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GB 26400-2011: National food safety standards of food additives docosahexaenoic acid grease (fermentation)
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National food safety standards of food additives docosahexaenoic acid grease (fermentation)
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GB 26400-2011
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Basic data
| Standard ID | GB 26400-2011 (GB26400-2011) |
| Description (Translated English) | National food safety standards of food additives docosahexaenoic acid grease (fermentation) |
| Sector / Industry | National Standard |
| Classification of Chinese Standard | C54 |
| Classification of International Standard | 67.220.20 |
| Word Count Estimation | 10,134 |
| Date of Issue | 2011-03-15 |
| Date of Implementation | 2011-05-15 |
| Regulation (derived from) | Ministry of Health Bulletin No. 7 of 2011 |
| Issuing agency(ies) | Ministry of Health of the People's Republic of China |
| Summary | This Chinese standard applies to the use of crack pot algae (Schizochytrium sp.) Or I Ken's pot algae (Ulkenia amoeboida) or Karber hidden dinoflagellate (Crypthecodinium cohnii) strains, obtained by fermentation docosahexaenoic acid (DHA) oils. |
GB 26400-2011: National food safety standards of food additives docosahexaenoic acid grease (fermentation)
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National food safety standards of food additives docosahexaenoic acid grease (fermentation)
National Standards of People's Republic of China
National standards for food safety
Food additives
Docosahexaenoic acid oil (fermentation method)
2011-03-15 release
2011-05-15 implementation
Issued by the Ministry of Health of the People's Republic of China
National standards for food safety
Food additives
Docosahexaenoic acid oil (fermentation method)
1 Scope
This standard is applicable to the use of Schizochytrium sp. Or Ulkeniaamoeboida or
(Crypthecodiniumcohnii), produced by bio-fermentation of docosahexaenoic acid (DHA) grease.
2 structural formula and relative molecular mass
2.1 Structural formula
2.2 Relative molecular mass
328.5 (according to.2007 International Relative Atomic Quality)
3 technical requirements
3.1 sensory requirements. should be consistent with the provisions of Table 1.
Table 1 sensory requirements
The project requires a test method
Color light yellow to orange yellow
Odor has the unique smell of this product
Organizational state Oily liquid
Take the sample 10mL, placed in a test tube, in the natural light using the head
Measured the method to observe the color and appearance of the form, the use of nasal sniffing method
Check the smell
3.2 Physical and chemical indicators. should be consistent with the provisions of Table 2.
Table 2 Physical and chemical indicators
Item Index Test Method
Content (based on C22H32O2 triglycerides), w /% ≥ 35.0 Appendix A, A.3
No saponification, w /% ≤ 4.0 GB/T 5535.1 Extraction of ether
Moisture, w /% ≤ 0.1 GB 5009.3 Decompression drying method
Insoluble impurities, w /% ≤ 0.2 GB/T 15688
Solvent Residue/(mg/kg) ≤ 1.0 GB/T 5009.37
Acid value (in KOH)/(mg/g) ≤ 1.0 GB/T 5009.37
Peroxide value/(meq/kg) ≤ 5.0 GB/T 5009.37 Titration
Trans fatty acids, w /% ≤ 1.0 GB 5413.36
Aflatoxin B1/(μg/kg) ≤ 5.0 GB/T 5009.22
Total arsenic (in As)/(mg/kg) ≤ 0.1 GB/T 5009.11 Hydride atomic fluorescence spectrophotometry
Lead (Pb)/(mg/kg) ≤ 0.1 GB 5009.12 Graphite Furnace Atomic Absorption Spectrometry
4 other requirements
The product should be installed in a suitable dark container or nitrogen storage or vacuum preservation, the product of the best storage temperature of -5 ℃ below.
Appendix A
Testing method
A.1 Safety requirements
Some of the reagents used in this test method are toxic and corrosive and should be handled with care. Such as splashing on the skin should be immediately water
Rinse, severe should be treated immediately. When using flammable products, it is forbidden to use open flame.
A.2 General provisions
Unless otherwise stated, the reagents used are of analytical grade, and the test water conforms to the tertiary water specified in GB/T 6682-2008. Inspection
Standard titration solution, standard solution for the determination of impurities, preparations and products, in the absence of other requirements, according to GB/T 601,
GB/T 602 and GB/T 603.
A.3 Determination of docosahexaenoic acid (DHA) triglycerides
A.3.1 Reagents and materials
A.3.1.1 Docosahexaenoic acid (DHA) triglyceride standards. purity ≥99%.
A.3.1.2 tridecanoic acid triglyceride standards. purity ≥99%.
A.3.1.3 Potassium hydroxide.
