbal teas are ground to 100 mesh; roselle (calyx) can be ground to 120 mesh. B3. Pre-fermentation treatment: The finished culture medium is uniformly sterilized by moist heat at 115°C for 10 minutes; (3) Inoculate the seed culture medium with 2% of the seed culture medium and culture it at 40℃ and 120 r / min for 6 days.

[0045] The fermentation medium consisted of: 25 g / L black beans, 18 g / L herbal tea, 25 g / L glycerol, 22.5 g / L yeast extract, 0.08 g / L K2HPO4, and 0.08 g / L MgSO4; pH was 7.

[0046] (4) Add the carrier to the fermentation medium with a content of 18%, wherein the carrier is 10% inulin and 8% oligomeric semi-solids, and then freeze-dry or spray-dry at low temperature to obtain fermented soybean powder rich in vitamin K2 and tea polyphenols.

[0047] Total vitamin K2 and tea polyphenols are the bioactive components of fermented soybean flour; the higher the content, the better the fermentation effect and the higher the quality of the soybean flour. Results are as follows... Figure 3 As shown, the vitamin K2 obtained by fermenting black beans and mint was 87.6 mg / Kg, and the tea polyphenols were 201.4 mg / Kg.

[0048] Example 4 In this embodiment, the soybean flour raw material was selected as broad bean, and the herbal tea raw materials were selected as chrysanthemum, mint, chamomile or roselle, respectively, in order to study the effect of adding broad bean and different herbal tea raw materials to the fermentation medium on the fermentation effect.

[0049] A method for preparing fermented soybean powder fortified with vitamin K2 and tea polyphenols, comprising the following steps: (1) Before being added to the culture medium, broad beans are treated through the following steps: A1. Cleaning and Impurity Removal: Remove dust, impurities, and broken beans; control moisture content to ≤13% of the original bean moisture content. A2. Soaking and cooking: Material-to-water ratio 1:2 (w / w), 20℃ for 10 h; then steam at 121℃ for 20 min until there is no hard center; A3. Drying, pulverizing, and sieving: Dry with hot air at 60°C until moisture content ≤8%; pulverize to 100 mesh; A4. Pre-fermentation treatment: Before preparation, dry powder of raw materials is heated at 85°C for 15 minutes. (2) The herbal teas in this embodiment are chrysanthemum, mint, chamomile or roselle, and are treated with the following steps before being added to the culture medium: B1. Raw material preparation: Remove impurities and select intact flower / leaf parts; dry with hot air at 60°C until moisture content ≤8%; B2. Grinding and sieving: Most herbal teas are ground to 100 mesh; roselle (calyx) can be ground to 120 mesh. B3. Pre-fermentation treatment: The finished culture medium is uniformly sterilized by moist heat at 115°C for 10 min; (3) Inoculate the seed culture medium with 2% of the seed culture medium and culture it at 40℃ and 120 r / min for 6 days.

[0050] The fermentation medium consisted of: broad bean 25 g / L, herbal tea 18 g / L, glycerol 25 g / L, yeast extract 22.5 g / L, K2HPO4 0.8 g / L, and MgSO4 0.08 g / L; pH 7.

[0051] (4) Add the carrier to the fermentation medium to a content of 18%, wherein the carrier is 10% xylooligosaccharide and 8% isomaltooligosaccharide, and then freeze-dry or spray-dry at low temperature to obtain fermented soybean powder rich in vitamin K2 and tea polyphenols.

[0052] Total vitamin K2 and tea polyphenols are the bioactive components of fermented soybean flour; the higher the content, the better the fermentation effect and the higher the quality of the soybean flour. Results are as follows... Figure 4 As shown, the vitamin K2 obtained by fermenting both broad beans and mint was 31.7 mg / Kg, and the tea polyphenols were 209.2 mg / Kg.

[0053] Example 5 In this embodiment, the soybean flour raw material was selected as adzuki beans, and the herbal tea raw materials were selected as chrysanthemum, mint, chamomile, or roselle, respectively, in order to study the effect of adding adzuki beans and different herbal tea raw materials to the fermentation medium on the fermentation effect.

