CN113951280A - Preparation method of lysozyme-polysaccharide nano-composite with antibacterial property - Google Patents
Preparation method of lysozyme-polysaccharide nano-composite with antibacterial property Download PDFInfo
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- CN113951280A CN113951280A CN202111358055.6A CN202111358055A CN113951280A CN 113951280 A CN113951280 A CN 113951280A CN 202111358055 A CN202111358055 A CN 202111358055A CN 113951280 A CN113951280 A CN 113951280A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/50—Isolated enzymes; Isolated proteins
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
- A01N25/22—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests containing ingredients stabilising the active ingredients
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Abstract
The invention relates to a preparation method of lysozyme-polysaccharide nano-composite with antibacterial property, which takes lysozyme and polysaccharide as raw materials and utilizes a high-pressure homogenization and alkali liquor combined treatment technology to prepare the lysozyme-polysaccharide nano-composite and maintain higher activity of lysozyme. Compared with lysozyme without polysaccharide protection, the lysozyme structure can be protected from being damaged by adding the polysaccharide, and the prepared lysozyme-polysaccharide nano composite has uniform particle size, good dispersibility and high antibacterial activity. The method is simple to operate and low in cost, and can realize large-scale preparation of the nano antibacterial compound in a short time.
Description
Technical Field
The invention belongs to the technical field of food processing and antibacterial preservation, and particularly relates to a preparation method of a lysozyme-polysaccharide nano composite with antibacterial property.
Background
Lysozyme is also called muramidase and is a natural antibacterial substance widely existing in human bodies and animal bodies, and the lysozyme is considered as a non-toxic, harmless and safe food additive by the world health organization and the food and agriculture organization of the United nations in 1992. Subsequently, lysozyme was used to preserve various foods for extended shelf life. Lysozyme is known to lyse bacteria to death primarily by disrupting the beta-1, 4 glycosidic bonds in the bacterial and fungal cell walls. In the application process of the lysozyme, the enzyme activity is reduced to different degrees and the stability and the activity are deteriorated due to over-long storage or unfavorable storage conditions.
Polysaccharides are carbohydrates which are distributed very widely in nature and have complex molecular structures. The natural polysaccharide has good biocompatibility and degradability, so that the natural polysaccharide is widely applied to the fields of food, biological medicine and the like. Research shows that under alkaline condition, the anionic polysaccharide has a great amount of negative charges and can be wound on the surface of the lysozyme molecule with positive charges through electrostatic interaction. The lysozyme-polysaccharide complex is formed, which is beneficial to improving the stability of lysozyme under different environmental conditions.
The smaller the particle size of the lysozyme-polysaccharide complex is, the more beneficial the lysozyme is to contact with bacterial cell walls, thereby improving the sterilization efficiency of the lysozyme. High pressure homogenization is a conventional non-thermal treatment technique. It has the characteristics of mild processing conditions, short processing time and the like. The high-pressure homogenization does not influence the lysozyme activity, and can reduce the particle size of the complex and improve the particle stability. Therefore, the lysozyme-polysaccharide nano antibacterial compound prepared by the high-pressure homogenization technology is an effective method for improving the stability and the activity of lysozyme, and has important significance for widening the application of lysozyme in the food industry.
Disclosure of Invention
The invention aims to solve the problem that lysozyme has poor stability and activity in the sterilization process, and provides a lysozyme-polysaccharide nano antibacterial compound and a preparation method thereof.
The purpose of the invention is realized by the following technical scheme:
a lysozyme-polysaccharide nano antibacterial compound is prepared by utilizing the electrostatic interaction between lysozyme and polysaccharide and adopting a high-pressure homogenization method, wherein the particle size of the nano antibacterial compound is 200nm, and the enzyme activity of lysozyme is improved by more than 25%.
Preferably, the polysaccharide is one of carboxymethyl cellulose, carrageenan, xanthan gum and pectin.
More preferably, the polysaccharide is carboxymethyl cellulose.
Mixing the lysozyme and the polysaccharide solution, adjusting the mixture to be alkaline by using sodium hydroxide, preparing the mixture under the condition of high-pressure homogenization, removing impurities by using a filter membrane, and performing vacuum freeze drying to obtain lysozyme-polysaccharide nano antibacterial compound dry powder.
Preferably, in the final reaction system, the pH value of the mixed solution is 10-11, and the ratio of the lysozyme to the polysaccharide is 1-4 g/L: 0.2-2 g/L.
Preferably, the homogenization pressure for high-pressure homogenization is 10-40 MPa, and the homogenization time is 3-15 min.
Preferably, the aperture of the filter membrane is 0.6-0.8 μm, the vacuum degree of vacuum freeze drying is 5-20 Pa, the temperature is-40 to-60 ℃, and the time is 24-72 h.
