Mycotoxin Research, Vol. 3 (1987)

Reduction ofaflatoxin B, levels by sheep saliva

Saad M Magdi

Mycotoxins Lab, National Research Centre,Dokki, Cairo, Egypt

Abstract

This study determined the decrease of aflatoxin 8, by sheep saliva at concentrationsof 150 and 300 M9 aflatoxin 8,/L saliva. Analyses for aflatoxins 8" M" and afla­toxicol (Ral were performed after 2, 4, 6, 24, and 48 hours of incubation. Afla­toxin M, and Ro were not detected and only residues of aflatoxin 8, were found.4 to 13% of aflatoxin B, were decomposed by sheep's saliva within 2 hrs and 33 to43 % of aflatoxin 8, after 24 hrs. Decomposition was affected by the aflatoxinconcentration.

Decrease of aflatoxin 8, at 2, 4, 6 hrs was nearly three times higher at the lowconcentration (150 ppbl compared to the high concentration (300 ppb], After48 hrs incubation more than 80% of the initial aflatoxin B, had been decomposedby the saliva.

Introduction

Mycotoxins are toxic compounds produced by fungi, and aflatoxin B1 is the metabo­lite produced by Aspergillus flavus and A. parasiticus (1). Aflatoxins were detectedat biologically significant levels in a wide spectrum of foods and feedstuffs fromvarious parts of the world and especially from Asia and Africa (2). Aflatoxin B1 isthe most toxic and carcinogenic form and has been used experimentally in variousforms (3). Man and animals are exposed to aflatoxins through oral ingestion ofcommodities contaminated with aflatoxins or by consuming products of animals thathave ingested aflatoxin contaminated feedstuffs (4).

This study was conducted to determine the effect of sheep saliva on the detoxi­cation of aflatoxin B1 as the first line of defense against contaminated foods andfeedstuffs.

Materials and Methods

Sheep saliva

Saliva of adult sheep was collected from canulated submaxillary glands. The pH valueof the collected saliva was 7.4. Its chemical composition is given in Table 1.

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Table l: The chemical composition of sheep's salivaaccording to McDougall, 1948 (5)

Component mmol/L gm/L

NaHG03 117.00 9.80NaHP04 26.00 9.30

NaGI 8.00 0.47KGI 8.00 0.57

GaGI 0.20 0.04MgGI 2 0.30 0.06Mucus Trace TraceUrea Trace Trace

Aflatoxin standards

Aflatoxin HI, aflatoxin MI, and aflatoxicol (Ra) were received as crystals (Sigma,St Louis, Mo, USA). The purity was checked by comparison of molar absorptionand absorption peak ratios with values given in the AOAC tables (9). Aflatoxin HIwas dissolved in acetone: water (1 : 100 v/v) to give a total concentration of 50 Jlgaflatoxin HI/mL acetone-water (stock solution). Aflatoxin MI and aflatoxicol (Ra)were dissolved separately in benzene: acetonitrile (98 : 2 v/v) to give a concentrationof 5 -10 Jlg/mL.

Procedure

Sixty tubes of 200 mL volume each were filled with 30 mL saliva and divided intotwo equal groups. Aflatoxin HI was added at a concentration of 150 Jlg/L to thethe first group and at a concentration of 300 Jlg/L to the second group. This wasequivalent to 4.5 and 9 Jlg aflatoxin HI per tube of saliva, respectively. As control,another 60 tubes were filled with 30 mL distilled water, divided into two equalgroups, and likewise received aflatoxin HI at the two studied concentrations of 150and 300 ppb. The tubes were covered with plugs, incubated at 38 -39°C and shakenevery hour. Qualitative assay was carried out for aflatoxin MI and aflatoxicol (Ro) bytwo-dimensional thin layer chromatography (TLC) as described by Stubblefieldet al. (6) and high performance liquid chromatography (HPLC) as recommendedby Pons et al. (7). Quantitative analysis for these aflatoxins was conducted after2, 4, 6, 24, and 48 hrs of incubation in 6 replicates/ period. The intensity of fluores­cence of the aflatoxin spots were measured with a densitometer (TLD-lOO, vitatron,Holland) at an excitation wavelength of 365 nm and an emission wavelength of443 nm. To confirm the obtained data, aflatoxin HI was determined using HPLC(Waters Associates) with fluorescence detector (Model 420) and aflatoxin lamp.

Results and Discussion

Two hydroxy derivatives of aflatoxin HI (aflatoxin MI and Ra) were included inthe chemical assay as species differences exist in the in vitro metabolism of afla­toxin HI through hydroxylation and demethylation (8). No traces of aflatoxin MIor aflatoxicol (Ra) were found. All the aflatoxin residues were in the form of afla­toxin HI.

Aflatoxin HI was quantitatively determined after dilution and then added tosaliva media at zero time. The initial concentration of aflatoxin HI (4.5 or 9 Jlgaflatoxin HI/tube for the low and high concentration, respectively) was recordedas 100 %at zero time.

