阅读说明：本技术 一种纳豆菌发酵菜用大豆等外品产黄酮类物质的方法 (Method for producing flavonoid substances from external products such as soybeans for bacillus natto fermented vegetables ) 是由 孙庆申 杨宸 李秀凉 靳文凤 于 2020-04-27 设计创作，主要内容包括：本发明涉及微生物发酵技术领域,具体地涉及一种纳豆菌发酵菜用大豆等外品产黄酮类物质的方法；包括挑选菜用大豆等外品,洗净灭菌蒸熟后使用纳豆菌发酵后熟得纳豆产品,将冻干发酵好的纳豆经石油醚超声除脂后用一定浓度的乙醇溶液热回流提取纳豆中的黄酮类物质,将粗提液过大孔树脂纯化后,旋干收集液后蒸馏水溶解,冷冻干燥得黄酮粉末。本发明可作为降脂产品进行应用,本发明利用从纳豆中提取纯化的黄酮类物质灌胃高脂小鼠,发现黄酮类物质可以降低高脂小鼠血清中血脂浓度,提高血脂代谢清除能力,同时还具有调节免疫的保健功能。本发明具有工艺简单、流程短、成本低的特点,适合大中小不同规模豆制品加工企业。(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.)

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.

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.