Compared with the prior art, the invention has the following beneficial effects:
(1) according to the invention, the antibacterial compound is prepared by utilizing the electrostatic interaction between lysozyme and polysaccharide, and the molecular chain of the polysaccharide is wound on the surface of lysozyme molecules, so that the structure of lysozyme can be protected from being damaged, and the stability of lysozyme under different environmental conditions is improved.
(2) The invention adopts high-pressure homogenization and alkali liquor treatment technologies, the particle size of the lysozyme-polysaccharide complex is obviously reduced, the particle size of the prepared nano antibacterial complex is 200nm, and the sterilization effect of the lysozyme can be effectively improved.
(3) The high-pressure homogenizing treatment technology adopted by the invention is a traditional non-heat treatment technology, and can improve the functional characteristics of protein, thereby improving the activity of lysozyme.
(4) The preparation method adopted by the invention is simple to operate, no additional reagent is added in the preparation process, the mass preparation of the nano antibacterial compound can be realized in a short time, and the preparation method is suitable for industrial production.
Drawings
FIG. 1 is a particle size diagram of lysozyme-carboxymethyl cellulose nano-antibacterial composites prepared in examples 1 to 4 and comparative example 1;
FIG. 2 is a potential diagram of lysozyme-carboxymethyl cellulose nano-antibacterial complexes prepared in examples 1 to 4 and comparative example 1.
Detailed Description
The following examples are presented to further illustrate the practice of the invention, but the practice and protection of the invention is not limited thereto. It is noted that the processes described below, if not specifically described in detail, are all realizable or understandable by those skilled in the art with reference to the prior art. The reagents or apparatus used are not indicated to the manufacturer, and are considered to be conventional products available by commercial purchase.
Example 1
(1) Respectively dissolving 200mg of lysozyme and 200mg of carboxymethyl cellulose in 200mL of ultrapure water, and stirring for 4 hours to prepare lysozyme and carboxymethyl cellulose solutions;
(2) adding 50mL of the lysozyme solution obtained in the step (1) into 50mL of carboxymethyl cellulose solution, adjusting the pH value of the mixed solution to 10.8, and uniformly stirring to obtain a final reaction system;
(3) homogenizing the mixed solution in a final reaction system by a high-pressure homogenizer under the pressure of 10MPa for 5min, filtering by using a filter membrane of 0.8 mu m to remove impurities, and carrying out vacuum freeze drying (the vacuum degree is 20Pa, the temperature is-50 ℃ and the time is 48h) to obtain the lysozyme-carboxymethyl cellulose nano antibacterial compound dry powder.
Example 2
(1) Respectively dissolving 200mg of lysozyme and 200mg of carboxymethyl cellulose in 200mL of ultrapure water, and stirring for 4 hours to prepare lysozyme and carboxymethyl cellulose solutions;
(2) adding 50mL of the lysozyme solution obtained in the step (1) into 50mL of carboxymethyl cellulose solution, adjusting the pH value of the mixed solution to 10.8, and uniformly stirring to obtain a final reaction system;
(3) homogenizing the mixed solution in a final reaction system by a high-pressure homogenizer under the pressure of 20MPa for 5min, filtering by using a filter membrane of 0.8 mu m to remove impurities, and carrying out vacuum freeze drying (the vacuum degree is 20Pa, the temperature is-50 ℃ and the time is 48h) to obtain the lysozyme-carboxymethyl cellulose nano antibacterial compound dry powder.
Example 3
(1) Respectively dissolving 200mg of lysozyme and 200mg of carboxymethyl cellulose in 200mL of ultrapure water, and stirring for 4 hours to prepare lysozyme and carboxymethyl cellulose solutions;
(2) adding 50mL of the lysozyme solution obtained in the step (1) into 50mL of carboxymethyl cellulose solution, adjusting the pH value of the mixed solution to 10.8, and uniformly stirring to obtain a final reaction system;
(3) homogenizing the mixed solution in a final reaction system by a high-pressure homogenizer under the pressure of 30MPa for 5min, filtering by using a filter membrane of 0.8 mu m to remove impurities, and carrying out vacuum freeze drying (the vacuum degree is 20Pa, the temperature is-50 ℃ and the time is 48h) to obtain the lysozyme-carboxymethyl cellulose nano antibacterial compound dry powder.
