- Email: [email protected]
Toxicity of Aspergillus flavus Cultures Isolated from Egypt
Zbl. Bald. Abt. II, Bd.131, S. 501l-009 (19ill)
[Food and Dairy Research Laboratories, National Research Centre, Dokki, Cairo, and Department of Phytopathology, Faculty of Agriculture, Tanta University, Kafr EI-Sheik, Egypt]
Toxicity of A8pergillu8 flavu8 Cultures Isolated from Egypt M. M. Naguib and M. EI-Khadem Summary Aflatoxin produced by 61.14% of the AspergillW! jlavW! cultures, isolated from Egypt. A wide potential range for aflatoxins Band G groups was obvious among the isolates. The concentration of aflatoxin B, produced by toxigenic cultures, ranged from 1,500 to 81,000 p .p.b. (pg/kg substrate), corresponding to 1,900 to 184,000 p .p.b . for aflatoxin G. However, 7.59 % of the isolates produced more than 50,000 p.p.b. of aflatoxins B + G.
Zusammenfassung Die Toxizitat del' Aspergillus jlavus-Stamme in Agypten ist unterschiedlich und reicht von un· giftigen, d. h . kein Toxin abscheidenden, bis zu hochtoxischen mit einer Bildung von mehr als 50000 p.p.b. (pg/kg) Aflatoxin (7.59 % del' untersuchten Stamme). Die toxischen Stamme produzierten Aflatoxine in Mengen von 1500 bis 81000 p. p. b. an Aflatoxin B und 1900 bis 184000 an Aflatoxin G.
The high incidence of mortality, observed among turkey poults and ducklings, was due to a toxic substances produced by certain strains of Aspergillus flav1ts (SARGEANT et a1. 1961). These hepatotoxic compounds of certain Aspergilli were named aflatoxins (designated as Bv B z, G1, and G2 ). These compounds are acutely toxic for most animal species (CARNAGHAN et al. 1963, ALCROFT 1969) and plants (SCHOENTAL and WHITE 1965, EL-KHADEM 1968). The growth of fungi and the production of aflatoxins was reported on several agricultural products. The hydroxylated aflatoxins M1 and M2 are secreted in milk (HOLZAPFEL and STEYN 1966, PURCHASE 1967) and aflatoxins B1 and B2 can develop in cheddar and brick cheeses (LIE and MARTH 1967, SHIH and MARTH 1971) . The problem of aflatoxins in relation to plant, animal, and food industries did not arise in Egypt. This may be due to either the unadequate quality control for detecting such problem or to the lower frequency of the toxin· producing strains in this country. However, this point has not been touched. Therefore, the object of this investigation is the qualitative and quantitative measurements of aflatoxin-producing strains of A./lavu8, isolated from different sources in Egypt.
Materials and Methods The Aspergilli cultures were isolated from different sources in Egypt on Czapek's Dox agar medium. Identification of A. flavu8 was carried out according to RAPER and FENNEL (1965) and NAGARAJAN and BRAT (1973).
Toxi(·ity of .·L~per!liIl1!8 JlctVU8 Cultures Isolated from Egypt
507
The identified strains were grown on rice medium with the following composition: 100 g rice flour, 10 g yeast extract, and 10 g agar in 1 liter distilled water. The medium was tubed in 5 ml amounts. The slants were inoculated with the test organisms and incubated at 29 DC for 10 days. Chloroform extracts (5 ml) of aflatoxins were filtered and concentrated to 1.0 ml quantities. Aflatoxins were determined by thin-layer chromatography, using a chloroform: methanol solvent (95: 5) and plates with 0.25 mm layer silica-gel G. Extractes were spotted in 100,u1 samples on TLC plates. Developed plates were dried in the dark for 5 minutes and examined with the aid of long-wave ultraviolet light. Blue and green fluorescence was located and eluted in 3 ml methanol. Readings for the methanolio solutions were taken, using a Beckman spectrophotometer, Model B, at 363 and 420 nm. Aflatoxin concentrations were calculated according to the equation of NABNEY and NESBITT (1965).
