GB/T 5413.16-1997 Determination of folic acid (folate activity) in infant formula and milk powder
Some standard content:
GB/T5413.16-1997
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 Promotion Center. The responsible drafting unit of this standard: National Dairy Quality Supervision and Inspection Center. The participating drafting units of this standard: Food Hygiene Supervision and Inspection Institute of the Ministry of Health, Zhejiang Light Industry Research Institute, Harbin Morinaga Dairy Co., Ltd., Nestle (China) Investment Service Co., Ltd. The main drafters of this standard: Yin Xiaohong, Wang Yun, Yang Jinbao, Zhang Yujie. 287
National Standard of the People's Republic of China
Infant formula foods and milk powder
Determinatfon of folic acid (folate activity)
Milk powder and formula foods for infant and young children--Determinatfon of folic acid (folate activity)1 Scope
This standard specifies the method for determining folic acid (folate activity) by microbiological method. This standard is applicable to the determination of folic acid (folate activity) in infant formula foods and milk powder. 2 Principle of the method
The activity of folate is evaluated by the growth of Lactobacillus casei. 3 Reagents, strains and culture media
GB/T 5413.16—1997
Replaces GB5413—85
All reagents, if the specifications are not specified, are of analytical grade; all experimental water, if no other requirements are specified, are of grade tertiary water. 3.1 Chicken pancreas: weigh 100 mg of dried chicken pancreas, 20 mL of distilled water, stir for 15 min, centrifuge for 10 min (3000 r/min), and take the supernatant. Prepare it before use.
3.2 Physiological saline: weigh 0.9 g of sodium fluoride (analytical grade) in a 100 mL volumetric flask, dilute to the mark, and oscillate to dissolve. Dispense 10 mL into each culture tube, cover, sterilize at 121°C for 20 min, and prepare it once a week. 3.3 Phosphate buffer: c(H,PO,) is 0.05 mol/L, and contains ascorbic acid. Dissolve 50 mg of ascorbic acid in 100 mL of 0.05 mol/L phosphate buffer, and prepare it before use.
3.4 Folic acid, standard.
3.5 Concentrated ammonia.
3.6 Toluene.
3.7 Bacterial strain: Lactobacillus casei. 3.8 Culture medium.
3.8.1 Lactobacillus agar culture medium: 15g aged milk, 5g yeast extract, 10g glucose, 100mL tomato juice, 2g potassium dihydrogen phosphate, 1g polysorbate monooleate, 10g agar, add distilled water to 1000mL, pH 6.8±0.2 (25℃). 3.8.2 Lactobacillus broth culture medium: 15g aged milk, 5g yeast extract, 10g glucose, 100mL tomato juice, 2g potassium dihydrogen phosphate, 1g polysorbate monooleate, add distilled water to 100mL, pH 6.8±0.2 (25℃). 3.8.3 Culture medium for folic acid determination: casein 10 g, glucose 40 g, sodium acetate 40 g, dipotassium hydrogen phosphate 1 g, potassium dihydrogen phosphate 1 g, DL-tryptophan 0.2 g, L-aspartic acid 0.6 g, L-cysteine hydrochloride 0.5 g, adenine sulfate 10 mg, guanine hydrochloride 10 mg, uracil 10 mg, xanthine 20 mg, polysorbate 0.1 g, glutathione 5 mg, magnesium sulfate 0.4 g, sodium fluoride 20 mg, ferrous sulfate 20 mg, manganese sulfate 15 mg, riboflavin 1 mg, p-aminobenzoic acid 2 mg, vitamin B4 mg, thiamine hydrochloride 400 μg, calcium pantothenate 800 μg, niacin 800 μg, biotin 20 μg, add distilled water to 1000 mL, pH 6.7 ± 0.1 (25°C). 3.9 Sodium hydroxide solution: c(NaOH) is 0.1 mol/L. Approved by the State Administration of Technical Supervision on May 28, 1997 288
Implemented on September 1, 1998
4 Instruments
Common laboratory instruments and:
4.1 Incubator.
4.2 Sterilizer.
4.3 Culture tubes and caps.
4.4 Test tube shaking rack.
4.5 Inoculation needle and inoculation loop.
4.6 pH meter.
