CN1132517C - Compound lysostaphin enzyme disinfectant - Google Patents
Compound lysostaphin enzyme disinfectant Download PDFInfo
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- CN1132517C CN1132517C CN 00116398 CN00116398A CN1132517C CN 1132517 C CN1132517 C CN 1132517C CN 00116398 CN00116398 CN 00116398 CN 00116398 A CN00116398 A CN 00116398A CN 1132517 C CN1132517 C CN 1132517C
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Abstract
The present invention discloses a compound lysostaphin disinfectant which is capable of effectively killing gram-positive bacteria and especially has notable sterilization effect on golden staphylococcus with drug resistance. The disinfectant of the present invention is a biologic disinfectant, has the advantages of no toxic and side effects, no environment pollution and simple and convenient preparation method, and is suitable for large-scale industrial production.
Description
Technical field
The present invention relates to biologic product, be specifically related to compound lysostaphin enzyme disinfectant.
Background technology
Usually the disinfectant of usefulness mainly contains following a few class:
1, the disinfectant of coagulated protein class:
It is by making albuminous degeneration reach sterilisation purpose.As phenol, lysol, acids, alcohols.Mentioned reagent generally is used for the sterilization of food, fruit, experiment equipment, hospital, skin etc.
2, the disinfectant of soluble protein class
Can make the microorganism dissolving and then play killing action.As sodium hydroxide, lime etc.
3, the disinfectant of oxidation protein class
Be that strong oxidation by oxidant kills pathogene.Comprise bleaching powder, Peracetic acid etc.Generally be used for the sterilization of periphery environment, water, room and article.
4, cation surface-active disinfectant:
This type of medicine is mainly quaternary amine, and this medicine is coagulated protein when high concentration, suppresses the metabolism of bacterium during low concentration, and such medicine is all effective to gram-positive bacteria and negative bacterium.
5, other
Formalin: can combine with the amino of albumen, make albuminous degeneration, play the effect of sterilizing.Be usually used in the fur goods of sterilizing.
Glutaraldehyde: its aldehyde radical can with the albumino reaction of microorganism, to reach sterilisation purpose.Virus, the reproduction type of bacterium, tubercle bacillus, fungi, gemma all there is bactericidal effect preferably.
Oxirane: be the bactericide that a kind of penetration power is very strong, have strong sterilizing ability.Can carry out disinfection to fur, fiber etc.
Hibitane, An Er iodine: be homemade disinfectant, it is good to have a Disinfection Effect, and usage is easy, is one of disinfectant preferably at present, and it has stronger bactericidal action to gram-positive bacteria.
But above disinfectant is chemosterilant.The chemical classes disinfectant has certain pollution to environment, can bring very big toxic and side effect in the clinical disinfection, as to skin and mucous membrane irritation, to blood flow wound and the stimulation of the blood stains surface of a wound etc.These all influence the application of the sterilizing of chemical classes disinfectant.
Summary of the invention
Purpose of the present invention is overcoming the weak point of above-mentioned disinfectant, develops a kind of having no side effect, and is difficult for producing drug resistance, biotype disinfectant efficiently.
The invention provides a kind of biotype disinfectant, specifically is a kind of compound lysostaphin enzyme disinfectant.Can efficiently kill gram-positive bacteria, especially remarkable to the bactericidal effect of staphylococcus aureus, it is 0.045u/ml (≈ 0.001 μ g) that minimum presses down bacteriocidal concentration MIC.
Compound lysostaphin enzyme disinfectant of the present invention is formed (percentage by weight) by following prescription:
Main component: (1) staphylococcus lysozyme (lysostaphin) 25-35%
(2) lysozyme (lysozyme) 50-65%
Auxiliary material: (3) potassium phosphate (potassium phosphate) 7-10%
(4) sodium chloride (sodium chloride) 0.5-1%
(5) chitin (chitin) 0.2-0.3%
The virus killing principle of compound lysostaphin enzyme disinfectant of the present invention is as follows:
Staphylococcus lysozyme is a kind of bacteriolysin of gram-positive bacteria.Form the amino acid of the tetrapeptide side chain of such gram-positive bacteria (for example staphylococcus aureus) whole cell peptidoglycan, be followed successively by the L-alanine, D-glutamic acid, L-lysine, D-alanine; And the first L-alanine links to each other with muramic acid by an amido link, and the L-lysine that this polysaccharide chains tetrapeptide side chain is the 3rd is attached on the adjacent polysaccharide chains tetrapeptide side chain D-alanine carboxyl by pentapeptide (five glycine) cross-bridge.Intersect in length and breadth thus, about connect and constitute very tough and tensile 3 D stereo loose structure, and aggregate into thicker peptide glycan layer.Staphylococcus lysozyme can cut off the Gly-Gly key in the peptide glycan, and another kind of biology enzyme can cut off key between-acetylmuramic acid and the N-acetylglucosamine in the molten Portugal coccus complex enzyme, thereby make bactericidal activity stronger, reach the purpose of dissolving and killing bacteria, and can avoid the generation of bacterial drug resistance.
