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Increased Aflatoxin Production by Aspergillus flavus Via Cobalt Irradiation
Increased Aflatoxin Production by Aspergillus Via Cobalt Irradiation
flavus
KENNETH L. APPLEGATE AND JOHN R. CHIPLEY Department of Poultry Science, The Ohio State University, 674 West Lane Avenue, Columbus, Ohio 43210 (Received for publication November 8, 1972) ABSTRACT Exposure of toxinogenic A. flavus spores to specific gamma radiation levels induces higher yields of aflatoxin per unit of time, while non-toxinogenic aspergilli remain unaltered with respect to aflatoxin production. POULTRY SCIENCE 52:1492-1496,1973
INTRODUCTION
objectives of the present study were to determine the effects of gamma irradiation FLATOXINS are a series of secondary >• metabolites produced by some strains upon toxinogenic and non-toxinogenic of Aspergillus flavus when grown on wheat, strains of A. flavus. corn, peanuts and other food crops or MATERIALS AND METHODS artificial media (Arseculeratne et al., 1969; The aspergilli tested in this study (AsChristensen and Kennedy, 1971; Strzelicki et al, 1969; Van Walbeek et al, 1968; pergillus flavus NRRL-3145 and NRRLWehanm, 1946). The aflatoxins produce A-12268) were obtained from the United pathological or undesirable physiological States Department of Agriculture, Northresponses in various biological systems, in- ern Utilization Research and Developcluding man (Oettle, 1964, 1965). In ment Division, Peoria, Illinois. These culgeneral, the pathological responses of tures were initially grown on potato dexthese toxins are manifested as tumoro- trose agar (PDA) slants for 24 hrs. at genic or carcinogenic foci with possible 25°C. The reference stock cultures were accompaniment of bile duct proliferation stored at room temperature under sterile and hemorrhagic involvement (Allcroft mineral oil according to the method of et al., 1967; Archibald et al., 1962; Asplin Werhanm (1946). Working stock cultures and Carnaghan, 1961; Halver, 1965). At- were obtained by subculturing mycelia tempts to reduce or eliminate the toxicity from the mineral oil covered slants to of aflatoxin-containing foods and feed- 16 X 125 mm. screw-cap vials, each vial stuffs by heating, cooking, or autoclaving containing a 10 ml. PDA slant. Cultures were reported by Blount (1961) to be used for spore development and subselargely ineffective. Sensitivity of the quent substrate inoculation were obtained saprophytic fungi to gamma irradiation from the working stock cultures after 14 has not been studied in detail. Only a few days of incubation. All active cultures reports are available on the production of were maintained in loosely fitted screwaflatoxins by A. flavus following ionizing cap vials at 28°C. in an atmosphere havirradiation. Jemmali and Guilbot (1969) ing a relative humidity of 98 + 2% (Epreported 7 strains of non-toxinogenic stein et al., 1970). spores of A. flavus were induced to proSpore suspensions were prepared by duce significant levels of aflatoxins. The adding sterile distilled water to 14-dayold sporulated cultures. The spore-laden distilled water was decanted into sterile Ohio Agricultural Research Development Center screw-cap vials, centrifuged for 5 minutes Journal Article No. 45-73.
A
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at 3,000 r.p.m., and the supernatant removed. The resultant "spore b u t t o n s " were then washed twice, centrifuged, and the final wash decanted prior to spore irradiation. Individual groups of five screw-cap vials containing the respective "spore b u t t o n s " were exposed to predetermined radiation levels of 25, 50, 100, 150, 200, 300, 400 or 600 krads. from a 60 Co source. Following irradiation, spore densities were adjusted by serial dilutions and Petroff-Hausser and Helber counting chamber (C. A. Hausser and Son, Philadelphia, Pa.) to 1.6 to 1.79 X 10" spores per ml. The maximum time lapse between irradiation of spores and inoculation of substrates was approximately 1.5 hours.
or synthetic medium. All inoculated substrates were incubated for 1 to 10 days a t 28°C. in a sealed glass tank enclosed in a Gyro-rotary incubator (New Brunswick Scientific Company). T a n k and contents were rotated a t 150 r.p.m., while a relative humidity of 98 + 2 % was maintained within the t a n k by use of saturated solutions of (NHd) 2 S04. I n conjunction with mechanical rotation, all wheat samples were manually shaken each day to facilit a t e grain separation and maximum aflatoxin production. To retain a 2 5 % moisture level in the inoculated wheat, one ml. of sterile distilled water was added to each flask at 24 and 48 hours, of incubation (Stubblefield et al, 1967).