Results and Discussion A number of 175 cultures belonging to A. flavU8 were isolated from different sources in Egypt. The qualitative screening of the cultures on TLC plates showed that 61.14% produced aflatoxins (Table 1). On examining TLC plates with ultraviolet lamp, aflatoxin B group displays blue fluorescence and G group shows green fluorescence. Chloroform: methanol solvent (95: 5) separated aflatoxins of the B group form the G group (JONES 1972). For the qualitative and quantitative determination of aflatoxins, further separation of the components of each group was not required. However, aflatoxins produced by different isolates were collected, and Bl and G1 toxins were separated, using the method described by DAVIS et al. (1966). The spectrum of both Bl and Gl were determined and were found to be identical to those reported by ROBERTSON et al. (1967). A comparatively higher percentage of the non-toxin producing strains were found among the isolates as compared to the finding of TABER and SCHROEDER (1967) and JOFFE (1969), who reported that 4.8 and 1.3 % (or 10.4 %), repectively, of their isolates did not produce aflatoxins. This might be due to strains and test condition differences. Table 1. Qualitative production of aflatoxins for Aspergillus flavu8 cultures, isolated from different sources in Agypt. Components of aflatoxins
No. of isolates
Non-producers Toxin-producers B producers B + G producers
68 107 75 32
38.86 61.14 42.85 18.28
Total isolates
175
100.00
Percentage of isolates
The majority of toxigenic strains produced only the B group (70.09 %), while a rather small amount of isolates (29.91 %) produced the Band G groups. None was found to produce the G group alone. This is in accordance with the findings of JOFFE (1969); on the other hand, TABER and SCHROEDER (1967) found that none of the A. flavu8 strains, isolated from peanuts, produced G group aflatoxins. Data presented in Table 2 show that a wide potential range for toxin production was obvious among the tested cultures. Some strains had the ability of producing both groups in a relatively high concentration. Strain 40 produced as much as 81,000 p.p.b. of aflatoxin Band 184,000 p.p.b. of G. The minimum B toxins produced was 1,500 p.p.b. and for G toxin it was 1,900 p.p.b. The percentage frequency distribution of the isolates for group B, G, and B + G aflatoxin concentrations are represented in Table 2. It is obvious from the results 34*
508
!VI. M.
XA(TIB
and:VI.
EL-KHAIJKH
Table 2. Percentage distribution of aflatoxin concentrations, produced by toxigenic cultures of A8pergillu8 jlavu8 isolated from different sources in Egypt Aflatoxin concentration (p.p.b.)
Percentage of isolates producing B group
G group
B
+ G group
1,000 to less than 5,000
25.30
35.00
5,000 to less than 10,000
18.99
20.00
18.99 21.52
10,000 to less than 15,000
22.78
20.00
17.73
15,000 to less than 20,000
11.39
5.00
13.91
20,000 to less than 25,000
11.39
0
12.65
25,000 to less than 50,000
6.33
15.00
7.59
Over 50,000
3.79
5.00
7.59
Maximum Minimum
B group 81,000 1,500
G group 184,000 1,900
B + Ggroup 265,000 1,500
that a rather small amount of the isolates is highly toxic, as only 5 % of the strains produced more than 50,000 p.p.b. of aflatoxin G, 3.79% of B, and 7.59% of B + G. TABER and SCHROEDER (1967) reported that 2.4 % of A. flavus-oryzae strains, isolated from peanuts, produced aflatoxins in concentrations of more than 50,000 p.p.b. on rice substrate. JOFFE (1969) found that 2:l.2 % of the isolates produced 125,000 to 1,500,000 p.p.b. of aflatoxin Bl and more than 60% of the isolates produced aflatoxin in excess of 15,000 p.p.b. The isolation of a potent toxic strain of A. flavus from Egypt supports the view that the toxigenic Aspergilli appear to be ubiquitous in nature. The elimination of such organisms in dairy and food products is the function of controlling the sources of contamination as well as favourable conditions for Aspergilli growth and production of toxin. However, the standards and grading of agricultural commodities in Egypt ignored any stipulations regarding the presence of aflatoxins. Therefore, this should ca.ll the attention of the authorities to put through additional acts to the food standards for controlling the limits of aflatoxins concentrations in various foods for the safe human and animal consumption.