Because the sterilization mechanism of staphylococcus lysozyme uniqueness makes it be different from general antibiotic.Except that producing beta-lactamase, also there is other resistance mechanism in the resistance mechanism complexity of drug resistance staphylococcus aureus.To antibacterials commonly used, more antibody-resistant bacterium is also arranged according to clinical observation MRSA as penicillin, oxacillin one, third generation cephalosporin class antibiotic and some Novel Quinolone antibacterials.From above-mentioned situation; though new antibiotic is arranged constantly to be developed and comes into operation; produce but just have new antibody-resistant bacterium in very short time, this is because most of antibiotic bacteria growing inhibiting impels new R-plasmid of bacterium generation or new enzyme to stop the effect of antibiotic.The mechanism of action of staphylococcus lysozyme is different from general antibiotic, and antibiotic effect only is a bacteria growing inhibiting, and this enzyme then is a cracking bacterium killing bacteria, and the staphylococcus aureus of methicillin-resistance (MRSA) is had stronger sterilizing ability equally.
Staphylococcus lysozyme can produce bacteriolysis by hydrolysis Gram-positive bacteria whole cell peptidoglycan, thereby thorough killing bacteria has been avoided the generation of bacterial drug resistance.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 staphylococcus lysozyme.Experimental result shows: staphylococcus lysozyme is to gram-positive bacteria, as common clinically staphylococcus aureus, Staphylococcus epidermidis, Diplococcus pneumopniae, D group enterococcus, tetrads, product monokaryon Listeria, streptococcus and stomach Helicobacter pylori etc. all have tangible inhibitory or killing effect.
Experiment also proves staphylococcus lysozyme to endurance strain, and as feeling most thorny methicillin-resistant staphylococcus aureus on the present clinical medicine, drug resistance Diplococcus pneumopniae, the effect of having a liking for the narrow food sporangium of Fructus Hordei Germinatus more are better than common antibiotics.
The bactericidal effect of compound lysostaphin enzyme disinfectant of the present invention (FE complex enzyme disinfectant I) is as follows through Shanghai City health and epidemic prevention station assay: one, test method: bacterial strain: the degreasing calico microbiological contamination method of staphylococcus aureus (ATCC6538) microbiological contamination carrier: 1.0cm * 1.0cm is for dripping the method for dying.The preparation of TE enzyme disinfectant:
Accurately take by weighing FE enzyme disinfectant 320mg (specific enzyme activity 900u/g), add sterile purified water 100ml, be made into the thimerosal of specific enzyme activity 2.88u/ml, mixing, suitably the dilution back is standby.
Test has been observed under 20 ℃ ± 2 and 37 ℃ ± 2 conditions FE disinfectant respectively to the staphylococcus aureus bactericidal effect, and support methods is adopted in test, during test, thimerosal is through pre-temperature, and by each microbiological contamination sheet 5ml thimerosal, print is added in the thimerosal, act on to the stipulated time, again print is moved in the test tube of 5ml neutralizer solution, in and 10 minutes, get neutralization back suspension 1ml and pour into inoculation as ordinary nutrient agar, duplicate, put in 37 ℃ of incubators and cultivated 48 hours, observe final result.Two, result:
Under two kinds of temperature to the average kill ratio (%) of the killing rate test temperature decontaminant concentration effect different time (min) of staphylococcus aureus (℃) (specific enzyme activity u/ml)
5 10 15 3020 2.88 99.82 99.87 99.98 99.99
1.44 99.53 99.83 99.92 99.95
0.72 98.99 99.70 99.83 99.9332 2.88 99.96 99.99 99.99 99.99
1.44 99.93 99.98 99.99 99.99
0.72 99.84 99.89 99.94 99.98 annotate: the several 20 ℃ of conditions 7.7 * 10 of the average bacterium of control group
5The cfu/ sheet
37 ℃ of conditions 6.5 * 10
5The attached neutralizer test of cfu/ sheet:
Neutralizer: 0.02% lauryl sodium sulfate PBS
Bacterial strain: staphylococcus aureus
Group survival clump count (cfu/ sheet)
Disinfectant+bacterium sheet 40
(disinfectant+bacterium sheet)+neutralizer 1.1 * 10
4
Neutralizer+bacterium sheet 1.5 * 10
4
(disinfectant+neutralizer)+bacterium sheet 1.4 * 10
4
PBS+ bacterium sheet 1.5 * 10
4
Test PBS 0
Test neutralizer 0
Test medium 0
The toxicity of compound lysostaphin enzyme disinfectant of the present invention is as follows through Shanghai City chemicals toxicological detection institute test result: one, skin irritatin is tested: test result:
Positive controls part skin has erythematous response, and average stimulation index damaged skin is 2.3, and intact skin is 0.6, and negative group does not all have the reaction of erythema oedema with sample sets and sees Table
Damaged skin reaction score intact skin reaction score group redness/oedema redness/oedema
1 24 48 (my god) 1 24 48 (my god)
1 0/0 0/0 0/0 0/0 0/0 0/0 negative controls 2 0/0 0/0 0/0 0/0 0/0 0/0
3 0/0 0/0 0/0 0/0 0/0 0/0
1 0/0 0/0 0/0 0/0 0/0 0/0 samples 2 0/0 0/0 0/0 0/0 0/0 0/0
3 0/0 0/0 0/0 0/0 0/0 0/0 evaluation of result:
This sample is 0 to the average PiI of complete sum damaged skin.Meet standard-required.Test event: primary cutaneous irritant test censorship unit: Gaoke Bio-Engineering Co Ltd, Shanghai's submitted sample: general proterties of FE complex enzyme disinfectant sample and purposes: test method:
With reference to method requirement among the WS 5-1-87 " medical heat cure methyl vinyl silicone rubber "
Animal subject: new zealand rabbit, 3 every group
Given the test agent: submitted sample is made into 200u/ml with physiological saline.With the negative contrast of physiological saline, the positive contrast of 3% formaldehyde.
