GB/T 4789.34-2003 Microbiological examination of food hygiene - Bifidobacterium examination
Some standard content:
ICS_07.100.30
National Standard of the People's Republic of China
GB/T4789.34—2003
Microbiological examination of food hygiene-Examination of Bifidobacterium2003-08-11Promulgated
Ministry of Health of the People's Republic of China
Standardization Administration of the People's Republic of China
2004-01-01Implementation
GB/T4789.34—2003
Appendix A and Appendix B of this standard are normative appendices. This standard is proposed and managed by the Ministry of Health of the People's Republic of China. Drafting units of this standard: Institute of Nutrition and Food Safety, Chinese Center for Disease Control and Prevention, Beijing Municipal Health and Epidemic Prevention StationMain drafters of this standard: Yang Baolan, Ran Lu, Li Zhigang, Wang Shuzhen, Yang Dajin, Jia Zhenzhen. 264
1 Scope
Microbiological Examination of Food Hygiene
Examination of Bifidobacterium
This standard specifies the examination method of Bifidobacterium. This standard applies to the examination of Bifidobacterium in food. 2 Normative References
GB/T4789.342003
The clauses in the following documents become the clauses of this standard through reference in this standard. For dated referenced documents, all subsequent amendments (excluding errata) or revisions are not applicable to this standard. However, the parties to an agreement based on this standard are encouraged to study whether the latest versions of these documents can be used. For undated referenced documents, the latest versions shall apply to this standard. GB/T4789.28--2003 Microbiological Examination of Food Hygiene Staining Methods, Culture Media and Reagents 3 Terms and Definitions
The following terms and definitions apply to this standard. 3.1
Bifidobacterium
A group of Gram-positive rods that can decompose glucose and produce a large amount of acetic acid and lactic acid. They are anaerobic, acid-intolerant, do not form spores, do not move, and have cells with various morphologies.
Enumeration of Bifidobacterium
The number of Bifidobacterium colonies contained in 1mL (g) of the sample obtained after cultivation under certain conditions. 4 Equipment and Materials
4.1 Constant temperature incubator: 36℃±1℃.
4.2 Constant temperature water bath: 46℃±1℃.
4.3 Microscope: 10×~100×.
Anaerobic culture equipment: anaerobic tank, anaerobic box. 4.4
4.5 Centrifuge: 3000z/min.
4.6 Gas chromatograph: (with hydrogen flame detector FID). Rack-plate pharmaceutical balance: 0g~500g, precision 0.5g. 4.7
Sterile pipette: 1mL (with 0.01 scale), 10mL (with 0.1 scale). 4.8
Sterile flat blood: diameter 90mm.
Wide-mouth bottle or conical flask: 300mL, 500mL. 4.10
5 Culture medium and reagents
5.10.85% sterile saline.
5.2TPY agar medium: see Chapter A.3. 5.3BL agar medium: see Chapter A.2. 265
GB/T4789.34—2003
5.4BBL agar medium: see Chapter A,1. 5.5 Basic culture medium for biochemical analysis of bifidobacteria: see Chapter A.4. 5.6 PYG liquid culture medium: see Chapter A.5. Gram staining solution: in accordance with 2.2 of GB/T4789.28-2003. 5.7
5.8 Sterilized liquid paraffin.
5.9 Methanol: analytical grade.
Chloroform: analytical grade.
Sulfuric acid: analytical grade.
Glacial acetic acid: analytical grade.
5.13 Lactic acid: analytical grade.
5.14 Acetic acid standard solution: take 5.7mL of analytical grade glacial acetic acid, transfer to a 100mL volumetric flask, add water to the mark, and calibrate. The calibration method is shown in Appendix B. The concentration of this solution is about 1mol/L. 5.15 Acetic acid standard working solution: dilute the calibrated acetic acid standard solution with water to 0.01mol/L. 5.16 Lactic acid standard solution: Pipette 8.4mL of analytically pure lactic acid, transfer to a 100mL volumetric flask, add water to the mark, and calibrate. The calibration method is shown in Appendix B. The concentration of this solution is about 1mol/L. 5.17 Lactic acid standard working solution: Dilute the calibrated lactic acid standard solution with water to 0.01mol/L. 6 Test procedure
The test procedure is shown in Figure 1.
