CN111850074A - Preparation method and application of bitter gourd polypeptide - Google Patents
Preparation method and application of bitter gourd polypeptide Download PDFInfo
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- CN111850074A CN111850074A CN202010664891.6A CN202010664891A CN111850074A CN 111850074 A CN111850074 A CN 111850074A CN 202010664891 A CN202010664891 A CN 202010664891A CN 111850074 A CN111850074 A CN 111850074A
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Abstract
The invention discloses a preparation method and application of bitter gourd polypeptide, which comprises the following steps: 1) crushing a bitter gourd raw material, adding the crushed bitter gourd raw material into water according to the proportion of 1: 1-10, adjusting and filtering to obtain a bitter gourd protein liquid; 2) adjusting the bitter gourd protein liquid obtained by filtering in the step 1) to be washed with water and precipitated to be neutral, adding a complex enzyme, carrying out enzymolysis on the bitter gourd protein, after the enzymolysis is finished, carrying out inactivation treatment on the complex enzyme, naturally cooling the inactivated bitter gourd protein liquid to room temperature, and removing insoluble substances and precipitated impurities to obtain clear crude bitter gourd polypeptide after enzymolysis; 3) and screening the molecular weight of the crude momordica charantia polypeptide subjected to enzymolysis by adopting a membrane filtration technology to obtain the momordica charantia polypeptide with the molecular weight of less than 3kDa, concentrating the volume, and drying to obtain the momordica charantia polypeptide powder. It has improved effective polypeptide molecule content and improved bitter gourd polypeptide taste. Can be used for preparing special diet, health food, and medicine with blood sugar metabolism regulating effect. Is suitable for industrial production.
Description
Technical Field
The invention belongs to the technical field of preparation of bioactive peptides, and relates to a preparation method and application of bitter gourd polypeptide.
Background
With the continuous improvement of living standard and the rapid development of social economy, the diabetic patients in China present a rapid growth situation, and the number of the diabetic patients accounts for about one third of the total number of the diabetic patients in the world. At present, the incidence rate of diabetes in China is up to 9.6 percent, the number of diabetic patients is over 1.1 hundred million, and the number of the diabetic patients is increased year by year. Diabetes is a great hazard, and the diabetic complications accompanying diabetes also pose a great threat to the health and life of patients. Diabetes mellitus is known as diabetes, and refers to a comprehensive disease mainly characterized by polydipsia, diuresis, polyphagia, emaciation, fatigue and urine sweetness, and is mainly characterized by hyperglycemia and urine glucose through laboratory examination. Diabetes is a disease characterized by polydipsia, polyphagia, diuresis and emaciation due to yin deficiency, dryness-heat and deficiency of five internal organs. Modern medical research shows that diabetes is caused by the fact that insulin sensitivity of a body is reduced due to damaged islet cells, insufficient insulin secretion or dysfunction of the body, and the diabetes is caused by the fact that the diabetes cannot achieve the function of regulating blood sugar.
The balsam pear is also called as summer melon, is a cucurbitaceae plant, is a medicine and food dual-purpose vegetable, and has the functions of clearing heat and relieving summer heat, nourishing blood and tonifying qi, tonifying kidney and spleen, and nourishing liver and improving eyesight according to records of Ben Cao gang mu and Dian nan Ben Cao. The main chemical components of balsam pear comprise charantin, various amino acids and lipoid. The previous experimental research shows that the charantin and the polypeptide in the balsam pear have better hypoglycemic activity. In 1981, Khannal uses acid, alcohol and other reagents, and adopts a thin layer chromatography method to extract bitter gourd polypeptide from bitter gourd fruit and bitter gourd, the bitter gourd polypeptide can reduce blood sugar of experimental animals after subcutaneous injection, and the bitter gourd polypeptide has 166 amino acid residues.
After the bitter gourd becomes old and yellow, the bitter gourd seeds become large and hard and can not be directly eaten, the bitter gourd seeds can be taken out and dried in the sun to be used as a medicine, the bitter gourd seeds are bitter and sweet in taste after being used as the medicine, mild in property and have the important effect of warming and recuperating kidney yang, the bitter gourd seed wine is usually mainly used for conditioning kidney yang deficiency of people, and has a good treatment effect on frequent urination, enuresis, male impotence and other symptoms caused by kidney yang deficiency of people. The balsam pear seeds can also reduce blood sugar and can be used for treating hyperglycemia and diabetes of human beings, and the balsam pear seeds contain a large amount of balsam pear glycosides, which not only can reduce the content of the blood sugar, but also can improve the function of pancreatic islets. However, most people eat fresh bitter gourds, the bitter gourds are thrown away generally after becoming yellow and old, and bitter gourds are fewer due to the particularity of the bitter gourds, so that certain waste is caused.
