阅读说明：本技术 一种从高粱黑粉菌中分离蛋白的方法及其应用 (Method for separating protein from ustilago sorghum and application of method ) 是由 鲁明 付欣 吴兴壮 史琳 宋梦雪 解梦汐 于 2021-09-14 设计创作，主要内容包括：本发明公开了一种具有降脂活性蛋白的制备方法,特别是涉及一种从高粱黑粉菌中分离蛋白的方法及其应用,它属于食品或药品技术领域。一种从高粱黑粉菌中分离蛋白的方法,其制备步骤包括浸提、粗提物制备和分离提纯。本发明提供了所述蛋白的制备方法,经内外降脂实验证明本发明提供的蛋白具有降低体脂含量、降低血清脂质水平和减轻体重的作用,对于开发新型的减肥、降血脂等天然食品和治疗药物具有重要意义。本发明的制备方法简单,成本低,提取纯度高,重现性好,可实现工业化生产。(The invention discloses a preparation method of protein with lipid-lowering activity, in particular relates to a method for separating protein from ustilago sorghum and application thereof, and belongs to the technical field of food or medicines. A method for separating protein from ustilago sorghum comprises the steps of leaching, crude extract preparation and separation and purification. The invention provides a preparation method of the protein, and internal and external lipid-lowering experiments prove that the protein provided by the invention has the effects of reducing body fat content, reducing serum lipid level and reducing weight, and has important significance for developing novel natural foods and therapeutic drugs for losing weight, reducing blood fat and the like. The preparation method of the invention is simple, has low cost, high extraction purity and good reproducibility, and can realize industrial production.)

1. A method for separating protein from ustilago sorghum is characterized by comprising the following steps:

(1) leaching: mixing defatted powder of ustilago sorghum with distilled water, adjusting the pH value by using NaOH solution, and magnetically stirring to obtain a crude extract;

(2) preparing a crude extract: taking the crude extract, adjusting the pH value by adopting HCl solution, standing for 8-12h, centrifuging, and collecting precipitate to obtain a crude extract;

(3) separation and purification: and (3) taking the crude extract, adjusting the pH value to 7, dialyzing in a dialysis bag, and freeze-drying in a vacuum freeze-drying machine to obtain the sorghum smut protein dry powder.

2. The method for separating protein from ustilago sorghum according to claim 1, wherein the defatted flour of ustilago sorghum in step (1) is prepared by the following steps: removing impurities from fresh sorghum smut, drying in a 60-DEG C oven, crushing by a crusher, mixing with petroleum ether with a boiling range of 60-90 ℃ in a material-liquid ratio of 1:5 to separate oil, volatilizing in a ventilated kitchen, and sieving by a 60-mesh sieve to obtain the degreased powder.

3. The method for separating protein from ustilago sorghum according to claim 1, wherein the defatted powder of ustilago sorghum is mixed with distilled water at a mass ratio of 1:10 in step (1), and the pH is adjusted to 8.0.

4. The method for separating protein from ustilago sorghum according to claim 1, wherein the magnetic stirring in step (1) is magnetic stirring at room temperature for 30min, and centrifugation at 8000r/min for 10 min.

5. The method for separating protein from ustilago sorghum according to claim 1, wherein the NaOH solution in the step (1) is a solution having a concentration of 1mol/L by mass.

6. The method for separating protein from ustilago sorghum according to claim 1, wherein the pH value of step (2) is adjusted to 4.6, the centrifugal speed is 8000r/min, and the time is 10 min.

7. The method for separating protein from ustilago sorghum according to claim 1, wherein the HCl solution in the step (2) is a solution having a concentration of 1mol/L by mass.

8. The method for separating protein from ustilago sorghum according to claim 1, wherein the dialysis method in step (3) is dialysis in a 6-8 KD dialysis bag at 4 ℃ for 72 h.

9. Use of the protein isolated from ustilago sorghum according to claim 1 for the preparation of a food or pharmaceutical product for the treatment of obesity and obesity.

Technical Field

The invention relates to a preparation method of protein with lipid-lowering activity, in particular to a method for separating protein from ustilago sorghum and application thereof, belonging to the technical field of functional food or medicines.

