CN111560406B - Method for producing flavonoid substances from external products such as soybean for vegetable by fermenting bacillus natto - Google Patents
Method for producing flavonoid substances from external products such as soybean for vegetable by fermenting bacillus natto 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 from external products such as vegetable soybeans and the like by fermenting bacillus natto; comprises selecting vegetable soybean and other external products, cleaning, sterilizing, steaming, fermenting with Bacillus natto, fermenting to obtain natto product, removing fat of lyophilized and fermented natto with petroleum ether, extracting flavonoids in natto with ethanol solution with certain concentration under reflux, purifying the crude extract with macroporous resin, spin-drying the collected liquid, dissolving with distilled water, and freeze-drying to obtain flavone powder. The invention can be used as a lipid-lowering product, and the invention utilizes the extracted and purified flavonoid substances from natto to perfuse the stomach into the high-lipid mice, and discovers that the flavonoid substances can reduce the blood lipid concentration in serum of the high-lipid mice, improve the blood lipid metabolism and remove the capacity, and 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 with different scales in large, medium and small.
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 and the like by fermenting bacillus natto.
Background
The natto has rich nutrition and has the functions of preventing diseases, protecting health and the like, and also contains various physiological active substances such as nattokinase, isoflavone, superoxide dismutase, tocopherol and the like, can strengthen physique and improve organism immunity, and is a preferred medicine and food homologous food for preventing and improving cardiovascular and cerebrovascular diseases. The current research shows that the isoflavone has the protective effect on blood vessels, the effect similar to female estrogen, the anti-hormone effect, the antibacterial activity, the osteoporosis prevention and the like; the flavone can also effectively reduce blood sugar and blood fat, and can be used as a plant extract source of the functional food for assisting blood sugar and blood fat reduction.
Vegetable soybean is a legume plant containing abundant vegetable proteins, various beneficial minerals, vitamins, dietary fibers, and the like. At present, most of vegetable soybeans are processed in China through preliminary processing, the research and development of deep processed products are relatively backward, the technical content is low, and the added value of the products is low. Therefore, it is necessary to design a method for fermenting flavonoids in vegetable soybeans and other external products by using bacillus natto, fermenting the vegetable soybeans as raw materials, and extracting and purifying flavonoids from the fermented soybeans to solve the problems of residual vegetable soybeans and other external products and deep processing products of the vegetable soybeans.
Disclosure of Invention
The invention aims to provide a method for producing flavonoid substances from external products such as vegetable soybeans by fermenting bacillus natto, which takes the vegetable soybeans as raw materials to ferment the natto and extract and purify the flavonoid substances from the fermented natto.
The technical scheme adopted for solving the technical problems is as follows:
a method for producing flavonoids from semen glycines and other external products by fermenting with Bacillus natto comprises:
(1) And (3) raw material treatment: selecting and washing vegetable and soybean, weighing, sterilizing with high pressure steam at 121deg.C for 20min, and steaming;
(2) Inoculating: preparing bacillus natto powder and vegetable soybeans and other foreign products into bacterial liquid according to the weight ratio of 2%, and inoculating;
(3) Fermentation: fermenting the vegetable soybean and other foreign matters poured with the bacterial liquid at a constant temperature of between 35 and 41 ℃ for 12 to 48 hours;
(4) Post-ripening: after fermentation, standing for 6-24 hours at the temperature of 4 ℃ to obtain natto products;
(5) Extracting: removing fat after freeze-drying and pulverizing, and obtaining flavonoid crude extract after hot reflux extraction;
(6) And (3) separating and purifying: purifying by macroporous resin;
(7) And (3) drying: obtaining freeze-dried powder.
Further, the temperature is kept at 45-55 ℃ after the step (1) is steamed.
Further, the inoculation amount of the step (2) is 2%.
And (2) pouring the bacterial liquid when the temperature is 48-52 ℃ after the bean sterilization and steaming are finished.
Further, the fermentation temperature in the step (3) is 37 ℃ and the fermentation time is 24 hours.
Further, the post-ripening time of the step (4) is 12 hours.
