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GB 1886.355-2022 PDF in English


GB 1886.355-2022 (GB1886.355-2022) PDF English
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GB 1886.355-2022: PDF in English

GB 1886.355-2022
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National food safety standard - Food additive - Stevia
ISSUED ON: JUNE 30, 2022
IMPLEMENTED ON: DECEMBER 30, 2022
Issued by: National Health Commission;
State Administration for Market Regulation.
Table of Contents
Foreword ... 3 
1 Scope ... 4 
2 Molecular formula, structural formula, relative molecular mass ... 4 
3 Technical requirements ... 6 
Appendix A Testing method ... 8 
Appendix B Reference chromatogram for mixed standard solution ... 15 
National food safety standard - Food additive - Calcium
Stevia
1 Scope
This standard applies to the food additive stevia, which is obtained by extracting and
refining the leaves of Stevia Rebaudiana Bertoni as raw materials. The known
glycosides include stevioside, rebaudioside A, rebaudioside B, rebaudioside C,
rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside M, rebaudioside N,
rebaudioside O, dulcoside A, rubusoside, steviolbioside.
2 Molecular formula, structural formula, relative molecular
mass
2.1 Molecular formulas of 13 glycosides
Stevioside: C38H60O18
Rebaudioside A: C44H70O23
Rebaudioside B: C38H60O18
Rebaudioside C: C44H70O22
Rebaudioside D: C50H80O28
Rebaudioside E: C44H70O23
Rebaudioside F: C43H68O22
Rebaudioside M: C56H90O33
Rebaudioside N: C56H90O32
Rebaudioside Q: C62H100O37
Dulcoside A: C38H60O17
Rubusoside: C32H50O13
Steviolbioside: C32H50O13
2.3 Relative molecular mass of 13 glycosides
Stevioside = 804.87 (according to 2018 International Relative Atomic Mass)
Rebaudioside A: 967.01 (according to 2018 international relative atomic mass)
Rebaudioside B: 804.87 (according to 2018 international relative atomic mass)
Rebaudioside C: 951.01 (according to 2018 international relative atomic mass)
Rebaudioside D: 1129.15 (according to 2018 international relative atomic mass)
Rebaudioside E: 967.01 (according to 2018 international relative atomic mass)
Rebaudioside F: 936.99 (according to 2018 international relative atomic mass)
Rebaudioside M: 1291.29 (according to 2018 international relative atomic mass)
Rebaudioside N: 1275.29 (according to 2018 international relative atomic mass)
Rebaudioside O: 1437.44 (according to 2018 international relative atomic mass)
Dulcoside A: 788.87 (according to 2018 international relative atomic mass)
Rubusoside = 642.73 (according to 2018 international relative atomic mass)
Steviolbioside = 642.73 (according to 2018 international relative atomic mass)
3 Technical requirements
3.1 Sensory requirements
Sensory requirements shall meet the requirements of Table 2.
3.2 Physical-chemical indicators
Physical-chemical indicators shall meet the requirements of Table 3.
Appendix A
Testing method
A.1 General provisions
The reagents and water, which are used in this standard, refer to analytically pure
reagents and grade-3 water, which are specified in GB/T 6682, unless otherwise
specified. The standard titration solution, standard solution for impurity determination,
preparations, products, which are used in the test, shall be prepared in accordance with
the provisions of GB/T 601, GB/T 602, GB/T 603, unless otherwise specified. The
solution, which is used in the test, refers to the aqueous solution, when the solvent is
not specified.
A.2 Identification test
A.2.1 Chromatographic test of stevia
In the stevia content test, the chromatographic peaks of the 13 glycosides, in the
chromatogram of the sample solution, shall correspond to the mixed standard solution.
A.3 Determination of stevia content (on a dry basis)
A.3.1 Reagents and materials
A.3.1.1 Acetonitrile: Chromatographically pure.
A.3.1.2 Sodium dihydrogen phosphate: Chromatographically pure.
