CN1364866A - Composite lysostaphin washings and preparing method - Google Patents
Composite lysostaphin washings and preparing method Download PDFInfo
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Abstract
The present invention discloses a kind of composite lysostaphin washings and its preparation process. The product as a kind disinfecting biological preparation can kill candida albicans, Neisseria gonorrhoeae, streptococcus hemolyticus, anaerobic bacteria, bacillus pyocyaneus and bacteria coli effectively, and may also kill methoxycelin resisting staphylcoccus aureaus and other drug ressiting bacteria. It has the features of wide bactericidal spectrum, no toxic side effect and no residual. It is suitable for large-scale production.
Description
The class biotechnological formulation that the present invention relates to sterilize is specifically related to a kind of composite lysostaphin washings (high section prozyme washing lotion) and preparation method.
Washing lotion is the disinfecting articles for washing that a class acts on mucocutaneous and vulva, and according to the difference of its prescription, it infects or cause of disease Gram-positive or negative bacterium causing, fungi, yeast and virus have killing and restraining effect in various degree.Used washing lotion has following a few class: (1) general chemistry class washing lotion only belongs to common articles for washing, and the pH value is a slightly acidic, can play certain cleaning action, but pathogenic bacteria is not had killing action.(2) oxidation protein class washing lotion is by the oxygenizement of oxygenant pathogenic agent to be killed, as iodine.But it has to a certain degree stimulation and damage to mucous membrane.(3) its major function composition of Chinese medicine class washing lotion is a traditional Chinese medicine, and its mechanism of action is heat-clearing and damp-drying drug, killing parasites to relieve itching.As Fructus Cnidii, golden cypress, kuh-seng etc.Non-stimulated to mucous membrane, but it is slower to take effect, and the course of treatment is longer.
The objective of the invention is to overcome above-mentioned weak point, provide a kind of and have no side effect, non-stimulated, be difficult for producing resistance, biotype washing lotion efficiently.
The invention discloses a kind of composite lysostaphin washings (high section prozyme washing lotion), this washing lotion is formed 100% form as major ingredient and glycerine (glycerol) 0.2～0.3% and distilled water as auxiliary material by staphylococcus lysozyme (Lysostaphin) 0.001～0.002% and N,O-Diacetylmuramidase (Lysozyme) 0.02～0.05%, Tubulicid 0.1-0.2%.
Because the bacteriolysin that a kind of composition staphylococcus lysozyme in the composite lysostaphin is a kind of gram-positive microorganism.Form the amino acid of the tetrapeptide side chain of such gram-positive microorganism (for example streptococcus aureus) whole cell peptidoglycan, be followed successively by the L-L-Ala, D-L-glutamic acid, L-Methionin, D-L-Ala; And the first L-L-Ala links to each other with teichoic acid by an amido linkage, and the L-Methionin that this polysaccharide chains tetrapeptide side chain is the 3rd is attached on the adjacent polysaccharide chains tetrapeptide side chain D-L-Ala carboxyl by pentapeptide (five glycine) cross-bridge.Intersect in length and breadth thus, about connect and constitute very tough and tensile 3 D stereo vesicular structure, and aggregate into thicker peptidoglycan layer.Staphylococcus lysozyme can cut off the Gly-Gly key in the peptidoglycan, and another kind of biological enzyme can cut off key between-acetylmuramic acid and the N-acetylglucosamine in the high section composite lysozyme, thereby make fungicidal activity stronger, reach the purpose of dissolving and killing bacteria, and can avoid the generation of bacterial drug resistance.
Because the sterilization mechanism of composite lysostaphin makes it be different from general microbiotic, common antibiotics is a bacteria growing inhibiting, and this enzyme then is a cracking bacterium killing bacteria.The bacterium of strain more than 400 that units such as the Huashan, Shanghai City hospital institute of Antibiotics, The 2nd Army Medical College Changhai hospital laboratory, Shanghai City Sixth People's Hospital clinical laboratory, Shanghai City the 9th the People's Hospital clinical laboratory once collected clinical each section has carried out the sterilization experiment that presses down of high section composite lysozyme.Experimental result shows: composite lysostaphin is to common clinically streptococcus aureus, and staphylococcus epidermidis, pneumococcus, D group faecalis, tetrads, product monokaryon listeria bacteria, suis and stomach Helicobacter pylori, intestinal bacteria, Pseudomonas aeruginosa etc. all have tangible inhibitory or killing effect.
Experimental results show that through Shanghai City preventive medicine research institute composite lysostaphin washings has very strong killing action to Diplococcus gonorrhoeae.Prove in the result of 60 routine test in places of hospital for obstetrics and gynaecology of Fudan University through Shanghai City Huangpu District disease prevention and control center that again composite lysostaphin washings is to perineal position effect 1 minute, the killing rate of bacterium is reached more than 90%.
Experiment also proves staphylococcus lysozyme to endurance strain, and as feeling most thorny methicillin-resistant staphylococcus aureus on the present clinical medicine, resistance pneumococcus, the effect of having a liking for the narrow food sporangium of Fructus Hordei Germinatus more are better than common antibiotics.
Another object of the present invention has provided the preparation method of above-mentioned composite lysostaphin washings, and this method is in sterilisable chamber or Bechtop, presses recipe quantity staphylococcus lysozyme (is bought from U.S. Sigma company,), N,O-Diacetylmuramidase, Tubulicid add in the clean glass or stainless steel container in proportion successively, stir gently, after fully mixing evenly, the glycerine and the distilled water of ratio shown in adding again, abundant mixing, can is in plastics or vial, warehouse-in, room temperature preservation gets final product.
