CN111560406A - Method for producing flavonoid substances from external products such as soybeans for bacillus natto fermented vegetables - Google Patents
Method for producing flavonoid substances from external products such as soybeans for bacillus natto fermented vegetables Download PDFInfo
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Abstract
The invention relates to the technical field of microbial fermentation, in particular to a method for producing flavonoid substances by fermenting vegetable soybeans and other external products with bacillus natto; selecting vegetable soybeans and other external products, cleaning, sterilizing, steaming, fermenting by using bacillus natto, then obtaining a natto product, ultrasonically degreasing freeze-dried and fermented natto by using petroleum ether, then extracting flavonoid substances in the natto by using an ethanol solution with a certain concentration through hot reflux, purifying a crude extract by using macroporous resin, then carrying out spin drying on a collected solution, dissolving in distilled water, and carrying out freeze drying to obtain flavone powder. The invention can be applied as a lipid-lowering product, and the flavonoid extracted and purified from natto is used for gastric perfusion of a high-fat mouse, so that the flavonoid can reduce the blood lipid concentration in the serum of the high-fat mouse, improve the blood lipid metabolism clearance capability and simultaneously have the health-care function of regulating immunity. The invention has the characteristics of simple process, short flow and low cost, and is suitable for bean product processing enterprises of large, medium and small scales.
Description
Technical Field
The invention relates to the technical field of microbial fermentation, in particular to a method for producing flavonoid substances from external products such as vegetable soybeans through bacillus natto fermentation.
Background
Natto is rich in nutrition, has the functions of preventing diseases, protecting health and the like, also contains various physiological active substances such as nattokinase, isoflavone, superoxide dismutase, tocopherol and the like, can enhance physique and improve the immunity of organisms, and is a first-choice medicine-food homologous food for preventing and improving cardiovascular and cerebrovascular diseases. The current research shows that isoflavone has the functions of protecting blood vessels, being similar to female estrogen, resisting hormone, inhibiting bacteria activity, preventing osteoporosis and the like; the flavone can also effectively reduce blood sugar and blood fat, and can be used as a plant extract source of a functional food for assisting in reducing blood sugar and blood fat.
The vegetable soybean is a legume plant which is rich in vegetable protein, various beneficial mineral substances, vitamins, dietary fibers and the like. At present, the vegetable soybeans are mostly processed primarily in China, the research and development of finely and deeply processed products are laggard, the technical content is low, and the added value of the products is low. Therefore, there is a need to design a method for producing flavonoids in external products such as vegetable soybeans by fermenting natto with vegetable soybeans as a raw material and extracting and purifying the flavonoids from the fermented natto, so as to solve the problem that the residual external products such as the vegetable soybeans and the deep-processed products of the kidney beans lag behind in research and development.
Disclosure of Invention
The invention aims to provide a method for producing flavonoids in external products such as vegetable soybeans by fermenting natto bacteria, and the like.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a method for producing flavonoids from exogenous products such as soybean for vegetable fermented by Bacillus natto comprises:
(1) raw material treatment: selecting vegetable soybean, washing, weighing, sterilizing with high pressure steam at 121 deg.C for 20min, and steaming;
(2) inoculation: preparing bacillus subtilis natto powder and vegetable soybeans and other external products into a bacterial liquid according to the weight ratio of 2%, and inoculating;
(3) fermentation: fermenting the vegetable soybeans and other external products poured with the bacterial liquid at the constant temperature of 35-41 ℃ for 12-48 hours;
(4) after-ripening: standing the fermented soybean at 4 ℃ for 6-24 hours to obtain a natto product;
(5) extraction: freeze-drying, pulverizing, removing fat, and extracting under reflux to obtain crude extract of flavonoids;
(6) separation and purification: purifying by macroporous resin;
(7) and (3) drying: obtaining the freeze-dried powder.
Further, the temperature is kept between 45 and 55 ℃ after the steaming in the step (1).
Further, the inoculation amount of the step (2) is 2%.
Further, in the step (2), the bacterial liquid is poured when the temperature is 48-52 ℃ after the beans are sterilized and steamed.
Further, the fermentation temperature of the step (3) is 37 ℃, and the fermentation time is 24 hours.
Further, the after-ripening time in the step (4) is 12 hours.
