CN112841226A - Novel bacteriophage antibacterial gel and preparation method and application thereof - Google Patents

Abstract

The invention belongs to the technical field of biology, and particularly relates to a novel bacteriophage antibacterial gel, and a preparation method and application thereof. The volume ratio of the coliphage bacteria liquid to the staphylococcus aureus phage bacteria liquid in the phage antibacterial gel is 1: 1, the total effective content of the phage is more than or equal to 1010 PFU/mL; the bacteriophage antibacterial gel contains a gelling agent; the gel is gamma-PGA-CS gel; the pH value of the antibacterial gel is 6-8; the antibacterial gel also contains calcium citrate and saponin. The gamma-PGA-CS-phage antibacterial gel disinfectant prepared by the invention has good safety, no corrosive and pungent odor of a chemical disinfectant, is safe and nontoxic to environment and people, has broad-spectrum bactericidal effect on escherichia coli and staphylococcus aureus, and has strong specificity and long-acting sterilization.

Description

Novel bacteriophage antibacterial gel and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a novel bacteriophage antibacterial gel, and a preparation method and application thereof.

Background

The germs are everywhere in daily life, so the disinfectant is widely applied to the disinfection work in various places. Traditional chemical disinfectants such as peroxide disinfectants, chlorine disinfectants, aldehyde disinfectants and the like have unstable chemical properties and strong irritation, can damage eyes, respiratory mucosa and the like after long-term use, and have carcinogenic risks for certain metabolites. In view of the large toxic and side effects of the chemical disinfectant, the development of the disinfectant which has good safety and strong sterilizing capability and can exist in the environment for a long time is particularly important.

Bacteriophages are a class of viruses that infect bacteria, causing lysis of the host bacteria, and are called bacteriophages. Bacteriophages are obligate intracellular parasites that can exist outside the bacterial cell, but can only multiply inside the cell (i.e., genetic material must enter the cell to multiply). The phage is usually separated from natural environment samples such as sewage or excrement, and the like, has host specificity and only attacks one or more bacteria, so the phage has no infection to human or animals and plants, does not pollute the environment and has higher safety. Bacteriophages can be divided into two broad categories, temperate bacteriophages and virulent bacteriophages, of which virulent bacteriophages can cause lysis of sensitive bacteria in a short time. In the aspect of sterilization effect, the virulent phage can crack bacteria and has the characteristic of exponential proliferation, and is a biological disinfectant with antibacterial effect.

Chitosan is a natural high molecular compound, is a natural alkaline polysaccharide with positive charges obtained by deacetylation of chitin, and is abundant in shells of shrimps and crabs. The chitosan has good biocompatibility, biodegradability and nontoxicity, and also has good antibacterial performance, and can inhibit the growth of some pathogenic microorganisms (such as escherichia coli and staphylococcus aureus). The chitosan has positive electricity and is crosslinked with poly-gamma-glutamic acid with negative charge in a liquid medium by utilizing the characteristic.

Poly gamma-glutamic acid, called gamma-PGA for short, is a non-ribosomal polypeptide formed by linking L-and D-glutamic acid monomers through gamma-glutamyl bonds under the catalytic action of enzymes, and a molecular chain has a large number of side chain carboxyl groups which can form hydrogen bonds in the molecule or among the molecules. Has biodegradability, easy modification, water retention property, solubilization property and slow release property.

Coli is a normal colonizing bacterium in the intestinal tract of animals and is a gram-negative facultative anaerobic rod-shaped bacterium. Coli is the most predominant and abundant bacterium in the intestine of humans and many animals, and it is mainly parasitic in the large intestine. When it invades some parts of the human body, it may cause infections such as peritonitis, cholecystitis, cystitis and diarrhea. Symptoms in humans after infection with E.coli are stomach ache, vomiting, diarrhea and fever. Infections can be fatal, especially for children and the elderly. The serotype of escherichia coli can cause gastrointestinal infections of human or animals, mainly caused by infection with specific pilus antigens, pathogenic toxins and the like, and can cause urinary tract infection, arthritis, meningitis, sepsis type infection and the like besides gastrointestinal tract infection.