A large number of experimental studies show that the balsam pear polypeptide in the balsam pear seeds has a better function of reducing blood sugar, but in general, the balsam pear polypeptide content in the balsam pear seeds is less, so that few people think of extracting the balsam pear polypeptide from the balsam pear seeds. According to the reference documents, the balsam pear is eagerly used for treating and preventing diabetes at home and abroad, and a plurality of products like 'momordicin', 'balsam pear total glycoside', 'balsam pear tea', 'balsam pear powder' and 'balsam pear protein powder' are developed in a successive research, but the products have the common defect that the molecular weight is large and the products are not easy to absorb and utilize by a human body. According to the theory of traditional Chinese medicine, people with cold constitution should not eat more or eat bitter gourd vegetables frequently, but the human body needs to take certain bitter gourd peptide to regulate sugar in the body. However, the conventional momordica charantia polypeptide can not meet the requirement of the momordica charantia polypeptide in the market to a great extent.
Disclosure of Invention
The invention aims to solve the technical problems and provides a preparation and purification method and application of momordica charantia polypeptide.
The invention is realized by the following technical scheme:
a method for preparing Momordica charantia polypeptide comprises the following steps:
1) crushing a bitter gourd raw material into 10-60-mesh small particles, adding the particles into water according to the proportion of 1: 1-10, heating the crushed bitter gourd raw material in water bath, adjusting the pH value to be alkaline, controlling the extraction temperature to be about 45-60 ℃, extracting for about 1.5-3h, and filtering to obtain bitter gourd protein liquid;
2) adjusting the pH value of the balsam pear protein liquid obtained by filtering in the step 1) to 3-5 to precipitate protein, washing the precipitate with water to be neutral, adding a complex enzyme solution, carrying out enzymolysis on the balsam pear protein, wherein the enzymolysis time is 4-6h, the enzymolysis temperature is 45-65 ℃, after the enzymolysis is finished, carrying out inactivation treatment on the enzyme in the balsam pear protein liquid for 1-1.5h under the condition of water bath at 85-90 ℃, naturally cooling the inactivated balsam pear protein liquid to room temperature, standing until insoluble substances and impurities are completely precipitated, and removing the insoluble substances and precipitated impurities to obtain clarified enzymolyzed crude balsam pear polypeptide;
3) And screening and determining the molecular weight of the crude momordica charantia polypeptide after enzymolysis by adopting a membrane filtration technology, removing impurities from macromolecular non-enzymolysis protein to obtain the momordica charantia polypeptide with the molecular weight of less than 3kDa, concentrating the volume, and drying to obtain the momordica charantia polypeptide powder.
The step 1) is specifically as follows: crushing bitter gourd raw materials into 10-60-mesh small particles, adding the particles into water according to the proportion of 1: 1-5, heating the crushed bitter gourd in water bath, adjusting the pH to 7.5-8.5, controlling the extraction temperature to be about 45-70 ℃, extracting for 2-3h, and filtering to obtain bitter gourd protein liquid.
The complex enzyme in the step 2) consists of soybean protease, trypsin, pectinase and cellulase according to the proportion of 2-4:1-3:1-3: 1-4.
The balsam pear protein solution in the step 1) is a balsam pear protein solution obtained by heating and extracting a balsam pear raw material in water bath, adjusting the pH of the solution and precipitating protein.
The molecular weight of the momordica charantia polypeptide in the crude momordica charantia polypeptide obtained in the step 2) is distributed between 1kDa and 68 kDa.
The step 3) is specifically as follows: screening the molecular weight of the bitter gourd crude polypeptide with the molecular weight of 1-68 kDa obtained in the step 2) by adopting an ultrafiltration membrane, wherein the preset molecular cut-off amounts of the ultrafiltration membrane are 10kDa, 3kDa and 1kDa, respectively collecting cut-off solutions with different molecular weights, and finally obtaining 4 bitter gourd polypeptide solutions with different molecular weights: 10kDa or more, 10kDa-3kDa, 3kDa-1kDa, or less than 1 kDa;
Selecting fructus Momordicae Charantiae polypeptide with molecular weight below 3kDa from 4 fructus Momordicae Charantiae polypeptide solutions with different molecular weights, concentrating to solid content of 50%, and drying to obtain fructus Momordicae Charantiae polypeptide powder.
The temperature of the ultrafiltration membrane is 35-40 ℃, and the operating pressure is 0.3-0.5 MPa.
The drying is vacuum drying.
The raw materials of the bitter gourd comprise fresh bitter gourd, dried bitter gourd, bitter gourd seeds, dried bitter gourd and bitter gourd meat.
Application of fructus Momordicae Charantiae polypeptide in preparing food additive adjuvant, health food or medicine for preventing type II diabetes is provided.
