阅读说明：本技术 一种快速提取红薯中多肽类物质的方法 (Method for rapidly extracting polypeptide substances from sweet potatoes ) 是由 舒友菊 刘锦琼 薛冬 王自力 刘萍 贾晓慧 王青云 郭永仓 于 2021-09-17 设计创作，主要内容包括：本发明涉及一种快速提取红薯中多肽类物质的方法,将新鲜洗干净的红薯用捣碎机制成红薯粉末,取一定量红薯粉末,加入纤维素酶溶液混合,再加入水,混合搅拌,在常温下酶解3小时,过滤除渣,得到混合液；将壳聚糖海藻酸钠溶液缓慢滴加到混合液中直到沉淀完毕,倒去上清液,得到沉淀；将所得沉淀清洗3-5次,入植物蛋白酶,再加水搅拌,然后在40-65℃酶解5-10小时,即得红薯中多肽类物质。本发明利用生物酶解法快速提取红薯中的多肽类物质,该多肽类物质具有优异的抗氧化活性,可以实现红薯的高值化利用,同时可以减少红薯加工过程中肽流失到废水中对生产过程的环境污染,为红薯中多肽类物质的提取开发提供了理论依据,为以后产业开发及升级奠定了基础。(The invention relates to a method for rapidly extracting polypeptide substances from sweet potatoes, which comprises the steps of preparing sweet potato powder from fresh and clean sweet potatoes by a pounding machine, taking a certain amount of sweet potato powder, adding a cellulase solution for mixing, adding water, mixing and stirring, carrying out enzymolysis at normal temperature for 3 hours, filtering and removing slag to obtain a mixed solution; slowly dripping a chitosan sodium alginate solution into the mixed solution until the precipitation is finished, and pouring out a supernatant to obtain a precipitate; washing the obtained precipitate for 3-5 times, adding plant protease, adding water, stirring, and performing enzymolysis at 40-65 deg.C for 5-10 hr to obtain polypeptide substance in sweet potato. The invention utilizes the biological enzymolysis method to rapidly extract the polypeptide substances in the sweet potatoes, the polypeptide substances have excellent antioxidant activity, can realize high-valued utilization of the sweet potatoes, can reduce the environmental pollution to the production process caused by the loss of peptides into wastewater in the processing process of the sweet potatoes, provides a theoretical basis for the extraction and development of the polypeptide substances in the sweet potatoes, and lays a foundation for the later industrial development and upgrading.)

1. A method for rapidly extracting polypeptide substances from sweet potatoes is characterized by comprising the following steps:

(1) preparing fresh and clean sweet potatoes into sweet potato powder by using a pounding machine, taking a certain amount of the obtained sweet potato powder, adding a cellulase solution, mixing, adding water, mixing and stirring to prepare a mixture with the solid mass content of 20-30%, and performing enzymolysis on the obtained mixture for 3 hours under normal temperature stirring; filtering and removing slag after enzymolysis is finished to obtain mixed liquor;

(2) slowly dripping a chitosan sodium alginate solution into the mixed solution obtained in the step (1) until the precipitation is finished, and stirring at a low speed while dripping the chitosan sodium alginate solution; after the precipitation is finished, pouring out the supernatant to obtain a precipitate;

(3) and (3) washing the precipitate obtained in the step (2) with water for 3-5 times, adding plant protease, adding water, stirring, performing enzymolysis at 40-65 ℃ for 5-10 hours, and stirring uninterruptedly during enzymolysis to finally obtain the polypeptide substance in the sweet potatoes.

2. The method for rapidly extracting polypeptide substances from sweet potatoes as claimed in claim 1, wherein the mass concentration of the cellulase solution in step (1) is 0.2-0.6%, and the cellulase solution is metered in according to the proportion of 0.2-1ml cellulase solution added per gram sweet potato powder.

3. The method for rapidly extracting polypeptide substances from sweet potatoes as claimed in claim 1 or 2, wherein the mass concentration of the sodium alginate chitosan solution in the step (2) is 10%.

