SN/T 5216-2020Commercial kit detection method Nisin Method 1 (English PDF)

Foreword

This standard was drafted in accordance with the rules given in GB/T 1.1-2009.

This standard was proposed and managed by the General Administration of Customs of the People's Republic of China.

Drafting organizations of this standard. Urumqi Customs of the People's Republic of China, Beijing Qinbang Biotechnology Co., Ltd.

1 Scope

This standard specifies the enzyme-linked immunoassay method for nisin in food.

This standard is applicable to the detection of the content of nisin A and nisin Z in foods such as milk-containing beverages and meat products.

2 Normative references

The following documents are indispensable for the application of this document. For dated reference documents, only the dated version applies to this article Pieces. For undated reference documents, the latest version (including all amendments) is applicable to this document.

GB/T 6682 Analytical laboratory water specifications and test methods SN/T 2775 Evaluation Method of Commercial Food Testing Kit

3 Terms and definitions

The following terms and definitions apply to this document.

Nisin

4 Method summary

The nisin in the sample and the antigen pre-coated on the microwell strip in the kit compete for the nisin antibody, add enzyme label two After anti-immunization, use the substrate color developing solution to develop the color. The sample absorbance value is negatively correlated with the content of nisin, which is compared with the standard curve and then multiplied by the corresponding dilution factor can be used to obtain the content of nisin in the sample.

5 Materials and reagents

Unless otherwise specified, all reagents are analytically pure, and the water is the first grade water specified in GB/T 6682.

5.1 Nisin test kit. Refer to SN/T 2775 for kit evaluation, and refer to Appendix A for specific evaluation results. Kit composition as follows.

a) 96-well microtiter plate. 12 strips × 8 wells (coated with coupled antigen);

b) Nisin standard. 0 mg/L, 0.1 mg/L, 0.5 mg/L, 2.5 mg/L, 12.5 mg/L, 62.5 mg/L;

c) High-concentration standard. 100 mg/L;

d) Enzyme marker concentrate;

e) Antibody working solution;

f) Concentrated cleaning solution;

g) Sample extraction solution;

h) Chromogenic solution. Substrate A, Substrate B;

i) Stop solution.

The kit should be stored at 4 °C ~ 8 °C protected from light, and the dissolved enzyme label should be frozen at -15 °C.

5.2 Hydrochloric acid.

5.3 Sodium hydroxide.

5.4 n-hexane.

6 Instruments

6.1 Balance. the range is 100 g, and the sensitivity is 0.01 g.

6.2 Microplate reader. wavelength 450 nm.

6.3 Vortex apparatus.

6.4 Centrifuge.

6.5 Hybrid oscillator.

6.6 Single channel pipette. 20 µL~200 µL, 100 µL~1 000 µL, 500 µL~5 000 µL.

6.7 Multi-channel pipette. 30 µL ~ 300 µL.

6.8 Polystyrene centrifuge tube. 2 mL, 10 mL, 50 mL.

6.9 Mortar or crushing device.

7 Preparation and extraction of samples

7.1 Milk and beverage liquid samples. Dilute the sample 10 times with the sample extract and take 50 µL for analysis. The sample dilution factor is 10.

7.2 Solid samples of dairy products and meat products. Weigh 25 g (accurate to 0.01 g) samples, and use a mortar or crushing device to make the samples in powder form or granular form with a diameter of less than 0.3 mm, weigh 1 g (accurate to 0.01 g) of the prepared sample in powder form to a 50 mL centrifuge tube Add 5 mL of sample extract, vortex and mix for 5 min, centrifuge at 3,000 r/min for 5 min, then stand for 10 min, and extract the supernatant Put 1 mL into the centrifuge tube (be careful not to suck the oil on the surface and the sediment on the bottom layer), and take 50 µL for analysis. The sample dilution factor is 5.

8 Analysis steps

9 Result calculation

9.1 Read the OD value, press (1) to calculate the percent absorbance value (B) of each standard solution and sample.

9.2 Take the percentage absorbance of the standard product as the ordinate (%) and the concentration of nisin standard solution (mg/L) as the abscissa, draw Standard working curve, substitute the percent absorbance of the sample into the standard curve, and read the nisin corresponding to the sample from the standard curve After the concentration of, the result is calculated according to formula (2).

9.3 If the B value of the sample is higher than the B value range of the standard working solution, the sample should be diluted and tested again.

10 Confirmation test

If the content of nisin in the tested sample exceeds the limit requirement, other methods shall be used for confirmation.

11 Method performance indicators