GB/T 39101-2020.Determination of antimicrobial activity for polypeptides-Inhibition zone method.
1 Scope
GB/T 39101 specifies the method for determining the inhibition zone of antimicrobial activity of polypeptides.
GB/T 39101 is applicable to the determination of the antibacterial and anti-filamentous fungi properties of polypeptides.
2 Normative references
The following documents are indispensable for the application of this document. For any dated reference, only the dated version applies to this document. For any undated reference, the latest version (including all amendments) applies to this document.
GB 4789.2 National food safety standard Determination of total bacterial count for food microbiological examination
GB/T 6682 Specifications and test methods for water for analytical laboratories
3 Terms and definitions
The following terms and definitions apply to this document.
3.1
Polypeptides
A class of compounds between amino acids and proteins, formed by two or more amino acids connected by peptide bonds.
3.2
Inhibition zone
A zone of sterile growth at the contact boundary between the surface of the solid culture medium and the sample.
3.3
Antibacterial activity
The ability to inhibit the growth of microorganisms, expressed as the antibacterial titer U value (AU).
4 Principle
When the polypeptide contacts the test indicator bacteria in the solid plate, the growth of the surrounding strains is inhibited to form a sterile growth zone. The activity is determined by calculating the antibacterial titer U value based on the diameter of the inhibition zone shown by the test bacteria on the surface of the solid plate.
5 Reagents or materials
Unless otherwise specified, the reagents used in this method are analytically pure, and the experimental water is the first-grade water specified in GB/T 6682.
5.1 Physiological saline
0.85% physiological saline: weigh 8.5 g sodium chloride into a volumetric flask, dissolve it with first-grade water and make up to 1 000 mL. Sterilize by high pressure steam at 121 °C±1 °C for 20 min.
5.2 Culture medium
Nutrient broth medium (NB), nutrient agar medium (NA), potato-dextrose agar medium (PDA), preparation see Appendix A.
This standard specifies the method for determining the inhibition zone of antibacterial properties of polypeptides. This standard is applicable to the determination of the antibacterial and anti-filamentous fungi properties of polypeptides.

ICS07.080
National Standard of the People's Republic of China
GB/T39101—2020
Determination of antimicrobial activity for polypeptides-Inhibition zone methodIssued on 2020-09-29
State Administration for Market Regulation
National Administration of Standardization
Issued
Implementation on 2021-04-01
Foreword
This standard was drafted in accordance with the rules given in GB/T1.1-2009. This standard was proposed and managed by the National Technical Committee for Standardization of Biochemical Testing (SAC/TC387). GB/T39101—2020
The drafting units of this standard are: Beijing Technology and Business University, Hebei University of Science and Technology, Shenzhen Institute of Metrology and Quality Inspection, Hebei Agricultural University, Beijing Sambo Technology Co., Ltd., Wuhan Mingliao Biotechnology Co., Ltd., Zhejiang Dongjie Biotechnology Co., Ltd., Food Evaluation Center of the State Administration for Market Regulation, and Biological Research Institute of China Testing Technology Research Institute. The main drafters of this standard are: Jia Yingmin, Ma Aijin, Gan Zhixin, Tian Yiling, Hao Shuai, Peng Hai, Zhou Lihua, Yang Zhijian, Zheng Yanyan, Zheng Gang, Chen Jing, Jiang Yu, and Yang Guowu.
