GB 4789.35-2023National food safety standard - Food microbiological examination - Lactic acid bacteria (English PDF)

2.1 lactic acid bacteria

A general name for a group of bacteria that ferment sugars and mainly produce large

amounts of lactic acid. It is a bacterium that cannot liquefy gelatin, does not produce

indole, is Gram-positive, non-motile, non-spore-bearing, catalase-negative, nitrate

reductase-negative and cytochrome oxidase-negative.

The lactic acid bacteria in this Standard are mainly Lactobacillus, Bifidobacterium and

Streptococcus thermophilus.

3.1 Constant temperature incubator. 36 degrees C+/-1 degrees C.

3.2 Anaerobic culture device. anaerobic incubator, anaerobic tank, anaerobic bag or

device that can provide equivalent anaerobic effect.

3.3 Refrigerator. 2 degrees C~8 degrees C.

3.4 Homogenizer and sterile homogenization bag, homogenization cup or sterilized

mortar.

3.5 Vortex mixer.

3.6 Electronic balance. division is 0.001 g.

3.7 Real-time PCR machine.

3.8 Thermostatic water bath or metal bath.

3.9 Centrifuge. centrifugal force >10000xg.

3.10 Sterile test tube. 18 mmx180 mm, 15 mmx100 mm.

3.11 Sterile pipette. 1 mL (with 0.01 mL graduation), 10 mL (with 0.1 mL graduation).

3.12 Micropipette and sterilized tips. 2 µL, 10 µL, 100 µL, 200 µL, 1000 µL.

4.1 Diluent. see A.1 in Annex A.

4.2 MRS (Man Rogosa Sharpe) agar medium. see A.2 in Annex A.

4.3 Li-Mupirocin and Cysteine Hydrochloride modified MRS agar medium. see A.3 in

Annex A.

4.4 MC (Modified Chalmers) agar medium. see A.4 in Annex A.

4.5 0.5% sucrose fermentation tube. see A.5 in Annex A.

4.6 0.5% cellobiose fermentation tube. see A.5 in Annex A.

4.7 0.5% maltose fermentation tube. see A.5 in Annex A.

4.8 0.5% mannitol fermentation tube. see A.5 in Annex A.

4.9 0.5% salicin fermentation tube. see A.5 in Annex A.

4.10 0.5% sorbitol fermentation tube. see A.5 in Annex A.

4.11 0.5% lactose fermentation tube. see A.5 in Annex A.

4.12 Esculin fermentation tube. see A.6 in Annex A.

4.13 Gram stain solution. see A.7 in Annex A.

4.14 Normal saline. see A.8 in Annex A.

6.1 Sample preparation

6.1.1 All sample preparation processes should follow sterile operating procedures.

6.1.2 The diluent should be fully preheated at 36 degrees C+/-1 degrees C for 15 min~30 min before

testing.

6.1.3 Frozen samples can be thawed at 2 degrees C~5 degrees C first. The time does not exceed 18 h.

It can also be defrosted at a temperature not exceeding 45°C. The time does not exceed

6.1.4 Solid and semi-solid samples. Weigh 25 g of sample aseptically. Place in a sterile

homogenizing cup containing 225 mL of diluent. Homogenize at 8000 x g ~ 10000 x g

for 1 min ~ 2 min to prepare a 1.10 sample homogeneous solution. Or place in a sterile

homogenization bag with 225 mL of diluent. Use a slap homogenizer to beat for 1 min

~ 2 min to prepare a 1.10 sample homogeneous solution.

6.1.5 Liquid sample. The liquid sample should be shaken thoroughly first. Then use a

sterile straw to take 25 mL of the sample and put it into a sterile Erlenmeyer flask

containing 225 mL of diluent (an appropriate number of sterile glass beads are preset

in the bottle) or a homogeneous bag. Shake thoroughly or beat with a slap homogenizer

for 1 min ~ 2 min to prepare a 1.10 sample homogeneous solution.

6.1.6 Food samples containing lactic acid bacteria that have been processed by special

technologies (such as embedding technology) should be effectively pre-treated under

the corresponding technical/process requirements.

6.2 Dilution and culture

6.3 Lactic acid bacteria counting

6.3.1 Total number of lactic acid bacteria

The selection of culture conditions for counting the total number of lactic acid bacteria

and the explanation of the results are shown in Table 1.

6.3.2 Bifidobacteria counting

Based on the estimate of the bifidobacteria content of the sample to be tested, 2 to 3

consecutive appropriate dilutions are selected. Pipette 1 mL of sample homogeneous

solution for each dilution into a sterilized plate. Make two plates for each dilution. After

the dilution is transferred to the plate, pour 15 mL ~ 20 mL of mupirocin lithium salt

and cysteine hydrochloride modified MRS agar medium that have been cooled to

48 degrees C~50 degrees C into the plate. Swirl the dish to mix evenly.

6.3.3 Streptococcus thermophilus counting

Based on the estimate of the viable number of streptococcus thermophilus in the sample

to be tested, 2 to 3 consecutive appropriate dilutions are selected. Pipette 1 mL of

sample homogeneous solution for each dilution into a sterilized plate. Make two plates

for each dilution. After the diluent is transferred to the plate, pour 15 mL ~ 20 mL of

MC agar medium cooled to 48 degrees C~50 degrees C into the plate in time. Swirl the dish to mix

evenly. After the culture medium solidifies, place it upside down for aerobic culture at

36°C+/-1°C. According to the growth characteristics of streptococcus thermophilus,

culture is generally selected for 48 h. If the colony does not grow or grows small, you

can choose to culture it for 72 h. The colony characteristics of streptococcus

thermophilus on the MC agar medium plate are. the colonies are medium to small, red

colonies with neat and smooth edges, 2 mm+/-1 mm in diameter, and the back of the

colonies is pink.