Test method: test preceding 24 hours backs and shave hair, go out 3 * 3cm in the unhairing zoning
2Two at four diagonal angles, test region scratch skin with the sterilization blade and become # shape wound, each treated animal is at preserved skin district (containing damaged skin) coated sample liquid, physiological saline and 3% formalin, cover with antiseptic gauze etc. immediately after being coated with and fixing, cleaning sample applying area after 24 hours, and the erythema at viewing test position, the oedema skin reaction.
Test result: in the observation period, the appearance activity in 1 hour of injection 20u/ml mouse reduces, and body weight has decline slightly, and the 2u/ml mouse does not see obvious poisoning symptom and death.Body weight and negative control group indifference.
After injecting before the injection of body weight change (gram) group before and after the mouse peritoneal injection
24 48 72(h)
1 20 22 23 25
2 20 21 22 24 negative control group 3 21 22 23.5 25
4 22 22 23 24
5 21 22 23 23.5
1 21 19 18.5 19.5
2 23.5 21.5 19 20.520u/ml 3 22 21 21 21.5
4 22.5 20 20.5 22
5 21.5 20 19.5 21
1 21 21 21.5 22
2 21 21 21.5 232u/ml 3 20 22.5 24 26
4 22 23 23.5 25
5 22.5 23 23 24.5
In 72 hour observation period, the reaction of 2u/ml sample sets animal is not more than control animals.Meet standard-required.Two, lumbar injection toxicity test: test event: lumbar injection test censorship unit: Gaoke Bio-Engineering Co Ltd, Shanghai's submitted sample: general proterties of FE complex enzyme disinfectant sample and purposes: test method:
Require to carry out with reference to method among the GB/T 16175-1996 " medical organosilicon material biological assessment test method ".Animal subject: cleaning level Kunming mouse, body weight 20～23 grams.Given the test agent: submitted sample is made into 20u/ml with physiological saline, 2u/ml.Test method: variable concentrations test agent constant temperature 1 hour in 37 ℃ of insulating boxs, make lumbar injection by the 50ml/kg amount, reach at once after the observed and recorded injection and occur symptom in 24,48,72 hours and weigh.Test result: recording the mean light absorbency sample sets is 0.016, and feminine gender is 0.007, and the positive is 0.767, and calculating hemolysis rate is 1.2%.Evaluation of result: this product hemolysis rate is 1.2%, meets the requirement of biological assessment standard＜5% of biomaterial and goods.Three, hemolytic test: test event: hemolytic test censorship unit: Gaoke Bio-Engineering Co Ltd, Shanghai's submitted sample: general proterties of FE complex enzyme disinfectant sample and purposes: test method: require to carry out with reference to method among the GB/T 16175-1996 " medical organosilicon material biological assessment test method ".Given the test agent: submitted sample is made into 200u/ml with physiological saline.Test method: it is stand-by to gather the hemodilution of fresh anti-freezing rabbit by standard-required.