Staphylococcus aureus belongs to the genus Staphylococcus, is a representative of gram-positive bacteria, and is a common food-borne pathogenic microorganism. Staphylococcus aureus is commonly parasitic on the skin, nasal cavity, throat, intestines and stomach, carbuncle, suppurative sore of human and animals, and is ubiquitous in the air, sewage and other environments. Staphylococcus aureus is the most common pathogenic bacterium in human pyogenic infection, and can generate enterotoxin under proper conditions, can cause local pyogenic infection, and can also cause pneumonia, pseudomembranous enteritis, pericarditis and the like, even septicemia, sepsis and other systemic infections. The more severely food is contaminated with staphylococcus aureus, the faster it will multiply and the more susceptible it will be to the formation of toxins.

With the wide application of antibiotics, the emergence and prevalence of drug-resistant bacterial strains are large, and the treatment effect of antibiotics is severely challenged. In recent years some "superbacteria" have emerged, which are insensitive to almost all antibiotics, resulting in human infection with such bacteria being almost non-pharmaceutically available, and new anti-infective weapons are needed to replace antibiotics.

Chinese patent 201810868504.3 discloses a bacteriophage with environment disinfection capability and application thereof, the bacteriophage has strong cracking effect on Pseudomonas aeruginosa, and provides a bacteriophage source for industrial production of the bacteriophage and disinfection and sterilization. But the targeted strains are single and the application range is small.

Chinese patent 200880006630.2 discloses a method for disinfecting the exterior of an animal comprising applying phage to the exterior layer to control the microbial population on the exterior layer of the animal. But this patent is not suitable for disinfection in the environment.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a novel bacteriophage antibacterial gel which is a bacteriophage mixed preparation based on two most common pathogenic bacteria in life, namely escherichia coli and staphylococcus aureus and has good safety and bactericidal capability, so that the pathogenic bacteria hidden in the environment can be killed permanently.

The invention also provides a preparation method of the novel bacteriophage antibacterial gel.

The invention also provides application of the phage antibacterial gel prepared by the preparation method.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

the invention provides a novel bacteriophage antibacterial gel, wherein the volume ratio of colibacillus bacteriophage bacterial liquid to staphylococcus aureus bacteriophage bacterial liquid in the bacteriophage antibacterial gel is 1: 1, the total effective content of the phage is more than or equal to 1010 PFU/mL; the bacteriophage antibacterial gel contains a gelling agent; the gel is gamma-PGA-CS gel; the pH value of the antibacterial gel is 6-8; the antibacterial gel also contains calcium citrate and saponin.

Further, the pH regulator of the antibacterial gel is one or more aqueous solutions of citric acid, sodium citrate, sodium carbonate and sodium bicarbonate.

Further, the adding amount of the calcium citrate is 0.1-0.2g per mL of the mixed bacterial liquid; the mass concentration of the saponin in the antibacterial gel is 3-5%.

The gamma PGA-CS-gel used in the invention is prepared by the following method: dissolving 5g of chitosan in 20ml of 10% acetic acid solution, stirring uniformly at normal temperature, and diluting with deionized water to obtain a chitosan solution; respectively dripping 10mL of 2 g/L gamma-PGA into 50 mL of chitosan solution by using a micro-injection pump according to the speed of 6 mL/h, stirring and carrying out ultrasound while adding, dialyzing for 1-3h to remove the unbound small molecular polymer, fully mixing, and freeze-drying to obtain the poly-gamma-glutamic acid/chitosan dry powder.

The invention also provides a preparation method of the novel bacteriophage antibacterial gel, which comprises the following steps:

(1) preparing mixed bacteria liquid of coliphage and staphylococcus aureus phage according to a proportion, adding calcium citrate into the mixed bacteria liquid, and standing for 20-30 min;

(2) adding gamma-PGA-CS dry powder and saponin into the mixed bacterial liquid, and uniformly mixing to obtain colloid;

(3) dripping a pH regulator to regulate the pH value of the colloid;

(4) and (4) filling to obtain the gamma-PGA-CS-phage antibacterial gel disinfectant.

