阅读说明：本技术 高α-淀粉酶抑制活力芸豆提取物及其制备方法和应用 (Kidney bean extract with high alpha-amylase inhibition activity and preparation method and application thereof ) 是由 刘丽娅 佟立涛 周素梅 王丽丽 汪云吉 周闲容 于 2020-12-07 设计创作，主要内容包括：本发明公开了无凝集活力的高α-淀粉酶抑制活力芸豆提取物的制备方法,包括依次进行的如下步骤：在pH2-4和温度30-70℃条件下,利用蛋白酶处理芸豆水溶液0.5-2h,之后回调pH并取上清液；将上清液处理得到粉末状的芸豆α-淀粉酶抑制剂提取物。本发明还公开了无凝集活力的高α-淀粉酶抑制活力芸豆提取物。本发明还公开了酸性水解酶于无凝集活力的高α-淀粉酶抑制活力芸豆提取物的提取中的应用。本发明可以在保留α-淀粉酶抑制活力的同时去除凝集素活力,还具有一定的体外抗氧化能力,操作简单,经济价值高,易于工艺化生产,在满足普通消费者控制体重的同时,也可以满足糖尿病患者的辅助治疗需求。(The invention discloses a preparation method of a kidney bean extract with high alpha-amylase inhibition activity and no agglutination activity, which comprises the following steps in sequence: treating kidney bean water solution with protease at pH2-4 and temperature 30-70 deg.C for 0.5-2h, adjusting pH, and collecting supernatant; treating the supernatant to obtain a powdered kidney bean alpha-amylase inhibitor extract. The invention also discloses a kidney bean extract with high alpha-amylase inhibition activity and no agglutination activity. The invention also discloses application of the acid hydrolase in extraction of the kidney bean extract with high alpha-amylase inhibition activity and no agglutination activity. The invention can remove the activity of agglutinin while keeping the inhibition activity of alpha-amylase, has certain in vitro oxidation resistance, simple operation, high economic value and easy process production, and can meet the requirement of auxiliary treatment of patients with diabetes while meeting the requirement of controlling weight of common consumers.)

1. The preparation method of the kidney bean extract with high alpha-amylase inhibition activity and no agglutination activity is characterized by comprising the following steps of:

treating kidney bean water solution with protease at pH2-4 and temperature 30-70 deg.C for 0.5-2h, adjusting pH, and collecting supernatant;

and treating the supernatant to obtain a powdery kidney bean alpha-amylase inhibitor extract.

2. The method for preparing kidney bean extract with high alpha-amylase inhibitory activity and no agglutination activity as claimed in claim 1, wherein the amount of protease is 1000-1500U/g sample.

3. The method for preparing kidney bean extract with high alpha-amylase inhibitory activity and no agglutination activity according to claim 1, wherein said protease is acid protease.

4. The method for preparing kidney bean extract with high alpha-amylase inhibition activity and no agglutination activity according to claim 1, wherein the method for preparing the kidney bean aqueous solution comprises the following steps:

step one, pretreatment of kidney beans: preparing kidney beans into 40-80-mesh kidney bean powder, extracting the kidney bean powder with 400-1000mL of distilled water at the stirring speed of 200-300rpm at room temperature for 1-4h, and filtering to remove residues to obtain filtrate;

and step two, standing the obtained filtrate at room temperature, centrifuging and taking supernatant fluid to obtain the kidney bean water solution.

5. The method for preparing kidney bean extract with high alpha-amylase inhibitory activity and no agglutination activity according to claim 4, wherein the step two further comprises the following steps: and performing microwave treatment on the supernatant for 10-12min under the pressure of 40-50MPa, wherein the microwave power is 80-100W, so as to obtain the kidney bean aqueous solution.

6. The method for preparing the kidney bean extract with high alpha-amylase inhibitory activity and without agglutination according to claim 1, wherein the kidney bean alpha-amylase inhibitor extract is obtained in powder form by drying the supernatant and then performing cyclone milling, wherein the drying method comprises freeze drying and spray drying.

7. The method for preparing kidney bean extract with high alpha-amylase inhibitory activity and without aggregation activity according to claim 1, wherein the kidney bean is a kidney bean having flower shape.

8. Kidney bean extract with high alpha-amylase inhibition activity and no agglutination activity, characterized in that the kidney bean extract with high alpha-amylase inhibition activity is prepared by the method of any one of claims 1 to 7.