Background

The sorghum head smut (Sphacelotheceareiliana), commonly known as ustilago sorghum, belongs to the phylum Basidiomycota, the class ustilago, and is an important plant pathogenic fungus. The sorghum smut is a plant pathogenic bacterium, but has high nutritive value and health-care effect as a typical black food and edible fungus. The sorghum smut is also applied to folk common traditional Chinese medicines and has a plurality of effects, such as menstruation regulation, hemostasis, colitis prevention and coronary heart disease prevention; the sorghum head smut has strong anti-tumor activity, has strong inhibition on tumor cells, has anti-aging effect on human bodies, promotes the synthesis of protein and nucleic acid, resists the damage of radioactive rays and increases the content of white blood cells, and has the effects of resisting ulcer, resisting inflammation, reducing blood sugar, enhancing memory and resisting coagulation. The sorghum head smut is an edible fungus with high nutritive value, and contains abundant polysaccharides, dietary fibers, trace elements, vitamins, proteins and amino acids, and especially contains abundant essential amino acids for human bodies.

However, the focus of research on smut bacteria at home and abroad is mainly on the infection mechanism and classification of phytopathogen, and the development and utilization of the sorghum smut bacteria are mainly processed into various foods and additives. Patent No. 200410082983.4, Chinese patent with application date of 2004, 12 months and 15 days, discloses a preparation method of a sorghum smut fungus nutritional additive and a product thereof. Chinese patent No. 200810229729.0, having application date of 2008, 12 months and 15 days, discloses a sorghum smut fungus rice sausage. However, the compounds separated from ustilago sorghum and used for losing weight and regulating blood fat and used for preparing lipid-lowering medicaments are not reported in a public way.

The incidence of obesity and hyperlipidemia is increasing worldwide, and it is a disease that develops progressively and in the later stages seriously harms the health and even life. Research has shown that lowering blood lipid levels is an effective way to improve hyperlipidemia. In the research of in-vitro and in-vivo lipid-lowering experiments, the preparation method and lipid-lowering application of the ustilago sorghum protein with lipid-lowering activity are discovered and invented. The research proves that the in vivo and in vitro lipid-lowering effect of the compound has a certain dose-effect relationship. The ustilago sorghum protein belongs to protein of natural plant sources, can reduce the absorption of fat and carbohydrate, and has the effect of reducing blood fat.

Disclosure of Invention

The invention aims to provide a method for separating protein from ustilago sorghum and application thereof, which separates substances with lipid-lowering activity from ustilago sorghum, adopts a water extraction and alcohol precipitation method, researches the lipid-lowering bioactivity of the materials and develops a new application problem of edible and medicinal values of ustilago sorghum.

The invention is realized by the following technical scheme in order to achieve the aim.

A method for separating protein from ustilago sorghum comprises the following steps:

(1) leaching: mixing defatted powder of ustilago sorghum with distilled water, adjusting the pH value by using NaOH solution, and magnetically stirring to obtain a crude extract;

(2) preparing a crude extract: taking the crude extract, adjusting the pH value by adopting HCl solution, standing for 8-12h, centrifuging, and collecting precipitate to obtain a crude extract;

(3) separation and purification: and (3) taking the crude extract, adjusting the pH value to 7, dialyzing in a dialysis bag, and freeze-drying in a vacuum freeze-drying machine to obtain the sorghum smut protein dry powder.

Preferably, the preparation method of the defatted powder of the ustilago sorghi in the step (1) is as follows: removing impurities from fresh sorghum smut, drying in a 60-DEG C oven, crushing by a crusher, mixing with petroleum ether with a boiling range of 60-90 ℃ in a material-liquid ratio of 1:5 to separate oil, volatilizing in a ventilated kitchen, and sieving by a 60-mesh sieve to obtain degreased powder.

Preferably, the sorghum smut spent meal and the distilled water in the step (1) are mixed according to the mass ratio of 1:10, and the pH value is adjusted to 8.0.

Preferably, the magnetic stirring in step (1) is magnetic stirring at room temperature for 30min, and centrifuging at 8000r/min for 10 min.

Preferably, the NaOH solution in the step (1) is a solution with a mass percentage concentration of 1 mol/l.