Further, the specific process of the step (5) is as follows: freeze-drying and pulverizing natto product, weighing, adding 5 times of petroleum ether, performing ultrasonic treatment for 1h, removing petroleum ether to remove fat, adding 20 times of 60% ethanol solution, and performing hot reflux extraction at 80 deg.C for 3h to obtain crude extract of flavonoids.
Further, the macroporous resin of the step (6) is purified into: and (3) soaking the macroporous resin subjected to pretreatment for 12h by using a 95% ethanol solution for purification, preferably D101 macroporous resin, wherein the sample loading amount is 250mL, the sample loading flow rate is 1.5mL/min, the eluent is a 70% ethanol solution, and the elution flow rate is 1.5 mL/min.
Further, the drying in the step (7) is vacuum freeze drying to obtain freeze-dried powder.
The invention has the technical effects that:
compared with the prior art, the method for producing flavonoids by fermenting vegetable soybeans and other external products with bacillus natto, disclosed by the invention, is used for producing flavonoids by fermenting vegetable soybeans and other external products, and fermentation substrates are new and sufficient; the fermentation condition is simple and quick; the invention provides a new idea for producing flavonoid substances and a solution for the problem of processing foreign products of companies and the like. The flavonoids extracted from fermented natto product can reduce blood lipid in serum of high-fat animals, enhance metabolic clearance of blood lipid, reduce weight of fat mice, and regulate immunity. The invention has the characteristics of simple process, short flow and low cost, is suitable for bean product processing enterprises of different sizes and is widely applied.
Drawings
FIG. 1 is a graph showing the influence of fermentation time on the content of flavonoids in natto according to the present invention;
FIG. 2 is a graph showing the influence of fermentation temperature on the content of flavonoids in natto according to the present invention;
FIG. 3 is a graph showing the effect of the inoculation amount of the present invention on the content of flavonoids in natto;
FIG. 4 is a graph showing the effect of after-ripening time on the content of flavonoids in natto according to the present invention;
FIG. 5 is a graph showing triglyceride changes in mice of various groups according to the present invention;
FIG. 6 is a graph showing the change in total cholesterol in various groups of mice according to the present invention;
FIG. 7 is a graph showing the change in high density lipoprotein in each group of mice according to the present invention;
FIG. 8 is a graph showing the change in hepatic lipase in various groups of mice according to the present invention;
FIG. 9 is a graph showing TNF- α changes in mice of various groups according to the present invention;
FIG. 10 is a graph showing the change of Baijie-6 in mice of each group according to the present invention;
FIG. 11 is a graph showing the change of Baijie 10 in each group of mice according to the present invention;
FIG. 12 is a graph showing the change in body weight of mice in each group according to the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. The components of embodiments of the present invention generally described and illustrated in the figures herein may be arranged and designed in a wide variety of different configurations. Thus, the following detailed description of the embodiments of the present invention, presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
It should be noted that: like reference numbers and letters refer to like items in the following figures, and thus, once an item is defined in one figure, it need not be further defined and explained in subsequent figures.
The invention is further illustrated by the following specific examples in combination with the accompanying drawings.
Example 1:
a method for producing flavonoids from exogenous products such as soybean for vegetable fermented by Bacillus natto comprises:
(1) raw material treatment: selecting vegetable soybean, washing, weighing, sterilizing with 121 deg.C high pressure steam for 20min, and steaming;
(2) inoculation: preparing bacillus subtilis natto powder and vegetable soybeans and other external products into a bacterial liquid according to a proportion of 2%, and pouring the bacterial liquid when the temperature is 50 ℃ after the beans are sterilized and steamed;
(3) fermentation: fermenting the vegetable soybeans poured with the bacterial liquid in a constant-temperature incubator at 37 ℃ for 24h, and after fermentation, putting the vegetable soybeans and other external products in a refrigerator at 4 ℃ for after-ripening for 12h to obtain natto products;
(4) after-ripening: after the fermentation is finished, putting the fermented soybean in a refrigerator at 4 ℃ for after-ripening for 18h to obtain a natto product;
(5) extraction: freeze-drying natto product, pulverizing, weighing, adding 5 times of petroleum ether, ultrasonic treating for 1 hr, removing petroleum ether to remove fat, adding 20 times of 60% ethanol solution, and extracting at 80 deg.C under reflux for 3 hr to obtain crude extract of flavonoids;
(6) separation and purification: pouring macroporous resin pretreated by soaking in 95% ethanol for 12h into a chromatographic column, washing with distilled water until no odor exists, pouring crude flavone extract at a flow rate of 1.5mL/min and a sample amount of 250mL, balancing for 30min, washing with 70% ethanol solution at a flow rate of 1.5mL/min until colorless, and collecting eluate;
(7) and (3) drying: and (3) rotatably evaporating the eluent to dryness, dissolving the eluent in distilled water, pre-freezing the eluent in a refrigerator with the temperature of-20 ℃, and freeze-drying the eluent by using a vacuum freeze dryer to obtain the purified flavonoid substance powder.
