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Hygienic requirement for lysostaphin and lysozyme disinfectants
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WS/T 647-2019
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Basic data Standard ID | WS/T 647-2019 (WS/T647-2019) | Description (Translated English) | Hygienic requirement for lysostaphin and lysozyme disinfectants | Sector / Industry | Health Industry Standard (Recommended) | Classification of Chinese Standard | C59 | Classification of International Standard | 11.080 | Word Count Estimation | 13,157 | Date of Issue | 2019 | Date of Implementation | 2019-07-01 | Issuing agency(ies) | National Health Commission |
WS/T 647-2019: Hygienic requirement for lysostaphin and lysozyme disinfectants---This is a DRAFT version for illustration, not a final translation. Full copy of true-PDF in English version (including equations, symbols, images, flow-chart, tables, and figures etc.) will be manually/carefully translated upon your order.
Hygienic requirement for lysostaphin and lysozyme disinfectants
ICS 11.080
C 59
WS
People's Republic of China Health Industry Standard
Hygienic requirements for lysostaphin and lysozyme disinfectants
Published on.2019-01-30
2019-07-01 Implementation
National Health and Wellness Committee of the People's Republic of China
Content
Foreword...1
1 Scope...2
2 Normative references... 2
3 Terms and Definitions...2
4 Raw material requirements...3
5 Technical requirements...3
6 Application range...4
7 How to use...4
8 Transportation, storage and packaging...5
9 Labels, signs and instructions...5
10 Inspection method...5
Appendix A (Normative) Determination of Staphylococcus aureus Enzyme Activity...7
Appendix B (Normative Appendix) Determination of Lysozyme Enzyme Activity...9
Appendix C (informative) Preparation of Staphylococcus aureus Enzyme Source Substrate...11
Foreword
This part is drafted in accordance with the rules given in GB/T 1.1-2009.
This section drafted by. Shanghai Municipal Center for Disease Control and Prevention, China Center for Disease Control and Prevention, Environmental and Health-related Product Safety Institute,
Fudan University.
The main drafters of this section. Tian Yi, Zhu Renyi, Yuan Zhengan, Shen Wei, Shen Yi, Qiu Xia, Lu Yiying, Huang Qingshan, Li Guodong, Lu Yi,
Zhao Xiaowei, Huang Jinjiang, Wu Hongyu.
Hygienic requirements for lysostaphin and lysozyme disinfectants
1 Scope
This standard specifies the raw material requirements, technical requirements, application range, use methods, transportation and storage of lysostaphin and lysozyme disinfectants.
Storage and packaging, labeling and instructions, inspection methods.
This section applies to enzyme disinfectants with lysostaphin and/or lysozyme as the main bactericidal ingredients.
2 Normative references
The following documents are indispensable for the application of this document. For dated references, only the dated version applies to this document.
For undated references, the latest edition (including all amendments) applies to this document.
GB/T 191 packaging storage and transportation icon
GB 27950 Hand Sanitizer Hygienic Requirements
GB 27951 Skin Disinfectant Hygienic Requirements
GB 27954 mucosal disinfectant hygiene requirements
Pharmacopoeia of the People's Republic of China (2015 Edition)
Disinfection Technical Specification (2002 Edition) Ministry of Health (Wei Jian Jian Fa [2002] No. 282)
Disinfection product label specification management regulations Ministry of Health (Wei Supervision issued [2005] No. 426)
3 Terms and definitions
The following terms and definitions apply to this document.
3.1
Enzyme disinfectant
A disinfectant with enzyme as the main bactericidal ingredient.
3.2
Lysostaphin
An enzyme capable of hydrolyzing a glycine peptide bond in a bacterial cell wall peptidoglycan to cause bacteria to die.
3.3
Lysozyme lysozyme
An enzyme that hydrolyzes mucopolysaccharides in bacteria. Also known as cell wall enzyme, N-acetyl cell wall glycan hydrolase.
3.4
Enzyme activity unit
A unit of measure of enzyme activity. An enzyme activity unit refers to the conversion of a unit substrate (or transformation substrate) per unit time under specific conditions.
