J.Vet. Med. B 39, 692-694 (1992) 8 1992 Paul Parey Scientific Publishers, Berlin and Hamburg ISSN 0931-1793
Faculty of Veterinary Medicine, Beni Sue5 Cairo University, Egypt
Reduction of Aflatoxin MI in Milk Using Hydrogen Peroxide and Hydrogen peroxide Plus Heat Treatment I. AMAN Address of author: Dr. IBRAHIMAMAN,Alexandria Mohoram Beck, 59 Bawaleno street, Egypt
With 3 tables (Received for publication February 14, 1992)
Summary Use of hydrogen peroxide (H202) under various conditions of temperature and time to inactivate aflatoxin MI in artificially contaminated raw milk was examined. The degree of inactivation was measured by Enzym Linked Immunosorbent Assay (ELISA). It appeared that there is no change in the content of aflatoxin MI in milk contained H202 and examined after 24 hours without heat treatment and in contaminated milk without add of H20, and heat treated treatment at (63°C for 30min, 75°C for 15sec) but slight inactivation (4.3 Yo) was obtained in milk boiled for 5min without H 2 0 2 .Maximum inactivation (27.8 %, 28.8 70 and 45.1 %) were obtained using 1 % H202 followed by heat treatment at 36 "C for 30 min, 75 "C for 15 sec and boiling for 5 min respectively.
Introduction Ingestion of aflatoxin B1 by lactating animals results in the excretion of the hydroxy1972). Aflatoxin MI has been lated derivative called aflatoxin M1 in the milk (PURCHASE, found in commercial samples of milk (POLZHOFER, 1977) and cheese (KIERMEIER et al., 1977). Since it has been proved that the toxicity of aflatoxin MI is nearly the same as the 1967) and that aflatoxin M1 is carcinogenic parent compound aflatoxin Bl (PURCHASE, (CANTONet al., 1975), its presence in milk is undesirable. It is well known that raw milk is processed mostly before human consumption. In connection with this it is of interest to know how far the processes common to the dairy industry influence the aflatoxin M1 content of milk. STOLOPF and TRUCKSESS (1974), VAN EGMONDet al. (1977) observed no loss aflatoxin M1 neither during storage nor after pasteurization of raw milk, spiked or naturally contamined with aflatoxin. The previous authors used chemicals in combination with heat treatment. SREENIVASANMURTHLY et al. (1967) treated aqueous suspension of defatted peanut meal contained 90ppm of aflatoxin with 6 % hydrogen peroxide (H202) for 30min at 80°C. Results indicated 97 YO of the aflatoxin was inactivated. APPLEBAUM and MARTH(1982) used 1 % of hydrogen peroxide plus riboflavin or lactoperoxidase followed by heat treatment to inactivate aflatoxin MI in milk. Results indicated about 98% of AFMl inactivated. The object of this study was to determine if hydrogen peroxide at lower concentrations plus physical treatments could be used to detoxify AFMl -contaminated milk. US.Copyright Clearance Center Code Statement: 0931 - 1793/92/3909-0692$02.50/0
Reduction of Aflatoxin MI in Milk Using Hydrogen Peroxide
693
Material and Methods Preparation of toxic milk Aflatoxin M1-free milk was artificially contaminated with a solution of aflatoxin MI (with stirring) to provide a calculated concentration of M , in the milk of 23.0ngIliter. The contaminated milk was then divided into three portions I, I1 and 111. H202(w/v) was then added to the portions I and I11 at various concentrations of 0.125, 0.250, 0.50 and 1.0%. Heating was done for milk in the portions I1 and 111by placing flasks in a water bath and holding them at the specified temperature and for a defined time.
Quantitation of AFMI Amounts of AFM, were determined by Enzyme Linked Immunosorbent Assay (ELISA) according to the method described by STEIMERet al. (1988).