Further, the specific process of the step (5) is as follows: and (3) freeze-drying and powdering the natto product, weighing, adding petroleum ether with the volume being 5 times of that of the natto product, carrying out ultrasonic treatment for 1h, discarding petroleum ether to remove fat, adding ethanol solution with the volume being 60% of that of the natto product after the fat removal is completed, and carrying out thermal reflux extraction for 3h at 80 ℃ to obtain flavonoid substance crude extract.
Further, the macroporous resin in the step (6) is purified as follows: the macroporous resin which is pretreated by soaking in 95% ethanol solution for 12 hours is preferably D101 macroporous resin, the loading amount is 250mL, the loading flow rate is 1.5mL/min, the eluent is 70% ethanol solution, and the eluting flow rate is 1.5mL/min.
Further, the drying in the step (7) is vacuum freeze drying, and freeze-dried powder is obtained.
The invention has the technical effects that:
compared with the prior art, the method for producing flavonoid substances by fermenting vegetable soybeans and other external products by using bacillus natto has the advantages that the vegetable soybeans and other external products are adopted to ferment and produce flavonoid substances, and the fermentation substrate is new and sufficient; the fermentation condition is simple and quick; the invention provides a new idea for producing flavonoid substances and a solution for processing external products such as companies and the like. The flavonoid substances extracted from the fermented natto product can reduce blood fat in serum of high-fat animals, enhance the metabolism and clearance capacity of the blood fat, have the weight-reducing effect on obese mice, and have the health-care function of regulating immunity. The invention has the characteristics of simple process, short flow and low cost, is suitable for bean product processing enterprises with different scales in large, medium and small, and has wide application.
Drawings
FIG. 1 is a graph showing the effect of fermentation time on the flavonoid content in natto;
FIG. 2 is a graph showing the effect of fermentation temperature on the flavonoid content in natto;
FIG. 3 is a graph showing the effect of the inoculation amount of the invention on the flavonoid content in natto;
FIG. 4 is a graph showing the effect of post-maturation time on the flavonoid content of natto;
FIG. 5 is a graph showing triglyceride change in mice of each group according to the present invention;
FIG. 6 is a graph showing the total cholesterol change in mice of each group according to the present invention;
FIG. 7 is a graph showing the change of the high density lipoprotein of each group of mice according to the present invention;
FIG. 8 is a graph showing liver lipase changes in mice of each group according to the invention;
FIG. 9 is a graph showing the change of TNF- α in each group of mice according to the present invention;
FIG. 10 is a graph showing the changes in interleukin-6 among groups of mice according to the present invention;
FIG. 11 is a graph showing the changes in the mouse interface 10 of each group according to the present invention;
FIG. 12 is a graph showing the weight change of mice in each group according to the present invention.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments of the present invention. The components of the 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 invention, as 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 made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
It should be noted that: like reference numerals and letters denote like items in the following figures, and thus once an item is defined in one figure, no further definition or explanation thereof is necessary in the following figures.
The invention will now be further illustrated by means of specific examples in connection with the accompanying drawings.