25mL (g) sample
Add 225mL of sterile saline to make several appropriate multiples of dilution. Select 2 to 3 appropriate dilutions, take 0.1mL of each and spread on BL, BBL, TPY culture medium at 36℃±1℃
Inoculate PYG anaerobic culture
37℃72h
Determine acetic acid and lactic acid
7 Operation steps||tt| |Oxygen culture
Biochemical culture observation for 10 days
Strain identification report
72h±3h
Gram staining, catalase test
Colony count report
7.1Use aseptic operation to put 25g (mL) of the thoroughly mixed sample into a sterile conical bottle containing 225mL of sterile saline to make a 266
1:10 uniform dilution.
GB/T4789.34—2003
7.2 Use a 1mL sterile pipette to draw 1mL of the 1:10 dilution, slowly inject it into a test tube containing 9mL of sterile saline along the tube wall, shake and mix, and make a 1:100 dilution. 7.3 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.
7.4 Select 2 to 3 or more appropriate dilutions. While making 10-fold incremental dilutions, take 0.1mL of each and add it to the counting culture medium to spread evenly. Make two plates for each dilution. It is best to use 2 to 3 culture media (BL, BBL, TPY culture media) at the same time, and make a sterile saline blank control at the same time. 7.5 After the agar surface is dry, turn the plate over and put it in the anaerobic tank. The whole operation must be completed within 20 minutes. 7.6 Place the anaerobic tank in a 36℃±1℃ incubator for 72h±3h, and observe the characteristics of bifidobacterium colonies as shown in Table 1. Select plates with colony counts between 30 and 300 to count suspicious colonies, and randomly select five suspicious colonies for Gram staining, microscopic examination and catalase test. Catalase negative, Gram stain positive, no spores, uneven coloring, "Y" or "V" shaped bifurcated, or rod-shaped polymorphic bacilli can be identified as bifidobacteria. Table 1 Morphological characteristics of bifidobacterium colony growth on different culture media Culture medium
BL culture medium is yellow
BBL culture medium is yellow
TPY culture medium is yellow
Characteristics of bifidobacteria
Colony is medium-sized, shiny surface, neat edges are porcelain white, cream color, soft and delicate textureColony is medium-sized, smooth and raised surface, neat edges are cream color, soft and delicate textureColony is smooth and raised surface, neat edges are cream color, porcelain white, soft and delicate texture7.7 Colony count: According to the number of colonies confirmed as bifidobacteria, calculate the number of bifidobacteria in the plate, and then multiply it by the dilution factor of the sample to get the number of bifidobacteria per milliliter of sample. The highest count among the three culture media is taken as the final result. For example, 35 suspicious colonies are generated on the BBL agar plate when the sample is diluted 1X10* times. 5 colonies are taken for identification, and 4 colonies are confirmed to be bifidobacteria. Then the number of bifidobacteria in 1mL sample is:
10×35×4/5×10*=2.8×106
8 Bifidobacterium strain identification
8.1 Biochemical test
8.1.1 Preparation of strains: Pick colonies from the plate, inoculate them on BL, BBL or TPY agar plates, put them in anaerobic tanks, and culture them anaerobically at 36℃±1℃ for 72h±3h. Scrape the bacterial moss and prepare bacterial suspension with a concentration of 10°cfu/mL~101cfu/mL, and conduct tests respectively. 8.1.2 Biochemical identification test: Bifidobacterium generally does not reduce nitrate (but can reduce nitrate when there are dissolved red blood cells in the culture medium), and does not produce indigo matrix and hydrogen sulfide. The carbohydrate reaction of common bifidobacteria is shown in Table 2. Table 2 Key points for identification of species within the genus Bifidobacterium
Common bifidobacteria
1. Bifidobacterium bifidum
(B.bifidum)Www.bzxZ.net
2. Bifidobacterium longum
(B.longum)
3. Bifidobacterium infantis
(B.infantis)
4. Bifidobacterium breve
(B.breve)
5. Bifidobacterium adolescentis
(B,adolescentis)
L-arabinose
D-ribose
Note: d means some strains are positive and some are negative. Lactose
【Sorbitol
Cellobiose
Melezitose
Raffinose
Gluconate
GB/T4789.34——2003
8.2 Gas chromatography analysis of organic acid metabolites of bifidobacteriaPick colonies from the plate, inoculate them in PYG liquid culture medium, culture them anaerobically at 36℃±1℃, 72h±3h, and take the sterilized supernatant to analyze the organic acid metabolites of bifidobacteria. Bifidobacteria decompose glucose to produce acetic acid and lactic acid, with a ratio of about 3:2. After the sample is acidified and centrifuged, the supernatant is directly fed into the gas chromatograph to determine the acetic acid therein. The sample is methylated with sulfuric acid and methanol, extracted with chloroform, and the chloroform layer is taken into the gas chromatograph to determine the lactic acid therein. 8.2.1 Analysis steps
8.2.1.1 Sample processing
8.2.1.1.1 Acetic acid determination: Take 2mL~3mL of sterilized supernatant, add 0.1mL of 50% sulfuric acid, mix well and centrifuge at 4000/min for 5min, and take the supernatant for analysis.
