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GB 8270-2014: National Food Safety Standard -- Food Additives -- Stevioside
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Basic data
Standard ID: GB 8270-2014 (GB8270-2014)
Description (Translated English): National Food Safety Standard -- Food Additives -- Stevioside
Sector / Industry: National Standard
Classification of Chinese Standard: X41
Classification of International Standard: 67.220.20
Word Count Estimation: 11,120
Date of Issue: 1/28/2015
Date of Implementation: 7/28/2015
Older Standard (superseded by this standard): GB 8270-1999
Regulation (derived from): National Health and Family Planning Committee Announcement 2015 No. 2
Issuing agency(ies): National Health and Family Planning Commission of the People's Republic of China
Summary: This Standard applies to stevia (Sterna Rebaudiana Bertoni) dry leaves for raw materials, extraction, refining derived food additives stevioside. The main glycoside is stevioside and rebaudioside A, other known glycosides include rebaudioside B, rebaudios
GB 8270-2014: National Food Safety Standard -- Food Additives -- Stevioside
---This is a DRAFT version for illustration, not a final translation. Full copy of true-PDF in English version (including equations, symbols, images, flow-chart, tables, and figures etc.) will be manually/carefully translated upon your order.
(National food safety standards for food additives stevia)
National Standards of People's Republic of China
National Food Safety Standard
Food additives steviol glycosides
Published 2015-01-28
2015-07-28 implementation
People's Republic of China
National Health and Family Planning Commission issued
Foreword
This standard replaces GB 8270-1999 "food additives Stevioside."
Compared with this standard GB 8270-1999, the main changes are as follows.
--- standard name was changed to "national food safety standards for food additives stevia";
--- modified the standard structure;
--- modified structural formula, molecular weight and molecular formula and other related content;
--- canceled the physical and chemical indicators of the level requirements, harmonized indicators;
--- modify the identification test;
--- modify the physical and chemical indicators stevioside content, loss on drying and ignition residue index;
--- removed the sweetness of the physical and chemical indicators, specific rotation, and heavy metals than the absorbance indicators;
--- increase the lead, methanol and ethanol indicators.
National Food Safety Standard
Food additives steviol glycosides
1 Scope
This standard applies to stevia (SteviaRebaudianaBertoni) dry leaves as raw material, extraction, refining derived food additives
Stevioside. The main glycoside is stevioside and rebaudioside A, other known glycosides include rebaudioside B, rebaudioside C, rebaudioside D,
Rebaudioside F, dulcoside A, rubusoside, and steviolbioside.
Formula 2, Structure and molecular weight
2.1 The main glycosides formula
Stevioside. C38H60O18
Rebaudioside A. C44H70O23
2.29 glycosidic structural formula
9 kinds of pressure glycoside compound name, R1 and R2 groups substituted-substituents are shown in Table 1.
Table 19 Compound Name pressure glycosidic is, R1 and R2 groups substituted-substituents
Compound Name
Chinese name English name
Position substituent R2 R1-substituents
Stevioside stevioside β-Glc β-Glc-β-Glc (2 → 1)
Rebaudioside A rebaudiosideA β-Glc
β-Glc-β-Glc (2 → 1)
-Glc (3 → 1)
Rebaudioside B rebaudiosideB H
β-Glc-β-Glc (2 → 1)
-Glc (3 → 1)
Rebaudioside C rebaudiosideC β-Glc
β-Glc-α-Rha (2 → 1)
-Glc (3 → 1)
Rebaudioside D rebaudiosideD β-Glc-β-Glc (2 → 1)
β-Glc-β-Glc (2 → 1)
-Glc (3 → 1)
Rebaudioside F rebaudiosideF β-Glc
β-Glc-β-Xyl (2 → 1)
-Glc (3 → 1)
TABLE 1 (cont.)
Compound Name
Chinese name English name
Position substituent R2 R1-substituents
Dulcoside A dulcosideA β-Glc β-Glc-α-Rha (2 → 1)
Rubusoside rubusoside β-Glc β-Glc
Steviolbioside steviolbioside H β-Glc-β-Glc (2 → 1)
Relative molecular mass of the major glycoside 2.3
Stevioside. 804.88 (according to 2007 international relative atomic mass)
Rebaudioside A. 967.03 (according to 2007 international relative atomic mass)
3 Technical requirements
3.1 Sensory requirements
Shall be in accordance with Table 2.
Table 2 Sensory requirements
Project requires test methods
Color
status
White to pale yellow
Powder or crystal
Proper amount of sample is placed in a clean, dry porcelain dish, from the
Observe the natural color and light state
3.2 Physical and Chemical Indicators
Shall be in accordance with Table 3.
