阅读说明：本技术 一种固定化聚半乳糖醛酸酶制备具有抑菌性的芒果皮果胶低聚糖的方法 (Method for preparing mango peel pectin oligosaccharides with antibacterial activity by using immobilized polygalacturonase ) 是由 金征宇 龙杰 薛琳 谢正军 周星 王金鹏 田耀旗 柏玉香 赵建伟 焦爱权 徐学明 于 2019-11-20 设计创作，主要内容包括：一种固定化聚半乳糖醛酸酶制备具有抑菌性的芒果皮果胶低聚糖的方法,属于天然防腐剂制备及酶工程技术领域。本发明首先从芒果皮中提取芒果皮果胶,并用醋酸钠缓冲液溶解芒果皮果胶配成果胶溶液；将聚半乳糖醛酸酶稀释后加入溶胶-凝胶体系中反应融合制成固定化酶；将固定化酶与果胶溶液按比例混合后反应,得到抑菌性的芒果皮果胶低聚糖。本发明首次以固定化聚半乳糖醛酸酶水解芒果皮果胶,增加了酶的重复使用及热稳定性；本发明以芒果皮为原料,减轻了环境污染,还加大资源利用,降低了工业生产成本,在天然防腐剂的研发等领域具有广泛的应用前景。(A method for preparing mango peel pectin oligosaccharide with antibacterial activity by immobilized polygalacturonase belongs to the technical field of natural preservative preparation and enzyme engineering. Firstly, extracting mango peel pectin from mango peel, and dissolving the mango peel pectin by using a sodium acetate buffer solution to prepare a pectin solution; diluting polygalacturonase, adding the diluted polygalacturonase into a sol-gel system, and reacting and fusing to prepare immobilized enzyme; mixing the immobilized enzyme and pectin solution in proportion, and reacting to obtain antibacterial mango peel pectin oligosaccharide. The method uses the immobilized polygalacturonase to hydrolyze mango peel pectin for the first time, so that the repeated use and the thermal stability of the enzyme are increased; the invention takes the mango peel as the raw material, thereby reducing the environmental pollution, increasing the resource utilization, reducing the industrial production cost and having wide application prospect in the fields of the research and development of natural preservatives and the like.)

1. A method for preparing mango peel pectin oligosaccharides with bacteriostasis by immobilized polygalacturonase is characterized by comprising the following steps: firstly, extracting mango peel pectin from mango peel, and dissolving the mango peel pectin by using a sodium acetate buffer solution to prepare a pectin solution; diluting polygalacturonase, adding the diluted polygalacturonase into a sol-gel system, and reacting and fusing to prepare immobilized enzyme; mixing the immobilized enzyme and pectin solution in proportion, and reacting to obtain antibacterial mango peel pectin oligosaccharide.

2. The method for preparing mango peel pectin oligosaccharides with bacterial inhibition by using the immobilized polygalacturonase of claim 1, which is characterized by the following steps:

(1) preparation of mango peel pectin solution: taking mango peel, cleaning, boiling, inactivating enzyme, leaching with acid, leaching with hot water, filtering to obtain supernatant extract, precipitating with ethanol, washing with anhydrous ethanol, filtering, freeze-drying to obtain mango peel pectin, and dissolving the mango peel pectin with sodium acetate buffer solution to obtain pectin solution;

(2) preparation of immobilized polygalacturonase: adding a PVA solution, a NaF solution and deionized water into a polygalacturonase solution under a stirring state, stirring to obtain a uniform solution, slowly adding n-Octyl Triethoxysilane (OTES) and a tetraethyl orthosilicate (TEOS) solution, continuously stirring, centrifuging to pour out a supernatant, and washing with deionized water for a plurality of times to obtain an immobilized enzyme;

(3) and (3) hydrolyzing, namely putting the immobilized enzyme prepared in the step (2) into a centrifugal tube, adding the mango peel pectin solution prepared in the step (1) as a substrate, wherein the mass-volume ratio of the immobilized enzyme to the substrate is 1:1 ~ 10, and reacting and centrifuging to obtain the bacteriostatic mango peel pectin oligosaccharide.