[0054] A method for preparing fermented soybean powder fortified with vitamin K2 and tea polyphenols, comprising the following steps: (1) Before being added to the culture medium, the red beans were treated through the following steps: A1. Cleaning and Impurity Removal: Remove dust, impurities, and broken beans; control moisture content to ≤13% of the original bean moisture content. A2. Soaking and cooking: Material-to-water ratio 1:2 (w / w), 20℃ for 10 h; then steam at 121℃ for 20 min until there is no hard center; A3. Drying, pulverizing, and sieving: Dry with hot air at 60°C until moisture content ≤8%; pulverize to 100 mesh; A4. Pre-fermentation treatment: Before preparation, dry powder of raw materials is heated at 85°C for 15 minutes. (2) The herbal teas in this embodiment are chrysanthemum, mint, chamomile or roselle, and are treated with the following steps before being added to the culture medium: B1. Raw material preparation: Remove impurities and select intact flower / leaf parts; dry with hot air at 60°C until moisture content ≤8%; B2. Grinding and sieving: Most herbal teas are ground to 100 mesh; roselle (calyx) can be ground to 120 mesh. B3. Pre-fermentation treatment: The finished culture medium is uniformly sterilized by moist heat at 115°C for 10 min; (3) Inoculate the seed culture medium with 2% of the seed culture medium and culture it at 40℃ and 120 r / min for 6 days.

[0055] The fermentation medium consisted of: 25 g / L red beans, 18 g / L herbal tea, 25 g / L glycerol, 22.5 g / L yeast extract, 0.08 g / L K2HPO4, and 0.08 g / L MgSO4; pH was 7.

[0056] (4) Add the carrier to the fermentation medium to a content of 18%, wherein the carrier is 10% mannitol and 8% maltitol. Then, perform low-temperature freeze drying or spray drying to obtain fermented soybean flour rich in vitamin K2 and tea polyphenols.

[0057] Total vitamin K2 and tea polyphenols are the bioactive components of fermented soybean flour; the higher the content, the better the fermentation effect and the higher the quality of the soybean flour. Results are as follows... Figure 5 As shown, the vitamin K2 obtained by fermenting both red beans and mint was 19.6 mg / Kg, and the tea polyphenols were 204.1 mg / Kg.

[0058] Example 6 In this embodiment, chickpeas were selected as the raw material for soybean flour, and chrysanthemum, mint, chamomile, or roselle were selected as the raw materials for herbal tea, respectively, in order to study the effect of adding chickpeas and different herbal tea raw materials to the fermentation medium on the fermentation effect.

[0059] A method for preparing fermented soybean powder fortified with vitamin K2 and tea polyphenols, comprising the following steps: (1) Chickpeas were treated with the following steps before being added to the culture medium: A1. Cleaning and Impurity Removal: Remove dust, impurities, and broken beans; control moisture content to ≤13% of the original bean moisture content. A2. Soaking and cooking: Material-to-water ratio 1:2 (w / w), 20℃ for 10 h; then steam at 121℃ for 20 min until there is no hard center; A3. Drying, pulverizing, and sieving: Dry with hot air at 60°C until moisture content ≤8%; pulverize to 100 mesh; A4. Pre-fermentation treatment: Before preparation, dry powder of raw materials is heated at 85°C for 15 minutes. (2) The herbal teas in this embodiment are chrysanthemum, mint, chamomile or roselle, and are treated with the following steps before being added to the culture medium: B1. Raw material preparation: Remove impurities and select intact flower / leaf parts; dry with hot air at 60°C until moisture content ≤8%; B2. Grinding and sieving: Most herbal teas are ground to 100 mesh; roselle (calyx) can be ground to 120 mesh. B3. Pre-fermentation treatment: The finished culture medium is uniformly sterilized by moist heat at 115°C for 10 min; (3) Inoculate the seed culture medium with 2% of the seed culture medium and culture it at 40℃ and 120 r / min for 6 days.

[0060] The fermentation medium consisted of: chickpeas 25 g / L, herbal tea 18 g / L, glycerol 25 g / L, yeast extract 22.5 g / L, K2HPO4 0.08 g / L, and MgSO4 0.08 g / L; pH 7.

[0061] (4) Add the carrier to the fermentation medium to a content of 18%, wherein the carrier is 10% resistant dextrin and 8% sucrose. Then, perform low-temperature freeze drying or spray drying to obtain fermented soybean flour rich in vitamin K2 and tea polyphenols.