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Mycotoxin Research, Vol. 3 (1987)

Table2: Mean values of aflatoxin B,8 reduction (~g/tube)

by sheep's saliva andmean percentages (% of the initial)at different incubation sampling

Low levelb High levelcTest

period Blank Saliva Blank Saliva~g/tube % ~/tube % ~g/tube % ~g/tube %

ohr 4.5 0 4.5 0 9.0 0 9.0 02 hrs 0.2 3.3 0.6 13.1 0.3 3.2 0.4 4.34 hrs 0.2 4.0 0.8 17.1 0.3 3.4 0.5 7.16 hrs 0.2 4.7 1.5 35.3 0.3 3.7 1.0 11.1

24 hrs 0.4 7.1 1.9 42.4 0.5 5.2 3.0 33.548 hrs 0.8 8.9 4.1 91.3 0.5 5.8 7.2 80.2

a Aflatoxin M1 and aflatoxicol were negative on detectionb Low level of aflatoxin B1 (150~g/L saliva or blank)c High level of aflatoxin B1 (300~g/L salivaor blank)

Table2 shows that the rate of aflatoxin B1 detoxication was higher in the lowtoxin level (4.5 ~g) than in the high level (9 ~g) in the blank media. However, themaximum values of aflatoxin B1 degradation in blank media were obtained duringthe first two hours of incubation. During the period from 2 to 24 hrs the percentagesof aflatoxin B1 detoxication did not exceed 1.99 and 3.76 for the high and lowconcentrations, respectively.

Generally, the detoxication of aflatoxin B1 from blank media of both aflatoxinconcentrations had nearly the same trend. These findings correspond to those al­ready reported (10).

Data presented in Table2 indicate that maximum absolute and relative values ofaflatoxin B1 detoxication were recorded during the period of 24 to 48 hrs of incu­bation with sheep's saliva. On the other hand, the minimum values of aflatoxinB1 detoxication were obtained from 2 to 4 hrs of incubation. During the first twohours of incubation, the aflatoxin B1 detoxication was equivalent to 4.33 and 13.11 %(from the initial dose) for both high and low toxin levels, respectively. Kiesslinget al. (11) reported that aflatoxin B1 decomposition was about 4 %without indicat­ing the level during the first 2 hrs of incubation with buffer (not specified) and 5 %from 2 to 4 hrs of incubation.

More than 80 % of the initial aflatoxin B1 were decomposed within 48 hrs ofincubation with aflatoxin B1 concentrations of 150 and 300 ppb. Taking into con­sideration that the time, the aflatoxin B1 would be exposed to ruminant saliva nomore than 48 hrs, these results are in agreement with those obtained by Saad (10),working with the artificial saliva recommended by McDougall (5).

The results indicate that the effect of water on the detoxication of aflatoxinB1 did not exceed 8.86 % after 48 hrs of incubation. Since, sheep saliva contains98.5 % water and the rest represents the cation and anion content. These findingswere confirmed by Saad (10) using the same levels of aflatoxin B1 and artificialruminant's saliva. As aflatoxin B1 level increased the percentage decompositiondecreased (Table2).

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Acknowledgement

The author is grateful to Dr Douglas Park, Dept of Health and Human Service, FDA,USA for providing aflatoxicol and other standards.

References

1 Edds T (1979) Aflatoxins. Conference of mycotoxins in animal feeds and grainsrelated to animal health. FAD, Rockville, Maryland, June, 1979.

2 Tulpule P, Manabe M, Jemmali P, Hamilton P, and Patterson D (1977) Panel onaflatoxin; Mycotoxins in human and animal health, Rodricks, JV, Pathotox-Publ,pp 181-186.

3 Goldblatt LD and Stoloff L (1983) History and natural occurrence of aflatoxins.Proc of the Int Symp on Mycotoxins, Cairo, Egypt.

4 Hsieh PH (1981) Mycotoxins: Metabolism and transmission. Proc of the Int Work­shop & Symp on Mycotoxins Sep 1981,National Res Centre, Cairo, Egypt.

5 McDougall EI (1984) The composition and output of sheep's saliva. J Biochem,Vo143, 1948.

6 Stubblefield RD, Pier AC, Richard JL, and Shotwall OL (1983) Fate of aflatoxinsin tissues, fluids, and excrements from cows dosed orally with AFB1• Am J ofVet Res, 44 (9) : 1750-1752.

7 Pons WA, Lee 18, and Stollof L (1980) Revised method for aflatoxins in cotton­seed products, and comparison of thin layer and high performance liquid chro­matography determination steps. Collaborative study. J Assoc Offic Anal Chern,63: 899-906.

8 Emafo PO (1976) Species differences in the metabolism of aflatoxin B1 • Afr JMed Sci, 5 (1):55-62.

9 AOAC (1980) Official Methods of Analysis of the Association of Official Ana­lytical Chemists. 11 th Ed, Benjamin Franklin Station, Washington, DC,20044.

10 Saad M Magdi (1984) Studies on the metabolism of aflatoxin B1 in buffaloes.Ph D Thesis Ain Shams Univ, Cairo, Egypt.

11 Kiessling KH, Pettersson H, Olsen M, and Tideman K (1982) Metabolism of afla­toxin B1 , zearalenone, and trichothecenes diacetoxyscirpenol in rumen fluid.Proc of Phycotoxin and Mycotoxins, Vienna, 1982.

Manuscript received March 30, 1986; accepted Dec 19, 1986.

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