GB Translated English of Chinese Standard: GB NATIONAL STANDARD

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1 Translated English of Chinese Standard: GB GB NATIONAL STANDARD OF THE PEOPLE S REPUBLIC OF CHINA GB National food safety standard -- Determination of fat in foods 食品安全国家标准食品中脂肪的测定 Issued on: December 23, 2016 Implemented on: June 23, 2017 Issued by: National Health and Family Planning Commission of the PRC; State Food and Drug Administration. Page 1 of 21

2 Table of contents Foreword Scope Principles Reagents and materials Instruments and equipment Analytical procedures Analysis results expression Precision Principles Reagents and materials Instruments and equipment Analytical procedures Analysis results expression Precision Principles Reagents and materials Instruments and equipment Analytical procedures Analysis results expression Precision Principles Page 2 of 21

3 21 Reagents and materials Instruments and equipment Analytical procedures Precision Appendix A Operation procedures using fat extraction tube equipped with siphon or washing bottle Page 3 of 21

4 Foreword This standard replaces GB/T Determination of fat in food,gb/t Meat and meat products - Determination of free fat content, GB National food safety standard - Determination of fat in foods for infants and young children, milk and milk products, GB/T Meat and meat products - Determination of total fat content, GB/T Determination of crude fat in foods, GB/T Inspect of grain and oilseeds - Determination of crude fat in content in grain, GB/T Determination of crude fat in edible mushroom, GB/T Starches native or modified - Determination of total fat content, GB/T Oilseed meals - Determination of oil content - Part 1: Extraction method with hexane (or light petroleum). This standard compared with GB/T , the main changes are as follows: - CHANGE the standard name into National food safety standard - Determination of fat in foods ; - MODIFY the acid hydrolysis and extraction procedures of meat products and starch; - ADD the alkali hydrolysis method and Gerber method. Page 4 of 21

5 National food safety standard -- Determination of fat in foods 1 Scope This standard specifies the method for the determination of fat content in food. The first method of this standard applies to the determination of the free fat content of fruits, vegetables and their products, food and food products, meat and meat products, eggs and egg products, aquatic products and their products, baked goods, candy and other foods. The second method of this standard applies to the determination of the free fat and bound fat of fruits, vegetables and their products, food and food products, meat and meat products, eggs and egg products, aquatic products and their products, baked goods, candy and other foods. The third method of this standard applies to the determination of fat in milk and dairy products AND infant formula food. The fourth method of this standard applies to the determination of fat in milk and dairy products AND infant formula food. Method I: Soxhlet extraction method 2 Principles Fat is easily soluble in organic solvents. After the sample is directly extracted with solvent such as anhydrous ether or petroleum ether, the solvent is removed by evaporation and dried to obtain the content of free fat. 3 Reagents and materials Unless otherwise stated, the reagents used in this method are of analytical pure AND the water is level III water as specified in GB/T Reagents Anhydrous ether (C4H10O). Page 5 of 21

6 3.1.2 Petroleum ether (CnH2n+2): petroleum ether boiling range of 30 C ~ 60 C. 3.2 Materials Quartz sand Absorbent cotton. 4 Instruments and equipment 4.1 Soxhlet extractor. 4.2 Constant temperature water bath. 4.3 Analytical balance: sensitivity of g and g. 4.4 Electric blast oven. 4.5 Dryer: built-in effective desiccant, such as silica gel. 4.6 Filter tube. 4.7 Evaporation pan. 5 Analytical procedures 5.1 Sample treatment Solid sample: WEIGH 2 g ~ 5 g of the fully mixed solid sample, accurate to g; TRANSFER all of them into the filter tube Liquid or semi-solid sample: WEIGH 5 g ~ 10 g of the uniformly mixed sample, accurate to g; PLACE it into the evaporation pan; ADD about 20 g of quartz sand; EVAPORATE it dry on the boiling water bath; PLACE it in the electric blast oven at 100 C ± 5 C to dry it for 30 min; TAKE it out; GRIND it fine; TRANSFER all of it into the filter tube. As for the evaporation pan and the glass rod attached with samples, USE the absorbent cotton dipped with ether to wipe it clean; PLACE the cotton into the filter tube. 5.2 Extraction PLACE the filter tube into the extraction tube of the Soxhlet extractor; CONNECT the reception bottle which had been dried to constant weight; from the upper end of the condensate pipe of the extractor, ADD anhydrous ether or Page 6 of 21