Sample sets given the test agent 1ml+ physiological saline 9ml, negative control group physiological saline 10ml, positive controls distilled water 10ml, do parallel sample 3 pipes for every group, respectively organize test tube then after 37 ℃ of insulating boxs are incubated 30 minutes, add diluted fresh anti-freezing rabbit blood 0.2ml in each pipe, it is centrifugal after 60 minutes to continue 37 ℃ of insulations, gets supernatant and surveys absorbance down in the 545nm wavelength.Test result:
Every 20u/ml specimen, cellular morphology is observed and cell counting sees Table: the cell counting of group cellular morphology
247 (my god) 247 (my god)
1 25 54 87 negative control group, 2 form normal growths good 23 57 83
3 20 53 88
1 17 43 77 samples, 2 form normal growths good 20 47 79
3 16 45 74
1 positive control 2 whole dead 0
3
According to the reaction hierarchical table relatively degree of propagation be 89% to belong to the I level.Evaluation of result: cellular morphology is normal, and adherent growth is good, and cell degree of propagation relatively belongs to the I level, meets standard-required.Four, cell toxicity test: test event: cell toxicity test censorship unit: Gaoke Bio-Engineering Co Ltd, Shanghai's submitted sample: general proterties of FE complex enzyme disinfectant sample and purposes: test method: with reference to the medical organosilicon material biological assessment of GB/T16175-1996 test method.Cell line: the L929 fibroblast is provided by cell biological research institute of the Chinese Academy of Sciences, goes down to posterity to cultivate 2 times, is mixed with the cell suspension that concentration is 40,000/ml.Given the test agent: submitted sample is mixed with 20u/ml with cell culture fluid.Negative control group: 15% calf serum cell culture fluid.Positive controls: the cell culture fluid that contains the calf serum of 6.4% phenol.Test method: cell proliferation degree method.Five, disinfectant of the present invention is tested through Shanghai The 2nd Army Medical College toxicity test and pyrogen:
Acute toxicity experience 1, nomenclature of drug: FE enzyme disinfectant (providing) by Gaoke Bio-Engineering Co Ltd, Shanghai.2, test method:
(1) 60 of Kunming kind small white mouses are adopted in this experiment, male and female half and half, and body weight 33.5 ± 0.25g is provided by the The 2nd Army Medical College Experimental Animal Center.
(2) FE presses continuous once a day 7 days of 10u/kg dosage through the intramuscular injection small white mouse, observes animal toxic reaction under maximum dose.
(3) result of the test:
After animal via intramuscular injection FE7 days, no abnormal reaction, movable normal, to observe through 7 days, 60 small white mouses all survive.(4) conclusion (of pressure testing):
This experiment Kunming kind small white mouse is with 10u/kg dosage intramuscular injection every day successive administration 7 days, no abnormal reaction, FE non-toxic reaction.
Excitant and pyrogen test 1, nomenclature of drug: FE enzyme disinfectant (providing) 2, eye irritant reaction by Gaoke Bio-Engineering Co Ltd, Shanghai:
(1) test method: 3 of the new zealand rabbits that this experiment employing The 2nd Army Medical College Experimental Animal Center provides, with FE stoste (1000u/ml), splash into one of rabbit right eye, with the left eye contrast, observe 1h respectively, 12h, 24h, 48h, the variation of 72h.
(2) criterion: conjunctival congestion, oedema situation.
(3) result of the test: no abnormal reaction behind 3 new zealand rabbits of FE eye stimulation, all negative.3, skin irritatin reaction:
(1) test method: a mao 5cm is shaved on rabbit dorsal column right side
2Be test area, the left side is contrast, is being tried to be coated with 0.1mlFE stoste (1000u/ml) observation 1h, 2h, 24h, 48h skin reaction situation on the area.
(2) criterion: whether red swelling of the skin has inflammatory reaction.
(3) result of the test: 3 new zealand rabbits all do not have any abnormal response, total negative through the FE skin irritation test.4, pyrogen test:
(1) medicine: FE enzyme disinfectant stock solution (1000u/ml) is provided by bioengineering product Co., Ltd of high section.
(2) animal: 3 of new zealand rabbits, female body weight 2.35 ± 0.1kg is provided by the The 2nd Army Medical College Experimental Animal Center.
(3) test method: get 3 of new zealand rabbits, measure its caecum normal body temperature after, slowly inject FE stoste from auricular vein, dosage is the 330u/kg body weight, takes temperature once every 1 hour, surveys altogether three times, sees its body temperature situation of change.
(4) result of the test: 3 new zealand rabbits are measured the body temperature of its injection front and back behind intravenous injection FE, all do not have significant difference.