8.2.1.1.2 Lactic acid determination: Take 2mL~3mL of sterilized supernatant, put it in a 10mL colorimetric tube, bathe at 100℃ for 10min, add 0.2mL of 50% sulfuric acid and 1mL of methanol, bathe at 58℃ for 30min, then add 1mL of water, add 0.5mL of chloroform, shake for 3min, centrifuge at 3000r/min for 5min, and take the chloroform layer for analysis. 8.2.1.2 Determination
8.2.1.2.1 Gas chromatograph reference conditions
8.2.1.2.1.1 Chromatographic column: 3.2mm inner diameter, 2m long stainless steel column, filled with 60-mesh 80-mesh GDX401 coated with 30g/L phosphoric acid.
8.2.1.2.1.2 Hydrogen flame detector (FID): Vaporization chamber, detector temperature: 200℃; nitrogen flow rate: 60mL/min, hydrogen flow rate: 55mL/min, air flow rate: 600mL/min, paper speed: 0.5mm/min. 8.2.1.2.3 Qualitative determination based on retention time, quantitative determination by external standard method. Calculation is as follows: A
Wherein:
X——the content of organic acid in the sample, in micromoles per milliliter (μmol/mL); A--the content of organic acid in the sample solution to be measured, in micromoles (μmol); V--the volume of the sample, in milliliters (mL). Report the results with two significant figures of the arithmetic mean of the parallel determinations. 8.2.1.2.4 See Figure 2 for the chromatogram.
8.2.1.2.5 Allowable difference: relative difference ≤15%. 8.2.2 Minimum detection concentration
-Acetic acid is 0.6μmol/mL;
Lactic acid is 0.8μmol/mL.
（1）
A.1BBL agar medium
A.1.1 Ingredients
Protein Chen
Yeast powder
Glucose
Soluble starch
Sodium chloride
5% cysteine
Tomato extract
Tween-80.
Liver extract
Distilled water
pH 7.0±0.1
A.1.2 Preparation method
Appendix A
(Normative Appendix)
Special culture medium and reagents
GB/T4789.34-2003
A.1.2.1 Preparation of tomato extract: Wash and weigh fresh tomatoes, chop them, add an equal amount of distilled water and heat them in a 100C water bath, stirring from time to time for about 90 minutes, then filter with flannel, adjust the pH to 7.0, divide into conical bottles, and sterilize at 115℃ for 15min20min. A.1.2.2 Preparation of liver extract: Weigh 1000g of fresh pork liver, cut into small pieces or mince, add distilled water to 2000mL, mix well, and place in the refrigerator overnight. Boil for 15min~20min the next day, filter with flannel, squeeze and collect all the filtrate, and add water to make up the original amount. Divide into conical bottles. A.1.2.3 Prepare according to the ingredients in A.1.1, adjust the pH to 7.0, dispense into conical bottles, and sterilize by high pressure at 115℃ for 15min~20min. A.2BL agar medium
A.2.1 Ingredients
Protein
Plant
Yeast powder
Glucose
Soluble starch
Beef extract
Tween-80
Liver extract
5% Cysteine
GB/T4789.34—2003
Distilled water
PH7.2±0.1
A.2.2 Preparation method
A.2.2.1 Preparation of liquid A: Weigh 10g of potassium dihydrogen phosphate and 10g of dipotassium hydrogen phosphate, add distilled water to 100mL, mix well, and sterilize under high pressure at 115℃ for 15min~20min.