Results and Discussion Results presented in Table 1 show that H202have no effect on aflatoxin M1 in milk when it used and not followed by heat treatment Table 2 lists results from inactivation experiments using heat treatment. The observation that heat-processing of milk except boiling for 5 min did not change the aflatoxin M1 content of milk. The results agree with those of STOLOFF and TRUCKNESS (1974) and VAN EGMOND et al. (1977) and contradict the results of PURCHASE et al. (1972). Results obtained in Table3 show that with the increasing of the concentration of H202and heat treatment at 63 "C for 30 min, the reduction of AFMl in milk increased from 7.7 to 27.8 %, while with heat treatment at 75 "C for 15 sec slight reduction of AFMl content in milk till conc. 0.5 % of H2O2and the percentage of reduction reached 28.8 at 1.0 of H20z.Boiling the milk for 5 min has shown promise for reduction of AFMl in which the percentage of reduction reached to 22.6 and 45.1 with 0.5 and 1.0 H202. Initial studies on reduction of AFMl were done with artificially and naturally contaminated milk (APPLEBAUM and MARTH,1982). Results showed that the maximum reduction at conc. of 1 % H202plus 0.5 mM riboflavin followed by heat treatment of 63 "C
Table 1. Effect of hydrogen peroxide on AFMl in milk Treatment
% of reduction
0.125 % H2Oz 0.250% HZ02 0.50 % H202 1.0 Yo H202
0 1 0.7 0
Milk samples examined after 24 hours from the addition of Samples examined in duplicate.
H20Z
Table2. Effect of heat treatment on AFMl in milk Treatment
% of reduction
at 63 "C for 30 min (PI) at 75 "C for 15 sec (P2) boiling for 5 min (P3)
0.8 0 4.3
694
AMAN Table 3. Reduction of AFMl in milk using H z 0 2and heat treatment Treatment
Hz0, 0.125 Yo 0.250 Yo 0.50 Yo 1.0 Yo
PI % of reduction
P2
P3
Yo of reduction
% of reduction
7.7 11.7 18.5 27.8
4.1 5.1 7.0 28.8
8.6 8.9 22.6 45.1
for 30 min. The authors used 1 % of H202 without riboflavin followed by heat treatment for 63°C for 30min and they found that the maximum inactivation value 11 YO was achieved. The mechanism by which H202 act to reduce A F M l is explained by KORYCKA-DAHL and RICHARDSON (1978). They explained this by the ability of H202 to generate singlet O2which is highly electrophilic and reacts readily with the terminal double and MARTH(1978) demonstrated that bond in the dihydrofuran ring of AFMI. Also DOYLE the chloride ions and lactoperoxidase oxidase enzyme in milk in the presence of H202 could degrade aflatoxin B1 and G1. SREENIVAS~MURTHY et al. (1967) studied the oxidative destruction of H202 with heat treatment at 80°C for 30min on defatted peanut meal containing 90ppm of aflatoxin B1. Results indicated that 97% of the aflatoxin was inactivated. References 1. APPLEBAUM, R.S., and MARTH,E.H. 1982: Inactivation of Aflatoxin MI in Milk Using Hydrogen Peroxide and Hydrogen Peroxide plus Riboflavin or Lactoperoxidase. J. Fd. Protection 45, 6, 557. 