CN104606672A - Staphylococcus-dissolving composite enzyme sterilization spraying agent and preparation method thereof - Google Patents

Abstract

A disclosed staphylococcus-dissolving composite enzyme sterilization spraying agent comprises, in parts by mass, 0.01-1 part of staphylococcus lysozyme, 0.1-1 part of antibacterial peptide, 0.01-1.5 parts of chlorhexidine acetate, 1-5 parts of sorbitol, 0.01-1 part of sodium dihydrogen phosphate, 0.01-1 part of disodium hydrogen phosphate, and 90-100 parts of distilled water. The sterilization spraying agent has the advantages that the agent is long disinfection action time and small stimulation, a wound is fast healing speed, and a patient does not generate drug resistance. The invention also discloses a preparation method for the sterilization spraying agent. The preparation method comprises: 1) dissolving staphylococcus lysozyme, antibacterial peptide and chlorhexidine acetate in distilled water and shaking uniformly; 2) dissolving sorbitol in distilled water and shaking uniformly; 3) dissolving sodium dihydrogen phosphate and disodium hydrogen phosphate in distilled water and shaking uniformly; and 4) mixing the solutions obtained in the step 1), 2) and 3), and stirring, and loading after the solutions are mixed uniformly. The preparation method has the advantages of simple technology and high operationality.

Description

A kind of composite lysostaphin disinfectant spray and preparation method thereof

Technical field

The invention belongs to field of biological, be specifically related to a kind of composite lysostaphin disinfectant spray and preparation method thereof.

Background technology

Disinfectant mainly refers to for killing pathogenic microorganism on communication media, makes it reach the preparation of innoxious requirement.

At present, for the hydrogen peroxide of disinfectant preparation normally 3% concentration of skin and wound surface sterilization, the ethanol of 70% concentration and iodine tincture, concrete using method dips bottled above-mentioned disinfectant preparation by common swab stick, again to skin, wound surface carries out disinfection, although such disinfectant preparation can play disinfective action, but also there is following shortcoming: 1, the disinfective action persistent period of disinfectant preparation is not long, 2, only can kill limited strain, 3, disinfectant is larger to tissue irritation, when scouring wound surface is as wound, mucosal infections place, during skin ulceration place, patient can be made to produce pain and the damage of stimulation, great misery is caused to patient, 4, in routine use process, if improper use, the swab stick of use is put into disinfectant bottle again, will produce price differential and infect, Disinfection Effect reduces greatly.

For oral cavity, the sterilization of throat, oral problem is primarily of oral ulcer, dental caries, halitosis, pharyngolaryngitis, periodontitis etc. cause, if these problems can not solve timely and effectively, the problem that patient is more serious will be caused, as heart disease, diabetes, septicemia etc., therefore oral cavity, the sterilization of throat also result in the attention of medical circle, make troubles to the life of patient as driven etc. containing spirituous ethanol in the oral disinfecting spray of current use, patient can produce drug resistance simultaneously, and then affect the gastrointestinal system of patient, therapeutic effect is not good, can not life-time service.

Summary of the invention

For the above-mentioned technical problem that prior art exists, the object of this invention is to provide a kind of composite lysostaphin germicidal spray, it may be used for the sterilization of skin, wound surface, there is disinfection time long, little to the stimulation of skin, wound surface, can not cross infection be caused, the advantage of quick wound healing; This composite lysostaphin germicidal spray also may be used for the sterilization of oral cavity, throat simultaneously, and it is high that it has disinfection efficiency, can not produce drug resistance, the advantage of colorless and odorless.

In order to solve the problems of the technologies described above, the present invention adopts following technical scheme:

A kind of composite lysostaphin germicidal spray, comprise the component of following mass fraction: staphylococcus lysozyme 0.01 ~ 1 part, antibacterial peptide 0.1 ~ 1 part, chlorhexidine acetate 0.01 ~ 1.5 part, sorbitol 1 ~ 5 part, sodium dihydrogen phosphate 0.01 ~ 1 part, sodium hydrogen phosphate 0.01 ~ 1 part and distilled water 90 ~ 100 parts.