6.4 Colony counting

See the colony counting part of GB 4789.2.

6.5 Result expression

See the calculation method part of GB 4789.2.

6.6 Report of colony counting

See the reporting part of total bacterial counting in GB 4789.2.

8.1 Method one -- Biochemical identification

8.1.1 Pure culture

Pick 3 or more single colonies and inoculate streptococcus thermophilus on MC agar

plate. Place at 36°C +/-1°C and incubate aerobically for 48 h. Lactobacillus are

inoculated on MRS agar plates. Place at 36°C +/-1°C and incubate anaerobically for 48 h.

8.1.2 Identification of bifidobacteria

Operate according to the provisions of GB 4789.34.

8.1.3 Smear microscopy

Streptococcus thermophilus cells are spherical or club-shaped under a microscope. The

diameter is 0.5 µm~2.0 µm. They are arranged in pairs or chains, no spores, Gram stain

positive. Lactobacillus have various cell shapes under the microscope and are long rod-

shaped, curved rod-shaped or short rod-shaped, without spores, and Gram stain positive.

8.1.4 Main biochemical reactions of lactic acid bacteria

The main biochemical reactions of lactic acid bacteria strains are shown in Table 2 andTable 3.

8.2 Method two -- Real-time fluorescence PCR identification

8.2.1 Pure culture

Same as 8.1.1.

8.2.2 DNA template preparation

Use an inoculating loop to scrape 2~10 colonies on the MC agar plate or MRS agar

plate. Suspend in 200 µL of sterilized physiological saline. Mix thoroughly. Centrifuge

at 10000xg to 12000xg for 3 min. Discard the supernatant. Add 50 µL of DNA

extraction solution and vortex to mix. Place in 100 degrees C water bath or metal bath for 10

min and then cool quickly. Centrifuge at 10000xg ~ 12000xg for 3 min. Pipette the

supernatant into a new PCR reaction tube and use it as a DNA template. The extracted

8.2.3 PCR reaction system

The total reaction system volume is 25 µL. 2.5 µL of 10 x PCR buffer, 1 µL of upstream

and downstream primers (10 µmol/L), 0.5 µL of probe (10 µmol/L), 3 µL of dNTPs (2.5

µmol/L), Taq DNA polymerase (5 U/µL) 0.5 µL, 1 µL of template DNA. Sterilized

deionized water are added to 25 µL. Each reaction should be run in at least 2 parallels.

8.2.4 PCR reaction conditions

50°C for 5 min, 95°C for 3 min, denaturation at 94°C for 5 s, and 60°C for annealing

and extension for 40 s (collecting FAM fluorescence at the same time) for 40 cycles.

8.2.5 Control settings

During the testing process (including DNA extraction), positive controls, negative

controls, and blank controls should be set for each reaction. The positive control

template is the positive clone molecule DNA or positive strain DNA of the amplified

fragment. The negative control template is non-lactic acid bacteria strain DNA. The

blank control template is sterile water.

8.2.6 Interpretation of results

Annex A

Culture medium and reagents

A.1 Diluent

A.1.1 Ingredients

A.1.2 Preparation method

Add the above ingredients to 1000 mL of distilled water. Heat to dissolve. After

dispensing, autoclave at 121°C for 15 min.

A.2 MRS agar medium

A.2.1 Ingredients

A.2.2 Preparation method

Add the above ingredients to 1000 mL of distilled water. Heat to dissolve. Adjust the

pH to 6.2+/-0.2.After dispensing, autoclave at 121°C for 15 min.

A.3 Mupirocin lithium salt and cysteine hydrochloride modified MRS agar

medium

A.3.1 Preparation of mupirocin lithium salt stock solution. Weigh 50 mg of mupirocin

lithium salt and add it to 5 mL of distilled water. Sterilize by filtration with 0.22 µm

microporous filter membrane. Prepare when needed.

A.3.2 Preparation of cysteine hydrochloride stock solution. Weigh 500 mg of cysteine

hydrochloride and add it to 10 mL of distilled water. Sterilize by filtration using a 0.22

µm microporous filter membrane. Prepare when needed.

A.3.3 Preparation method

Add the ingredients in A.2.1 to 985 mL of distilled water. Heat to dissolve. Adjust the

pH to 6.2+/-0.2.After dispensing, autoclave at 121°C for 15 min. Heat and melt the agar

before use. Cool to 48 degrees C~50 degrees C in a water bath. Use a sterile syringe to prepare the

mupirocin lithium salt stock solution and cysteine hydrochloride stock solution. Add

them to the molten agar. Make the concentration of mupirocin lithium salt in the culture

medium 50 µg/mL and the concentration of cysteine hydrochloride 500 µg/mL.

A.4 MC agar medium

A.4.1 Ingredients

A.4.2 Preparation method

Add the first 7 ingredients to the distilled water. Heat to dissolve. Adjust the pH to

6.0+/-0.2.Add neutral red solution. After dispensing, autoclave at 121°C for 15 min.

A.5 Lactobacillus sugar fermentation tube

A.5.1 Ingredients

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