Example 1:
a method for producing flavonoids from semen glycines and other external products by fermenting with Bacillus natto comprises:
(1) And (3) raw material treatment: selecting and washing vegetable soybean and other external products, weighing, placing into a sterilizing pot, sterilizing with high-pressure steam at 121deg.C for 20min, and steaming;
(2) Inoculating: preparing bacillus natto bacterial powder and vegetable soybeans and other foreign products into bacterial liquid according to the weight ratio of 2%, and pouring the bacterial liquid when the temperature is 50 ℃ after the sterilization and steaming of the beans are completed;
(3) Fermentation: placing vegetable soybeans poured with bacterial liquid into a constant temperature incubator at 37 ℃ for fermentation for 24 hours, and placing vegetable soybeans and other external products into a refrigerator at 4 ℃ for after-ripening for 12 hours after the fermentation is completed to obtain natto products;
(4) Post-ripening: after fermentation is completed, placing the fermented product in a refrigerator at the temperature of 4 ℃ for after-ripening for 18 hours to obtain a natto product;
(5) Extracting: freeze-drying natto product, pulverizing into powder, weighing, adding petroleum ether with volume of 5 times for 1 hr, removing petroleum ether to remove fat, adding ethanol solution with volume of 60% 20 times after removing fat, and extracting under reflux at 80deg.C for 3 hr to obtain flavonoid crude extract;
(6) And (3) separating and purifying: pouring macroporous resin which is soaked and pretreated for 12 hours by using 95% ethanol into a chromatographic column, washing with distilled water until the macroporous resin is odorless, pouring a coarse flavone extract into the chromatographic column according to the sample loading of 250mL and the flow rate of 1.5mL/min, balancing for 30min, washing with 70% ethanol solution to be colorless according to the flow rate of 1.5mL/min, and collecting eluent;
(7) And (3) drying: rotary evaporating the eluent to dryness, dissolving in distilled water, pre-freezing in a refrigerator at-20deg.C, and lyophilizing with vacuum lyophilizing machine to obtain purified flavonoid powder.
Example 2: determination of flavone content
Measuring flavone content by sodium nitrite-aluminum nitrate-sodium hydroxide method
1. Drawing a standard curve: accurately sucking 0, 0.50, 1.00, 2.00, 3.00 and 4.00mL (corresponding to 0, 75, 150, 300, 450 and 600 mug rutin) of rutin standard solution, transferring into a 10mL graduated colorimetric tube, adding 30% ethanol solution to 5mL, respectively adding 0.3mL of 5% sodium nitrite solution, shaking for 5min, adding 0.3mL of 10% aluminum nitrate solution, shaking for 6min, adding 2mL of 1.0mol/L sodium hydroxide solution, fixing volume to scale with 30% ethanol, shaking for 15min, measuring absorbance at a wavelength of 510nm, taking a zero tube as a blank, drawing a standard curve by taking rutin content (mug) as an abscissa and absorbance as an ordinate: y=5.02x+0.0007, r 2 ＝0.9996。
2. Determination of flavone content: and (3) concentrating the flavone crude extract, measuring the absorbance value according to a standard curve preparation method, and substituting the absorbance value into a linear regression equation to calculate the flavone content in the crude extract.
Example 3: single factor test for producing flavonoid substances by fermenting vegetable soybean with bacillus natto
Inoculating bacillus natto into steamed vegetable soybean for fermentation, and respectively examining the influence of four factors of fermentation time, inoculum size, fermentation temperature and post-maturation time on the content of flavonoid substances produced by the bacillus natto. After screening by each single factor test, a preliminary test basis is provided for the next orthogonal optimization test.
1. Influence of fermentation time on flavonoid content in natto
Inoculating 2% of bacillus natto liquid into sterilized steamed vegetable soybeans, placing into a constant temperature incubator with preset temperature of 37 ℃, respectively culturing for 12 hours, 24 hours, 36 hours and 48 hours, placing into a refrigerator with 4 ℃ for after-ripening for 12 hours after fermentation, performing three groups of parallel tests on each group, and then performing freeze drying and powdering on the obtained finished products. Adding petroleum ether 10 times volume, removing fat twice by ultrasonic treatment, adding 30 times volume of 60% ethanol, refluxing at 80deg.C for 3 hr, collecting extractive solution to obtain coarse extractive solution of flavone, concentrating, and measuring flavone content. As can be seen from FIG. 1, the content of flavonoids in natto changes from low to high and from low as the fermentation time is prolonged. At the stage of 12-24 h, the content of flavonoid substances in natto is in an increasing trend, at the moment, along with the extension of fermentation time, the flavonoid substances produced by the fermentation of bacillus natto are increased, and the content of the flavonoid substances in the natto reaches the highest when the fermentation time is 24 h; the fermentation time exceeds 24 hours, the nutrients in the vegetable soybeans may not be enough to maintain the metabolism of the bacillus natto, and the secondary metabolites excessively inhibit the primary metabolites, so that the flavonoid content in the natto gradually decreases along with the extension of the fermentation time.