References ALCROFT, R.: In "Aflatoxin". L. A. Goldblatt (Ed.), Acad. Press, New York and London 1969, p.237-264. CARNAGHAN, R. B. A., HARTLEY, R. D., and O'KELLY, J.: Nature 200 (1963), llO1. DAVIS, N. D., HAYES, A. W., ALDRIDGE, D. W., and DIENER, U. L.: J. Ass. Off. Anal. Chem. 49 (1966),1224-1225. EL-KHADEM, M.: Phytopathol. Z. 61 (1968) 218-223. HOLZAPFEL, C. W., and STEYN, P. S.: Tetrahendron Lett.ers No. 25 (1966), 2799-2803. JOFFE, A. Z.: Nature 221 (1969),492. JONES, B. D.: Trop. Prod. Inst. (1972). LIE, J. L., and MARTH, E. H.: J. Dairy Sci. 50 (1967),1708-1710. NABNEY, J., and NESBITT, B. F.: Analyst 90 (1965),155-160. NAGARAJAN, V., and BHAT, R. V.: Appl. Microbiol. 25 (1973),319-321. PURCHASE, 1. F. H.: J. Ass. Off. Anal. Chem. aO (1967),363-366. RAPER, K. B., and FENNEL, D. L.: The Genus A8pergillu8. The Williams and Wilkins Comp., Baltimore, U.S.A. 1965.
T""wily of .·hpl'l'flillllsflrn.'lls Cultures lsolatpd frm n Egypt
,j09
ROBERTSON, J. A., Po"", W. A. ,iI'., and GOLDBLATT, L. A.: Agr. Fd. Chern. 15 (1967), 798-80l. SARGEANT, K., SHERIDAN, A., and O'KELLY, J.: Nature 192 (1961), 1096-1097. SCHOENTAL, R., and WHITE, A. F.: Nature 205 (1965), 57 - 58. SHIN, C. N., and MARTH, E. H.: J. Dairy Sci. 54 (1971),749. TABER, R. A., and SCHROEDER, H. W.: Appl. :\1icrobiol. 15 (1967),140-144. Author's address: Dr. M. M. NAGUIB, Food and Dairy Research Laboratories, National Research Centre, Dokki, Cairo, Egypt.
[Food and Dairy Research Laboratories, National Research Centre, Dokki, Cairo, and Department of Phytopathology, Faculty of Agriculture, Tanta University, Kafr EI-Sheik, Egypt]
Toxicity of A8pergillu8 flavu8 Cultures Isolated from Egypt M. M. Naguib and M. EI-Khadem Summary Aflatoxin produced by 61.14% of the AspergillW! jlavW! cultures, isolated from Egypt. A wide potential range for aflatoxins Band G groups was obvious among the isolates. The concentration of aflatoxin B, produced by toxigenic cultures, ranged from 1,500 to 81,000 p .p.b. (pg/kg substrate), corresponding to 1,900 to 184,000 p .p.b . for aflatoxin G. However, 7.59 % of the isolates produced more than 50,000 p.p.b. of aflatoxins B + G.
Zusammenfassung Die Toxizitat del' Aspergillus jlavus-Stamme in Agypten ist unterschiedlich und reicht von un· giftigen, d. h . kein Toxin abscheidenden, bis zu hochtoxischen mit einer Bildung von mehr als 50000 p.p.b. (pg/kg) Aflatoxin (7.59 % del' untersuchten Stamme). Die toxischen Stamme produzierten Aflatoxine in Mengen von 1500 bis 81000 p. p. b. an Aflatoxin B und 1900 bis 184000 an Aflatoxin G.
The high incidence of mortality, observed among turkey poults and ducklings, was due to a toxic substances produced by certain strains of Aspergillus flav1ts (SARGEANT et a1. 1961). These hepatotoxic compounds of certain Aspergilli were named aflatoxins (designated as Bv B z, G1, and G2 ). These compounds are acutely toxic for most animal species (CARNAGHAN et al. 1963, ALCROFT 1969) and plants (SCHOENTAL and WHITE 1965, EL-KHADEM 1968). The growth of fungi and the production of aflatoxins was reported on several agricultural products. The hydroxylated aflatoxins M1 and M2 are secreted in milk (HOLZAPFEL and STEYN 1966, PURCHASE 1967) and aflatoxins B1 and B2 can develop in cheddar and brick cheeses (LIE and MARTH 1967, SHIH and MARTH 1971) . The problem of aflatoxins in relation to plant, animal, and food industries did not arise in Egypt. This may be due to either the unadequate quality control for detecting such problem or to the lower frequency of the toxin· producing strains in this country. However, this point has not been touched. Therefore, the object of this investigation is the qualitative and quantitative measurements of aflatoxin-producing strains of A./lavu8, isolated from different sources in Egypt.