9. The application of acid hydrolase in the extraction of kidney bean extract with high alpha-amylase inhibition activity and no agglutination activity.

Technical Field

The invention belongs to the technical field of preparation of plant active ingredients, and relates to a preparation method of a kidney bean extract with high alpha-amylase inhibition activity and no agglutination activity.

Background

Kidney beans, the scientific name kidney beans (Phaseolus vulgaris lin.sp), belong to the genus Phaseolus (Phaseolus L.) of the family Leguminosae (Leguminosae), are widely planted and are one of the major beans consumed in the world. The kidney bean cultivation area of China is about 800 ten thousand mu year round, the total output is about 80-90 ten thousand tons, and the kidney bean cultivation area is mainly distributed in black, inner Mongolia, Ji, jin, sweet, New, Sichuan, Dian, Qian and other places.

Kidney beans contain a glycoprotein, an alpha-amylase inhibitor, which is a pure natural active substance, belongs to one of glycoside hydrolase inhibitors, and has been proved to effectively inhibit amylase activity, hinder carbohydrate hydrolysis and digestion, and reduce human body weight by forming an enzyme-inhibitor complex with enzymes. Purified kidney bean extracts have been shown to reduce blood glucose levels in humans and experimental animals. In addition, the kidney bean is rich in natural antioxidant substances, such as polyphenol and flavonoid compounds, and the active substances have the effects of resisting inflammation, resisting cancer, preventing cardiovascular diseases and the like. However, kidney beans also contain lectins, which have the ability to agglutinate human red blood cells, can destroy the integrity of intestinal epithelial cells, influence the digestive absorption of nutrients, and even induce the growth of pancreas, causing ulcer and necrosis of intestinal epithelial cells. Food poisoning may result from consumption of raw kidney beans, which are generally cooked to denature lectins for the purpose of consumption in daily life. Since both the alpha-amylase inhibitor and the lectin are proteins and the thermal stability of the alpha-amylase inhibitor is lower than that of the lectin, the alpha-amylase inhibitor is denatured and loses the inhibitory activity while the kidney beans are subjected to heat treatment to remove the activity of the lectin.

In 2019, about 4.63 million adults aged 20-79 years worldwide suffer from diabetes (1 out of 11 people is diabetic). It is estimated that by 2030, diabetics will reach 5.784 billion; it is expected that by 2045 years, diabetics will reach 7.002 billion. China is the country with the most diabetic patients, the number of the diabetic patients in the age of 20-79 years is about 1.164 hundred million, and the disease trend is gradually younger. Therefore, research and development of products for preventing and controlling diabetes mellitus have high social value and economic value.

The extraction method of the pure kidney bean alpha-amylase inhibitor depends on a chromatographic column, the process is complex, the time consumption is long, the economic value is not high, the urgent needs of the current society cannot be met, relatively speaking, the preparation method of the kidney bean crude extract is simple, the kidney bean crude extract has the effect of inhibiting alpha-amylase, but the extract is a mixture containing the lectin harmful to human bodies, and on the basis, an appropriate method is urgently needed, so that the activity of the lectin can be reduced while the alpha-amylase inhibiting effect of the extract is kept to the maximum extent.

Disclosure of Invention

An object of the present invention is to solve at least the above problems and/or disadvantages and to provide at least the advantages described hereinafter.

It is still another object of the present invention to provide a method for preparing a kidney bean extract having high alpha-amylase inhibitory activity without aggregation activity.

Another object of the present invention is to provide the use of acid hydrolase in the extraction of kidney bean extracts with high alpha-amylase inhibitory activity without aggregation activity.

It is still another object of the present invention to provide kidney bean extract having high alpha-amylase inhibitory activity without aggregation activity.

Therefore, the technical scheme provided by the invention is as follows:

the preparation method of the kidney bean extract with high alpha-amylase inhibition activity and no agglutination activity comprises the following steps in sequence:

treating kidney bean water solution with protease at pH2-4 and temperature 30-70 deg.C for 0.5-2h, adjusting pH, and collecting supernatant;

and treating the supernatant to obtain a powdery kidney bean alpha-amylase inhibitor extract.