The amount of enzyme in the unit concerned group.
4 raw material requirements
4.1 lysostaphin
White or yellowish powder, odorless, soluble in water; calculated according to dry product, enzyme activity unit should be ≥100 U/mg.
4.2 Lysozyme
Meet the requirements of lysozyme in national drug standards.
4.3 Production water
Comply with the requirements of purified water in the Pharmacopoeia of the People's Republic of China (2015 Edition).
4.4 Other raw materials
Raw materials that are food grade or chemically pure or that meet the requirements of the Pharmacopoeia are in compliance with relevant national regulations and may not be used at the industrial level.
4.5 Prohibited substances
It should not contain antibiotics, antifungal drugs, hormones, and other prohibited substances prescribed by the state.
5 Technical requirements
5.1 Appearance
No delamination, no precipitation and suspended matter, no odor.
5.2 Physical and chemical indicators
5.2.1 pH
The pH is 5.0 to 8.0.
5.2.2 Effective content
The activity of the lysostaphin enzyme in the disinfectant ranges from 0.5 U/mL to 20 U/mL, and the activity range of lysozyme is 10000 U/mL~
200000 U/mL.
The active ingredient content of the product is within 80% to 150% of the set value or the indicated center value.
5.2.3 Stability
≥12 months.
5.3 Killing microbial indicators
The concentration and time of action shall be in accordance with the requirements of the product labeling instructions. The microbiological indicators shall comply with the requirements of Table 1.
For hand disinfection should meet the requirements of GB 27950; for skin disinfection should meet the requirements of GB 27951; for mucosal disinfection should be consistent
GB 27954 requirements.
Table 1 Killing microbial indicators
project
Killing the logarithm
Suspension carrier method
E. coli (8099) ≥5.00 ≥3.00
Staphylococcus aureus (ATCC 6538) ≥ 5.00 ≥ 3.00
Pseudomonas aeruginosa (ATCC 15442) ≥5.00 ≥3.00
Candida albicans (ATCC 10231) ≥4.00 ≥3.00
Natural bacteria (field test a) ≥1.00
a Used for hand disinfection hand-on-site test for skin or mucous membrane disinfection for skin field test.
6 Application range
Suitable for disinfection of hands, skin and mucous membranes.
Products that are based on product specifications and have experimental basis can add other applications.
7 How to use
7.1 hand disinfection
7.1.1 Sanitary hand disinfection
Take appropriate amount of disinfectant in the palm of your hand, hands rub each other to evenly coat each part, the action time meets the requirements of GB 27950.
7.1.2 Surgical hand disinfection
After surgical washing, apply proper amount of disinfectant to the skin of the lower third of the hands, forearms and upper arm. The action time is in accordance with GB 27950.
Claim.
7.2 Skin disinfection
7.2.1 Complete skin
Remove the poison stock solution, or use the disinfectant stock solution in purified water or sterile water that meets the Pharmacopoeia of the People's Republic of China (2015 Edition)
Release to the specified concentration of the instructions, evenly spray or wipe the skin surface with a medical cotton swab, the action time should meet the requirements of GB 27951.
7.2.2 Broken skin
Dispose of it from the original packaging and use it once. Remove the poison stock solution, or use the disinfectant stock solution in accordance with the Pharmacopoeia of the People's Republic of China (2015)
The sterilized water of the annual edition) is diluted to the specified concentration of the specification, and the surface of the damaged skin is washed, and the action time should meet the requirements of GB 27951.
7.3 mucosal disinfection
7.3.1 Oral mucosa
Dispose of it from the original packaging and use it once. Use disinfectant stock solution or use disinfectant stock solution in accordance with the Pharmacopoeia of the People's Republic of China (2015)
Dilute water or sterile water diluted to the specified concentration in the manual, evenly spray or wipe with medical cotton swab or sputum, the action time is
According to the instructions, the maximum duration of action is 5 min.
