This standard specifies the technical requirements for the inspection of lactic acid bacteria in lactic acid bacteria beverages. This standard applies to active lactic acid bacteria beverages with corresponding flavors made from fresh milk, milk powder or soybeans as raw materials through lactic acid bacteria fermentation. GB/T 4789.35-2003 Food Hygiene Microbiological Inspection Lactic Acid Bacteria Inspection GB/T4789.35-2003 Standard Download Decompression Password: www.bzxz.net

1CS07.100.30
National Standard of the People's Republic of China
GB/T4789.35-2003
Replaces GB/T16347-1996
Microbiological examination of food hygiene
Examination of Lactic acid bacteria in yoghurt beverage
Microbiological examination of food hygiene-Examination of Lactic acid bacteria in yoghurt beverageIssued on August 11, 2003
Ministry of Health of the People's Republic of China
Standardization Administration of the People's Republic of China
Implementation on January 1, 2004
GB/T4789.35-—2003
This standard revise GB/T16347-1996 "Microbiological Examination of Lactic Acid Bacteria in Yoghurt Beverages". Compared with GB/T16347--1996, this standard has the following major revisions: The format and text of the standard text are revised in accordance with GB/T1.1-2000. 1. Modify and standardize the "equipment and materials" in the original standard method. From the date of implementation of this standard, GB/T16347--1996 will be abolished at the same time. Appendix A of this standard is a normative appendix.
This standard is proposed and managed by the Ministry of Health of the People's Republic of China. The drafting units of this standard: Nanjing Health and Epidemic Prevention Station, Guangdong Food Hygiene Supervision and Inspection Institute, Shanghai Food Hygiene Supervision and Inspection Institute, Nutrition and Food Safety Institute of China Center for Disease Control and Prevention. The main drafters of this standard: Chen Xiaowei, Liu Min, Yang Ming, Zhu Mandan, Zhai Mingjuan, Zhou Beijun, Yang Baolan, Li Zhigang. The original standard was first issued in 1996, and GB/T4789.35-2003 replaced GB/T16347-1996. 274
1 Scope
Microbiological examination of food hygiene
Examination of lactic acid bacteria in lactic acid bacteria beverages
This standard specifies the technical requirements for the examination of lactic acid bacteria in lactic acid bacteria beverages. GB/T4789.35-2003
This standard applies to active lactic acid bacteria beverages with corresponding flavors made from fresh milk, milk powder or soybeans as raw materials and processed by lactic acid bacteria fermentation.
2 Normative references
The clauses in the following documents become the clauses of this standard through reference in this standard. For all dated references, all subsequent amendments (excluding errata) or revisions are not applicable to this standard. However, parties to agreements based on this standard are encouraged to study whether the latest versions of these documents can be used. For any undated referenced document, the latest version shall apply to this standard. GB/T4789.1 General principles for food hygiene microbiological examination methods GB/T4789.28 Food hygiene microbiological examination methods Staining methods, culture media and reagents 3 Terms and definitions
The following terms and definitions apply to this standard. 3.1
Lactic acid bacteria lactic acid bacteria
A group of aerobic and facultative anaerobic bacteria that can decompose glucose or lactose to produce lactic acid, are mostly non-motile, catalase negative, and Gram-positive non-spore-forming bacilli and cocci.
Total number of lactic acid bacteria colonies The total number of lactic acid bacteria colonies contained in 1 mL (g) of the obtained sample after the sample is cultured under certain conditions. 4 Equipment and materials
4.1 Refrigerator: 0℃~4℃.
4.2 Constant temperature incubator: 36℃±1℃.
4.3 Constant temperature water bath: 46℃±1℃.
4.4 Microscope: 10×~100×.
4.5 Homogenizer or sterile mortar.
4.6 Plate-mounted pharmaceutical balance: 0g～500g, accurate to 0.5g. 4.7 Conical flask: 500mL.
4.8 Wide-mouth bottle: 500mL.
4.9 Sterile pipette: 1mL (with 0.01mL scale), 10mL (with 0.1mL scale). 4.10 Sterile blood, diameter 90mm.
4.11 Sterile knife, scissors, and plating.
5 Culture medium and reagents
5.1 Modified TJA medium (modified tomato juice agar medium). 275
GB/T4789.35-2003
5.2 Modified MC medium (Modified Chalmers medium). 5.3 0.1% methylene blue milk medium.
5.4 6.5% sodium azide broth.