The invention also provides application of the novel bacteriophage antibacterial gel prepared by the preparation method, wherein the application amount of the antibacterial gel is 100mL/m²。

The preparation method of the mixed bacterial liquid of the coliphage and the staphylococcus aureus phage comprises the following steps: inoculating 5mL of bacteriophage into the broth culture medium of susceptible bacteria, and after 18-24h, making the turbid culture transparent again, sterilizing and filtering. The formula of the broth culture medium is as follows: 10g/L of peptone, 3g/L of beef extract powder and 5g/L of sodium chloride, and the pH value is 7.4.

The invention has the beneficial effects that:

1. the gamma-PGA-CS-phage antibacterial gel disinfectant prepared by the invention has good safety, no corrosive and pungent odor of a chemical disinfectant, is safe and nontoxic to environment and people, has broad-spectrum bactericidal effect on escherichia coli and staphylococcus aureus, and has strong specificity and long-acting sterilization.

2. The preparation process provided by the invention is simple and convenient, and is suitable for large-scale production.

Detailed Description

The present invention will be further illustrated in detail with reference to the following specific examples, which are not intended to limit the present invention but are merely illustrative thereof. The experimental methods used in the following examples are not specifically described, and the materials, reagents and the like used in the following examples are generally commercially available under the usual conditions without specific descriptions.

Example 1

(1) Dissolving 5g of chitosan in 20ml of 10% acetic acid solution, stirring uniformly at normal temperature, and diluting with deionized water to obtain a chitosan solution; respectively dripping 10mL of 2 g/L gamma-PGA into 50 mL of chitosan solution by using a micro-injection pump according to the speed of 6 mL/h, stirring and carrying out ultrasound while adding, dialyzing for 1-3h to remove unbound small molecular polymers, fully mixing, and freeze-drying to obtain poly gamma-glutamic acid/chitosan dry powder;

(2) activating the phage to enable the titer to be more than or equal to 1010PFU/mL, mixing 40mL of escherichia coli phage bacterial liquid and 40mL of staphylococcus aureus phage bacterial liquid, adding 8g of calcium citrate into the mixed bacterial liquid, and standing for 30 min;

(3) adding 40g of gamma-glutamic acid/chitosan dry powder and saponin accounting for 3% of the total amount into the mixed bacterial liquid, uniformly mixing to obtain colloid, dropwise adding 3.8% of sodium citrate aqueous solution to adjust the pH value of the colloid to 7.4, and filling to obtain the gamma PGA-CS-phage antibacterial gel disinfectant.

Example 2

(1) Dissolving 5g of chitosan in 20ml of 10% acetic acid solution, stirring uniformly at normal temperature, and diluting with deionized water to obtain a chitosan solution; respectively dripping 10mL of 2 g/L gamma-PGA into 50 mL of chitosan solution by using a micro-injection pump according to the speed of 6 mL/h, stirring and carrying out ultrasound while adding, dialyzing for 1-3h to remove unbound small molecular polymers, fully mixing, and freeze-drying to obtain poly gamma-glutamic acid/chitosan dry powder;

(2) activating the phage to enable the titer to be more than or equal to 1010PFU/mL, mixing 40mL of colibacillus phage bacterial liquid and 40mL of staphylococcus aureus phage bacterial liquid, adding 10g of calcium citrate into the mixed bacterial liquid, and standing for 30 min;

(3) adding 40g of gamma-glutamic acid/chitosan dry powder and saponin accounting for 5% of the total amount into the mixed bacterial liquid, uniformly mixing to obtain colloid, dropwise adding 3.8% of sodium citrate aqueous solution to adjust the pH value of the colloid to 7.4, and filling to obtain the gamma PGA-CS-phage antibacterial gel disinfectant.