[0062] Total vitamin K2 and tea polyphenols are the bioactive components of fermented soybean flour; the higher the content, the better the fermentation effect and the higher the quality of the soybean flour. Results are as follows... Figure 6 As shown, the vitamin K2 obtained by fermenting chickpeas and mint was 79.6 mg / Kg, and the tea polyphenols were 197.7 mg / Kg.

[0063] The results from Examples 2-6 show that the highest vitamin K2 content was achieved using soybean flour as the raw material, followed by black beans > chickpeas > broad beans > red beans; the highest tea polyphenol content was achieved using peppermint as the raw material, followed by chamomile > roselle > chrysanthemum.

[0064] To compare the effects of adding soybean flour on the enrichment of tea polyphenols in herbal tea culture medium and the effects of adding herbal tea ingredients on the enrichment of vitamin K2 in soybean flour culture medium, control examples 1 and 2 were set up in this embodiment of the invention as blank control groups.

[0065] Fermentation effect of fermentation medium containing only soybean flour (as in Example 1) In this control example, only soybean flour was added to the fermentation medium. The study aimed to investigate the effect of different soybean flours on the fermentation effect and to achieve high production of vitamin K2 through combination screening of soybean flours.

[0066] A method for preparing vitamin K2-fortified fermented soybean powder, comprising the following steps: (1) Various types of bean flour include, but are not limited to, any one or more of soybeans, black beans, broad beans, red beans and chickpeas, and are specifically grouped as follows: yellow / black, yellow / broad bean, yellow / red, yellow / chickpea, black / broad bean, black / red, black / chickpea, broad bean / red, broad bean / chickpea, red / chickpea, yellow / black / broad bean, yellow / black / red, yellow / black / chickpea, black / broad bean / red, black / broad bean / chickpea, broad bean / red / chickpea, yellow / black / broad bean / red, yellow / black / broad bean / chickpea, yellow / black / broad bean / red / chickpea, yellow / black / broad bean / red / chickpea.

[0067] These soybean flours undergo the following multiple processes before being added to the culture medium: A1. Cleaning and Impurity Removal: Remove dust, impurities, and broken beans; control moisture content to ≤13% of the original bean moisture content. A2. Soaking and cooking: Material-to-water ratio 1:2 (w / w), 20℃ for 10 h; then steam at 121℃ for 20 min until there is no hard center; A3. Drying, pulverizing, and sieving: Dry with hot air at 60°C until moisture content ≤8%; pulverize to 100 mesh; A4. Pre-fermentation treatment: Before preparation, dry powder of raw materials is heated at 85°C for 15 minutes. (2) The seed culture solution was inoculated into the fermentation medium at an inoculation rate of 2% and cultured at 40℃ and 120 r / min for 6 days.

[0068] The fermentation medium consisted of 25-30 g / L of various soybean flours, 25 g / L of glycerol, 22.5 g / L of yeast extract, 0.08 g / L of K2HPO4, and 0.08 g / L of MgSO4; with a pH of 7.

[0069] In the two-component soybean flour, each soybean flour component is 12.5 g / L; in the three-component soybean flour, each soybean flour component is 10 g / L; in the four-component soybean flour, each soybean flour component is 7 g / L; and in the five-component soybean flour, each soybean flour component is 6 g / L. (3) Add the carrier to the fermentation medium to a content of 18%, wherein the carrier is 10% maltodextrin and 8% fructooligosaccharides. Then, freeze-dry or spray-dry at low temperature to obtain fermented soybean flour rich in vitamin K2.

[0070] Total vitamin K2 is a bioactive component of fermented soybean flour; the higher the content, the better the fermentation effect and the higher the quality of the soybean flour. Results are as follows... Figure 7As shown, the highest vitamin K2 yield was obtained by fermenting a combination of soybean, black bean, and chickpea flour, with a yield of 181.6 mg / kg.

[0071] Compare with Example 2: Fermentation effect of fermentation medium containing only herbal tea. In this control example, only herbal tea was added to the fermentation medium. The study aimed to investigate the effects of different herbal teas on the fermentation effect, with the goal of achieving high yield of tea polyphenols by combining and screening different types of herbal teas.