5.5 pH 9.6 glucose broth.
5.6 40% bile broth.
5.7 Starch hydrolysis medium: see Chapter A.7. 5.8 Arginine hydrolysis medium: see Chapter A.8. 5.9 Esculin medium: see Chapter A.10. 5.10
Gram staining solution: in accordance with GB/T4789.28. 13% hydrogen peroxide solution: in accordance with GB/T4789.28. 5.11
Protein water, indigo matrix reagent: in accordance with GB/T4789.28. Gelatin culture medium: in accordance with GB/T4789.28. Nitrate culture medium, nitrate reagent: in accordance with GB/T4789.28. 0.85% sterile saline.
6 Determination of total lactic acid bacteria colony count
6.1 Test procedure
The test procedure for total lactic acid bacteria colony count is shown in Figure 1. Test sample
Make several dilutions of appropriate multiples
Select 2-3 appropriate dilutions and add 1mL of each to sterile culture dishes. Add appropriate modified TJA or modified MC culture medium to each dish and incubate at 36℃±172h±3h
Count colonies
6.2 Operation steps
6.2.1Use aseptic operation to put 25mL (g) of the test sample that has been fully shaken into a sterile wide-mouth bottle containing 225mL of sterile saline to make a 1:10 uniform dilution.
6.2.2Use a 1mL sterile pipette to draw 1mL of the 1:10 dilution and slowly inject it into the test tube containing sterile saline along the wall of the tube (be careful not to let the tip of the pipette touch the dilution in the tube), shake the test tube and mix evenly. 6.2.3 Take another 1mL sterile pipette and make 10-fold incremental dilutions according to the above operation sequence. Each time the dilution increases, use a 1mL sterile pipette.
6.2.4 Select 2 to 3 or more appropriate dilutions. While making 10-fold incremental dilutions, use the pipette that draws the dilution to transfer 1mL of the dilution into the sterile plate. Make two plate IIIs for each dilution. 276
GB/T4789.35—2003
6.2.5 After the dilution is transferred into the plate III, about 15mL of lactic acid bacteria counting medium (modified TJA or modified MC) cooled to 50℃ should be injected into the plate in time, and the plate III should be rotated to mix evenly. At the same time, the lactic acid bacteria counting medium is poured into the sterile plate with 1mL of sterile physiological saline for dilution sample inspection as a blank control. The above operation should be completed within 20 minutes from the addition of the culture to the inoculation result.
6.2.6 After the agar solidifies, turn the plate over and place it in a 36℃±1℃ incubator for 72h±3h. Observe the characteristics of the lactic acid bacteria colonies (see Table 1) and select plates with colony counts between 30 and 300 for counting. After calculation, randomly select five colonies for Gram staining, microscopic examination and catalase test. Gram-positive, catalase-negative, non-spore cocci or bacilli can be identified as lactic acid bacteria. Calculate the number of lactic acid bacteria in the blood based on the confirmed lactic acid bacteria colonies, and then multiply by the dilution factor to obtain the number of lactic acid bacteria per milliliter of sample. For example, the dilution of sample 10 generates 35 suspicious colonies on the modified TJA agar plate. Five of them are taken for identification, and four are confirmed to be lactic acid bacteria. Then the number of lactic acid bacteria in 1mL of sample is:
35×4/5×10*=2.8×105
The morphological characteristics of lactic acid bacteria colony growth on modified TJA and modified MC medium are shown in Table 1. 6.3
Table 1 Colony characteristics of lactic acid bacteria on different culture media Improved TJA
The bottom of the plate is yellow, the colonies are medium-sized, slightly white, and moist. Improved MC
The bottom of the plate is pink, the colonies are small, round, red, with irregular edges, a diameter of 3mm±1mm, like cotton wool. The colonies are star-shaped, with a diameter of 2mm±1mm, and may have a faint halo. The bottom of the plate is yellow, the colonies are smooth, moist, slightly white, and have neat edges.