Comparative example 1

(1) Dissolving 5g of chitosan in 20ml of 10% acetic acid solution, stirring uniformly at normal temperature, and diluting with deionized water to obtain a chitosan solution; respectively dripping 10mL of 2 g/L gamma-PGA into 50 mL of chitosan solution by using a micro-injection pump according to the speed of 6 mL/h, stirring and carrying out ultrasound while adding, dialyzing for 1-3h to remove unbound small molecular polymers, fully mixing, and freeze-drying to obtain poly gamma-glutamic acid/chitosan dry powder;

(2) activating the phage to enable the titer to be more than or equal to 1010PFU/mL, adding 8g of calcium citrate into 40mL of colibacillus phage bacterial liquid, standing for 30min, adding 20g of gamma-glutamic acid/chitosan dry powder and saponin accounting for 3% of the total amount, uniformly mixing to obtain colloid, dropwise adding 3.8% of sodium citrate aqueous solution to adjust the pH value of the colloid to 7.4, and filling to obtain the gamma PGA-CS-phage antibacterial gel disinfectant.

Comparative example 2

(1) Dissolving 5g of chitosan in 20ml of 10% acetic acid solution, stirring uniformly at normal temperature, and diluting with deionized water to obtain a chitosan solution; respectively dripping 10mL of 2 g/L gamma-PGA into 50 mL of chitosan solution by using a micro-injection pump according to the speed of 6 mL/h, stirring and carrying out ultrasound while adding, dialyzing for 1-3h to remove unbound small molecular polymers, fully mixing, and freeze-drying to obtain poly gamma-glutamic acid/chitosan dry powder;

(2) activating the phage to enable the titer to be more than or equal to 1010PFU/mL, mixing 40mL of colibacillus phage bacterial liquid and 40mL of staphylococcus aureus phage bacterial liquid, and standing for 30 min;

(3) and adding 40g of gamma-glutamic acid/chitosan dry powder into the mixed bacterial liquid, uniformly mixing to obtain colloid, dropwise adding 3.8% sodium citrate aqueous solution to adjust the pH value of the colloid to 7.4, and filling to obtain the gamma PGA-CS-phage antibacterial gel disinfectant.

Comparative example 3

(2) Activating the phage to enable the titer to be more than or equal to 1010PFU/mL, mixing 40mL of escherichia coli phage bacterial liquid and 40mL of staphylococcus aureus phage bacterial liquid, adding 8g of calcium citrate into the mixed bacterial liquid, and standing for 30 min;

(3) adding 40g of chitosan and saponin accounting for 3% of the total amount into the mixed bacterial liquid, uniformly mixing to obtain colloid, dropwise adding 3.8% of sodium citrate aqueous solution to adjust the pH value of the colloid to 7.4, and filling to obtain the gamma PGA-CS-phage antibacterial gel disinfectant.

Effect (I) examples

1. Preparing host bacterium liquid.

One colony was picked on the plate, and the single colony was streaked on a common agar plate and cultured in a 37 ℃ incubator for 12-16 hours. Respectively selecting escherichia coli and staphylococcus aureus, respectively inoculating the escherichia coli and the staphylococcus aureus into a test tube containing 5mL of LB culture medium, and performing shake culture at 37 ℃ and 200rpm for 12 hours to obtain bacterial suspension of host bacteria.

2. And setting an experimental group and a control group.

4 test pieces and 4 control pieces each having a size of 2.0cm × 3.0cm were taken, the test pieces were soaked with the disinfecting gel prepared in example 1, the control pieces and the test pieces were of the same material and the same size but containing no antibacterial material, and were subjected to sterilization treatment, and the test pieces and the control pieces were divided into 4 groups and placed in 4 sterilization plates. And (2) taking the suspension of the escherichia coli, the suspension of the staphylococcus aureus and the sterilized LB culture medium in the step (1), respectively dropwise adding 10 mu L of the suspension to each test sample and each control sample, uniformly coating, starting timing, acting for 1min, respectively putting the samples into a test tube containing 5mL of a neutralizer (PBS buffer solution) by using sterile forceps, and fully and uniformly mixing.