2. CANTON,J. H., R. KROES,M. J. VAN LOGTEN,M. VAN SCHOTHORST, J. C.F. STAVENUITER, and C.A.H. VERHULSDONK, 1975: The carcinogenicity of aflatoxin M1 in rainbow trout. Fd. Cosmet. Toxicol. 13, 441. 3. DOYLE,M. P., and E. H. MARTH,1978: Degradation of aflatoxin by lactoperoxidase. Z. Lebensm. Unters. Forsch. 166, 271 -273. 4. KIERMEIER, F., G. WEISE,G. BEHRINGER, and M. MILLER,1977: Uber das Vorkommen und den Gehalt von Aflatoxin MI in Kasen des Handels. Z. Lebensm. Unters. Forsch. 163, 268. 5. KORYCKA-DAHL, M. B., and T. RICHARDSON, 1978: Activated oxygen species and oxidation of food constituents. CRC Crit. Rev. Food Sci. Nutr. 10, 209. 6. POLZHOPER, K., 1977: Aflatoxinbestimmung in Milch und Milchprodukten. Z. Lebensm. Unters. Forsch. 163, 175. 7. PURCHASE, I. F. H., 1967: Acute toxicity of aflatoxin M1 and M2 in one-day-old duckling. Fd. Cosmet. Toxicol. 5, 585. 8. PURCHASE, I. F. H., 1972: Aflatoxin residues in food of animal origin. Fd. Cosmet. Toxicol. 10, 531. 9. PURCHASE, I. F. H., M. STEYN,R. RINSMA,and R. C. TUSTIN,1972: Reduction of aflatoxin, M 1 content of milk by processing. Fd. Cosmet. Toxicol. 10, 383. 10. SREENIVASAMURTHY, V., H. A. B. PARPIA,S. SRIKANTA, and A. SHANKARMURTI, 1967 Detoxification of aflatoxin in peanut meal by hydrogen peroxide. J. Assoc. Of. Agric. Chem. 50,350. 11. STOLOFP,L., and M. TRUCKNESS, 1974: Studies on the stability of aflatoxin MI in milk. Manuscript FDA. Bureau of Foods Washington D. C. 12. STEIMER, J., G. HAHN,W. HEESCHEN, and A. BLUTHGEN, 1988: Zum enzymimmunologischen
Nachweis von Aflatoxin MI in der Milch: Entwicklung eines schnellen Aflatoxin-M,-Suchtests unter Verwendung von Polystyrol-Perlen als Festkorperphase. Milchwissenschaft 43, 12, 772. 13. VANEGMOND, H. P., W. E. PAULSCH,H. A. VERINGA, and P. L SCHULLER, 1977: The effect of processing on the aflatoxin M1 content of milk and milk products. Archives de I'Institut Pasteur de Tunis 3-4, 381.
Faculty of Veterinary Medicine, Beni Sue5 Cairo University, Egypt
Reduction of Aflatoxin MI in Milk Using Hydrogen Peroxide and Hydrogen peroxide Plus Heat Treatment I. AMAN Address of author: Dr. IBRAHIMAMAN,Alexandria Mohoram Beck, 59 Bawaleno street, Egypt
With 3 tables (Received for publication February 14, 1992)
Summary Use of hydrogen peroxide (H202) under various conditions of temperature and time to inactivate aflatoxin MI in artificially contaminated raw milk was examined. The degree of inactivation was measured by Enzym Linked Immunosorbent Assay (ELISA). It appeared that there is no change in the content of aflatoxin MI in milk contained H202 and examined after 24 hours without heat treatment and in contaminated milk without add of H20, and heat treated treatment at (63°C for 30min, 75°C for 15sec) but slight inactivation (4.3 Yo) was obtained in milk boiled for 5min without H 2 0 2 .Maximum inactivation (27.8 %, 28.8 70 and 45.1 %) were obtained using 1 % H202 followed by heat treatment at 36 "C for 30 min, 75 "C for 15 sec and boiling for 5 min respectively.