In this programme, staphylococcus lysozyme is adopted to be that germicidal spray prepared by raw material, wherein staphylococcus lysozyme is a kind of bacteriolysin of gram positive bacteria, the aminoacid of the tetrapeptide side chain of the whole cell peptidoglycan of staphylococcus lysozyme is followed successively by ALANINE, D-Glu, C-lysine, D-alanine, wherein the first ALANINE is connected with 3-O-.alpha.-carboxyethyl-D-glucosamine. by amido link, the 1B of this polysaccharide chains tetrapeptide side chain the 3rd is connected by the D-alanine base of five glycine cross-bridges with adjacent polysaccharide chains tetrapeptide side face, formed thus and intersect in length and breadth, left and right connects, tough and tensile 3 D stereo loose structure, and aggregate into thicker peptidoglycan layer, the Gly-Gly that staphylococcus lysozyme can cut off in Peptidoglycan is good for, and another kind of enzyme can cut off the key between N-acetyl cell Teichaic acid and N-Acetyl-D-glucosamine in composite lysostaphin, make bacteria lysis, thus reach the object of sterilization.

In the present invention, the mass fraction of each component is: staphylococcus lysozyme 0.01 ~ 1 part, antibacterial peptide 0.1 ~ 1 part, chlorhexidine acetate 0.001 ~ 1 part, sorbitol 1 ~ 5 part, sodium dihydrogen phosphate 0.01 ~ 1 part, sodium hydrogen phosphate 0.01 ~ 1 part and distilled water 90 ~ 100 parts.In the bacteriolyze process of staphylococcus lysozyme, because the effect for antibacterial peptide after dissolving staphylococcal bacteria ferment treatment creates condition thus makes antibacterial peptide show obvious enhancement effect, make bacteria lysis dead, make the two play synergistic function, thus final germicidal spray have higher germicidal efficiency.Sorbitol can make staphylococcus lysozyme keep greater activity, thus disinfectant better can play disinfective action; Potassium dihydrogen phosphate and dipotassium hydrogen phosphate can make the germicidal spray obtained have suitable pH value, there is the longer holding time, make the active component in spray in suitable acid-base value environment simultaneously, can carry out disinfection to skin, wound surface, oral cavity, throat etc. for a long time, effectively ensure that bactericidal effect; Germicidal spray can be avoided to cause corrosion to sterile interface simultaneously.

In this programme, composite lysostaphin germicidal spray contains the distilled water of 90 ~ 100 parts, each like this component can be dissolved completely and be formed an entirety, each active component is made to give full play to synergistic function, it is the usefulness performance that dissolve medium can not affect each component with distilled water simultaneously, also can not produce wound surface, skin, oral cavity, throat etc. and stimulate, make the acceptant such disinfection way of patient's energy.

In this programme, the content of staphylococcus lysozyme is 0.01 ~ 1 mass parts, and the staphylococcus lysozyme of such content, for the sterilization in the places such as wound surface, skin, oral cavity, throat, can not cause too large stimulation, even if as the sterilization in hospital, also can directly use; Such component proportion is the result after applicant carries out test of many times checking, if the content of staphylococcus lysozyme is less be less than 0.01 mass parts, sterilization, sterilization functions completely can not be played, if the content of contrary staphylococcus lysozyme is greater than 1 mass parts, then can produce larger stimulation to wound surface, skin, oral cavity, throat etc., simultaneously for hospital sterilization can not directly use, could use after needing dilution, so Disinfection Effect when the content of staphylococcus lysozyme is 0.01 ~ 1 mass parts, sterilizing efficiency are the highest, easy to use.

As optimization, described composite lysostaphin germicidal spray comprises the component of following mass fraction: staphylococcus lysozyme 0.02 part, antibacterial peptide 0.5 part, chlorhexidine acetate 0.08 part, sorbitol 3 parts, sodium dihydrogen phosphate 0.01 part, sodium hydrogen phosphate 0.04 part and pure water 96.9 parts.

In this programme, the component of composite lysostaphin germicidal spray is: staphylococcus lysozyme 0.02 part, antibacterial peptide 0.5 part, chlorhexidine acetate 0.08 part, sorbitol 3 parts, sodium dihydrogen phosphate 0.01 part, sodium hydrogen phosphate 0.04 part and pure water 96.9 parts, applicant is in the such component proportion of verification experimental verification, and sorbitol can ensure that staphylococcus lysozyme fully plays the disinfective action of self and keeps active for a long time; The ratio of staphylococcus lysozyme, antibacterial peptide and chlorhexidine acetate has the highest germicidal efficiency; Sodium dihydrogen phosphate and sodium hydrogen phosphate make the pH value of the germicidal spray obtained in optimal scope, ensure that the performance of each component usefulness in spray.Such composite lysostaphin germicidal spray can kill rapidly in hospital and infect common bacteria, pathogenic entero becteria, pyococcus, saccharomytes and Hemolytic streptococcus.

