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GB 5009.88-2014 English PDF (GB5009.88-2014)

GB 5009.88-2014 English PDF (GB5009.88-2014)

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GB 5009.88-2014: National Food Safety Standard -- Determination of dietary fiber in food

This Standard specifies the determination of dietary fiber in food (enzymatic gravimetric method). This Standard is applicable to the determination of total, soluble and insoluble dietary fiber in all vegetable foods and their products; but does not include the dietary fiber components such as oligofructose, galacto-oligosaccharide, polydextrose, resistant maltodextrin, resistant starch, etc.
GB 5009.88-2014
NATIONAL STANDARD OF THE
PEOPLE REPUBLIC OF CHINA
National Food Safety Standard -
Determination of Dietary Fiber in Food
ISSUED ON: SEPTEMBER 21, 2015
IMPLEMENTED ON: MARCH 21, 2016
Issued by: National Health and Family Planning Commission of PRC
Table of Contents
Foreword ... 3
1 Scope ... 4
2 Terms and Definitions ... 4
3 Principle ... 4
4 Reagents and Materials ... 5
5 Apparatus ... 7
6 Analytical Procedures ... 8
Appendix A Activity Measurement and Determination Criteria of Heat-Stable Amylase, Protease and Amyloglucosidase ... 13
National Food Safety Standard -
Determination of Dietary Fiber in Food
1 Scope
This Standard specifies the determination of dietary fiber in food (enzymatic gravimetric method).
This Standard is applicable to the determination of total, soluble and insoluble dietary fiber in all vegetable foods and their products; but does not include the dietary fiber components such as oligofructose, galacto-oligosaccharide, polydextrose, resistant maltodextrin, resistant starch, etc.
2 Terms and Definitions
2.1 Dietary fiber (DF)
A carbohydrate polymer that is not digested by the human small intestine but is of health significance, naturally occurring in plants or by extraction/synthesis, has a degree of polymerization of DP???3. It includes cellulose, hemicellulose, pectin and the other monomer components.
2.2 Soluble dietary fiber (SDF)
Part of dietary fiber that is soluble in water, including oligosaccharides and partially indigestible polysaccharides.
2.3 Insoluble dietary fiber (IDF)
Part of the dietary fiber that is insoluble in water, including lignin, cellulose, partially hemicellulose and the like.
2.4 Total dietary fiber (TDF)
The sum of soluble dietary fiber and the insoluble dietary fiber.
3 Principle
The dried specimen is enzymatically hydrolyzed and digested with heat-stable ??- 4.1.11 Heat-stable ??-amylase solution: CAS 9000-85-5, IUB 3.2.1.1, 10000U/mL ?? 1000U/mL; no glycerol stabilizer; store in refrigerator at 0??C~5??C; the enzyme activity measurement and determination criteria shall meet the requirements of Appendix A. 4.1.12 Protease solution: CAS 9014-01-1, IUB 3.2.21.14, 300U/mL~400U/mL; no glycerol stabilizer; store in refrigerator at 0??C~5??C; the enzyme activity measurement and determination criteria shall meet the requirements of Appendix A.
4.1.13 Amyloglucosidase solution: CAS 9032-08-0, IUB 3.2.1.3, 2000U/mL~3300U/mL; store in refrigerator at 0??C~5??C; the enzyme activity measurement and determination criteria shall meet the requirements of Appendix A.
4.1.14 Diatomaceous earth: CAS 68855-54-9.
4.2 Reagent preparation
4.2.1 Ethanol solution (85%, volume fraction): take 895mL of 95% ethanol; dilute with water and make constant volume of 1L; mix evenly.
4.2.2 Ethanol solution (78%, volume fraction): take 821mL of 95% ethanol; dilute with water and make constant volume of 1L; mix evenly.
