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GB 5009.261-2016 English PDF (GB5009.261-2016)

GB 5009.261-2016 English PDF (GB5009.261-2016)

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GB 5009.261-2016: Determination of neurotoxic shellfish poisons in shellfish for export -- Mouse biology method

This Standard specifies the method of mouse biology for the determination of neurotoxic shellfish poisoning (NSP) in shellfish. This Standard is applicable to the determination of neurotoxic shellfish poisoning (NSP) in shellfish.
GB 5009.261-2016
GB
NATIONAL STANDARD OF THE
PEOPLE REPUBLIC OF CHINA
National Food Safety Standard - Determination of
neurotoxic shellfish poisoning in shellfish
ISSUED ON. DECEMBER 23, 2016
IMPLEMENTED ON. JUNE 23, 2017
Issued by. National Health and Family Planning Commission of PRC;
China Food and Drug Administration.
Table of Contents
Foreword ... 3
1 Scope ... 4
2 Principle ... 4
3 Reagents and materials ... 4
4 Apparatus ... 5
5 Analysis steps ... 5
6 Expression of analysis results ... 7
Annex A Conversion between mouse death time and mouse unit ... 9
Foreword
This Standard replaces SN/T 1573-2013 ?€?Inspection of neurotoxin shellfish poison in shellfish - Method of mouse biology?€?.
Compared with SN/T 1573-2013, the main deviation in this Standard is as follow. - changed the standard name as ?€?National Food Safety Standard -
Determination of neurotoxic shellfish poisoning in shellfish?€?.
National Food Safety Standard - Determination of
neurotoxic shellfish poisoning in shellfish
1 Scope
This Standard specifies the method of mouse biology for the determination of neurotoxic shellfish poisoning (NSP) in shellfish.
This Standard is applicable to the determination of neurotoxic shellfish poisoning (NSP) in shellfish.
2 Principle
Use ether to extract the neurotoxic shellfish poisoning in shellfish. After the extract is pressure-reduced and evaporated to dryness, use 1%Tween-60
saline as dispersion medium to prepare NSP-1%Tween-60 saline suspension. Inject this suspension into abdominal cavity. Observe the survival of the mouse. Calculate its virulence.
3 Reagents and materials
Unless otherwise stated, the reagents used in this method are analytically pure; the water is grade one water specified in GB/T 6682.
3.1 Reagents
3.1.1 Hydrochloric acid (HCl).
3.1.2 Anhydrous ether (C4H10O).
3.1.3 Tween-60 (C64H126O26).
3.1.4 Sodium chloride (NaCl).
3.1.5 Sodium hypochlorite (NaClO).
3.2 Reagent preparation
3.2.1 Sodium chloride solution (0.85%). weigh 0.85 g of NaCl, add water to dissolve and set volume to 100 mL.
3.2.2 1%Tween-60. weigh 1.0 g of Tween-60, use 0.85% sodium chloride
solution to dissolve and set volume to 100 mL.
3.2.3 Sodium hypochlorite solution (5%). weigh 50 g of sodium hypochlorite, use water to dissolve and dilute to 1000 mL.
3.3 Standard product
Short naked toxin analog (brevetoxin-2), purity ??? 90%.
3.4 Materials
Mouse. healthy ICR strain male mouse weighing 19g ~ 21g.
4 Apparatus
4.1 Rotary evaporator.
4.2 Homogenizer.
4.3 Balance. resolution of 0.1 g.
4.4 Separating funnel.
4.5 Round-bottomed flask.
4.6 Brinell funnel.
4.7 Disposable syringe. 1mL.
4.8 Centrifuge. rotating speed ??? 6000 r/min.
4.9 Metal screen. aperture of about 2mm.
5 Analysis steps
NOTE. The used equipment such as rubber gloves, glass products, and discarded extracts shall be soaked in sodium hypochlorite solution (5%) for more than 1h so as to decompose toxins.
5.1 Specimen preparation
5.1.1 Sample collection
At least 10 shellfish shall be taken for each analysis sample to make the shellfish more than 200g.
The frozen sample is placed in an insulation box sent for inspection. Or ensure it is at a low temperature state (0??C ~ 10??C) sent for inspection. If it is a shell sample, it shall be opened to remove moisture and then sent for inspection. 5.1.2 Specimen preparation
5.1.2.1 Fresh shell sample
Use clean water to wash the shell surface thoroughly. Cut off the muscle. Open shell. Use water to rinse the interior so as to remove sediment and other foreign objects. Separate the occipital muscle from the tissue attached to the gluing. Take out the shellfish. Never cut the flesh. Do not heat or use anesthetic before opening the shell. Collect 200g of shellfish and drain it on a metal sieve with a pore size of about 2mm for 5min. Pick up broken shells and other debris. Homogenize the shellfish, for use.
5.1.2.2 Frozen sample
At room temperature, make the frozen sample semi-frozen. For shell frozen sample, according to the method in 5.1.2.1, clean, open shell, rinse and take the meat. Remove the borneol attached to the outside of the shellfish, wipe off the water, and then relax at room temperature. Collect 200g of shellfish and drain it on a metal sieve with a pore size of about 2mm for 5min. Homogenize the shellfish, for use.
5.1.2.3 Canned shellfish
Drain the contents of the can. Pour into the homogenizer for full homogenization, for use.