Example 2: measurement of flavone content
Method for determining flavone content by adopting sodium nitrite-aluminum nitrate-sodium hydroxide method
1. Drawing a standard curve: accurately absorbing 0, 0.50, 1.00, 2.00, 3.00 and 4.00mL of rutin standard solution (equivalent to 0, 75, 150, 300, 450 and 600 μ g of rutin), transferring the rutin standard solution into a 10mL graduated colorimetric tube, adding 30% ethanol solution to 5mL, adding 0.3mL of 5% sodium nitrite solution respectively, shaking and placing for 5min, adding 0.3mL of 10% aluminum nitrate solution, shaking uniformly and placing for 6min, adding 2mL of 1.0mol/L sodium hydroxide solution, fixing the volume to the graduation by using 30% ethanol, shaking uniformly and placing for 15min, measuring absorbance at a wavelength of 510nm, drawing a standard curve by using a zero tube as a blank, using the content (μ g) of rutin as a horizontal coordinate and using the absorbance as a vertical coordinate: y is 5.02x +0.0007, R2＝0.9996。
2. And (3) measuring the flavone content: concentrating the crude extract of flavone, determining its absorbance value according to standard curve preparation method, substituting into linear regression equation, and calculating flavone content in the crude extract.
Example 3: single-factor test for producing flavonoids from soybeans for bacillus natto fermented vegetables
The bacillus natto is inoculated in steamed vegetable soybeans for fermentation, and the influence of four factors, namely fermentation time, inoculation quantity, fermentation temperature and after-ripening time, on the content of flavonoid substances generated by the bacillus natto is respectively considered. After screening by each single-factor test, a preliminary test basis is provided for the next orthogonal optimization test.
First, the influence of fermentation time on the content of flavonoids in natto
Inoculating 2% bacillus natto liquid into the sterilized and steamed vegetable soybeans, putting the vegetable soybeans into a constant-temperature incubator with the preset temperature of 37 ℃, respectively culturing for 12h, 24h, 36h and 48h, after fermentation, putting the vegetable soybeans into a 4 ℃ refrigerator for after-ripening for 12h, carrying out three-group parallel tests on each group, and then freeze-drying and powdering the obtained finished product. Adding 10 times volume of petroleum ether, removing fat twice by ultrasonic treatment, adding 30 times volume of 60% ethanol, refluxing at 80 deg.C for 3 hr, collecting extractive solution to obtain crude flavone extractive solution, concentrating, and measuring flavone content. As can be seen from FIG. 1, the content of flavonoids in natto changes from low to high to low with the increase of fermentation time. At the stage of 12-24 h, the content of the flavonoid substances in the natto is in a rising trend, the flavonoid substances produced by fermenting the bacillus natto are increased along with the prolonging of the fermentation time, and the content of the flavonoid substances in the natto reaches the highest level when the fermentation time is 24 h; when the fermentation time exceeds 24 hours, the nutrient substances in the vegetable soybeans may not be enough to maintain the metabolism of the bacillus natto, and meanwhile, secondary metabolites excessively inhibit primary metabolites, so that the content of flavonoids in the natto gradually decreases along with the extension of the fermentation time.
