GB 5413.25-2010 English PDF

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GB 5413.25-2010: National food safety standard -- Determination of inositol in foods for infants and young children, milk and milk products
Status: Obsolete

GB 5413.25: Historical versions

Standard ID	USD	BUY PDF	Lead-Days	Standard Title (Description)	Status
GB 5413.25-2010	399	Add to Cart	3 days	National food safety standard -- Determination of inositol in foods for infants and young children, milk and milk products	Obsolete
GB/T 5413.25-1997	199	Add to Cart	2 days	Milk powder and formula foods for infant and young children--Determination of inositol	Obsolete

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Basic data

Standard ID: GB 5413.25-2010 (GB5413.25-2010)
Description (Translated English): National food safety standard -- Determination of inositol in foods for infants and young children, milk and milk products
Sector / Industry: National Standard
Classification of Chinese Standard: C53;X82
Classification of International Standard: 67.100.10
Word Count Estimation: 10,122
Date of Issue: 2010-03-26
Date of Implementation: 2010-06-01
Older Standard (superseded by this standard): GB/T 5413.25-1997
Quoted Standard: GB/T 6682
Regulation (derived from): Circular of the Ministry of Health (2010)7
Issuing agency(ies): Ministry of Health of the People's Republic of China
Summary: This Chinese standard specifies the infant foods and milk products Determination of inositol. This standard applies to infant foods and milk products Determination of inositol.

GB 5413.25-2010: National food safety standard -- Determination of inositol in foods for infants and young children, milk and milk products

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National food safety standard.Determination of inositol in foods for infants and young children, milk and milk products National Standards of People's Republic of China People's Republic of China Ministry of Health issued Issued on. 2010-03-26 2010-06-01 implementation National Food Safety Standard Determination of infant foods and dairy products inositol National food safety standard Determination of inositol in foods for infants and young children, milk and milk products

Foreword

This standard replaces GB/T 5413.25-1997 "infant formula milk powder and inositol determination." This standard compared with GB/T 5413.25-1997, the first major change in law as follows. - A media culture collections have been corrected; - Sample processing by the hydrochloric acid distillation method to the high pressure hydrochloric acid hydrolysis; - By the way of inoculation broth mixed with the culture medium after dispensing tube to the dropping of the bacteria to the tube; - Concentration of standard working solution has been adjusted; - Sterilization temperature from 100 ℃ adjusted to 121 ℃; - Clear requirements and method for controlling the concentration of the bacterial suspension inoculation; - Improve the applicability of the calculation formula; - Increase the detection limit. The second major change in law as follows. - The use of inositol silylation derivatization; - Increase the detection limit. Appendix A of this standard and Appendix B is an informative annex. This standard replaces the standards previously issued as follows. --GB 5413-1985, GB/T 5413.25-1997. National Food Safety Standard Determination of infant foods and dairy products inositol

1 Scope

This standard specifies the method for determination of infant foods and dairy products inositol. This standard applies to food and dairy products Determination of inositol infants.

2 Normative references

The standard file referenced in the application of this standard is essential. For dated references, only the edition date of the note This applies to this standard. For undated references, the latest edition (including any amendments) applies to this standard. The first method microbiological method Principle 3 The use of grape juice yeast (Saccharomyces uvarum) of inositol specificity and sensitivity, quantitative determination of the sample to be tested Substance content. In the medium containing the test substance, except for all nutrients, the content of the test substance with growth of microorganisms linearly Relations, according to the light transmittance curve is compared with the standard of work, we can calculate the amount of the substance to be detected in the sample.

