CN107988301B

CN107988301B - Preparation method and application of chickpea bean cotyledon polypeptide

Info

Publication number: CN107988301B
Application number: CN201810025491.3A
Authority: CN
Inventors: 阿布力米提·伊力; 艾合米丁·外力; 阿吉艾克拜尔·艾萨; 高彦华; 夏热菲鼎·米日扎艾合米多瓦
Original assignee: Xinjiang Technical Institute of Physics and Chemistry of CAS
Current assignee: Xinjiang Technical Institute of Physics and Chemistry of CAS
Priority date: 2018-01-11
Filing date: 2018-01-11
Publication date: 2021-04-27
Anticipated expiration: 2038-01-11
Also published as: CN107988301A

Abstract

The invention relates to a preparation method and application of chickpea bean cotyledon polypeptide, wherein the method comprises the steps of fully degreasing grease components of chickpea bean cotyledon, extracting by using a phosphoric acid buffer solution, hydrolyzing by using four enzymes of neutral protease, alkaline protease, trypsin or papain, preparing polypeptide with molecular weight of 6-10kDa, 3.5-6kDa and 1-3.5kDa by using an ultrafiltration membrane technology, and then refining by using spray drying or freeze drying. The polypeptide powder is proved to have antibacterial and fungal activities, has wide antimicrobial activity, has the functions of enhancing humoral immunity and systemic immunity, and is rich in polypeptide and protein which are easily digested and absorbed by human bodies and are purely natural and plant-derived. The protein is characterized in that the original bioactivity of the chickpea is kept, and the purposes of changing waste into valuable and fully utilizing resources are realized; the protein can be used as natural antibacterial agent, food additive or medicine, and can be used in food and medicine industry.

Description

Preparation method and application of chickpea bean cotyledon polypeptide

Technical Field

The invention relates to a preparation method and application of chickpea bean cotyledon polypeptide.

Background

Chickpeas (Cicer arietinum L.), also known as peach beans, chicken peas, leguminous genus Cicer, a representative plant of leguminous subfamily Papilionaceae, annual or perennial herbaceous plants, are named because the shape of the hawk head has a coracoid protrusion near the hilum. Chickpeas are native to the southern Siberian ear, are imported into Xinjiang in China as early as 2500 years ago, are cultivated all over the world at present, are mainly distributed in arid and semiarid regions such as the west, the north and the like, are very drought-resistant and barren and have the nitrogen fixation effect. The statistical data of FAO in 1994 shows that the planting area of chickpeas accounts for 15% of the total planting area of economic crops in the whole world, is about 1020 ten thousand hectares, has the yield of 7900 ten thousand tons, accounts for 14% of the total yield, belongs to the second most consumed beans in the world, and has the yield behind soybeans and peas and is third to beans in the world.

The chickpeas contain abundant nutritional ingredients, which greatly exceed other beans in terms of variety and quantity, and are natural plant protein resources. The protein content is about 15% -30% of dry weight of the grains, and is similar to fish, meat and eggs, and twice of oat. The protein efficacy ratio is about 2.8, which is far higher than that of pea, soybean, kidney bean and other bean products, and the digestibility can reach 79-88%. The chickpea protein has high amino acid content and balanced composition, comprises 18 amino acids and 8 essential amino acids which are necessary for human bodies, and contains a large amount of lysine which has the effects of promoting the growth of bones and the intelligence development of children, so the chickpea protein can be considered as a good amino acid supplement, has higher medical health care value and has immeasurable effect on the body building of children and middle-aged and old people; in addition, the chickpeas are rich in edible fiber, trace elements, vitamins and a plurality of active substances with different functions, wherein the content of calcium is as high as 0.35g/100g, phosphorus is 0.32g/100g, the content of vitamins is as high as 0.013g/100g, the content of iron is 0.047g/100g which is higher than that of most beans, and the content of iron is 90% higher than that of other beans.

