GB 4789.34-2016 PDF in English
GB 4789.34-2016 (GB4789.34-2016) PDF English
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| GB 4789.34-2016 | English | 85 |
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National Food Safety Standard -- Food Microbiological Examination -- Examination of Bifidobacterium
| Valid |
| GB 4789.34-2012 | English | 519 |
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National food safety standards -- Microbiological examination of food -- Identification of bifidobacteria
| Obsolete |
| GB/T 4789.34-2008 | English | 479 |
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Microbiological examination of food hygiene -- Examination of bifidobacterium
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| GB/T 4789.34-2003 | English | 399 |
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Microbiological examination of food hygiene -- Examination of Bifidobacterium
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Standards related to (historical): GB 4789.34-2016
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GB 4789.34-2016: PDF in English GB 4789.34-2016
GB
NATIONAL STANDARD OF
THE PEOPLE’S REPUBLIC OF CHINA
National Food Safety Standard - Food Microbiological
Examination - Examination of Bifidobacterium
ISSUED ON. DECEMBER 23, 2016
IMPLEMENTED ON. JUNE 23, 2017
Issued by. National Health and Family Planning Commission of the
People 's Republic of China;
China Food and Drug Administration.
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Table of Contents
Foreword ... 3
1 Scope ... 4
2 Equipment and materials ... 4
3 Medium and reagent ... 4
4 Inspection procedures ... 5
5 Operation steps ... 6
Annex A Medium and reagent ... 12
Annex B Detection of organic acid metabolites of Bifidobacterium ... 14
Foreword
This Standard replaces GB 4789.34-2012 National Food Safety Standard -
Food Microbiological Examination - Identification of Bifidobacterium.
Compared with GB 4789.34-2012, the main changes in this Standard are as
follows.
- added the counting method for Bifidobacterium;
- added MRS medium;
- modified the application scope of this Standard;
- modified Annex B as optional.
National Food Safety Standard - Food Microbiological
Examination - Examination of Bifidobacterium
1 Scope
This Standard specifies the identification and counting method for
Bifidobacterium.
This Standard applies to the identification and counting of pure bacteria strain
of Bifidobacterium. This Standard applies to the identification of bacteria
when the food only contains single Bifidobacterium. This Standard applies to
the counting when the food only contains Bifidobacterium, i.e., the food may
contain one or more different Bifidobacterium species.
2 Equipment and materials
In addition to microbial laboratory routine sterilization and training equipment,
other equipment and materials are as follows.
2.1 Constant temperature incubator. 36°C ± 1°C.
2.2 Refrigerator. 2°C ~ 5°C.
2.3 Balance. resolution of 0.01 g.
2.4 Sterile test tube. 18mm × 180mm, 15mm × 100mm.
2.5 Sterile pipettes. 1mL (with 0.01mL scale), 10mL (with 0.1mL scale) or
micro-pipettes (200μL ~ 1000μL) and matching tips.
2.6 Sterile petri dish. 90 mm in diameter.
3 Medium and reagent
3.1 Bifidobacterium culture medium. see A.1.
3.2 PYG medium. see A.2.
3.3 MRS medium. see A.3.
3.4 Methanol. analytically pure.
5 Operation steps
5.1 Sterile requirements
All operating procedures shall follow the sterile procedures.
5.2 Identification of Bifidobacterium
5.2.1 Pure bacteria species
5.2.1.1 Sample processing. semi-solid or liquid species are directly
inoculated on Bifidobacterium agar plates or MRS agar plates. For solid
bacteria or vacuum freeze-dried bacteria, it shall add an appropriate amount
of sterilized saline or other suitable diluent to dissolve the bacteria powder.
5.2.1.2 Vaccination. inoculate on Bifidobacterium agar plates or MRS agar
plates. Perform anaerobic culture at 36°C ± 1°C for 48h ± 2h that can be
extended to 72h ± 2h.
5.2.2 Food sample
5.2.2.1 Sample processing. take 25.0g (mL) of sample into a sterile conical
flask or homogeneous bag containing 225.0mL of physiological saline,
homogenizing at 8000r/min ~ 10000r/min for 1min ~ 2min or using slapping
homogenizer to beat 1min ~ 2min to make 1.10 sample homogenizing
solution. The frozen sample may be thawed at 2°C ~ 5°C for not more than
18h or at a temperature not greater than 45°C for not more than 15min.
5.2.2.2 Inoculation or coating. inoculate the above sample homogenizing
solution on a Bifidobacterium agar plate or MRS agar plate OR take 0.1mL of
sample homogenizing solution of appropriate dilution to evenly coat on a
Bifidobacterium agar plate or MRS agar plate. Perform anaerobic culture at
36°C ± 1°C for 48h ± 2h that can be extended to 72h ± 2h.
5.2.2.3 Pure culture. pick up three or more individual colonies to inoculate
on Bifidobacterium agar plates or MRS agar plates. Perform anaerobic
culture at 36°C ± 1°C for 48h ± 2h that can be extended to 72h ± 2h.
5.2.3 Identification of bacteria
5.2.3.1 Smear microscopy. pick up Bifidobacterium single colony growing in
Bifidobacterium plate or MRS plate for dyeing. Bifidobacterium is
Gram-positive, short rod-shaped, slender rod-shaped or spherical, can form a
variety of branches or bifurcation and other forms, non-acid, no spores, no
power.