Introduction Ingestion of aflatoxin B1 by lactating animals results in the excretion of the hydroxy1972). Aflatoxin MI has been lated derivative called aflatoxin M1 in the milk (PURCHASE, found in commercial samples of milk (POLZHOFER, 1977) and cheese (KIERMEIER et al., 1977). Since it has been proved that the toxicity of aflatoxin MI is nearly the same as the 1967) and that aflatoxin M1 is carcinogenic parent compound aflatoxin Bl (PURCHASE, (CANTONet al., 1975), its presence in milk is undesirable. It is well known that raw milk is processed mostly before human consumption. In connection with this it is of interest to know how far the processes common to the dairy industry influence the aflatoxin M1 content of milk. STOLOPF and TRUCKSESS (1974), VAN EGMONDet al. (1977) observed no loss aflatoxin M1 neither during storage nor after pasteurization of raw milk, spiked or naturally contamined with aflatoxin. The previous authors used chemicals in combination with heat treatment. SREENIVASANMURTHLY et al. (1967) treated aqueous suspension of defatted peanut meal contained 90ppm of aflatoxin with 6 % hydrogen peroxide (H202) for 30min at 80°C. Results indicated 97 YO of the aflatoxin was inactivated. APPLEBAUM and MARTH(1982) used 1 % of hydrogen peroxide plus riboflavin or lactoperoxidase followed by heat treatment to inactivate aflatoxin MI in milk. Results indicated about 98% of AFMl inactivated. The object of this study was to determine if hydrogen peroxide at lower concentrations plus physical treatments could be used to detoxify AFMl -contaminated milk. US.Copyright Clearance Center Code Statement: 0931 - 1793/92/3909-0692$02.50/0
Reduction of Aflatoxin MI in Milk Using Hydrogen Peroxide
693
Material and Methods Preparation of toxic milk Aflatoxin M1-free milk was artificially contaminated with a solution of aflatoxin MI (with stirring) to provide a calculated concentration of M , in the milk of 23.0ngIliter. The contaminated milk was then divided into three portions I, I1 and 111. H202(w/v) was then added to the portions I and I11 at various concentrations of 0.125, 0.250, 0.50 and 1.0%. Heating was done for milk in the portions I1 and 111by placing flasks in a water bath and holding them at the specified temperature and for a defined time.
Quantitation of AFMI Amounts of AFM, were determined by Enzyme Linked Immunosorbent Assay (ELISA) according to the method described by STEIMERet al. (1988).
Results and Discussion Results presented in Table 1 show that H202have no effect on aflatoxin M1 in milk when it used and not followed by heat treatment Table 2 lists results from inactivation experiments using heat treatment. The observation that heat-processing of milk except boiling for 5 min did not change the aflatoxin M1 content of milk. The results agree with those of STOLOFF and TRUCKNESS (1974) and VAN EGMOND et al. (1977) and contradict the results of PURCHASE et al. (1972). Results obtained in Table3 show that with the increasing of the concentration of H202and heat treatment at 63 "C for 30 min, the reduction of AFMl in milk increased from 7.7 to 27.8 %, while with heat treatment at 75 "C for 15 sec slight reduction of AFMl content in milk till conc. 0.5 % of H2O2and the percentage of reduction reached 28.8 at 1.0 of H20z.Boiling the milk for 5 min has shown promise for reduction of AFMl in which the percentage of reduction reached to 22.6 and 45.1 with 0.5 and 1.0 H202. Initial studies on reduction of AFMl were done with artificially and naturally contaminated milk (APPLEBAUM and MARTH,1982). Results showed that the maximum reduction at conc. of 1 % H202plus 0.5 mM riboflavin followed by heat treatment of 63 "C
Table 1. Effect of hydrogen peroxide on AFMl in milk Treatment
% of reduction
0.125 % H2Oz 0.250% HZ02 0.50 % H202 1.0 Yo H202
0 1 0.7 0
Milk samples examined after 24 hours from the addition of Samples examined in duplicate.