4 Reagents and materials

Unless otherwise specified, the reagents used in this method are analytically pure water GB/T 6682 provisions of secondary water. 4.1 Strain. grape juice yeast (Saccharomyces uvarum), ATCC 9080. 4.2 inositol (myo-Inositol) Standard. Formula C6H12O6, purity ≥99%. 4.3 Sodium chloride (NaCl). 4.4 Sodium hydroxide (NaOH). 4.5 Medium 4.5.1 malt extract agar medium (Malt Extract Agar). See Appendix A. 4.5.2 inositol assay medium. See Appendix A. 4.6 sodium chloride solution (9 g/L). Weigh 9.0 g of sodium chloride was dissolved in 1000 mL of water, packaging tubes, each tube 10 mL. 121 ℃ Sterilized 15 min. 4.7 hydrochloric acid (1 mol/L). Measure 82.0 mL of hydrochloric acid dissolved in water, cool and dilute to 1000 mL. 4.8 hydrochloric acid (0.44 mol/L). Measure 36.6 mL of hydrochloric acid dissolved in water, cool and dilute to 1000 mL. 4.9 sodium hydroxide solution (600 g/L). Weigh 300 g of sodium hydroxide dissolved in water, cool and dilute to 500 mL. 4.10 sodium hydroxide solution (1 mol/L). Weigh 40 g of sodium hydroxide dissolved in water, cool and dilute to 1000 mL. 4.11 standard solution of inositol 4.11.1 inositol standard stock solution (0.2 mg/mL). inositol standards set containing phosphorus pentoxide desiccator 24 h or more, Weigh 50 mg of inositol standard (4.2) (accurate to 0.1 mg), water and fully dissolved, the volume to 250 mL, stored in a refrigerator. 4.11.2 inositol intermediate standard solution (10 μg/mL). draw 5 mL inositol stock standard solution (4.11.1) to volume with water to 100 mL, Stored in a refrigerator. 4.11.3 inositol working standard solution (1 μg/mL and 2 μg/mL). draw 10 mL inositol intermediate standard solution (4.11.2) twice, Water volume to 100 mL and 50 mL. The working fluid required each prepared before use. 4.12 Desiccant. phosphorus pentoxide (P2O5). 4.13 glass beads. diameter of about 5 mm. 5. Apparatus In addition to the microbiological laboratory conventional sterilization and cultivation equipment, other equipment and materials as follows. 5.1 Balance. a sense of the amount of 0.1 mg. 5.2 pH meter. Accuracy ≤0.02. 5.3 Spectrophotometer. 5.4 vortex mixer. 5.5 Centrifuge. ≥2000 rpm rev/min. 5.6 incubator. 30 ℃ ± 1 ℃. 5.7 Shaking Incubator. 30 ℃ ± 1 ℃, shaking speed 140 times/min ~ 160 times/min. 5.8 Refrigerator. 2 ℃ ~ 5 ℃. 5.9 sterile pipette. 10 mL (0.1 mL with scale) or micropipette or suction head. 5.10 bottle dispenser. 0 mL ~ 10 mL. 5.11 Erlenmeyer flasks.200 mL. 5.12 flask (A class). 100 mL, 250 mL, 500 mL. 5.13 single pipette (A Class). capacity of 5 mL. 5.14 funnel. a diameter of 90 mm. 5.15 Quantitative filter paper. diameter 90 mm. 5.16 test tube. 18 mm × 180 mm. Note. The front glass instrument, with an active agent for hard glass measuring tube and other necessary glassware cleaning, cleaning after dry heat at 200 ℃ 2 h. Step 6 Analysis Suspension 6.1 Preparation of inoculum 6.1.1 species recovery The strain (4.1) upon activation inoculated into malt extract agar slant (4.5.1) on, 30 ± 1 ℃ ℃ 16 h ~ 24 h after incubation, Then transfer 2 or 3 generations generations to enhance the vitality of bacteria into stock, store in a refrigerator (8.5), the shelf life of not more than two weeks. Then just before use Species to the new malt extract agar slant. 6.1.2 Preparation of inoculum suspension Before using the stock will be transferred to one day strain on malt extract agar slant freshly prepared and cultured at 30 ℃ ± 1 ℃ 16 h ~ 24 h. By inoculating loop scraping lawn to a sterile sodium chloride solution (4.6) in a test tube. In 2000 r/min centrifugation 2 min ~ 3 min, The supernatant was decanted, 10 mL of sodium chloride solution (4.6), Shakers, and then centrifuged 2 min ~ 3 min, so clean 3 to 4 times. Draw a certain amount of bacteria transferred to the sodium chloride solution containing 10 mL (4.6) in a test tube, inoculated with bacterial suspension prepared. With a spectrophotometer, with sodium chloride solution (4.6) as a blank, measurement of the bacterial suspension inoculation transmittance at 550 nm wavelength adjustment Or the amount of bacteria added a certain amount of sodium chloride solution to make bacterial suspension light transmittance of 60% to 80%. 