1 Scope
Determination of antibacterial activity of polypeptides
This standard specifies the method for determining the antibacterial zone of antibacterial activity of polypeptides. Inhibition zone method
This standard is applicable to the determination of the antibacterial and anti-filamentous fungi properties of dry polypeptides. Normative references
GB/T39101—2020
The following documents are indispensable for the application of this document. For any dated reference, only the dated version applies to this document. For any undated reference, the latest version (including all amendments) applies to this document. GB4789.2 National Food Safety Standard Food Microbiology Test Determination of Total Colony Count GB/T6682 Specifications and Test Methods for Water in Analytical Laboratories 3 Terms and Definitions
The following terms and definitions apply to this document. 3.1
Polypeptides
polypeptides
A class of compounds between amino acids and proteins, formed by two or more amino acids connected by peptide bonds. 3.2
Inhibition zone
The ring zone of sterile growth at the boundary between the surface of the solid culture medium and the sample 3.3
antimicrobial activity
Antibacterial activity
The ability to inhibit the growth of microorganisms, expressed as the antibacterial potency U value (AU). Principle
After the polypeptide contacts the test indicator bacteria in the solid plate, the growth of the surrounding strains is inhibited to form a sterile reproduction area. According to the diameter of the inhibition zone shown by the test bacteria on the surface of the solid plate, the antibacterial titer U value is calculated to determine the activity. Reagents or materials
Unless otherwise specified, the reagents used in this method are analytically pure, and the experimental water is the first-class water specified in GB/T6682. Physiological saline
0.85% physiological saline: weigh 8.5g sodium chloride in a volumetric flask, dissolve it with first-class water and make up to 1000mL. High steam sterilization at 121℃±1℃ for 20min.
GB/T39101—2020
Culture medium
Nutrient broth medium (NB), nutrient agar medium (NA), potato-dextrose agar medium (PDA), preparation see Appendix A.
5.3 Standard strains of indicator bacteria
Gram-positive bacteria: Staphylococcus aureus CICC10473 (CGMCC1.8721, ATCC29213), Micrococcus luteus CICC10269 (CGMCC1.3749, ATCC10240), Bacillus cereus CICC10277 (CICC10468, ATCC11778). Gram-negative bacteria: Escherichia coli CICC10305 (CICC23657, CGMCC1.2385, ATCC25922), Salmonella typhimurium CICC22956 (ATCC14028), Pseudomonas aeruginosa CICC10351 (CICC23694CGMCC1.2421, ATCC9027). Filamentous fungi: Aspergillus niger CICC2463 (CGMCC3.5487, CICC40372, ATCC16404), Penicillium chrysogenum CICC40654 (CGMCC3.7894, ATCC10106), Penicillium citrinum CICC2478 (ATCC9849). 6 Instruments and equipment
Constant temperature incubator: temperature accuracy 1°C.
Electronic balance: sensitivity 0.0001g and 0.01g. 6.3 Spectrophotometer: wavelength range 190nm~900nm. 6.4
Vernier caliper or inhibition zone measuring instrument: accuracy 0.02mm~0.1mm. 6.5 Blood cell counting plate: 16 grids × 25 grids or 25 grids × 16 grids. 6.6
Glass flat cup: diameter 90mm, flat bottom. 6.7
Stainless steel Oxford cup: inner diameter 6.0mm±0.1mm, outer diameter 7.8mm±0.1mm, height 10.0mm±0.1mm7 Test steps
Determination of antibacterial performance
7.1.1
Sample preparation
Weigh 10.0mg of polypeptide sample, dissolve and dilute the sample with good water solubility in sterile buffer, dissolve the sample with poor water solubility in anhydrous ethanol and dilute to 1.0mL with sterile buffer to make the concentration 10.0mg/mL, as the stock solution, store in a refrigerator at 4℃～6℃, and use within one week. When used, the polypeptide stock solution is diluted with sterile buffer to prepare at least 5 test solutions of different concentrations. The concentration should be selected so that the diameter of the inhibition zone is 8.00mm~25.00mm and the linear equation R2≥0.9900. 7.1.2 Preparation of indicator bacteria suspension
7.1.2.1 Activation of bacteria
Inoculate the indicator bacteria in a test tube of 5.0mL NB culture medium, and culture at 36℃±1℃, 200r/min±1r/min for 18h~24h for the first subculture; take the first generation culture solution and inoculate it in a test tube of 5.0mL NB culture medium, and culture at 36℃±1℃, 200r/min±1r/min for 18h~24h for the second subculture; take the second generation culture solution and inoculate it in a test tube of 5.0mL NB culture medium or a conical flask of 50.0mL NB culture medium, and culture at 36℃±1℃, 200r/min±1r/min until the stable period of the indicator bacteria, as the third generation culture solution. 2
7.1.2.2 Preparation of bacterial suspension
GB/T39101—2020
Pre-count the colonies of indicator bacteria according to GB4789.2, and establish the corresponding relationship between the number of colonies and the absorbance value (ODoo); adjust the third-generation culture fluid to an appropriate absorbance value so that the concentration of the bacterial suspension is 1×10°CFU/mL5×10°CFU/mL. 7.1.3 Preparation of test plates
Heat and dissolve the prepared NA culture medium, cool to 45℃～50℃, add the prepared indicator bacteria suspension to the NA culture medium at a rate of 1%, mix well, measure 20mL and pour it into a sterile plate, gently shake the plate to make it evenly spread, and wait for it to solidify before use.