Compared with the prior art, the invention has the following beneficial technical effects:
the invention provides a preparation method and application of bitter gourd polypeptide, in particular to a preparation method for extracting protein from bitter gourd and obtaining the bitter gourd polypeptide through complex enzyme enzymolysis of the protein. The invention aims to further improve the content of effective polypeptide molecules and improve the taste of the momordica charantia polypeptide by preparing and purifying the polypeptide in the momordica charantia. Can be used for preparing special diet, health food, food additive, and medicine with blood sugar metabolism regulating effect. Is suitable for industrial production.
The molecular weight of the prepared bitter gourd polypeptide product is below 3kDa, the bitter gourd polypeptide product is easy to absorb by a human body, can be quickly utilized by human tissues, quickly balances blood sugar and can be taken for a long time.
The product of the invention can be widely used as a medicine raw material, a medicine, a health product and a food additive. The method is a product which has the advantages of easy holding of technology, simple equipment, simple and easy process, less investment, short period, high added value and no environmental pollution and is suitable for industrial technical production.
Drawings
FIG. 1 is a process flow diagram of a method for preparing and purifying Momordica charantia polypeptide.
Detailed Description
The present invention will be described in further detail with reference to specific examples, and the technical solutions of the present invention will be clearly and completely described with reference to the animal function tests, but it is obvious that the description is only a part of the present invention, rather than the whole, and all other examples obtained by one of ordinary skill in the art without any inventive work based on the examples of the present invention are within the scope of the present invention, and the following is an explanation and not a limitation of the present invention.
Example 1, referring to fig. 1, a process for the preparation of momordica charantia polypeptide comprising the steps of:
1) crushing a bitter gourd raw material into 10-60-mesh small particles, adding the particles into water according to the proportion of 1: 1-10, heating the crushed bitter gourd raw material in water bath, adjusting the pH value to be alkaline, controlling the extraction temperature to be about 45-60 ℃, extracting for about 1.5-3h, and filtering to obtain bitter gourd protein liquid; the balsam pear protein solution in the step 1) is prepared by heating balsam pear raw materials in a water bath, extracting, adjusting the pH value of the solution, and precipitating protein.
2) Adjusting the pH value of the balsam pear protein liquid obtained by filtering in the step 1) to 3-5 to precipitate protein, washing the precipitate with water to be neutral, adding a complex enzyme solution, carrying out enzymolysis on the balsam pear protein, wherein the enzymolysis time is 4-6h, the enzymolysis temperature is 45-65 ℃, after the enzymolysis is finished, carrying out inactivation treatment on the complex enzyme in the balsam pear protein liquid after the enzymolysis for 1-1.5h under the condition of water bath at 85-90 ℃, naturally cooling the balsam pear protein liquid after the inactivation treatment to the room temperature, standing until insoluble substances and impurities are completely precipitated, and removing the insoluble substances and the precipitated impurities to obtain clear crude balsam pear polypeptide after the enzymolysis; wherein, the molecular weight of the momordica charantia polypeptide in the crude momordica charantia polypeptide obtained in the step 2) is distributed between 1kDa and 68 kDa; the complex enzyme in the step 2) consists of soybean protease, trypsin, pectinase and cellulase according to the proportion of 2-4:1-3:1-3: 1-4.
3) Screening and determining the molecular weight of the crude momordica charantia polypeptide after enzymolysis by adopting a membrane filtration technology, removing impurities from macromolecular non-enzymolysis protein to obtain momordica charantia polypeptide with the molecular weight of below 3kDa, concentrating the volume, and drying in vacuum to obtain momordica charantia polypeptide powder; wherein the temperature of ultrafiltration membrane is 35-40 deg.C, and the operation pressure is 0.3-0.5 MPa.
Specifically, the step 1) specifically comprises the following steps: crushing bitter gourd raw materials into 10-60-mesh small particles, adding the particles into water according to the proportion of 1: 1-5, heating the crushed bitter gourd in water bath, adjusting the pH to 7.5-8.5, controlling the extraction temperature to be about 45-70 ℃, extracting for 2-3h, and filtering to obtain bitter gourd protein liquid.
The step 3) is specifically as follows: screening the molecular weight of the bitter gourd crude polypeptide with the molecular weight distribution of 1-68 kDa obtained in the step 2) by adopting an ultrafiltration membrane, wherein the preset molecular cut-off amount of the ultrafiltration membrane is 10kDa, 3kDa and 1kDa, respectively collecting cut-off solutions with different molecular weights, and finally obtaining 4 bitter gourd polypeptide solutions with different molecular weights: 10kDa or more, 10kDa-3kDa, 3kDa-1kDa, or less than 1 kDa;
selecting fructus Momordicae Charantiae polypeptide with molecular weight below 3kDa from 4 fructus Momordicae Charantiae polypeptide solutions with different molecular weights, concentrating to solid content of 50%, and drying to obtain fructus Momordicae Charantiae polypeptide powder.