Six, disinfectant of the present invention is as follows through Shanghai Changhai Hospital test bactericidal effect: FE complex enzyme disinfectant is to the observation sample title of mouse burn wound staphylococcus aureus killing effect: FE complex enzyme disinfectant censorship unit: Gaoke Bio-Engineering Co Ltd, Shanghai's censorship date: in April, 1991 the sample source: Gaoke Bio-Engineering Co Ltd, Shanghai's test item: measure FE complex enzyme disinfectant to mouse burn wound staphylococcus aureus killing effect
| Rabbit number | Body weight (kg) | Normal body temperature (℃) | Injection back body temperature ℃ 1h 2h 3h |
| 1 | 2.4 | 39.7 | 39.6 39.8 39.8 |
| 2 | 2.25 | 39.8 | 39.7 39.7 39.7 |
| 3 | 2.5 | 39.8 | 39.7 39.7 39.8 |
Method one, infection of burn model: animal is a Kunming kind small white mouse, male and female half and half, body weight 25-30g.Animal is divided into three groups at random, causes mouse back skin I II degree burn (75 ℃, 15 seconds), burn surface area (about 5cm with the circular flatiron of diameter 2.6cm
2) account for the 6-7% of mouse corpus surface area, to hinder back 30 minutes, surface of a wound inoculum density is 10
9The staphylococcus aureus bacterium liquid 0.1ml of cfu/ml, bacterial strain is staphylococcus aureus ATCC25923.Two, burn wound disinfects
Behind burn wound inoculation staphylococcus aureus 3-4 hour, pick the physiological saline that contains 0.02% lauryl sodium sulfate with aseptic cotton swab, embrocating the surface of a wound and do to sample before the sterilization, is the FE complex enzyme disinfectant of 2 μ/ml with enzyme concentration alive then, the burn wound of applying ointment or plaster (1-2ml/ only).Disinfective action 30 minutes remakes the sterilization post-sampling.Through the vibration washing, sampling liquid 0.1ml smears the agar plate inoculation, and 2 flat boards of each sample liquid inoculation were cultivated 48 hours in 37 ℃ of incubators, observed final result.
The result
The result proves through 24 hours of the sterilization of FE complex enzyme disinfectant, to have only 2 to have only staphylococcus aureus to detect, and 19 mouse of control group then all have staphylococcus aureus to detect (subordinate list).
Subordinate list FE complex enzyme disinfectant is to burn wound staphylococcus aureus Disinfection Effect
| Group | Detect number of animals (only) | Positive number of animals (only) | Positive rate (%) |
| FE complex enzyme disinfectant | 24 | 2 | 8.30 |
| Control group | 19 | 19 | 100.00 |
2 are detected positive mouse after FE complex enzyme disinfectant is sterilized, and its staphylococcus aureus bacterium number is respectively 2.2 * 10
4Cfu/cm
2With 9.8 * 10
3Cfu/cm
2, and control group mice staphylococcus aureus number is 0.98 * 10
5Cfu/cm
2-1.82cfu/cm
2Between, far above using FE complex enzyme disinfectant processed group.
Conclusion
Prove that through 24 artificial burned mice simulation sterilizing tests FE enzyme disinfectant has good killing effect to the staphylococcus aureus of artificial contamination's burn wound.
Examining report sample title: FE complex enzyme disinfectant censorship unit: Gaoke Bio-Engineering Co Ltd, Shanghai's sample source: Gaoke Bio-Engineering Co Ltd, Shanghai's properties of samples: white powder, tasteless, test item soluble in water: FE complex enzyme disinfectant is to the mensuration of the practical Disinfection Effect of burn wound staphylococcus aureus
The preparation of method one, FE complex enzyme disinfectant
Accurately take by weighing FE complex enzyme disinfectant and be made into the thimerosal that concentration is 2.0u/ml, place 4 ℃ to preserve standby down with sterile distilled water.Two, practical sterilizing test
Test is 2.0u/ml with FE enzyme in the FE complex enzyme disinfectant than concentration alive, and used neutralizer is the physiological saline that contains 0.02% lauryl sodium sulfate.Before the sterilization, soak in the test tube that contains neutralizer physiological saline with aseptic cotton swab, and after inboard wall of test tube is extracted, embrocate sampling in the sample region that patient's burn wound delimited, sampling area is 1cm
2, after the sampling, cotton swab is put back to former sample solution in vitro, as contrast before the sterilization.During sterilization, with the FE complex enzyme disinfectant of the concentration 2.0u/ml patient's burn wound of applying ointment or plaster, after the disinfective action 2 hours, do the sterilization sampling, sample solution is through beaing washing, the sampling liquid stoste or suitably dilute after, make high salt mannite agar and pour into inoculation, every ware 0.1ml, duplicate, put and cultivate 48h in 37 ℃ of incubators, observe final result.
The result
Prove that with the practical sterilizing test of 11 routine fire victims through FE complex enzyme disinfectant was applied ointment or plaster sterilization to burn wound after, surface of a wound staphylococcus aureus number was by 1850-55650cfu/cm
2Drop to 0-2250cfu/cm
2, the killing rate scope is 91.66-100.00%.Killing rate to the staphylococcus aureus of patient's burn wound reaches more than 98.00%, shows that the burn wound that FE complex enzyme disinfectant is used for infection of staphylococcus aureus has tangible Disinfection Effect (subordinate list).