7.3.2 Vaginal and external genital mucosa
Dispose of it from the original packaging and use it once. Use disinfectant stock solution or use disinfectant stock solution in accordance with the Pharmacopoeia of the People's Republic of China (2015)
The purified water or sterile water of the annual edition) is diluted to the specified concentration in the manual, and wiped or irrigated or rinsed with a medical cotton swab.
Mingshu, the longest action time is 5 min.
8 Transportation, storage and packaging
8.1 Transportation
Avoid sun and rain when transporting products. Do not mix and transport with items that have odor or affect product quality.
8.2 Storage
Store at room temperature or in a cool place, protected from light, airtight, and dry. Do not store in the same place as odor or products that affect product quality.
8.3 Packaging
The packaging of the product is non-toxic and clean, and the packaging material meets the cosmetic packaging requirements of the corresponding materials. The product is sealed and sealed to ensure the product
Stability and safety during storage, transportation and use. The packaging and transportation pictorial signs shall comply with the requirements of GB ∕T l91.
9 Identification requirements
9.1 Labels and instructions
Comply with the "Management Specifications for Labeling Instructions for Disinfecting Products"
9.2 Precautions
The instructions include at least the following.
-- Avoid contact with antagonists. Cannot be used together with anionic surfactants, Ba2, Ca2, Mg2, etc.
- placed in a place where children are not easily accessible.
-- Use with caution for people who are allergic to protein.
10 Test methods
10.1 Appearance
The color and shape were observed with the naked eye, and the smell was identified by the sense of smell.
10.2 pH determination
Determined according to the method specified in the "Disinfection Technical Specifications (2002 Edition)".
10.3 Determination of effective content
10.3.1 Determination of lysostaphin activity
Determine according to the method specified in Appendix A.
10.3.2 Determination of lysozyme activity
Determine according to the method specified in Appendix B.
10.4 Stability determination
The stability test was carried out by the microbiological method according to the method of "Disinfection Technical Specification (2002 Edition)". Valid for < 24 months, with nature
The sample retention method is valid; the validity period is 24 months. According to the product performance, the method of storage at 37 °C for 90 days or the natural sample retention method is used.
During the period of validity, the active ingredient content shall not be lower than the lower limit of the labeled amount.
10.5 Killing microbial test
According to the relevant provisions of the "Disinfection Technical Specifications (2002 Edition). It is advisable to use the suspension method; if the carrier method is used, non-adsorption should be used.
Materials (metal, glass, pigskin, etc.) are used as carriers.
AA
Appendix A
(normative appendix)
Determination of lysostaphin enzyme activity
A.1 Principle
S. aureus cell wall peptidoglycan (KNR-PG) coupled with reactive brilliant blue dye KNR as a color source substrate, according to the action of the enzyme
The small molecule soluble fragment product with KNR dye group is quantitatively released during the process, and the supernatant is removed after removing the unreacted insoluble substrate.
The solution was subjected to colorimetric determination of lysostaphin enzymatic activity.
The absorbance of KNR-PG solution is increased every minute at a temperature of 37 ° C at a temperature of 37 ° C at a temperature of 595 nm at a pH of 10.0 in a 1.2 mL reaction system.
The amount of enzyme added with 0.35 is one unit of lysostaphin enzyme activity.
A.2 reagent
A.2.1 Staphylococcus aureus enzyme standard solution. The lysostaphin lyophilized powder is calibrated according to the lysostaphin enzyme activity unit definition.
If necessary, dissolve in Tris-HCl buffer solution, prepare 0.9 Sa/mL standard solution, store at -20 °C, use 1 test for each test to avoid
Repeated freezing and thawing.
A.2.2 Glycine. Analytically pure.
A.2.3 Sodium hydroxide. analytically pure.
A.2.4 Tris. Analytically pure.
A.2.5 95% ethanol. analytically pure.
A.2.6 Glycine-sodium hydroxide buffer. 0.2 mol/L, pH 10.0.
A.2.7 Tris-HCl buffer (Tris-HCl buffer). 0.05 mol/L, pH 7.5.