Chickpea is a Chinese medicine and traditional Chinese medicine of Viburnum, can clear away heat and toxic materials and is mainly used for treating diabetes, hepatitis and beriberi. In addition, the chickpea also has the pharmacological activities of resisting osteoporosis, resisting oxidation, reducing blood sugar and blood fat, regulating cholesterol level and the like. The semen Ciceris Arietini isoflavone can also prevent proliferation of cancer cells, and has effect in promoting cancer cell death, especially gonad related cancer such as breast cancer, prostate cancer, etc.

With the development of economy, people solve the problems of satiety, improvement of living standard, change of dietary structure, increase of rich diseases and increase of senile diseases. In developed areas, the pace of life is accelerated, the working pressure is increased, sub-health people are increased, and people begin to pay attention to health care. Modern nutritional studies have demonstrated that: in the diet, major nutrients (glucose, fat, amino acids) and micronutrients (e.g., iron, vitamins) coordinate rapidly with gene expression involved in regulation and nutritional changes, and functional foods are produced in this context. Uygur medicine commonly uses medicine and food dual-purpose medicinal material chickpea (Cicer arietinum L.) has the functions of eliminating abnormal body fluid, opening body fluid block, regulating organism and the like. Can be used for treating emaciation, hyposexuality, anorexia, skin pruritus and diabetes. The chickpeas have balanced nutrition proportion, are easy to digest and absorb by human bodies, have fewer anti-nutritional factors, and are known as one of the most healthy foods in the world. In India, Pakistan and Utziblestan, chickpea is an important food and vegetable, and some special nutritional foods taking chickpea as raw materials are developed abroad and are mainly used for diet conditioning of people with high blood sugar and blood fat. In europe, the product of chickpeas is used as a food supplement for diabetics, insulin-resistant patients and hypoglycemic patients.

On the basis of earlier-stage theories and research works of Xinjiang physicochemical technology research of the Chinese academy of sciences, the extraction separation and preparation process research of functional factors such as flavone and saponin are carried out on the chickpeas which are specific resources of Xinjiang, the preparation production line is established, and a national health food production batch with independent intellectual property rights is obtained to realize industrialization;

at present, there are several enterprises engaged in producing chickpea series products in Xinjiang, and the enterprises which have been developed and produced the chickpea products include Xinjiang Dalong food company, Wu mu qi Arman commercial company, Aksu Shuishi Yanshan Saint spring agricultural technology company, and the like;

the Dalong food finite responsibility company starts to cooperate with Xinjiang physicochemical technology research institute of Chinese academy of sciences as early as 2002, starts to produce, process and sell chickpea series products in a production management mode of company + scientific research + base, and has advantages in the aspects of chickpea raw material supply, research and development force, new product development force, equipment and tooling, production technology and the like.

Through the on-site investigation of the people, the enterprises with the chickpeas generate about 10 tons of chickpeas by-products each year during the processing, packaging, classification and the like, which is a great waste of the chickpeas resource rarely in Xinjiang;

therefore, it is very practical to extract and separate the protein and polypeptide in the chickpea bean cotyledon and combine the distinctive and rich nutrient component to develop the polypeptide powder with the antimicrobial function.

Chickpea protein is an important source of vegetable protein due to its balanced amino acid composition, high bioavailability and low anti-nutritional factor, and is also a good resource for obtaining bioactive peptides. When hydrolyzed by proper protease, the activity is released, which is not only easy to digest and absorb, but also has various functions of regulating metabolism and physiological functions of human body, and is a natural resource treasure house for developing functional foods and screening medicines. In China, chickpea is a natural plant protein resource, rich in nutrition and has multiple physiological functions, is a Uygur nationality medicine and a traditional Chinese medicine, and chickpea peptide also becomes one of the research hotspots of plant-derived active peptides. The nutritional powder can be used as natural emulsifier and food additive in food industry.