4.2.3 Sodium hydroxide solution (6mol/L): take 24g of sodium hydroxide; dilute with water to 100mL; mix evenly.
4.2.4 Sodium hydroxide solution (1mol/L): take 4g of sodium hydroxide; dilute with water to 100mL; mix evenly.
4.2.5 Hydrochloric acid solution (1mol/L): take 8.33mL of hydrochloric acid; dilute with water to 100mL; mix evenly.
4.2.6 Hydrochloric acid solution (2mol/L): take 167mL of hydrochloric acid; dilute with water to 1L; mix evenly.
4.2.7 MES-TRIS buffer solution (0.05mol/L): take 19.52g of 2-(N-Morpholino) ethane- sulfonic acid and 12.2g of tris (hydroxymethyl) aminomethane; dissolve in 1.7L of water; use 6mol/L sodium hydroxide solution to adjust the pH at the room temperature; so that pH is 8.3 at 20??C, 8.2 at 24??C, and 8.1 at 28??C; pH shall be corrected by interpolation at the temperature between 20??C and 28??C. Add water to dilute to 2L. 4.2.8 Protease solution: use 0.05mol/L MES-TRIS buffer solution to prepare the protease solution with concentration of 50mg/mL; prepare currently before use; temporarily store at 0??C~5??C.
4.2.9 Pickling diatomaceous earth: take 200g of diatomaceous earth into 600mL of 2mol/L hydrochloric acid solution; soak overnight; filter, wash with water till the filtrate is neutral; burn ash at the 525??C??5??C muffle furnace for later-use.
6 Analytical Procedures
6.1 Specimen preparation
NOTE: The specimen treatment shall be appropriately implemented and dried according to the moisture content, fat content and sugar content; pulverized, mixed and sieved. 6.1.1 Specimen with fat content < 10%
If the specimen has a low moisture content (< 10%), the specimen shall be directly repeated for pulverizing until it is completely sieved. Mix for later-use. If the specimen has a high moisture content (???10%), after mix the specimen evenly, take appropriate amount of sample (mC, no less than 50g); place into 70??C??1??C vacuum drying oven to dry and make constant weight. Transfer the post-dried specimen into desiccator; weigh it (mD) when the specimen temperature drops to room temperature. According to the specimen mass before and after drying, calculate the mass loss factor (f) of the specimen. After drying, the specimen shall be repeated for pulverizing till completely sieved; place into desiccator for later-user. NOTE: If the specimen is not suitable for heating, use the freeze-drying method. 6.1.2 Specimen with fat content ???10%
The specimen needs to be degreased. Take appropriate amount of specimen (mC, no less than 50g); place into the funnel; wash by petroleum ether in a ratio of 25mL per gram of specimen; continuously wash for 3 times. After degreasing, mix the specimen, then dry and weigh it (mD) as per the 6.1.1; record the mass loss factor (f) after degreasing and drying. The specimen shall be repeated for pulverizing till completely sieved; place into the desiccator for later-use.
NOTE: If the fat content in the specimen is unknown, it shall be treated by first degreasing and then drying and pulverizing.
6.1.3 Specimen with sugar content ???5%
The specimen shall be subjected to de-sugar treatment. Take appropriate amount of specimen (mC, no less than 50g); place into the funnel; wash by 85% ethanol solution in a ratio of 10mL per gram of specimen; discharge the ethanol solution for 3 continuous times. After de-sugar treatment, place the specimen in 40??C drying oven to dry overnight; weigh it (mD); record the mass loss factor (f) of specimen after de-sugar and drying. The specimen shall be repeated for pulverizing till completely sieved; place into the desiccator for later-use.
6.2 Enzymatic hydrolysis
the room temperature.
6.3.1.2 Sucking filtration: take the crucible added with diatomaceous earth, dried and weighed; use 15mL of 78% ethanol to wet and flatten the diatomaceous earth; connect it with vacuum sucking filtration device; suck the ethanol; so that the diatomaceous earth in the crucible pave on the filtering plate. Transfer the specimen ethanol precipitate into the crucible for sucking filtration; use spatula and 78% ethanol to transfer all residues from the tall beaker into the crucible.
6.3.1.3 Washing: separately use 15mL of 78% ethanol to wash the residue for twice; use 15mL of 95% ethanol to wash the residue for twice; use 15mL of acetone to wash the residue for twice; after sucking and filtering the washing solution, dry the crucible together with residue at 105??C overnight. Place the crucible into the desiccator to cool off for 1h; weigh it (mGR, including the mass of treated crucible and residue), accurate to 0.1mg. Subtracting the mass of treated crucible, calculate the mass of specimen residue (mR).
