GB/T 5413.19-1997 Determination of free biotin in infant formula and milk powder

This standard specifies the method for the determination of free biotin by microbiological method. This standard is applicable to the determination of free biotin in infant formula and milk powder. GB/T 5413.19-1997 Determination of free biotin in infant formula and milk powder GB/T5413.19-1997 Standard download decompression password: www.bzxz.net

GB/T5413.19-1997
This standard is equivalent to the method of the Association of Public Analytical Chemists (AOAC). This series of standards will replace GB5413-85 from the date of implementation. This standard is proposed by the China Light Industry Federation.
This standard is under the jurisdiction of the National Dairy Standardization Center. The responsible drafting unit of this standard is the National Dairy Quality Supervision and Inspection Center. The participating drafting units of this standard are: Food Hygiene Supervision and Inspection Institute of the Ministry of Health, Zhejiang Light Industry Research Institute, Harbin Morinaga Dairy Co., Ltd., and Nestlé (China) Investment Services Co., Ltd. The main drafters of this standard are: Zhang Yujie, Wang Yun, and Yang Jinbao. 300
National Standard of the People's Republic of China
Infant formula foods and milk powder
Determination of free biotin content1Scope
This standard specifies the method for determining free biotin by microbiological method. This standard applies to the determination of free biotin in infant formula and milk powder. 2 Principle of the method
GB/T 5413.19—1997
Replaces GB5413-85
The free biotin content is determined by the growth of Lactobacillus plantarum. 3 Reagents, strains and culture media
All reagents, if the specifications are not specified, refer to analytical pure. All experimental water, if no other requirements are specified, refers to grade tertiary water. 3.1 Biologist: standard.
3.2 The volume fraction of ethanol is 50%.
3.3 Strain: Lactobacillus plantorum. 3.4 Culture medium
3.4.1 Lactobacillus agar culture medium: 15g photolyzed Chen, 5g yeast extract, 10g glucose, 100mL tomato juice, 2g potassium dihydrogen phosphate, 1g polysorbate monooleate, 10g agar, 1000mL distilled water, pH 6.8±0.2 (25℃). 3.4.2 Lactobacillus broth culture medium: 15g photolyzed Chen, 5g yeast extract, 10g glucose, 100mL tomato juice, 2g potassium dihydrogen phosphate, 1g polysorbate monooleate, 1000mL distilled water, pH 6.8±0.2 (25℃). 3. 4.3 Culture medium for biotin determination: 12g casamino acids for vitamin determination, 49g glucose, 20g sodium acetate, 0.2g L-cystine, 0.2g DL-tryptophan, 20mg adenine sulfate, 20mg guanine hydrochloride, 20mg uracil, 2mg thiamine hydrochloride, 2mg riboflavin, 2mg niacin, 2mg calcium pantothenate, 2mg pyridoxine hydrochloride, 200μg p-aminobenzoic acid, 1g dipotassium hydrogen phosphate, 1g potassium dihydrogen phosphate, 0.4g magnesium sulfate, 20mg sodium chloride, 20mg ferrous sulfate, 20mg manganese sulfate, add distilled water to 1000mL, pH 6.7±0.1 (25℃). 4 Instruments
Commonly used microbiological laboratory instruments and pH meter. 5 Preparation
5.1 Preparation of strains
5.1.1 Preparation of liquid culture medium
Pipette a fixed amount of basic liquid culture medium, add an equal amount of distilled water, and dispense 10mL into each culture tube, cover it, and sterilize it at 121℃ for 15min. Cool the sterilized tube quickly to avoid color formation. Store in a refrigerator. Approved by the State Administration of Technical Supervision on May 28, 1997, and implemented on September 1, 1998
GB/T 5413.19—1997
5.1.2 Preparation of stock tubes of Lactobacillus plantarum Inoculate the used microbial strains into more than two agar culture tubes, select a constant temperature (±0.5℃) between 28 and 40℃, and culture for 6 to 24 hours. After completion, put it in the refrigerator. When using a new strain for a single assay, make several subculture transfers within 1 to 2 weeks. Do not inoculate with culture medium that has been stored for more than a week, and do not open the culture medium during storage. 5.1.3 Preparation of inoculationWww.bzxZ.net
Transfer Lactobacillus planlarum (3.3) to a sterile 10mL liquid culture medium to obtain a bacterial suspension.