Preferably, in the preparation method of the kidney bean extract with high alpha-amylase inhibition activity and no agglutination activity, the amount of the protease is 1000-1500U/g of the sample.

Preferably, in the method for preparing the kidney bean extract with high alpha-amylase inhibitory activity and no agglutination activity, the protease is an acid protease.

Preferably, in the preparation method of the kidney bean extract with high alpha-amylase inhibition activity and no agglutination activity, the preparation method of the kidney bean aqueous solution comprises the following steps:

step one, pretreatment of kidney beans: preparing kidney beans into 40-80-mesh kidney bean powder, extracting the kidney bean powder with 400-1000mL of distilled water at the stirring speed of 200-300rpm at room temperature for 1-4h, and filtering to remove residues to obtain filtrate;

and step two, standing the obtained filtrate at room temperature, centrifuging and taking supernatant fluid to obtain the kidney bean water solution.

Preferably, in the preparation method of the kidney bean extract with high alpha-amylase inhibition activity and no agglutination activity, the following treatment is further included in the step two: and performing microwave treatment on the supernatant for 10-12min under the pressure of 40-50MPa, wherein the microwave power is 80-100W, so as to obtain the kidney bean aqueous solution.

Preferably, in the preparation method of the kidney bean extract with high alpha-amylase inhibition activity and no agglutination activity, the supernatant is dried and then is subjected to cyclone milling to obtain the kidney bean alpha-amylase inhibitor extract in powder, wherein the drying method comprises freeze drying and spray drying.

Preferably, in the preparation method of the kidney bean extract with high alpha-amylase inhibition activity and no agglutination activity, the kidney bean is a flower kidney bean.

Kidney bean extract with high alpha-amylase inhibition activity and no agglutination activity, which is prepared by the method in any one of the above manners.

The application of acid hydrolase in the extraction of kidney bean extract with high alpha-amylase inhibition activity and no agglutination activity.

The invention at least comprises the following beneficial effects:

according to the invention, the kidney bean water extract is treated by the acid protease at a certain pH and temperature, so that the activity of the agglutinin can be removed while the alpha-amylase inhibition activity is kept, a certain in vitro oxidation resistance is provided, the operation is simple, the economic value is high, the process production is easy, the weight control of common consumers is met, and the auxiliary treatment requirement of the diabetic patient can also be met.

Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.

Drawings

FIG. 1 is a graph showing the measurement of lectin activity by the 96-well plate method according to each example of the present invention.

Detailed Description

The present invention is further described in detail below with reference to the attached drawings so that those skilled in the art can implement the invention by referring to the description text.

It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.

The invention provides a preparation method of a kidney bean extract with high alpha-amylase inhibition activity and no agglutination activity, which comprises the following steps in sequence:

treating kidney bean water solution with protease at pH2-4 and temperature 30-70 deg.C for 0.5-2h, adjusting pH, and collecting supernatant;

and treating the supernatant to obtain a powdery kidney bean alpha-amylase inhibitor extract.

In the above scheme, the amount of the protease is preferably 1000-1500U/g of the sample.

In one embodiment of the present invention, preferably, the protease is an acid protease.

In one embodiment of the present invention, preferably, the preparation method for obtaining the kidney bean aqueous solution comprises the following steps:

step one, pretreatment of kidney beans: preparing kidney beans into 40-80-mesh kidney bean powder, extracting the kidney bean powder with 400-1000mL of distilled water at the stirring speed of 200-300rpm at room temperature for 1-4h, and filtering to remove residues to obtain filtrate;

and step two, standing the obtained filtrate at room temperature, centrifuging and taking supernatant fluid to obtain the kidney bean water solution.

In one embodiment of the present invention, preferably, step two further includes the following processing: and performing microwave treatment on the supernatant for 10-12min under the pressure of 40-50MPa, wherein the microwave power is 80-100W, so as to obtain the kidney bean aqueous solution. According to the method, the kidney bean aqueous solution is subjected to microwave treatment under the pressure condition, the kidney bean aqueous solution is better dissolved and more uniform and consistent under the synergistic effect of the pressure and the microwaves, and the inhibition activity of an alpha-amylase inhibitor in the kidney bean extract is remarkably improved.

In one embodiment of the present invention, preferably, the kidney bean alpha-amylase inhibitor extract is obtained in a powder form by performing a cyclone milling process after drying the supernatant, wherein the drying process comprises freeze drying and spray drying.