The bitter gourd raw materials comprise fresh bitter gourd, dried bitter gourd, bitter gourd seeds, dried bitter gourd and bitter gourd meat. Preferably, the preparation method of the invention adopts balsam pear seeds as raw materials for extracting the balsam pear polypeptide, thus changing waste into valuable and having multiple purposes.
The method is characterized in that the used complex enzyme is completed through two steps, firstly, the enzymolysis condition with the best enzymolysis effect of single enzyme is screened through a single-factor experiment, and secondly, the input proportion of the complex enzyme is determined through an orthogonal experiment.
Further, the application of the momordica charantia polypeptide is used as a raw material for preparing or preparing food additive auxiliary materials, health-care food or medicines for preventing type II diabetes.
Example 2, referring to fig. 1, a process for the preparation of momordica charantia polypeptide comprising the steps of:
1) crushing bitter gourd raw materials into 10-mesh small particles, adding the particles into water according to the proportion of 1:5, heating the crushed bitter gourd raw materials in water bath, adjusting the pH value to be alkaline, controlling the extraction temperature to be about 50 ℃, extracting for about 2 hours, and filtering to obtain bitter gourd protein liquid; the balsam pear protein solution in the step 1) is prepared by heating balsam pear raw materials in a water bath, extracting, adjusting the pH value of the solution, and precipitating protein.
2) Adjusting the pH value of the balsam pear protein liquid obtained by filtering in the step 1) to 4 to precipitate protein, washing the precipitate with water to be neutral, adding a complex enzyme solution, carrying out enzymolysis on the balsam pear protein, wherein the enzymolysis time is 5h, the enzymolysis temperature is 50 ℃, after the enzymolysis is finished, carrying out inactivation treatment on the complex enzyme in the balsam pear protein liquid after the enzymolysis for 1h under the condition of 90 ℃ in a water bath, naturally cooling the balsam pear protein liquid after the inactivation treatment to the room temperature, standing until insoluble substances and impurities are completely precipitated, and removing the insoluble substances and the precipitated impurities to obtain clear crude balsam pear polypeptide after the enzymolysis; wherein, the molecular weight of the momordica charantia polypeptide in the crude momordica charantia polypeptide obtained in the step 2) is distributed between 1kDa and 68 kDa; the complex enzyme in the step 2) is composed of soybean protease, trypsin, pectinase and cellulase according to the proportion of 2:1:1: 1.
3) Screening and determining the molecular weight of the crude momordica charantia polypeptide after enzymolysis by adopting a membrane filtration technology, removing impurities from macromolecular non-enzymolysis protein to obtain momordica charantia polypeptide with the molecular weight of below 3kDa, concentrating the volume, and drying in vacuum to obtain momordica charantia polypeptide powder; wherein the temperature of ultrafiltration membrane is 35-40 deg.C, and the operation pressure is 0.3-0.5 MPa.
Specifically, the step 1) specifically comprises the following steps: crushing bitter gourd raw materials into 10-mesh small particles, adding the particles into water according to the proportion of 1:5, heating the crushed bitter gourd in water bath, adjusting the pH to 8, controlling the extraction temperature to be about 50 ℃, extracting for 2h, and filtering to obtain bitter gourd protein liquid.
The step 3) is specifically as follows: screening the molecular weight of the bitter gourd crude polypeptide with the molecular weight distribution of 1-68 kDa obtained in the step 2) by adopting an ultrafiltration membrane, wherein the preset molecular cut-off amount of the ultrafiltration membrane is 10kDa, 3kDa and 1kDa, respectively collecting cut-off solutions with different molecular weights, and finally obtaining 4 bitter gourd polypeptide solutions with different molecular weights: 10kDa or more, 10kDa-3kDa, 3kDa-1kDa, or less than 1 kDa;
selecting fructus Momordicae Charantiae polypeptide with molecular weight below 3kDa from 4 fructus Momordicae Charantiae polypeptide solutions with different molecular weights, concentrating to solid content of 50%, and drying to obtain fructus Momordicae Charantiae polypeptide powder.
The bitter gourd raw materials comprise fresh bitter gourd, dried bitter gourd, bitter gourd seeds, dried bitter gourd and bitter gourd meat. Preferably, the preparation method of the invention adopts balsam pear seeds as raw materials for extracting the balsam pear polypeptide, thus changing waste into valuable and having multiple purposes.
The method is characterized in that the used complex enzyme is completed through two steps, firstly, the enzymolysis condition with the best enzymolysis effect of single enzyme is screened through a single-factor experiment, and secondly, the input proportion of the complex enzyme is determined through an orthogonal experiment.
Further, the application of the momordica charantia polypeptide is used as a raw material for preparing or preparing food additive auxiliary materials, health-care food or medicines for preventing type II diabetes.