Table 3 Physical indicators
Item Index Test Method
Stevioside content (dry basis, w) /% ≥ 85 Appendix A A.4
Residue on ignition (w) /% ≤ 1 GB 5009.4
Loss on drying (w) /% ≤ 6 GB 5009.3 a direct drying method
Lead (Pb)/(mg/kg) ≤ 1 GB 5009.12
Total arsenic (As)/(mg/kg) ≤ 1 GB 5009.11
Methanol/(mg/kg) ≤
Ethanol/(mg/kg) ≤
A.4
NOTE. stevioside commercialized product should comply with the standard stevioside as a raw material, starch and the like may be added for the purpose of standardization of food material.
a drying temperature and time were ± 2 ℃ and 2h 105 ℃.
Appendix A
Testing method
A.1 General Provisions
This standard reagents and water, in the absence of other requirements specified, refer to three predetermined water analytical reagents and GB/T 6682 in.
As the test standard titration solution, standard solution, the determination of impurities, products and preparations, in the absence of other requirements specified use, are by
GB/T 601, GB/T 602, a predetermined preparation GB/T 603 a. Was used in the test does not indicate when formulated with solvents which are meant water
Solution.
A.2 Identification Test
A.2.1 stevioside and rebaudioside A Test
In stevioside content of the test, the liquid chromatograph of the sample solution stevioside and rebaudioside A solution to be mixed with two main peaks standard
Liquid chromatogram corresponds.
sample solution pH A.2.2
Weigh 1g sample was dissolved in 100mL of water, with the pH of the sample solution measured acidity of 4.5 to 7.0.
Content Determination A.3 stevioside
A.3.1 method
A.3.1.1 Reagents and materials
A.3.1.1.1 Acetonitrile. chromatographically pure.
A.3.1.1.2 sodium dihydrogen phosphate. chromatography.
A.3.1.1.3 phosphate. chromatography.
A.3.1.1.4 Water. GB/T 6682-2008 in a predetermined water.
A.3.1.1.5 aqueous acetonitrile. water volume ratio of acetonitrile and 30.70.
A.3.1.1.6 sodium phosphate buffer (pH2.6). Weigh 1.20g sodium dihydrogen phosphate (NaH2P04), was dissolved in 800mL of water, adjusted with phosphoric acid
To pH to 2.6.
A.3.1.1.7 Stevioside standard. stevioside content (mass fraction, dry basis) ≥99.0%.
A.3.1.1.8 rebaudioside A standard. rebaudioside A content (mass fraction, dry basis) ≥99.0%.
A.3.1.1.9 other seven glycosidic standards. rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside F, dulcoside A, rubusoside
And steviolbioside.
A.3.1.2 instruments and equipment
HPLC. equipped with an ultraviolet detector, or other equivalent detectors.
A.3.1.3 Reference chromatographic conditions
A.3.1.3.1 Column. C18 reversed-phase column, 250mm × 4.6mm, 5 m particle size; column or other equivalent.
A.3.1.3.2 mobile phase. acetonitrile. sodium phosphate buffer = 32.68.
A.3.1.3.3 mobile phase flow rate. 1.0mL/min.
A.3.1.3.4 Detection wavelength. 210nm.
A.3.1.3.5 Injection volume. 2μL ~ 10μL.
A.3.1.3.6 Column temperature. 40 ℃.
A.3.1.4 analysis step
Preparation A.3.1.4.1 standard mixture solution
Were weighed amount of stevioside, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside F., Dulcoside A, sweet
Tea glycosides, steviolbioside standards, placed in the same flask, after complete dissolution volume aqueous acetonitrile, to give a mixed standard solution.
Mixed standard solution for determining the relative retention time of 9 kinds glycoside.
Preparation of standard solution A.3.1.4.2
Weigh 0.05g standard stevioside and rebaudioside A standard, accurate to 0.001 g of, were placed in 50mL volumetric flask
And diluted to the mark with an aqueous solution of acetonitrile, to give a standard solution stevioside and rebaudioside A standard solution.
Preparation of sample solution A.3.1.4.3
Weigh 0.05g ~ 0.1g sample, accurate to 0.001 g of, placed in 50mL volumetric flask, dilute to the mark with acetonitrile aqueous solution of
Degrees, to obtain a sample solution.
A.3.1.5 Determination
In reference A.3.1.3 chromatographic conditions were mixed standard solution, the standard solution and sample solution chromatographed. The sample
Chromatogram was compared with the chromatogram of a mixed standard solution, sample solution to determine the peak chromatogram corresponding to the components. Record test
Sample solution chromatogram stevioside, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside F., Dulcoside A, sweet tea
Glycoside, and the peak area of chromatogram of the standard solution steviolbioside of stevioside and rebaudioside A the peak area.