The bottom of the plate III is pink, the colonies are small, round, red, with neat edges, and may have a faint halo.
Note that Lactobacillus casei on improved TJA medium is round and smooth, with neat edges and diamond-shaped sides. Identification of lactic acid bacteria
When the lactic acid bacteria isolated above need to be identified, the following tests are performed: 7.1 Preparation of bacterial strains: Pick colonies from the plate, inoculate them on modified TJA or modified MC agar slant, culture at 36℃±1℃ for 24h~48h, scrape the bacterial moss, and perform the following tests respectively. 7.2 Identification test of lactic acid bacteria rarely reduces nitrates, does not liquefy gelatin, and does not produce indigo matrix and hydrogen sulfide. 7.3 Carbohydrate reactions of common species in the genus Lactobacillus, see Table 2 Table 2 Carbohydrate reactions of common species in the genus Lactobacillus Seven leaf moss
Lactobacillus casei
Caseus subspecies
Lactobacillus bulgaricus
Lactobacillus acidophilus
Lactobacillus lactis
Cellobiose
Maltose
Note: d Some strains are positive, some are negative; # Weak, slow or negative. 7.4
For identification test of lactic acid-producing streptococci, see Table 3. Mannitol
Table 3 Identification table of lactic acid-producing streptococci
Growth test
Thermophilus Streptococcus
Lactococcus
Cream Streptococcus
Methylene blue milk
Note: dSome strains are positive, some are negative. 6.5%
Sodium chloride
Salicin
Behenol
Arginine
GB/T4789.35—2003
Appendix A
(Normative Appendix)
Special culture medium and reagents
A.1 Improved tomato juice agar medium (improved TJA medium) A.1.1 Ingredients
Tomato juice
Yeast extract||tt| |Beef extract
Glucose
Potassium hydrogen phosphate (K,HPO.)
Toluene-80
Sodium acetate
Distilled water
pH6.8±0.2
A.1.2 Preparation method
Add to 1000mL
Preparation of tomato juice: Wash and chop fresh tomatoes (do not crush them), put them into a conical flask, put them in a 4℃ refrigerator for 8h~12h, take them out and filter them with gauze. If they are not used up at one time, they can be placed in a 0℃ refrigerator and can be stored for four months. Let them dissolve naturally at room temperature when using. Preparation method: Add all ingredients to the steamed stuffing water, heat to dissolve, and adjust the pH to 6.8±0.2. Package and sterilize at high pressure at 121℃ for 15min~20min. Heat to melt agar before use and use it when it cools to 50℃. A.2 Improved MC medium
A.2.1 Ingredients
Soy protein
Beef extract
Yeast extract
Glucose
Calcium carbonate
Distilled water
1% neutral red solution
1000mL
Polymyxin B sulfate (add as appropriate) 100,000 IUA.2.2 Preparation method: Add the previous seven ingredients into distilled water, heat to dissolve, adjust pH to 6, and add neutral red solution. Divide into flasks and sterilize by high pressure at 121℃ for 15min~20min. Heat to dissolve agar before use, cool to 50℃, add or not add polymyxin B sulfate as appropriate. If the sample has fat or odor after opening the can, etc., which is contaminated by foreign bacteria, polymyxin B can be added and used after mixing). A.30.1% methylene blue milk culture medium
Fresh skim milk
1% methylene blue aqueous solution
A.46.5% sodium chloride broth
Meat paste (pH7.6)
Sodium chloride
5pH9.6 glucose broth
Ordinary broth adjusted to pH9.6 with 0.2% glucoseA.640% bile broth
pH7. 6 Meat extract
Glucose
Ox bile
A.7 Starch hydrolysis medium
pH7.6 Meat extract agar
Sheep serum
GB/T4789.35—2003
3% starch solution
Dissolve the meat extract agar, wait until it cools to about 50℃, add starch solution and sterile sheep serum under aseptic operation, mix, and pour into the plate. A.8 Arginine hydrolysis medium
Protein Chen
Sodium chloride
Dipotassium hydrogen phosphate