Composite lysostaphin washings of the present invention (high section prozyme washing lotion) is as follows through Shanghai City preventive medicine research institute detected result: I PH determination test one, material:
Sterilizing agent: high section prozyme washing lotion
PH determinator: PYS-3C type digital ph.Two, method:
Measure this sample stoste with PHS-3C type digital ph, measure three duplicate samples, every part of replication 3 times is got its mean value.Three, result:
The pH value measurement result
Four, conclusion: the pH value average out to 6.90 of this sample.II chlorhexidine acetate assay one, material: 1, sterilizing agent: high section prozyme washing lotion 2, reagent: this standardized solution of the high chlorine of 0.0956mol/L
| Sample number into spectrum | PH value |
| ????1-1 ????1-2 ????l-3 ????2-1 ????2-2 ????2-3 ????3-1 ????3-2 ????3-3 | ????6.70 ????6.70 ????6.70 ????6.68 ????6.68 ????6.68 ????6.69 ????6.69 ????6.69 |
| Mean value | ????6.69 |
Acetone
Glacial acetic acid
The saturated acetone soln two of tropeolin-D, method:
Get chlorhexidine acetate 30.0ml, add acetone 30ml and Glacial acetic acid 2ml in the 100ml iodine flask, after jolting made dissolving, the saturated acetone soln 1.0ml of methylate orange was with the titration of perchloric acid reference liquid.Treat that solution shows orange, record perchloric acid solution consumption.Repeat aforesaid operations (blank) with acetone and the glacial acetic acid solution that does not contain chlorhexidine acetate simultaneously.Repeat to survey three times, get its mean value and carry out following calculating.
Three, result:
Four, conclusion:
| Sample number into spectrum | Chlorhexidine acetate content (%) |
| ????1-1 ????1-2 ????1-3 ????2-1 ????2-2 ????2-3 ????3-1 ????3-2 ????3-3 | ????0.1047 ????0.1047 ????0.1047 ????0.1047 ????0.1047 ????0.1047 ????0.1047 ????0.1047 ????0.1047 |
| On average | ????0.1047 |
The chlorhexidine acetate content of this sample is 0.1047%.III stability test (effective component content) one, material: 1, sterilizing agent: high section prozyme washing lotion 2, reagent: 0.0956mol/L perchloric acid standardized solution
Acetone
Glacial acetic acid
The saturated acetone soln two of tropeolin-D, method:
Three parts of toxic agent of cancellation are put 37 ℃ of incubators (relative humidity＞75%) three months in placing the forward and backward chlorhexidine acetate content of measuring respectively.(method is with the chlorhexidine acetate assay), every duplicate samples repeats 3 times, get its average its, calculate rate of descent.Three, result:
The stability test result
Annotate: test conditions is 37 ℃ and placed three months.Four, conclusion: this sample was through 37 ℃ of placements three months, and chlorhexidine acetate content average rate of decrease is 4.78%.The IV escherichia coli vector soaks quantitative disinfecting test one, material 1, bacterial strain: intestinal bacteria (8099) 2, sterilizing agent: high section prozyme washing lotion 3, neutralizing agent: the PBS of 0.5% Sulfothiorine, 2% tween-80,0.5% Yelkin TTS.Two, method 1, the preparation of intestinal bacteria suspension
| Sample number into spectrum | Chlorhexidine acetate content (%) before placing | Place back chlorhexidine acetate content (%) | Rate of descent (%) |
| ????1-1 ????1-2 ????1-3 ????2-1 ????2-2 ????2-3 ????3-1 ????3-2 ????3-3 | ????0.1047 ????0.1047 ????0.1047 ????0.1047 ????0.1047 ????0.1047 ????0.1047 ????0.1047 ????0.1047 | ????0.0997 ????0.0997 ????0.0997 ????0.0997 ????0.0997 ????0.0997 ????0.0997 ????0.0997 ????0.0997 | ??4.78 ??4.78 ??4.78 ??4.78 ??4.78 ??4.78 ??4.78 ??4.78 ??4.78 |
| On average | ????/ | ????/ | ??4.78 |
Open intestinal bacteria (8099) freeze-drying lactobacillus pipe, after separation and Culture, get 4-14 for the fresh slant cultures of cultivating 24h through 37 ℃, wash lawn with 5mlPBS, it is standby to do the suitable back of diluting with the PBS that contains 1% peptone then.2, bacterium sheet preparation:
Drip the bacteria suspension that the suitable bacterium of 0.02ml is measured through 1 * 1cm of degreasing, cleaning sterilizing cloth sheet, standby behind 37 ℃ of dry 30min.3, carrier quantitation method neutralizing agent qualification test:
Test is that stoste, neutralizing agent are the PBS that contains 0.5% Sulfothiorine, 2% tween-80,0.5% Yelkin TTS with thimerosal concentration, and the neutralizing agent test divides 8 groups and carries out.(1) the 5.0ml thimerosal adds 1 microbiological contamination sheet, and effect 1min takes out the bacterium sheet and moves among the 5.0mlPBS.(2) the 5.0ml thimerosal adds 1 microbiological contamination sheet effect 1min, takes out the bacterium sheet and moves in the 5.0ml neutralizing agent, effect 10min.(3) the 5.0ml neutralizing agent adds 1 microbiological contamination sheet, effect 10min.Take out the bacterium sheet and move in the 5.0ml neutralizing agent, with neutralizing agent dilution back inoculation.(4) 5.0ml neutralized reaction product solution (carrier that will be soaked with sterilizing agent is put effect 10min in the 5.0ml neutralizing agent) adds 1 of microbiological contamination sheet, effect 10min.Taking out the bacterium sheet moves in the 5.0ml neutralized reaction product, to inoculate behind the neutralized reaction product solution dilution.(5) 5.0mlPBS adds 1 microbiological contamination sheet, acts on 10 minutes, takes out the bacterium sheet and moves among the 5.0mlPBS, with PBS dilution back inoculation.(6) (7) (8) group is respectively PBS, neutralizing agent, substratum negative control group.Cultivate the 48h observations for 37 ℃.Test repeats 3 times, 20 ℃ ± 2 ℃ of test temperatures (pre-temperature 5min before the test).4, carrier soaks quantitative disinfecting test:
Get the thimerosal of three different concns respectively, inject the sterilization plate with the amount of every 5.0ml, in 20 ℃ ± 2 ℃) behind the water-bath 5min, add intestinal bacteria bacterium sheet, when acting on to the specified time, take out the bacterium sheet respectively and move in the 5.0ml neutralizing agent, shake well washes bacterium on the bacterium sheet behind the effect 10min, draw the 0.5ml washing lotion and carry out 37 ℃ of cultivations of viable bacteria cultivation counting 48h observationss, replace sterilizing agent to make positive controls with PBS simultaneously.Inoculation neutralizing agent, PBS and with batch negative contrast of substratum, test repeats 3 times, calculates killing rate and average kill ratio.Three, result: 1, carrier quantitation method neutralizing agent qualification test:
Table 1 neutralizing agent qualification test result
2, carrier soaks quantitative disinfecting test (cloth sheet carrier)
| Group | Deposit viable count (cfu/ sheet) | ||
| ????1 ????2 ????3 ????4 ????5 ????6 ????7 ????8 | ??0 ??1.75×10 4??1.21×10 6??1.15×10 6??1.24×10 6??0 ??0 ??0 | ????0 ????2.05×10 4????1.25×10 6????1.21×10 6????1.28×10 6????0 ????0 ????0 | ????0 ????2.50×10 4????1.29×10 6????1.25×10 6????1.30×10 6????0 ????0 ????0 |
Table 2 pair colibacillary killing effect
Annotate: the average bacterium number of positive controls is 1.30 * 106cfu/ sheet (1.28-1.33 * 10
6The cfu/ sheet)
| Weaker concn (%) | The average kill ratio (%) of effect different time (min) | |||
| ????2 | ????5 | ????10 | ????20 | |
| Stoste 50 25 | ??98.95 ??(98.76-99.07) ??89.94 ??(88.76-91.30) ??55.76 ??(53.84-56.92) | ?99.89 ?(99.8％99.90) ?91.89 ?(91.30-92.53) ?65.76 ?(63.84-68.07) | 99.98 (99.98-99.98) 93.29 (92.92-93.69) 82.69 (78.84-86.53) | 99.99 (99.99-99.99) 99.72 (99.68-99.76) 97.42 (97.15-97.69) |
Negative control group: neutralizing agent, PBS, the acellular growth of substratum.Four, conclusion: 1, repeat the neutralizing agent qualification test and show through 3 times, but the sterilizing agent of PBS neutralization test concentration that contains 0.5% Sulfothiorine, 2% tween-80,0.5% Yelkin TTS is to colibacillary residual action, and neutralized reaction product does not have the growth that is tried bacterium and influences.2, through 3 revision tests, the thimerosal effect of this sample stoste was 99.98% to colibacillary killing rate in 10 minutes.V streptococcus aureus carrier soaks quantitative disinfecting test one, material: 1, bacterial strain: streptococcus aureus (ATCC 6538) 2, sterilizing agent: the PBS of high section prozyme washing lotion 3, neutralizing agent 0.5% Sulfothiorine, 2% tween-80,0.5% Yelkin TTS.Two, method: 1, streptococcus aureus suspension preparation:
Open streptococcus aureus (ATCC 6538) freeze-drying lactobacillus pipe, after separation and Culture, get 4-14 for the fresh slant cultures of cultivating 24h through 37 ℃, wash lawn with 5mlPBS, it is standby to do the suitable back of diluting with the PBS that contains 1% peptone then.2, bacterium sheet preparation:
Drip the bacteria suspension that the suitable bacterium of 0.02ml is measured through 1 * 1cm of degreasing, cleaning sterilizing cloth sheet, standby behind 37 ℃ of dry 30min.3, carrier soaks quantitative disinfecting test;
Get the thimerosal of three different concns respectively, inject the sterilization plate with the amount of every 5.0ml, behind 20 ℃ ± 2 ℃ water-bath 5min, add streptococcus aureus bacterium sheet, act on and take out the bacterium sheet respectively to the specified time and move in the 5.0ml neutralizing agent, shake well washes bacterium on the bacterium sheet behind the effect 10min, draws the 0.5ml washing lotion and carries out viable bacteria cultivation counting.Cultivate the 48h observationss for 37 ℃, replace sterilizing agent to make positive controls with PBS simultaneously, inoculation neutralizing agent, PBS and with batch negative contrast of substratum.Test repeats 3 times, calculates killing rate and average kill ratio.Three, result:
The result that kills to streptococcus aureus
Annotate: the average bacterium number of positive controls is 1.12 * 10
6Cfu/ sheet (1.28-1.33 * 10
6The cfu/ sheet)
| Weaker concn (%) | The average kill ratio (%) of effect different time (min) | |||
| ????2 | ????5 | ????10 | ????20 | |
| Stoste 50 25 | ??98.48 ??(98.25-99.70) ??87.19 ??(86.83-87.63) ??55.76 ??(48.66-54.46) | 99.71 (99.66-99.75) 89.36 (88.92-89.73) 65.76 (58.03-65.17) | 99.93 (99.93-99.94) 91.71 (91.25-9200) 82.69 (77.67-86.16) | 99.97 (99.99-99.99) 99.57 (99.52-99.62) 97.42 (96.83-97.36) |
Negative control group: neutralizing agent, PBS, the acellular growth of substratum.Four, conclusion: 1, through 3 revision tests, 10 minutes killing rates to streptococcus aureus of the thimerosal effect of this sample stoste are 99.93%.VI Candida albicans carrier soaks quantitative disinfecting test one, material: 1, bacterial strain: Candida albicans (ATCC 10231) 2, sterilizing agent: the PBS of high section prozyme washing lotion 3, neutralizing agent 0.5% Sulfothiorine, 2% tween-80,0.5% Yelkin TTS.Two, method: 1, Candida albicans suspension preparation:
Open Candida albicans (ATCC 10231) freeze-drying lactobacillus pipe, after separation and Culture, get 4-14 for the fresh slant cultures of cultivating 24h through 37 ℃, wash lawn with 5mlPBS, it is standby to do the suitable back of diluting with the PBS that contains 1% peptone then.2, bacterium sheet preparation:
Drip the bacteria suspension that the suitable bacterium of 0.02ml is measured through 1 * 1cm of degreasing, cleaning sterilizing cloth sheet, standby behind 37 ℃ of dry 30min.3, carrier quantitation method neutralizing agent qualification test:
Test is that stoste, neutralizing agent are the PBS that contains 0.5% Sulfothiorine, 2% tween-80,0.5% Yelkin TTS with thimerosal concentration, and the neutralizing agent test divides 8 groups and carries out.(1) the 5.0ml thimerosal adds 1 microbiological contamination sheet, and effect 1min takes out the bacterium sheet and moves among the 5.0mlPBS.(2) the 5.0ml thimerosal adds 1 microbiological contamination sheet effect 1min, takes out the bacterium sheet and moves in the 5.0ml neutralizing agent, effect 10min.(3) the 5.0ml neutralizing agent adds 1 microbiological contamination sheet, effect 10min.Take out the bacterium sheet and move in the 5.0ml neutralizing agent, with neutralizing agent dilution back inoculation.(4) 5.0ml neutralized reaction product solution (carrier that will be soaked with sterilizing agent is put effect 10min in the 5.0ml neutralizing agent) adds 1 of microbiological contamination sheet, effect 10min.Take out the bacterium sheet and move in the 5.0ml neutralized reaction product, release the back inoculation with neutralized reaction product solution.(5) 5.0mlPBS adds 1 microbiological contamination sheet, acts on 10 minutes, takes out the bacterium sheet and moves among the 5.0mlPBS, with PBS dilution back inoculation.(6) (7) (8) group is respectively PBS, neutralizing agent, substratum negative control group.Cultivate the 72h observations for 37 ℃.Test repeats 3 times, 20 ℃ ± 2 ℃ of test temperatures (pre-temperature 5min before the test).4, carrier soaks quantitative disinfecting test:
Get the thimerosal of three different concns respectively, inject the sterilization plate with the amount of every 5.0ml, in 20 ℃ ± 2 ℃) behind the water-bath 5min, add intestinal bacteria bacterium sheet, when acting on to the specified time, take out the bacterium sheet respectively and move in the 5.0ml neutralizing agent, shake well washes bacterium on the bacterium sheet behind the effect 10min, draw the 0.5ml washing lotion and carry out 37 ℃ of cultivations of viable bacteria cultivation counting 72h observationss, replace sterilizing agent to make positive controls with PBS simultaneously.Inoculation neutralizing agent, PBS and with batch negative contrast of substratum, test repeats 3 times, calculates killing rate and average kill ratio.Three, result: 1, carrier quantitation method neutralizing agent qualification test:
Table 1 neutralizing agent qualification test result
2, carrier soaks quantitative disinfecting test (cloth sheet carrier)
| Group | Deposit viable count (cfu/ sheet) | ||
| ????1 ????2 ????3 ????4 ????5 ????6 ????7 ????8 | ??0 ??4.75×10 4??7.95×10 5??7.50×10 5??8.15×10 5??0 ??0 ??0 | ??0 ??4.90×10 4??8.40×10 5??7.95×10 5??8.50×10 5??0 ??0 ??0 | ????0 ????4.15×10 4????7.60×10 5????7.40×10 5????7.80×10 5????0 ????0 ????0 |
The average kill ratio (%) of the oidiomycetic killing effect weaker concn of table 2 pair white effect different time (min) (%) 25 10 20
97.11 (99.33-99.81) (99.90-99.91) (99.97-99.98) for 99.55 99.90 99.98 stostes (95.37-98.58)
82.48???????????????84.93???????????????89.44???????????????99.4150????????????(81.79-82.96)???????(84.50-85.61)???????(88.64-90.00)???????(99.33-99.46)
50.00 56.68 78.61 94.5325 (45.29-53.08) (53.70-59.25) (72.22-79.62) (94.07-94.87) annotate: the average bacterium number of positive controls is 8.10 * 10
5Fu/ sheet (7.80-1.33 * 10
5The fu/ sheet)
Negative control group: neutralizing agent, PBS, the acellular growth of substratum.Four, conclusion: 1, repeat the neutralizing agent qualification test and show through 3 times, but the sterilizing agent of PBS neutralization test concentration that contains 0.5% Sulfothiorine, 2% tween-80,0.5% Yelkin TTS is to the oidiomycetic residual action of white, and neutralized reaction product does not have the growth that is tried bacterium and influences.2, through 3 revision tests, the thimerosal effect of this sample stoste was 99.90% to the oidiomycetic killing rate of white in 10 minutes.
Composite lysostaphin of the present invention (high section composite lysozyme) washing lotion is as follows through Shanghai City preventive medicine research institute assay to the bactericidal assay of perineal position bacterium and gonorrhoea:check situation sample title:high section prozyme washing lotion sample number into spectrum:the yellow disease control 20004345 existing 60/1-60 censorship unit that disappears:Gaoke Bio-Engineering Co Ltd; Shanghai please test project:sterilizing rate test foundation:GB15981-1995 " disinfection technology standard "-2000 assay:subjects and quantity:perineal position; Totally 60.Sterilization concentration (dosage): stoste; Action time:1 minute neutralizing agent title concentration (or consumption) 0.5% Sulfothiorine; 2% tween-80; 0.5% Yelkin TTS PBS test ambient temperature (℃): 22; Relative humidity (%): 79.The method of sampling:the aseptic cotton swab that will be soaked with above-mentioned neutralizing agent sample solution evenly is coated with several times back and forth in the perineal position left side; Then cotton swab is turned back in the test tube as sampling before the sterilization.The cotton swab that will be soaked with high section prozyme washing lotion more evenly is coated with several times back and forth on the perineal position right side; Embrocates with aforesaid method after 1 minute; 37 ℃ of of of of of agar of as the sterilization post-sampling again.Then with wash-out in the cotton end insertion degree pipe.Sampling is totally 60 before and after the sterilization.The method of inspection:washing lotion is poured into plates; cultivates 48 hours, and live bacterial count calculates sterilizing rate. ( ) ( ) ( ) ( ％ ) 1 1 2900 90 96.90 2 1 1100 10 99.09 3 1 2600 20 99.23 4 1 3400 50 98.53 5 1 4700 250 94.68 6 1 3400 210 93.82 7 1 8800 630 92.84 8 1 8700 490 94.37 9 1 2900 190 93.45 10 1 3700 290 92.16 11 1 6500 570 91.23 12 1 4200 170 95.95 13 1 1500 30 98.00 14 1 2400 40 98.33 15 1 4700 220 95.32 16 1 2700 200 95.59 17 1 3500 210 94.00 18 1 4600 200 95.65 19 1 3200 200 93.75 20 1 1700 60 96.47 21 1 3300 290 91.21 22 1 880 20 97.73 23 1 6900 520 92.46 24 1 2900 210 92.76 25 1 1100 70 93.64 26 1 2800 280 90.00 27 1 3700 370 90.00 28 1 4700 420 91.06 29 1 4900 290 94.08 30 1 4200 320 92.38：