4. The method for rapidly extracting polypeptide substances from sweet potatoes as claimed in claim 1, wherein the plant protease in the step (3) is papain, and the mass ratio of the added mass of the papain to the precipitate obtained in the step (2) is 1: 100000.

5. The method for rapidly extracting polypeptide substances from sweet potatoes as claimed in claim 1, wherein the mass ratio of the added water in the step (3) to the sweet potato powder used in the step (1) is 1: 1.

Technical Field

The invention relates to the technical field of biology, in particular to a method for quickly extracting polypeptide substances from sweet potatoes.

Background

Sweet potatoes (Ipomoea batatas (L.)) are native to Central America, are Convolvulaceae plants of Tulipae, and are named sweet potatoes, and other sweet potatoes, sweet potatoes and the like are commonly used. The Chinese characters are introduced from the early stage of the Ming Dynasty perpetual calendar, and the Chinese characters have profound influence on agriculture and economy in China. According to FAO (world food and agricultural organization) statistics, the total yield of Chinese sweet potatoes is 5.20 multiplied by 10 in 2019⁷Ton, harvest area 2.37X 10⁶Hectare, 2.19 tons per hectare. Currently, sweet potatoes remain one of the most prominent food crops.

Scientific research shows that each 100g of sweet potatoes contains 1.8g of protein, 29.5g of sugar, 0.2g of fat, 20mg of phosphorus, 18mg of calcium, 0.4mg of iron and 1.1mg of carotene, and also contains nicotinic acid, vitamins, linoleic acid, dietary fibers, lysine, polysaccharide, polypeptide and the like, wherein the content of the vitamins is 3-6 times higher than that of normal grains, and the vitamins are rich in lysine compared with the grains.

Sweet potato polypeptide is a substance with very strong biological activity, has very strong water solubility and very high nutritional value, can be applied to the medical care and food processing industries, is ignored as waste treatment and disposal in the sweet potato processing industry at present, so the polypeptide in the sweet potato is developed and used as a future health care and nutrient, and has a certain application prospect for the upgrading of the sweet potato industry and the increasing of the additional value of the sweet potato.

According to the current situation of sweet potato processing, most sweet potatoes are processed into sweet potato starch, vermicelli and vermicelli, and polypeptide substances in the sweet potatoes are discharged as waste liquid, so that the problems of serious resource waste and environmental pollution are caused.

Disclosure of Invention

The invention aims to provide a method for quickly extracting polypeptide substances from sweet potatoes, which utilizes a biological enzymolysis method to quickly extract the polypeptide substances from the sweet potatoes, has excellent antioxidant activity, can realize high-valued utilization of the sweet potatoes, can reduce environmental pollution to the production process caused by peptide loss to wastewater in the processing process of the sweet potatoes, provides a theoretical basis for extraction and development of the polypeptide substances from the sweet potatoes, and lays a foundation for later industrial development and upgrading.

The invention is realized by the following technical scheme, and the method for rapidly extracting the polypeptide substances from the sweet potatoes provided by the invention specifically comprises the following steps:

(1) preparing fresh and clean sweet potatoes into sweet potato powder by using a pounding machine, taking a certain amount of the obtained sweet potato powder, adding a cellulase solution, mixing, adding water, mixing and stirring to prepare a mixture with the solid mass content of 20-30%, and performing enzymolysis on the obtained mixture for 3 hours under normal temperature stirring; filtering and removing slag after enzymolysis is finished to obtain mixed liquor;

(2) slowly dripping a chitosan sodium alginate solution into the mixed solution obtained in the step (1) until the precipitation is finished, and stirring at a low speed while dripping the chitosan sodium alginate solution; after the precipitation is finished, pouring out the supernatant to obtain a precipitate;

(3) and (3) washing the precipitate obtained in the step (2) with water for 3-5 times, adding plant protease, adding water, stirring, performing enzymolysis at 40-65 ℃ for 5-10 hours, and stirring uninterruptedly during enzymolysis to finally obtain the polypeptide substance in the sweet potatoes.