L-arginine
1.6% bromocresol purple ethanol solution
Distilled water
Lactobacillus sugar fermentation tube
A.9.1 Basic ingredients
Beef extract
Protein Chen
Yeast extract
Tween-80
1.6% bromocresol purple ethanol solution
Distilled water
1000mL
A.9.2 Add the required sugar at 0.5% and divide into small test tubes. A.10
Aesculin culture medium
Protein Chen
GB/T4789.35—2003
Dipotassium hydrogen phosphate
Aesculin
Iron citrate
1.6% bromocresol purple alcohol solution
Distilled water
1000mL2 Preparation method: Add the previous seven ingredients into distilled water, heat to dissolve, adjust pH to 6, and add neutral red solution. Divide into flasks and sterilize under high pressure at 121℃ for 15min~20min. Heat to dissolve agar before use, cool to 50℃, add or not add polymyxin B sulfate as appropriate. If the sample has fat or odor after opening the can, etc., which is contaminated by bacteria, polymyxin B can be added and mixed before use. A. 30.1% methylene blue milk culture medium
Fresh skim milk
1% methylene blue aqueous solution
A. 46.5% sodium chloride broth
Meat paste (pH7.6)
Sodium chloride
5pH9.6 glucose broth
Ordinary broth adjusted to pH9.6 and added with 0.2% glucoseA. 640% bile broth
pH7. 6 Meat extract
Glucose
Ox bile
A.7 Starch hydrolysis medium
pH7.6 Meat extract agar
Sheep serum
GB/T4789.35—2003
3% starch solution
Dissolve the meat extract agar, wait until it cools to about 50℃, add starch solution and sterile sheep serum under aseptic operation, mix, and pour into the plate. A.8 Arginine hydrolysis medium
Protein Chen
Sodium chloride
Dipotassium hydrogen phosphate
L-arginine
1.6% bromocresol purple ethanol solution
Distilled water
Lactobacillus sugar fermentation tube
A.9.1 Basic ingredients
Beef extract
Protein Chen
Yeast extract
Tween-80
1.6% bromocresol purple ethanol solution
Distilled water
1000mL
A.9.2 Add the required sugar at 0.5% and divide into small test tubes. A.10
Aesculin culture medium
Protein Chen
GB/T4789.35—2003
Dipotassium hydrogen phosphate
Aesculinbzxz.net
Iron citrate
1.6% bromocresol purple alcohol solution
Distilled water
1000mL2 Preparation method: Add the previous seven ingredients into distilled water, heat to dissolve, adjust pH to 6, and add neutral red solution. Divide into flasks and sterilize under high pressure at 121℃ for 15min~20min. Heat to dissolve agar before use, cool to 50℃, add or not add polymyxin B sulfate as appropriate. If the sample has fat or odor after opening the can, etc., which is contaminated by bacteria, polymyxin B can be added and mixed before use. A. 30.1% methylene blue milk culture medium
Fresh skim milk
1% methylene blue aqueous solution
A. 46.5% sodium chloride broth
Meat paste (pH7.6)
Sodium chloride
5pH9.6 glucose broth
Ordinary broth adjusted to pH9.6 and added with 0.2% glucoseA. 640% bile broth
pH7. 6 Meat extract
Glucose
Ox bile
A.7 Starch hydrolysis medium
pH7.6 Meat extract agar
Sheep serum
GB/T4789.35—2003
3% starch solution
Dissolve the meat extract agar, wait until it cools to about 50℃, add starch solution and sterile sheep serum under aseptic operation, mix, and pour into the plate. A.8 Arginine hydrolysis medium
Protein Chen
Sodium chloride
Dipotassium hydrogen phosphate
L-arginine
1.6% bromocresol purple ethanol solution
Distilled water
Lactobacillus sugar fermentation tube
A.9.1 Basic ingredients
Beef extract
Protein Chen
Yeast extract
Tween-80
1.6% bromocresol purple ethanol solution
Distilled water
1000mL
A.9.2 Add the required sugar at 0.5% and divide into small test tubes. A.10
Aesculin culture medium
Protein Chen
GB/T4789.35—2003
Dipotassium hydrogen phosphate
Aesculin
Iron citrate
1.6% bromocresol purple alcohol solution
Distilled water
1000mL
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