Subordinate list is to the effect of burn wound staphylococcus aureus
| The test sequence number | Bacterium number (cfu/cm before the sterilization 2) | Sterilization back bacterium number (cfu/cm 2) | Killing rate (%) |
| 1 | 24500 | 250 | 98.97 |
| 2 | 27000 | 2250 | 91.66 |
| 3 | 10750 | 0 | 100.00 |
| 4 | 2000 | 0 | 100.00 |
| 5 | 1850 | 0 | 100.00 |
| 6 | 45250 | 0 | 100.00 |
| 7 | 29625 | 287 | 99.03 |
| 8 | 3765 | 60 | 98.41 |
| 9 | 29850 | 410 | 98.63 |
| 10 | 27460 | 650 | 97.63 |
| 11 | 55650 | 580 | 98.96 |
Conclusion
FE enzyme disinfectant is used to wash burn wound, and surface of a wound staphylococcus aureus is had killing effect preferably, and its average kill ratio can reach more than 98.00%.Seven, to measure Disinfection Effect through hospital of the General Logistics Department of Chinese People's Liberation Army engineering army unit as follows for disinfectant of the present invention: sample title: FE disinfectant censorship unit: Gaoke Bio-Engineering Co Ltd, Shanghai's censorship date: on April 10th, 1997 sample source: Gaoke Bio-Engineering Co Ltd, Shanghai's properties of samples: white powder, tasteless, test item soluble in water: FE enzyme disinfectant is to the mensuration of the practical Disinfection Effect of burn wound staphylococcus aureus
The preparation of method one, FE enzyme disinfectant
Accurately taking by weighing FE enzyme disinfectant, to be made into concentration of enzymatic activity with anhydrous bacterium distilled water be 2.88u/ml, and used neutralizer is the physiological saline that contains 0.02% lauryl sodium sulfate.Before the sterilization, soak in the test tube that contains neutralizer physiological saline with aseptic cotton swab, and after inboard wall of test tube is extracted, embrocate sampling in the sample region that patient's burn wound delimited, sampling area is 2 * 2cm
2, after the sampling, cotton swab is put back to former sample solution in vitro, as contrast before the sterilization.During sterilization, FE enzyme disinfectant flushing patient burn wound with concentration of enzymatic activity 2.88u/ml, act on 2 hours post-samplings that carry out disinfection, sample solution, is made high sugared mannite agar and is poured into inoculation after getting sample solution stoste or doing suitably dilution through beaing washing, every ware 0.1ml, the same form two minutes is put 37 ℃ of incubators and is cultivated 48h, observes final result.
The result
Practical sterilizing test through 30 routine patients proves that after FE enzyme disinfectant was to the burn surface irrigation and disinfection, surface of a wound staphylococcus aureus detected number by the preceding 987～41000cfu/cm of sterilization
2Drop to 0～1630cfu/cm
2, the killing rate scope is 94.24～100.00%.Show that the burn wound that FE enzyme disinfectant is used for infection of staphylococcus aureus has tangible Disinfection Effect (subordinate list).
Conclusion
FE enzyme disinfectant is used for the burn wound irrigation and disinfection, can reach more than 94.24% the killing rate of staphylococcus aureus.
Subordinate list is to the killing effect of burn wound staphylococcus aureus
Eight, to measure Disinfection Effect through Wuxi City No.3 People's Hospital as follows for disinfectant of the present invention: sample title: FE complex enzyme disinfectant censorship unit: Gaoke Bio-Engineering Co Ltd, Shanghai's sample date: on July 10th, 1997 sample source: Gaoke Bio-Engineering Co Ltd, Shanghai's properties of samples: white powder, tasteless, test item soluble in water: FE complex enzyme disinfectant is to the mensuration of the practical Disinfection Effect of burn wound staphylococcus aureus
| The test sequence number | Bacterium number (cfu/cm before the sterilization 2) | Sterilization back bacterium number (cfu/cm 2) | Killing rate (%) |
| 1 | 4800 | 230 | 95.20 |
| 2 | 9500 | 530 | 94.42 |
| 3 | 1050 | 0 | 100.00 |
| 4 | 1450 | 0 | 100.00 |
| 5 | 1500 | 100 | 93.33 |
| 6 | 9750 | 0 | 100.00 |
| 7 | 9650 | 210 | 94.73 |
| 8 | 3790 | 160 | 95.28 |
| 9 | 18500 | 240 | 98.70 |
| 10 | 24550 | 750 | 96.94 |
| 11 | 35600 | 580 | 98.37 |
| 12 | 35750 | 410 | 98.85 |
| 13 | 46500 | 378 | 99.18 |
| 14 | 17500 | 290 | 96.13 |
| 15 | 16500 | 950 | 94.24 |
| 16 | 27300 | 398 | 98.54 |
| 17 | 18350 | 5 | 99.97 |
| 18 | 32140 | 1560 | 95.15 |
| 19 | 2880 | 67 | 97.67 |
| 20 | 1930 | 3 | 99.84 |
| 21 | 24610 | 300 | 98.78 |
| 22 | 29360 | 1630 | 94.45 |
| 23 | 29120 | 9 | 99.96 |
| 24 | 1850 | 0 | 100.00 |
| 25 | 2010 | 19 | 99.05 |
| 26 | 3570 | 20 | 99.43 |
| 27 | 27530 | 375 | 98.63 |
| 28 | 1370 | 0 | 100.00 |
| 29 | 987 | 0 | 100.00 |
| 30 | 11000 | 538 | 98.68 |