The synthetic medium (Adyes and Mateles, 1964) was prepared immediately before inoculation with spores and was sterilized by the membrane filter technique using a filter pore size of 0.45 u. T h e synthetic medium was dispensed into sterile 250 ml. cotton-stoppered erlenmeyer flasks, each flask receiving 25 ml. of medium. For the wheat substrate, a cracked hard red wheat, too large to pass through an 18 X 14 sieve mesh screen, was used. T h e wheat was initially cracked in a 10-C International hammer mill. The wheat medium was contained in 250 ml. cottonstoppered erlenmeyer flasks, each flask containing 25 gm. of medium. Distilled water (12.5 ml.) was added to each flask of wheat prior to autoclaving at 121°C. for 20 minutes. To obtain proper consistency, each flask was vigorously shaken by hand prior to and immediately after autoclaving.
Aflatoxin standard was prepared with a benzene-acetonitrile solution according to the method of Rodricks and Stoloff (1970). All standards, when not in use, were stored at — 18°C. in 5-dram glass teflon-lined screw-cap vials. A TLR-5 thermoluminescent dosimeter (Eberline) was used for determination of irradiation potential of the 60 Co source. An average dose rate of 10.2 krad. per min. was calculated following trial exposures of L i F crystals to the Cobalt source. Each crystal was contained in a glass vial later used in the spore irradiation studies.
The autoclaved wheat remained at room temperature for 18 hours to allow for proper moisture distribution prior to spore inoculations. One ml. of irradiated spore suspension was aseptically added to each of 10 flasks containing either wheat
Mycelial growth was separated from the synthetic medium by filtering and the filtrate collected in a 250 ml. separatory funnel. The mycelia-free liquid was then extracted according to the method of Adyes and Mateles (1964) with the following exception: phase extractions of the growth-free liquid was accomplished b y two 25 ml. washes of chloroform instead of a single 1:1 chloroform-liquid wash. T h e two aflatoxin-containing chloroform phases were pooled and brought to dryness under nitrogen. T h e resulting residue was transferred to 10 X 75 mm. glass vials via three 2 ml. chloroform washes
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and returned to dryness. All dried extracts were stored at — 18°C. Primary wheat extractions of each wheat sample was accomplished according to Stoloff (1971). The primary chloroform extract was subsequently reduced to dryness under nitrogen. The resulting residue was eluted according to the method of Pons (1969), returned to dryness on a rotary flask, evaporator, and the residue transferred to vials by three 2 ml. chloroform washes, redried, and stored at -18°C. The respective crude extracts representing the different incubation times, irradiation levels, and sample media were spotted on thin-layer glass plates coated with MN Kieselgel silica gel to a thickness of 250 mm. and activated at 80°C. for 2 hours prior to use. Aflatoxin Bi was collected into extraction soxlets via a microvacuum zone collector and eluted by three 10 ml. "flow-through" washes of chloroform-acetone (4:1) into 16 X 125 mm. test tubes and brought to dryness under nitrogen gas. The dried residues were later resuspended in 2.2 ml. of benzene-acetonitrile (98:2), agitated for 30 seconds on a Vortex Jr. mixer (Scientific Products), and quantitatively determined by use of a DU-2 spectrophotometer at 358 nm. (Rodricks and Stoloff, 1970).