In one embodiment of the present invention, preferably, the kidney bean is a flower kidney bean.

The invention also provides a kidney bean extract with high alpha-amylase inhibition activity and no agglutination activity, and the kidney bean extract with high alpha-amylase inhibition activity is prepared by the method as described in any one of the above.

The invention also provides application of the acid hydrolase in extraction of the kidney bean extract with high alpha-amylase inhibition activity and no agglutination activity.

In order to make the technical solutions of the present invention better understood, the present invention is further described in detail with reference to the following examples, but the scope of the present invention is not limited to the following examples:

the invention provides a preparation method of kidney bean alpha-amylase inhibitor extract without agglutination activity, which comprises the steps of treating a flower kidney bean water extract by acid protease under the conditions of certain pH and temperature, removing agglutinin, simultaneously retaining alpha-amylase inhibition activity, and simultaneously having a certain in vitro oxidation resistance;

the method specifically comprises the following steps:

s1, pretreating kidney beans, grinding, sieving, extracting with water, and filtering to remove residues to obtain filtrate;

s2, standing the obtained filtrate at room temperature to fully settle the starch, and centrifuging to obtain a supernatant;

s3, adjusting the pH value of the filtrate, adding protease, carrying out water bath at a certain temperature, adjusting the pH value back, and centrifuging to obtain a supernatant;

and S4, drying the obtained supernatant, and performing cyclone grinding to obtain a powdery kidney bean alpha-amylase inhibitor extract.

Optionally, in step S1, the kidney bean pretreatment refers to 20-80 mesh flower kidney bean powder, and the kidney bean powder is extracted with water at room temperature at a stirring speed of 200-300rpm for 1-4h, with an amount of 400-1000mL of distilled water per 100g of kidney bean powder. Filtering with 8 layers of gauze to remove residue to obtain filtrate.

Optionally, in step S2, performing microwave treatment on the supernatant at a pressure of 40-50MPa for 10-12min, wherein the microwave power is 80-100W, so as to obtain the kidney bean aqueous solution.

Alternatively, in step S3, the pH may be adjusted with an acid such as hydrochloric acid, and the final pH adjustment may be between pH2 and pH 4.

Optionally, in step S3, the enzyme added to the aqueous extract of kidney bean is a protease such as acid protease.

Optionally, in step S3, the amount of the enzyme for digesting lectin added is 500-1500U/g sample and higher.

Optionally, the water bath temperature in step S3 may be between 30-70 ℃ and the water bath time may be between 0.5-2 h.

Alternatively, in step S4, the drying includes freeze drying and spray drying but is not limited to these two drying methods.

The present disclosure provides a method for preparing a phaseolus vulgaris alpha-amylase inhibitor extract without agglutination activity, which is prepared using the above method.

According to the technical scheme, the kidney bean extract prepared by the method adopts the acid protease to treat the kidney bean water extract at a certain pH and temperature, can remove the activity of agglutinin while keeping the alpha-amylase inhibition activity, also has a certain in vitro oxidation resistance, is simple to operate, high in economic value and easy for process production, and can meet the requirement of the common consumers on weight control and the auxiliary treatment of the diabetic patients.

The methods described in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like, if not specifically mentioned, are commercially available.

Determination of alpha-amylase inhibition activity of kidney bean extract:

properly diluting the kidney bean extract, adding 0.25mL of alpha-amylase solution (1.5U/mL) and 0.25mL of diluted sample to be detected into 0.5mL of PBS (pH6.9, 100mmol of phosphate buffer solution, the same below), adding 0.25mL of 1% (w/w) soluble starch solution after water bath at 37 ℃ for 10min, accurately reacting for 5min, adding 1mL of DNS reagent, cooling in an ice bath after boiling water bath for 10min, adding 5mL of deionized water, and measuring the light absorption value at the wavelength of 540 nm. In the measurement process, blank tubes, blank control tubes, and inhibition control tubes were set, and the measurement system is shown in table 1.

Table 1: activity measuring system for alpha-amylase inhibitor

The alpha-amylase inhibition ratio of the kidney bean extract can be calculated according to the following formula:

inhibition ratio (%) - (1- (a)₃-A₄)/(A₁-A₂))*100％

In the formula A₁、A₂、A₃、A₄The absorbance values of blank tube, blank control tube, inhibition tube and inhibition control tube at 540nm respectively.