Example 3, referring to fig. 1, a process for the preparation of momordica charantia polypeptide comprising the steps of:
1) crushing bitter gourd raw materials into 35-mesh small particles, adding the particles into water according to the proportion of 1:1, heating the crushed bitter gourd raw materials in water bath, adjusting the pH value to be alkaline, controlling the extraction temperature to be about 45 ℃ and the extraction time to be about 1.5, and filtering to obtain bitter gourd protein liquid; the balsam pear protein solution in the step 1) is prepared by heating balsam pear raw materials in a water bath, extracting, adjusting the pH value of the solution, and precipitating protein.
2) Adjusting the pH value of the balsam pear protein liquid obtained by filtering in the step 1) to 3 to precipitate protein, washing the precipitate with water to be neutral, adding a complex enzyme solution, carrying out enzymolysis on the balsam pear protein, wherein the enzymolysis time is 4h, the enzymolysis temperature is 45 ℃, after the enzymolysis is finished, carrying out inactivation treatment on the complex enzyme in the balsam pear protein liquid after the enzymolysis for 1.2h under the condition of water bath 87 ℃, then naturally cooling the balsam pear protein liquid after the inactivation treatment to the room temperature, standing until insoluble substances and impurities are completely precipitated, and removing the insoluble substances and the precipitated impurities to obtain clarified crude balsam pear polypeptide after the enzymolysis; wherein, the molecular weight of the momordica charantia polypeptide in the crude momordica charantia polypeptide obtained in the step 2) is distributed between 1kDa and 68 kDa; the complex enzyme in the step 2) is composed of soybean protease, trypsin, pectinase and cellulase according to the proportion of 3:2:2: 2.5.
3) Screening and determining the molecular weight of the crude momordica charantia polypeptide after enzymolysis by adopting a membrane filtration technology, removing impurities from macromolecular non-enzymolysis protein to obtain momordica charantia polypeptide with the molecular weight of below 3kDa, concentrating the volume, and drying in vacuum to obtain momordica charantia polypeptide powder; wherein the temperature of ultrafiltration membrane is 35-40 deg.C, and the operation pressure is 0.3-0.5 MPa.
Specifically, the step 1) specifically comprises the following steps: crushing bitter gourd raw materials into 35-mesh small particles, adding the particles into water according to the proportion of 1:1, heating the crushed bitter gourd in water bath, adjusting the pH to 7.5, controlling the extraction temperature to be about 45 ℃, extracting for 2.5h, and filtering to obtain bitter gourd protein liquid.
The step 3) is specifically as follows: screening the molecular weight of the bitter gourd crude polypeptide with the molecular weight distribution of 1-68 kDa obtained in the step 2) by adopting an ultrafiltration membrane, wherein the preset molecular cut-off amount of the ultrafiltration membrane is 10kDa, 3kDa and 1kDa, respectively collecting cut-off solutions with different molecular weights, and finally obtaining 4 bitter gourd polypeptide solutions with different molecular weights: 10kDa or more, 10kDa-3kDa, 3kDa-1kDa, or less than 1 kDa;
selecting fructus Momordicae Charantiae polypeptide with molecular weight below 3kDa from 4 fructus Momordicae Charantiae polypeptide solutions with different molecular weights, concentrating to solid content of 50%, and drying to obtain fructus Momordicae Charantiae polypeptide powder.
The bitter gourd raw materials comprise fresh bitter gourd, dried bitter gourd, bitter gourd seeds, dried bitter gourd and bitter gourd meat. Preferably, the preparation method of the invention adopts balsam pear seeds as raw materials for extracting the balsam pear polypeptide, thus changing waste into valuable and having multiple purposes.
The method is characterized in that the used complex enzyme is completed through two steps, firstly, the enzymolysis condition with the best enzymolysis effect of single enzyme is screened through a single-factor experiment, and secondly, the input proportion of the complex enzyme is determined through an orthogonal experiment.
Further, the application of the momordica charantia polypeptide is used as a raw material for preparing or preparing food additive auxiliary materials, health-care food or medicines for preventing type II diabetes.
Example 4, a method for preparing a momordica charantia polypeptide, comprising the steps of:
1) crushing bitter gourd raw materials into 60-mesh small particles, adding the particles into water according to the proportion of 1:10, heating the crushed bitter gourd raw materials in water bath, adjusting the pH value to be alkaline, controlling the extraction temperature to be about 60 ℃, extracting for about 3 hours, and filtering to obtain bitter gourd protein liquid; the balsam pear protein solution in the step 1) is prepared by heating balsam pear raw materials in a water bath, extracting, adjusting the pH value of the solution, and precipitating protein.
