阅读说明：本技术 一种溶菌酶复合物 (Lysozyme complex ) 是由 李秋雁 徐志良 张咪 仲倩蕊 于 2020-05-28 设计创作，主要内容包括：本发明公布了一种溶菌酶复合物,由溶菌酶和羧化壳聚糖进行复配,其特征在于：各组分的质量体积浓度(g/L)：溶菌酶1-5,羧化壳聚糖0.1-5；其中溶菌酶和羧化壳聚糖的质量比为10：1-10：20。本发明将溶菌酶和壳聚糖衍生物按照一个优选的比例进行复配,制备所得溶菌酶/羧化壳聚糖复合物,较溶菌酶或羧化壳聚糖单独作为抑菌剂使用时抑菌率有所提高；优选配比的复合物溶解性、稳定性好,解决了溶菌酶稳定性差、对革兰氏阴性菌抑制效果差的问题。(The invention discloses a lysozyme compound, which is compounded by lysozyme and carboxylated chitosan, and is characterized in that: mass volume concentration (g/L) of each component: 1-5 parts of lysozyme and 0.1-5 parts of carboxylated chitosan; wherein the mass ratio of the lysozyme to the carboxylated chitosan is 10: 1-10: 20. according to the invention, lysozyme and chitosan derivatives are compounded according to a preferable proportion, and the prepared lysozyme/carboxylated chitosan compound has higher bacteriostatic rate than that of lysozyme or carboxylated chitosan which is used as a bacteriostatic agent alone; the compound with the optimal proportion has good solubility and stability, and solves the problems of poor lysozyme stability and poor inhibition effect on gram-negative bacteria.)

1. A lysozyme complex is prepared by compounding lysozyme and carboxylated chitosan, and is characterized in that: mass volume concentration (g/L) of each component: 1-5 parts of lysozyme and 0.1-5 parts of carboxylated chitosan; wherein the mass ratio of the lysozyme to the carboxylated chitosan is 10: 1-10: 20.

2. the lysozyme complex of claim 1, wherein: the mass ratio of the lysozyme to the carboxylated chitosan is 10: 1-10: 1.5.

3. the lysozyme complex of claim 1 or 2, wherein: the carboxylated chitosan is carboxymethyl chitosan or carboxyethyl chitosan.

Technical Field

The invention belongs to the technical field of biological agents, and particularly relates to a lysozyme complex.

Background

Lysozyme is a natural antimicrobial protein, also known as muramidase or acetylmuramyl peptide glycan hydrolase, widely distributed in nature, and found in egg white, tears, saliva, plasma, milk and histiocytes, as well as in bacterial and plant latex. Peptidoglycan is a major component of bacterial cell walls and is composed of N-acetylglucosamine (NAG), N-acetylmuramic acid (NAM) and a tetrapeptide, with two sugar molecules alternately linked by β -1,4 glycosidic bonds. The lysozyme can effectively hydrolyze peptidoglycan on bacterial cell walls, and because the content of the peptidoglycan in the cell walls of gram-positive bacteria is higher, and the content of the peptidoglycan in gram-negative bacteria is lower and is buried in an outer membrane lipopolysaccharide layer, the lysozyme is prevented from acting on the peptidoglycan, so the lysozyme has strong bactericidal action on the gram-positive bacteria, but has almost no action on the gram-negative bacteria. Meanwhile, lysozyme is an alkaline protease, has strong thermal stability under acidic conditions, but has easy damage to the activity under alkaline conditions; under the conditions of drying and low temperature, the activity of the lysozyme is not influenced, and the lysozyme can be stored for a long time, but the activity of the lysozyme can be reduced when the aqueous solution is stored for a long time or is repeatedly frozen and thawed.

The chitosan is a product of chitosan deacetylation, is the only alkaline polysaccharide in natural polysaccharide, has no toxicity, no antigenicity and biodegradability, has the effects of bacteriostasis, cancer resistance, immunity enhancement and the like, is a new hemostatic material in recent years, and is widely applied to the fields of medicine, chemical industry, food, biotechnology and the like. Chitosan can only be dissolved in acidic environment, the antibacterial spectrum is narrow, and the antibacterial effect is related to the deacetylation degree, the molecular weight, the pH value of the environment and other factors, so that modification of chitosan to improve the solubility of chitosan, the antibacterial activity and the hemostatic and wound healing effects of chitosan become important directions for developing chitosan derivatives.