2. Influence of fermentation temperature on flavonoid substances in natto
Inoculating 2% bacillus natto solution into sterilized steamed vegetable soybean, culturing in a constant temperature incubator at 35deg.C, 37deg.C, 39deg.C and 41 deg.C for 24 hr, fermenting, aging in a refrigerator at 4deg.C for 12 hr, performing three parallel tests, freeze drying, and pulverizing. Adding petroleum ether 10 times volume, removing fat twice by ultrasonic treatment, adding 30 times volume of 60% ethanol, refluxing at 80deg.C for 3 hr, collecting extractive solution to obtain coarse extractive solution of flavone, concentrating, and measuring flavone content. As can be seen from fig. 2, as the fermentation temperature increases, the flavonoid content in natto increases and then decreases. The fermentation temperature is 37 ℃, the content of flavonoid substances in the natto is highest, and when the fermentation temperature is higher or lower than 37 ℃, the shape of the natto is slightly shrunken, and the content of flavonoid substances in the natto is reduced. The growth and metabolism process of the microorganism need proper temperature, when the reaction of the bacillus natto for producing the flavonoid substances is at the optimal temperature, the content of the flavonoid substances in the natto reaches the highest, and once the temperature exceeds the optimal temperature, the content of the flavonoid substances can be reduced. Meanwhile, the temperature is too high, so that the color of the natto is dull, and the shape is flat and soft.
3. Effect of inoculum size on Nattokinase Activity
Inoculating 1%, 2%, 3% and 4% of the inoculated soybeans into sterilized steamed soybeans, culturing in a constant-temperature incubator with a preset temperature of 37 ℃ for 24 hours, after fermentation, aging in a refrigerator with a temperature of 4 ℃ for 12 hours, performing three groups of parallel tests on each group, and freeze-drying and powdering the obtained finished product. Adding petroleum ether 10 times volume, removing fat twice by ultrasonic treatment, adding 30 times volume of 60% ethanol, refluxing at 80deg.C for 3 hr, collecting extractive solution to obtain coarse extractive solution of flavone, concentrating, and measuring flavone content. As can be seen from FIG. 3, the amount of inoculation has a certain effect on the production of flavonoids during fermentation. The flavonoid content has the trend of increasing and then decreasing with the increase of the inoculation amount. As the inoculation amount increases, the mucosa on the natto surface increases and then decreases. When the inoculation amount is 1%, the natto has insufficient viscosity, and the flavonoid content in the natto is low, probably due to the fact that the inoculation amount is too small, the growth and propagation rate of strains is low, and the fermentation is incomplete; when the inoculation amount is 2%, the cell growth rate is moderate, the secondary metabolic process is smoothly carried out, and the flavonoid content in natto reaches the maximum; when the inoculation amount is more than 2%, the content of flavonoid substances in natto is reduced along with the increase of the inoculation amount, and at the moment, the bacteria propagation speed is too high, so that the nutritional ingredients in the vegetable soybean are rapidly exhausted, primary metabolism is vigorous, secondary metabolism is inhibited, and the content of flavonoid substances in the natto is reduced, and the ammonia odor is increased.
4. Influence of the after-ripening time on the Nattokinase Activity
Inoculating 2% bacillus natto solution into sterilized steamed vegetable soybean, culturing in a constant temperature incubator with preset temperature of 37 ℃ for 24 hours, after fermentation, respectively after-ripening in a refrigerator with temperature of 4 ℃ for 6 hours, 12 hours, 18 hours and 24 hours, performing three groups of parallel tests on each group, and freeze-drying and powdering the obtained finished product. Adding petroleum ether 10 times volume, removing fat twice by ultrasonic treatment, adding 30 times volume of 60% ethanol, refluxing at 80deg.C for 3 hr, collecting extractive solution to obtain coarse extractive solution of flavone, concentrating, and measuring flavone content. As can be seen from fig. 4, the increase in flavonoid content followed by maturation time showed a trend of increasing followed by decreasing. The content of flavonoid substances in the natto is highest when the after-ripening time is 12 hours, and the unique ammonia taste of the natto is less, the viscosity is lower and the content of flavonoid substances in the natto is low when the after-ripening time is less than 12 hours; when the after-ripening time is more than 12 hours, the ammonia odor of the natto is too great, and the content of flavonoid substances in the natto is gradually reduced.