Secondly, the influence of the fermentation temperature on the flavonoid substances in the natto
Inoculating 2% Bacillus natto bacterial solution into sterilized and steamed vegetable soybean, culturing in a constant temperature incubator at preset temperature of 35 deg.C, 37 deg.C, 39 deg.C and 41 deg.C for 24h, aging in a 4 deg.C refrigerator for 12h after fermentation, performing three-group parallel test for each group, freeze drying the obtained product, and pulverizing. Adding 10 times volume of petroleum ether, removing fat twice by ultrasonic treatment, adding 30 times volume of 60% ethanol, refluxing at 80 deg.C for 3 hr, collecting extractive solution to obtain crude flavone extractive solution, concentrating, and measuring flavone content. As can be seen from FIG. 2, the content of flavonoids in natto increases and then decreases with the increase of fermentation temperature. The content of flavonoid in natto is highest at a fermentation temperature of 37 deg.C, and when the fermentation temperature is higher than or lower than 37 deg.C, the natto is slightly shriveled, and the content of flavonoid in natto is reduced. The growth and metabolism process of the microorganism needs proper temperature, when the reaction of producing the flavonoid by the bacillus natto is in the proper temperature, the flavonoid content in the natto reaches the highest, and once the optimum temperature is exceeded, the flavonoid content is reduced. Meanwhile, too high temperature can cause dark color and luster of natto and shrivelled shape.
Third, the influence of the inoculation amount on the activity of the nattokinase
Inoculating 1%, 2%, 3% and 4% of the sterilized and steamed vegetable soybeans with bacteria, respectively, culturing in a constant-temperature incubator at 37 ℃ for 24h, after fermentation, after-ripening in a refrigerator at 4 ℃ for 12h, performing three-group parallel test on each group, freeze-drying the obtained finished product, and powdering. Adding 10 times volume of petroleum ether, removing fat twice by ultrasonic treatment, adding 30 times volume of 60% ethanol, refluxing at 80 deg.C for 3 hr, collecting extractive solution to obtain crude flavone extractive solution, concentrating, and measuring flavone content. As can be seen from FIG. 3, the amount of the inoculated substance has a certain influence on the production of flavonoids during the fermentation process. The flavonoid content shows the trend of increasing firstly and then decreasing with the increase of the inoculation amount. With the increase of the inoculation amount, the mucous membrane on the surface of the natto is increased and then reduced. When the inoculation amount is 1%, the viscosity of the natto is not enough, the content of flavonoid substances in the natto is low, and probably the inoculation amount is too small, the growth and reproduction rate of strains is low, and the fermentation is incomplete; when the inoculation amount is 2%, the cell growth rate is moderate, the secondary metabolism process is smoothly carried out, and the content of the flavonoid substances in the natto is maximum; when the inoculation amount is more than 2%, the content of the flavonoids in the natto is reduced along with the increase of the inoculation amount, at the moment, the rapid exhaustion of the nutrient components in the vegetable soybeans is caused due to the excessively high propagation speed of bacteria, the generation of the primary metabolism is vigorous, and the secondary metabolism is inhibited, so that the content of the flavonoids in the natto is reduced and the ammonia odor is increased.
Fourth, the influence of the after-ripening time on the activity of the enzyme Nattokinase
Inoculating 2% bacillus natto liquid into the sterilized and steamed vegetable soybeans, putting the vegetable soybeans into a constant-temperature incubator with the preset temperature of 37 ℃ for culturing for 24h, putting the vegetable soybeans into a refrigerator with the temperature of 4 ℃ for respectively after-ripening for 6h, 12h, 18h and 24h after fermentation is finished, carrying out three-group parallel tests on each group, and then freeze-drying and powdering the obtained finished product. Adding 10 times volume of petroleum ether, removing fat twice by ultrasonic treatment, adding 30 times volume of 60% ethanol, refluxing at 80 deg.C for 3 hr, collecting extractive solution to obtain crude flavone extractive solution, concentrating, and measuring flavone content. As can be seen from FIG. 4, the increase in flavonoid content followed by maturation time shows a tendency of increasing first and decreasing second. The content of flavonoid in natto is highest when the after-ripening time is 12h, and when the after-ripening time is less than 12h, the unique ammonia taste of natto is less, the viscosity is lower, and the content of flavonoid in natto is low; when the after-ripening time is longer than 12h, the ammonia odor of natto is too big, and the content of flavonoids in natto is gradually reduced.