As optimization, the mass ratio of described di(2-ethylhexyl)phosphate staphylococcus lysozyme hydrogen sodium and sodium hydrogen phosphate is 1:4.

In this programme, such proportioning enables the pH value of each component of disinfectant remain in suitable scope, make the convenient preservation of its stable performance, and in the process of preserving, the problem that disinfectant lost efficacy or disinfection effect weakens can not occur because of the change of condition.

As optimization, the mass ratio of described staphylococcus lysozyme, described antibacterial peptide and described chlorhexidine acetate is 1:25:4.

The present invention also provides the preparation method of above-mentioned composite lysostaphin disinfectant spray, comprises following preparation process:

1) staphylococcus lysozyme, antibacterial peptide and chlorhexidine acetate are dissolved in distilled water, shake up;

2) sorbitol is dissolved in distilled water, shakes up;

3) sodium dihydrogen phosphate and sodium hydrogen phosphate are dissolved in distilled water, shake up;

4) by step 1), 2) and 3) the solution mix and blend that obtains, mix homogeneously carries out fill.

In this programme, staphylococcus lysozyme, antibacterial peptide and chlorhexidine acetate are dissolved in distilled water, make the abundant mix homogeneously of component with bactericidal effect like this, and in the bacteriolyze process of staphylococcus lysozyme, bactericidal action for antibacterial peptide after dissolving staphylococcal bacteria ferment treatment creates condition, thus making antibacterial peptide show obvious enhancement effect, three is with the use of antibacterial rapid cleavage can be made dead; Sorbitol is dissolved in distilled water the solution system forming stable homogeneous, is conducive to sorbitol and mixs homogeneously with other components; Sodium dihydrogen phosphate and sodium hydrogen phosphate are dissolved in distilled water and form the stable buffer solution of pH value, then mix with other components, the performance of each component in mixed process can not be changed because pH value must change, be of value to component property and stablize, ensure that germicidal spray has good bactericidal effect.

Compared to existing technology, technical scheme of the present invention has following beneficial effect:

1) take staphylococcus lysozyme as raw material, it is biological enzyme component, and safety and environmental protection can kill antibacterial fast, simultaneously can not damaged tissue cell; Simultaneously with antibacterial peptide and chlorhexidine acetate with the use of, adopt dual breaking cellular wall biological antibiotic mechanism, make the germicidal spray obtained have higher disinfection efficiency, promote the advantage of the quickly-healing of wound surface, there is no toxic and side effects simultaneously, also can not produce drug resistance.

2) contain sorbitol component in germicidal spray of the present invention, make effective ingredient can have higher activity in the long period, during for wound, wound surface sterilization, there is good Disinfection Effect.

3) germicidal spray of the present invention has stable performance, is easy to the advantage that stores, the holding time is long.

4) have employed zinc protease component in germicidal spray of the present invention, it can promote the quickly-healing of wound surface.

5) germicidal spray of the present invention is the one of disinfectant, and it designs for aerosol type, easy to use, can not cause secondary pollution.

6) preparation method of composite lysostaphin germicidal spray of the present invention has that technique is simple, the advantage of strong operability.

Detailed description of the invention

Below in conjunction with specific embodiment, the present invention is described in further detail.

one, specific embodiment:

As shown in table 1,1# ~ 4# embodiment is the composite lysostaphin germicidal spray adopting method of the present invention to prepare, wherein the mass fraction of each component is: staphylococcus lysozyme 0.01 ~ 1 part, antibacterial peptide 0.1 ~ 1 part, chlorhexidine acetate 0.01 ~ 1.5 part, sorbitol 1 ~ 5 part, sodium dihydrogen phosphate 0.01 ~ 1 part, sodium hydrogen phosphate 0.01 ~ 1 part and distilled water 90 ~ 100 parts.5# ~ 7# is composite lysostaphin germicidal spray prepared by comparative example.

The preparation method of above-mentioned composite lysostaphin germicidal spray, comprises following preparation process:

1) staphylococcus lysozyme, antibacterial peptide and chlorhexidine acetate are dissolved in distilled water, shake up;

2) sorbitol is dissolved in distilled water, shakes up;

3) sodium dihydrogen phosphate and sodium hydrogen phosphate are dissolved in distilled water, shake up;

4) by step 1), 2) and 3) the solution mix and blend that obtains, mix homogeneously carries out fill.