7.1.4 Determination
Place 5 to 6 Oxford cups at an equal distance on the test plate containing the indicator bacteria, press gently, measure 100μL of the polypeptide sample, and slowly add it to the Oxford cup. Repeat each treatment 3 times. Move the plate to a 4℃~6℃ refrigerator for pre-diffusion for 4h~10h, take out the plate, put it in a 36℃±1℃ constant temperature incubator, and culture it upright until the inhibition zone is clear. Use a vernier caliper or an inhibition zone measuring instrument to measure the diameter of the inhibition zone. Measure each inhibition zone 3 times in different directions and record the average value. Use the logarithm of the reciprocal of the polypeptide dilution multiple as the horizontal axis and the diameter of the inhibition zone as the vertical axis to establish a linear equation (R\≥0.9900). For the linear relationship diagram of bacitracin against S. aureus ATCC25923, see Figure B.1 in Appendix B. 7.2 Determination of anti-filamentous fungi performance
7.2.1 Sample preparation
Same as 7.1.1.
7.2.2 Preparation of indicator bacteria spore suspension
7.2.2.1 Activation of bacteria
Inoculate the indicator bacteria or bacterial suspension on a solid plate of PDA medium and culture it at a constant temperature of 29℃±1℃ for 48h~72h as the first generation spore culture; take the first generation spore culture and inoculate it on a solid plate of PDA medium and culture it at a constant temperature of 29℃±1℃ for 48h~72h as the second generation spore culture; take the second generation spore culture and inoculate it on a solid plate of PDA medium and culture it at a constant temperature of 29℃±1℃ for 48h~72h as the third generation spore culture. 7.2.2.2 Preparation of spore suspension
Elute the spores from the third generation spore culture with 0.85% saline, count them with a hemocytometer, and adjust the spore concentration to 1×10°CFU/mL~5×10°CFU/mL.
7.2.3 Preparation of test plate
Heat the prepared PDA medium to dissolve, cool to 45℃~50℃, add the prepared indicator bacteria spore suspension to the PDA medium at a rate of 1%, mix thoroughly, measure 20mL and pour it into a sterile plate, gently shake the plate to make it evenly flat, and wait for it to solidify before use.
7.2.4 Determination
Place 5~6 Oxford cups at an equal distance on the test plate containing the indicator bacteria, press gently, measure 100μL of the polypeptide sample, and slowly add it to the Oxford cup. Repeat each treatment 3 times. Move the plate to a 4℃～6℃ refrigerator for pre-diffusion for 4h～10h, take out the plate, put it in a constant temperature incubator at 29℃±1℃, and culture it upright for 18h～24h until the inhibition zone is clear. At this time, the indicator bacteria is in the form of moss, and basically no bacteria 3
GB/T39101—2020
filaments have been formed; use a vernier caliper or an inhibition zone measuring instrument to measure the diameter of the inhibition zone. Each inhibition zone is measured 3 times in different directions, and the average value is recorded. Use the logarithm of the reciprocal of the peptide dilution multiple as the horizontal axis and the diameter of the inhibition zone as the vertical axis to establish a linear equation (R2≥0.9900). See Figure B.2 for the linear relationship diagram of polymyxin B sulfate against A. niger ATCC16404. 8
Test data processing
The antibacterial activity is calculated according to formula (1):
V×10x×c
Wherein:
U——antibacterial potency, unit is AU per milligram (AU/mg); V—the measured volume of the sample, unit is milliliter (mL); X. —the X-axis intercept of the linear equation;
the initial measured concentration of the sample, unit is milligram per milliliter (mg/mL). The calculation result is expressed as the arithmetic mean of 3 parallel determination values, retaining two significant figures. 4
A.1 Nutrient Broth (NB) Appendix A
(Informative Appendix)
Culture Medium
GB/T39101—2020
A.1.1 Put 10g of egg white, 3g of beef extract and 5g of sodium chloride into 1000mL of first-grade water, boil and dissolve, adjust the pH value to 7.2~7.4, and sterilize in a high-pressure steam sterilizer at 121℃±1℃ for 20min after packaging. 2 Commercial culture media available on the market can be used, and the manufacturer's instructions for use shall be strictly followed when using. A.1.2
A.2 Nutrient agar medium (NA) A.2.1 Put 10g egg white, 3g beef extract, 5g sodium chloride and 15g agar into 1000mL first-grade water, boil to dissolve, adjust pH to 7.2-7.4, and sterilize in a high-pressure steam sterilizer at 121℃±1℃ for 20min. Commercial culture media available on the market can be used, and the manufacturer's instructions are strictly followed when using. A.2.2
3 Potato-Dextrose Agar (PDA) A.3
A.3.1 Put 200g peeled and diced potatoes into 1000mL first-grade water, boil for 10min-20min, filter with gauze, and add first-grade water to 1000mL. Add 20g glucose and 15g agar, heat and dissolve, adjust the pH to 5.4-5.8, and sterilize at 115℃±1℃ for 20 min.