CHIPLEY
Exposure of this organism to 25, 50, 100, 150, 200, 300, 400 or 600 krad. did not induce the production of aflatoxin Bi by this organism on either wheat or in the synthetic medium. However, the quantities of aflatoxin Bi produced from the irradiated toxinogenic strain (NRRL3145) were greater in both wheat and synthetic media following a 10-day incubation period at 28°C. than were nonirradiated toxinogenic controls (Fig. 1). Aflatoxin Bi production on wheat medium was increased by exposing spores from this strain to 150 or 200 krad. Of the six doses of ionizing irradiation studied (25, 50, 100, 150, 200 and 300 krad.), only the 150 krad. doses induced higher Bi production by the toxinogenic strain than by nonirradiated toxinogenic controls following a 10-day incubation in synthetic medium (Fig. 2). DISCUSSION
These results contradict those by Jemmali and Guilbot (1969). These investigators reported that exposure of seven strains of non-toxinogenic A. flavus to 100
RESULTS
The growth and sporulation of Aspergillus flavus NRRL-A-12268 were greatly reduced in both the wheat and synthetic media by exposure of the fungus to 300 krad., with complete growth inhibition resulting from exposures to 400 krad. of irradiation. These results are similar to those reported by Bridges et al. (1956) who observed that spores of A. flavus were inactivated by an ionizing radiation dose of 300 krad.
Time (days)
60
FIG. 1. Effects of Co irradiation on aflatoxin Bi production by Aspergillus flavus on 25 grams of wheat.
A F L A T O X I N PRODUCTION
FIG. 2. Effects of 60Co irradiation on aflatoxin Bi production by Aspergillusfiavusin 25 ml. of synthetic medium.
1495
were conducted since the Rf values of these spots were significantly different from those of the Bi standards. Although results of this investigation indicate t h a t exposure to gamma irradiation does not induce aflatoxin production by non-toxinogenic aspergilli, toxinogenic aspergilli appear to be induced to produce significantly larger amounts of aflatoxin Bi when exposed to irradiation levels of 150 or 200 krad. Since cobalt irradiation is presently sanctioned by the United States D e p a r t m e n t of Agriculture for irradiation of wheat, the induced production of anatoxins by irradiated toxinogenic strains of fungi could be of public health significance. ACKNOWLEDGMENT
krad. induced the individual strains to produce varying amounts of aflatoxin Bi. I t is possible t h a t this difference in results between the present study and those of Jemmali and Guilbot (1969) m a y be due to difference in test organisms since the strains of A. flavus which they used were not available for comparison with A.flavus NRRL-A-12268. I t is also possible t h a t the strains of A. flavus which they used were, in fact, aflatoxin-producing organisms b u t produced too little aflatoxin for detection by the techniques employed. Irradiation then m a y have increased the level of aflatoxin production to a detectable level rather t h a n induced anatoxin production by their strains. I t is also possible t h a t substances which exhibit the same blue fluorescent properties of aflatoxin on thin-layer chromatography (TLC) plates m a y have been mistaken b y Jemmali and Guilbot (1969) as anatoxins (Mislivec et al., 1968). I n the present study, similar faint blue spots were observed periodically on T L C plates after growth of irradiated N R R L A-12268 spores on wheat. No bioassays
The senior author wishes to acknowledge the assistance of Dr. J. F. Stephens in this study. REFERENCES Adyes, J., and R. I. Mateles, 1964. Incorporation of labeled compounds into anatoxins. Biochem. Biophys. Acta, 86: 418-420. AUcroft, R., B. A. Roberts and W. H. Butler, 1967. Aflatoxin in milk. Food Cosmet. Toxicol. 5: 597. Archibald, R. McG., H. J. Smith and J. D. Smith, 1962. Brazilian ground-nut toxicosis in Canadian broiler chickens. Can. Vet. J. 3: 322-325. Arseculeratne, S. N., L. M. Desilva, S. Wijesundera and C. H. S. R. Banduntha, 1969. Coconut as a medium for experimental production of aflatoxin. Appl. Microbiol. 18: 88-94. Asplin, F. D., and R. B. A. Carnaghan, 1961. The toxicity of certain groundnut meals for poultry with special reference to their effect on ducklings and chickens. Vet. Res. 73: 1215-1218. Blount, W. P., 1971. Turkey "X" disease. Turkeys (J. Brit. Turkey Federation) 9 (2): 52, 55-58, 61, 77. Bridges, A. E., J. P. Olive and V. L. Chandler, 1965. Relative resistance of microorganisms to cathode rays. Appl. Microbiol. 4: 147-152. Christensen, C. M., and B. W. Kennedy, 1971. Filamentous fungi and bacteria in macaroni and spaghetti products. Appl. Microbiol. 21:144-146. Epstein, E., M. P. Steinberg, A. I. Nelson and L. S. Wei, 1970. Aflatoxin production as affected by