Preferably, the pH value of the step (2) is adjusted to be 4.6, the centrifugal speed is 8000r/min, and the time is 10 min.

Preferably, the HCl solution in the step (2) is a solution with a mass percentage concentration of 1 mol/l.

Preferably, the dialysis method in the step (3) is dialysis in a dialysis bag with 6-8 KD at 4 ℃ for 72 hours.

An application of the protein separated from the ustilago sorghum in preparing the food or the medicine for reducing fat and losing weight.

Due to the adoption of the technical scheme, the invention has the following characteristics and effects:

the invention provides a preparation method of the protein, and the protein interferes lipid metabolism and reduces blood fat by promoting liver AMPK phosphorylation. Internal and external lipid-lowering experiments prove that the protein provided by the invention has the effects of reducing body fat content, reducing serum lipid level and reducing weight, and has important significance for developing novel natural foods and therapeutic drugs for losing weight, reducing blood fat and the like. The preparation method of the invention is simple, the cost is low, the extraction purity is high, the reproducibility is good, the industrial production can be realized, and the protein has good application prospect in the aspect of preparing lipid-lowering functional foods and medicines.

Drawings

FIG. 1 shows the protein WM-P of ustilago sorghum₁Comparison of purity before and after purification.

FIG. 2 is a photograph of a blank group of pathological sections of mouse liver tissue (HE staining, X200).

FIG. 3 is a graph of a mouse liver histopathological section (HE staining, X200) model set.

FIG. 4 is a graph of a mouse liver histopathological section (HE staining, x 200) low dose group.

FIG. 5 is a graph of dose groups in pathological sections of mouse liver tissue (HE staining, x 200).

FIG. 6 is a high dose set of pathological sections of mouse liver tissue (HE staining, X200).

FIG. 7 is a graph showing the condition of TC in the blood of a mouse.

FIG. 8 is a graph showing TG in mouse blood.

FIG. 9 is a graph of HDL levels in mouse blood.

FIG. 10 is a graph showing LDL in the blood of mice.

FIG. 11 is a graph of the effect of ustilago sorghum protein on p-AMPK in mouse liver.

Detailed Description

The present invention will be further illustrated with reference to the following examples. The following examples are only a few specific examples of the present invention, but the design concept of the present invention is not limited thereto, and any insubstantial modifications made by the design concept should fall within the scope of infringing on the protection scope of the present invention.

The methods in the following examples and test examples are conventional methods unless otherwise specified.

A method for separating protein from ustilago sorghum comprises the following steps:

(1) leaching: removing impurities from fresh ustilago sorghum, drying in a 60-DEG C oven, crushing by a crusher, mixing with petroleum ether with a boiling range of 60-90 ℃ in a material-liquid ratio of 1:5 to separate grease, volatilizing in a ventilated kitchen, sieving by a 60-mesh sieve to obtain defatted powder, mixing 100 parts by mass of the defatted powder of ustilago sorghum with 1000 parts by mass of distilled water, adjusting the pH value to 8.0 by using a NaOH solution with the mass percent concentration of 1mol/l, magnetically stirring for 30min at room temperature, and centrifuging for 10min at 8000r/min to obtain a crude extract;

(2) preparing a crude extract: taking the crude extract, adjusting the pH value to 4.6 by adopting a HCl solution with the mass percentage concentration of 1mol/l, standing for 8-12h, centrifuging at the rotating speed of 8000r/min for 10min, and collecting the precipitate to obtain a crude extract;

(3) separation and purification: and (3) taking the crude extract, adjusting the pH value to 7, dialyzing in a dialysis bag with 6-8 KD at the temperature of 4 ℃ for 72 hours, and freeze-drying in a vacuum freeze-drying machine to obtain the sorghum smut protein dry powder.

The protein separated from the ustilago sorghum can be applied to preparing food or medicine with the functions of reducing fat and losing weight.

The characteristics and effects of the present invention will be further described below by test data, and in the following test examples, test methods not specifically described are all conventional methods. The proteins used in the following examples are all proteins prepared by the method of the present invention.