6.3.1.4 Determination of protein and ash: take one of the two specimen residues, determine the nitrogen (N) content as per the GB 5009.5; take 6.25 as the conversion factor, calculate the protein mass (mP). Take the other specimen to determine the ash; namely, ash for 5h at 525??C; cool off in the desiccator; accurately weigh the total mass of the crucible (accurate to 0.1mg); subtracting the mass of treated crucible, calculate the ash mass (mA).
6.3.2 Determination of insoluble dietary fiber (IDF)
6.3.2.1 Take specimen as per 6.1; conduct the enzymatic hydrolysis as per 6.2. 6.3.2.2 Sucking filtration and washing: Take the treated crucible; use 3mL of water the wet and flatten the diatomaceous earth; suck the water, so that the diatomaceous earth in the crucible is paved on the filtering plate. Transfer all the specimen enzymatic hydrolysate into the crucible for sucking filtration; use 10mLof 70??C hot water to wash the residue for twice; collect and combine the filtrate; transfer to the other 600mL tall beaker; prepare for determining the soluble dietary fiber. Wash, dry and weigh the residue as per 6.3.1.3; record the residue weight.
6.3.2.3 Determine the protein and ash as per the 6.3.1.4.
6.3.3 Determination of soluble dietary fiber (SDF)
6.3.3.1 Calculate the filtrate volume: collect the filtrate generated by the sucking filtration of insoluble dietary fiber to the pre-weighed 600mL tall beaker. Estimate the filtrate volume by subtracting the beaker mass from the total mass of ?€?beaker + filtrate?€?. 6.3.3.2 Precipitation: add 4 times of the amount of 95% ethanol preheated to 60??C according to the volume of the filtrate; precipitate at the room temperature for 1h. The Appendix A
Activity Measurement and Determination Criteria of Heat-Stable
Amylase, Protease and Amyloglucosidase
A.1 Requirements of enzyme activity
A.1.1 Heat-stable amylase
A.1.1.1 Amylase activity tested by Nelson/Somogyi reducing sugar, using starch as substrate: 10000U/mL+1000U/mL; 1U represents the amount of enzyme required to release 1??mol of reducing sugar per minute at 40??C, pH 6.5.
A.1.1.2 Amylase activity tested with p-nitrophenyl-maltoside as the substrate: 3000Ceralpha U/mL + 300Ceralpha U/mL. 1CeralphaU represents the amount of enzyme required to release 1??mol of p-nitrophenyl per minute at 40??C, pH 6.5. A.1.2 Protease
A.1.2.1 Protease activity tested with casein as a substrate: 300U/mL ~ 400U/mL. 1U represents the amount of enzyme required to hydrolyze 1??mol of tyrosine soluble in trichloroacetic acid per minute from soluble casein at 40??C, pH 8.0.
A.1.2.2 Protease activity measured by Folin-Ciocalteau chromogenic assay using casein as a substrate: 7U/mg ~ 15U/mg. 1U represents the amount of enzyme required for the color change caused by 1.0??mol of tyrosine in the color reaction is obtained by hydrolysis form casein per minute at 37??C, pH 7.5.
A.1.2.3 Endopeptidase activity tested by azo-casein: 300U/mL ~ 400U/mL. 1U represents the amount of enzyme required to hydrolyze 1??mol of tyrosine per minute from the soluble casein at 40??C, pH 8.0.
A.1.3 Amyloglucosidase
A.1.3.1 Amyloglucosidase activity measured by starch/glucose oxidase-peroxidase method: 2000U/mL ~ 3300U/mL. 1U represents the amount of enzyme required to release 1??mol of glucose per minute at 40??C, pH 4.5.
A.1.3.2 Amyloglucosidase activity measured by p-nitrophenyl-??-maltoside (PNPBM) method: 130PNP U/mL ~ 200 PNP U/mL. 1PNP U represents the enzyme required to release 1??mol of p-nitrophenyl per minute from p-nitrophenyl-??-maltoside at 40??C in the present of excessive ??-glucosidase.
A.2 Enzyme interference

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