High section prozyme washing lotion to the minimal bactericidal concentration of Diplococcus gonorrhoeae be 3.9 *
10
-5u/ml。
The high section prozyme washing lotion of different concns (0.5u/ml, 1u/ml, 2u/ml) all has killing action to Diplococcus gonorrhoeae at different operative temperatures (10 ℃, 20 ℃, 37 ℃) under the condition of different action times (2 ', 5 ', 10 ', 15 ', 30 ', 45 ').VII organic substance influence test one, material: 1, bacterial strain: streptococcus aureus (ATCC 6538) 2, sterilizing agent: the PBS of high section prozyme washing lotion 3, neutralizing agent 0.5% Sulfothiorine, 2% tween-80,0.5% Yelkin TTS.Two, method: 1, streptococcus aureus suspension preparation:
Streptococcus aureus suspension and aseptic calf serum be made into contain 50% calf serum group, contain 25% calf serum group and do not contain three groups of calf serums, drip respectively and dye the sterilizing cloth sheet and prepare the microbiological contamination sheet, the bacterium amount reaches 5 * 10
5-5 * 10
6The cfu/ sheet.Carry out carrier with the original liquid concentration thimerosal behind 37 ℃ of dry 30min and soak quantitative disinfecting test.Test repeats 3 times, calculates killing rate and average kill ratio.Three, result:
The organic substance influence test-results
Four, conclusion:
| Weaker concn (%) | The average kill ratio (%) of effect different time (min) | Contrast bacterium number (cfu/ sheet) |
| 10?????????????20?????????????30?????????????40 | ||
| Stoste 50 25 | 97.11??????????99.55??????????99.90??????????99.98 (95.37-98.58)??(99.33-99.81)??(99.90-99.91)??(99.97-99.98) 82.48??????????84.93??????????89.44??????????99.41 (81.79-82.96)??(84.50-85.61)??(88.64-90.00)??(99.33-99.46) 50.00??????????56.68??????????78.61??????????94.53 (45.29-53.08)??(53.70-59.25)??(72.22-79.62)??(94.07-94.87) | 9.50×10 5(9.10-9.90×10 5) 1.50×10 6(1.03-1.08×10 6) 1.25×10 6(1.22-1.28×10 6) |
Repeat organic substance influence through 3 times and test, when containing 50% calf serum in bacteria suspension, the effect of this sample being killed streptococcus aureus has moderate influence; When containing 25% calf serum in bacteria suspension, the effect of this sample being killed streptococcus aureus has slight effect.Conclusion:
High section prozyme washing lotion pH value is 6.69.Chlorhexidine acetate content is 0.1047%.Sample was through 37 ℃ of placements three months, and chlorhexidine acetate content rate of descent is 4.78%.10 minutes killing rates to intestinal bacteria, streptococcus aureus and Candida albicans of this sample stoste effect are respectively 99.98%, 99.93% and 99.90%.When 50% calf serum existed, the effect of this sample being killed streptococcus aureus had moderate influence; Slight effect when existing, 25% calf serum is arranged.
Dissolving staphylococcal bacteria washing lotion of the present invention (high section prozyme washing lotion) is as follows through test-results such as Shanghai City preventive medicine research institute detection acute toxicities: I acute oral toxicity test one, test objective: detect and tried the toxicity of thing when the short period of time per os is contaminated, its result can be used as the foundation of test doses such as toxicity grading and definite subchronic toxicity.Two, material and animal
Tried thing
Proterties: submitted sample is a colourless liquid
Compound method: accurately take by weighing sample, add distilled water and be made into 0.25g, ml is for examination.
Laboratory animal: Kunming mouse, cleaning level, body weight 18-22 gram is provided by laboratory animal portion of Shanghai Medical Univ, conformity certification number: 02-22-1.
22 ± 2 ℃ of receptacle temperature, relative humidity 50-80 gram, the qualified 02-28 of animal housing.The mouse breeding feed, technology ﹠ development Co. provides by the Su Hang laboratory animal, conformity certification number: 9905084.Three, test method: 1, test basis: in the disinfection technology standard (third edition) 1999.11 3.4.2, dosage and grouping: behind the animal overnight fasting, divide two groups of male and female at random, 10 every group by body weight.Dosage is 5.0g/kg.3, testing sequence: adopt a per os to irritate stomach contamination method, press the 0.2ml/10g body weight, calculate the contamination amount.Observation period was two weeks.Put to death animal to expiring and dissect, visual inspection animal general pathology changes.4, calculate: use once method calculating LD to greatest extent
50Be worth, and carry out toxicity grading according to acute toxicity grading criteria.Four, test-results:
Various dose treated animal body weight change, death condition sees Table.
Contamination back: do not see obvious toxicity symptom
Gross anatomy:
Expire and put to death animal, each internal organs no abnormality seen.
Chmice acute Oral toxicity result of the test dosage treated animal is counted dead other g/kg (only) 07 14 (d) number of body weight X ± SD (g) dead animal (only) rate (%) ♂ 5.0 10 20.0 ± 1.3 24.1 ± 1.2 27.5 ± 1.4 0 00 ♀ 5.0 10 19.5 ± 1.4 23.7 ± 1.1 26.9 ± 1.2 005, conclusion:
Chmice acute per os LD50:
Female:＞5.0g/kg
Male:＞5.0g/kgII skin irritation test one, test objective: as to detect and tried whether to produce local excitation reaction and degree thereof after thing contacts animal skin.Two, material and animal:
Tried thing
Proterties: submitted sample is a colourless liquid
Compound method: stoste is directly tested.