A.3.1.4 anhydrous methanol.
A.3.1.5 n-heptane, chromatographic pure.
A.3.1.6 petroleum ether, boiling range, 30 ℃ ~ 60 ℃.
A.3.1.7 Potassium hydroxide solution (4 mol/L). Weigh 22.4 g of potassium hydroxide (A.3.1.3) In a beaker, add an appropriate amount of anhydrous A
Alcohol (A.3.1.4) to dissolve and transfer to a 100 mL volumetric flask. When the potassium hydroxide methanol solution is cooled to room temperature, it is diluted with anhydrous methanol
Release to the scale, shake.
A.3.1.8 Docosahexaenoic acid (DHA) triglyceride stock solution (10 mg/mL). 250 mg (accurate to 0.0001 g)
Dicyclohexaenoic acid (DHA) triglyceride (A.3.1.1) In a 25 mL volumetric flask, add n-heptane (A.3.1.5) to dissolve and dilute
Release to the scale, shake.
A.3.1.9 Tridecanoic acid triglyceride internal standard stock solution (10 mg/mL). Weigh 0.25 g (accurate to 0.0001 g) tridecanoic acid
Triglyceride (A.3.1.2) In a 25 mL volumetric flask, add n-heptane (A.3.1.5) to dissolve it and dilute it to the mark and shake it.
A.3.1.10 Preparation of standard working solutions. Appropriate amount of docosahexaenoic acid (DHA) triglyceride stock solution
(A.3.1.8) were transferred to 2.0 mL of tridecanoic acid triglyceride internal standard stock solution (A.3.1.9) in 6 10 mL volumetric flasks, respectively
Into n-heptane (A.3.1.5) diluted to the mark, shake.
Table A.1 Contents of docosahexaenoic acid (DHA) triglycerides in standard working solutions
Numbering
The volume of docosahexaenoic acid (DHA) triglyceride stock solution was added
mL
Concentration of docosahexaenoic acid (DHA) triglycerides in standard working solutions
mg/mL
2 0.1 0.1
3 0.5 0.5
4 1.0 1.0
5 2.0 2.0
6 3.0 3.0
A.3.1.11 nitrogen, purity ≥99.999%.
A.3.1.12 helium, purity ≥99.9999%.
A.3.2 Instruments and equipment
A.3.2.1 Gas Chromatography - Mass Spectrometer.
A.3.2.2 Gas Chromatograph with Hydrogen Flame Ionization Detector.
A.3.2.3 Analysis of balance. 0.01g, 0.1mg.
A.3.2.4 Centrifuge.
A.3.2.5 Shaker.
A.3.3 Qualitative analysis
A.3.3.1 Docosahexaenoic acid (DHA) grease
Weigh 0.25g (accurate to 0.01g) sample in a 50mL volumetric flask, add proper amount of n-heptane (A.3.1.5) to dissolve and dilute to
Scale, shake. Accurate absorption of 2mL n-heptane dissolved in 10mL covered with a centrifuge tube, add 0.2mL potassium hydroxide methanol solution
(A.3.1.7), oscillating more than 1 min for 10 min (methylation reaction). Such as organic layer turbidity, can be centrifuged to clarify. Take on
Layer of organic phase solution.
A.3.3.2 Determination
A.3.3.2.1 Gas chromatographic conditions
a) Column. DB-5ms (30.0m × 0.25mm × 0.25μm) quartz capillary column, or equivalent;
b) column temperature program. 100 ℃ for 3min, to 10 ℃/min rose to 260 ℃, keep 8min;
c) Carrier gas. helium (A.3.1.12);
d) Flow rate. 1.0 mL/min;
e) Inlet temperature. 250 ° C;
f) Injection mode. Split injection (split ratio 1.10);
g) Injection volume. 1 μL.
A.3.3.2.2 Mass spectrometry conditions
a) ion source. EI source, 70 eV;
b) Interface temperature. 280 ° C;
c) ion source temperature. 230 ° C;
d) quadrupole temperature. 150 ° C;
e) data acquisition mode. full scan;
f) Quality scanning range. 20m/z ~ 330m/z;
g) solvent delay 3.5 min.
A.3.3.3 Determination of results
The mass spectrum of the sample should be consistent with the mass spectrum of the docosahexaenoic acid (DHA) methyl ester standard (Appendix B, Figure B.1).
A.3.4 Quantitative analysis
Principle of A.3.4.1
Dodecahexaenoic acid (DHA) in the sample under the action of potassium hydroxide methanol to produce docosahexaenoic acid (DHA) methyl ester, with a
Hydrogen flame ionization detector was measured by gas chromatograph, and quantified by internal standard method. As a result, dicosylhexaenoic acid (DHA)
Ester.