Example 4
(1) Respectively dissolving 200mg of lysozyme and 200mg of carboxymethyl cellulose in 200mL of ultrapure water, and stirring for 4 hours to prepare lysozyme and carboxymethyl cellulose solutions;
(2) adding 50mL of the lysozyme solution obtained in the step (1) into 50mL of carboxymethyl cellulose solution, adjusting the pH value of the mixed solution to 10.8, and uniformly stirring to obtain a final reaction system;
(3) homogenizing the mixed solution in a final reaction system by a high-pressure homogenizer under the pressure of 40MPa for 5min, filtering by using a filter membrane of 0.8 mu m to remove impurities, and carrying out vacuum freeze drying (the vacuum degree is 20Pa, the temperature is-50 ℃ and the time is 48h) to obtain the lysozyme-carboxymethyl cellulose nano antibacterial compound dry powder.
Comparative example 1
(1) Respectively dissolving 200mg of lysozyme and 200mg of carboxymethyl cellulose in 200mL of ultrapure water, and stirring for 4 hours to prepare lysozyme and carboxymethyl cellulose solutions;
(2) adding 50mL of the lysozyme solution obtained in the step (1) into 50mL of carboxymethyl cellulose solution, adjusting the pH value of the mixed solution to 10.8, and uniformly stirring to obtain a final reaction system;
(3) in the final reaction system, the mixed solution is filtered by a filter membrane with the diameter of 0.8 mu m to remove impurities, and is frozen and dried in vacuum (the vacuum degree is 20Pa, the temperature is 50 ℃ below zero, and the time is 48 hours) to obtain the lysozyme-carboxymethyl cellulose nano antibacterial compound dry powder.
In summary, the nano antibacterial compound dry powder prepared in examples 1 to 4 and comparative example 1 was dissolved in phosphate buffer (0.1mol/L) for performance testing, wherein the enzyme activity of lysozyme was calculated by the following formula:
in the formula: | A1-A2The | is the change in absorbance per minute at 450nm, and m is the mass (mg) of the enzyme solution.
TABLE 1 enzyme Activity of lysozyme in examples 1 to 4 and comparative example 1
| Group of | Enzyme activity (U/mg) | Relative enzyme activity (%) |
| Example 1 | 176.36 | 25.03 |
| Example 2 | 179.51 | 27.26 |
| Example 3 | 183.27 | 30.98 |
| Example 4 | 184.75 | 29.93 |
| Comparative example 1 | 148.82 | 5.50 |
As can be seen from comparative example 1, the addition of carboxymethyl cellulose has little effect on the activity of lysozyme, and the relative enzyme activity of lysozyme is 5.50%. The results show that carboxymethyl cellulose is a good polysaccharide material, and can be used for protecting the structure of lysozyme and improving the stability of lysozyme. As can be seen from examples 1-4, the relative enzyme activity of lysozyme increases to more than 25% with the increase of high-pressure homogenization pressure. The result shows that high-pressure homogenization is an effective processing technology, the activity of the lysozyme can be obviously improved, and the sterilization effect of the lysozyme is enhanced.
The above examples are only preferred embodiments of the present invention, which are intended to be illustrative and not limiting, and those skilled in the art should understand that they can make various changes, substitutions and alterations without departing from the spirit and scope of the invention.
Claims (5)
1. A preparation method of lysozyme-polysaccharide nano-composite with antibacterial property is characterized in that a mixture system is adjusted to be alkaline through a sodium hydroxide solution, a high-pressure homogenization method is combined to prepare the nano-antibacterial composite by utilizing electrostatic interaction between lysozyme and polysaccharide, impurities are removed through a filter membrane, and then vacuum freeze drying is carried out to obtain lysozyme-polysaccharide nano-antibacterial composite particles.
2. The method of claim 1, wherein the polysaccharide is one of carboxymethylcellulose, carrageenan, xanthan gum, or pectin.
3. The method for preparing lysozyme-polysaccharide nanocomposite with antibacterial property according to claim 1, wherein the pR value of the reaction system is 10-11, and the ratio of lysozyme to polysaccharide is 1-4 g/L: 0.2-2 g/L.
4. The method for preparing lysozyme-polysaccharide nanocomposite with antibacterial property as claimed in claim 1, wherein the high-pressure homogenization pressure is 10-40 MPa, the homogenization time is 3-15 min, the pore size of the filter membrane is 0.6-0.8 μm, the vacuum degree of vacuum freeze drying is 5-20 Pa, the temperature is-40 to-60 ℃, the time is 24-72 h, and the particle size is kept at about 200 nm.
5. The method for preparing lysozyme-polysaccharide nanocomposite with antibacterial property according to claim 1, wherein the enzyme activity of lysozyme is relatively increased by more than 25%.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115736241A (en) * | 2022-12-02 | 2023-03-07 | 武汉轻工大学 | Preparation method of nanogel |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115736241A (en) * | 2022-12-02 | 2023-03-07 | 武汉轻工大学 | Preparation method of nanogel |
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