Further, the mass concentration of the cellulase solution in the step (1) is 0.2-0.6%, and the addition amount of the cellulase solution is metered according to the proportion of adding 0.2-1ml of cellulase solution into each gram of sweet potato powder.

Further, the mass concentration of the chitosan sodium alginate solution in the step (2) is 10%.

Further, the plant protease in the step (3) is papain, and the mass ratio of the added mass of the papain to the mass of the precipitate obtained in the step (2) is 1: 100000.

Further, the mass ratio of the added water in the step (3) to the sweet potato powder used in the step (1) is 1: 1.

The biological enzymolysis technology makes the substances fully decomposed by the action of enzyme to obtain the extract required by the biological engineering. Meanwhile, the characteristics of different active ingredients of the substances are mastered by utilizing different catalytic actions of different enzymes, the active substances can be extracted, compared with the traditional mode of adopting high-temperature decomposition or low-temperature quick freezing and the like, the biological enzymolysis technology can well prevent most active substances from being damaged, and the efficacy of the extracted sweet potato polypeptide substances is ensured. The biological enzyme is a special biological catalyst which is generated by living cells of organisms and exists in the form of protein, can decompose plant cell walls under the conditions of normal temperature, normal pressure and mild acid and alkali, greatly improves the extraction rate of effective components in natural plants, improves the filtration speed and purification effect in the production process, and improves the product purity and the quality of a preparation.

The cellulase adopted in the step (1) is a general name of a group of enzymes for degrading cellulose to generate glucose, comprises 3 components of endoglucanase, cellobiohydrolase and beta-glucosidase, has the effects of decomposing, softening cellulose, destroying cell walls and increasing the dissolution amount of plant cell contents, can destroy fibrous substances such as cell walls in sweet potatoes, and is beneficial to the dissolution and release of sweet potato protein.

The chitosan-sodium alginate adopted in the step (2) is a green skeleton-type structure biomacromolecule, and can efficiently capture soluble polypeptide and protein in water. The substance is formed by quaternary ammonification of chitosan and soluble alginic acid under the action of sodium hydroxide. The chitosan is prepared by deacetylating chitin widely existing in the nature, is a natural high molecular substance, has excellent biological functionality and compatibility, blood compatibility, safety, microbial degradability and the like, is prepared by extracting chitin serving as a raw material, is insoluble in water, can be dissolved in dilute acid, and can be absorbed by a human body. The chitosan sugar acid is a high molecular basic polysaccharide polymer with cations and has unique physical and chemical properties and biological activation function. Alginic acid is a natural substance with a skeleton structure, can efficiently identify and capture metal ions in a water body, and has targeting effect. The chitosan-sodium alginate has important functions on protein and polypeptide.

The plant protease added in the step (3) is papain, one or more effective components in the plant are directionally obtained and concentrated by taking papaya as a raw material through a physical and chemical extraction and separation process, the structure of the effective components is not changed to form a low-specificity proteolytic enzyme, the active center of the papain contains cysteine, the papain belongs to thiol protease, and the papain has the characteristics of high enzyme activity, good thermal stability, nature, sanitation, safety and the like; the sweet potato polypeptide can be obtained by using papain to catalyze the hydrolysis of protein, and the enzymolysis rate of the protein and cellulose of the sweet potato reaches more than 90 percent.

According to the invention, through a three-step enzymolysis method, the sweet potato protein is released by utilizing the hydrolysis wall breaking of cellulase, the sweet potato protein is precipitated by utilizing a chitosan sodium alginate solution, and then the sweet potato protein is hydrolyzed by utilizing papain, so that the polypeptide substance in the sweet potato is finally obtained. The method can quickly extract the polypeptide substances in the sweet potatoes, improves the utilization rate of sweet potato protein, and retains the higher natural biological activity of the polypeptide substances in the sweet potatoes. The method has the advantages of low requirements on process equipment, easy industrial application and low extraction cost.