A.3.4.2 Preparation of test solution
A.3.4.2.1 Preparation of docosahexaenoic acid (DHA) grease sample solution
Weigh 0.04g sample (accurate to 0.0001g) in 10mL volumetric flask, add 2.0mL tridecanoic acid triglyceride internal standard storage
(A.3.1.9), the following as A.3.3.1 "to join the appropriate amount of n-heptane (A.3.1.5) take the upper organic phase solution standby" operation. Each
Try to do at least two parallel samples.
A.3.4.2.2 Preparation of blank solution
Except that the sample is not treated as described in A.3.4.2.1.
A.3.4.3 Standard working solution methylation treatment
Respectively, accurately absorb A.3.1.10 series of standard working solution 2mL in 10mL covered with a centrifuge tube, the following according to A.3.3.1 from
"Add 0.2mL potassium hydroxide methanol solution (A.3.1.7) to take the upper organic phase solution for" operation.
A.3.4.4 Determination
A.3.4.4.1 Gas chromatographic conditions
a) Column. DB-23 (30.0m × 0.25mm × 0.25μm) quartz capillary column, or equivalent;
b) Column temperature program. 90 ℃ for 5min, 9 ℃/min rose to 240 ℃, keep 5min, 3 ℃/min rose to 250 ℃;
c) Carrier gas. nitrogen (A.3.1.11);
d) Flow rate. 1.33 mL/min;
e) Inlet temperature. 250 ° C;
f) Injection mode. Split injection (split ratio 1. 100);
g) detector temperature. 300 ° C;
h) Injection volume. 1 μL.
A.3.4.4.2 Drawing of standard curves
The standard working solution after methyl esterification was sequentially injected under the measurement conditions listed in A.3.4.4.1 to measure docosahexaenoic acid (DHA)
Methyl ester and tridecanoic acid methyl ester. The peak area of docosahexaenoic acid (DHA) methyl ester is different from that of the standard working solution
Hexahydrate (DHA) triglyceride is proportional to the concentration. The concentration of docosahexaenoic acid (DHA) triglycerides in standard working solutions
(mg/mL) as the abscissa, with the peak area ratio of docosahexaenoic acid (DHA) methyl ester and tridecanoic acid methyl ester as the ordinate,
curve. The chromatograms of docosahexaenoic acid (DHA) methyl ester and methyl tridecanoate are shown in Appendix C, C.1.
Calculate the regression parameters according to formula (A.1).
y = (a x x) b (A.1)
Where.
y - docosahexaenoic acid (DHA) methyl ester with tetramethylcarboxylic acid methyl ester;
a - the slope of the regression curve;
x - Concentration of docosahexaenoic acid (DHA) triglycerides in standard working solutions in milligrams per milliliter (mg/mL);
b - the intercept of the regression curve.
A.3.4.4.3 Determination of test solution
The blank solution (A.3.4.2.2) and the sample solution (A.3.4.2.1) were sequentially injected and the blank value was deducted to obtain docosahexaenoic acid
(DHA) methyl ester with tridecanoic acid methyl ester.
A.3.4.5 Calculation of results
The content of docosahexaenoic acid (DHA) triglyceride in the sample is calculated according to formula (A.2)
X1 = y-ba × V ×
m x 100%
(A.2)
Where.
X1 - content of docosahexaenoic acid (DHA) triglyceride in sample,%;
y - the peak area ratio of methyl dicitahydrohexaenoic acid (DHA) methyl ester to methyl tridecanoate after methyl esterification;
b - the intercept of the regression curve;
a - the slope of the regression curve;
V - the final volume of the test solution, in milliliters (mL);
m --- the quality of the sample in milligrams (mg).
Such as the amount of docosahexaenoic acid (DHA), multiplied by the conversion factor of 0.9597.
The calculated results are expressed as the arithmetic mean of the parallel measured values, leaving two digits after the decimal point.
A.3.4.6 Repeatability
The absolute difference between the two independent determinations obtained under repeatability shall not exceed 10% of the arithmetic mean.
Appendix B
(Docosahexaenoic acid (DHA) methyl ester standard gas chromatography - mass spectrometry
Note. The spectrum is derived from the NIST library.
Figure B.1 Standard map of docosahexaenoic acid (DHA) methyl ester
Appendix C.
Standard Practice for Gas Chromatography after Mixed Standard Working Solution Methylation
1 --- tridecanoic acid methyl ester (9.9 min);
2 - docosahexaenoic acid (DHA) methyl ester (18.8 min).
Figure C.1 Gas chromatogram of mixed standard working solution after methyl esterification
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