Measuring the agglutination activity of the kidney bean extract:

the assay was performed by 96-well hemagglutination plate method, 50. mu.L PBS (10mmol, pH7.4 phosphate buffer, the same as below) was added to each well, 50. mu.L of the extract was added to the first well of each row, mixed well, 50. mu.L of the mixture was aspirated from the 1 st well, added to the 2 nd well, mixed well, 50. mu.L of the mixture was aspirated from the 2 nd well, added to the 3 rd well, mixed well, and so on, diluted in multiples, 50. mu.L of the mixture was aspirated from the 12 th well, and discarded. mu.L of rabbit red blood cell suspension was added to each well, and the mixture was allowed to stand at 4 ℃ for 1.5 hours to observe the coagulation results.

The kidney bean extract agglutination activity can be calculated according to the following formula:

(2) agglutination activityⁿ)/(C_(pr)*V)(n≥2)

Wherein n represents the maximum number of wells showing hemagglutination in a 96-well plate, C_(pr)Represents the protein concentration of the sample in (mg/mL), and V represents the volume of the sample added in (mL)

In vitro antioxidant capacity determination:

measurement of DPPH.radical scavenging ability: 1mL of the appropriately diluted extract or standard was added to a centrifuge tube containing 1mL of DPPH working solution (0.2mmol/L) in a total volume of 2 mL. The mixture was reacted at room temperature for 30 minutes in the absence of light, and then the absorbance was measured at 517 nm. The results are expressed as water-soluble VE equivalents (. mu.mol TE/g DW).

Determination of ABTS +. free radical scavenging energy: mu.L of the appropriately diluted sample extract or standard was mixed with 2mL of the ABTS working solution (7mmol of ABTS solution was mixed with 2.45mmol of potassium persulfate solution (1: 1; v: v), the mixture was left in the dark for 12 hours to give an absorbance at 734nm of 0.70. + -. 0.05), reacted at room temperature with exclusion of light for 30 minutes, and the absorbance at 734nm was read. The results are expressed as water-soluble VE equivalents (. mu.mol TE/g DW).

Determination of iron ion reduction oxidation resistance FRAP: mu.L of an appropriately diluted sample extract or standard was mixed with 1.8mL of FRAP reagent (0.3mol sodium acetate buffer: 20mmol FeCl)₃Solution: 10mmol TPTZ solution ═ 10: 1: 1; v: v: v) mixing, reacting for 10 minutes under 37 ℃ water bath condition, and reading the absorbance at 593 nm. Results expressed as μmolFe (II) equivalents (μmolFe (II)/g DW)

Example 1

Crushing five common kidney beans (black kidney bean, white kidney bean, milky kidney bean, red kidney bean and flower kidney bean) by a cyclone mill, and sieving by a 60-mesh sieve, wherein the material-liquid ratio is 1: 5(w/v) extracting with deionized water at room temperature for 2h, stirring at 200rpm during extraction, filtering with 8 layers of gauze to remove residue after extraction is completed to obtain filtrate, standing the filtrate at room temperature for 1h, collecting supernatant after starch precipitation, centrifuging at 10000rpm for 30min, collecting supernatant, freezing at-20 ℃, and freeze-drying.

Table 2 shows the results of measurements of alpha-amylase inhibition and in vitro antioxidant capacity of 5 different kidney beans. The data show that the inhibitor rates of the red kidney bean and the flower kidney bean on alpha-amylase are the highest and the difference is not obvious, and the inhibitor rates of the black kidney bean, the white kidney bean and the milk flower kidney bean are lower; the white kidney bean has the lowest in-vitro antioxidant capacity, the milky flower kidney bean has the highest in-vitro antioxidant capacity, and by combining the results, the flower kidney bean has the highest alpha-amylase inhibition rate and higher antioxidant capacity, so the flower kidney bean is selected as the best variety for extraction.