Laboratory animal: new zealand rabbit, regular grade, four, body weight 2.4～2.8kg is provided by laboratory animal portion of Shanghai Medical Univ, conformity certification number: 05-52-1.20 ± 2 ℃ of receptacle temperature, relative humidity 50-70%, Animal House conformity certification number: 02-28, feed for rabbit is provided by Su Hang laboratory animal technology ﹠ development Co., conformity certification number: 9905084.Three, test method: 1, detect foundation: in the disinfection technology standard (third edition) 1999.11 3.6.2, test was cut each about 3 * 3cm of unhairing scope with back part of animal backbone diamond wool in preceding 24 hours
23, second day, on the intact skin of unhairing in left side, mark 2.5 * 2.5cm
2The test site will be tried thing 0.2ml and will be evenly coated on the test site skin, and cover with one deck glass, use non-stimulated immobilization with adhesive tape again, make to be tried thing and directly contact 4h with skin, and the right side skin of unhairing compares.4, remove coverture after the off-test, and with warm water clean remove residual tried thing after.Respectively at remove residual tried behind the thing 1,24 and 48h observe and smear the position skin reaction, by the standards of grading scoring of skin irritation response intensity with to the classification of skin irritation response intensity.Four, test-results:
Tried after thing and the skin contact local skin not show spot, oedema, do not seen other toxic action.An irritant reaction scoring of rabbit skin sees Table.
Total to the total red water of irritant reaction grade form of the animal skin moving not same product control sample of gonosome 1h 24h 48h thing control sample contrast volume total red water of the total red water of the total red water of the total red water of (kg) red water
Spot is swollen to divide that spot is swollen to divide that spot is swollen to divide that spot is swollen to divide that spot is swollen to divide spot swollen 1 female 2.4 00000000000000000 02 male 2.4 00000000000000000 03 female 2.4 00000000000000000 04 male 2.4 000000000000000000 total mark averages 000000 five, the conclusion of dividing:
Being tried thing is 0 to rabbit skin irritation (the highest) total mark average, belongs to nonirritant.III mouse polychromatic erythrocytes micronucleus test one, test objective: detect and tried thing, estimate genetoxic in its body to the influence that the mouse polychromatic erythrocytes micronucleus forms.Two, material and animal: tried the rerum natura shape: submitted sample is the colourless liquid compound method: take by weighing sample 5.0,2.5,1.0g, respectively adding distil water is to 20ml, fully behind the mixing as given the test agent.Positive control: endoxan is produced by Hualian Pharmaceutical Co., Ltd., Shanghai.Compound method: take by weighing endoxan 40mg adding distil water to 20ml, fully standby behind the mixing.
Laboratory animal: Kunming mouse, regular grade, body weight 25-30 gram is provided by laboratory animal portion of Shanghai Medical Univ, conformity certification number: 02-22-1.20 ± 2 ℃ of receptacle temperature, relative humidity 50-70%, Animal House conformity certification number: 02-28, the mouse breeding feed is provided by Su Hang laboratory animal technology ﹠ development Co., conformity certification number: 9905084.Three, test method: 1, detect foundation: 3.11.4 bar in the disinfection technology standard (third edition) 1999.11.2, animal is divided into 5 groups at random, 10 every group, male and female half and half are respectively three dosage groups of sample and negative control group and endoxan positive controls.3, adopt 30h secondary administration by gavage, with the different concns sample 0.4mg/20g body weight of preparation, respectively each treated animal is irritated stomach, positive controls adopts irritates stomach, and negative control group adopts irritates stomach.4, after for the second time irritating stomach 6 hours, animal was put to death in the cervical vertebra dislocation, got femur bone marrow and added the calf serum mixing, routine smear, fixing Giemsa stained preparation.5, microscopy is observed, and every mouse is counted 1000 polychromatic erythrocytes (PCE), and calculates and wherein contain the micronucleated cell number, represents with permillage ‰.Also observe the ratio of PCE/NCE (mature erythrocyte) simultaneously.6, respectively organizing the micronucleated cell rate compares with negative control group with Poisson's distribution U check carrying out statistical treatment.Four, test-results:
Mouse polychromatic erythrocytes microkernel incidence group dosage number of animals is examined PCE and is contained micronucleus micronucleated cell PCE/ statistics
(mg/kg) (only) number (individual) PCE (individual) rate ‰ NCE check is tried 5,000 10 10,000 10 1.0 1.07＞0.05 thing groups 2,500 10 10,000 9 0.9 1.06＞0.05
1,000 10 10,000 8 0.8 1.08＞0.05 negative distilled water
10 10,000 9 0.9 1.06 control group 20000mg/kg male hoop phosphorus stack acid amides
10 10,000 28.2 28.2 0.96＜0.01 control group 40mg/kg five, conclusion:
It is negative to mouse polychromatic erythrocytes micronucleus test result to be tried thing.IV vaginal mucomembranous irritant test one, test objective: detect and tried hormesis and the intensity of thing to the experimental animal vaginal mucosa.Two, material and animal: tried the rerum natura shape: closing the sample product is the colourless liquid compound method: get sliver 0.01, soak into the back as being tried thing with submitted sample.Laboratory animal: the SD rat, female, regular grade, 12, body weight 240-260g divides two groups, is tried thing group and control group, 6 every group.Provide conformity certification number by laboratory animal portion of Shanghai Medical Univ: 02-22-2.20 ± 2 ℃ of receptacle temperature, relative humidity 50-70%, Animal House conformity certification number: 02-28, the rat feeding material is provided by Su Hang laboratory animal technology ﹠ development Co., conformity certification number: 9905084.Three, test method: 1, detect foundation: in the disinfection technology standard (third edition) 1999.11 3.8.2, will be tried thing and put the animal intravaginal, and contact 4 hours with vaginal mucosa, control group soaks with sterile saline with sliver 0.01g does same test.3, put to death animal in 24h, take out local vagina tissue, observation has or not phenomenons such as hyperemia, water bracelet.4, with reference to the standards of grading and the skin irritation strength grading of skin irritation response intensity in the disinfection technology standard (third edition) 1999.11, carry out evaluation to vaginal mucosa irritation intensity.Four, test-results:
The scoring of animal vaginal mucosa irritation reaction scoring treated animal body weight irritant reaction Bian Hao (g) erythema oedema total points
1 240 000 are subjected to 2 245 000 examinations, 3 252 000 things 4 254 000 group 5 260 000
6 255 000 total mark averages 0
7?????????243???????0?????????0?????????0
8 245 000 pairs 9 251 000 according to 10 260 000 group 11 248 000
12 250 000 total mark averages 0 five, conclusion:
Being tried thing is 0 to the highest total mark average of rat vagina mucous membrane irritation reaction, belongs to nonirritant.V skin allergic reaction test one, test objective: detection is tried thing and is repeated to contact the skin allergic reaction that the back produces animal.Two, material and animal: tried the rerum natura shape: submitted sample is the colourless liquid compound method: stoste is directly tested.Laboratory animal: albino guinea-pig is planted by Britain, regular grade, and male and female half and half, body weight 200-240g, totally 48, be divided into and tried three groups of thing groups, negative control group, positive controls, 16 every group, provide conformity certification number by laboratory animal portion of Shanghai Medical Univ: 02-52-2.20 ± 2 ℃ of receptacle temperature, relative humidity 50-70%, Animal House conformity certification number: 02-28, the rat feeding material is provided by Su Hang laboratory animal technology ﹠ development Co., conformity certification number: 9905084.Three, test method: 1, detect foundation: in the disinfection technology standard (third edition) 1999.11 3.9.2,24h cuts the about 3 * 3cm of area with guinea pig back left side hair before the test
23, induce contact: will induce with being tried thing 0.2ml at 2 * 2cm
2On the two-layer gauze, and it is sticked on tried to cover with one deck glassine paper on unhairing district, the treated animal left side skin, use non-stimulated immobilization with adhesive tape again, make and tried thing and directly contact 6h at skin.In kind repeat once in 7d and 14d.4, excite contact: induce contact back 14d at last, to excite with being tried thing 0.2ml on the two-layer gauze of 2 * 2cm2, and it is sticked on be subjected on examination group and the negative control treated animal right side skin of unhairing, cover with one deck glassine paper, use non-stimulated immobilization with adhesive tape again, make direct contact 6h.The thing that tried that 5, will stick behind the 6h takes down.Observe skin reaction in 24, behind the 48h, by skin reaction strength grading standard bisection and calculate the sensitization rate and by sensitization strength assessment standard evaluation sensitization intensity.6, positive controls contacts with exciting with the contact of inducing of 5% drinking 2%2,4-dinitro-chlorine stupid work of generation is same respectively.Four, test-results:
Guinea pig skin transformation reactions result
Annotate: skin erythema reaction, oedema response intensity are 0,1,2,3,4 o'clock the reaction number of animals and the ratio of experimental animal number at integration promptly.Five, conclusion:
| Group | Number of animals | Induced concentration | Excite concentration | Observing time | Erythematous response intensity | Oedema response intensity sensitization rate (%) |
| 0??????1??????2??????3??????4 | 0???????1??????2??????3 | |||||
| Tried the thing group | ??16 | Stoste | Stoste | ??24h ??48h | 16/16??0/16???0/16???0/16???0/16 16/16??0/16???0/16???0/16???0/16 | 16/16???0/16???0/16???0/16????0 16/16???0/16???0/16???0/16????0 |
| Positive controls | ??16 | ??/ | Stoste | ??24h ??48h | 16/16??0/16???0/16???0/16???0/16 16/16??0/16???0/16???0/16???0/16 | 16/16???0/16???0/16???0/16????0 16/16???0/16???0/16???0/16????0 |
| Positive controls | ??16 | ??5％/ | ??2％ | ??24h ??48h | 6/16???8/16???2/16???0/16???0/16 4/16???5/16???4/16???3/16???0/16 | 14/16???2/16???0/16???0/16????0 10/16???6/16???0/16???0/16????0 |
Tried thing to guinea pig skin transformation reactions test-results, the sensitization rate is 0 (%), and sensitization intensity belongs to extremely light.VI cumulative toxicity test---dosage escalation cumulative coefficient method one, test objective: detect and to be tried thing, and select to provide parameter for the dosage of subchronic test and other relevant toxicity tests in experimental animal body accumulation toxicity.Two, material and animal: tried the rerum natura shape: submitted sample is the colourless liquid collocation method: accurately take by weighing sample, add distilled water, be made into that concentration is respectively 50,75,110,250mg/kg, tried thing as each phase.Laboratory animal: Kunming mouse, cleaning level, body weight 20-21g is provided by laboratory animal portion of Shanghai Medical Univ, conformity certification number: 02-22-1.20 ± 2 ℃ of receptacle temperature, relative humidity 50-80%, Animal House conformity certification number: 02-28.The mouse breeding feed, technology ﹠ development Co. provides by the Su Hang laboratory animal, conformity certification number: 9905084.Three, test method: 1, detect foundation: 3.10.3 bar in the disinfection technology standard (third edition) 1999.11.2, according to the acute oral toxicity test result, this sample its mouse oral LD
50In 5.0g/kg.3, animal is divided at random two groups of male and female, 10 every group, per os is irritated gastric capacity with the 0.1ml/10g body weight.Once a day, per 4 days is first phase.Downpayment dosage 0.1 LD
50Be 0.5g/kg, continuous 4 days.Later each issue dosage once increases progressively 50%, heavily weighs increasing progressively simultaneously, proofreaies and correct the stomach amount of irritating by body weight.Four, test-results:
Do not see that during the cumulative toxicity test contamination obvious toxicity symptom occurs.Tried the thing poisoning dosage, integral dose and animal dead number see Table.