3. The method for preparing mango peel pectin oligosaccharides with bacterial inhibition by using immobilized polygalacturonase according to claim 2, wherein the mango peel pectin solution in step (1) is prepared by the following steps:

a. boiling Mangifera Indica pericarp, inactivating enzyme, oven drying at 40 ~ 60 deg.C, pulverizing, and sieving with 40 ~ 60 mesh sieve;

b. adding water according to a solid-to-liquid ratio of 1: 10 ~ 50, adjusting pH to 2 ~ 5 with 0.01 ~ 1mol/L hydrochloric acid, leaching in a water bath at 70 ~ 90 deg.C for 1 ~ 5h, and filtering to obtain extractive solution;

c. mixing the obtained extractive solution with 95% ethanol at a volume ratio of 1:1 ~ 3, stirring, standing at 4 deg.C for 6 ~ 12h, and filtering to obtain precipitate;

d. washing the obtained precipitate with anhydrous ethanol for 2 ~ 3 times, and freeze drying at-40 ~ -60 deg.C for 48 ~ 72h to obtain mango peel pectin;

e. sodium acetate buffer solution is adopted to prepare mango peel pectin solution with the mass concentration of 0.1% ~ 2%.

4. The method for preparing mango peel pectin oligosaccharides with bacteriostasis by using the immobilized polygalacturonase as claimed in claim 2, wherein the extraction rate of mango peel pectin in step (1) is 7% ~ 15%.

5. The method for preparing mango peel pectin oligosaccharides with bacterial inhibition by using the immobilized polygalacturonase according to claim 2, wherein the immobilized polygalacturonase in step (2) is prepared by the following specific process:

f. diluting polygalacturonase by 10 ~ 50 times to obtain diluted pure enzyme solution;

g. adding pure enzyme solution obtained in the step f, PVA solution with the mass volume ratio of 2% ~ 5%, NaF solution with the mass volume ratio of 0.1 ~ 1mol/L and deionized water with the volume ratio of 4 ~ 10: 2: 1:1 ~ 2 under the stirring state, and stirring for 30 ~ 60min to obtain uniform solution;

h. slowly adding n-octyl triethoxysilane and tetraethyl orthosilicate in a volume ratio of 1: 2 ~ 5, adding n-octyl triethoxysilane and tetraethyl orthosilicate solution, and continuing stirring for 8 ~ 12 h;

i. and (3) centrifuging at 6000-.

6. The method for preparing mango peel pectin oligosaccharides with bacterial inhibition by using the immobilized polygalacturonase of claim 5, wherein the method comprises the following steps: in the step g, the volume concentration of the PVA solution is 4%; the concentration of the NaF solution is 1 mol/L.

7. The method for preparing mango peel pectin oligosaccharides with bacteriostasis by using the immobilized polygalacturonase as claimed in claim 2, wherein the protein loading rate in the immobilized enzyme in the step (2) is 60% ~ 90%.

8. The method for preparing mango peel pectin oligosaccharides with bacteriostasis by using immobilized polygalacturonase according to claim 2, wherein the hydrolysis in step (3) is carried out by putting immobilized enzyme into a centrifuge tube, adding pectin solution into the immobilized enzyme, wherein the mass-to-volume ratio of the immobilized enzyme to the pectin solution is 1:1 ~ 10, reacting at 30 ~ 60 ℃ and pH 3 ~ 7 for 5 ~ 60min, and centrifuging at 5000-10000r/min for 5-30min to obtain the mango peel pectin oligosaccharides with bacteriostasis.

9. The method for preparing mango peel pectin oligosaccharides with bacterial inhibition by using immobilized polygalacturonase according to claim 8, wherein the method comprises the following steps: the polymerization degrees of the bacteriostatic mango peel pectin oligosaccharides are different; the immobilized enzyme can be repeatedly used.

Technical Field

The invention relates to a method for preparing antibacterial mango peel pectin oligosaccharide by immobilized polygalacturonase, and belongs to the technical field of natural preservative preparation and enzyme engineering.

Background

The food additive is a catalyst and a base of modern food industry, is known as soul of the modern food industry, has permeated into various fields of food processing, and plays a decisive role in the development of the food industry in a certain sense. With the improvement of the living standard of people and the update of consumption concept, people pay more and more attention to the safety of food, and the harm of microorganisms is the primary problem of food safety. In order to solve the problem, the preservation and freshness of food are enhanced, the shelf life of the food is prolonged, various preservatives are commonly used in the food industry, wherein, the chemically synthesized preservatives are more, the problems of carcinogenicity, teratogenicity and easy food poisoning of the chemically synthesized preservatives cause people's worry, and meanwhile, the chemically artificially synthesized food preservatives can also generate certain substances harmful to human bodies. Therefore, the development trend of food preservatives is moving towards nature, nutrition and functionalization, and the search for broad-spectrum, efficient, safe and economical natural preservatives has become a hot spot in the food field.