A.2.8 Color source substrate KNR-PG solution. Prepare the color source substrate KNR-PG according to the method of Appendix C, using glycine-sodium hydroxide buffer to 1.
5 mass to volume ratio is evenly suspended.
A.3 Test procedure
A.3.1 Preparation of standard curve
Take 6 clean and dry microcentrifuge tubes, label the corresponding amount of lysostaphin standard solution in each tube according to the order in Table A.1.
(A.2.1), add different amounts of 0.2 mol/L glycine-sodium hydroxide buffer (A.2.6). Then add the color source substrate KNR-PG by serial number.
Solution (A.2.8) 130 μL, quickly add to the vortex mixer after adding the substrate to each tube. Move the microcentrifuge tube with the solution quickly into the 37 °C
Timely reaction in a constant temperature water bath for 20 min. Remove the microcentrifuge tube from the water bath and add 300 μL of 95% ethanol to each tube to stop the reaction.
After centrifugation for 10 min at r/min, the supernatant was taken at 595 nm, and the absorbance was measured with a tube No. 0 as a blank. According to the measured absorbance
Value and corresponding standard lysostaphin enzymatic activity (U/mL), and obtain a linear regression curve.
C0=(AB)/K(A.1)
In the formula.
C0--Standard lysostaphin enzymatic activity (U/mL);
A--absorbance;
B--intercept;
K--The slope of the standard curve.
Table A.1 Standard curve preparation
Pipe number
Staphylococcus aureus enzyme standard solution/μL 0 20 40 60 80 100
Glycine-Sodium Hydroxide Buffer/μL 770 750 730 710 690 670
Substrate/μL 130 130 130 130 130 130
95% ethanol/μL 300 300 300 300 300 300
Staphylococcus aureus enzyme standard solution final concentration/U/mL 0 0.02 0.04 0.06 0.08 0.1
A.3.2 Determination of samples
The measurement method of the sample to be tested is the same as the standard curve, and 50 μL is taken for the measurement. The measurement was performed three times in parallel, and the average value was calculated.
A.4 Calculation of results
Calculate the activity of lysostaphin in unknown solution according to the standard curve, see the following formula.
C = C0 × V inverse/V sample × N (A. 2)
In the formula.
C--sample lysostaphin enzymatic activity (U/mL);
C0--Standard lysostaphin enzymatic activity (U/mL);
V anti-reaction system volume, 0.9 mL;
V sample - sample volume added, 0.05 mL;
N--sample dilution factor.
Appendix B
(normative appendix)
Lysozyme activity assay
B.1 Principle
Lysozyme dissolves bacteria by dissolving the cell wall of Gram-positive bacteria, and the absorbance of the bacterial liquid in the visible range is reduced.
As a substrate, the lysozyme activity was determined by spectrophotometry at a decrease in absorbance per unit time at a wavelength of 450 nm.
At room temperature 25 ° C, pH 6.2, at a wavelength of 450 nm, the absorbance decreased by 0.001 per minute to 1 lysozyme enzyme activity unit.
B.2 reagent
B.2.1 Phosphate buffer. Take 10.4 g of sodium dihydrogen phosphate and 7.86 g of disodium hydrogen phosphate and 0.37 g of disodium edetate, dissolved in water.
Adjust the pH to 6.2 to 1000 mL.
B.2.2 Micrococcus lysodeik, CGMCC 1.0634.
B.3 Test procedure
B.3.1 Preparation of sample solution
The sample was weighed and diluted to a solution of about 1000 U/mL with phosphate buffer, and the dilution was recorded as N.
B.3.2 Preparation of substrate suspension
Prepare before use. Weigh 15 mg to 20 mg of M. solani, add 0.5 mL to 1 mL of phosphate buffer, and grind in a mortar for 3 min.
Add an appropriate amount of phosphate buffer to a total volume of about 50 mL. The absorbance of the suspension at 25 ° C ± 0.1 ° C at 450 nm is
0.70 ± 0.05.