At present, many researches have been made on adding lysozyme and chitosan as antibacterial components into products, and improving the antibacterial activity and stability of the lysozyme and chitosan by compounding with different components, for example, Chinese patent No. CN102138570A discloses a lysozyme combined bacteriostatic agent, and the problem of poor inhibition effect of lysozyme on gram-negative bacteria is solved by compounding and combining lysozyme, glycine and ethylenediamine tetraacetic acid; chinese patent No. CN107737024A discloses an additive composition containing biological lysozyme, which combines biological lysozyme with modified vegetable gum, EDTA disodium and deionized water according to specific content to protect the activity of biological lysozyme from being influenced by other components in the product; chinese patent No. CN107997984A discloses an antibacterial peptide-chitosan complex, its preparation method and its application, wherein the antibacterial peptide is used to modify chitosan, so as to improve its antibacterial activity. Because chitosan contains beta-1, 4 glycosidic bonds, lysozyme can degrade chitosan, and the lysozyme and chitosan are simply compounded to fail to achieve ideal effects, even the stability of the system can be damaged, so that a proper proportion needs to be found to improve the antibacterial activity, the stability and the like of the lysozyme and chitosan compounded, and the research on related aspects is less at present.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provide a lysozyme compound which can improve the stability of the lysozyme/carboxylation chitosan compound on the one hand and improve the bacteriostatic activity of the lysozyme/carboxylation chitosan compound on the other hand, thereby solving the problem of poor inhibition effect on gram-negative bacteria.

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

a lysozyme complex is prepared by compounding lysozyme and carboxylated chitosan, and is characterized in that: mass volume concentration (g/L) of each component: 1-5 parts of lysozyme and 0.1-5 parts of carboxylated chitosan; wherein the mass ratio of the lysozyme to the carboxylated chitosan is 10: 1-10: 20. the lysozyme and carboxylated chitosan compound solution in the proportion has the bacteriostatic effect on staphylococcus aureus over 60.4 percent; the bacteriostatic effect on Candida albicans reaches more than 95.4 percent. The proportion can also effectively slow down the reduction of enzyme activity.

Preferably: the mass ratio of the lysozyme to the carboxylated chitosan is 10: 1-10: 1.5. the lysozyme and carboxylated chitosan compound solution in the proportion has the bacteriostatic effect on staphylococcus aureus reaching about 98.7 percent; the bacteriostatic effect on candida albicans reaches 100 percent; the bacteriostatic effect on the escherichia coli reaches more than 50 percent. After the compound solution with the proportion is placed at 37 ℃ for 1 month, the lysozyme activity is reduced by 14.3 percent, and the reduction of the enzyme activity is effectively slowed down.

Further: the carboxylated chitosan is carboxymethyl chitosan or carboxyethyl chitosan.

According to the invention, lysozyme and chitosan derivatives are compounded according to a preferable proportion, and the prepared lysozyme/carboxylated chitosan compound has higher bacteriostatic rate than that of lysozyme or carboxylated chitosan which is used as a bacteriostatic agent alone; the compound with the optimal proportion has good solubility and stability, and solves the problems of poor lysozyme stability and poor inhibition effect on gram-negative bacteria.

Drawings

FIG. 1 is a comparison graph of the bacteriostatic effect of the antibacterial agent on staphylococcus aureus.

FIG. 2 is a comparison graph of the bacteriostatic effect of the invention on Candida albicans.

FIG. 3 is a comparison of the bacteriostatic effect of the invention on Escherichia coli.

Detailed Description

The present invention will be described in further detail with reference to examples, which are provided for illustration only and are not intended to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.

1. Preparation of lysozyme/carboxylation chitosan complex

Under the condition of high-speed magnetic stirring, the lysozyme solution is dropwise added into the carboxylated chitosan solution, and the mixture is stirred for 10min, wherein the mixture ratio is shown in table 1.