The preparation of method one, FE complex enzyme disinfectant
Accurately take by weighing FE complex enzyme disinfectant and be made into the thimerosal of concentration 2.0u/ml, place 4 ℃ to preserve standby down with sterile distilled water.Two, practical sterilizing test
Test is 2.0u/ml with FE enzyme in the FE complex enzyme disinfectant than concentration alive, and used neutralizer is the physiological saline that contains 0.02% lauryl sodium sulfate.Before the sterilization, soak in the test tube that contains neutralizer physiological saline with aseptic cotton swab, and after inboard wall of test tube is extracted, embrocate sampling in the sample region that patient's burn wound delimited, sampling area is 1cm
2, after the sampling, cotton swab is put back to former sample solution in vitro, as contrast before the sterilization.During sterilization, with the FE complex enzyme disinfectant of the concentration 2.0u/ml patient's burn wound of applying ointment or plaster, the sterilization post-sampling is made in disinfective action 2 hours, and sample solution is through beaing washing, the sampling liquid stoste or suitably dilute after, make high salt mannite agar and pour into inoculation, every ware 0.1ml, duplicate, put and cultivate 48h in 37 ℃ of incubators, observe final result.
The result
Prove that through the practical sterilizing test of 10 routine fire victims through FE complex enzyme disinfectant was applied ointment or plaster sterilization to burn wound after, surface of a wound staphylococcus aureus number was by 22000-340000cfu/cm
2Drop to 0-2720cfu/cm
2, the killing rate scope is 96-100%.Show that the burn wound that FE complex enzyme disinfectant is used for infection of staphylococcus aureus has tangible Disinfection Effect (subordinate list).
Subordinate list is to the preceding bacterium number (cfu/cm of killing effect test sequence number sterilization of burn wound staphylococcus aureus
2) sterilization back bacterium number (cfu/cm
2) killing rate (%) 1 35,000 0 1,002 42,000 0 1,003 22,000 440 98.04 38,000 418 98.95 75,000 2,250 97.06 49,000 1,960 96.07 52,000 676 98.78 67,000 2,000 97.09 340,000 2,720 99.210 85,000 340 99.6
Conclusion
FE enzyme disinfectant is used to wash burn wound, and surface of a wound staphylococcus aureus is had killing effect preferably, and its average kill ratio can reach more than 96%.
Another object of the present invention has provided the preparation method of compound lysostaphin enzyme disinfectant, and this method is in desinfection chamber or superclean bench, with staphylococcus lysozyme (buying from U.S. Sigma company), lysozyme, potassium phosphate, sodium chloride, chitin, add in the clean glass or stainless steel container in proportion successively, stir gently, fully mixed evenly after, be packaged in the papery bag, warehouse-in, room temperature preservation gets final product.
The Main Ingredients and Appearance of staphylococcus lysozyme disinfectant of the present invention is staphylococcus lysozyme and lysozyme, and they are protein, can not produce stimulation to skin and mucous membrane, body is not produced any toxic and side effect, and can thoroughly degrade by metabolism, do not have residually, do not influence environmental protection.Its advantage is as follows:
1) wholesomeness.Compound lysostaphin enzyme disinfectant (I) demonstrates special efficacy killing aspect the methicillin-resistant staphylococcus aureus.Bactericidal effect is splendid.
2) fungicidal spectrum is wider.Compound lysostaphin enzyme disinfectant (II) and composite lysostaphin enzyme spray for oral cavity can not only be killed methicillin-resistant staphylococcus aureus, and can effectively kill common malignant bacterias such as Escherichia coli, Candida albicans, hemolytic streptococcus, anaerobic bacteria, Pseudomonas aeruginosa.
3) bactericidal activity and stability are much higher than staphylococcus lysozyme.Because composite lysostaphin is to be main component with staphylococcus lysozyme (lysostaphin), being equipped with effective biotic component such as stabilizing agent, synergist is composited, has bigger stability than pure staphylococcus lysozyme, stable in temperature active maintenance below 60 ℃, PH accommodation 3-14,3 years enzymic activitys of freeze-dried powder keep stable.
Particularly the drug resistance staphylococcus aureus is effective especially to the gram positive bacteria infection of the cross-infection in the hospital, burn and operation patients for the molten Portugal of the present invention coccus complex enzyme disinfectant, and other disinfectants and medicine can't be mentioned in the same breath with it.