5.2 Preparation of standard solution
5.2.1 Biotin standard stock solution: concentration 50μg/mL. Accurately weigh 50-60mg of biotin standard (3.1) (from the desiccator), dilute with ethanol (3.2) to obtain a 50μg/mL biotin solution, and store in a refrigerator.
5.2.2 Biotin standard working solution: concentration 0.1ng/mL. Pipette 1mL of standard stock solution (5.2.1), dilute to 1000mL with water, and then pipette 1mL of each of the diluted solutions, dilute one to 500mL as a high concentration standard solution, and dilute the other to 1000mL as a low concentration standard solution. Prepare freshly prepared samples each time. 5.3 Preparation of glassware
Use flame activator to clean hard glass test tubes and other necessary glassware III (sodium laurel sulfate is a suitable cleaning agent). The test microorganisms are highly sensitive to a few growth factors and many cleaning agents. Therefore, after cleaning, they need to be heated at 250℃ for 1 to 2 hours.
6 Operation steps
6.1 Sample processing
6.1.1 Dry powder sample
Accurately weigh a certain amount of sample, which should contain 0.2 to 0.5 mg of biotin. Continue with step 6.1.3. 6.1.2 Liquid sample
Aspirate a certain amount of liquid sample, which should contain 0.2 to 0.5 mg of biotin. Continue with step 6.1.3. 6.1.3 Sample extraction
Add water to make the total sample volume 150mL. After mixing, adjust the pH to 4.5~4.6 with 1mol/L hydrochloric acid, transfer quantitatively to a 250mL volumetric flask, make up to volume with water, mix thoroughly, filter with filter paper, discard the first few milliliters, aspirate 5mL of filtrate, and make up to 100mL with water. 6.2 Preparation of standard curve
Add distilled water, standard solution and culture medium for vitamin B12 determination into culture tubes in the order of Table 1, in triplicate. Table 1
Test tube No.
Distilled water, ml.
Standard solution\, mL
Culture medium, mL
1) Add low concentration standard solution to test tubes No.3~7, and high concentration standard solution to test tubes No.8~10. 6.3 Assay solution
Add distilled water, sample solution and vitamin B12 assay medium to the culture tube in the order of Table 2, in triplicate. 302
Test tube No.
Distilled water, mL
Sample, mL
Culture medium, mL
6.4 Sterilization
GB/T5413.19--1997
Directly sterilize all test tubes, cool to the culture temperature or quickly put them into a circulating water bath to make the color form the lightest. Ensure uniform heating and cooling conditions (too many sterilized tubes or too close distances in the autoclave can have adverse effects). Sterilize at 121℃ for 10 minutes. 6.5 Inoculation
Aseptically inoculate each tube and add one drop of appropriate inoculum. Except for the 0.0mL standard solution (uninoculated blank tube) in group two (or three). Cover the tubes and shake them thoroughly to mix. 6.6 Incubation
Choose a constant temperature between 28~~40℃ (±0.5℃) and incubate for 60~72h. 6.7 Determination (acidity method)
The test tube is contaminated by any foreign microorganisms, and the test is invalid. Through visual inspection of each test tube, the reaction prediction is carried out. The uninoculated tube is clear, and no other microorganisms should grow in the standard solution and the sample. 6.7.1 Titration
Use bromothymol blue as an indicator and titrate the solution in each tube with 0.1mol/L sodium hydroxide, or use pH 6.8 as the potentiometric titration endpoint. If the inoculated blank titration reaction is equal to or higher than 1.5mL of the uninoculated blank level, the test result should be ignored. Usually, the reaction of 5.0mL of the standard solution is equivalent to the titration of 8~~12mL of 0.1mol/L sodium hydroxide. 6.7.2 Determination of pH value
The test tube is contaminated by any foreign microorganisms, and the test is invalid. The reaction prediction is made by visual inspection of each test tube. The uninoculated tube is clear and no other microorganisms should grow in the standard solution and the sample. After incubation, the pH value of the tube contents is read to the nearest 0.01 pH unit. 7 Analysis results
The content of biotin in the sample (μg/100g or μg/100ml.) = Where: X is the average value of biotin content in each milliliter of the test solution, ml; F is the dilution factor;
m--the mass or volume of the sample, g or mL. 8 Allowable difference
The difference between two determinations of the same sample shall not exceed 10% of the average value of the two determinations. X
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