The membrane separation technology is an emerging edge discipline established on the basis of polymer materials science. Because the separation process is not heated, has no phase state and chemical change, has the characteristics of simple process, easy automation control and the like, is widely applied to the industries of metal, textile, leather making, paper making, chemical industry, food, biochemistry, medicine, health care, water treatment, national defense and the like, and becomes one of the most important means in the separation science at present. The membrane separation is a technology which utilizes natural or artificial synthetic polymer membranes with selective permeability to transfer mass between membrane phases by means of different driving forces so as to achieve separation, classification, purification and concentration of different components. The membrane separation process can be roughly divided into two types according to the difference of driving force. One is a membrane process that uses pressure as a driving force, such as ultrafiltration; the other is a membrane process using electricity as driving force, and in the field of dairy industry, the main application is a membrane separation process using pressure as driving force. The membrane separation process using pressure as driving force can be divided into the following steps according to the interception capacity of the membrane to different components: reverse Osmosis (RO), Nanofiltration (NF), Ultrafiltration (UF), Microfiltration (MF). Membranes of different pore sizes or molecular weight cut-off may be used depending on the size of the isolate.

The invention develops a method for separating polypeptide by using an ultrafiltration membrane in order to fully utilize local plant resources, realize full utilization of resources and change waste into valuable, and the method is suitable for the preparation industry of separating polypeptide powder by using the ultrafiltration membrane technology of chickpea bean petal protein zymolyte in industrial production; the implementation of the patent promotes product varieties, expands social employment and drives sustainable development of local economy; the method has great promotion effects on improving the comprehensive utilization rate of dominant natural resources in the Muramon county and even the east three counties of Changji state, promoting the development of pollution-free characteristic agricultural production, increasing the income of farmers, increasing the economic benefit of enterprises, building a harmonious society, promoting the construction of new rural areas and the like.

Disclosure of Invention

The invention aims to provide a preparation method and application of chickpea bean cotyledon polypeptide, wherein fat and oil components of chickpea bean cotyledon are fully degreased, extracted by phosphoric acid buffer solution, hydrolyzed by four enzymes of neutral protease, alkaline protease, trypsin or papain, and prepared into polypeptide with molecular weight of 6-10kDa, 3.5-6kDa and 1-3.5kDa by ultrafiltration membrane technology, and then spray-dried or freeze-dried and refined. The polypeptide powder is proved to have antibacterial and fungal activities, has wide antimicrobial activity, has the functions of enhancing humoral immunity and systemic immunity, and is rich in polypeptide and protein which are easily digested and absorbed by human bodies and are purely natural and plant-derived. The protein is characterized in that the original bioactivity of the chickpea is kept, and the purposes of changing waste into valuable and fully utilizing resources are realized; the protein can be used as natural antibacterial agent, food additive or medicine, and can be used in food and medicine industry.

The preparation method of the chickpea bean cotyledon polypeptide provided by the invention comprises the following steps:

a. crushing the dried bean cotyledon after sprouting and peeling of the chickpeas, sieving with a 80-mesh sieve, adding petroleum ether into w/v bean powder, namely petroleum ether 1:5, oscillating at room temperature for 4h for degreasing, repeatedly extracting for 3-4 times until the bean cotyledon is colorless, naturally settling the chickpea bean cotyledon powder, separating the petroleum ether from the chickpea bean cotyledon powder, pouring out the upper layer of petroleum ether for recycling, combining precipitates, naturally drying to obtain degreased chickpea bean cotyledon powder, and storing in a refrigerator at the temperature of-20 ℃ for later use;

b. b, adding 350ml of 0.1M phosphate buffer solution with the pH value of 7.4 into 100g of the chick pea bean powder degreased in the step a, wherein the material-liquid ratio is 1:3.5, and the temperature is 4 ℃, and extracting for 48 hours;