B.3.3 Sample determination
Measure 3 mL of substrate suspension at 25 °C ± 0.1 °C and set the absorbance at 450 nm in the cuvette as a reading of 0 s
A0, then measure 0.15 mL (equivalent to 7.5 μg of lysozyme) at 25 °C ± 0.1 °C, add to the cuvette, mix quickly, use
The stopwatch is timed, and the absorbance A is measured at 60 s. At the same time, the phosphate buffer solution is 0.15 mL, and the same method is used as a blank test.
0 s reading A'0 and 60 s reading A'.
The measurement was performed three times in parallel, and the average value was calculated.
B.4 Calculation of results
Calculate the lysozyme activity in an unknown solution, see the following formula.
C=
0.15
A'-A'-AA 00 )()(
×N×10
3 (B.1)
In the formula.
C--sample lysozyme activity (U/mL);
A0 - the absorbance at 0s of the sample;
A - the absorbance of the sample at 60 s;
A'0--the absorbance at blank 0s;
A' - the absorbance at 60s blank;
N -- dilution factor.
Appendix C
(normative appendix)
Preparation of Staphylococcus aureus enzyme source substrate
C.1 reagent
C.1.1 Trichloroacetic acid (TCA)
10% TCA. 10 g of trichloroacetic acid was dissolved in 100 mL of pure water.
5% TCA. Take 5 g of trichloroacetic acid and dissolve in 100 mL of pure water.
C.1.2 0.05mol/L Tris-HCl Tris-HCl
6.0 g of tris (hydroxymethyl) aminomethane was taken, dissolved in 800 mL of water, adjusted to pH 7.5 with hydrochloric acid, and diluted to 1000 mL with water.
C.1.3 0.25mol/L sodium hydroxide solution
Take 10.0 g of sodium hydroxide and dissolve it by adding 1000 mL of water.
C.1.4 Other reagents
Staphylococcus aureus, physiological saline, trypsin, brilliant blue dye (KNR dye), sodium azide, pure water.
C.2 Preparation method
C.2.1 Preparation of Staphylococcus aureus suspension in accordance with the relevant provisions of the "Disinfection Technical Specifications (2002 Edition)".
C.2.2 After sterilizing the bacterial suspension at 121 ° C for 20 min, centrifuge to collect the cells.
C.2.3 Precipitation of the cells was washed twice with physiological saline.
C.2.4 The cells were suspended with 7 times the bacterial mass of 10% TCA solution and placed at 4 ° C for 72 h.
C.2.5 Centrifuge and centrifuge the cells once with physiological saline.
C.2.6 The cells were suspended in a 5% TCA solution of 5 times the bacterial mass, and a water bath at 90 ° C for 10 min.
C.2.7 The precipitate was collected by centrifugation and washed by physiological saline 3 times.
C.2.8 The cells were suspended with 5 times bacterial mass of Tris-HCl, trypsin was added at a concentration of 0.5 mg/mL, and placed in a water bath at 37 ° C for 24 h.
C.2.9 The cells were inactivated by trypsin in a water bath at 90 ° C for 10 min.
C.2.10 The precipitate was collected by centrifugation and washed three times with pure water.
C.2.11 The cells were suspended in a small amount of pure water and dialyzed against pure water at 4 ° C for 24 h.
C.2.12 Centrifuge and collect the precipitate.
C.2.13 According to the 7-fold bacterial mass, prepare an appropriate amount of 0.25N sodium hydroxide solution.
C.2.14 The cells were suspended in half volume of the prepared 0.25 N sodium hydroxide solution.
C.2.15 KNR dye is weighed according to the cell mass of 1/7, and dissolved with the remaining sodium hydroxide solution.
C.2.16 Add the prepared dye to the suspended bacterial solution, and bath at 37 ° C for 24 h.
C.2.17 Centrifuge, wash with pure water until the supernatant is colorless, suspend the cells with a small amount of pure water, transfer to a storage container, and add at a concentration of 10 mg/L.
Add sodium azide and store at 4 °C.
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