Materials and Methods The Aspergilli cultures were isolated from different sources in Egypt on Czapek's Dox agar medium. Identification of A. flavu8 was carried out according to RAPER and FENNEL (1965) and NAGARAJAN and BRAT (1973).
Toxi(·ity of .·L~per!liIl1!8 JlctVU8 Cultures Isolated from Egypt
507
The identified strains were grown on rice medium with the following composition: 100 g rice flour, 10 g yeast extract, and 10 g agar in 1 liter distilled water. The medium was tubed in 5 ml amounts. The slants were inoculated with the test organisms and incubated at 29 DC for 10 days. Chloroform extracts (5 ml) of aflatoxins were filtered and concentrated to 1.0 ml quantities. Aflatoxins were determined by thin-layer chromatography, using a chloroform: methanol solvent (95: 5) and plates with 0.25 mm layer silica-gel G. Extractes were spotted in 100,u1 samples on TLC plates. Developed plates were dried in the dark for 5 minutes and examined with the aid of long-wave ultraviolet light. Blue and green fluorescence was located and eluted in 3 ml methanol. Readings for the methanolio solutions were taken, using a Beckman spectrophotometer, Model B, at 363 and 420 nm. Aflatoxin concentrations were calculated according to the equation of NABNEY and NESBITT (1965).
Results and Discussion A number of 175 cultures belonging to A. flavU8 were isolated from different sources in Egypt. The qualitative screening of the cultures on TLC plates showed that 61.14% produced aflatoxins (Table 1). On examining TLC plates with ultraviolet lamp, aflatoxin B group displays blue fluorescence and G group shows green fluorescence. Chloroform: methanol solvent (95: 5) separated aflatoxins of the B group form the G group (JONES 1972). For the qualitative and quantitative determination of aflatoxins, further separation of the components of each group was not required. However, aflatoxins produced by different isolates were collected, and Bl and G1 toxins were separated, using the method described by DAVIS et al. (1966). The spectrum of both Bl and Gl were determined and were found to be identical to those reported by ROBERTSON et al. (1967). A comparatively higher percentage of the non-toxin producing strains were found among the isolates as compared to the finding of TABER and SCHROEDER (1967) and JOFFE (1969), who reported that 4.8 and 1.3 % (or 10.4 %), repectively, of their isolates did not produce aflatoxins. This might be due to strains and test condition differences. Table 1. Qualitative production of aflatoxins for Aspergillus flavu8 cultures, isolated from different sources in Agypt. Components of aflatoxins
No. of isolates
Non-producers Toxin-producers B producers B + G producers
68 107 75 32
38.86 61.14 42.85 18.28
Total isolates
175
100.00
Percentage of isolates
The majority of toxigenic strains produced only the B group (70.09 %), while a rather small amount of isolates (29.91 %) produced the Band G groups. None was found to produce the G group alone. This is in accordance with the findings of JOFFE (1969); on the other hand, TABER and SCHROEDER (1967) found that none of the A. flavu8 strains, isolated from peanuts, produced G group aflatoxins. Data presented in Table 2 show that a wide potential range for toxin production was obvious among the tested cultures. Some strains had the ability of producing both groups in a relatively high concentration. Strain 40 produced as much as 81,000 p.p.b. of aflatoxin Band 184,000 p.p.b. of G. The minimum B toxins produced was 1,500 p.p.b. and for G toxin it was 1,900 p.p.b. The percentage frequency distribution of the isolates for group B, G, and B + G aflatoxin concentrations are represented in Table 2. It is obvious from the results 34*
508
!VI. M.
XA(TIB
and:VI.
