GB/T 5413.25-1997 Determination of inositol in infant formula and milk powder
Some standard content:
GB/T5413.25—1997
This standard is equivalent to the method of the American Association of Public Analytical Chemists (AOAC) and measures the content of inositol with biological value. 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: National Dairy Quality Supervision and Inspection Center. The participating drafting units of this standard: Food Sanitation Supervision and Inspection Institute of the Ministry of Health, Zhejiang Light Industry Research Institute, Harbin Morinaga Dairy Co., Ltd., and Shengchao (China) Investment Service Co., Ltd. The main drafters of this standard: Zhang Yujie, Wang Yun, Fang Yuguo, and Wang Kexin. 322
National Standard of the People's Republic of China
Infant formula foods and milk powder
Determination of inositol
Milk powder and formula foods for infant and young children--Determination of inositol
1 Sample
This standard specifies the determination method of inositol. GB/T5413.25—1997
Replaces GB5413-85
Method 1 of this standard is applicable to the determination of inositol in infant formula foods and milk powder, and method 2 is applicable to the determination of inositol in milk powder. Method 1 Microbiological method
2 Principle of the method
The content of inositol is evaluated by the growth of Saccharomyces uvarum. 3 Reagents, strains and culture media
All reagents, if the specifications are not specified, are analytically pure, and all experimental water, if no other requirements are specified, is grade tertiary water. 3.1 Hydrochloric acid: volume ratio is 1:1
Dilute 50mL of concentrated hydrochloric acid with a volume fraction of 37% with water to 100mL. 3.2 Sodium hydroxide: c(NaOH) is 1mol/L. Dissolve 40g of sodium hydroxide in water and dilute to 1000mL. 3.3 Inositol standard: anhydrous crystals.
3.4 Bacterial species
Saccharomyces uvarum.
3.5 Culture medium
3.5.1 Neurospora agar culture medium: 5g No. 3 protein Chen, 5g yeast extract, 40g maltose, 15g agar. 3.5.2 Culture medium for inositol determination: glucose 100 g, potassium citrate 10 g, citric acid 2 g, potassium dihydrogen phosphate 1.1 g, potassium chloride 0.85 g, magnesium sulfate 0.25 g, calcium chloride 0.25 g, manganese sulfate 50 mg, ferrous fluoride 50 mg, DL-tryptophan 80 mg, L-cystine 0.1 g, L-isoleucine 0.5 g, L-lysine 0.5 g, L-methionine 0.2 g, DL-phenylalanine 0.2 g, L-tyrosine 0.2 g, L-asparagine 0.8 g, DL-aspartic acid 0.2 g, DL-serine 0.1 g, glycine 0.2 g, DL-threonine 0. 4g, L-valine 0.5g, L-histidine 0.124g, L-proline 0.2g, DL-alanine 0.4g, L-glutamic acid 0.6g, L-arginine 0.48g, thiamine hydrochloride 500pg, biotin 16μg, calcium pantothenate 5mg, pyridoxine hydrochloride 1mg, add distilled water to 1000mL, pH5.2±0.2 (25℃). 4 Instruments
Common laboratory instruments and:bzxZ.net
4.1 pH meter.
Guohao Technical Supervision Bureau approved on May 28, 1997 and implemented on September 1, 1998
GB/T5413.25-1997
4.2 250mL round bottom flask, conical drum glass joint connected to the condenser. 4.3 Colorimeter.
5 Sample preparation
5.1 Preparation of stock culture
5.1.1 Transfer two or more prepared malt extract agar slants from the pure strain S. uvarum. Incubate at 30℃ for 16-24h. Store in a refrigerator for no more than 2 weeks, then transfer to a new agar slants. 5.1.2 Preparation of inoculation
It is best to transfer from the stock culture to a newly prepared malt extract agar slants on the day of use, and incubate at 30℃ for 16-24h. Aseptically transfer the strain into a sterile physiological saline with an inoculation loop until a suspension of bacterial cells of a certain concentration is obtained. For determination, prepare a yeast suspension solution with a yeast concentration of 0.1mg/mL. The spectrophotometer is adjusted to 100% transmittance with water in advance, and then the transmittance of this "standard" yeast is measured. Transfer the bacterial cells from the newly inoculated slant into sterile physiological saline until the concentration reaches the same transmittance as the above standard solution. 5.2 Standard solution
5.2.1 Standard stock solution, the concentration of inositol is 0.2mg/mL. Box.
Weigh 100mg of inositol (the inositol standard has been placed in a desiccator for 24h), dilute it to 500mL with water in a volumetric flask. Store on ice 5.2.2 Standard working solution, the concentration of inositol is 2ug/mL. Take 5mL (5.2.1) solution, dilute it to 500mL with water, and make up to volume. Prepare on the same day. 6 Operation steps
6.1 Sample treatment
6.1.1 Dry powder sample
Accurately weigh a quantitative sample, which contains about 0.5 mg of inositol, and transfer it to a 250 mL round-bottom flask. Add 100 mL of hydrochloric acid (1:1) (3.1) and continue with step 6.1.3.