Compared with the prior art, the invention has the following advantages:

the invention takes sweet potato as raw material, and utilizes biological enzymolysis method to extract polypeptide substance in sweet potato, the method has mild reaction condition, little influence on protein, and high natural biological activity, and can well control reaction condition. Factors such as enzymolysis time, temperature, enzyme dosage, substrate concentration and the like are controlled in the extraction process to investigate the influence on the polypeptide extraction rate and the antioxidant activity, the enzymolysis rate of protein and cellulose of sweet potatoes reaches over 90 percent, cheap raw materials are provided for the biological health industry, the extraction cost is reduced, high-valued utilization of the sweet potatoes is realized, and meanwhile, the environmental pollution of the production process caused by the loss of peptide into wastewater in the sweet potato processing process is reduced.

Drawings

FIG. 1 is an infrared image of polypeptide substances in sweet potatoes obtained by the invention;

FIG. 2 is a nuclear magnetic resonance image of polypeptide substances in the sweet potatoes obtained by the invention;

FIG. 3 is a graph showing the effect of the polypeptide substances in the sweet potato of the present invention on cell growth.

Detailed Description

For a better understanding of the contents of the invention, reference will now be made to the following examples and accompanying drawings which illustrate the invention. The present embodiment is implemented based on the technology of the present invention, and a detailed implementation manner and operation steps are given, but the scope of the present invention is not limited to the following embodiments.

The method for rapidly extracting the polypeptide substances from the sweet potatoes specifically comprises the following steps:

(1) preparing fresh and clean sweet potatoes into sweet potato powder by using a pounding machine, taking a certain amount of the obtained sweet potato powder, adding a cellulase solution with the mass concentration of 0.2-0.6%, mixing, adding a certain amount of water, mixing and stirring to prepare a mixture with the solid mass content of 20-30%, and carrying out enzymolysis on the obtained mixture for 3 hours under normal temperature stirring; filtering and removing slag after enzymolysis is finished to obtain mixed liquor;

wherein the addition amount of the cellulase solution is metered according to the proportion of adding 0.2-1ml of cellulase solution into each gram of sweet potato powder.

(2) Slowly dropwise adding a 10% chitosan sodium alginate solution into the mixed solution obtained in the step (1) until precipitation is finished, and stirring at a low speed while dropwise adding the chitosan sodium alginate solution; after the precipitation is finished, pouring out the supernatant to obtain a precipitate;

(3) and (3) washing the precipitate obtained in the step (2) with water for 3-5 times, adding plant protease, adding water, stirring, performing enzymolysis at 40-65 ℃ for 5-10 hours, and stirring uninterruptedly during enzymolysis to finally obtain the polypeptide substance in the sweet potatoes. Wherein the mass ratio of the added plant protease to the precipitate obtained in the step (2) is 1:100000, and the plant protease is specifically papain. The mass ratio of the added water to the sweet potato powder used in the step (1) is 1: 1.

The chitosan sodium alginate solution can be prepared according to the following method:

putting 1g of alginic acid into a three-neck flask, adding 10ml of PBS buffer solution with the molar concentration of 50mM and the pH value of 5.5, stirring and dissolving at normal temperature, adding quaternary ammoniated chitosan after complete dissolution, stirring for 30 minutes, adding a sodium hydroxide solution with the mass fraction of 10%, and adjusting the pH value to about 8.5 in a water bath at 45 ℃ to obtain the chitosan-sodium alginate solution.

The following is a detailed description of specific embodiments.

Example 1:

(1) preparing sweet potato powder from fresh and clean sweet potatoes by using a pounding machine, taking 500g of the obtained sweet potato powder, adding 0.2ml of cellulase solution into each gram of sweet potato powder according to the proportion of adding 0.2ml of cellulase solution, adding a certain amount of water after mixing, mixing and stirring to prepare a mixture with the solid mass content of 20%, and carrying out enzymolysis on the obtained mixture for 3 hours under normal temperature stirring; filtering and removing slag after enzymolysis is finished to obtain mixed liquor;

(2) slowly dripping the prepared chitosan sodium alginate solution with the mass concentration of 10% into the mixed solution obtained in the step (1) until the precipitation is finished, stirring at a low speed while dripping the chitosan sodium alginate solution, and pouring out the supernatant after the precipitation is finished to obtain the precipitate;

(3) washing the precipitate obtained in the step (2) with water for 3-5 times, adding papain, adding 500g of water, stirring, performing enzymolysis at 40 ℃ for 10 hours, and stirring uninterruptedly while performing enzymolysis to obtain polypeptide substances in the sweet potatoes; wherein the mass ratio of the added amount of the papain to the precipitate obtained in the step (2) is 1: 100000.

