阅读说明：本技术 一种抗氧化广谱抑菌剂的制备方法 (Preparation method of antioxidant broad-spectrum bacteriostatic agent ) 是由 许威 郑树青 李志帆 罗登林 葛佩佩 金伟平 孙玉青 徐路阳 于 2019-09-20 设计创作，主要内容包括：本发明提供一种抗氧化广谱抑菌剂及其制备方法,该抑菌剂为利用抗坏血酸/过氧化氢作为反应体系偶联溶菌酶和茶多酚得到的溶菌酶-茶多酚共聚物。制备方法包括1：取溶菌酶溶于超纯水中,在氮气保护下,加入过氧化氢与抗坏血酸,水浴锅中搅拌,得混合溶液A；2：向混合液A中加入茶多酚溶液,搅拌得混合溶液B,然后将混合溶液B搅拌；3：将步骤2所得溶液进行透析,得溶菌酶-茶多酚复合体系；4：将步骤3所得溶菌酶-茶多酚复合体系置于冷冻干燥器中进行冷冻干燥,得到溶菌酶-茶多酚共聚物,即为抗氧化广谱抑菌剂。本发明方法采用抗坏血酸作为还原剂,过氧化氢作为氧化剂,制备过程中无需添加有毒有害的试剂,制备过程简单、温和、绿色。(The invention provides an antioxidant broad-spectrum bacteriostatic agent and a preparation method thereof, wherein the bacteriostatic agent is lysozyme-tea polyphenol copolymer obtained by coupling lysozyme and tea polyphenol by using ascorbic acid/hydrogen peroxide as a reaction system. The preparation method comprises the following steps of 1: dissolving lysozyme in ultrapure water, adding hydrogen peroxide and ascorbic acid under the protection of nitrogen, and stirring in a water bath kettle to obtain a mixed solution A; 2: adding a tea polyphenol solution into the mixed solution A, stirring to obtain a mixed solution B, and then stirring the mixed solution B; 3: dialyzing the solution obtained in the step 2 to obtain a lysozyme-tea polyphenol composite system; 4: and (3) placing the lysozyme-tea polyphenol composite system obtained in the step (3) into a freeze dryer for freeze drying to obtain a lysozyme-tea polyphenol copolymer, namely the antioxidant broad-spectrum bacteriostatic agent. The method adopts the ascorbic acid as a reducing agent and the hydrogen peroxide as an oxidizing agent, does not need to add toxic and harmful reagents in the preparation process, and has simple, mild and green preparation process.)

1. An antioxidant broad-spectrum bacteriostatic agent is characterized in that the bacteriostatic agent is lysozyme-tea polyphenol copolymer obtained by coupling lysozyme and tea polyphenol by using ascorbic acid/hydrogen peroxide as a reaction system.

2. The preparation method of the antioxidant broad-spectrum bacteriostatic agent is characterized by comprising the following steps:

step 1: dissolving lysozyme in ultrapure water, adding hydrogen peroxide and ascorbic acid under the protection of nitrogen, and stirring at constant temperature to obtain a mixed solution A;

step 2: adding a tea polyphenol solution into the mixed solution obtained in the step (1), stirring to obtain a mixed solution B, and then putting the mixed solution B into a water bath kettle to continue stirring and reacting;

and step 3: dialyzing the solution obtained in the step 2 to obtain a lysozyme-tea polyphenol composite system;

and 4, step 4: and (3) placing the lysozyme-tea polyphenol composite system obtained in the step (3) into a freeze dryer for freeze drying to obtain a lysozyme-tea polyphenol copolymer, wherein the lysozyme-tea polyphenol copolymer is the antioxidant broad-spectrum bacteriostatic agent.