c. filtering the extracting solution in the step b, centrifuging at 4 ℃ and 10000r/min for 10min at 4 ℃, dialyzing for 48h at 4 ℃, performing freeze-drying at-80 ℃ under the pressure of 9.8Pa, and performing freeze-drying for 24h to obtain 10.27g of crude protein powder, wherein the protein extraction rate is 10.27%;

d. c, performing electrophoresis on the crude protein powder in the step c, and measuring the antibacterial activity and the protein content to be 19.14%;

e. d, hydrolyzing the crude protein powder in the step d by neutral protease, alkaline protease, trypsin or papain to obtain the chickpea bean cotyledon total polypeptide, and then performing ultrafiltration, separation, enzymolysis and freeze drying to obtain polypeptide powder;

f. and e, centrifuging the zymolyte in the step e, taking supernatant, carrying out interception separation on the supernatant by using a 10kDa regenerated fiber filter membrane, keeping the concentrated solution at the temperature of 25 ℃ and the flow rate of 35m/s, carrying out interception separation on the filtrate by using 6kDa and 3.5kDa regenerated fiber filter membranes respectively, sequentially preparing polypeptides with the molecular weights of 6-10kDa, 3.5-6kDa and 1-3.5kDa, taking polypeptide solutions of all parts, carrying out vacuum drying, and detecting the polypeptides with different molecular weights by using a liquid chromatography-mass spectrometry (LC/MS) instrument.

The chickpea bean cotyledon polypeptide obtained by the method is used for preparing a medicine with antibacterial bioactivity.

The chickpea bean petal polypeptide obtained by the method is used for preparing foods with antifungal and biological activities.

The chickpea bean cotyledon polypeptide obtained by the method is used for preparing food additives.

The preparation method and the application of the chickpea bean cotyledon polypeptide have the advantages that the content of chickpea protein in the method is greatly different from 15 to 30 percent due to different varieties. Chickpea protein is an important source of vegetable protein due to its balanced amino acid composition, high bioavailability and low anti-nutritional factor, and is also a good resource for obtaining bioactive peptides. The polypeptide powder is prepared by taking chickpea bean cotyledon dregs as raw materials, crushing, degreasing, extracting total protein by adopting a traditional phosphoric acid buffer solution, hydrolyzing by neutral protease, alkaline protease, trypsin or papain to obtain chickpea bean cotyledon total polypeptide, finally performing ultrafiltration separation on enzymolysis polypeptide, and freeze-drying. Centrifuging the zymolyte, collecting supernatant, intercepting and separating with 10kDa regenerated fiber filter membrane, retaining concentrated solution, intercepting and separating filtrate with 6kDa and 3.5kDa regenerated fiber filter membrane respectively, and sequentially preparing polypeptide with molecular weight of 6-10kDa, 3.5-6kDa and 1-3.5 kDa. And then, taking each part of polypeptide solution for vacuum drying, and detecting the polypeptides with different molecular weights by using a liquid chromatography-mass spectrometry (LC/MS) instrument.