Example 2:

(1) preparing sweet potato powder from fresh and clean sweet potatoes by using a pounding machine, taking 500g of the obtained sweet potato powder, adding 0.5ml of cellulase solution into each gram of sweet potato powder according to the proportion, adding a certain amount of water after mixing, mixing and stirring to prepare a mixture with the solid mass content of 25%, and carrying out enzymolysis on the obtained mixture for 3 hours under normal temperature stirring; filtering and removing slag after enzymolysis is finished to obtain mixed liquor;

(2) slowly dripping the prepared chitosan sodium alginate solution with the mass concentration of 10% into the mixed solution obtained in the step (1) until the precipitation is finished, stirring at a low speed while dripping the chitosan sodium alginate solution, and pouring out the supernatant after the precipitation is finished to obtain the precipitate;

(3) washing the precipitate obtained in the step (2) with water for 3-5 times, adding papain, adding 500g of water, stirring, performing enzymolysis at 50 ℃ for 8 hours, and stirring uninterruptedly while performing enzymolysis to obtain polypeptide substances in the sweet potatoes; wherein the mass ratio of the added amount of the papain to the precipitate is 1: 100000.

Example 3:

(1) preparing sweet potato powder from fresh and clean sweet potatoes by using a pounding machine, taking 500g of the obtained sweet potato powder, adding 1ml of cellulase solution into each gram of sweet potato powder, adding a certain amount of water after mixing, mixing and stirring to prepare a mixture with the solid mass content of 30%, and carrying out enzymolysis on the obtained mixture for 3 hours under normal temperature stirring; filtering and removing slag after enzymolysis is finished to obtain mixed liquor;

(2) slowly dripping the prepared chitosan sodium alginate solution with the mass concentration of 10% into the mixed solution obtained in the step (1) until the precipitation is finished, stirring at a low speed while dripping the chitosan sodium alginate solution, and pouring out the supernatant after the precipitation is finished to obtain the precipitate;

(3) washing the precipitate obtained in the step (2) with water for 3-5 times, adding papain, adding 500g of water, stirring, performing enzymolysis at 65 ℃ for 5 hours, and stirring uninterruptedly while performing enzymolysis to finally obtain polypeptide substances in the sweet potatoes; wherein the mass ratio of the added amount of the papain to the precipitate is 1: 100000.

FIG. 1 is an infrared chart of polypeptide substances in sweet potatoes obtained by the invention, and it can be seen from the chart that 3400-3500 is a telescopic vibration absorption peak of NH, 1550-1510, 1680-1630 is a carbonyl absorption peak, and about 1260 is an absorption peak of CN.

FIG. 2 is a nuclear magnetic resonance image of the polypeptide substance in the sweet potato obtained by the present invention, and it can be seen from the image that the amino acid sequence of the polypeptide substance in the sweet potato obtained by sequencing under the amino acid equilibrium point is Trp1-MeG 2-Asn 3-Thr 4-Gly 5-Orn 6-Ala 7.

FIG. 3 is a graph of the effect of different concentrations of polypeptide on epidermal cell growth, from which it can be seen that: the epidermal cells are all green, which indicates that the cells grow well, and the polypeptide has good immunity to the epidermal cells.

The above description is only an embodiment of the present invention, and is not intended to limit the present invention in any way, and the present invention may also have other embodiments according to the above structures and functions, and is not listed again. Therefore, any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present invention by those skilled in the art can be made within the technical scope of the present invention.