6.2 sample handling 6.2.1 Weigh about inositol containing 0.5 mg ~ 2.0 mg sample (accurate to 0.1 mg) in 250 mL Erlenmeyer flask, add the dry sample Into 80 mL of hydrochloric acid (4.8), the liquid sample was added 100 mL of hydrochloric acid (4.8), mix dry powder sample was dissolved. 6.2.2 The flask covered with aluminum foil, digestion in an autoclave 125 ℃ 1 h... After cooling to room temperature, about 2 mL hydroxide A solution of sodium (4.9), and cooled. Adjusted with sodium hydroxide solution (4.10) or hydrochloric acid solution (4.7) pH to 5.2, into 250 mL capacity Flask, dilute to the mark. Mixing, filtration, the filtrate was collected, the filtrate as the test solution. Dilution adjustment, so that the concentration of myo-inositol in the test solution 1 μg/mL ~ 10 μg/mL range. 6.3 standard curve Table 1. Add distilled water, inositol standard working solution (4.11.3) and inositol assay medium (4.5.2) in culture tubes, a formula Triplicate. Table 1 standard curve Tube No. S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 Distilled water (mL) 5 5 4 3 2 1 0 2 1 0 Inositol standard working solution 1 μg/mL (mL) Inositol standard working solution 2 μg/mL (mL) Medium (mL) 5 5 5 5 5 5 5 5 5 5 6.4 test solution for production Add distilled water in Table 2, the test solution (6.2.2) and inositol assay medium (4.5.2) in culture tubes in triplicate. Table 2 production test solution Tube No. 1234 Distilled water (mL) 4 3 2 1 Test solution (mL) 1 2 3 4 Medium (mL) 5 5 5 5 6.5 Sterilization Was added to each tube a glass beads (4.13), cover the tube cap, 121 ℃ sterilization 5 min (according to label instructions into the commodity media Line sterilization). 6.6 inoculation Said tubes is rapidly cooled to below 30 ℃. Above with a dropper or pipette to each test tube a drop (about 50 μL) was inoculated bacteria Suspension (6.1.2), except where the standard curve tubes inoculated blank tube S1. 6.7 Training The tubes were fixed in a shaking incubator, with about 140 beats/min ~ 160 times/min of shaking speed, at 30 ℃ ± 1 ℃ shaking culture 22 h ~ 24 h. 6.8 Determination Visual inspection of each tube, the tube blank inoculated culture medium S1 should be clarified if turbid, the result is invalid. 6.8.1 removed from the shaking incubator tube into the autoclave, 100 ℃ to maintain 5 min, stop the growth of microorganisms. 6.8.2 inoculation blank tube S1 (6.3) as a blank, the spectrophotometer adjusted to 100% transmittance (or absorbance A is 0), read The inoculated blank test tube S2 (6.3) readings. Then inoculated blank blank test tube S2, adjust the light transmittance was 100% (or A is 0), Sequentially read out each other tube transmittance (or absorbance A). 6.8.3 thoroughly mixed with a vortex mixer for each one tube (you can also add a drop of anti-foaming agent) immediately after the culture was transferred into the cuvette than the inner Line measurement wavelength of 540 nm ~ 660 nm, a stable reading 30 s after the read-out light transmittance, each tube settling time be the same. In muscle Alcohol content standard as the horizontal, the vertical axis as the light transmittance of the standard curve. 6.8.4 According to the test solution transmittance, Richard from the standard curve the concentration of the test solution inositol, according to the dilution factor and sample weight meter Content of the sample is calculated inositol. Transmittance curve beyond the standard tube sample tube S3 ~ S10 range to rounding. 6.8.5 test tube test solution of each number, calculate the concentration per ml of the test solution inositol number with each tube of light transmittance, and count This number is considered average concentrations of inositol test solution, the concentration is measured in each tube shall not exceed ± 15% of the average, than those who want to round go with. If the number of tubes that meets this requirement is less than the manifold number of test solution all four numbers of 2/3 of the sample used to calculate the content of data Is not sufficient, we need to re-test. If the number of tubes to meet the requirements of more than two-thirds of the original number of tubes, re-calculate the effective sample for each number of The average per ml tube inositol content was measured in order to calculate the average number of total average of all sample tubes Cx, it is used to calculate the sample Inositol content. NOTE. The standard curve can read transmittance (T%), can also read the absorbance (A).