Table 1 lysozyme in different ratios: carboxylated chitosan

2. And (3) testing the antibacterial performance of the lysozyme/carboxylated chitosan compound:

collecting freshly cultured slant culture of Escherichia coli, Staphylococcus aureus, Candida albicans, and Streptococcus mutans, washing thallus Porphyrae with PBS (pH 7.2), and adjusting OD₆₀₀＝0.And 5, diluting by 1000 times, adding 0.1mL of bacterial suspension into lysozyme/carboxylation chitosan compound solutions with different ratios respectively, placing the lysozyme/carboxylation chitosan compound solutions into a shaking table to shake for 20 hours at 37 ℃, coating the flat plate with 0.1mL of treated solutions with different ratios, culturing for 18-24 hours at 37 ℃, and observing the bacteriostatic effect of the lysozyme/carboxylation chitosan compound.

Carry out colony count discovery to 3 kinds of bacteria through dilution coating plate method, 2mg/mL lysozyme and 2mg/mL chitosan have inhibitory action of different degree to 3 kinds of bacteria, and after both are compounded, the antibacterial effect of lysozyme carboxylation chitosan complex solution is not the stack of simple lysozyme, two antibacterial effects of chitosan, and when lysozyme and carboxylation chitosan mass ratio be 10: 1.25 hours later, the lysozyme/carboxylated chitosan compound has the best antibacterial effect, which is shown in figures 1-3.

The bacteriostatic effect of staphylococcus aureus is as follows:

in FIG. 1, (a) shows the number 303 of staphylococcus aureus colonies in a control group, and (b) shows the number 120 of staphylococcus aureus colonies treated by a 2mg/mL carboxylated chitosan solution, wherein the bacteriostasis rate is 60.4%; (c) the bacterial colony number of the staphylococcus aureus treated by the lysozyme solution of 2mg/mL is 25, and the bacteriostasis rate is 91.7 percent; (d) the mass ratio is shown as 10: 1.25, the colony number of staphylococcus aureus treated by the carboxylated chitosan solution is 4, and the bacteriostasis rate is 98.7 percent.

Bacteriostatic effect of candida albicans:

in FIG. 2, (a) shows the number of Candida albicans colonies 1120 in the control group, and (b) shows the number of Candida albicans colonies 51 treated with 2mg/mL carboxylated chitosan solution, with a bacteriostasis rate of 95.4%; (c) the colony number of candida albicans treated by 2mg/mL lysozyme solution is 3, and the bacteriostasis rate is 99.7%; (d) the colony number of the candida albicans treated by the carboxylated chitosan solution with the mass ratio of 10:1.125 is 0, and the bacteriostasis rate is 100%.

The antibacterial effect of the escherichia coli is as follows:

in FIG. 3, (a) shows the colony count 303 of Escherichia coli in the control group, (b) shows 2mg/mL of carboxylated chitosan solution and (c) shows that 2mg/mL of lysozyme solution had no inhibitory effect on Escherichia coli; (d) the carboxylation chitosan solution with the mass ratio of 10:1.125 has the bacteriostasis rate of more than 50 percent to escherichia coli.

3. Lysozyme/carboxylated chitosan complex stability assay

The lysozyme activity is measured by taking Micrococcus muralis CGMCC1.0634 as a substrate and using a spectrophotometry method according to the absorbance reduction degree of bacteria liquid at the wavelength of 450nm in unit time. The decrease in absorbance at a wavelength of 450nm of 0.001 per minute at 25 ℃ at pH 6.2 at room temperature was 1 lysozyme activity unit, and the results are shown in Table 2.

TABLE 2 lysozyme activity assay

In table 2, the lysozyme activity of the lysozyme solution was reduced by 28.6% after being placed at 37 ℃ for 1 month, and the lysozyme activity of the lysozyme/carboxylated chitosan complex solution was reduced by 14.3%, which effectively slowed down the reduction of the enzyme activity.

As can be seen from the results in FIGS. 1-3 and Table 2, the lysozyme/carboxylated chitosan composite with the mass ratio of 10:1.125 can improve the bacteriostatic activity to Escherichia coli and Staphylococcus aureus, slow down the reduction of the lysozyme activity and improve the stability of the lysozyme.