TABLE 25 inhibition ratio and in vitro antioxidant capacity of kidney bean alpha-amylase

	Black kidney bean	White kidney bean	Daihua kidney bean	Red kidney bean	Flower kidney bean
						Alpha-amylase inhibition (%)	53.58	53.50	52.54	60.20	60.17
DPPH(μmol TE/g DW)	12.52	3.46	17.93	10.24	11.47
						ABTS(μmol TE/g DW)	45.98	13.09	49.77	28.55	33.84
FRAP(μmol Fe(II)/g DW)	20.61	9.11	27.53	16.94	19.43

Example 2

Crushing the kidney bean in a cyclone mill, and sieving the crushed kidney bean with a 60-mesh sieve according to a material-liquid ratio of 1: 5(w/v) extracting with deionized water at room temperature for 2h, stirring at 200rpm during extraction, filtering with 8 layers of gauze to remove residue after extraction is completed to obtain filtrate, standing the filtrate at room temperature for 1h, collecting supernatant after starch precipitation, centrifuging at 10000rpm for 30min, collecting supernatant, freezing at-20 ℃, and freeze-drying.

Example 3

Crushing kidney beans by using a universal mill, and sieving by using a 60-mesh sieve, wherein the material-liquid ratio is 1: 5(w/v) extracting with deionized water at room temperature for 2h, stirring at 200rpm during extraction, filtering with 8 layers of gauze to remove residues after extraction is completed, obtaining filtrate, standing the filtrate at room temperature for 1h, taking supernatant after starch precipitation, centrifuging at 10000rpm for 30min, taking supernatant, adjusting pH to 3 with 1M hydrochloric acid, carrying out water bath at 70 ℃ for 1h, cooling in ice bath, adjusting pH back to 6.4 with 1M NaOH, centrifuging at 10000rpm for 30min, taking supernatant, freezing at-20 ℃, and freeze-drying.

Example 4

Crushing kidney beans by using a universal mill, and sieving by using a 60-mesh sieve, wherein the material-liquid ratio is 1: 5(w/v) extracting with deionized water at room temperature for 2h, stirring at 200rpm during extraction, filtering with 8 layers of gauze to remove residues after extraction is completed, obtaining filtrate, standing the filtrate at room temperature for 1h, taking supernatant after starch precipitation, centrifuging at 10000rpm for 30min, taking supernatant, adjusting pH to 3 with 1M hydrochloric acid, adjusting the addition amount of acid protease to 1000U/g, carrying out water bath at 50 ℃ for 1h, cooling in ice bath, adjusting pH to 6.4 with 1M NaOH, centrifuging at 10000rpm for 30min, taking supernatant, freezing at-20 ℃, and freeze-drying.

Example 5

Crushing kidney beans by using a universal mill, and sieving by using a 60-mesh sieve, wherein the material-liquid ratio is 1: 5(w/v) extracting with deionized water at room temperature for 2h, stirring at 200rpm during extraction, filtering with 8 layers of gauze to remove residues after extraction is completed, obtaining filtrate, standing the filtrate at room temperature for 1h, taking supernatant after starch precipitation, centrifuging at 10000rpm for 30min, taking supernatant, adjusting pH to 3 with 1M hydrochloric acid, adjusting the addition amount of acid protease to 1000U/g, carrying out water bath at 50 ℃ for 2h, cooling in ice bath, adjusting pH to 6.4 with 1M NaOH, centrifuging at 10000rpm for 30min, taking supernatant, freezing at-20 ℃, and freeze-drying.

Example 6

Crushing kidney beans by using a universal mill, and sieving by using a 60-mesh sieve, wherein the material-liquid ratio is 1: 5(w/v) extracting with deionized water at room temperature for 2h, stirring at 200rpm during extraction, filtering with 8 layers of gauze to remove residues after extraction is completed, obtaining filtrate, standing the filtrate at room temperature for 1h, taking supernatant after starch precipitation, centrifuging at 10000rpm for 30min, taking supernatant, adjusting pH to 3 with 1M hydrochloric acid, adjusting the addition amount of acid protease to 1000U/g, carrying out water bath at 60 ℃ for 1h, cooling in ice bath, adjusting pH to 6.4 with 1M NaOH, centrifuging at 10000rpm for 30min, taking supernatant, freezing at-20 ℃, and freeze-drying.