7 expression analysis

Inositol content of the sample according to the formula (1). ×× = f CX x (1) Where. Inositol content X-- sample, the unit is milligram per hundred grams (mg/100 g); Cx - 6.8.5 overall average calculated in micrograms (μg); m-- sample mass, in grams (g); f - dilution. Infant foods and dairy f 250. The arithmetic mean of two under the same condition of independent determination results indicated that the results to three significant figures.

8 Precision

Two independent determination results under the absolute difference in repeatability condition must not exceed 10% of the arithmetic mean. Gas Chromatography Method II Principle 9 Inositol in the sample after extraction with water and ethanol, and the silylating agent derived hexane extraction, separation by chromatography, external standard the amount. 10 Reagents and materials Unless otherwise specified, the reagents used in this method are analytically pure water GB/T 6682 provided a water. 10.1 absolute ethanol (C2H6O). 10.2 n-hexane (C6H14). Ethanol 10.3 (95%). Ethanol 10.4 (70%). 10.5 trimethylchlorosilane (C3H9ClSi). 10.6 hexamethyldisilazane amine alkoxylates (C6H19NSi2). 10.7 N, N- dimethylformamide (C3H7NO). 10.8 silylating agents. Pipette volume ratio of 1. 2. 8 trimethylchlorosilane, hexamethyldisilazane alkyl amine, N, N- dimethylformamide Amides, ultrasonic mixing, prepared before use. 10.9 inositol Standard. Purity ≥99%. 10.10 inositol standard solution (0.010 mg/mL). Weigh 100 mg (accurate to 0.1 mg) After 105 ± 1 ℃ ℃ drying 2 h of Inositol standards (10.9) in 100 mL volumetric flask with 25 mL of water to dissolve completely. With ethanol (10.3) to volume, and mix. Take 1 mL of this solution to 100 mL volumetric flask with ethanol (10.4) to volume, and mix. 11 instruments and equipment 11.1 balance. a sense of the amount of 0.1 mg. 11.2 GC with FID detector. 11.3 Centrifuge. ≥5000 rpm rev/min. 11.4 rotary evaporator. 11.5 ultrasonic cleaner. 11.6 thermostatic water bath. 11.7 with rib cover 25 mL tube. 12 analysis steps 12.1 sample processing and Derivatives 12.1.1 sample processing. mixing uniformly solid sample Weigh 1 g (accurate to 0.0001 g), in 50 mL volumetric flask, add 12 mL The sample was dissolved in warm water about 40 ℃; direct liquid sample weighed 12 g (accurate to 0.0001 g) in 50 mL volumetric flask. Ultrasound of the sample Extract about 10 min, washed with ethanol (10.3) to volume, and mix. After standing for about 20 min, 10 mL of in 15 mL centrifuge tube, Not less than 4000 r/min centrifuged for about 5 min. 5 mL supernatant was concentrated in a rotary evaporator flask. 12.1.2 Drying and derivatives. To the concentrate was added 10 mL vial anhydrous ethanol (10.1), at 80 ℃ ± 2 ℃ when the rotation was concentrated to near dryness Added 5mL of anhydrous ethanol (10.1) concentration was continued to dry thoroughly (water will make the next step silylation is not complete). Silane reagent was added (10.8) 10 mL, 5 min sonication and transferred to a 25 mL screw cap centrifuge tube and placed in a water bath at 80 ℃ ± 2 ℃ derived trans Should be about 75 min, during intervals of about 20 min removed once the shock, then remove to cool to room temperature. 5mL was added n-hexane (10.2), Zhen Layered still swing after mixing. Upper liquid 3 mL in advance plus a little anhydrous sodium sulfate with a screw cap centrifuge tube, after the oscillation of not less than 4000 r/min centrifugation, this is the sample measurement solution. 12.2 Preparation of the standard solution measured Pipette 0.0 mL, 2.0 mL, 4.0 mL, 6.0 mL, 8.0 mL, 10.0 mL standard solution of inositol (10.10) in the concentrated bottle , Press 12.1.2 steps. 12.3 Determination 12.3.1 reference chromatographic conditions Column. filler is 50% cyanopropyl - methyl silicone capillary column (column length 60 m, an inner diameter of 0.25 mm, film thickness 0.25 μm) Or equivalent column. Inlet temperature. 280 ℃. Detector temperature. 300 ℃. Split ratio. 10. 1. Injection volume. 1.0 μL. Temperature program shown in Table 3. Table 3 temperature-programmed Heating rate (/ min ℃) Temperature (℃) Duration (min) 12.3.2 standard curve The standard solution were measured liquid (12.2) is injected into the gas chromatograph (chromatogram see Appendix B), to the measured peak area (or Peak height) for the vertical axis, to determine the content of the standard solution of inositol inositol as the horizontal calibration curve. 12.3.3 Determination of the sample solution The samples were measured liquid (12.1.2) is injected into the gas chromatograph to obtain the peak area (or peak height), to obtain a sample from the standard curve Determination of liquid inositol (mg). 13 analysis results presentation Inositol content of the sample according to equation (2). 100 ×× = is fCX (2) Where. X-- inositol content of the sample, the unit is milligram per hundred grams (mg/100 g); Cs-- content of the test sample solution obtained from the standard curve of inositol, in milligrams (mg). mi-- sample mass, in grams (g). fi-- sample was measured in terms of the sample contained inositol inositol contained coefficient is 10. The arithmetic mean of two under the same condition of independent determination results indicated that the results to three significant figures. 14 Precision Two independent determination results under the absolute difference in repeatability condition must not exceed 10% of the arithmetic mean. Other 15 This standard is the first method, the second method detection limits were 2.0 mg/100 g.