5.2.3.2 Biochemical identification. pick up Bifidobacterium single colony
5.3.1.2 Preparation of liquid sample. weigh aseptically 1.0mL of sample;
place in 9.0mL of diluent; well mix to make 1.10 sample homogenizing
solution.
5.3.2 Food sample
5.3.2.1 Sample processing. take 25.0g (mL) of sample; place in a sterile
conical flask or homogeneous bag containing 225.0mL of physiological saline,
homogenizing at 8000r/min ~ 10000r/min for 1min ~ 2min, or using slapping
homogenizer to beat 1min ~ 2min to make 1.10 sample homogenizing
solution. The frozen sample may be thawed at 2°C ~ 5°C for not more than
18h or at 45°C for not more than 15min.
5.3.3 Series dilution and culture
Use a 1mL sterile pipet or micro pipette to prepare 10 times serial sample
homogenizing solution, homogenizing at 8000r/min ~ 10000r/min for 1min ~
2min, or using slapping homogenizer to beat 1min ~ 2min. Dilute once for
every increment, i.e., switch a 1mL sterile pipet or pipet head once. According
to the estimation of the sample concentration, select two to three sample
homogenizing solution of appropriate dilution. When performing 10 times
increment dilution, pipette 1.0mL of sample in the sterile plate. Do two plates
for each dilution. Meanwhile, respectively pipette 1.0mL of blank diluent into
two sterile plates for blank control. Timely pour 15mL ~ 20mL of
Bifidobacterium agar medium cooled to 46°C or MRS agar medium (can be
placed in 46°C ± 1°C constant temperature water bath for insulation) into the
plate, rotate the plate to make it evenly mixed. From sample dilution to plate
pouring, it requires 15 min. After agar solidification, flip the plate, perform
anaerobic culture at 36°C ± 1°C for 48h ± 2h that can be extended to 72h ± 2h.
Count the number of colonies on the plate after incubation.
5.3.4 Colony counting
5.3.4.1 It can be observed with the naked eye, if necessary, with a
magnifying glass or colony counter. Record the dilution factor and the
corresponding number of colonies. The colony counting is expressed in
colony-forming units (CFU).
5.3.4.2 Select the total number of colony colonies counted between 30CFU
and 300CFU, and the total number of colonies without flake colonies. Record
the number of specific colonies on plates below 30CFU. When it exceeds
300CFU, it can be recorded as countless. The number of colonies per dilution
shall be the average of two plates.
5.3.4.3 If one of the plates is growing with large flake colonies, it shall not be
used. It shall use the plate without flake colonies as the number of colonies of
this dilution. If the number of flake colonies is less than half of the plate, and
Annex A
Medium and reagent
A.1 Bifidobacterium agar medium
A.1.1 Ingredients
Peptone 15.0 g
Yeast extract 20.0 g
Glucose 20.0 g
Soluble starch 0.5 g
Sodium chloride 5.0 g
Tomato extract 400.0 mL
Tween 80 1.0 mL
Liver powder 0.3 g
Agar powder 20.0 g
Adding distilled water to 1000.0 mL
A.1.2 Preparation
A.1.2.1 Preparation of cysteine solution. weigh 0.5 g of cysteine solution,
add into 1.0 mL of hydrochloric acid to make cysteine all dissolved and
prepare cysteine salt solution.
A.1.2.2 Preparation of tomato extract. wash the fresh tomatoes and scrape
them; add the same amount of distilled water and heat in a 100°C water bath,
stir for 90 min, then filter with gauze, calibrate to pH 7.0 ± 0.1; after
sub-packaging the leaching solution, autoclave at 121°C for 15 min ~ 20min.
A.1.2.3 Preparation. add all components of A.1.1 into distilled water,
dissolve by heating, and then add into the cysteine solution, calibrate to pH
6.8 ± 0.1; after sub-packaging the leaching solution, autoclave at 121°C for
15 min ~ 20min.
A.2 PYG liquid medium
A.2.1 Composition
Peptone 10.0 g
Glucose 2.5 g
Yeast 5.0 g
Cysteine-HCl 0.25 g
Salt solution 20.0 mL
Vitamin K1 solution 0.5 mL
Hemoglobin solution 5 mg/mL 2.5 mL
Adding distilled water to 500.0 mL
Annex B
Detection of organic acid metabolites of Bifidobacterium
B.1 Preparation of Bifidobacterium culture medium
Inoculate the Bifidobacterium cultured on Bifidobacterium agar plate or MRS
agar plate into PYG liquid medium. Then use non-inoculated PYG liquid
medium for blank control, anaerobic, culture at 36°C ± 1°C for 48h.
B.2 Preparation of standard solution
B.2.1 Acetic acid standard solution. accurately pipette 5.7 mL of pure acetic
acid, add water and dilute to 100.0 mL, shake and calibrate, prepare about
1.0 mol/L acetic acid standard solution. Calibration method. accurately weigh
3.0 g of acetic acid, add 15.0 mL of water, 2 drops of phenolphthalein
indicator solution, use 1.0 mol/mL sod...
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Source: Above contents are excerpted from the PDF -- translated/reviewed by: www.chinesestandard.net / Wayne Zheng et al.
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