At present, the nosocomial infection of drug resistance staphylococcus aureus MRSA is difficult to treatment, and this bacterioid all has pesticide resistance to the most antibiotics except that vancomycin.But the vancomycin toxic and side effect is very big, and after patient used, the liver renal toxicity was big.In addition, vancomycin can not external application, and clinical SD-Ag commonly used and hibitane are strong to the damaged skin excitant, and MRSA is not had effect substantially.And existing recently people reports the drug resistance staphylococcus aureus of finding anti-vancocin.So external preparation that clinical especially burn urgent need can be killed the drug resistance staphylococcus aureus.The molten Portugal coccus complex enzyme disinfectant of Gaoke Bio-Engineering Co Ltd, Shanghai's development and production can the hydrolysis gram-positive bacteria particularly the drug resistance staphylococcus aureus peptide glycan glycine key and produce bacteriolyze, thereby thorough killing bacteria, be difficult for producing endurance strain, there is not any skin irritation again, so these product are a kind of very promising skin disinfectant for external use.
The surface of a wound of MRSA infection is the residual wound in late period of healing difficulty mostly clinically, and secretion obviously reduces behind the molten Portugal of the external application coccus complex enzyme disinfectant, and the surface of a wound begins healing.After continuous three uses, most surface of a wound MRSA turn out cloudy, and total sterilizing rate reaches more than 98%, promotes wound healing, reduces cross infection in hospital.
Specific embodiments
Example: the compound method of every manufacturing 1 gram staphylococcus lysozyme disinfectant (I).
A. respectively with staphylococcus lysozyme 0.3 gram, lysozyme 0.4 restrains, potassium phosphate 0.088 gram, sodium chloride 0.01 gram, chitin 0.002 gram mixing encapsulation.
B. respectively with staphylococcus lysozyme 0.35 gram, lysozyme 0.55 restrains, potassium phosphate 0.09 gram, sodium chloride 0.007 gram, chitin 0.003 gram mixing encapsulation.This prescription is to staphylococcus aureus sterilizing rate 99.1%
C. respectively with staphylococcus lysozyme 0.25 gram, lysozyme 0.64 restrains, potassium phosphate 0.10 gram, sodium chloride 0.008, chitin 0.002 gram mixing encapsulation.This prescription is to staphylococcus aureus sterilizing rate 99.0%.
Claims (2)
1, a kind of compound lysostaphin enzyme disinfectant is characterized in that this disinfectant is made up of following percentage by weight prescription:
Main Ingredients and Appearance: staphylococcus lysozyme 25～35%
Lysozyme 55～65%
Auxiliary material potassium phosphate 7～10%
Sodium chloride 0.5～1%
Chitin 0.2～0.3%
2, a kind of preparation method of compound lysostaphin enzyme disinfectant, it is characterized in that this method is in desinfection chamber or superclean bench, with staphylococcus lysozyme 25～35%, lysozyme 55～65%, potassium phosphate 7～10%, sodium chloride 0.5～1%, chitin 0.2～0.3%, add successively in the clean glass or stainless steel container, stir gently, after fully mixing evenly, be packaged in the papery bag.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 00116398 CN1132517C (en) | 2000-06-08 | 2000-06-08 | Compound lysostaphin enzyme disinfectant |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 00116398 CN1132517C (en) | 2000-06-08 | 2000-06-08 | Compound lysostaphin enzyme disinfectant |
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| CN1327742A CN1327742A (en) | 2001-12-26 |
| CN1132517C true CN1132517C (en) | 2003-12-31 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN 00116398 Expired - Lifetime CN1132517C (en) | 2000-06-08 | 2000-06-08 | Compound lysostaphin enzyme disinfectant |
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Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100450546C (en) * | 2002-01-28 | 2009-01-14 | 上海高科生物工程有限公司 | Compound preparation for dissolving staphyloase and preparation method |
| CN102671190A (en) * | 2012-04-26 | 2012-09-19 | 安徽省芬格欣普蓝生物药业有限公司 | Enzymic preparations for improving human body immunity and enhancing gastrointestinal and respiratory functions |
| CN103877569B (en) * | 2014-04-14 | 2016-01-06 | 李志臣 | A kind of compound lysostaphin enzyme disinfectant |
| CN104780459A (en) * | 2015-04-16 | 2015-07-15 | 美国掌赢信息科技有限公司 | Method and electronic equipment for loading effects in instant video |
| CN106561716A (en) * | 2016-11-02 | 2017-04-19 | 厦门华厦学院 | Agricultural antibacterial complex enzyme preparation and preparation method thereof |
| CN112641532A (en) * | 2020-12-22 | 2021-04-13 | 江苏瞪羚科技有限公司 | Application method of compound enzyme disinfectant |
| CN112931536A (en) * | 2021-02-01 | 2021-06-11 | 长沙道勤生物科技有限公司 | Dry powder type environmental disinfectant for livestock and poultry breeding and preparation method thereof |
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