Appendix A

(Normative) Media and reagents A.1 malt extract agar medium (Malt Extract Agar) A.1.1 ingredients Maltose 12.75 g, dextrin 2.75 g, glycerol 2.35 g, peptone 0.78 g, agar 15.0 g, distilled water 1000 mL, pH 4.7 ± 0.2 (25 ± 5 ℃ ℃). A.1.2 Method First Additional components other than agar were dissolved in distilled water, the pH adjusted, and then agar was added, heated to boiling, agar melt. mix After closing evenly dispensing tubes, each tube 10 mL. 121 ℃ autoclaving 15 min, put beveled spare. A.2 inositol assay medium A.2.1 ingredients Glucose 100 g, and potassium citrate 10 g, citric acid 2 g, potassium dihydrogen phosphate 1.1 g, potassium chloride 0.85 g, magnesium sulfate 0.25 g, chloro Calcium 0.25 g, manganese sulfate 50 mg, ferrous chloride 50 mg, DL- tryptophan 80 mg, L- cystine 0.1 g, L- isoleucine 0.5 g, L- leucine 0.5 g, L- lysine 0.5 g, L- methionine 0.2 g, DL- phenylalanine 0.2 g, L- tyrosine 0.2 g, L- aspartic Acid 0.8 g, DL- aspartate 0.2 g, DL- serine 0.1 g, glycine 0.2 g, DL- threonine 0.4 g, L- valine 0.5 g, L- histidine 0.124 g, L- proline 0.2 g, DL- alanine 0.4 g, L- glutamic acid 0.6 g, L- arginine 0.48 g, thiamine hydrochloride 500 μg, biotin 16 μg, calcium pantothenate 5 mg, pyridoxine hydrochloride 1 mg, distilled water 1000 mL, pH 5.2 ± 0.2 (25 ± 5 ℃ ℃). A.2.2 Method The above ingredients were dissolved in water, the pH adjusted, the standby. Note. Some commercially available synthetic medium to good effect, the commercialization of synthetic medium formulated according to label instructions.

Appendix B

(Informative) Inositol standard derivative gas chromatogram B.1 standard inositol derivative gas chromatogram Inositol standard derivative gas chromatogram B.1. Figure B.1 standard inositol derivative gas chromatogram ......

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