Example 4: orthogonal optimization test for producing flavonoids from soybeans for bacillus natto fermented vegetables
According to the single-factor test result, three main factors of fermentation time, inoculation amount and after-ripening time are selected for orthogonal test, a three-factor three-level orthogonal analysis table is designed, and the process for producing the flavonoid substances from the soybeans for the bacillus natto fermented vegetables is further optimized: the results are shown in Table 1.
TABLE 1 results of orthogonal experiments
And (3) processing and analyzing the orthogonal test result, wherein the sequence of the influence of the 3 factors on the content of the flavonoid substances in the vegetable soybean fermented natto is A > B > C, namely the fermentation time > the ratio of the bacterial powder > the after-ripening time. A1B2C2 is a fermentation scheme with the highest content of flavonoids in natto, namely, vegetable soybean is fermented for 24h, the ratio of bacillus natto powder is 2%, the after-ripening time is 12h, and the content of flavonoids in natto is 4.30mg/g under the fermentation scheme.
Example 5: research on in-vitro oxidation resistance of purified flavone
1. Determination of hydroxyl radical scavenging Capacity
Taking 1mL of PBS buffer solution with the concentration of 0.2M/LpH7.4, 1mL of safranine with the concentration of 300ug/mL, 0.5mL (5mg/mL) of a sample, reacting in a water bath with the temperature of 3% H2O2 ℃ for 30min, measuring the light absorption value at 520nm, replacing the sample with 0.5mL of distilled water in a blank group, and replacing EDTA-Fe (II) and the sample to be measured with 1.5mL of distilled water in a control group.
Clearance ═ [ (sample a-blank)/(control a-blank) ] × 100%
Wherein: sample A, blank A, control A, absorbance values for sample, blank and control respectively.
2. Determination of antioxidant Activity in DPPH System
Taking 2mL of the aqueous solution of flavone with the concentration of 1, 2, 3, 4 and 5mg/mL, adding 2mL of 0.1mM/L DPPH solution, uniformly mixing, reacting for 20min in the dark at room temperature, centrifuging at 10000r/min for 10min to obtain a sample group, and replacing the DPPH solution with 2mL of 95% ethanol in the blank group. The control group was 2mL of DPPH and 2mL of deionized water. The zero-adjustment group was 2mL of deionized water and 2mL of 95% ethanol. Absorbance was measured at 517 nm.
Clearance ═ 1- (sample a-blank)/control a ] × 100%
Determination of Total reducing Capacity
0.5mL of sample solution (5mg/mL) is taken, 2.5mL of 0.2M/L PBS buffer solution with pH 6.6 and 2.5mL of 1% potassium ferricyanide solution are added, water bath at 50 ℃ is carried out for 20min, 2.5mL of 10% TCA solution is added, after uniform mixing, centrifugation is carried out at 4000r/min for 10min, 2.5mL of supernatant is taken, 2.5mL of distilled water and 0.5mL of 1% FeCl3 solution are added, after standing for 10min, the system solution is changed from yellow to green, the sample solution is replaced by equal amount of deionized water for zero adjustment, and the absorbance value is measured at 700 nm.
TABLE 2 measurement results of in vitro antioxidant activity of flavones
As can be seen from Table 2, at this concentration, the ability of the flavonoid solution and ascorbic acid to scavenge DPPH radicals is essentially the same; the capacity of eliminating hydroxyl free radicals of the flavone solution is obviously stronger than that of the ascorbic acid solution; the total reducing power of the ascorbic acid solution is stronger than that of the flavone solution.
Example 6: verification of blood fat reducing effect of freeze-dried flavone powder
Placing test mice in an animal feeding room, adaptively feeding the test mice for one week, wherein the period is 12h of illumination, 12h of darkness and free diet drinking water, randomly dividing 48 mice into 6 groups of 8 mice each, continuously feeding common feed or high-fat feed for 8 weeks to establish a high-fat mouse model, and grouping the mice according to the following scheme in the ninth week after successful modeling:
a: normal control group (fed with common feed)
B: positive control group (fed with high fat feed plus orlistat)
C: negative control group (fed with high fat feed)
D: flavone high dose group (feeding high fat feed and adding 100 mg/(kg. d) flavone water solution)
E: flavone medium dose group (feeding high fat feed and 50 mg/(kg. d) flavone water solution)
F: flavone low dose group (feeding high fat feed and adding 20 mg/(kg. d) flavone water solution)
Each mouse in each group was fed a dose of 0.2 mL.