Integral dose and animal dead numerical table
Mouse was contaminated 20 days continuously, did not see death, and integral dose is 26.20g/kg, cumulative coefficient＞5.Five, conclusion:
| Contamination fate (d) | Poisoning dosage (g/kg) | Integral dose (g/kg) | Animal dead number (only) | Remarks | ||
| ??LD 50× | ??G/kg | ??LD 50× | ????g/kg | ????♀???♂ | ||
| ????1-4 | ??0.10 | ??0.50 | ??0.40 | ????2.00 | ????0????0 | |
| ????5-8 | ??0.15 | ??0.75 | ??1.00 | ????5.00 | ????0????0 | |
| ????9-12 | ??0.22 | ??1.10 | ??1.88 | ????9.40 | ????0????0 | |
| ????13-16 | ??0.34 | ??1.70 | ??3.24 | ????16.20 | ????0????0 | |
| ????17-20 | ??0.50 | ??2.50 | ??5.24 | ????26.20 | ????0????0 | |
| ????21- | ??0.75 | |||||
Tried thing to the mouse cumulative toxicity test, its cumulative coefficient (K value)＞5 is according to the property accumulated a little less than the cumulative toxicity classification genus.Assay gathers: one, acute oral toxicity test
Female mice LD
50＞5.0g/kg
Male mice LD
50＞5.0g/kg
The nontoxic level two in true border, skin irritation test: belong to nonirritant.Three, micronucleus test: feminine gender four, vaginal mucomembranous irritant test: belong to nonirritant five, skin allergic reaction: belong to extremely light by six, cumulative toxicity test: cumulative coefficient K＞5, the property accumulated a little less than the genus.
Major ingredient of the present invention-Gao Ke composite lysozyme is protein in essence, can not produce stimulation to skin and mucous membrane, body is not produced any type of toxic side effect, and can thoroughly degrade, do not have residual by metabolism, can enrichment, so this washing lotion in use need not to wash.
Its basic characteristics are:
1) fungicidal spectrum is wide.Can not only effectively kill common malignant bacterias such as Candida albicans, Hemolytic streptococcus, anerobe, Pseudomonas aeruginosa, intestinal bacteria, Diplococcus gonorrhoeae is also had very strong killing action.To some drug tolerant bacterias, also demonstrate extremely strong sterilization effect in addition as methicillin-resistant staphylococcus aureus etc.
2) fungicidal activity and stability are much higher than staphylococcus lysozyme.Because high section prozyme washing lotion is to be main component with staphylococcus lysozyme (lysostaphin), being equipped with compositions such as another kind of N,O-Diacetylmuramidase and stablizer, synergistic agent is composited, have bigger stability than pure staphylococcus lysozyme, prove that after tested this washing lotion can be preserved 2 years at normal temperatures.
Example 1,100 milliliters of composite lysostaphin washings components of manufacturing: Portugal, molten Portugal coccus enzyme 0.001 gram
N,O-Diacetylmuramidase 0.02 gram
Tubulicid 0.1 gram
0.2 milliliter of glycerine
99.7 milliliters of methods of distilled water:
With staphylococcus lysozyme 0.001 gram, N,O-Diacetylmuramidase 0.02 restrains respectively, and Tubulicid 0.1 gram mixing adds glycerine 0.2ml distilled water 99.7ml, the mixing can again.
Example 2: make 100 milliliters of composite lysostaphin washings components: staphylococcus lysozyme 0.002 gram
N,O-Diacetylmuramidase 0.02 gram
Tubulicid 0.1 gram
0.2 milliliter of glycerine
99.7 milliliters of methods of distilled water:
With staphylococcus lysozyme 0.002 gram, N,O-Diacetylmuramidase 0.02 restrains respectively, and Tubulicid 0.1 gram mixing adds glycerine 0.2ml distilled water 99.7ml, the mixing can again.
Example 3,100 milliliters of composite lysostaphin washings components of manufacturing; Staphylococcus lysozyme 0.002 gram
N,O-Diacetylmuramidase 0.02 gram
Tubulicid 0.15 gram
0.2 milliliter of glycerine
99.6 milliliters of methods of distilled water:
With staphylococcus lysozyme 0.002 gram, N,O-Diacetylmuramidase 0.02 restrains respectively, and Tubulicid 0.15 gram mixing adds glycerine 0.2ml distilled water 99.6ml, the mixing can again.
Claims (2)
1, a kind of composite lysostaphin washings is characterized in that this washing lotion is by forming 100% form as major ingredient and glycerine 0.2～0.3% and distilled water as auxiliary material by staphylococcus lysozyme 0.001～0.002% and N,O-Diacetylmuramidase 0.02～0.05%, Tubulicid 0.1-0.2%.
2, a kind of preparation method of composite lysostaphin washings as claimed in claim 1, it is characterized in that this method is in sterilisable chamber or Bechtop, presses recipe quantity with staphylococcus lysozyme, N,O-Diacetylmuramidase, Tubulicid adds in the clean glass or stainless steel container in proportion successively, stir gently, fully mixed evenly after, the glycerine and the distilled water of ratio shown in adding again, abundant mixing, can is put room temperature preservation in storage in plastics or vial.
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|---|---|---|---|
| CNB01105204XA CN1137979C (en) | 2001-01-11 | 2001-01-11 | Composite lysostaphin washings and preparing method |
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|---|---|---|---|
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| CN1137979C CN1137979C (en) | 2004-02-11 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104524557A (en) * | 2014-12-16 | 2015-04-22 | 杨陈 | Lysostaphin compound disinfection gel |
| CN106590949A (en) * | 2016-12-11 | 2017-04-26 | 深圳市美益洁生物科技有限公司 | Clothes washing tank biological enzyme sterilization cleaning agent and preparing method thereof |
| WO2019059804A1 (en) | 2017-09-25 | 2019-03-28 | Георгий Георгиевич ЧУМБУРИДЗЕ | Thermostable composition with antiviral and antibacterial activity and use thereof |
-
2001
- 2001-01-11 CN CNB01105204XA patent/CN1137979C/en not_active Expired - Lifetime
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104524557A (en) * | 2014-12-16 | 2015-04-22 | 杨陈 | Lysostaphin compound disinfection gel |
| CN106590949A (en) * | 2016-12-11 | 2017-04-26 | 深圳市美益洁生物科技有限公司 | Clothes washing tank biological enzyme sterilization cleaning agent and preparing method thereof |
| WO2019059804A1 (en) | 2017-09-25 | 2019-03-28 | Георгий Георгиевич ЧУМБУРИДЗЕ | Thermostable composition with antiviral and antibacterial activity and use thereof |
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| Publication number | Publication date |
|---|---|
| CN1137979C (en) | 2004-02-11 |
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