A.3.1.3 Phosphoric acid: Chromatographically pure.
A.3.1.4 Water: Grade-1 water specified in GB/T 6682.
A.3.1.5 Aqueous solution of acetonitrile: The volume ratio of acetonitrile and water is
30:70.
A.3.1.6 Sodium phosphate buffer (pH 2.6): Weigh 1.20 g of sodium dihydrogen
phosphate (NaH2PO4); dissolve it in 800 mL of water; use phosphoric acid to adjust the
pH to 2.6.
A.3.1.7 Rebaudioside A standard: Rebaudioside A content (mass fraction, on a dry basis)
≥ 99.0%.
A.3.1.8 Rebaudioside D standard: Rebaudioside D content (mass fraction, on a dry basis)
≥ 95.0%.
times of the 13 glycosides.
A.3.4.2 Preparation of standard solutions
Weigh 5.0 mg, 10.0 mg, 20.0 mg, 25.0 mg of rebaudioside A standard and 2.5 mg, 5.0
mg, 10.0 mg, 12.5 mg of rebaudioside D standard, accurate to 0.1 mg, respectively.
Place them in a 25 mL volumetric flask. Use acetonitrile aqueous solution to dissolve
it. Dilute it to the mark, to obtain the rebaudioside A standard solutions, which have
concentrations of 200 mg/L, 400 mg/L, 800 mg/L, 1000 mg/L, AND rebaudioside D
standard solutions, which have concentrations of 100 mg/L, 200 mg/L, 400 mg/L, 500
mg/L.
A.3.4.3 Preparation of specimen solution
Weigh 25.0 mg ± 5.0 mg of specimen (dry basis), accurate to 0.1 mg. Put it in a 50 mL
volumetric flask. Use acetonitrile aqueous solution to dissolve it. Dilute it to the mark,
to obtain a specimen solution.
A.3.5 Determination
A.3.5.1 Drawing of standard curve
Under the reference chromatographic conditions of A.3.3, carry out chromatographic
analysis, for 200 mg/L, 400 mg/L, 800 mg/L, 1000 mg/L rebaudioside A standard
solution and 100 mg/L, 200 mg/L, 400 mg/L, 500 mg/L rebaudioside D standard
solution, respectively. Record the peak area values, corresponding to the standard
solutions of each concentration. Take the peak area of rebaudioside A or rebaudioside
D, in the chromatogram of the standard solution, as the Y-axis; take the corresponding
solution concentration (mg/L) as the X-axis, to draw a standard curve, to obtain the
rebaudioside A standard curve and rebaudioside D standard curve, respectively, (forced
through the origin). From the standard curve, obtain the rebaudioside A linear function
slope (Ka) and the rebaudioside D linear function slope (Kd), respectively.
A.3.5.2 Determination of contents
Under the reference chromatographic conditions of A.3.3, perform chromatographic
analysis, on the mixed standard solution and specimen solution, respectively. Compare
the chromatogram of the specimen solution with the chromatogram of the mixed
standard solution (see Appendix B), to determine the peaks, which are corresponding
to each component in the chromatogram of the specimen solution. Record the peak area
of rebaudioside A, rebaudioside D, stevia, rebaudioside B, rebaudioside C, rebaudioside
E, rebaudioside F, rebaudioside M, rebaudioside N, rebaudioside O, dulcoside A,
steviolbioside, in the chromatogram of the specimen solution.
A.3.6 Result calculation
The mass fraction of the 8 glycoside contents (on a dry basis) is calculated, according
A.4.1 Instruments and equipment
PH meter.
A.4.2 Reagents and materials
Weigh 1 g of specimen. Dissolve it in 100 mL of carbon dioxide-free water. Use an acid
meter, to measure the pH of the specimen solution.
A.5 Determination of methanol and ethanol
A.5.1 Reagents and materials
A.5.1.1 Methanol: Chromatographically pure.
A.5.1.2 Ethanol: Chromatographically pure.