6.1.2 Liquid sample.
Pipette a certain amount of sample, which contains about 0.5 mg of inositol, and add 100 mL of hydrochloric acid (3.1). Continue with step 6.1.3. 6.1.3 Connect the round-bottom beaker containing the sample to a condenser. After 6 hours, remove the hydrochloric acid by distillation. Rinse the residue into a beaker with distilled water and adjust the pH value of the solution to 5.1±0.1 with 1 mol/L sodium hydroxide. Dilute to 250 mL, filter, and discard the first few milliliters. The concentration of inositol in this solution is 2 μg/mL. 6.2 Standard curve
Add distilled water and standard solution to the culture tube in the order of Table 1, in triplicate. Table 1
Test tube No.
Distilled water, ml
Standard solution, mL
6.3 Determination solution
Add distilled water and sample solution to the culture tube in the order of Table 2, in triplicate. Table 2
Test tube No.
Distilled water, mL
Sample solution, mL
6.4 Heat treatment
GB/T 5413.25—1997
Cover all test tubes with cotton plugs or stainless steel caps and steam at 100℃ for 10 minutes. 6.5 Inoculation
: Cool the test tubes to below 30℃. Aseptically add the inoculum to the basal stock culture medium at a ratio of 2 mL per 100 mL, mix thoroughly, and aseptically add the inoculated culture medium to each test tube in 6.2 and 6.3, adding 5 mL to each tube (of which only 5 mL of uninoculated culture medium is added to the No. 1 test tube in the standard curve). 6.6 Culture
Fix the test tube on a mechanical oscillator, and culture at a constant temperature (±0.5°C) between 28 and 30°C for 22 to 24 hours with a reciprocating motion of 100 to 200 times per minute. 6.7 Determination
Remove the test tube from the oscillator and steam at 100°C for 5 min or add 1 drop of disinfectant (Thoral) to each tube to stop growth. The test tube is invalid if it is contaminated by any foreign microorganism. Predict the reaction by visual inspection of each test tube. The uninoculated tube is clear and no other microorganisms should grow in the standard solution and sample. Mix each test tube thoroughly (you can also add a drop of defoamer), add the reaction solution to the colorimetric blood, stir the contents, put the colorimetric blood into the colorimetric cell of the spectrophotometer, the wavelength is 540-660nm, read the transmittance or absorbance, and stabilize for 30s. The stabilization time for each test tube should be the same.
For the uninoculated tube, adjust its transmittance to 100% (or absorbance to 0) and read the readings of the other inoculated blank tubes. Adjust the transmittance of the inoculated blank tube to 100% (or absorbance to 0) and read each other tube in turn. For each level of test solution, calculate the content of vitamins per milliliter, calculate the average of the obtained values, and the value measured in each tube shall not exceed ± 15% of the average value. If the number of tubes obtained is less than 2/3 of the number of tubes of the four levels of dilutions tested, the data used to calculate the sample concentration is insufficient. If the number of remaining tubes is 2/3 or more of the original number of tubes, the content of the sample can be calculated based on the average value.
Note: Metal caps (except stainless steel caps) cannot be used. This is due to the possibility of metal contamination of the test tube during incubation and shaking. 7. Degradation of analytical results
The content of inositol in the sample (mg/100g or mg/100mL) = Where: X is the average value of the inositol content in each milliliter of the test solution in the test tube, ug; - the mass or volume of the sample, g or ml.
8. Allowable difference
The difference between two measured values of the same sample shall not exceed 10% of the average value of the two measurements. Method 2 Gas chromatography
9 Method summary
After the inositol in the sample reacts with the derivatizing agent, it is separated and quantified by gas chromatography. 10 Reagents
10.1 N-trimethylsilyl imidazole.
10.2 Trimethylsilane.
11 Instruments
11.1 Gas chromatograph: FID detector.
(1)
GB/T 5413.25—1997
11.2 Chromatographic column: 50% Phenylmethylcloxane, Db-17 (J&w). 0.32mmidX30m0.25μm, 12 Operating steps
12.1 Accurately weigh 1g of sample in a beaker, dissolve it in warm water, transfer it to a 100mL volumetric flask, and make up to volume. Take 5.0mL of the solution and put it into a small test tube as the test solution.
12.2 Accurately weigh 50mg of inositol standard into a small beaker, dissolve it with water, transfer it to a 100mL volumetric flask, and make up to volume. Use a pipette to take 5mL of the solution into a 100mL volumetric flask and make up to volume with water. Take 2mL of this solution into a small test tube. 12.3 Freeze-dry the test tubes containing the sample and standard solution mentioned above. Add 1mL of internal standard solution (0.05mg of anthracene per milliliter of pyridine) to each test tube, cover the stopper tightly, and let it stand for 15 minutes. Add 0.5mL of N-trimethylsilyl imidazole and 0.2mol/L to each test tube, mix, let it stand for 15 minutes, and then inject. 12.4 Gas chromatography conditions:
a) Column temperature: 140~260℃ (5℃/min) b) Detector: FID,
c) Detector temperature: 280℃,
d) Injector: split (1:40),
e) Injector temperature: 280℃,
f) Injection volume: luL;
g) Flow rate: 1.4mL/min.
13 Analysis of the nesting
Inositol content in the sample (mg/100g)
Concentration of standard working solution, μg/mL,
Where: c—
A. Sample peak area,
-Standard sample peak area,
Sample internal standard peak area,
Standard sample internal standard peak area;
-Sample mass, g.
14 Allowable difference
The difference between two measured values of the same sample shall not exceed 5% of the average value of the two measurements. 326
200×cX200
（6）
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