H20Z
Table2. Effect of heat treatment on AFMl in milk Treatment
% of reduction
at 63 "C for 30 min (PI) at 75 "C for 15 sec (P2) boiling for 5 min (P3)
0.8 0 4.3
694
AMAN Table 3. Reduction of AFMl in milk using H z 0 2and heat treatment Treatment
Hz0, 0.125 Yo 0.250 Yo 0.50 Yo 1.0 Yo
PI % of reduction
P2
P3
Yo of reduction
% of reduction
7.7 11.7 18.5 27.8
4.1 5.1 7.0 28.8
8.6 8.9 22.6 45.1
for 30 min. The authors used 1 % of H202 without riboflavin followed by heat treatment for 63°C for 30min and they found that the maximum inactivation value 11 YO was achieved. The mechanism by which H202 act to reduce A F M l is explained by KORYCKA-DAHL and RICHARDSON (1978). They explained this by the ability of H202 to generate singlet O2which is highly electrophilic and reacts readily with the terminal double and MARTH(1978) demonstrated that bond in the dihydrofuran ring of AFMI. Also DOYLE the chloride ions and lactoperoxidase oxidase enzyme in milk in the presence of H202 could degrade aflatoxin B1 and G1. SREENIVAS~MURTHY et al. (1967) studied the oxidative destruction of H202 with heat treatment at 80°C for 30min on defatted peanut meal containing 90ppm of aflatoxin B1. Results indicated that 97% of the aflatoxin was inactivated. References 1. APPLEBAUM, R.S., and MARTH,E.H. 1982: Inactivation of Aflatoxin MI in Milk Using Hydrogen Peroxide and Hydrogen Peroxide plus Riboflavin or Lactoperoxidase. J. Fd. Protection 45, 6, 557. 2. CANTON,J. H., R. KROES,M. J. VAN LOGTEN,M. VAN SCHOTHORST, J. C.F. STAVENUITER, and C.A.H. VERHULSDONK, 1975: The carcinogenicity of aflatoxin M1 in rainbow trout. Fd. Cosmet. Toxicol. 13, 441. 3. DOYLE,M. P., and E. H. MARTH,1978: Degradation of aflatoxin by lactoperoxidase. Z. Lebensm. Unters. Forsch. 166, 271 -273. 4. KIERMEIER, F., G. WEISE,G. BEHRINGER, and M. MILLER,1977: Uber das Vorkommen und den Gehalt von Aflatoxin MI in Kasen des Handels. Z. Lebensm. Unters. Forsch. 163, 268. 5. KORYCKA-DAHL, M. B., and T. RICHARDSON, 1978: Activated oxygen species and oxidation of food constituents. CRC Crit. Rev. Food Sci. Nutr. 10, 209. 6. POLZHOPER, K., 1977: Aflatoxinbestimmung in Milch und Milchprodukten. Z. Lebensm. Unters. Forsch. 163, 175. 7. PURCHASE, I. F. H., 1967: Acute toxicity of aflatoxin M1 and M2 in one-day-old duckling. Fd. Cosmet. Toxicol. 5, 585. 8. PURCHASE, I. F. H., 1972: Aflatoxin residues in food of animal origin. Fd. Cosmet. Toxicol. 10, 531. 9. PURCHASE, I. F. H., M. STEYN,R. RINSMA,and R. C. TUSTIN,1972: Reduction of aflatoxin, M 1 content of milk by processing. Fd. Cosmet. Toxicol. 10, 383. 10. SREENIVASAMURTHY, V., H. A. B. PARPIA,S. SRIKANTA, and A. SHANKARMURTI, 1967 Detoxification of aflatoxin in peanut meal by hydrogen peroxide. J. Assoc. Of. Agric. Chem. 50,350. 11. STOLOFP,L., and M. TRUCKNESS, 1974: Studies on the stability of aflatoxin MI in milk. Manuscript FDA. Bureau of Foods Washington D. C. 12. STEIMER, J., G. HAHN,W. HEESCHEN, and A. BLUTHGEN, 1988: Zum enzymimmunologischen
Nachweis von Aflatoxin MI in der Milch: Entwicklung eines schnellen Aflatoxin-M,-Suchtests unter Verwendung von Polystyrol-Perlen als Festkorperphase. Milchwissenschaft 43, 12, 772. 13. VANEGMOND, H. P., W. E. PAULSCH,H. A. VERINGA, and P. L SCHULLER, 1977: The effect of processing on the aflatoxin M1 content of milk and milk products. Archives de I'Institut Pasteur de Tunis 3-4, 381.