The formula for calculating the drug feeding amount of orlistat in the positive control group is as follows: dm ═ dh/hw ═ k ═ mw
dm and dh respectively represent the daily dose of the mice and the daily dose of the human; mw represents the body weight of the mouse, hw represents the body weight of the human, the body weight of the human is generally 70kg, and the body weight of the mouse when the drug is administered is 40 g; k represents a conversion factor of 9 between human and mouse. The drug concentration of orlistat calculated as 0.8mL dosing volume per mouse should be 1.5 mg/mL.
Blood lipid measurements were performed 4 weeks after continuous gavage: the mice are fasted for 12 hours, water is freely drunk, an eyeball-picking blood-taking method is adopted, blood is collected by a 1.5mL centrifuge tube without pyrogen and endotoxin, serum and red blood cells are rapidly and carefully separated after centrifugation for 10 minutes at 3000 rpm, the upper layer serum is sucked into a microcentrifuge tube, and the marked serum is stored in a refrigerator at the temperature of minus 20 ℃ for standby. After thawing at room temperature and ensuring that the sample was uniformly and sufficiently thawed, the sample was tested according to the kit instructions. Determination of the content of Triglyceride (TG), Total Cholesterol (TC), High Density Lipoprotein (HDL), Hepatic Lipase (HL), tumor necrosis factor-alpha (TNF-alpha), IL-6, IL-10 in serum and the change in mouse body weight (see FIG. 5, FIG. 6, FIG. 7, FIG. 8, FIG. 9, FIG. 10, FIG. 11 and FIG. 12).
1. Fig. 12 shows the body weight changes of mice at week 0, week 8 and week 12, and the body weight of the normal control group (a) is significantly lower than that of the other five groups at week eight, with significant difference (P <0.05), which proves the successful establishment of the high fat animal model. After the modeling is successful, the intragastric flavonoid is continuously infused for 4 weeks, and the weight change difference of each group of mice is obvious. As can be seen from the figure, the body weight of the negative control group (C) was significantly higher than that of the other five groups at the end of week 12, with significant difference (P < 0.05); the weight of the flavone high-dose group (D), the flavone medium-dose group (E) and the flavone low-dose group (F) is reduced compared with that of the group at the 8 th week end, and the weight of the group is not obviously different from that of the normal control group (A) and the positive control group (B) (P is more than 0.05). In conclusion, the flavone has the weight-losing effect on the fat mice induced by high-fat diet.
2. The contents of TG and TC in serum of three groups of mice with high, medium and low dose of flavone are obviously lower than that of a negative control group (C); the contents of TG and TC in the serum of the three groups with high and low flavone contents are slightly lower than those of a normal control group (A) and a positive control group (B). In conclusion, the contents of triglyceride and total cholesterol in the mouse are increased by long-term high-fat diet, the blood fat in the mouse is higher, and the contents of triglyceride and total cholesterol in the mouse blood serum are reduced after the gastric lavage of the flavone aqueous solution, which shows that the flavone aqueous solution has the probiotic health-care function of reducing the blood fat in the blood.
3. The contents of HDL and HL in the serum of mice in the negative control group (C) are obviously lower than those of the other five groups; the contents of HDL and HL in the serum of mice in the three groups of mice with high and medium-low flavone doses are higher than those of a normal control group (A), a positive control group (B) and a negative control group (C). The long-term high-fat diet reduces the content of high-density lipoprotein and liver lipase in mice, the capacity of removing blood fat in mice is reduced, and after the gastric lavage of the aqueous flavone solution, the content of high-density lipoprotein and liver lipase in the blood serum of the mice is increased, which shows that the aqueous flavone solution has the health-care function of enhancing the capacity of removing blood fat in vivo.
4. The content of inflammatory factors IL-6 and TNF-alpha in the serum of the mice in the negative control group (C) is obviously higher than that of the mice in the other five groups, and the content of the flavone high-dose group (D), the flavone medium-dose group (E) and the flavone low-dose group (F) is lower than that of the normal control group (A) and the positive control group (B). The content of the anti-inflammatory factor IL-10 in the serum of the mice in the negative control group (C) is obviously lower than that in the normal control group (A), and the content of the flavone high-dose group (D), the flavone medium-dose group (E) and the flavone low-dose group (F) is obviously higher than that in the normal control group (A), the positive control group (B) and the negative control group (C). In conclusion, the content of IL-6 and tumor necrosis factor-alpha in a body can be increased and the content of IL-10 in the body can be reduced by long-term high-fat diet, which indicates that the long-term high-fat diet can cause immune disorder in a mouse body, and the content of flavone tends to be normal after the gastric lavage of the flavone aqueous solution, thereby indicating that the flavone has the health care function of regulating immunity.