EL-KHAIJKH
Table 2. Percentage distribution of aflatoxin concentrations, produced by toxigenic cultures of A8pergillu8 jlavu8 isolated from different sources in Egypt Aflatoxin concentration (p.p.b.)
Percentage of isolates producing B group
G group
B
+ G group
1,000 to less than 5,000
25.30
35.00
5,000 to less than 10,000
18.99
20.00
18.99 21.52
10,000 to less than 15,000
22.78
20.00
17.73
15,000 to less than 20,000
11.39
5.00
13.91
20,000 to less than 25,000
11.39
0
12.65
25,000 to less than 50,000
6.33
15.00
7.59
Over 50,000
3.79
5.00
7.59
Maximum Minimum
B group 81,000 1,500
G group 184,000 1,900
B + Ggroup 265,000 1,500
that a rather small amount of the isolates is highly toxic, as only 5 % of the strains produced more than 50,000 p.p.b. of aflatoxin G, 3.79% of B, and 7.59% of B + G. TABER and SCHROEDER (1967) reported that 2.4 % of A. flavus-oryzae strains, isolated from peanuts, produced aflatoxins in concentrations of more than 50,000 p.p.b. on rice substrate. JOFFE (1969) found that 2:l.2 % of the isolates produced 125,000 to 1,500,000 p.p.b. of aflatoxin Bl and more than 60% of the isolates produced aflatoxin in excess of 15,000 p.p.b. The isolation of a potent toxic strain of A. flavus from Egypt supports the view that the toxigenic Aspergilli appear to be ubiquitous in nature. The elimination of such organisms in dairy and food products is the function of controlling the sources of contamination as well as favourable conditions for Aspergilli growth and production of toxin. However, the standards and grading of agricultural commodities in Egypt ignored any stipulations regarding the presence of aflatoxins. Therefore, this should ca.ll the attention of the authorities to put through additional acts to the food standards for controlling the limits of aflatoxins concentrations in various foods for the safe human and animal consumption.
References ALCROFT, R.: In "Aflatoxin". L. A. Goldblatt (Ed.), Acad. Press, New York and London 1969, p.237-264. CARNAGHAN, R. B. A., HARTLEY, R. D., and O'KELLY, J.: Nature 200 (1963), llO1. DAVIS, N. D., HAYES, A. W., ALDRIDGE, D. W., and DIENER, U. L.: J. Ass. Off. Anal. Chem. 49 (1966),1224-1225. EL-KHADEM, M.: Phytopathol. Z. 61 (1968) 218-223. HOLZAPFEL, C. W., and STEYN, P. S.: Tetrahendron Lett.ers No. 25 (1966), 2799-2803. JOFFE, A. Z.: Nature 221 (1969),492. JONES, B. D.: Trop. Prod. Inst. (1972). LIE, J. L., and MARTH, E. H.: J. Dairy Sci. 50 (1967),1708-1710. NABNEY, J., and NESBITT, B. F.: Analyst 90 (1965),155-160. NAGARAJAN, V., and BHAT, R. V.: Appl. Microbiol. 25 (1973),319-321. PURCHASE, 1. F. H.: J. Ass. Off. Anal. Chem. aO (1967),363-366. RAPER, K. B., and FENNEL, D. L.: The Genus A8pergillu8. The Williams and Wilkins Comp., Baltimore, U.S.A. 1965.
T""wily of .·hpl'l'flillllsflrn.'lls Cultures lsolatpd frm n Egypt
,j09
ROBERTSON, J. A., Po"", W. A. ,iI'., and GOLDBLATT, L. A.: Agr. Fd. Chern. 15 (1967), 798-80l. SARGEANT, K., SHERIDAN, A., and O'KELLY, J.: Nature 192 (1961), 1096-1097. SCHOENTAL, R., and WHITE, A. F.: Nature 205 (1965), 57 - 58. SHIN, C. N., and MARTH, E. H.: J. Dairy Sci. 54 (1971),749. TABER, R. A., and SCHROEDER, H. W.: Appl. :\1icrobiol. 15 (1967),140-144. Author's address: Dr. M. M. NAGUIB, Food and Dairy Research Laboratories, National Research Centre, Dokki, Cairo, Egypt.