Test example 1

Coomassie brilliant blue method test for protein content

The instrument comprises the following steps: visible spectrophotometer, centrifuge, pipette

Sample preparation: tissue sample, weighing about 0.03g of sorghum smut protein powder in example, adding 0.3ml of extracting solution (distilled water), homogenizing in ice bath, centrifuging at 12000rpm at 4 ℃ for 10min, and taking supernatant, namely to-be-detected liquid.

The crude extract was diluted to 10 times and measured, the wavelength was set to 600nm, and distilled water was added to the EP tube in order of zero setting, as shown in Table 1.

TABLE 1 Main test reagent dosage and configuration method TABLE

Tissue sample:

protein content (mg/g) [ (. DELTA.A +0.0044)/8.3429 [)]/(W×V₁/V)×D＝32.825mg/g

Sample pretreatment: preparation of 6mol/L HCl solution approximately 0.4g of sorghum smut protein powder is weighed, 2ml of 6mol/L hydrochloric acid is added into a bottle. Placing the mixture in a drying box, hydrolyzing for 24h at the temperature of 110-120 ℃, pouring the hydrolysate into an evaporating dish, and placing the evaporating dish on a boiling water bath for heating to remove hydrochloric acid. A small amount of distilled water was added to the evaporation pan to dissolve the residue, and heating was continued on a boiling water bath to remove water.

Test example 2

The protein before and after purification is subjected to purity analysis by SDS-PAGE electrophoresis. Preparing separation gel and concentrated gel with SDS concentration of 12% and 5%, taking 15-20 μ g of quantified protein sample, adding loading buffer solution, DTT and marker according to volume, boiling with boiling water for 3-10min, taking supernatant, performing electrophoresis until casein blue indicator runs to the bottom of gel, and stopping electrophoresis.

As can be seen from FIG. 1, the purified sorghum smut protein WM-P₁The purity can reach more than 80 percent, and the method is proved to be capable of obtaining the sorghum smut protein WM-P with higher purity₁。

Test example 3

Evaluation of lipid-lowering Activity

Lipid-lowering activity test, constructing an obesity model of a high-fat induced C57BL/6 mouse, calculating the weight, water intake and feed eating condition of the mouse by observing and measuring the change of liver, kidney and epididymis adipose tissues, and discussing the ustilago sorghum protein WM-P by combining the constitution indexes, histopathological conditions and blood biochemical indexes of various groups of mice₁Reducing blood lipid of mice.

The sample used in the experiment is the extracted sorghum smut protein WM-P₁. The Liaoning Biotechnology GmbH provides clean-grade male C57BL/6 mice, which are 7-8 weeks old, 18-20g in weight, and SPF grade. Production license number of experimental animal: SCXK (Liao) 2015-0001. The mice were kept in a suitable warm humid chamber for 12h light/dark cycle, ensuring a suitable amount of feed and sufficient water. All mice were allowed at least one week of acclimation to the environmental conditions prior to the experiment.

After one week of adaptive feeding of 40 SPF grade male C57BL/6 mice, they were randomly divided into 5 groups: blank set, model set, WM-P₁Low dose group (10mg/kg), WM-P1 low dose group (25mg/kg) and WM-P₁High dose groups (50mg/kg), 8 per group. During the feeding period, the mice freely drink water to provide sufficient feed, and the formula of the high-fat feed comprises 70% of common feed, 15% of lard and 15% of cane sugar. Changes in body weight of mice were recorded every 2 d.

1. Mouse model establishment

And (3) feeding the high-fat animal model by using a high-fat feed, and when the weight gain of the animal after 6w is 20% higher than the weight before the experiment, the obese mouse is considered to be successfully modeled.

TABLE 2 mouse weight Change

Comparison P between left and blank groups < 0.05

Comparison of # with model group P < 0.05

2. Influence on mouse body fat

The experiment was carried out until the last day after week 6, and body fat, muscle, free water, and total water content of each group of mice were measured using a living animal constitution analyzer EchoMRI-1100H. The results showed that the body fat content of the high-fat diet mice was significantly higher than that of the blank group and the sample group, while there was no significant difference in muscle, free water and total water, and it was presumed that the body weight difference was probably caused by the difference in fat content. All indexes of the sample group are obviously different from those of the model group, and the percentage of the fat, the muscle, the free water and the total water accounting for the body weight is calculated to find that the contents of the muscle, the free water and the total water are not obviously different (P is more than 0.05) except the fat content, which indicates that other body indexes are not obviously influenced while the body fat of the mice of the sample group is reduced.

