GB 4789.34-2016National food safety standard - Food Microbiological Examination - Examination of Bifidobacterium (English PDF)

2.1 Constant temperature incubator. 36°C ± 1°C.

2.2 Refrigerator. 2°C ~ 5°C.

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.

3.1 Bifidobacterium culture medium. see A.1.

3.2 PYG medium. see A.2.

3.6 Sulfuric acid. analytically pure.

3.7 Sulfuric acid. analytically pure.

3.8 Lactic acid. analytically pure.

5.1 Sterile requirements

All operating procedures shall follow the sterile procedures.

5.2 Identification of Bifidobacterium

5.2.1 Pure bacteria species

5.2.2 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.

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.

5.3 Counting of Bifidobacterium

5.3.1 Pure bacteria species

5.3.1.1 Preparation of solid and semi - solid samples. weigh aseptically 2.0g

of sample; place into a sterile homogeneous cup containing 198.0mL of

diluent, homogenizing at 8000r/min ~ 10000r/min for 1min ~ 2min, OR place

in a sterile homogeneous bag containing 198.0mL of diluent;

5.3.2 Food sample

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).

5.3.5 Result representation

5.3.5.1 If the number of colonies on a dilution plate is within the appropriate

counting range, calculate the average of the two plate colonies and multiply

the average by the corresponding dilution factor as the total number of

colonies per gram or milliliter.

5.3.5.2 If there are two consecutive dilutions of the number of plate colonies

in the appropriate counting range, it shall calculate according to equation (1).

5.3.6 Report of the number of colonies

5.4 Results and report

According to the results of 5.2.3.1, 5.2.3.2, 5.2.3.3, the species name of

Bifidobacterium shall be reported. Issue the report according to the results of

5.3.6 colony counting, in CFU/g (mL).

Annex A

Medium and reagent

A.1 Bifidobacterium agar medium

A.1.1 Ingredients

A.1.2 Preparation

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

A.2.2 Preparation

A.3 MRS medium

A.3.1 Composition

A.3.2 Preparation

Add all components of A.3.1 to distilled water, dissolve by heating, calibrate to

pH 6.2 ± 0.1, autoclave at 121°C for 15min ~ 20min after sub-packaging.

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.3 Method

B.3.1 Treatment of acetic acid

Take 2.0ml ~ 3.0mL of Bifidobacterium culture medium into 10mL centrifuge

tube, add 0.2mL of 50% sulfuric acid solution (volume fraction), mix, add

2.0mL of acetone, mix and add excess sodium chloride, shake for 1min, then

add 2.0mL of ether, shake for 1min, centrifuge at 3000 r/min for 5min.

B.3.2 Treatment of lactic acid

Take 2.0mL ~ 3.0mL of Bifidobacterium culture medium into 10mL

colorimetric tube, place in a 100°C water bath for 10min, add 0.2mL of 50%

(volume fraction) sulfuric acid solution, mix, add 1.0mL of methanol, place in a

58°C water bath for 30min. Add 1.0 mL of water, add 1.0 mL of chloroform,

shake 3min, centrifuge at 3000 r/min for 5min, take chloroform layer for

analysis. Use same operation steps for lactic acid standard and blank culture

medium.

B.3.3 Liquid chromatography conditions

Chromatographic column. ZorbaxSb-Aq liquid chromatography column (4.6 x