Table 1

Two, performance Detection

1, the bactericidal effect test of oral cavity, throat:

Choose arbitrarily the patient that 35 have oral problem, wherein the range of age is 20 ~ 60 years old, and the sign of patient checks as shown in table 2; For the oral problem of patient, give the composite lysostaphin germicidal spray that every patient's dispenser every day embodiment 1 obtains respectively, concrete application method is: dispenser every day 4 times, every minor tick 3 hours, again oral problem inspection is carried out after using one week, check result is as shown in table 2, wherein in check result: effectively for symptom after dispenser disappears completely; Part effectively alleviates or partial remission for symptom after dispenser, wherein effective percentage=efficiently individual quantity/problem number; Invalid for using rear symptom still to exist, without any alleviating or disappearing; Wherein part effective percentage=part efficiently individual quantity/problem number.

Table 2

By above result of the test surface, composite lysostaphin germicidal spray prepared by the present invention has good biological antibiotic effect, fast to the healing speed of oral problem, there is the advantage of colorless and odorless, wherein for oral problem such as oral ulcer, halitosis, gingivitis, periodontitis, aphtha, pharyngolaryngitis, there is significant therapeutic effect and mitigation.

2, bacteriostatic test:

Bacteriostatic test mainly adopts inhibition zone method to test composite lysostaphin spray to staphylococcus aureus, colibacillary fungistatic effect, and concrete test method comprises the steps:

(1) preparation of bacteria suspension: bacteria suspension staphylococcus aureus and escherichia coli being made 5 × 106cfu/ml;

(2) preparation of bacteria inhibition tablet: the 1# ~ 7# composite lysostaphin germicidal spray 20 μ L getting preparation in table 1, then drip respectively on aseptic, dry filter paper, again filter paper is lain against in clean sterilized petri dishes, uncap put 37 DEG C incubator in dry, or put under room temperature for subsequent use after natural drying;

(3) preparation of negative control print: get aseptic dry filter paper sheet 4; Every sheet drips sterile distilled water 20 μ l, for subsequent use after dry;

(4) inoculation of test organisms: draw bacteria suspension prepared by 40 μ L steps (1), at nutrient agar base planar surface uniform application, builds plate, at room temperature dry 5min; Wherein the component of nutrient agar is: peptone 10g, Carnis Bovis seu Bubali cream 5g, sodium chloride 5g, agar 15g and distilled water 1000ml, except agar, other component dissolves are in distilled water, adjust ph is 7.2 ~ 7.4, add agar, heating for dissolving, subpackage, can use in 121 DEG C of pressuresteam sterilization 20min;

(5) antibacterial print is placed with: each test is placed with 1 microbiological contamination flat board, and each flat board is placed with 4 test prints, 1 negative control print, totally 5.Be placed with in planar surface with aseptic nipper coupongs.At a distance of more than 25mm between each print center, with periphery 15 more than the mm apart of flat board.After being placed with, with the light pressure-like sheet of aseptic nipper, it is made to be close to planar surface.Build plate, put 37 DEG C of incubators, cultivate 16h ~ 18h observed result, with the diameter (comprising paster) of vernier caliper measurement bacterial restrain, test result is as shown in table 3.

Table 3

Bacterial species

1#

2#

3#

4#

5#

6#

7#

Staphylococcus aureus

18mm

18.7 mm

17.6 mm

8.3 mm

6.7mm

5.9mm

5.3mm

Escherichia coli

16 mm

17.1 mm

17.3 mm

9.4 mm

6.8mm

6.1mm

5.5mm

Can be learnt by bacteriostatic test test result, 1# ~ 4# composite lysostaphin germicidal spray all has good fungistatic effect to staphylococcus aureus and escherichia coli, and wherein the diameter of bacterial restrain is all greater than 16mm, meets antibacterial requirement; And germicidal spray prepared by comparative example 5# ~ 7# to staphylococcus aureus and colibacillary fungistatic effect poor, do not meet antibacterial requirement.

3, destroying microorganisms test:

Destroying microorganisms tests the composite lysostaphin germicidal spray of main testing example 1 ~ 7 preparation to the insect killing effect of staphylococcus aureus, escherichia coli and Candida albicans, and concrete test method comprises the steps:

(1) be the bacteria suspension of 5 × 108cfu/ml by staphylococcus aureus, escherichia coli and Candida albicans configuration concentration;

(2) 4 aseptic Boiling tubes of sterilizing test are got, first often propping up in aseptic ambassador's pipe the bacteria suspension adding 0.5ml step 1) and prepare respectively, add 0.5ml organic interfering substance matter again, the two mixing mixing is placed on 5min in 20 DEG C ± 1 DEG C water-bath, inject wherein with composite lysostaphin germicidal spray 4.0 ml prepared by aseptic straw extraction embodiment 1 ~ 4 again, mix rapidly and clock immediately; Organic interfering substance matter generally adopts 3%(W/V) bovine serum albumin stock solution, get 0.5ml and join in disinfecting system and (dilute 10 times), carry out disinfection test.