TABLE 3 mouse body fat analysis

Note: compared among groups, the marked letters on the same line result indicate that the groups have significant difference, and P is less than 0.05.

3. Conditions of liver, kidney and epididymal fat in mice

After the physical experiment is completed, mice in each group are anesthetized on the same day (10% chloral hydrate is injected into the abdominal cavity), then the eyeballs are picked up to take blood, the whole blood is kept stand for 30min at room temperature, centrifugation is carried out for 15min at 3500r/min at 4 ℃, and supernatant serum is taken and stored in a refrigerator at minus 80 ℃. Mice were sacrificed by cervical dislocation and intact liver, kidney and epididymal adipose tissues were removed. After the dry interstitial fluid was cleared with filter paper, it was weighed and recorded.

TABLE 4 weight of liver, kidney and epididymal fat in mice

Note: compared among groups, the marked letters on the same line result indicate that the groups have significant difference, and P is less than 0.05.

The anatomical results show that the weight of the liver and the epididymal fat of the model group of mice is obviously higher than that of the other two groups of mice due to the accumulation of the fat, and the weight of the kidney is not obviously different, which indicates that the high-fat diet mainly increases the accumulation of the liver and the epididymal fat.

4. Liver pathological section

As can be seen from FIGS. 2 and 6, when the pathological section images of the livers of the experimental mice in each group are compared, the hepatocytes of the animals in the blank group are not changed in any pathological way, as shown in FIG. 2; model group, fatty vacuolar degeneration of a large number of hepatocytes visible around the central veins of the livers of animals, see fig. 3; through the low dose group, sorghum smut protein WM-P₁After treatment, the degeneration degree of the animal liver cells is obviously reduced, and only a small amount of liver cells have vacuolar degeneration, which is shown in figure 4; through the medium dose group, sorghum smut protein WM-P₁After the intervention treatment, the animal hepatocytes still had clearly visible fat vacuoles, as shown in fig. 5; high dose group, Sphaerotheca fuliginea protein WM-P₁The liver cells of the treated animals have no obvious vacuole, as shown in figure 6, and the result shows that the sorghum smut protein WM-P₁The intervention can reverse the state of the fatty degeneration of the liver cells of the experimental animal and is concentration-dependent.

5. Enzymatic assay of serum

An automatic blood analyzer is used to detect routine items of blood. Four blood biochemical indexes of TC, TG and HDL-C, LDL-C are detected by using a 7180 Hitachi automatic biochemical analyzer and a kit.

As shown in FIGS. 7 and 8, TC and TG were observed in the blood of the mice.

Low, medium and high dose groups of ustilago sorghum protein WM-P compared to the model group₁Significantly reduced levels of TC and TG (P)<0.05). Presumably, the sorghum smut protein WM-P₁Has the effects of reducing total cholesterol and total triglyceride of fat mice caused by high fat diet, and is dose-dependent.

The HDL and LDL levels in the blood of the mice are shown in FIGS. 9 and 10.

Low, medium and high dose groups of ustilago sorghum protein WM-P compared to the model group₁Has a significantly increased HDL level and a significantly decreased LDL level (P)<0.05) and has a clear dose-effect relationship. It is speculated that the low, medium and high dose groups of ustilago sorghum protein WM-P₁Has good cholesterol regulating effect on fat mice caused by high fat diet.

6 p-AMPK protein expression in liver tissue

As shown in FIG. 11, the sorghum smut protein WM-P was obtained 6 weeks after gavage₁For the expression quantity of the mouse liver gluconeogenesis key protease P-AMPK protein, compared with the model group, the content of the P-AMPK in the low-dose group and the medium-dose combination high-dose group can be obviously increased (P)<0.05), i.e. capable of increasing p-AMPK expression. Experiments prove that the sorghum smut protein WM-P₁AMPK is phosphorylated in animals and activated, and thus it is presumed that it can interfere with lipid metabolism in obese mice.