7 10 Instruments and equipment 10.1 Constant temperature water bath Hot plate: high temperature of 200 C Conical flask Analytical balance: sensitivity of 0.1 g and g Electric blast oven. 11 Analytical procedures 11.1 Sample acid hydrolysis Meat products WEIGH 3 g ~ 5 g of the uniformly mixed sample, accurate to g; PLACE it in the conical flask (250 ml); ADD 50 ml of 2 mol/l hydrochloric acid solution and a few pieces of glass beads; COVER the watch glass; PLACE it on the hot plate to heat to slight boiling; MAINTAIN for 1 h; ROTATE and SHAKE it once every 10 min. TAKE off the conical flask; ADD 150 ml of hot water; MIX it uniformly; FILTER it. USE hot water to wash the conical flask and the watch glass clean; FILTER the hot water together. USE hot water to wash the precipitate to neutral (USE blue litmus test paper for inspection, AND its color will not change after reaching to neutral). PLACE the precipitate and filter paper on the large watch glass; DRY it in the 100 C ± 5 C drying oven for 1 h; COOL it down Starch In accordance with the estimated value of the total fat content, WEIGH 25 g ~ 50 g of the uniformly mixed sample, accurate to 0.1 g; PLACE it in the beaker and ADD 100 ml of water. Slowly POUR 100 ml of hydrochloric acid into the 200 ml of water; PLACE this solution in the hot plate to make it boiling; ADD it into the sample solution; HEAT this mixed solution to boiling and MAINTAIN for 5 min; after stop heating, TAKE several drops of mixed solution into the test tube; after cooling down, ADD 1 drop of iodine solution; if no blue color appears, it may start the operation of the next procedure. If blue color appears, it shall continue boiling the mixed solution, AND use the aforementioned method to conduct continue inspection, until it is determined that the mixed solution does not contain starch; START the operation of the next procedure. Page 9 of 21

8 Iodine (I2) Reagent preparation Mixed solvent: MIX the ether and petroleum ether of equivalent volume; PREPARE it before use Iodine solution (0.1 mol/l): WEIGH 12.7 g of iodine and 25 g of potassium iodide; DISSOLVE it in water and MAKE the volume reach to 1 L Congo red solution: DISSOLVE 1 g of Congo red into water; DILUTE it to 100 ml. Note: it is optional. Congo red solution allows the solvent and water interface to be clear, and it may also use other solutions that allow the aqueous phase to be colored without affecting the determination results Hydrochloric acid solution (6 mol/l): MEASURE 50 ml of hydrochloric acid; slowly POUR it into 40 ml of water; MAKE the volume reach to 100 ml; MIX it uniformly. 16 Instruments and equipment 16.1 Analytical balance: sensitivity of g Centrifuge: it can be used to place the fat extraction bottle or tube, with the speed of 500 r/min ~ 600 r/min, AND it can produce the 80 g ~ 90 g gravity field at the outer end of the fat extraction bottle Electric blast oven Constant temperature water bath Dryer: built-in dry desiccant, such as silica gel Fat extraction bottle: the fat extraction bottle shall be fitted with cork or other stoppers (such as silicone or polytetrafluoroethylene) that do not affect the solvent. Cork shall be firstly soaked in ether, placed in 60 C or 60 C above water for at least 15 min, and cooled before use. When it is out of service, it shall be soaked in water, which shall be replaced once every day. Note: It may also use the fat extraction tube (or flask) with a siphon or washing bottle, BUT the operational procedure is different, as specified in Appendix A. The lower end of the inner long branch pipe of the joint may be in spoon shape. Page 12 of 21

9 extraction bottle for several times; MIX it uniformly; AND the rest operations are same as Cream, light cream Firstly PLACE the cream sample into the warm water bath to dissolve it; MIX it uniformly; WEIGH about 0.5 g of sample (accurate to g); WEIGH about 1 g of light cream into the fat extraction bottle; ADD 8 ml ~ 10 ml of about 45 C water. ADD 2 ml of ammonia; MIX it uniformly; AND the rest operations are same as Cheese WEIGH about 2 g of the crushed sample (accurate to g) in the fat extraction bottle; ADD 10 ml of 6 mol/l hydrochloric acid; MIX it uniformly; COVER the bottle plug; HEAT it in the boiling water for 20 min ~ 30 min; TAKE it out and COOL it to room temperature; LET it stand for 30 s Extraction ADD 10 ml of ethanol; MAKE it mixed gently but thoroughly; AVOID liquid too close to the bottle neck. If required, it may add 2 drops of Congo red solution ADD 25 ml of ether; COVER the bottle plug; MAKE the fat extraction bottle in horizontal position; MAKE the extension part of the pallet point upwards and be clamped onto the shaker; SHAKE it for 1 min at the frequency of 100 times/min; AND it may also use manual shaking method. However, it shall pay attention to avoid the formation of persistent emulsion. After the fat extraction bottle is cooled down, carefully OPEN the plug; USE a small amount of mixed solvent to rinse the plug and bottle neck; MAKE the rinsing solution flow into the fat extraction bottle ADD 25 ml of petroleum ether; COVER the re-wetted plug; gently SHAKE it for 30 s as specified in PLACE the plugged fat extraction bottle in the centrifuge; CENTRIFUGE it for 5 min at 500 r/min ~ 600 r/min; or otherwise let the fat extraction bottle stand for at least 30 min, until the supernatant is clear AND obviously separated from water Carefully OPEN the plug; USE a small amount of mixed solvent to rinse the plug and the inner wall of the bottle neck; MAKE the rinsing liquid flow into the fat extraction bottle. Page 14 of 21