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environmental conditions. J. Food Sci. 35: 389390. Halver, J. F., 1965. In: Mycotoxins in Foodstuffs G. N. Wogan, ed., M.I.T. Press, Cambridge, Mass. pp. 204-234. Jemmali, M., and A. Guilbot, 1969. Influence de l'irradiation des spores d'A. flavus sur la production d'aflatoxin Bi. C. R. Acad. Sc. Paris, T. 269, series D : 2271-2273. Mislivec, P. B., J. H. Hunter, and J. Tuile, 1968. Assay for anatoxin production by the genera Aspergillus and Penicillium. Appl. Microbiol. 16: 1053-1055. Oettle, A. G., 1964. Cancer in Africa, especially in regions south of the Sahara. J. Natl. Cancer Inst. 33:383-439. Oettle, A. G., 1965. The etiology of primary carcinoma of the liver in Africa: A critical appraisal of previous ideas with an outline of the mycotoxin hypothesis. South Africa Med. J. 39: 817-925. Pons, W. A., Jr., 1969. Collaborative study on the determination of anatoxins in cottonseed products. J. Assoc. Ofiic. Anal. Chemists, 52: 61-72.
Rodricks, J. V., and L. Stoloff, 1970. Determination of concentration and purity of anatoxin standards. J. Assoc. Offic. Anal. Chemists, 53:92-95. Stoloff, L., S. Nesheim, L. Yin, J. V. Rodricks, M. Stack and A. D. Campbell, 1971. A multimycotoxin detection method for anatoxins, ochratoxins, zearalenone, sterigmatocystin, and patulin. J. Assoc. Offic. Chemists, 54: 91-97. Strzelicki, E., H. S. Lillard and J. C. Ayres, 1969. Country cured ham as a possible source of anatoxin. Appl. Microbiol. 18: 938-939. Stubblefield, R. D., O. L. Shotwell, C. W. Hesseltine, M. L. Smith and H. H. Hall, 1967. Production of anatoxin on wheat and oats: measurement with a recording densitometer. Appl. Microbiol. 15: 186-190. Van Walbeek, W., P. M. Scott and F. S. Thatcher, 1968. Mycotoxins from food-borne fungi. Can. J. Microbiol. 14: 131-137. Werhanm, C. C , 1946. Mineral oil as a fungus culture preservative. Mycologia, 38: 691-692. Wogan, G. N., 1965. Anatoxin risks and control measures. Fed. Proc. 27: 932-938.
Moisture and Microwave Effects on Selected Characteristics of Turkey Pectoral Muscles GEORGIA W. CREWS 1 AND GRAYCE E. GOERTZ Department of Food Science and Institution Administration, College of Home Economics and Agricultural Experiment Station, The University of Tennessee, Knoxville, Tennessee 37916 (Received for publication November 10, 1972) ABSTRACT The effect of added water and microwave heating on the water holding capacity as indicated by expressible moisture index (EMI), total moisture, cooking losses and p H for U.S.D.A. Grade A turkey toms was studied. Samples (200 g.) of ground composites of pectoral muscles containing 0,15, or 30 ml. added water were heated in a microwave oven for 0, 70 or 130 sec. The 15 and 30 ml. samples represented 7 and 13% added water and end point temperatures for 70 and 130 sec. were 44 to 47°C. and 61 to 64°C. Expressible moisture index was the best measure of treatment effects. Percent variation was greater for the E M I than for measures of cooking losses and total moisture. Total moisture values indicated water loss during cooking but some added water was retained. Cooking time caused the greatest percent variation; added water also caused significant effects on all characteristics studied. POULTRY SCIENCE 52: 1496-1500, 1973
INTRODUCTION
H
EATING alters the chemical and physical characteristics of muscle protein, as evidenced by denaturation and :
Present Address: Department of Human Nutrition and Food, Virginia Polytechnic Institute and State University, Blacksburg, Virginia.