Example 4: orthogonal optimization test for producing flavonoid substances by fermenting vegetable soybeans with bacillus natto
According to the single-factor test result, three main factors of fermentation time, inoculum size and after-ripening time are selected for orthogonal test, a three-factor three-level orthogonal analysis table is designed, and the process for fermenting flavonoids in soybeans by using bacillus natto is further optimized: the results are shown in Table 1.
TABLE 1 results of orthogonal experiments
And (3) processing and analyzing the orthogonal test results, wherein the order of influence of the 3 factors on the flavonoid content in the fermented natto of the vegetable soybean is A > B > C, namely fermentation time > fungus powder proportion > after-ripening time. A1B2C2 is the fermentation scheme with the highest flavonoid content in natto, namely the fermentation time of vegetable soybean is 24 hours, the proportion of added natto bacterial powder is 2 percent, the post-ripening time is 12 hours, and the flavonoid content in natto is 4.30mg/g under the fermentation scheme.
Example 5: study of in vitro Oxidation resistance of purified flavone
1. Determination of the ability to scavenge hydroxyl radicals
1mL of 0.2M/L PBS buffer solution with pH of 7.4, 1mL of 300ug/mL saffron red, 0.5mL of sample (5 mg/mL), 3% H2O2 ℃ water bath reaction for 30min, absorbance at 520nm, 0.5mL of distilled water for the blank group instead of the sample, and 1.5mL of distilled water for the control group instead of EDTA-Fe (II) and the sample to be tested were taken.
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
2mL of flavone aqueous solution with the concentration of 1, 2, 3, 4 and 5mg/mL is taken, 2mL of DPPH solution with the concentration of 0.1mM/L is added, the mixture is uniformly mixed, the mixture reacts for 20min at room temperature in a dark place, the mixture is centrifuged for 10min at 10000r/min to obtain a sample group, and 2mL of 95% ethanol is used for replacing the DPPH solution in the blank group. The control group was 2mL DPPH and 2mL deionized water. The zeroing group was 2mL deionized water and 2mL 95% ethanol. Absorbance was measured at 517 nm.
Clearance = [1- (a sample-a blank)/a control ] ×100%
Determination of Total reducing Capacity
Taking 0.5mL of sample solution (5 mg/mL), adding 2.5mL of 0.2M/L PBS buffer solution with pH of 6.6, adding 2.5mL of 1% potassium ferricyanide solution, carrying out water bath for 20min at 50 ℃, adding 2.5mL of 10% TCA solution, centrifuging for 10min at 4000r/min after uniform mixing, taking 2.5mL of supernatant, adding 2.5mL of distilled water and 0.5mL of 1% FeCl3 solution, standing for 10min, changing the system solution from yellow to green, replacing the sample solution with equal amount of deionized water, zeroing, and measuring the absorbance at 700 nm.
TABLE 2 determination of in vitro antioxidant Activity of flavonoids
As can be seen from Table 2, at this concentration, the ability of the flavone solution and ascorbic acid to scavenge DPPH radicals is substantially the same; the ability of the flavone solution to remove hydroxyl free radicals is obviously stronger than that of the ascorbic acid solution; the total reducing power is stronger against the ascorbic acid solution than the flavone solution.