A.5.1.3 Water: Grade-1 water specified in GB/T 6682.
A.5.2 Instruments and equipment
Gas chromatograph: Equipped with hydrogen flame ionization detector (FID) and
headspace sampler.
A.5.3 Reference chromatographic conditions
A.5.3.1 Chromatographic column: Bonded polyethylene glycol fused silica capillary
column (column length is 30 m; column inner diameter is 0.25 mm; film thickness is
0.25 µm), or other equivalent chromatographic columns.
A.5.3.2 Carrier gas: Nitrogen (Purity ≥ 99.99%)
A.5.3.3 Carrier gas flow: 5.0 mL/min.
A.5.3.4 Column temperature: Hold at 40 °C for 5 min; heat up to 120 °C, at 10 °C/min;
hold for 2 min; finally heat up to 200 °C, at 16 °C/min; hold for 5 min.
A.5.3.5 Sample inlet temperature: 200 °C.
A.5.3.6 Detector temperature: 250 °C.
A.5.3.7 Injection volume: 1 mL.
A.5.3.8 Split ratio: 1:50.
A.5.4 Reference headspace conditions
A.5.4.1 Headspace bottle temperature: 80 °C.
A.5.4.2 Headspace bottle's equilibration time: 30 min.
A.5.5 Analytical procedures
A.5.5.1 Preparation of blank solution
Pipette 2 mL of water. Put it in a headspace bottle. Quickly press tightly the bottle cap.
Prepare for use.
A.5.5.2 Preparation of standard solutions
A.5.5.2.1 Preparation of methanol standard solution
Weigh 0.1 g of methanol, accurate to 0.001 g. Use water to dilute it. Transfer it into a
1000 mL volumetric flask. Add water to the mark. Shake well, to obtain 100 mg/L
methanol standard stock solution. Prepare this solution, into a series of methanol
standard solutions, which have a concentration of 2.5 mg/L, 5 mg/L, 10 mg/L, 20 mg/L,
50 mg/L. Pipette 2 mL of each of the above-mentioned series of concentration solutions,
place them in headspace bottle. Quickly press tightly the caps, to prepare for later use.
A.5.5.2.2 Preparation of ethanol standard solution
Weigh 0.1 g of ethanol, accurate to 0.001 g. Use water to dilute it. Transfer it into a 100
mL volumetric flask. Add water to the mark. Shake well, to obtain 1000 mg/L ethanol
standard stock solution. Prepare this solution, into a series of ethanol standard solutions,
which have a concentration of 50 mg/L, 100 mg/L, 200 mg/L, 500 mg/L, 750 mg/L.
Pipette 2 mL of each of the above-mentioned series of concentration solutions, place
them in headspace bottle. Quickly press tightly the caps, to prepare for later use.
A.5.5.3 Preparation of specimen solution
Weigh 1.0 g of specimen, accurate to 0.001 g. Use water to dissolve it. Transfer it into
a 10 mL volumetric flask. Ultrasonicate it for about 3 min, at room temperature. Add
water to dilute to the mark. Shake well. Pipette 2 mL of the solution, into a headspace
bottle. Quickly press tightly the caps, to prepare for later use.
A.5.6 Determination
Under the reference operating conditions A.5.3 and A.5.4, measure the blank solution,
standard series solution, specimen solution, respectively. Record the peak area value of
methanol or ethanol. Take the peak area of methanol or ethanol, in the chromatogram
of the standard series solution, as the Y-axis; take the corresponding solvent
concentration (mg/L) as the X-axis, to draw a standard curve, to obtain a methanol
standard curve or an ethanol standard curve. According to the peak area value of
methanol or ethanol, in the chromatogram of the specimen solution, obtain the
concentration (mg/L) of methanol or ethanol in the specimen solution, from the
standard curve.
......
Source: Above contents are excerpted from the PDF -- translated/reviewed by: www.chinesestandard.net / Wayne Zheng et al.