Determination of Natamycin1

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Natamycin is a polyene antifungal drug used as a food preservative. It is produced by certain Streptomyces bacteria and works by inhibiting the growth of yeasts and molds on food surfaces like cheese rinds.

Natamycin is used as a preservative in food to prevent superficial mold growth on cheese rinds and other foods. It has the advantage of not affecting taste or appearance.

Samples are extracted by adding methanol, shaking, incubating in the dark, centrifuging, and transferring the liquid layer for dilution and analysis.

Ultraviolet Spectrophotometric

Method for the Determination of


Natamycin in dairy products

Friday 27/3/2020
Rapid Ultraviolet Spectrophotometric and Liquid Chromatographic
Methods for the Determination of Natamycin in Lactoserum Matrix
JOURNAL OF AOAC INTERNATIONAL VOL. 83, NO. 4, 2000
Definition:
Natamycin, also known as pimaricin, is a polyene antifungal drug (Chemical
Abstracts Service Registry No. 7681-93-8) with the chemical formula
C33H47NO13. The polyene compounds are so named because of the conjugated
double bonds that constitute part of their macrolide ring structure.

The polyene antibiotics are all products of Streptomyces species; natamycin is


produced by the actinomycete S. natalensis and also by S. chattanoogensis. In
general, polyene antibiotics are unstable compounds that lose their biological
activity in solution when exposed to air, heat, light, and chemicals such as acids
and bases through oxidation.
Use:
Natamycin is active against both yeast and molds without interfering with bacterial
fermentation or ripening processes. The principal use of natamycin is as a preservative
in food to prevent superficial mold growth in cheese rind, with the advantage over
other preservatives that it does not affect the taste and appearance of the cheese.

Regulations and Norms:


The use of natamycin in lactoserum handling is not clearly addressed in the
regulations of many countries. The U.S. Food and Drug Administration specifies
that this microbial-derived ingredient is an additive that may be applied only to
the cheese surface with limitations. The European Union has set a draft limit (1
mg/dm2 ) on cheese rind, as reported in the International Dairy Federation
(IDF) standard. The Spanish regulation establishes a limit of 1.2 mg/dm2 on the
surfaces of sausages and cheeses However, no regulations have been
established.
Common methods of analysis:

1- UV VIS spectrophotometer

2- HPLC high performance liquid chromatography


Uv-Vis spectro methods: AOAC
1- Reagents:
• Water (Milli-Q) Or LC quality.
• Natamycin standard stock solution.—Dissolve 500 mg
natamycin (high purity) in 1 L methanol Store stock solution in
dark bottle at 4C; solution is stable fo 2 weeks.
• Working solutions.—Prepare by appropriate dilutions with
water–methanol (1 + 3, v/v).

2- Equipment's and glassware's:


• Spectrophotometer UV vis with quartz cuvette
• Centrifuge
• Volumetric flasks
• Micro-pipet
• Erlenmeyer
Extraction and Analysis:
Sample preparation:
A 2 mL portion of lactoserum or an appropriate dilution was collected in a
glass centrifuge tube, and 6 mL methanol was added. The tube was shaken
mechanically for 1 min and placed in the dark for 60 min. The tube was
then centrifuged at 4000 × g for 30 min. The liquid layer was transferred to
a 10 mL volumetric flask and diluted to volume with water–methanol (1 + 3,
v/v). This solution was analyzed by UV spectrophotometry.
2ml sample
+ Shake for 1min then store 60min in dark
6 ml methanol

Centrifuge for 30min at 4000rpm

Liquid layer transferred to 10ml volumetric flask and


we continue to mark line by methanol-water (3-1)
Analysis:

UV Spectrophotometry Procedure Appropriate dilutions of the natamycin stock


solution were made with water–methanol (1 + 3, v/v) to obtain standard solutions
containing natamycin at 0.5–8.0 mg/L in 10 mL volumetric flasks.

UV spectra of the standard solutions were recorded between 325nm against a 1 cm


quartz reference cell filled with methanol.

Calibration curve: absorbance vs concentration of working solutions


Linear equation with good correlation factor > 0.99
Determination area with
selectivity and good sensitivity
Performance analytical method:

Analysis by UV VIS:
• Blank with methanol
• Calibration curve linear withR2>0.992

 Extraction method: Recovery test

• Spike lank solution with 5mg/L ofNatamycin standard


• Extract accordind to described method
• Determine the concetration according calibration curve
𝑐𝑜𝑛𝑐 𝑜𝑏𝑡𝑎𝑖𝑛𝑒𝑑
• Calculate the recovery percentage = ∗ 100
𝑐𝑜𝑛𝑐 𝑠𝑝𝑖𝑘𝑒𝑑
Analysis of unknown samples:
 Extraction method described slide8
 Analysis by Uvvis Spectro at 325 nm
 Determination for the concentration in extract according calibration curve
 Determination of the concentration in dairy sample As:

10
 C mg/Kg=𝐶𝑐𝑎𝑙𝑖𝑏 𝑐𝑢𝑟𝑣𝑒 ∗
2
 10ml is the volume of volumetric flask so final volume of extract
 2 ml is the volume of sample
Principle of UV vis spectrophotometer;

𝐴 = 𝑒𝑝𝑠𝑖𝑙𝑜𝑛 ∗ 𝐶 ∗ 𝑙

Select one wavelength

Absorbance value
Deuterium lamp for UV range
Tungsten lamp for visible range
Cmg/L working solution Absorbance
0.5 0.02
1 0.042
2 0.08
4 0.145
5 0.185 Absorbance
0.35 y = 0.0355x + 0.0054
8 0.289 R² = 0.9993
0.3

0.25

Absorbance of unknown sample


0.2

0.15

0.1

0.05

0
0 1 2 3 4 5 6 7 8 9

Concentration calibration curve in mg/L

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