Food Hydrocolloids VolA no.3 pp.221-225 , 1990

Procedure for the detection of aflatoxins in gum arabic samples

C.J .Smith, P.A.Williams, M.Jones and G.O.Phillips

Faculty ofScience and Inno vation, The North East Wales Institute, Connah'sQ~y,ClwydCH54BR,UK

Abstract. In the light of recent warnings regarding the possible presence of aflatoxin in gum arabicsamples, the usefulness of a commercially available ELISA system for the detection of thesecompounds in food was studied as a screening technique for gum arabic. A series of gum arabicsamples were assayed, as were the same samples deliberately spiked with aflatoxin. The resultsconfirmed that the assay system was capable of determining aflatoxin in the concentration range2.0-200 .0 p.p .b . in the presence of gum arabic. None of the 16 untre ated samples contained anydetectable aflatoxin.

Introduction

Aflatoxins, the toxic metabolites of Aspergillus flavus and A.parasiticus, areconsidered to be the most potent carcinogens known. These compounds havebeen implicated in outbreaks of aflatoxicosis, chronic hepatitis , Reye's syn­drome in animals and human heptacellular carcinoma (1,2).

Contamination of foods , including peanuts, maize, cassava , rice, dried fish,beans , milk and beer often occurs in hot , humid climates (1,3-7), where theorganisms are indigenous. Recently A.flavus was identified on a twig from anAcacia senegal tree being tapped for gum arabic (8). Concern at the possibilitythat the organism might contaminate raw gum arabic has resulted in examinationof techniques for the detection of aflatoxins in gum arabic samples with theobjective of providing a reliable routine screening procedure. The existence ofan enzyme-linked immunosorbent assay (ELISA) kit for the detection ofaflatoxins in food (Biokits Ltd) meant that a system was available which mightbe applicable to gum arabic samples. It was, however , necessary to test thissystem since immunological assays are prone to non-specific interference whenused with systems other than those for which they were designed .

Materials and methods

Gum arabic

Samples of gum arabic (Table I) were donated by Agrisales Ltd, London, UK.Each sample was treated identically. Gum arabic (25 g) was weighed out and

transferred to a Waring blender bowl. Extraction buffer (5.0 mllg) , consisting ofacetonitrile:distilled water (50:50), was added to the sample. The mixture wasblended for 2 min in the covered bowl and the resultant slurry filtered (WhatmanNo. 4 filter paper) . The first 20 ml of filtrate was collected and used insubsequent analyses .

Preparation of test samples

The extracts, prepared as described, were diluted to 1:25 (sample: diluent

221

C.J.Smith et al,

Table I. Gum arabic samples examined

Code

ABCDEFGHIJKLMN

Table II. Gum arabic aflatoxin spiked standards

Sample

Negative controlPositive controlGum arabicGum arabic spiked with aflatoxinGum arabic spiked with aflatoxinGum arabic spiked with aflatoxinGum arabic spiked with aflatoxinSpray dried gum arabicSpray dried gum arabicspiked with aflatoxinSpray dried gum arabic spiked with aflatoxinSpray dried gum arabic spiked with aflatoxinSpray dried gum arabic spiked with aflatoxin

Expectedp.p.b.

<210

25

1020

25

1020

Meanabsorbance

1.5910.9821.5891.4341.1991.0160.6501.6191.5431.3531.1230.780

Description/source

Cleaned KordofanVerek/ChadMaliMauretanianNigerian Grade ICleaned KordofanNigerian grade 2MaliVerek/ChadNigerian grade 2Niger grade 1MauretanianNigerian grade 1Niger grade 1

Mean observedp.p.b.

<29.0 ± 1.768

<22.7 ± 0.4245.0 ± 0.4248.2 ± 0.0

23.0 ± 8.485<2

2.0 ± 0.0713.3 ± 1.988.0 ± 0.353

20.0 ± 1.414

buffer) by mixing 0.1 ml of sample with 2.4 ml of diluent buffer. The diluentbuffer is provided as part of the assay kit.

Preparation of 'spiked' samples

A series of aflatoxin standards in diluent buffer were prepared (see Table II forconcentrations) and used to dilute the gum arabic filtrates (1:25) to produce arange of 'spiked' gum arabic standards.

ELISA

The assay was carried out essentially as described in the manufacturer'sinstructions. The positive and negative controls provided with the assay kit wereused to confirm that all assay steps had been carried out correctly. All assayswere carried out in quadruplicate. Briefly, test and control samples (50 J.LI) wereadded to separate wells of a 96-well pre-sensitized microwell module followedby rat anti-aflatoxin antibody (50 J.LI per well). The microwell module was then

222

Detection of aflatoxins in gum arabic samples

2.0

1.8

1.6

1.4

1.2

r:n

~1.0

~

~0.8

0r:n~ 0.6

0.4

0.2

0.01 10

Standard (ppb)25

2050

200

100

Mean Absorbance (414nm)1.5471.2820.6940.3900.206

1000

AFlATOXIN (ppb)

Fig. 1. Total aflatoxin assay standard curve.

placed on an orbital plate shaker and shaken gently at room temperature for 2 h.After incubation the plate was emptied and the wells washed five times withwash buffer (0.02 mol/dnr' phosphate, 0.15 mol/dnr' NaCI, pH 7.2 containing 0.5mg/ml Tween 80 and 0.01 mg/ml thimerosal) and blot dried. To each well wasadded rabbit anti-rat IgG peroxidase conjugate (100 fLI). The plate wasincubated with shaking for a further 30 min then washed and blotted dryasabove. The enzyme substrate solution (100 fLI; 0.15 ul/ml HzOz, 0.6 mg/mI2,2'­azimobis-(3-ethylbenzthiazoline-6-sulphonate) in 0.1 mol/drrr' citrate buffer, pH4.0 was added and the plate was incubated for a further 30 min. Stop solution(50 fLI; 1.5% w/v sodium fluoride) was added to all wells and the absorbance(405 nm) was measured against a blank of enzyme substrate using a Multiskanplate reader (Flow Labs Ltd, Ayrshire, UK).