Pectin Oligosaccharides (POS), also known as polygalacturonic acids, are a new class of prebiotics made from 2 to 20 galacturonic acids bonded by alpha-1, 4-glycosidic bonds. In recent years, pectin oligosaccharides have been used in various fields due to their specific prebiotic activities, such as anti-tumor and anti-oxidation activities, but have little attention on their bacteriostatic activity. The natural preservatives such as chitosan currently studied are only suitable for acidic foods containing no protein, and thus the market of natural preservatives is still not mature at home and abroad. On the other hand, the pectin oligosaccharide is reported to have a good inhibition effect on the growth of bacteria, namely, the research of using the pectin oligosaccharide as a preservative is a new field and has great development value and development prospect.

Polygalacturonase (EC 3.2.1.15) is a major hydrolase in pectinase, and can randomly cut alpha-1, 4 glycosidic bonds between non-esterified galacturonic acids in the pectin backbone to generate pectin oligosaccharides with different polymerization degrees. But the possibility of industrialization is restricted by the characteristics of higher price of free enzyme, poor thermal stability and the like. The immobilization of the enzyme is a new technology developed by people aiming at the defects of free enzyme, can improve the thermodynamic stability of the enzyme after immobilization, regulate and control the activity and selectivity of the enzyme, is favorable for reaction in a substrate solution, can be conveniently separated and reused, and has important significance for improving the utilization efficiency of the enzyme, prolonging the service life and reducing the industrial cost. The sol-gel method is to mix silicon ester, water, catalyst and solvent, and then to perform immobilization by hydrolysis, condensation (including poly condensation), sol, aging and gel, and since silica has more chemical and thermal stability than organic polymer materials, it is widely used. The immobilized enzyme meets the strategic requirements of sustainable development due to the ecological environment effects of saving energy and resources and reducing pollution.

Mango is one of the important tropical fruits, and China is the second largest mango producing country next to India. Because of their extreme decay and deterioration, mangoes are also widely used in commercial processes such as: mango juice, mango jam, mango powder, dried mango and other deep-processed products. However, a large amount of mango peel is generated in the mango processing process, the mango peel accounts for 15 to 20 percent of the weight of the fruit, the mango peel has no commercial application value, and serious problems of resource waste, environmental pollution and the like are caused if the mango peel is not properly treated. Today, the use of plant cell wall polysaccharides as a source of novel high value-added oligosaccharides has become a trend.

Disclosure of Invention

The invention aims to provide a method for preparing mango peel pectin oligosaccharide with antibacterial activity by immobilized polygalacturonase.

The technical scheme of the invention is a method for preparing mango peel pectin oligosaccharide with bacteriostasis by immobilized polygalacturonase, which comprises the steps of firstly extracting mango peel pectin from mango peel, and dissolving the mango peel pectin by using a sodium acetate buffer solution to prepare a pectin solution; diluting polygalacturonase, adding the diluted polygalacturonase into a sol-gel system, and reacting and fusing to prepare immobilized enzyme; mixing the immobilized enzyme and pectin solution in proportion, and reacting to obtain antibacterial mango peel pectin oligosaccharide.

Further, the specific steps are as follows:

(1) preparation of mango peel pectin solution: taking mango peel, cleaning, boiling, inactivating enzyme, leaching with acid, leaching with hot water, filtering to obtain supernatant extract, precipitating with ethanol, washing with anhydrous ethanol, filtering, freeze-drying to obtain mango peel pectin, and dissolving the mango peel pectin with sodium acetate buffer solution to obtain pectin solution;

(2) preparation of immobilized polygalacturonase: adding a PVA solution, a NaF solution and deionized water into a polygalacturonase solution under a stirring state, stirring to obtain a uniform solution, slowly adding n-Octyl Triethoxysilane (OTES) and a tetraethyl orthosilicate (TEOS) solution, continuously stirring, centrifuging to pour out a supernatant, and washing with deionized water for a plurality of times to obtain an immobilized enzyme;

(3) and (3) hydrolyzing, namely putting the immobilized enzyme prepared in the step (2) into a centrifugal tube, adding the mango peel pectin solution prepared in the step (1) as a substrate, wherein the volume ratio of the immobilized enzyme to the substrate is 1:1 ~ 10, and reacting and centrifuging to obtain the bacteriostatic mango peel pectin oligosaccharide.