3. And (4) a bacteria counting method.

And (3) putting a sample needing bacteria counting into physiological saline with a certain proportion, and fully oscillating at room temperature to prepare the bacteria leaching solution. Diluting the bacterial leachate with sterile normal saline at a ratio of 10 times, adding 1mL of liquid with a certain dilution ratio into a sterile empty plate, adding an LB agar culture medium which is autoclaved and cooled to be not too hot to be scalded, and slightly shaking to uniformly distribute the liquid. Standing at room temperature for 30min for solidification, culturing at 37 deg.C for 10-12h, and counting the corresponding bacteria number by taking a plate with appropriate dilution ratio. Triplicate were set for each dilution, plate counts were performed, and kill rates were calculated.

And (3) performance testing:

the results are shown in Table 1.

TABLE 1

(II) detection of environmental Sterilization

Randomly selecting 10 points (each point is 1m2 x 20cm) as test areas in a biological laboratory, marking, uniformly mixing two strains of escherichia coli and staphylococcus aureus with the concentrations of 1 x 108PFU/mL respectively in an equal ratio, spraying the mixture to the test areas in the biological laboratory, uniformly smearing the gamma PGA-CS-phage antibacterial gel disinfectant obtained in example 1 to the test areas at the dosage of 100mL/m2, detecting the bacterium residue after 1h, finding that 2 of the test areas still have residues, detecting the bacterium residue after 4h, and detecting no strain residue in 10 test areas.

Claims (6)

1. The novel bacteriophage antibacterial gel is characterized in that the volume ratio of coliphage bacteria liquid to staphylococcus aureus phage bacteria liquid in the bacteriophage antibacterial gel is 1: 1, the total effective content of the phage is more than or equal to 1010 PFU/mL; the bacteriophage antibacterial gel contains a gelling agent; the gel is gamma-PGA-CS gel; the pH value of the antibacterial gel is 6-8; the antibacterial gel also contains calcium citrate and saponin.

2. The novel bacteriophage antimicrobial gel according to claim 1, wherein the pH adjusting agent of the antimicrobial gel is an aqueous solution of one or more of citric acid, sodium citrate, sodium carbonate and sodium bicarbonate.

3. The novel bacteriophage antibacterial gel according to claim 1, wherein the amount of calcium citrate added is 0.1-0.2g per mL of mixed bacterial liquid; the mass concentration of the saponin in the antibacterial gel is 3-5%.

4. A novel bacteriophage antimicrobial gel according to any one of claims 1 to 3, wherein said γ PGA-CS-gel is prepared by the following method: dissolving 5g of chitosan in 20ml of 10% acetic acid solution, stirring uniformly at normal temperature, and diluting with deionized water to obtain a chitosan solution; respectively dripping 10mL of 2 g/L gamma-PGA into 50 mL of chitosan solution by using a micro-injection pump according to the speed of 6 mL/h, stirring and carrying out ultrasound while adding, dialyzing for 1-3h to remove the unbound small molecular polymer, fully mixing, and freeze-drying to obtain the poly-gamma-glutamic acid/chitosan dry powder.

5. A method for preparing a novel bacteriophage antimicrobial gel according to any one of claims 1 to 3, comprising the steps of:

(1) preparing mixed bacteria liquid of coliphage and staphylococcus aureus phage according to a proportion, adding calcium citrate into the mixed bacteria liquid, and standing for 20-30 min;

(2) adding gamma-PGA-CS dry powder and saponin into the mixed bacterial liquid, and uniformly mixing to obtain colloid;

(3) dripping a pH regulator to regulate the pH value of the colloid;

(4) and (4) filling to obtain the gamma-PGA-CS-phage antibacterial gel disinfectant.

6. Use of a novel bacteriophage antibacterial gel prepared by the preparation method of claim 5, wherein the antibacterial gel is applied in an amount of 100mL/m²。