3. The method for preparing the anti-oxidation broad-spectrum bacteriostatic agent according to claim 2, wherein the lysozyme solution prepared in the step 1 is specifically prepared by the following steps: slowly adding lysozyme into water at the temperature of 25 ℃, and stirring for 2-4 hours at the speed of 200-800 rpm to obtain a lysozyme solution with the concentration of less than or equal to 1 mg/mL; then adding hydrogen peroxide and ascorbic acid, stirring at a constant temperature, wherein the stirring speed is 200-800 rpm, and the stirring time is 0.5-3 hours, so as to obtain a mixed solution A, wherein the concentration of the hydrogen peroxide in the mixed solution A is 0.05-30 mol/L, and the concentration of the ascorbic acid in the mixed solution A is 0.1-0.4%.

4. The method for preparing the antioxidant broad-spectrum bacteriostatic agent according to claim 2, wherein the concentration of the tea polyphenol in the mixed solution B in the step 2 is 0.1-1 mmol/L, the stirring speed is 200-800 rpm, and the stirring time is 12-48 h.

5. The method for preparing the anti-oxidation broad-spectrum bacteriostatic agent according to claim 2, wherein the dialysis in step 3 is specifically: dialyzing at 20 ℃ for 24-72 hours, and intercepting substances with the molecular weight of 3600 Da.

6. The method for preparing the anti-oxidation broad-spectrum bacteriostatic agent according to claim 2, wherein the step 4 of freeze-drying specifically comprises the following steps: and (3) storing the lysozyme-tea polyphenol complex system obtained in the step (3) at-4 ℃ for 1-3 days, and then putting the lysozyme-tea polyphenol complex system into a freeze-drying machine for freeze-drying, wherein the freeze-drying temperature is-50 to-80 ℃, the vacuum degree is 0.5-10 pa, and the freeze-drying time is 48 hours.

Technical Field

The invention belongs to the technical field of food processing, and particularly relates to an antioxidant broad-spectrum bacteriostatic agent and a preparation method thereof.

Background

Lysozyme is an alkaline enzyme that hydrolyzes mucopolysaccharides in pathogenic bacteria, and dissolves bacteria mainly by breaking the beta-1, 4 glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in the cell wall, breaking the cell wall insoluble mucopolysaccharide into soluble glycopeptides, causing the contents of the broken cell wall to escape. Lysozyme can also be directly combined with virus protein with negative charge, and forms double salt with DNA, RNA and apoprotein to inactivate virus. The lysozyme has stronger heat resistance. The lysozyme can not be inactivated by the treatment of an organic solvent, the activity of the lysozyme can be completely recovered when the lysozyme is transferred into an aqueous solution, and the lysozyme can be frozen or dried and has stable activity. The food additive society of FAO/WTO has identified lysozyme as safe for use in food in 1992. Therefore, the lysozyme has the functions of antibiosis, antiphlogosis, antivirus, and the like, and is a safe natural preservative. Tea polyphenols, which is the main component of green tea polyphenols, is catechin monomer separated from tea, and has antibacterial, antiviral, antioxidant, arteriosclerosis resisting, thrombosis resisting, vascular proliferation resisting, antiinflammatory and antitumor effects.

Ascorbic acid and hydrogen peroxide systems are redox initiation systems free of metal elements and are gaining widespread attention, and the initiators are of great significance in polymer synthesis.

The current applications of antioxidants and bacteriostats in Food are respectively played, for example, lysozyme is commonly used in the fresh-keeping and preservation work of Food, but lacks of antioxidant property (Wang, Guanying, Liu Shao, etc., Food science, 2018,39(11): 227-.

Disclosure of Invention

The invention aims to provide an antioxidant broad-spectrum bacteriostatic agent which is simple in preparation process and safe and edible and a preparation method thereof. The invention utilizes ascorbic acid and hydrogen peroxide systems to couple lysozyme and tea polyphenol, plays the antibacterial and antioxidant roles of the two, and prepares the antioxidant broad-spectrum bacteriostatic agent so as to meet the requirements of the fields of food, medicine and the like.

In order to achieve the purpose, the invention adopts the technical scheme that:

in a first aspect, the invention provides an antioxidant broad-spectrum bacteriostatic agent, which is lysozyme-tea polyphenol copolymer obtained by coupling lysozyme and tea polyphenol by using ascorbic acid/hydrogen peroxide as a reaction system.