A.3.2
Commercial culture media available on the market can be used. SAG
GB/T39101—2020
Determination of antibacterial performance
Appendix B
(Informative Appendix)
Linear relationship diagram of antibacterial performance determination
Bacillus against S. aureus
Bacillus was diluted in five times gradients, with a concentration range of 0.0625mg/mL to 2.0mg/mL. 2The linear relationship diagram of ATCC25923 is shown in Figure B.1. 26
·Test 1
Test 2
Test 3
-1.6-1.4 -1.2-1.00.80.6-0.4 -0.20.00.2Ig(1/n)）
Linear relationship diagram of bacitracin against s.aureusATCc25923 Figure B.1
2Determination of anti-filamentous fungi performance
Polymyxin B sulfate was diluted twice in a gradient manner to 5 concentrations, with a concentration range of 0.125mg/mL~4.0mg/mL. The linear relationship diagram of polymyxin B sulfate against A. niger ATCC16404 is shown in Figure B.2. 22
·Experiment
·Experiment 2
Experiment 3
1.6-1.4-1.2-1.00.80.60.40.20.0°0.2lg(1/n)
Figure B.2
Linear relationship diagram of polymyxin B sulfate against A. niger ATCC164042.8
Test data processing
Antibacterial activity was calculated according to formula (1):
V×10x×cWww.bzxZ.net
Wherein:
U——antibacterial potency, unit is AU per milligram (AU/mg); V—measurement volume of the sample, unit is milliliter (mL); X. —X-axis intercept of the linear equation;
initial measurement concentration of the sample, unit is milligram per milliliter (mg/mL). The calculation result is expressed as the arithmetic mean of 3 parallel determination values, retaining two significant figures. 4
A.1 Nutrient Broth (NB) Appendix A
(Informative Appendix)
Culture Medium
GB/T39101—2020
A.1.1 Put 10g of egg white, 3g of beef extract and 5g of sodium chloride into 1000mL of first-grade water, boil and dissolve, adjust the pH value to 7.2~7.4, and sterilize in a high-pressure steam sterilizer at 121℃±1℃ for 20min. 2 Commercial culture media available on the market can be used, and the manufacturer's instructions for use shall be strictly followed when using. A.1.2
A.2 Nutrient agar (NA) A.2.1 Put 10g egg white, 3g beef extract, 5g sodium chloride and 15g agar into 1000mL first-grade water, boil to dissolve, adjust pH to 7.2-7.4, and sterilize in a high-pressure steam sterilizer at 121℃±1℃ for 20min. Commercial culture media available on the market can be used, and the manufacturer's instructions are strictly followed when using. A.2.2
3 Potato-Dextrose Agar (PDA) A.3
A.3.1 Put 200g peeled and diced potatoes into 1000mL first-grade water, boil for 10min-20min, filter with gauze, and add first-grade water to 1000mL. Add 20g glucose and 15g agar, heat and dissolve, adjust the pH to 5.4-5.8, and sterilize at 115℃±1℃ for 20 min.