The invention relates to a preparation method and application of chickpea bean cotyledon polypeptide, wherein the polypeptide powder is an optimized process for separating and hydrolyzing polypeptide by ultrafiltration, wherein chickpea bean cotyledon dregs degreased by petroleum ether are used as a raw material, total protein is extracted by adopting a traditional phosphoric acid buffer solution, and then the chickpea bean cotyledon total polypeptide is obtained by hydrolyzing neutral protease, alkaline protease, trypsin and papain. Centrifuging the zymolyte, collecting supernatant, intercepting and separating with 10kDa hollow fiber filter membrane, retaining concentrated solution, intercepting and separating filtrate with 6kDa and 3.5kDa hollow fiber filter membrane respectively, and sequentially preparing polypeptide with molecular weight of 6-10kDa, 3.5-6kDa and 1-3.5 kDa. Then, carrying out vacuum drying on each part of polypeptide solution, measuring the yield of the polypeptides with different molecular weights, and detecting the molecular weights of the four enzyme hydrolysis parts by using a liquid chromatography-mass spectrometry (LC/MS) instrument; hydrolyzing chickpea beans with neutral protease to obtain 21 polypeptides with molecular weight of 1360, 2329, 2330, 2449, 2469, 3186, 3252, 3327, 3436, 3461, 3571, 3611, 3613, 3871, 3956, 4116, 4669, 4751, 4785, 4792, 6287 Da; hydrolyzing chickpea beans with alkaline protease to obtain 23 polypeptides with molecular weight of 1562, 1628, 1741, 1781, 2152, 2216, 2460, 2522, 2574, 3011, 3139, 3303, 3453, 3585, 4297, 4444, 4492, 4668, 4727, 4785, 4946, 5155, 5429Da, etc.; hydrolyzing chickpea bean cotyledon with trypsin to obtain 19 polypeptides with molecular weight of 1154, 1209, 1563, 1674, 2189, 2417, 2460, 2691, 2899, 2988, 3190, 3452, 3567, 3710, 3871, 4231, 4489, 5447, 5801Da, etc.; hydrolysis of chick pea bean cotyledon with papain yields 15 polypeptides with molecular weights of 1050, 1138, 1390, 2640, 2680, 3023, 3941, 4755, 4971, 5250, 5315, 5350, 5450, 5672, 6692Da, etc. When hydrolyzed by proper protease, the activity is released, which is not only easy to digest and absorb, but also has various functions of regulating metabolism and physiological functions of human body, and is a natural resource treasure house for developing functional foods and screening medicines. In China, chickpea is a natural plant protein resource, rich in nutrition and has multiple physiological functions, is a Uygur nationality medicine and a traditional Chinese medicine, and chickpea peptide also becomes one of the research hotspots of plant-derived active peptides. The nutritional powder can be used as natural emulsifier and food additive in food industry.

Drawings

FIG. 1 is a 12.5% SDS-PAGE Coomassie brilliant blue electrophoresis chart of the PBS extract of chickpea bean cotyledons of the invention hydrolyzed with different enzymes, wherein M is a standard molecular weight protein; 1. extracting a PBS (phosphate buffer solution) part of the chickpea bean cotyledon; 3. the part is enzymolyzed by alkaline enzyme; 4. neutral enzyme enzymolysis site; 5. (ii) a trypsin enzymatic site; 6. papain enzymatic hydrolysis part;

FIG. 2 is a graph showing the bacteriostatic action of the chick pea bean cotyledon protein hydrolysate on Candida albicans, wherein 6. the PBS extraction site of the chick pea bean cotyledon; 7. the part is enzymolyzed by alkaline enzyme; 8. neutral enzyme enzymolysis site; 9. (ii) a trypsin enzymatic site; 10. papain enzymatic hydrolysis part;

FIG. 3 is a liquid chromatography mass spectrometry (LC/MS) spectrum of the hydrolysis site of chickpea broad bean protein with neutral protease;

FIG. 4 is a liquid chromatography mass spectrometry (LC/MS) spectrum of the hydrolysis site of chickpea broad bean protein with alkaline protease;

FIG. 5 is a liquid chromatography mass spectrometry (LC/MS) map of the tryptic site of the chickpea bean cotyledon protein of the invention.

FIG. 6 is a liquid chromatography mass spectrometry (LC/MS) spectrum of the papain enzymatic hydrolysis part of chickpea bean cotyledon protein of the invention.