Example 7

Crushing kidney beans by using a universal mill, and sieving by using a 60-mesh sieve, wherein the material-liquid ratio is 1: 5(w/v) extracting with deionized water at room temperature for 2h, stirring at 200rpm during extraction, filtering with 8 layers of gauze to remove residues after extraction is completed, obtaining filtrate, standing the filtrate at room temperature for 1h, taking supernatant after starch precipitation, centrifuging at 10000rpm for 30min, taking supernatant, adjusting pH to 3 with 1M hydrochloric acid, adjusting the addition amount of acid protease to 1000U/g, carrying out water bath at 60 ℃ for 2h, cooling in ice bath, adjusting pH to 6.4 with 1M NaOH, centrifuging at 10000rpm for 30min, taking supernatant, freezing at-20 ℃, and freeze-drying.

Example 8

Crushing kidney beans by using a universal mill, and sieving by using a 60-mesh sieve, wherein the material-liquid ratio is 1: 5(w/v) extracting with deionized water at room temperature for 2h, stirring at 200rpm during extraction, filtering with 8 layers of gauze to remove residues after extraction is completed, obtaining filtrate, standing the filtrate at room temperature for 1h, taking supernatant after starch precipitation, centrifuging at 10000rpm for 30min, taking supernatant, adjusting pH to 3 with 1M hydrochloric acid, adjusting the addition amount of acid protease to 1000U/g, carrying out water bath at 70 ℃ for 1h, cooling in ice bath, adjusting pH to 6.4 with 1M NaOH, centrifuging at 10000rpm for 30min, taking supernatant, freezing at-20 ℃, and freeze-drying.

Example 9

Crushing kidney beans by using a universal mill, and sieving by using a 60-mesh sieve, wherein the material-liquid ratio is 1: 5(w/v) extracting with deionized water at room temperature for 2h, stirring at 200rpm during extraction, filtering with 8 layers of gauze to remove residues after extraction is completed, obtaining filtrate, standing the filtrate at room temperature for 1h, taking supernatant after starch precipitation, centrifuging at 10000rpm for 30min, taking supernatant, adjusting pH to 3 with 1M hydrochloric acid, adjusting the addition amount of acid protease to 1000U/g, carrying out water bath at 70 ℃ for 2h, cooling in ice bath, adjusting pH to 6.4 with 1M NaOH, centrifuging at 10000rpm for 30min, taking supernatant, freezing at-20 ℃, and freeze-drying.

Example 10

Crushing kidney beans by using a universal mill, and sieving by using a 80-mesh sieve, wherein the material-liquid ratio is 1: 10(W/v) extracting with deionized water at room temperature for 1h, stirring at the speed of 200rpm in the extraction process, filtering with 8 layers of gauze to remove residues after extraction is finished, obtaining filtrate, standing the filtrate at room temperature for 1h, taking supernatant after starch precipitation, centrifuging at 10000rpm for 30min, taking supernatant, and performing microwave treatment on the supernatant at the pressure of 40MPa for 12min, wherein the microwave power is 100W, thereby obtaining the kidney bean aqueous solution. Then adjusting pH to 2 with 1M hydrochloric acid, adding 1500U/g acid protease, water-bathing at 30 deg.C for 0.5h, cooling in ice bath, adjusting pH to 6.4 with 1M NaOH, centrifuging at 10000rpm for 30min, collecting supernatant, freezing at-20 deg.C, and lyophilizing.

The extracts obtained in the above examples were subjected to measurement of α -amylase inhibitory activity and agglutination activity, and the inhibitory activities were converted into relative inhibition ratios for comparison. The results are shown in Table 3.

Table 3: relative inhibition activity and agglutination activity of kidney bean extract alpha-amylase after different treatments

Note: relative inhibition rate of extract after treatment/inhibition activity of untreated extract

Relative agglutination activity after treatment/agglutination activity of untreated extract

DPPH and ABTS scavenging ability expressed as μmol TE/g DW

FRAP antioxidant ability is expressed in μmol Fe (II)/g DW

Table 2 and fig. 1 show that the kidney bean extract prepared by using the present disclosure has an agglutination activity of almost 0, and the inhibition ratio is maintained at about 81.87%, while the treatment conditions have no significant effect on the in vitro antioxidant capacity.

The use, modification and variation of the kidney bean extract having high alpha-amylase inhibitory activity without agglutination activity and the preparation method of the present invention will be apparent to those skilled in the art.

While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable in various fields of endeavor to which the invention pertains, and further modifications may readily be made by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein without departing from the general concept defined by the appended claims and their equivalents.