4.7 Centrifuge.
5 Preparation
5.1 Preparation of bacterial strains
GB/T 5413.16—1997
5.1. 1 Inoculate the pure strain Lactobacillus casei from the bacterial culture medium into three transfer medium test tubes, culture at 37℃ for 24h, inoculate once a month, label and store in a refrigerator. Inoculate one of the culture tubes (4.3) inoculated monthly into another transfer medium, culture at 37℃ for 24h, do this transfer once a week, inoculate 4 to 5 transfer medium inoculation tubes from the inoculation tube every week, culture at 37℃ for 24h, label and use for daily measurement. At the beginning of each month, inoculate 3 transfer tubes from the monthly inoculation tube to store new bacterial strains. 5.1.2 Inoculate one tube from the daily inoculation tube into the bacterial culture medium, culture at 37℃ for 24h. Centrifuge the culture solution for 10 min (2000 r/min) under sterile conditions, pour off the supernatant, wash the bacterial cells with 10 mL of saline (3.2), centrifuge again for 10 min (2000 r/min), pour off the supernatant, and wash with another 10 mL of saline (3.2). Centrifuge as before, discard the supernatant, and add 10 mL of saline (3.2). Pipette 1 mL of the bacterial suspension into 10 mL of saline (3.2) and mix well. 5.2 Preparation of standard solution
5.2.1 Standard stock solution, folic acid concentration 500 μg/mL. Accurately weigh 55~~56 mg of folic acid standard (3.4), quantitatively transfer it into a 100 mL volumetric flask with 50 mL of distilled water, and add 2 mL of ammonia water (3.5). After the solution is prepared, calculate the volume of the solution. The concentration of folate in the stock solution is required to be 500μg/mL: Stock solution volume (mL) = m2×, 1000×c
100×500
Or simplified as:
Stock solution volume (mL)
Where: m is the mass of the standard sample, mg;
c is the purity of the standard sample, this/100g.
Dilute the solution to the mark with distilled water, add distilled water to the required calculated volume with a pipette, mix thoroughly, put it in a red or brown bottle, and store it in the refrigerator. The shelf life is 4 months. 5.2.2 Standard intermediate solution, the concentration of folic acid is 50μg/mL. Accurately pipette 10mL of the standard stock solution (5.2.1) into a 100mL brown or red volumetric flask, dilute to the mark with distilled water, mix thoroughly, and store it in the refrigerator. The shelf life is 1 month. 5.2.3 Standard working solution, folic acid concentration 0.1 ng/mL. Pipette 1 mL of standard intermediate solution (5.2.2) into a 100 mL brown volumetric flask, dilute to volume with distilled water, and mix. Pipette 1 mL of the solution into a 100 mL brown volumetric flask, dilute to volume, and mix. Pipette 5 mL of the upper solution into a 250 mL brown volumetric flask, dilute to volume with 0.05 mol/L phosphate buffer, and mix to obtain the standard 289
Accurately weigh a quantitative sample (the sample contains about 5 μg of folic acid) into a 100 mL beaker, reconstitute the sample with 25-30 mL of water, and pipette 1 mL of the diluted sample and 1 mL of chicken pancreas (3.1) into a 18 mm × 150 mm culture tube with a screw cap, and mix thoroughly. Add 18 mL of 0.05 mol/L phosphate buffer (3.3) containing ascorbic acid, and then add 1 mL of toluene (3.5). Make a blank control tube, incubate the sample tube and blank tube at 37℃ for 16h, sterilize at 121℃ for 10min, centrifuge, dilute 1mL distilled water and 1mL chicken pancreas with 0.05mol/L phosphate buffer in the blank tube, and also add 18mL of 0.05mol/L buffer (3.3) and 1mL of toluene (3.5). 10
Pipette a quantitative sample containing about 5μg of folic acid into a 100mL volumetric flask, dilute to the mark with water, and continue 6.1.3. 5
Directly sterilize all test tubes, cool to the incubation temperature or quickly put them in a circulating water bath to make the color as light as possible. Ensure heating and sterilization. 1