[0072] A method for preparing fermented soybean powder fortified with tea polyphenols, comprising the following steps: (1) Various herbal teas include, but are not limited to, any one or more of chrysanthemum, mint, chamomile and roselle, specifically grouped as follows: Chrysanthemum / mint, Chrysanthemum / chamomile, Chrysanthemum / roselle, Mint / chamomile, Mint / roselle, Chamomile / roselle, Chrysanthemum / mint / chamomile, Chrysanthemum / mint / chamomile, Mint / chamomile / roselle, Chrysanthemum / mint / chamomile / roselle, Chrysanthemum / mint / chamomile / chamomile / chamomile / chamomile / mint ...chamomile / chamomile / cha

[0073] Before being added to the culture medium, the following steps are performed: B1. Raw material preparation: Remove impurities and select intact flower / leaf parts; dry with hot air at 60°C until moisture content ≤8%; B2. Grinding and sieving: Most herbal teas are ground to 100 mesh; roselle (calyx) can be ground to 120 mesh. B3. Pre-fermentation treatment: The finished culture medium is uniformly sterilized by moist heat at 115°C for 10 minutes; (2) The seed culture solution was inoculated into the fermentation medium at an inoculation rate of 2% and cultured at 40℃ and 120 r / min for 6 days.

[0074] The fermentation medium consisted of: herbal tea 6-20 g / L, glycerol 20-30 g / L, yeast extract 15-30 g / L, K2HPO4 0.06-0.1 g / L, and MgSO4 0.06-0.1 g / L; pH 6-8.

[0075] In the two-component herbal tea, each herbal tea component was 9 g / L; in the four-component herbal tea, each herbal tea component was 6 g / L; and in the four-component herbal tea, each herbal tea component was 5 g / L. (3) Add the carrier to the fermentation medium to a content of 18%, wherein the carrier is 10% maltodextrin and 8% fructooligosaccharides. Then, freeze-dry or spray-dry at low temperature to obtain fermented soybean flour rich in tea polyphenols.

[0076] Tea polyphenols are the bioactive components of fermented soybean flour; the higher the content, the better the fermentation effect and the higher the quality of the soybean flour. The results are as follows... Figure 8As shown, the tea polyphenols obtained from the fermentation of two groups of herbal teas were consistent. The tea polyphenols obtained from the combined fermentation of peppermint and chamomile were 255.1 mg / Kg, and the tea polyphenols obtained from the combined fermentation of peppermint and roselle were 259.6 mg / Kg.

[0077] By combining the fermentation mediums used in Comparative Examples 1 and 2 with both legume and herbal tea ingredients, the optimal fermentation combination was found to be a three-component soybean flour (soybean, black bean, and chickpea) and a two-component herbal tea (mint and roselle).

[0078] Based on the results of the effects of different soybean flour and herbal teas on the fermentation effect obtained from Comparative Examples 1 and 2, and Examples 2-6, it can be seen that when herbal tea ingredients were added to the original single soybean flour culture medium in Examples 2-6, the original vitamin K2 enrichment capacity was weakened to varying degrees. Similarly, when soybean flour ingredients were added to the original single herbal tea culture medium, the original tea polyphenol enrichment capacity was also weakened to varying degrees. This indicates that when a single soybean flour ingredient or a single herbal tea ingredient is added to the fermentation culture medium, the original vitamin K2 and tea polyphenol enrichment capacities are weakened to varying degrees, which is not conducive to the simultaneous enrichment of vitamin K2 and tea polyphenols. Therefore, the following examples of the present invention further combine the optimal soybean flour combination (soybeans, black beans, and chickpeas) and the optimal herbal tea combination (mint and chamomile, or mint and roselle) to study and explore the synergistic effect of the two optimal combinations on the fermentation culture.

[0079] Example 7 Based on the results of Comparative Examples 1 and 2, the soybean flour raw materials selected in this embodiment are soybeans, black beans and chickpeas, and the herbal tea raw materials are mint and chamomile, or mint and roselle, respectively. The aim is to study the effect of adding the best legume raw materials and the best herbal tea raw materials to the fermentation medium on the fermentation effect.