Detailed Description

Example 1

a. Crushing 613g of dried bean cotyledon after sprouting and peeling of chickpeas, sieving with a 80-mesh sieve, adding petroleum ether into w/v bean powder according to the ratio of 1:5 to the petroleum ether, oscillating for 4h at room temperature, repeatedly extracting for 3-4 times until the bean cotyledon is colorless, naturally settling the chickpea bean cotyledon powder, separating the petroleum ether from the chickpea bean cotyledon powder, pouring out the upper petroleum ether for recovery, combining precipitates, naturally drying to obtain 516.7g of degreased chickpea bean cotyledon powder, wherein the extraction rate of oil is 96.3/613: 15.7%, and storing in a refrigerator at the temperature of-20 ℃ for later use;

c. filtering the extracting solution in the step b, centrifuging at 4 ℃ for 10min at 10000r/min, dialyzing at 4 ℃ for 48h (the specification of a dialysis bag is 1000Da), performing freeze-drying at the pressure of 9.8Pa and the temperature of-80 ℃ for 24h to obtain 10.27g of crude protein powder, wherein the protein extraction rate is 10.27%;

e. d, hydrolyzing the crude protein powder in the step d by using alkaline protease, weighing 1.0g of the crude protein powder, dissolving the crude protein powder by using 30ml of NaOH with pH of 9.5 and 0.05N, adding 0.3% of alkaline protease by mass, extracting for 3h at 45 ℃ and pH of 9-11, centrifuging the extracting solution at 4 ℃ and 10000r/min for 10min, taking the supernatant at 4 ℃ and dialyzing for 48h, freeze-drying the sample at 9.8Pa at-80 ℃ for 24h to obtain 0.12g of hydrolyzed polypeptide, wherein the polypeptide extraction rate is 0.12g/1 g-12%;

f. centrifuging the alkaline protease zymolyte extracting solution obtained in the step e, taking supernate, carrying out interception separation on the supernate by using a 10kDa regenerated fiber filter membrane, keeping the concentrated solution at the temperature of 25 ℃ and the flow rate of 35m/s, carrying out interception separation on the filtrate by using 6kDa and 3.5kDa regenerated fiber filter membranes respectively, sequentially preparing polypeptides with the molecular weights of 6-10kDa, 3.5-6kDa and 1-3.5kDa, taking each part of polypeptide solution, carrying out vacuum drying, and detecting the molecular weights of 3.5-6kDa and the yields of 1-3.5kDa by using a liquid chromatography-mass spectrometry (LC/MS) instrument to be 56 percent and 44 percent;

the obtained polypeptide parts were compared in terms of molecular weight, antibacterial activity and protein content by polyacrylamide gel electrophoresis (SDS-PAGE) -Coomassie brilliant blue staining pattern, and the protein contents were 48% and 56%, respectively (see Table 1).

Example 2

e. d, hydrolyzing the crude protein powder in the step d by neutral protease: weighing 1.0g of crude protein powder, dissolving the crude protein powder by 30ml of NaOH with pH 7.5 and 0.05N, adding neutral protease with the mass ratio of 0.3%, extracting for 3h at 50 ℃ and pH 7.5, centrifuging the extracting solution at 10000r/min and 4 ℃ for 10min, taking supernatant, dialyzing at 1000Da and 4 ℃ for 48h, freeze-drying the sample at the pressure of 9.8Pa, at-80 ℃ and 24h to obtain 0.42g of hydrolyzed polypeptide, wherein the polypeptide extraction rate is 0.42g/1g which is 42%;

f. centrifuging the neutral protease zymolyte extracting solution in the step e, taking supernate, carrying out interception separation on the supernate by using a 10kDa regenerated fiber filter membrane, keeping the concentrated solution at the temperature of 25 ℃ and the flow rate of 35m/s, carrying out interception separation on the filtrate by using 6kDa and 3.5kDa regenerated fiber filter membranes respectively, sequentially preparing polypeptides with the molecular weights of 6-10kDa, 3.5-6kDa and 1-3.5kDa, taking each polypeptide solution, carrying out vacuum drying, and detecting the yields of the polypeptides with the molecular weights of 6-10kDa, 3.5-6kDa and 1-3.5kDa by using a liquid chromatography-mass spectrometry (LC/MS) instrument to be 4.7%, 47.65% and 47.65%;

the obtained polypeptide parts were compared in terms of molecular weight, antibacterial activity and protein content by polyacrylamide gel electrophoresis (SDS-PAGE) -Coomassie brilliant blue staining pattern, and the protein contents were 60%, 76% and 70%, respectively (see Table 1).

