ESTIMATION OF AFLATOXIN BY HPLC ON FOOD BY ARTIFICIALLY INDUCED CONDITION OF FUNGAL GROWTH

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Abstract:

Toxins are of many types that include the bacterial toxins, plant toxins and several other toxins produced by micro-organisms. But of all toxins, fungal toxins are the most potent one. The fungal toxins are produced by several pathogenic strains of fungi. Food that most human consume are mainly prone to, fungal contamination, if the preservation is not adequate. There are several factors that contributes to growth of fungi, from the environment, that includes moisture, pH, type of food, light etc. Aflatoxins of most toxins are produced by Aspergillus species of fungi, such as A.flavus and A. parasiticus. The umbrella term aflatoxin refers to four different types of mycotoxins produced, which are B1, B2, G1, and G2. Aflatoxin B1, the most toxic, is a potent carcinogen and has been directly correlated to adverse health effects, such as cancer, in many animal species. Our aim of study was to use different kind food source as media for growth of A.flavus in different environmental conditions with different growth parameters. The estimation of the toxin was done by HPLC and a comparative study of toxin produced was used to judge the storage and proper condition how the food needs to be stored.

Chemical Nature:

 The four most prevalent aflatoxins are B1, B2,G1, and G2, whose chemical structures are shown in Figure 1. Aflatoxins are quite stable in many foods and are fairly resistant to degradation. Collectively, the aflatoxins are chemical derivatives of difuranocoumarin. They fluoresce under UV radiation, and fluorescence detection is often used for trace analysis in HPLC. Aflatoxins have no polar functional groups, and can be separated by virtue of their hydrophobicity.

 

Standards for the estimation:

The standards of Aflatoxin (G2, G1, B2 and B1–Supelco) was diluted in methanol to 200 ng/mL solution of G2, 200 ng/mL of B2, 60 ng/mL of G1, and 60 ng/mL of B1. This solution is diluted 10 times (G2, B2 = 20 ppb, and G1, B1 = 6 ppb), 20 times (G2, B2 =10 ppb, andG1, B1= 3 ppb), and 50 times (G2, B2 =4 ppb, and G1, B1= 1.2 ppb). These solutions are considered as standard solutions.  

 

Method & Materials:

Condition for growth of the Fungi:

The fungi were grown in the laboratory condition with following parameters:

  • Sample (Bread & Multigrain) N2– 10 gms of bread and 10 gms of crushed multigrain was mixed together and exposed to room temperature in dark.
  • Sample (Multigrain) N1– 20 gms of crushed multigrain was taken & exposed to room temperature
  • Sample (Multigrain) PH1– pH has been lowered to acidic to enhance the growth of mould to 5.5.
  • Sample (Bread) B: Growth promoted at 25 oC in normal pH Condition.
  • Sample (Multigrain) PH2– pH has been lowered to acidic to enhance the growth of mould to 4.

Samples:

 

The samples obtained are extracted with 70/30 v/v  methanol solution by vigorous shaking and simultaneous filtration of the extract by Whatman filtration.In different storage condition some samples are prepared. Due to higher concentration, these samples are diluted 50 times and injected in HPLC.

 

Experimental Conditions:

 

HPLC Conditions:

Column: Brownlee C18, 4.6 mm ×250 mm,5 μm

Mobile phase: Water/MeOH; 30/70 (v/v)

Flow rate: 0.6 mL/min

Temperature: 30 0C.

Detector: Fluorescence detector.

Excitation-365 nm, Emmission-435 nm

Injection volume: 20 μL .

Analytes: Listed in order of elution

  1. aflatoxin G2,
  2. aflatoxin G1,
  3. aflatoxin B2,
  4. aflatoxin B1,

 

The instruments were controlled and the data analyzed using the TNV-data system. No step changes of the excitation and emission wavelengths were used during the run.

 

 

 

Results and Discussion:

 

All four aflatoxins were separated using an isocratic binary mixture of water and methanol. A baseline separation was achieved in less than 6 min.

Standard

G2

G1

B2

B1

Retention time (min)

3.496

4.551

5.291

5.764

Area of 10 times diln solution

609591.16

438225.08

1794826.93

3309818.05

Area of 20 times diln solution

332910.99

363231.81

708210.14

1366080.13

Area of 50 times diln solution

129755.63

267206.00

273719.41

567360.04

Recovery of 20 times solution

243233.73

446216.60

807085.22

1470000.70

Recovery:  G2 –73.0%,G1 – 123%, B2 – 114%, B1 -75.5%.

 

For Samples:

 

Samples

G2 (Area)

G1 (Area)

B2 (Area)

B1 (Area)

N1 X 50*

275803.21

264255.94

–

–

N2 X 50*

175934.08

303515.13

–

–

PH1 X 50*

155442.43

258532.52

–

–

BX 50*

1550129.33

1080556.16

21277.82

157687.97

PH2 X 50*

1112236.45

996755.05

76938.57

–

 

Results in ppb:

Results (ppb)

G2

G1

B2

B1

 N1 X 50*

414.22

109.12

–

–

N2 X 50*

264.23

125.34

–

–

PH1 X 50*

233.46

106.76

–

–

B X 50*

2.328

446.23

15.02

17.31

 PH2 X 50*

1670.47

411.61

28.16

–

(50* times diluted)

Following Chromatogram of the samples are given below:

 

 

Description of the Figure:

  Figure 1: Chromatogram of N1

  Figure 2: Chromatogram of N2

  Figure 3: Chromatogram of PH1

  Figure 4: Chromatogram of B

  Figure 5: Chromatogram of PH2

Discussion:

 

 So according to the data obtained, the Sample B contains 4 types of aflatoxin altogether with having optimum growth condition for fungal growth, whereas in the Sample pH2 do have high content of G2 , G1 due to lowered pH with high concentration of aflatoxin.

Contributed by: Mr. Dipan Roy, Mr. Sudip Roy