Natamycin is a natural antifungal compound produced by Streptomyces, inhibiting molds and yeasts without affecting bacteria. Natamycin is widely used for surface preservation in baked goods, mooncakes, meat products, cheese products, and more. This protocol is based on GB/T 21915-2008 and aims to establish a sensitive, accurate, and reproducible detection method to meet the routine monitoring needs of food manufacturers and regulatory authorities for natamycin content. This method exhibits excellent linearity (R=1.0000) within the range of 1-30 μg/mL and achieves a spiked recovery rate of 102.7%, enabling reliable assessment of natamycin residues in complex matrices such as cake.
Standards: Natamycin (content≥98.3%).
Reagents: Methanol (chromatographic grade), glacial acetic acid (analytical grade), deionized water (18.2MΩ).
Stock Solutions: Dissolve 10mg of natamycin in mobile phase and dilute to 10mL in a volumetric flask to obtain 1mg/mL.
Series Working Solutions: Dilute the standard solution to concentrations of 1, 2, 5, 10, 20, 30 μg/mL.
Sample: Accurately weigh 5g of sample into a 50mL centrifuge tube. Add 30mL of methanol, sonicate for 30min. Add 10mL of water, mix well, then centrifuge at 3500r/min for 5min. Filter the supernatant through a 0.45μm membrane filter before analysis.
Spiked Sample: Accurately weigh 5g of sample into a 50mL centrifuge tube. Add 0.4mL of 100μg/mL natamycin standard solution, then add 29.6mL of methanol. Subsequent steps are the same as for the sample.
Liquid Chromatography System: EClassical 3200 (including P3200 high-pressure constant flow pump, D3200 UV-vis detector, S3200 autosampler, O3220 column oven, etc.)
Pretreatment Equipment: Solvent filter apparatus, diaphragm vacuum pump, ultrasonic cleaner, precision electronic balance, vortex mixer, centrifuge, volumetric flasks, micropipettes, etc.
Standard Separation
Retention time for natamycin was 3.03 min, tailing factor 1.01 (Figure 1).
Figure 1. Chromatogram of 5μg/mL standard solution of natamycin
Linearity
Within 1~30μg/mL, linear equation y=43.862x+3.9647 for natamycin, R=1.0000.
Sample Analysis
Sample analysis results of a commercial Swiss roll cake sample was shown in Figure 2 and 3. The content of natamycin was 0.41 g/kg, with a spiked recovery rate of 102.7%.
Figure 2. Chromatogram of natamycin (peak 1) in Swiss roll cake sample
Figure 3. Chromatogram of natamycin (peak 1) in Swiss roll cake sample solution spiked with 1ug/mL of natamycin