223

C.J.Smith et al,

Results

Standard curve

The initial step was the preparation of a standard curve using five samples (nogum arabic) of known aflatoxin concentration. The test was carried out asdescribed and the absorbance determined. The results were plotted to yield astandard curve (Figure 1) from which it is possible to determine the aflatoxinconcentration of any unknown sample for which the absorbance is known.

Analysis of aflatoxin in the presence of gum arabic

Two samples of gum arabic, cleaned Kordofan and spray dried Kordofan, wereused to measure their effect, if any, on the assay parameters. The samples wereextracted as described previously and the extracts divided into four fractions.One fraction of each sample was diluted 1:25 with diluent buffer. The remainingfractions were diluted with diluent buffer plus aflatoxin to give final aflatoxinconcentrations of 2, 5 and 10 p.p.b. respectively. All eight samples were

Table III. Aflatoxin results 18/9/89

Sample Expected ~ean absorbance Mean observedp.p.b. 414 nm p.p.b.

NC <2 1.131 <2PC 10 0.836 9.6 ± 0.495Gum arabic A 1.200 <2Gum arabic A spiked 10 0.899 7.0 ± 0.353Gum arabic B 1.142 <2Gum arabic B spiked 10 0.854 8.8 ± 0.707Gum arabic C 1.111 <2Gum arabic C spiked 10 0.806 10.0 ± 0.707Gum arabic D 1.186 <2Gum arabic D spiked 10 0.912 7.0 ± 0.707Gum arabic E 1.131 <2Gum arabic E spiked 10 0.847 8.6 ± 0.353Gum arabic F 1.216 <2Gum arabic F spiked 10 0.910 7.0 ± 0.353Gum arabic G 1.275 <2Gum arabic G spiked 10 0.846 8.6 ± 1.061Gum arabic H 1.265 <2Gum arabic H spiked 10 0.897 7.2 ± 1.061Gum arabic I 1.231 <2Gum arabic I spiked 10 0.931 6.6 ± 0.353Gum arabic J 1.273 <2Gum arabic J spiked 10 0.912 7.0 ± 0.212Gum arabic K 1.148 <2Gum arabic K spiked 10 0.865 8.0 ± 0.707Gum arabic L 1.198 <2Gum arabic L spiked 10 0.832 9.4 ± 0.707Gum arabic M 1.198 <2Gum arabic M spiked 10 0.900 7.0 ± 1.273Gum arabic N 1.245 <2Gum arabic N spiked 10 0.830 9.6 ± 1.061Mean unspiked 1.200Mean spiked 0.876 7.92 ± 1.159

224

Detection of aflatoxins in gum arabic samples

analysed using the ELISA and the results are presented in Table II. There wasno detectable aflatoxin in the two test samples, both gave absorbancesequivalent to less than 2 p.p.b., the lowest detectable level. In all cases thespiked samples gave results equivalent to the expected values based on theadded aflatoxin alone, showing that the assay is accurate and that it is notaffected by the presence of gum arabic.

Finally, a range of gum arabic samples (Table I) were tested as received andmixed with aflatoxin to give a final concentration of 10 p.p.b. This gave 28samples each of which was tested in duplicate using the ELISA technique. Noneof the natural tested samples was shown to contain aflatoxin at a level detectableby the assay. In contrast, all of the spiked samples had detectable aflatoxin levelsclose to the expected value (Table III).

The results presented here show that a commercial ELISA system designed todetect aflatoxin in peanut samples is equally sensitive when used to detect theanalyte in the presence of gum arabic. There was no detectable aflatoxin in anyof the different samples tested and deliberately added aflatoxin was readilydetected in the presence of all these samples. It may be concluded that the assaycan be used routinely for screening gum arabic for contamination with aflatoxin.

References

1. Busby,W.F. and Wogan,G.N. (1984) Aflatoxins. In Searle,C.F. (ed.), Chemical Carcinogens(2nd edn). American Chemical Society, Washington, D.C., pp. 745-1136.

2. Denning,D.W. (1987) Aflatoxin and human disease. A review. In De Glanville.H, (ed.), Adversedrug reactions and acute poisoning reviews. In press.

3. Alpert,M.E., Hutt,M.S.R., Wogan,G.N. and Davidson,C.S. (1971) Association betweenaflatoxin content of food and hepatoma frequency in Uganda. Cancer, 28, 253-260.

4. Peers,F.G., Gilman,G.A. and Linsell,C.A. (1976) Dietary aflatoxins and human liver cancer. Astudy in Swaziland. Int. J. Cancer, 17, 167-176.

5. Nwokolo,C. and Okonkwo,P. (1978) Aflatoxin load of common food in savanna and forestregions of Nigeria. Trans. R. Soc. Trop. Med. Hyg., 72, 329-332.

6. Okonkwo,P.O. and Obinu,C.N. (1981) Implications of seasonal variations in aflatoxin B levels inNigerian market foods. Nutr. Cancer, 3, 35-39.

7. Alozie,T.C., Rotini,C.N. and Oyibo,B.B. (1980) Production of aflatoxin by Aspergillus flavus(UBMI) in some Nigerian indigenous beverages and foodstuffs. Mycopathologica, 70, 125-128.

8. Abdalla,M.H. (1988) Isolation of aflatoxin from Acacia twigs and the incidence of Aspergillusflavus in the Sudan. Mycopathologia, 104, 143-147.

225