2) Adjusting the pH value of the balsam pear protein liquid obtained by filtering in the step 1) to 5 to precipitate protein, washing the precipitate with water to be neutral, adding a complex enzyme solution, carrying out enzymolysis on the balsam pear protein, wherein the enzymolysis time is 6h, the enzymolysis temperature is 65 ℃, after the enzymolysis is finished, carrying out inactivation treatment on the complex enzyme in the balsam pear protein liquid after the enzymolysis for 1.5h under the condition of water bath at 85 ℃, then naturally cooling the balsam pear protein liquid after the inactivation treatment to room temperature, standing until insoluble substances and impurities are completely precipitated, and removing the insoluble substances and the precipitated impurities to obtain clarified crude balsam pear polypeptide after the enzymolysis; wherein, the molecular weight of the momordica charantia polypeptide in the crude momordica charantia polypeptide obtained in the step 2) is distributed between 1kDa and 68 kDa; the complex enzyme in the step 2) is composed of soybean protease, trypsin, pectinase and cellulase according to the proportion of 4:3:3: 4.
3) Screening and determining the molecular weight of the crude momordica charantia polypeptide after enzymolysis by adopting a membrane filtration technology, removing impurities from macromolecular non-enzymolysis protein to obtain momordica charantia polypeptide with the molecular weight of below 3kDa, concentrating the volume, and drying in vacuum to obtain momordica charantia polypeptide powder; wherein the temperature of ultrafiltration membrane is 35-40 deg.C, and the operation pressure is 0.3-0.5 MPa.
Specifically, the step 1) specifically comprises the following steps: crushing bitter gourd raw materials into 60-mesh small particles, adding the small particles into water according to the proportion of 1:10, heating the crushed bitter gourd in water bath, adjusting the pH to 8.5, controlling the extraction temperature to be about 70 ℃, extracting for 3h, and filtering to obtain bitter gourd protein liquid.
The step 3) is specifically as follows: screening the molecular weight of the bitter gourd crude polypeptide with the molecular weight distribution of 1-68 kDa obtained in the step 2) by adopting an ultrafiltration membrane, wherein the preset molecular cut-off amount of the ultrafiltration membrane is 10kDa, 3kDa and 1kDa, respectively collecting cut-off solutions with different molecular weights, and finally obtaining 4 bitter gourd polypeptide solutions with different molecular weights: 10kDa or more, 10kDa-3kDa, 3kDa-1kDa, or less than 1 kDa;
selecting fructus Momordicae Charantiae polypeptide with molecular weight below 3kDa from 4 fructus Momordicae Charantiae polypeptide solutions with different molecular weights, concentrating to solid content of 50%, and drying to obtain fructus Momordicae Charantiae polypeptide powder.
The bitter gourd raw materials comprise fresh bitter gourd, dried bitter gourd, bitter gourd seeds, dried bitter gourd and bitter gourd meat. Preferably, the preparation method of the invention adopts balsam pear seeds as raw materials for extracting the balsam pear polypeptide, thus changing waste into valuable and having multiple purposes.
The method is characterized in that the used complex enzyme is completed through two steps, firstly, the enzymolysis condition with the best enzymolysis effect of single enzyme is screened through a single-factor experiment, and secondly, the input proportion of the complex enzyme is determined through an orthogonal experiment.
Further, the application of the momordica charantia polypeptide is used as a raw material for preparing or preparing food additive auxiliary materials, health-care food or medicines for preventing type II diabetes.
Specifically, in example 5, after crushing bitter melon seeds into 10-mesh small particles, pretreating, adding water according to a water-material ratio of 5:1, adjusting the pH to 8, controlling the extraction temperature to be about 50 ℃, extracting for 2 hours, filtering, taking supernatant fluid to obtain bitter melon protein fluid, performing enzymolysis on the bitter melon protein fluid by using screened complex enzyme (soybean protease, trypsin, pectin and cellulase ═ 2: 1: 1: 1), performing enzyme deactivation treatment for 1 hour at 90 ℃ after the enzymolysis is finished, naturally cooling to room temperature, standing for a period of time until impurities and insoluble substances are all precipitated, and removing the insoluble substances and precipitated impurities. And screening and determining the molecular weight of the enzymolysis polypeptide by adopting a membrane filtration technology, removing impurities from macromolecular non-enzymolysis protein to obtain the bitter gourd polypeptide with the molecular weight of below 3kDa, concentrating the volume, and drying in vacuum to obtain the bitter gourd polypeptide powder.
Further, in example 6, after crushing fresh bitter gourds into 35-mesh small particles, pretreating, adding water according to a water-material ratio of 1:1, adjusting the pH to 7.5, controlling the extraction temperature to be about 45 ℃, extracting for 2.5 hours, filtering, taking the supernatant to obtain bitter gourd protein liquid, performing enzymolysis on the bitter gourd protein liquid by using screened complex enzymes (soybean protease, trypsin, pectin and cellulase are 3: 2: 2: 2.5), performing enzyme deactivation treatment for 1.2 hours at 87 ℃ after the enzymolysis is finished, naturally cooling to room temperature, standing for a period of time until impurities and insoluble substances are completely precipitated, and removing the insoluble substances and precipitated impurities. And screening and determining the molecular weight of the enzymolysis polypeptide by adopting a membrane filtration technology, removing impurities from macromolecular non-enzymolysis protein to obtain the bitter gourd polypeptide with the molecular weight of below 3kDa, concentrating the volume, and drying in vacuum to obtain the bitter gourd polypeptide powder.