(3) bacterium to be tested (staphylococcus aureus, escherichia coli and Candida albicans) and germicidal spray interacted to the scheduled time, then drew 0.5ml test organisms and composite lysostaphin germicidal spray prepared by embodiment 1 ~ 4 and be added on 4.5ml in the nertralizer of sterilizing and mix homogeneously;

(4) each aseptic Boiling tube and spray mixed liquor are after adding nertralizer effect 10min, draw 1.0ml sample liquid, and cultivate method of counting mensuration by viable bacteria and deposit viable count, often pipe sample liquid inoculates 2 plates.When clump count as grow on plates is more, after series 10 times dilution can be carried out, then carry out viable bacteria cultivation counting;

(5) replace germicidal spray with diluent simultaneously, carry out parallel test, as positive control;

(6) all test samples are all cultivated in 37 DEG C of incubators, cultivate 48h observe final result to bacterial propagule; 72h need be cultivated to bacterial spore and observe final result;

(7) calculate the viable bacteria concentration (cfu/ml) of each group, and be scaled logarithm value (N), be then calculated as follows kill oncomelania:

Logarithm value (the No)-test group viable bacteria concentration logarithm value (Nx) of kill oncomelania (KL)=matched group mean viable concentration

When wherein calculating kill oncomelania, get 2 significant digits value, can Figure correction be carried out.If sterilizing test group disinfects rear average production clump count, when being less than or equal to 1, its kill oncomelania, is namely more than or equal to the logarithm value of the front matched group mean viable concentration of test.Test result is as shown in table 4:

Table 4

From above test result: 1# ~ 4# composite lysostaphin germicidal spray has higher insect killing effect to staphylococcus aureus and escherichia coli, its have bacterial kill rate high, kill rapidly, advantage that insect killing effect is good.And the bactericidal effect of 5# ~ 7# germicidal spray to staphylococcus aureus, escherichia coli and Candida albicans is poor, does not meet sterilization requirement.

The above embodiment of the present invention is only for example of the present invention is described, and is not the restriction to embodiments of the present invention.For those of ordinary skill in the field, other multi-form change and variations can also be made on the basis of the above description.Here cannot give exhaustive to all embodiments.Every belong to technical scheme of the present invention the apparent change of amplifying out or variation be still in the row of protection scope of the present invention.

Claims (5)

1. a composite lysostaphin germicidal spray, it is characterized in that, comprise the component of following mass fraction: staphylococcus lysozyme 0.01 ~ 1 part, antibacterial peptide 0.1 ~ 1 part, chlorhexidine acetate 0.01 ~ 1.5 part, sorbitol 1 ~ 5 part, sodium dihydrogen phosphate 0.01 ~ 1 part, sodium hydrogen phosphate 0.01 ~ 1 part and distilled water 90 ~ 100 parts.

2. a composite lysostaphin germicidal spray, is characterized in that, comprises the component of following mass fraction: staphylococcus lysozyme 0.02 part, antibacterial peptide 0.5 part, chlorhexidine acetate 0.08 part, sorbitol 3 parts, sodium dihydrogen phosphate 0.01 part, sodium hydrogen phosphate 0.04 part and distilled water 96.9 parts.

3. composite lysostaphin germicidal spray according to claim 1, is characterized in that, the mass ratio of described sodium dihydrogen phosphate and sodium hydrogen phosphate is 1:4.

4. composite lysostaphin germicidal spray according to claim 1, is characterized in that, the mass ratio of described staphylococcus lysozyme, described antibacterial peptide and described chlorhexidine acetate is 1:25:4.

5. a preparation method for the composite lysostaphin germicidal spray as described in any one of Claims 1 to 4, is characterized in that, comprises following preparation process:

1) staphylococcus lysozyme, antibacterial peptide and chlorhexidine acetate are dissolved in distilled water, shake up;

2) sorbitol is dissolved in distilled water, shakes up;

3) sodium dihydrogen phosphate and sodium hydrogen phosphate are dissolved in distilled water, shake up;

4) by step 1), 2) and 3) the solution mix and blend that obtains, mix homogeneously carries out fill.