Inoculate each tube as sterile as possible and add a drop of appropriate inoculum. Except for standard tube No.7. Cover the lid and shake thoroughly to mix all 4
Add distilled water, standard solution and culture medium for folic acid determination to the culture tube in the order of Table 1, in triplicate. 5
If the test tube is contaminated by any foreign microorganism, the test is invalid. Predict the reaction by visual inspection of each test tube. The uninoculated tube is 2
Amounts are transferred to a 100mL volumetric flask and distilled to the mark with distilled water. Continue with step 6.1.3. 5
GB/T 5413. 16-1997
Cold conditions are uniform (too many sterilized tubes or too close distances can have adverse effects in the autoclave). Sterilize at 121℃ for 10min. 7
Add distilled water, sample solution and culture medium for folic acid determination to the culture tube in the order of Table 2, in triplicate. Standard working solution (0.0001ug/mL or 0.1ng/mL). Prepare before each determination. 0
6.1.3 The mass concentration of folic acid in the solution is about 0.05μg (50ng)/mL. 3
1) Add low concentration standard solution to test tubes No. 3~7; add high concentration standard solution to No. 8~10. Dissolve to obtain a solution with a mass concentration of folate of about .0.1ng/mL. 4
Select a constant temperature (±0.5℃) between 28～~40℃ and incubate for 60～72h. 5
6.1 Preparation of determination solution
6.1.1 Powdered sample
6.1.2 Liquid sample
6 Operation steps
6.2 Preparation of standard curve
Standard solution\,mL
Test tube No.
Clear, no other growth in standards and samples. Distilled water, mL
Culture medium, mL
6.3 Assay solutionbzxZ.net
Test tube No.
Distilled water, mL
Sample, mL
Culture medium, mL
6.4 Sterilization
6.5 Inoculation
6.6 Culture
6.7 Assay (acidity method)
6.7.1 Titration
GB/T5413.16—1997
Use bromothymol blue as an indicator and titrate the solution in each tube with 0.1 mol/L sodium hydroxide (3.9), or use pH 6.8 as the potentiometric titration endpoint. If the inoculated blank titration reaction is equal to or higher than 1.5 mL of the uninoculated blank level, the assay result should be ignored. Usually the reaction of the standard solution in 5.0 mL is equivalent to the titer of 8-12 mL of 0.1 mol/L sodium hydroxide. 6.7.2 Determination of pH
Read the pH of the tube contents after incubation to the nearest 0.01 pH unit. 6.8 Calculation
Make a concentration response curve (acidity, pH, transmittance or absorbance) for each standard solution, and each tube contains the corresponding reference standard content.
Quantitatively determine the vitamin content of each level of test solution, and discard any absorbance value below 0.5 mL of standard solution or above 4.5 mL of standard solution.
For each level of test solution, calculate the vitamin content per milliliter. Calculate the average of the values obtained, and the measured value of each tube shall not exceed ±15% of the average. If the number of tubes obtained is less than 2/3 of the number of tubes for the four aqueous dilutions tested, the data for calculating the sample concentration is insufficient. If the number of remaining tubes is 2/3 or more of the original number of tubes, the content in the sample can be calculated based on the average value.
7 Expression of analysis results
Folic acid content in sample (mg/100g or 100mL) = [(X× D)EB}×Where: X—Folic acid content per milliliter of test solution at different levels, ng; D—dilution of 1mL sample after enzyme treatment; EB—Folic acid content in blank tube of enzyme treatment solution, ng/mL; m
8 Allowable difference
The mass of the sample initially weighed (absorbed), g (or mL). The difference between two measured values of the same sample shall not exceed 10% of the average value of the two measurements. 100
(2)
Tip: This standard content only shows part of the intercepted content of the complete standard. If you need the complete standard, please go to the top to download the complete standard document for free.