10 23 Analytical procedures ADD 10 ml of sulfuric acid to the Gerber cream meter; then carefully ADD ml of sample along the tube wall, so that the sample is not mixed with sulfuric acid; then ADD 1 ml of isoamyl alcohol; COVER the rubber plug; MAKE the bottle mouth face downwards; USE cloth to wrap it to avoid the plug from pushing out; vigorously SHAKE it to make it in uniform brown liquid; LET it standard for several minutes (bottle mouth downwards); PLACE it into the 65 C ~ 70 C water bath for 5 min; TAKE it out and PLACE it into the cream centrifuge for 5 min at the rotation speed of 1100 r/min; then PLACE it in the 65 C ~ 70 C water bath for thermal insulation for 5 min (note that the water bath level shall be higher than the fat layer of the cream meter). TAKE it out; TAKE reading immediately, which is the percentage of fat. 24 Precision The absolute difference between the two independent determinations obtained under repeatability shall not exceed 5% of the arithmetic mean. Page 18 of 21

11 HEAT to dissolve the casein; USE hydrochloric acid to neutralize it; ADD 10 ml of hydrochloric acid; ADD 0.5 g of sea sand; COVER the glass plug; USE gentle fire to make it boil for 5 min; after cooling it down, TRANSFER the contents of the beaker into the bottom of the fat extraction tube; USE 25 ml of anhydrous ether to rinse the beaker; INCLUDE the rinsing solution into the fat extraction tube. A.2 Extraction A.2.1 ADD 10 ml of anhydrous ethanol; MAKE it gently and thoroughly mixed at the tube bottom; if necessary, ADD two drops of Congo red solution. A.2.2 ADD 25 ml of anhydrous ether; ADD the cork (saturated with water) or other plugs soaked in water; REVERSE it for 1 min; AVOID excessive operation to prevent the formation of permanent emulsion. If necessary, PLACE the tube in the flowing water to cool it down; then carefully OPEN the cock; USE a small amount of mixed solvent (use washing bottle) to rinse the plug and tube neck; MAKE the rinsing solution flow into the tube. A.2.3 ADD 25 ml of petroleum ether; COVER the plug (which is wetted by water again); gently SHAKE it for 30 s as specified in A.2.2. A.2.4 PLACE the plugged tube into the centrifuge; CENTRIFUGE it for 1 min ~ 5 min at the speed of 500 r/min ~ 600 r/min. Or otherwise LET it stand for at least 30 min, until the supernatant becomes clear and is clearly separated from the aqueous phase; COOL it down. A.2.5 Carefully OPEN the cork; USE a small amount of mixed solvent to rinse the plug and the tube neck; MAKE the rinsing solution flow into the tube. A.2.6 INSERT the siphon or washing bottle connector into the tube; PRESS downwards the long branch tube, until it reaches to the point 4 mm above the two-phase interface. The inner branch tube shall be parallel with the tube axis. Carefully move the supernatant into a fat collection bottle containing zeolite, or a metal dish. Avoid moving into any water phase. USE a small amount of mixed solvent to rinse the outlet of the long branch tube; COLLECT the rinsing solution into the fat collection bottle. A.2.7 RELEASE the joint at the neck; USE a small amount of mixed solvent to rinse the joint and the lower part of the internal long branch tube; INSERT the connector again; TRANSFER the rinsing solution into the fat collection bottle. USE a small amount of mixed solvent to rinse the outlet; COLLECT the rinsing solution in the bottle; if necessary, USE the distillation or evaporation as described in 17.3 to eliminate some solvent. Page 20 of 21

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