Example 3

e. d, hydrolyzing the crude protein powder in the step d by trypsin: weighing 1.0g of crude protein powder, dissolving the crude protein powder by 30ml of NaOH with pH of 8.1 and 0.05N, adding trypsin with the mass ratio of 0.3%, extracting at 37 ℃ and pH of 8.1 for 3h, centrifuging the extracting solution at 10000r/min and 4 ℃ for 10min, taking supernatant, dialyzing at 1000Da, at 4 ℃ and 48h, freeze-drying the sample at the pressure of 9.8Pa, at-80 ℃ and 24h to obtain 0.42g of hydrolyzed polypeptide, wherein the polypeptide extraction rate is 0.46g/1g which is 46%; (ii) a

f. Centrifuging the trypsin zymolyte extracting solution obtained in the step e, collecting supernate, intercepting and separating by using a 10kDa regenerated fiber filter membrane, keeping the concentrated solution at the temperature of 25 ℃ and the flow rate of 35m/s, then respectively intercepting and separating the filtrate by using 6kDa and 3.5kDa regenerated fiber filter membranes to sequentially prepare polypeptides with the molecular weights of 6-10kDa, 3.5-6kDa and 1-3.5kDa, then collecting polypeptide solutions of all parts for vacuum drying, and detecting that the molecular weights are 3.5-6kDa and the yields of 1-3.5kDa are 63% and 37% by using a liquid chromatography-mass spectrometry (LC/MS) instrument;

the obtained polypeptide sites were compared in terms of molecular weight, antibacterial activity and protein content by polyacrylamide gel electrophoresis (SDS-PAGE) -Coomassie blue staining pattern, and the protein content was 72% and 59%, respectively (see Table 1).

Example 4

e. d, hydrolyzing the crude protein powder in the step d by papain: weighing 1.0g of crude protein powder, dissolving the crude protein powder by 30ml of NaOH with pH 5.7 and 0.05N, adding papain with the mass ratio of 0.3%, extracting at 55-60 ℃ and pH 5.7 for 3h, centrifuging the extracting solution at 10000r/min and 4 ℃ for 10min, taking the supernatant, dialyzing the supernatant at 1000Da and 4 ℃ for 48h, freeze-drying the sample at the pressure of 9.8Pa and the temperature of-80 ℃ for 24h to obtain 0.12g of hydrolyzed polypeptide, wherein the polypeptide extraction rate is 0.12g/1g which is 12%;

f. centrifuging the papain zymolyte extracting solution obtained in the step e, taking supernate, intercepting and separating by using a 10kDa regenerated fiber filter membrane, keeping the concentrated solution at the temperature of 25 ℃ and the flow rate of 35m/s, intercepting and separating the filtrate by using 6kDa and 3.5kDa regenerated fiber filter membranes respectively, sequentially preparing polypeptides with the molecular weights of 6-10kDa, 3.5-6kDa and 1-3.5kDa, taking each part of polypeptide solution, carrying out vacuum drying, and detecting that the yields of the polypeptides with the molecular weights of 6-10kDa, 3.5-6kDa and 1-3.5kDa are 6.7%, 53% and 40% by using a liquid chromatography-mass spectrometry (LC/MS) instrument;

TABLE 1 molecular weight of chickpea bean cotyledon zymolyte prepared by ultrafiltration membrane technique

Example 5

Determination of protein content: determining the protein content of the chickpea bean paste obtained in the step 1 to 5 by using a biuret method;

drawing a standard curve: 6 test tubes were numbered and the reagents were added as per Table 2:

TABLE 2 preparation of bovine serum albumin at different concentrations

Shaking for 15min, standing at room temperature for 30min, carrying out color comparison at 540nm, and drawing a standard curve by taking the protein content (mg) as a horizontal coordinate and the absorbance as a vertical coordinate;

and (3) determination of a sample: determining the protein concentration of the unknown sample, taking care that the concentration of the sample does not exceed 10 mg/ml;

drying the ground and sieved grain sample at the temperature of 80 ℃ to constant weight, taking out and placing in a dryer for cooling for later use;

weighing two 2mg samples, respectively placing the two samples into two dry test tubes, respectively adding 5ml of 0.05mol/L NaOH solution into each test tube for wetting, then adding 4ml of biuret reagent, shaking for 15min, standing at room temperature for reaction for 30min, respectively filtering, taking filtrate for colorimetric analysis at 540nm wavelength, finding out corresponding protein content (mg) on a standard curve, and finding out the protein content (mg) from the standard curve.

Example 6

Determination of antibacterial Activity:

melting agar culture medium, cooling to 46 + -0.5 deg.C, adding cultured bacterial liquid to make test bacterial suspension concentration be 5 × 105cfu/ml-5 × 106cfu/ml, pouring into flat dish, 15-20 ml/dish, standing for 20min to solidify;

punching with agar puncher, with diameter of 5-6mm and 4-5 holes/dish, uniformly distributing, and making the distance between the centers of various sample sheets be 25mm and the distance between the centers of various sample sheets and the periphery of the flat plate be 15 mm;

the sample concentration is 100mg/ml (100mM), each hole is added with 20 mul of sample solution, the plate is covered, the plate is placed in an incubator at the temperature of 37 ℃ for 30-60min, the solution is completely absorbed, the inverted culture is carried out for 16-18 h, and the diameter of the antibacterial ring is measured by a vernier caliper and recorded;

evaluation:

judging the bacteriostatic action: if the diameter of the bacteriostatic ring is larger than 7mm, the bacteriostatic ring is judged to have bacteriostatic action; if the diameter of the antibacterial ring is less than or equal to 7mm, judging that the antibacterial ring has no antibacterial effect; the results are shown in Table 3:

TABLE 3 antibacterial action determining table for chickpea bean cotyledon zymolyte

As can be seen from the table: the antibacterial activity of the chickpea broad bean protein is obviously enhanced after the hydrolysis by four proteases, neutral protease hydrolysate has better inhibition rates on four bacteria such as staphylococcus epidermidis, escherichia coli, klebsiella and salmonella typhimurium, alkaline protease hydrolysate has better inhibition rates on two bacteria such as klebsiella and salmonella typhimurium, papain hydrolysate only has an inhibition effect on candida albicans, and trypsin hydrolysate has no inhibition effect on the 10 bacteria.

Claims

1. A preparation method of chickpea bean polypeptides is characterized by comprising the following steps:

a. crushing the dried bean cotyledon after sprouting and peeling of the chickpeas, sieving with a 80-mesh sieve, adding petroleum ether according to w/v bean powder: petroleum ether =1:5, oscillating for 4h at room temperature for degreasing, repeatedly extracting for 3-4 times until the bean cotyledon is colorless, naturally settling the chickpea bean cotyledon powder, separating the petroleum ether from the chickpea bean cotyledon powder, pouring out the upper layer petroleum ether for recycling, combining precipitates, naturally drying to obtain degreased chickpea bean cotyledon powder, and storing in a refrigerator at the temperature of-20 ℃ for later use;

2. Use of a chick pea bean polypeptide obtained according to the method of claim 1 in the preparation of a medicament against bacteria selected from the group consisting of staphylococcus epidermidis, escherichia coli, klebsiella pneumoniae and salmonella typhimurium.

3. Use of a chick pea bean polypeptide obtained according to the method of claim 1 in the preparation of an antifungal food product, said fungus being candida albicans.

4. Use of a chick pea bean polypeptide obtained according to the method of claim 1 for the preparation of a food additive.