A.3.2
Commercial culture media available on the market can be used. SAG
GB/T39101—2020
Determination of antibacterial performance
Appendix B
(Informative Appendix)
Linear relationship diagram of antibacterial performance determination
Bacillus against S. aureus
Bacillus was diluted in five concentrations with a concentration range of 0.0625mg/mL to 2.0mg/mL. 2The linear relationship diagram of ATCC25923 is shown in Figure B.1. 26
·Test 1
Test 2
Test 3
-1.6-1.4 -1.2-1.00.80.6-0.4 -0.20.00.2Ig(1/n)）
Linear relationship diagram of bacitracin against s.aureusATCc25923 Figure B.1
2Determination of anti-filamentous fungi performance
Polymyxin B sulfate was diluted twice in a gradient manner to 5 concentrations, with a concentration range of 0.125mg/mL~4.0mg/mL. The linear relationship diagram of polymyxin B sulfate against A. niger ATCC16404 is shown in Figure B.2. 22
·Experiment
·Experiment 2
Experiment 3
1.6-1.4-1.2-1.00.80.60.40.20.0°0.2lg(1/n)
Figure B.2
Linear relationship diagram of polymyxin B sulfate against A. niger ATCC164042.8
Test data processing
Antibacterial activity was calculated according to formula (1):
V×10x×c
Wherein:
U——antibacterial potency, unit is AU per milligram (AU/mg); V—measurement volume of the sample, unit is milliliter (mL); X. —X-axis intercept of the linear equation;
initial measurement concentration of the sample, unit is milligram per milliliter (mg/mL). The calculation result is expressed as the arithmetic mean of 3 parallel determination values, retaining two significant figures. 4
A.1 Nutrient Broth (NB) Appendix A
(Informative Appendix)
Culture Medium
GB/T39101—2020
A.1.1 Put 10g of egg white, 3g of beef extract and 5g of sodium chloride into 1000mL of first-grade water, boil and dissolve, adjust the pH value to 7.2~7.4, and sterilize in a high-pressure steam sterilizer at 121℃±1℃ for 20min. 2 Commercial culture media available on the market can be used, and the manufacturer's instructions for use shall be strictly followed when using. A.1.2
A.2 Nutrient agar (NA) A.2.1 Put 10g egg white, 3g beef extract, 5g sodium chloride and 15g agar into 1000mL first-grade water, boil to dissolve, adjust pH to 7.2-7.4, and sterilize in a high-pressure steam sterilizer at 121℃±1℃ for 20min. Commercial culture media available on the market can be used, and the manufacturer's instructions are strictly followed when using. A.2.2
3 Potato-Dextrose Agar (PDA) A.3
A.3.1 Put 200g peeled and diced potatoes into 1000mL first-grade water, boil for 10min-20min, filter with gauze, and add first-grade water to 1000mL. Add 20g glucose and 15g agar, heat and dissolve, adjust the pH to 5.4-5.8, and sterilize at 115℃±1℃ for 20 min.
A.3.2
Commercial culture media available on the market can be used. SAG
GB/T39101—2020
Determination of antibacterial performance
Appendix B
(Informative Appendix)
Linear relationship diagram of antibacterial performance determination
Bacillus against S. aureus
Bacillus was diluted in five concentrations with a concentration range of 0.0625mg/mL to 2.0mg/mL. 2The linear relationship diagram of ATCC25923 is shown in Figure B.1. 26
·Test 1
Test 2
Test 3
-1.6-1.4 -1.2-1.00.80.6-0.4 -0.20.00.2Ig(1/n)）
Linear relationship diagram of bacitracin against s.aureusATCc25923 Figure B.1
2Determination of anti-filamentous fungi performance
Polymyxin B sulfate was diluted twice in a gradient manner to 5 concentrations, with a concentration range of 0.125mg/mL~4.0mg/mL. The linear relationship diagram of polymyxin B sulfate against A. niger ATCC16404 is shown in Figure B.2. 22
·Experiment
·Experiment 2
Experiment 3
1.6-1.4-1.2-1.00.80.60.40.20.0°0.2lg(1/n)
Figure B.2
Linear relationship diagram of polymyxin B sulfate against A. niger ATCC16404
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