decreased ability of muscle to retain water. Factors that affect the chemical and physical composition of protein also affect water in ways that alter the juiciness, tenderness, and overall acceptability of meat. In previous studies, heat denaturation of tube-cooked beef began at 40°C. and was
flavus
KENNETH L. APPLEGATE AND JOHN R. CHIPLEY Department of Poultry Science, The Ohio State University, 674 West Lane Avenue, Columbus, Ohio 43210 (Received for publication November 8, 1972) ABSTRACT Exposure of toxinogenic A. flavus spores to specific gamma radiation levels induces higher yields of aflatoxin per unit of time, while non-toxinogenic aspergilli remain unaltered with respect to aflatoxin production. POULTRY SCIENCE 52:1492-1496,1973
INTRODUCTION
objectives of the present study were to determine the effects of gamma irradiation FLATOXINS are a series of secondary >• metabolites produced by some strains upon toxinogenic and non-toxinogenic of Aspergillus flavus when grown on wheat, strains of A. flavus. corn, peanuts and other food crops or MATERIALS AND METHODS artificial media (Arseculeratne et al., 1969; The aspergilli tested in this study (AsChristensen and Kennedy, 1971; Strzelicki et al, 1969; Van Walbeek et al, 1968; pergillus flavus NRRL-3145 and NRRLWehanm, 1946). The aflatoxins produce A-12268) were obtained from the United pathological or undesirable physiological States Department of Agriculture, Northresponses in various biological systems, in- ern Utilization Research and Developcluding man (Oettle, 1964, 1965). In ment Division, Peoria, Illinois. These culgeneral, the pathological responses of tures were initially grown on potato dexthese toxins are manifested as tumoro- trose agar (PDA) slants for 24 hrs. at genic or carcinogenic foci with possible 25°C. The reference stock cultures were accompaniment of bile duct proliferation stored at room temperature under sterile and hemorrhagic involvement (Allcroft mineral oil according to the method of et al., 1967; Archibald et al., 1962; Asplin Werhanm (1946). Working stock cultures and Carnaghan, 1961; Halver, 1965). At- were obtained by subculturing mycelia tempts to reduce or eliminate the toxicity from the mineral oil covered slants to of aflatoxin-containing foods and feed- 16 X 125 mm. screw-cap vials, each vial stuffs by heating, cooking, or autoclaving containing a 10 ml. PDA slant. Cultures were reported by Blount (1961) to be used for spore development and subselargely ineffective. Sensitivity of the quent substrate inoculation were obtained saprophytic fungi to gamma irradiation from the working stock cultures after 14 has not been studied in detail. Only a few days of incubation. All active cultures reports are available on the production of were maintained in loosely fitted screwaflatoxins by A. flavus following ionizing cap vials at 28°C. in an atmosphere havirradiation. Jemmali and Guilbot (1969) ing a relative humidity of 98 + 2% (Epreported 7 strains of non-toxinogenic stein et al., 1970). spores of A. flavus were induced to proSpore suspensions were prepared by duce significant levels of aflatoxins. The adding sterile distilled water to 14-dayold sporulated cultures. The spore-laden distilled water was decanted into sterile Ohio Agricultural Research Development Center screw-cap vials, centrifuged for 5 minutes Journal Article No. 45-73.