5.1.2.4 Dried shellfish product
Weigh 100g of dried product into sufficient water, soaking 24h ~ 48h
(refrigerated at 4??C). Drain, homogenize, for use.
5.1.2.5 Salted product
Use clean water to rinse. Use running water to desalt. Drain, homogenize, for use.
5.2 Specimen extraction
Take 100g of specimen into a 500mL beaker. Add 5g of sodium chloride and 1mL of concentrated hydrochloric acid. Mix them well. While stirring, heat the mixture to it is boiling. Boil it on a slow fire for 5min. Cool to room temperature. Transfer the mixture to a 500mL centrifuge tube. Use 50mL of ether to rinse the beaker. Move the rinse together into the centrifuge tube. Add 100mL of ether into the centrifuge tube. Cap a plug. Shake thoroughly. Centrifuge at 6000 r/min for 15min.
After centrifugation, carefully pour off the ether layer (upper layer) solution into a 1000mL separatory funnel. Use ether to repeat the extraction of the
precipitate in the centrifuge tube three times. The total amount of extraction for three times is 250 mL of ether. Transfer the ether layer liquid to the separatory funnel. Gently oscillate (cannot produce emulsion). Remove the water layer (lower layer) and shellfish fragments after static layering and stratification. Transfer the ether layer to a 500mL round bottom flask. Conduct concentration under reduced pressure (rotary evaporator, 35??C ?? 1??C) to remove ether. Use 1%Tween-60 saline to dilute the concentrate to 10 mL in a graduated tube. Make a full shake. Prepare it to a uniform NSP-1%Tween-60 saline suspension. At this time, the amount of 1 mL of liquid corresponds to the weight of 10 g of shelled shellfish measured in advance. Take this suspension as test stock solution for animal experiment.
5.3 Mouse test
Choose 6 healthy ICR strain male mice with a body weight of 19g ~ 21g. Divide them into 2 groups randomly. testing sample group and blank control group (1%Tween-60 saline), 3 mice/group.
Separately take 1 mL of testing solution or 1%Tween-60 saline for
intraperitoneal injection. If the extract overflows during the injection, the mouse must be discarded and another mouse shall be re-injected. Record the time of completion of injection and the time of death when the mouse stops breathing, and observe 930min continuously. If the median death time of the mice is within 2h, the test solution shall be diluted with 1%Tween-60 saline to re-inject until the death time of the mice is within 2h ~ 6h.
6 Expression of analysis results
6.1 Determination of the corrected mouse unit (CMU) of testing sample
According to the mouse death time of the testing sample (see Table A.1), the corresponding mouse unit number MU is found. Find the corresponding body weight correction coefficient based on the weight of the mouse (see Table A.2). The mouse unit of the same mouse is multiplied by the body weight correction coefficient to obtain the CMU of this mouse. Select the median CMU of 3 mice in the test group, which is the median CMU of the sample group. Use this value to conduct the calculation of 6.2.
6.2 Calculation of toxin virulence and expression of results
6.2.1 NSP virulence calculation
The NSP virulence in the sample is calculated according to formula (1). where,
X - NSP virulence in the sample, in mouse unit per gram (MU/g);
CMU - Median correction mouse unit of the testing group of the mice of testing sample, in mouse unit per milliliter (MU/mL);
DF - Dilution multiple.
NOTE. 10 - the unit is milliliter; 100 - the unit is gram.
6.2.2 Expression of results
In the case where the blank control mice are normal, the following judgments and expressions are made.
If the death time of the mouse is equal to 360min, the NSP virulence of the testing sample is equivalent to 0.2 MU/g.
If the median death time of the experimental group is less than 120min, the sample extract shall be diluted. Select another 3 mice for the test till the median death time is 120min ~ 360min. Calculate the mouse unit virulence of the sample based on the final dilution test results. Report the NSP virulence of this sample as. XXX MU/g.
If the median death time of the experimental group is greater than 360min, directly calculate and determine the virulence of the sample mouse. Report the NSP virulence of this sample as. XXX MU/g.
If all the mice in the experimental group do not die within 930min of observation, the NSP virulence of the sample shall be also reported as less than 0.1 MU/g. Annex A
Conversion between mouse death time and mouse unit
Refer to Table A.1 for the conversion between mouse death time and mouse unit.
Table A.1 -- Conversion between mouse death time and mouse unit
Table A.2 -- Correction coefficient for mouse weight
Death time / min Mouse unit / MU
Mouse weight / g Weight correction coefficient
Table A.2 (continued)
Mouse weight / g Weight correction coefficient

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