Example 6: verification of hypolipidemic efficacy of lyophilized flavone powder
The test mice are placed in an animal feeding room, firstly, the mice are adaptively fed for one week, the period is 12 hours of illumination, the darkness is 12 hours, free diet drinking water is carried out, 48 mice are randomly divided into 6 groups, 8 mice are fed with common feed or high-fat feed for 8 weeks, a high-fat mouse model is built, and the mice are grouped according to the following scheme in the ninth week after the modeling is successful:
a: normal control group (with common feed)
B: positive control group (high fat feed with orlistat)
C: negative control group (high fat feed)
D: huang Tonggao A group (fed with high-fat feed plus 100 mg/(kg. D) of aqueous flavone solution)
E: dosage group of flavone (high fat feed with 50 mg/(kg. D) flavone aqueous solution)
F: low dosage group of flavones (high fat feed with 20 mg/(kg. D) of aqueous flavones)
Each mouse of each group was fed a dose of 0.2mL.
The formula of the positive control group orlistat feeding amount is as follows: dm=dh/hw k mw
dm and dh represent the daily dose of mice and the daily dose of human, respectively; mw represents the weight of the mouse, hw represents the weight of the human, and the weight of the human is generally 70kg, and 40g when the mouse is dosed; k represents a human to mouse conversion factor of 9. The concentration of orlistat drug should be 1.5mg/mL calculated as 0.8mL of drug delivery per mouse.
Blood lipid determination was performed 4 weeks after continuous gastric lavage: the mice are fasted for 12 hours and drink water freely, blood is collected by adopting an eyeball-picking blood-taking method by using a 1.5mL centrifuge tube without pyrogen and endotoxin, serum and red blood cells are separated rapidly and carefully after centrifugation for 10 minutes at 3000 rpm, and the upper serum is sucked and placed into a microcentrifuge tube, marked and placed in a refrigerator at the temperature of minus 20 ℃ for standby. After thawing at room temperature and ensuring that the samples were uniformly and sufficiently thawed, the samples were tested according to the kit instructions. Serum Triglycerides (TG), total Cholesterol (TC), high Density Lipoprotein (HDL), liver lipase (HL), tumor necrosis factor- α (TNF- α), IL-6, IL-10 assay and changes in mouse body weight (see fig. 5, 6, 7, 8, 9, 10, 11 and 12).
1. Fig. 12 shows the weight change of mice at week 0, week 8 and week 12, and the weight of the positive control group (a) was significantly lower than that of the other five groups at week eight, and the variability was significant (P < 0.05), demonstrating that the establishment of the high-fat animal model was successful. After modeling is successful, the stomach flavone 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 negative control group (C) had significantly higher body weight than the other five groups on the 12 th weekend, with significant variability (P < 0.05); huang Tonggao (D), flavone medium (E) and flavone low (F) dose groups had a weight loss compared to the 8 th week, and the weights were not significantly different from the normal (A) and positive (B) control groups (P > 0.05). In conclusion, the flavone has weight reducing effect on obese 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 doses of flavone are obviously lower than those of a negative control group (C); the contents of TG and TC in serum of the three groups with high, medium and low flavone are slightly lower than those of the normal control group (A) and the positive control group (B). In conclusion, the long-term high-fat diet increases the content of triglyceride and total cholesterol in mice, the blood fat in the mice is higher, and after the stomach-filling flavone aqueous solution is infused, the content of triglyceride and total cholesterol in the serum of the mice is reduced, which indicates that the flavone aqueous solution has the probiotic health care function of reducing the blood fat in blood.
3. The HDL and HL content in the serum of the mice in the negative control group (C) is obviously lower than that in the other five groups; the serum of the three groups of mice with high, medium and low doses of flavone has higher HDL and HL content than that of the normal control group (A), the positive control group (B) and the negative control group (C). The long-term high-fat diet reduces the content of high-density lipoprotein and hepatic lipase in mice, reduces the scavenging capacity of blood fat in the mice, and increases the content of high-density lipoprotein and hepatic lipase in the serum of the mice after the gastric-lavage of the aqueous solution of flavone, which indicates that the aqueous solution of flavone has the health-care function of enhancing the scavenging capacity of blood fat in the mice.