The preparation process of the mango peel pectin solution in the step (1) is as follows:

a. boiling Mangifera Indica pericarp, inactivating enzyme, oven drying at 40 ~ 60 deg.C, pulverizing, and sieving with 40 ~ 60 mesh sieve;

b. adding water according to a solid-to-liquid ratio of 1: 10 ~ 50, adjusting pH to 2 ~ 5 with 0.01 ~ 1mol/L hydrochloric acid, leaching in a water bath at 70 ~ 90 deg.C for 1 ~ 5h, and filtering to obtain extractive solution;

c. mixing the obtained extractive solution with 95% ethanol at a volume ratio of 1:1 ~ 3, stirring, standing at 4 deg.C for 6 ~ 12h, and filtering to obtain precipitate;

d. washing the obtained precipitate with anhydrous ethanol for 2 ~ 3 times, and freeze drying at-40 ~ -60 deg.C for 48 ~ 72h to obtain mango peel pectin;

e. sodium acetate buffer solution is adopted to prepare mango peel pectin solution with the mass concentration of 0.1% ~ 2%.

The extraction rate of mango peel pectin in the step (1) is 7% ~ 15%.

The specific process for preparing the immobilized polygalacturonase in the step (2) is as follows:

f. diluting polygalacturonase by 10 ~ 50 times to obtain diluted pure enzyme solution;

g. adding pure enzyme solution obtained in the step f, PVA solution with the mass volume ratio of 2% ~ 5%, NaF solution with the mass volume ratio of 0.1 ~ 1mol/L and deionized water with the volume ratio of 4 ~ 10: 2: 1:1 ~ 2 under the stirring state, and stirring for 30 ~ 60min to obtain uniform solution;

h. slowly adding n-octyl triethoxysilane and tetraethyl orthosilicate according to the volume ratio to feed 1: 2 ~ 5, adding n-octyl triethoxysilane and tetraethyl orthosilicate solution, and continuing stirring for 8 ~ 12 h;

i. and (3) centrifuging at 6000-.

In the step g, the volume concentration of the PVA solution is 4%; the concentration of the NaF solution is 1 mol/L.

In the step (2), the protein loading rate of the immobilized enzyme is 60% ~ 90%.

The hydrolysis process in the step (3) comprises the steps of putting immobilized enzyme into a centrifuge tube, adding pectin solution, reacting for 5 ~ 60min at 30 ~ 60 ℃ and pH 3 ~ 7, and centrifuging for 5-30min at 5000-10000r/min to obtain the bacteriostatic mango peel pectin oligosaccharide, wherein the mass volume ratio of the immobilized enzyme to the pectin solution is 1:1 ~ 10.

The polymerization degrees of the bacteriostatic mango peel pectin oligosaccharides are different; the immobilized enzyme can be repeatedly used.

The method mainly uses sodium fluoride as an initiator, n-octyltriethoxysilane and tetraethyl orthosilicate as silane precursors, water as a solvent to uniformly mix raw materials in a liquid phase, a series of hydrolysis and condensation reactions are carried out, a stable transparent sol system is formed in the solution, the colloidal particles slowly polymerize after aging, long chains are continuously formed to form a gel network, and the enzyme is wrapped in the gel to finally obtain the gel with immobilized polygalacturonase.

Compared with free enzyme, the immobilized enzyme prepared by the invention has higher relative enzyme activity in an acid environment, is more resistant to the acid environment than the free enzyme, and has wider pH value tolerance. In the environment of pH 3.0, the free enzyme retains 75.72% of relative enzyme activity, and the immobilized enzyme retains 94.60% of enzyme activity.

The immobilized enzyme prepared by the invention has better heat resistance than free enzyme, when the reaction temperature is increased to 80 ℃, the free enzyme only retains 6.3 percent of enzyme activity, and the immobilized enzyme still retains 81.5 percent of enzyme activity.

The immobilized enzyme prepared by the invention has simple recycling steps, supernatant is centrifugally taken out after the reaction is finished, and new reaction liquid is added into the precipitate to realize the recycling of the enzyme, so that the production cost is greatly reduced, and the recovery rate of the enzyme activity reaches 93.4 percent; and the repeated stability is better, and the enzyme activity retention after repeated use for 6 times reaches 65 percent.

The invention has the beneficial effects that: the mango peel pectin oligosaccharide prepared by the method can solve the problem of pollution of the existing mango peel, realizes comprehensive utilization of resources, is safe, has no pollution, has broad-spectrum antibacterial activity, can inhibit the growth of gram-positive bacteria and gram-negative bacteria, and is a natural antibacterial agent with great development potential.

Detailed Description