A.2.2.2 Preparation of liquid B: weigh 10g magnesium sulfate, 0.5g ferrous sulfate, 0.5g sodium chloride, 0.337g manganese sulfate, add distilled water to 250mL, mix well, and sterilize by autoclave at 115℃ for 15min~20min. A.2.2.3 Prepare according to the ingredients in A, 2.1, adjust pH to 7.2, dispense into conical bottles, and sterilize by autoclave at 115℃ for 15min~20min. A.3TPY agar medium
A.3.1 Ingredients
Hydrolyzed casein
Plant sorghum
Yeast powder
Glucose
5% Cysteine
Dipotassium hydrogen phosphate (K,HPO,)
Magnesium chloride (MgCl.·6H,O)
Zinc sulfate (ZnSO,·7HO)
Calcium chloride (CaCl,)
Ferric chloride (FeCl
Tween-80
Steamed stuffing water
pH6.5±0.1
A.3.2 Preparation method
Heat and dissolve the ingredients in A.3.1, adjust to pH6.5±0.1, divide into conical bottles, and sterilize under high pressure at 115℃ for 15min~20min. A.4 Basic culture medium for biochemistry of Bifidobacterium
A.4.1 Ingredients
Protein Chen
Tween-80
Solution B
L-cysteine
1.6% bromocresol purple
pH7.2±0.1
A.4.1.1 Solution B: weigh 10g magnesium sulfate, 0.05g ferrous sulfate, 0.5g sodium chloride, 0.337g manganese sulfate, add distilled water to 250mL. A.4.1.2 Preparation of culture medium for sugar decomposition test: After dissolving the basic culture medium components (except indicator solution), adjust the pH to 7.2±0.1, and then add indicator solution. Add 0.5% of the type of sugar (0.25% for esculin), divide into small test tubes, 3mL per tube, and sterilize at 115℃ for 20min.
Biochemical tests must have sugar-free control tubes, and each tube is inoculated with 0.05 mL of bacterial suspension, which is directly inoculated to the bottom of the culture medium. Because bifidobacteria are 270
GB/T4789.34—2003
specifically anaerobic, a layer of liquid paraffin (0.8 mL) should be placed on the culture medium. Culture at 37°C for 7 to 10 days. A.4.2 Observation results
Observe the sugar decomposition performance twice on the 4th and 10th days. Determine by the color change of the indicator in the culture medium: slightly yellow is 10, partially yellow is 10, completely yellow is slightly transparent is 10, and completely yellow with obvious bacterial development is 100. A.5PYG liquid culture medium
A.5.1 Ingredients
Protein
Glucose
Yeast extract
Cysteine hydrochloride
Salt solution
Vitamin Ki solution
Hemin solution 5mg/mL
Distilled water
pH7.2±o.1
A.5.1.1 Preparation of salt solution: weigh 0.2g of anhydrous calcium chloride, 0.2g of magnesium sulfate, 1g of dipotassium hydrogen phosphate, 1g of potassium dihydrogen phosphate, 10g of sodium bicarbonate, 2g of sodium chloride, and add distilled water to 1000mL. A.5.1.2 Preparation of hemin solution (5mg/mL): weigh 0.5g is dissolved in 1mL of 1mol/L sodium hydroxide, distilled water is added to 100mL, autoclaved at 121℃ for 15min, and stored in a refrigerator. A.5.1.3 Preparation of vitamin K solution: Weigh 1g of vitamin K, add 99mL of anhydrous ethanol, filter and sterilize, and store in a cold and dark place. A.5.1.4 Preparation method: Mix the ingredients in A.5.1 and heat to dissolve, adjust the pH to 7.2 after cooling. Heat for 10min, filter, and autoclave at 121℃ for 15min.
GB/T4789.34—2003
Appendix B
(Normative Appendix)
Standardization of standard solution
Standardization of acetic acid: Accurately weigh 3g of acetic acid, add 15mL of water and two drops of phenolphthalein indicator solution, titrate with 1mol/L sodium hydroxide solution, and B.1
Correct the titration result with a blank test. 1mL 1mol/L sodium hydroxide solution is equivalent to 60.05mg acetic acid (C, H, O,). B.2 Calibration of lactic acid: Accurately weigh 1g of lactic acid, add 50mL of water, accurately add 25mL of sodium hydroxide titration solution (1mol/L), boil for 5min, add two drops of phenol indicator solution, titrate with 0.5mol/L sulfuric acid titration solution while hot, and correct the titration result with a blank test. Each 1mL 1mol/L sodium hydroxide titration solution is equivalent to 90.08mg of lactic acid (CgHgO,). 272
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