A.3.1.6 calculation results
Rebaudioside A content (dry basis) by the mass fraction wa of formula (A.1) is calculated.
wa =
mR
m ×
Aa
AR ×
100% (A.1)
Where.
mR --- rebaudioside A standard solution mass of rebaudioside A (dry basis), milligrams (mg);
--- mass m of the sample solution in the sample (dry basis), milligrams (mg);
AA --- sample solution chromatogram peak area of rebaudioside A;
Peak area value of the AR --- rebaudioside A standard solution chromatogram of rebaudioside A.
Eight other glycoside content (dry basis) by the mass fraction wi of formula (A.2) is calculated.
wi =
mS
m ×
fi × Ai
AS ×
100% (A.2)
Where.
i --- s, b, c, d, f, da, ru, sb, respectively stevioside, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside F.,
Dulcoside A, rubusoside, steviolbioside;
--- mass mS stevioside stevioside standard solution (dry basis), milligrams (mg);
--- mass m of the sample solution in the sample (dry basis), milligrams (mg);
fi --- i value component with the ratio of formula stevioside. 1.00 (stevioside), 1.00 (rebaudioside B), 1.18 (rebaudioside C), 1.40 (Rui
Bowditch glycoside D), 1.16 (rebaudioside F), 0.98 (dulcoside A), 0.80 (rubusoside), 0.80 (steviolbioside);
Peak area of component i --- AI in the chromatogram of the sample solution;
--- the AS peak area of stevioside standard stevioside solution chromatogram.
By the formula (A.1) and the formula (A.2) to obtain nine kinds of components calculated content wa, ws, wb, wc, wd, wf, wda, wru and wsb, taking the components
Is the sum of the content of stevia content in the sample.
A.3.2 Method Two
A.3.2.1 Reagents and materials
A.3.2.1.1 Acetonitrile. chromatographically pure.
A.3.2.1.2 sodium dihydrogen phosphate. chromatography.
A.3.2.1.3 phosphate. chromatography.
A.3.2.1.4 Water. GB/T 6682-2008 in a predetermined water.
A.3.2.1.5 aqueous acetonitrile. water volume ratio of acetonitrile and 30.70.
A.3.2.1.6 sodium phosphate buffer (pH2.6). Weigh 1.20g sodium dihydrogen phosphate (NaH2P04), was dissolved in 800mL of water, adjusted with phosphoric acid
To pH 2.6, diluted with water to 1000mL.
A.3.2.1.7 Stevioside standard. stevioside content (mass fraction, dry basis) ≥99.0%.
A.3.2.1.8 eight other glycoside standards. rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside F, Duke
Glycosides A, rubusoside, and steviolbioside.
A.3.2.2 instruments and equipment
With A.3.1.2.
A.3.2.3 Reference chromatographic conditions
With A.3.1.3.
A.3.2.4 analysis step
Preparation A.3.2.4.1 standard mixture solution
Were weighed amount of stevioside, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside F., Dulcoside A, sweet
Tea glycosides, steviolbioside standards, placed in the same flask, after complete dissolution volume aqueous acetonitrile, to give a mixed standard solution.
Mixed standard solution for determining the relative retention time of 9 kinds glycoside.
Preparation of standard solution A.3.2.4.2
Weigh 0.05g stevioside standard, accurate to 0.001 g of, placed in 50mL volumetric flask, and diluted with aqueous acetonitrile to dissolve
Scale, to give a standard stevioside solution.
Preparation of sample solution A.3.2.4.3
Weigh 0.05g ~ 0.1g sample, accurate to 0.001 g of, placed in 50mL volumetric flask, dilute to the mark with acetonitrile aqueous solution of
Degrees, to obtain a sample solution.
A.3.2.5 Determination
In reference A.3.2.3 chromatographic conditions were mixed standard solution, the standard solution and sample solution chromatographed. The sample
Chromatogram was compared with the chromatogram of a mixed standard solution, sample solution to determine the peak chromatogram corresponding to the components. Record test
Sample solution chromatogram stevioside, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside F., Dulcoside A, sweet tea
Glycosides, stevioside and peak areas of the standard solution chromatogram of steviolbioside.
A.3.2.6 calculation results
9 kinds glucoside content (dry basis) by the mass fraction wi of formula (A.3) is calculated.
wi =
mS
m ×
fi × Ai
AS ×
100% (A.3)
Where.
i --- s, a, b, c, d, f, da, ru, sb, respectively stevioside, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside
D, rebaudioside F., Dulcoside A, rubusoside, steviolbioside;
--- mass mS stevioside stevioside standard solution (dry basis), milligrams (mg);
--- mass m of the sample solution in the sample (dry basis), milligrams (mg);
fi --- i-type component with stevioside ratio value. 1.00 (stevioside), 1.20 (rebaudioside A), 1.00 (rebaudioside B), 1.18 (Rui
Bowditch glycosides C), 1.40 (rebaudioside D), 1.16 (rebaudioside F), 0.98 (dulcoside A), 0.80 (rubusoside), 0.80 (sweet
Ju double glucoside);
Peak area of component i --- AI in the chromatogram of the sample solution;
--- the AS peak area of stevioside standard stevioside solution chromatogram.