4. The serum content of inflammatory factors IL-6 and TNF-alpha in the negative control group (C) is obviously higher than that in the other five groups, and the content of inflammatory factors IL-6 and TNF-alpha in the Huang Tonggao dose group (D), the content of flavone in the flavone low dose group (E) and the content of flavone low dose group (F) are lower than those in the normal control group (A) and the positive control group (B). The content of 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 anti-inflammatory factor IL-10 in the serum of the mice in the Huang Tonggao dose group (D), the flavone in the flavone low dose group (E) and the flavone low dose group (F) are obviously higher than those in the normal control group (A), the positive control group (B) and the negative control group (C). In conclusion, the long-term high-fat diet can raise the content of IL-6 and tumor necrosis factor-alpha in the body and reduce the content of IL-10 in the body, which indicates that the long-term high-fat diet can cause the in-vivo immune disorder of mice, and the contents of the gastric lavage flavone aqueous solution are normal, thereby indicating that the flavone has the health care function of regulating the immunity.
According to the embodiment of the invention, the flavonoid substances extracted from the fermented natto product can be used for reducing blood fat in serum of a high-fat animal, enhancing the metabolic clearance capacity of the blood fat, reducing weight of obese mice, and regulating immunity.
The foregoing embodiments are merely examples of the present invention, and the scope of the present invention includes, but is not limited to, the forms and styles of the foregoing embodiments, and any suitable changes or modifications made by those skilled in the art, which are consistent with the claims of the present invention, shall fall within the scope of the present invention.
Claims (6)
1. A method for fermenting flavonoid substances produced by vegetable soybeans by using bacillus natto is characterized by comprising the following steps of: comprising the following steps:
(1) And (3) raw material treatment: vegetable soybeans are weighed after being selected and washed, and are sterilized by high-pressure steam for 20min at 121 ℃ and steamed;
(2) Inoculating: preparing bacillus natto powder and vegetable soybeans into bacterial liquid according to the weight ratio of 2%, and inoculating;
(3) Fermentation: fermenting vegetable soybean poured with bacterial liquid at a constant temperature of between 35 and 41 ℃ for 12 to 48 hours;
(4) Post-ripening: after fermentation, standing for 6-24 hours at the temperature of 4 ℃ to obtain natto products;
(5) Extracting: removing fat after freeze-drying and pulverizing, and obtaining flavonoid crude extract after hot reflux extraction;
(6) And (3) separating and purifying: purifying by macroporous resin;
(7) And (3) drying: obtaining freeze-dried powder;
the specific process of the step (5) is as follows: freeze-drying natto product, pulverizing into powder, weighing, adding petroleum ether with volume of 5 times, performing ultrasonic treatment for 1 hr, removing petroleum ether to remove fat, adding ethanol solution with volume of 60% 20 times after removing fat, and performing thermal reflux extraction at 80deg.C for 3 hr;
the macroporous resin in the step (6) is purified into: and (3) purifying the macroporous resin subjected to pretreatment 12h by adopting a 95% ethanol solution, wherein the loading amount is 250mL, the loading flow rate is 1.5mL/min, the eluent is 70% ethanol solution, and the eluting flow rate is 1.5mL/min.
2. The method for fermenting flavonoids from vegetable soybeans by using bacillus natto according to claim 1, wherein the method comprises the following steps of: and (3) keeping the temperature at 45-55 ℃ after steaming in the step (1).
3. The method for fermenting flavonoids from vegetable soybeans by using bacillus natto according to claim 1, wherein the method comprises the following steps of: and (2) pouring the bacterial liquid when the temperature is 48-52 ℃ after the vegetable soybeans are sterilized and steamed.
4. The method for fermenting flavonoids from vegetable soybeans by using bacillus natto according to claim 1, wherein the method comprises the following steps of: the fermentation temperature in the step (3) is 37 ℃ and the fermentation time is 24 hours.
5. The method for fermenting flavonoids from vegetable soybeans by using bacillus natto according to claim 1, wherein the method comprises the following steps of: and (3) the post-ripening time of the step (4) is 12 hours.
6. The method for fermenting flavonoids from vegetable soybeans by using bacillus natto according to claim 1, wherein the method comprises the following steps of: and (3) drying in the step (7) is vacuum freeze drying, so as to obtain freeze-dried powder.
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