The embodiment of the invention shows that the flavonoids extracted from the fermented natto product can reduce the blood fat in the serum of the hyperlipidemic animals and enhance the metabolic clearance capability of the blood fat, has the weight reducing effect on the obese mice, and simultaneously has the health care function of regulating the immunity.
The above embodiments are only specific examples of the present invention, and the protection scope of the present invention includes but is not limited to the product forms and styles of the above embodiments, and any suitable changes or modifications made by those skilled in the art according to the claims of the present invention shall fall within the protection scope of the present invention.
Claims (9)
1. A method for producing flavonoid substances from external products such as soybeans for bacillus natto fermented vegetables is characterized by comprising the following steps: the method comprises the following steps:
(1) raw material treatment: selecting vegetable soybean, washing, weighing, sterilizing with high pressure steam at 121 deg.C for 20min, and steaming;
(2) inoculation: preparing bacillus subtilis natto powder and vegetable soybeans and other external products into a bacterial liquid according to the weight ratio of 2%, and inoculating;
(3) fermentation: fermenting the vegetable soybeans and other external products poured with the bacterial liquid at the constant temperature of 35-41 ℃ for 12-48 hours;
(4) after-ripening: standing the fermented soybean at 4 ℃ for 6-24 hours to obtain a natto product;
(5) extraction: freeze-drying, pulverizing, removing fat, and extracting under reflux to obtain crude extract of flavonoids;
(6) separation and purification: purifying by macroporous resin;
(7) and (3) drying: obtaining the freeze-dried powder.
2. The method for producing flavonoid compounds from exo-products such as soybean for fermented vegetables with Bacillus natto according to claim 1, wherein the method comprises: after the steaming in the step (1), keeping the temperature at 45-55 ℃.
3. The method for producing flavonoid compounds from exo-products such as soybean for fermented vegetables with Bacillus natto according to claim 1, wherein the method comprises: the inoculation amount of the step (2) is 2%.
4. The method for producing flavonoid compounds from exo-products such as soybean for fermented vegetables with Bacillus natto according to claim 1, wherein the method comprises: and (2) after the beans are sterilized and steamed, pouring the bacterial liquid when the temperature is 48-52 ℃.
5. The method for producing flavonoid compounds from exo-products such as soybean for fermented vegetables with Bacillus natto according to claim 1, wherein the method comprises: the fermentation temperature of the step (3) is 37 ℃, and the fermentation time is 24 hours.
6. The method for producing flavonoid compounds from exo-products such as soybean for fermented vegetables with Bacillus natto according to claim 1, wherein the method comprises: the after-ripening time in the step (4) is 12 hours.
7. The method for producing flavonoid compounds from exo-products such as soybean for fermented vegetables with Bacillus natto according to claim 1, wherein the method comprises: the specific process of the step (5) is as follows: freeze-drying and pulverizing natto product, weighing, adding 5 times of petroleum ether, ultrasonically treating for 1h, removing petroleum ether to remove fat, adding 20 times of 60% ethanol solution, and extracting at 80 deg.C under reflux for 3 h.
8. The method for producing flavonoid compounds from exo-products such as soybean for fermented vegetables with Bacillus natto according to claim 1, wherein the method comprises: the macroporous resin in the step (6) is purified into: and (3) soaking the macroporous resin subjected to pretreatment 12h in a 95% ethanol solution for purification, wherein the sample loading amount is 250mL, the sample loading flow rate is 1.5mL/min, the eluent is a 70% ethanol solution, and the elution flow rate is 1.5 mL/min.
9. The method for producing flavonoid compounds from exo-products such as soybean for fermented vegetables with Bacillus natto according to claim 1, wherein the method comprises: and (4) the drying in the step (7) is vacuum freeze drying to obtain freeze-dried powder.
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