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GB 4789.43-2016: National Food Safety Standard -- Food Microbiological Examination -- Determination of Antibacterial Activity of Microbe-source Enzyme
GB 4789.43-2016
GB
NATIONAL STANDARD OF
THE PEOPLE’S REPUBLIC OF CHINA
Food safety national standard - Food microbiological
examination - Microbial source enzyme preparation
antibacterial activity determination
ISSUED ON. DECEMBER 23, 2016
IMPLEMENTED ON. JUNE 23, 2017
Issued by. National Health and Family Planning Commission;
China Food and Drug Administration.
3. No action is required - Full-copy of this standard will be automatically and
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Table of Contents
1 Scope ... 3 
2 Equipment and material ... 3 
3 Medium and reagent ... 4 
4 Test strain ... 4 
5 Inspection procedures ... 4 
6 Operation steps ... 5 
7 Antimicrobial activity report ... 7 
Annex A Mediums and reagents ... 8 
Food safety national standard - Food microbiological
examination - Microbial source enzyme preparation
antibacterial activity determination
1 Scope
This Standard specifies the method for the determination of antibacterial
activity of microbial source enzyme preparation.
This Standard is applicable to the determination of antibacterial activity of
enzyme preparation produced by microorganism.
2 Equipment and material
In addition to microbial laboratory routine sterilization and training equipment,
other equipment and materials are as follows.
2.1 biological safety cabinet.
2.2 refrigerator. 2°C ~ 5°C.
2.3 constant temperature incubator. 36°C ± 1°C.
2.4 constant temperature water bath. 46°C ± 1°C.
2.5 balance. resolution of 0.1 g.
2.6 oscillator.
2.7 sterile pipettes. 1 mL (with 0.01 mL scale), 10 mL (with 0.1 mL scale) or
micro pipette and suction head.
2.8 sterile Petri dish. 90mm in diameter.
2.9 sterile Erlenmeyer flask. capacity of 250 mL, 500 mL.
2.10 pH meter or precision pH test paper.
2.11 sterile paper. see A.8.
2.12 sterile tweezers.
6.2 Determination of concentration of bacteria suspension solution
6.2.1 Dilution of bacteria suspension stock solution
Respectively take the bacteria suspension stock solution prepared in 6.1 to
carry out ten times gradient dilution in order. Bacillus cereus and bacillus
circulans shall be respectively prepared to 10-4 dilution solution.
Staphylococcus aureus, escherichia coli, streptococcus pyogenes, serratia
marcescens shall be respectively prepared to 10-5 dilution solution.
6.2.2 Culture count
Respectively pipet 1 mL of each bacteria dilution solution prepared in 6.2.1
into sterile plate. Each dilution shall be done in two parallel. Use sterile saline
for blank control. Pour 15mL ~ 20mL of plate count agar that have been
cooled to around 46°C (can be placed at a 46°C ± 1°C constant temperature
water bath box for insulation) into the plate. Rotate the plate to mix evenly.
After agar solidification, turn over the plate. Cultivate at 36°C ± 1°C for 24h
then count. Use when the bacterial concentration of each suspension stock
solution is more than 106 CFU/mL.
6.3 Preparation of inspection plate
Pour 15 mL of TSA medium that is melted and cooled to 46°C ± 1°C into
sterile petri dish. Make it TSA plate after it is solidified, for use. Respectively
use TSA medium that is cooled to 46°C ± 1°C to dilute each culture after 6.1
second subculture according to a 1.10 ratio (streptococcus pyogenes ATCC
12344 shall be diluted according to 1.20). After well mixing, respectively take
10 mL into the TSA plate that is well prepared and insulated at 46°C ± 1°C.
Gently shake the plate to make bacteria solution evenly paved. Make it
inspection plate after solidification, for use.
NOTE To prevent bacterial culture medium is solidified when it immediately meets TSA
and cause uneven bacteria suspension solution pavement, it shall place the TSA plate at
46°C ± 1°C for 10 min insulation before bacteria suspension solution pavement.
6.4 Preparation of enzyme preparation paper
Accurately weigh (pipet) 1.0 g (mL) of enzyme preparation into 9 mL of sterile
saline. After even mixing, prepare it to 10% enzyme preparation solution.
Place the sterile paper in a sterile plate. Slowly add 100 µL of 10% enzyme
preparation solution on each piece of paper to make it completely absorbed.
Prepare 12 pieces of paper for each enzyme preparation sample.
6.5 Sticker and culture
A.4.1 Compositions
HCl 9 mL
Distilled water 991 mL
A.4.2 Preparation method
Pipet 9 mL of concentrated hydrochloric acid (mass concentration is 36%,
density is 1.17 g/m3). Use sterile distilled water to dilute to 1000 mL. Use 0.22
μm microporous membrane to filter and sterilize for use.
A.5 Sterile saline
A.5.1 Compositions
Sodium chloride 8.5 g
Distilled water 1000 mL
A.5.2 Preparation method
Pipet 8.5 g of Sodium chloride and dissolve into 1000 mL of distilled water.
Sterilize at 121°C for 15 min.
A.6 50.0 μg/mL ciprofloxacin (CIP)
A.6.1 Compositions
1000 μg/mL ciprofloxacin 0.5 mL
Sterile saline 9.5 mL
A.6.2 Preparation method
Weigh a certain mass of ciprofloxacin according to the product instruction
(percentage, water content or potency). Use an appropriate amount of 0.1
mol/L HCl to dissolve to make its concentration 1000 μg/mL. Under aseptic
conditions, use 0.22 μm microporous membrane to filter and pipet 0.5 mL.
Use sterile saline to dilute to 10 mL. Mix well.
A.7 5.0 μg/tablet ciprofloxacin paper (CIP paper)
A.7.1 Compositions
50.0 μg/mL ciprofloxacin 100 μL
Sterile paper 1 piece
A.7.2 Preparation method
Pipet 100 μL of 50.0 μg/mL ciprofloxacin solution. Slowly and evenly drop
onto sterile paper to make 5.0 μg/tablet ciprofloxacin paper.
A.8 Sterile paper