By the formula (A.3) is calculated to obtain nine kinds of components content ws, wa, wb, wc, wd, wf, wda, wru and wsb, take the sum of the concentrations of the components i.e.
Stevioside content of the sample.
A.4 determination of methanol and ethanol
A.4.1 Reagents and materials
A.4.1.1 Methanol. HPLC grade.
A.4.1.2 ethanol. chromatography.
A.4.1.3 water. a water.
A.4.2 Instruments and Equipment
Gas chromatograph. with a flame ionization detector (FID) and a headspace sampler.
A.4.3 Reference chromatographic conditions
A.4.3.1 Column. polyethylene glycol bonded fused silica capillary column (column length of 30m, an inner column diameter 0.25mm, film thickness
0.25μm), or other equivalent column.
A.4.3.2 Carrier gas. nitrogen gas (purity ≥99.99%).
A.4.3.3 carrier gas flow rate. 5.0mL/min.
A.4.3.4 Column temperature. 40 ℃ maintained 5min, at 10 ℃/min temperature was raised to 120 ℃, holding 2min, and finally at 16 ℃/min warmed to
200 ℃, maintained 5min.
A.4.3.5 Inlet temperature.200 ℃.
A.4.3.6 detector temperature. 250 ℃.
A.4.3.7 Injection volume. 1mL.
A.4.3.8 split ratio. 1.50.
Headspace Conditions Reference A.4.4
A.4.4.1 headspace vial temperature. 80 ℃.
A.4.4.2 vial Ping Heng time. 30min.
A.4.5 Analysis step
Preparation of blank solution A.4.5.1
Pipette 2mL water and placed in a vial, bottle quickly pressed aside.
Preparation of standard solution A.4.5.2
Preparation of standard solution of methanol A.4.5.2.1
Weigh 0.1g of methanol, accurate to 0.001 g of, diluted with water and transferred to a 1000mL volumetric flask, the water was added to the mark, i.e.,
To give 100mg/L standard stock solution in methanol. The solution was formulated as a range of concentrations of 50mg/L, 20mg/L, 10mg/L, 5mg/L
And 2.5mg/L in methanol standard solution. Pipette solution of the above-described series of concentrations 2mL, were placed in a vial, bottle quickly pressed aside.
Preparation of standard solution of ethanol A.4.5.2.2
Weigh 0.1g of ethanol, accurate to 0.001 g of, diluted with water and transferred to a 100mL volumetric flask, the water was added to the mark,
I.e., obtain 1000mg/L standard stock solution in ethanol. The solution was formulated as a range of concentrations of 750mg/L, 500mg/L,
200mg/L, 100mg/L and 50mg/L ethanol standard solution. Pipette solution of the above-described series of concentrations 2mL, were placed in vial
, The bottle quickly pressed aside.
Preparation of sample solution A.4.5.3
1.0g sample was weighed to the nearest 0.001 g of, transferred to a 10mL volumetric flask and dissolved in water, sonicated at room temperature for about
3min, diluted with water to the mark. The solution was pipetted placed on top of 2mL vial, bottle quickly pressed aside.
A.4.6 Determination
Under reference operating conditions (A.4.3 and A.4.4), each blank solution, the standard solution and the series of a sample solution, recording A
Peak area alcohol or ethanol. Solution Series standard chromatogram peak area value of ethanol, methanol or the Y-axis to correspond to the concentration of the solvent
(Mg/L) as the X-axis, the standard curve, a calibration curve of methanol or ethanol to obtain a standard curve. The sample solution chromatogram methanol or ethanol
Peak area alcohol concentration in the sample from the standard curve determined in methanol or ethanol solution (mg/L).
A.4.7 calculation results
W methanol or ethanol content in milligrams per kilogram (mg/kg) basis, according to formula (A.4) is calculated.
w =
c × V
(A.4)
Where.
--- C from the standard curve obtained from the sample solution methanol or ethanol concentration, in milligrams per liter (mg/L);
V --- volume of the sample solution, in milliliters (mL);
--- m sample mass, in grams (g).
Appendix B
Referring chromatogram of the standard mixture solution
Referring chromatogram of the standard mixture solution is shown in Figure B.1.
Note. The measured mixed standard solution, the concentration of rebaudioside F is about 0.1mg/mL, a concentration of the remaining components of 0.5mg/mL.
Figure B.1 schematic chromatography reference standard mixture solution
......
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