Further, in example 7, after crushing the dried bitter gourd slices into 60-mesh small particles, pretreating, adding water according to a water-material ratio of 10:1, adjusting the pH to 8.5, controlling the extraction temperature to be about 70 ℃, performing filtration, taking supernatant fluid, namely bitter gourd protein fluid, performing enzymolysis on the bitter gourd protein fluid by using screened complex enzymes (soybean protease, trypsin, pectin and cellulase are 4: 3: 3: 4), performing enzyme deactivation activity treatment for 1.5 hours at 85 ℃, naturally cooling to room temperature, standing for a period of time until impurities and insoluble substances are completely precipitated, and removing insoluble substances and precipitated impurities. And screening and determining the molecular weight of the enzymolysis polypeptide by adopting a membrane filtration technology, removing impurities from macromolecular non-enzymolysis protein to obtain the bitter gourd polypeptide with the molecular weight of below 3kDa, concentrating the volume, and drying in vacuum to obtain the bitter gourd polypeptide powder.
In the embodiment, the bitter gourd raw material can be fresh bitter gourd, dry bitter gourd slices or bitter gourd seeds, and can also be other bitter gourd raw materials, so that the raw materials have diversity, and the method is proved to have certain stability.
The invention discloses a preparation method of bitter gourd polypeptide, which relates to a preparation and separation and purification method of bitter gourd polypeptide. And (3) carrying out enzymolysis on the balsam pear protein liquid by using a complex enzyme, carrying out ultrafiltration treatment on the clear enzymolysis liquid, and concentrating and drying to obtain the balsam pear polypeptide.
Wherein, the balsam pear polypeptide enzymolysis liquid obtained by filtering is processed by ultrafiltration. The preset molecular cut-off amounts are 10kDa, 3kDa and 1 kDa. Respectively collecting trapped liquids with different molecular weights, and concentrating until the solid content is 50%.
Wherein, when the ultrafiltration process is carried out, the temperature of the ultrafiltration membrane purification system is 35-40 ℃ and the operating pressure is 0.3-0.5MPa when the ultrafiltration is carried out.
The invention provides a preparation method and application of bitter gourd polypeptide, in particular to a method for obtaining bitter gourd polypeptide by carrying out enzymolysis on bitter gourd protein by using a complex enzyme, which comprises the steps of crushing bitter gourd raw materials by using a protein extraction technology, a bioengineering enzymolysis technology and a membrane filtration technology, adding water according to a proportion, adjusting the pH, controlling the temperature at 50 ℃, heating for 2 hours, filtering, taking supernatant fluid to obtain bitter gourd protein fluid, carrying out enzymolysis on the bitter gourd protein fluid by using the screened complex enzyme, and screening the molecular weight of the enzymolyzed bitter gourd polypeptide by using the membrane filtration technology after the enzymolysis is finished to obtain the bitter gourd polypeptide with the molecular weight of below 3 kDa. The fructus Momordicae Charantiae polypeptide can be dried by vacuum drying method, and can be used as raw material for oral liquid, capsule, tablet, granule, etc. The small-molecule bitter gourd polypeptide consists of amino acid, has higher nutritional and hypoglycemic effects, is not added with chemical reagents, is green and safe, and has excellent quality.
The molecular weight of the prepared bitter gourd polypeptide product is below 3kDa, the bitter gourd polypeptide product is easy to absorb by a human body, can be quickly utilized by human tissues, quickly balances blood sugar and can be taken for a long time.
The invention provides a preparation method and application of bitter gourd polypeptide, in particular relates to a preparation method for extracting protein from bitter gourd and obtaining bitter gourd polypeptide through complex enzyme enzymolysis of the protein, and belongs to the technical field of biological medicine. The method comprises the steps of crushing bitter gourd raw materials, pretreating to obtain crude protein liquid, carrying out enzymolysis, and carrying out ultrafiltration treatment on enzymolysis liquid to obtain bitter gourd polypeptide. The complex enzyme used in the enzymatic hydrolysis step was determined by single factor and orthogonal assays (soy protease, trypsin, pectin, and cellulase ═ 2: 1: 1: 1). The invention aims to further improve the content of effective polypeptide molecules and improve the taste of the momordica charantia polypeptide by preparing and purifying the polypeptide in the momordica charantia. Can be used for preparing special diet, health food, food additive, and medicine with blood sugar metabolism regulating effect. Is suitable for industrial production.
The product of the invention can be widely used as a medicine raw material, a medicine, a health product and a food additive. The method is a product which has the advantages of easy holding of technology, simple equipment, simple and easy process, less investment, short period, high added value and no environmental pollution and is suitable for industrial technical production.