In a second aspect, the invention provides a preparation method of an antioxidant broad-spectrum bacteriostatic agent, which comprises the following steps:

step 1: dissolving lysozyme in ultrapure water, adding hydrogen peroxide and ascorbic acid under the protection of nitrogen, and stirring at constant temperature to obtain a mixed solution A;

step 2: adding a tea polyphenol solution into the mixed solution obtained in the step (1), stirring to obtain a mixed solution B, and then putting the mixed solution B into a water bath kettle to continue stirring and reacting;

and step 3: dialyzing the solution obtained in the step 2 to obtain a lysozyme-tea polyphenol composite system;

and 4, step 4: and (3) placing the lysozyme-tea polyphenol composite system obtained in the step (3) into a freeze dryer for freeze drying to obtain a lysozyme-tea polyphenol copolymer, wherein the lysozyme-tea polyphenol copolymer is the antioxidant broad-spectrum bacteriostatic agent.

Further, the preparation method of the lysozyme (Ly) solution in the step 1 specifically comprises the following steps: slowly adding lysozyme into water at the temperature of 25 ℃, and stirring for 2-4 hours at the speed of 200-800 rpm to obtain a lysozyme solution with the concentration of less than or equal to 1 mg/mL; then adding hydrogen peroxide and ascorbic acid, stirring at a constant temperature, wherein the stirring speed is 200-800 rpm, and the stirring time is 0.5-3 hours, so as to obtain a mixed solution A, wherein the concentration of the hydrogen peroxide in the mixed solution A is 0.05-30 mol/L, and the concentration of the ascorbic acid in the mixed solution A is 0.1-0.4%.

Further, the concentration of tea polyphenol (EGCG) in the mixed solution B in the step 2 is 0.1-1 mmol/L, the tea polyphenol solution with the concentration can be fully coupled with lysozyme, and is beneficial to removing redundant tea polyphenol, the stirring speed is 200-800 rpm, and the stirring time is 12-48 h.

Further, the dialysis in step 3 is specifically: dialyzing at 20 ℃ for 24-72 hours, and intercepting substances with the molecular weight of 3600 Da.

Further, the step 4 of freeze-drying specifically comprises: and (3) storing the lysozyme-tea polyphenol complex system obtained in the step (3) at-4 ℃ for 1-3 days, and then putting the lysozyme-tea polyphenol complex system into a freeze-drying machine for freeze-drying, wherein the freeze-drying temperature is-50 to-80 ℃, the vacuum degree is 0.5-10 pa, and the freeze-drying time is 48 hours.

The invention has the beneficial effects that:

1. according to the preparation method of the antioxidant broad-spectrum bacteriostatic agent, ascorbic acid/hydrogen peroxide is used as a reaction system, other substances such as a stabilizer are not required to be added in the preparation process, the preparation process is simple, mild, green and safe, and the prepared lysozyme-tea polyphenol has good broad-spectrum bacteriostatic activity and antioxidant capacity.

2. The method adopts a mild method to couple the lysozyme and the tea polyphenol, fully exerts respective antibacterial and antioxidant properties, and widens the antibacterial spectrum. The antioxidant broad-spectrum bacteriostatic agent provided by the invention has a certain inhibiting effect on escherichia coli and staphylococcus aureus.

Drawings

FIG. 1 shows the UV-VIS absorption spectrum of Ly-EGCG copolymer.

FIG. 2 is the steady state fluorescence spectrum of the Ly-EGCG copolymer.

FIG. 3 shows the radical scavenging (a) and total antioxidant capacity (b) of Ly-EGCG copolymer.

In the figure, LE represents Ly-EGCG copolymer, LE0.1, LE0.3, LE0.5, LE0.7 represents EGCG addition amount of 0.1mmol/L, 0.3mmol/L, 0.5mmol/L, 0.7mmol/L respectively.

Detailed Description

The following examples are intended to better illustrate the technical solutions of the present invention, but not to limit the scope of the present invention.