[0080] A method for preparing fermented soybean powder fortified with vitamin K2 and tea polyphenols, comprising the following steps: (1) Soybeans, black beans and chickpeas were treated with the following steps before being added to the culture medium: A1. Cleaning and Impurity Removal: Remove dust, impurities, and broken beans; control moisture content to ≤13% of the original bean moisture content. A2. Soaking and cooking: Material-to-water ratio 1:2 (w / w), 20℃ for 10 h; then steam at 121℃ for 20 min until there is no hard center; A3. Drying, pulverizing, and sieving: Dry with hot air at 60°C until moisture content ≤8%; pulverize to 100 mesh; A4. Pre-fermentation treatment: Before preparation, dry powder of raw materials is heated at 85°C for 15 minutes. (2) The herbal teas in this embodiment are chrysanthemum, mint, chamomile or roselle, and are treated with the following steps before being added to the culture medium: B1. Raw material preparation: Remove impurities and select intact flower / leaf parts; dry with hot air at 60°C until moisture content ≤8%; B2. Grinding and sieving: Most herbal teas are ground to 100 mesh; roselle (calyx) can be ground to 120 mesh. B3. Pre-fermentation treatment: All 8 finished culture media are uniformly sterilized by moist heat at 115°C for 10 min; (3) Inoculate the seed culture medium with 2% of the seed culture medium and culture it at 40℃ and 120 r / min for 6 days.

[0081] The fermentation medium consisted of 10 g / L each of soybean, black bean, and chickpea; 6 g / L each of peppermint, chamomile, and roselle; 25 g / L of glycerol; 22.5 g / L of yeast extract; 0.08 g / L of K2HPO4; and 0.08 g / L of MgSO4; with a pH of 7.

[0082] (4) Add the carrier to the fermentation medium to a content of 18%, wherein the carrier is 10% maltodextrin and 8% fructooligosaccharides. Then, perform low-temperature freeze drying or spray drying to obtain fermented soybean flour rich in vitamin K2 and tea polyphenols.

[0083] Total vitamin K2 and tea polyphenols are the bioactive components of fermented soybean flour; the higher the content, the better the fermentation effect and the higher the quality of the soybean flour. Results are as follows... Figure 9 As shown, the vitamin K2 obtained by multi-combination fermentation of soybeans, black beans, chickpeas, mint, and roselle was 179.3 mg / Kg, and the tea polyphenols were 251.2 mg / Kg.

[0084] The above results indicate that the fermentation medium used to enrich vitamin K2 and tea polyphenols has specific selectivity for soybean flour raw materials and herbal tea raw materials.

[0085] 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 a fortified vitamin K2 and tea polyphenol fermented soy flour, characterized by, The method comprises inoculating the activated liquid of Bacillus natto into a fermentation medium, and fermenting the medium at 35-40 DEG C and 120-140 r / min for 6 days to obtain fermented soybean powder containing vitamin K2 and tea polyphenols, wherein the fermentation medium contains 20-30 g of soybean powder, 6-20 g of herbal tea per 1 L, The soybean powder comprises soybean, black bean and chickpea. The herbal tea comprises mint, and further comprises chamomile and / or jasmine.

2. The method of claim 1, wherein, The inoculation amount of the activated liquid of Bacillus natto is 2%-4%.

3. The method of claim 1, wherein, The Bacillus natto is Bacillus natto ND-1-A27 preserved in China Center for Type Culture Collection, and the number is CCTCC NO:M 2018131.

4. The method of claim 1, wherein, The fermentation medium comprises 20-30 g of soybean powder, 6-30 g of herbal tea, 20-30 g of glycerol, 15-30 g of yeast extract, 0.06-0.1 g of K2HPO4 and 0.06-0.1 g of MgSO4 per 1 L, and the pH is 6-8.

5. The method of claim 1, wherein, The soybean powder further comprises broad bean, and the herbal tea further comprises chrysanthemum.

6. The method of claim 1, wherein, The fermentation medium is added with 15%-30% w / w carriers, which include but are not limited to any one or more of malt dextrin, fructooligosaccharide, inulin, galactooligosaccharide, xylooligosaccharide, isomaltooligosaccharide, mannitol, maltitol, resistant dextrin, sucrose, glucose and fructose.

7. A fermented soybean powder fortified with vitamin K2 and tea polyphenols, which is prepared according to any one of the methods of claims 1-6.

8. The vitamin K2 and tea polyphenol fortified fermented soy flour according to claim 7, characterized in that, The fermented soybean powder contains vitamin K2 not less than 160 mg / Kg and tea polyphenols not less than 240 mg / Kg. Preferably, the fermented soybean powder contains vitamin K2 not less than 170 mg / Kg and tea polyphenols not less than 250 mg / Kg.

9. A food or drug product comprising the fermented soybean powder fortified with vitamin K2 and tea polyphenols according to claim 8.

10. Use of the food or drug product according to claim 9 in the preparation of a medicament for treating or preventing bone metabolism or cardiovascular diseases.

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