According to the preparation method of the bitter gourd polypeptide, the bitter gourd raw materials adopted by the method can be bitter gourds which can not be eaten, are old and are yellow, so that bitter gourd seeds in the bitter gourd polypeptide can also be extracted, the bitter gourd seeds can be taken out for recycling, and the bitter gourd polypeptide is extracted.
The foregoing shows and describes the general principles and broad features of the present invention and advantages thereof. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (10)
1. A preparation method of bitter gourd polypeptide is characterized by comprising the following steps:
1) crushing a bitter gourd raw material into 10-60-mesh small particles, adding the particles into water according to the proportion of 1: 1-10, heating the crushed bitter gourd raw material in water bath, adjusting the pH value to be alkaline, controlling the extraction temperature to be about 45-60 ℃, extracting for about 1.5-3h, and filtering to obtain bitter gourd protein liquid;
2) Adjusting the pH value of the balsam pear protein liquid obtained by filtering in the step 1) to 3-5 to precipitate protein, washing the precipitate with water to be neutral, adding a complex enzyme solution, carrying out enzymolysis on the balsam pear protein, wherein the enzymolysis time is 4-6h, the enzymolysis temperature is 45-65 ℃, after the enzymolysis is finished, carrying out inactivation treatment on the enzyme in the balsam pear protein liquid for 1-1.5h under the condition of water bath at 85-90 ℃, naturally cooling the inactivated balsam pear protein liquid to room temperature, standing until insoluble substances and impurities are completely precipitated, and removing the insoluble substances and precipitated impurities to obtain clarified enzymolyzed crude balsam pear polypeptide;
3) and screening and determining the molecular weight of the crude momordica charantia polypeptide after enzymolysis by adopting a membrane filtration technology, removing impurities from macromolecular non-enzymolysis protein to obtain the momordica charantia polypeptide with the molecular weight of less than 3kDa, concentrating the volume, and drying to obtain the momordica charantia polypeptide powder.
2. The method for preparing momordica charantia polypeptide according to claim 1, wherein step 1) is specifically: crushing a bitter gourd raw material into 10-60-mesh small particles, adding the particles into water according to the proportion of 1: 1-5, heating the crushed bitter gourd in water bath, adjusting the pH to 7.5-8.5, controlling the extraction temperature at 45-70 ℃, extracting for 2-3h, and filtering to obtain the bitter gourd protein liquid.
3. The process for preparing momordica charantia polypeptide according to claim 1 wherein the complex enzyme in step 2) is comprised of soy protease, trypsin, pectinase and cellulase in the ratio of 2-4:1-3:1-3: 1-4.
4. The preparation method of the momordica charantia polypeptide according to claim 1, wherein the momordica charantia protein solution in the step 1) is a momordica charantia protein solution obtained by performing water bath heating extraction on a momordica charantia raw material, adjusting the pH of the solution, and precipitating and separating out proteins.
5. The process for the preparation of a momordica charantia polypeptide according to claim 1, wherein the molecular weight distribution of the momordica charantia polypeptide in the crude momordica charantia polypeptide obtained in step 2) is between 1kDa and 68 kDa.
6. The method for preparing momordica charantia polypeptide according to claim 1, wherein step 3) is specifically: screening the molecular weight of the bitter gourd crude polypeptide with the molecular weight of 1-68 kDa obtained in the step 2) by adopting an ultrafiltration membrane, wherein the preset molecular cut-off amounts of the ultrafiltration membrane are 10kDa, 3kDa and 1kDa, respectively collecting cut-off solutions with different molecular weights, and finally obtaining 4 bitter gourd polypeptide solutions with different molecular weights: 10kDa or more, 10kDa-3kDa, 3kDa-1kDa, or less than 1 kDa;
selecting fructus Momordicae Charantiae polypeptide with molecular weight below 3kDa from 4 fructus Momordicae Charantiae polypeptide solutions with different molecular weights, concentrating to solid content of 50%, and drying to obtain fructus Momordicae Charantiae polypeptide powder.
7. The process for the preparation of the momordica charantia polypeptide according to claim 6, wherein the temperature of the filtration using the ultrafiltration membrane is 35 to 40 ℃ and the operating pressure is 0.3 to 0.5 MPa.
8. The process for the preparation of a momordica charantia polypeptide according to claim 1 wherein the drying is vacuum drying.
9. The method for preparing the momordica charantia polypeptide according to claim 1, wherein the momordica charantia raw material comprises fresh momordica charantia, dried momordica charantia, momordica charantia seeds, dried momordica charantia and momordica charantia pulp.
10. The application of the momordica charantia polypeptide is characterized in that the momordica charantia polypeptide is used as a raw material for preparing or preparing food additive auxiliary materials, health-care food or medicines for preventing type II diabetes.
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