A
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A F L A T O X I N PRODUCTION
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at 3,000 r.p.m., and the supernatant removed. The resultant "spore b u t t o n s " were then washed twice, centrifuged, and the final wash decanted prior to spore irradiation. Individual groups of five screw-cap vials containing the respective "spore b u t t o n s " were exposed to predetermined radiation levels of 25, 50, 100, 150, 200, 300, 400 or 600 krads. from a 60 Co source. Following irradiation, spore densities were adjusted by serial dilutions and Petroff-Hausser and Helber counting chamber (C. A. Hausser and Son, Philadelphia, Pa.) to 1.6 to 1.79 X 10" spores per ml. The maximum time lapse between irradiation of spores and inoculation of substrates was approximately 1.5 hours.
or synthetic medium. All inoculated substrates were incubated for 1 to 10 days a t 28°C. in a sealed glass tank enclosed in a Gyro-rotary incubator (New Brunswick Scientific Company). T a n k and contents were rotated a t 150 r.p.m., while a relative humidity of 98 + 2 % was maintained within the t a n k by use of saturated solutions of (NHd) 2 S04. I n conjunction with mechanical rotation, all wheat samples were manually shaken each day to facilit a t e grain separation and maximum aflatoxin production. To retain a 2 5 % moisture level in the inoculated wheat, one ml. of sterile distilled water was added to each flask at 24 and 48 hours, of incubation (Stubblefield et al, 1967).
The synthetic medium (Adyes and Mateles, 1964) was prepared immediately before inoculation with spores and was sterilized by the membrane filter technique using a filter pore size of 0.45 u. T h e synthetic medium was dispensed into sterile 250 ml. cotton-stoppered erlenmeyer flasks, each flask receiving 25 ml. of medium. For the wheat substrate, a cracked hard red wheat, too large to pass through an 18 X 14 sieve mesh screen, was used. T h e wheat was initially cracked in a 10-C International hammer mill. The wheat medium was contained in 250 ml. cottonstoppered erlenmeyer flasks, each flask containing 25 gm. of medium. Distilled water (12.5 ml.) was added to each flask of wheat prior to autoclaving at 121°C. for 20 minutes. To obtain proper consistency, each flask was vigorously shaken by hand prior to and immediately after autoclaving.
Aflatoxin standard was prepared with a benzene-acetonitrile solution according to the method of Rodricks and Stoloff (1970). All standards, when not in use, were stored at — 18°C. in 5-dram glass teflon-lined screw-cap vials. A TLR-5 thermoluminescent dosimeter (Eberline) was used for determination of irradiation potential of the 60 Co source. An average dose rate of 10.2 krad. per min. was calculated following trial exposures of L i F crystals to the Cobalt source. Each crystal was contained in a glass vial later used in the spore irradiation studies.
The autoclaved wheat remained at room temperature for 18 hours to allow for proper moisture distribution prior to spore inoculations. One ml. of irradiated spore suspension was aseptically added to each of 10 flasks containing either wheat
Mycelial growth was separated from the synthetic medium by filtering and the filtrate collected in a 250 ml. separatory funnel. The mycelia-free liquid was then extracted according to the method of Adyes and Mateles (1964) with the following exception: phase extractions of the growth-free liquid was accomplished b y two 25 ml. washes of chloroform instead of a single 1:1 chloroform-liquid wash. T h e two aflatoxin-containing chloroform phases were pooled and brought to dryness under nitrogen. T h e resulting residue was transferred to 10 X 75 mm. glass vials via three 2 ml. chloroform washes
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K. L. APPLEGATE AND J. R.
and returned to dryness. All dried extracts were stored at — 18°C. Primary wheat extractions of each wheat sample was accomplished according to Stoloff (1971). The primary chloroform extract was subsequently reduced to dryness under nitrogen. The resulting residue was eluted according to the method of Pons (1969), returned to dryness on a rotary flask, evaporator, and the residue transferred to vials by three 2 ml. chloroform washes, redried, and stored at -18°C. The respective crude extracts representing the different incubation times, irradiation levels, and sample media were spotted on thin-layer glass plates coated with MN Kieselgel silica gel to a thickness of 250 mm. and activated at 80°C. for 2 hours prior to use. Aflatoxin Bi was collected into extraction soxlets via a microvacuum zone collector and eluted by three 10 ml. "flow-through" washes of chloroform-acetone (4:1) into 16 X 125 mm. test tubes and brought to dryness under nitrogen gas. The dried residues were later resuspended in 2.2 ml. of benzene-acetonitrile (98:2), agitated for 30 seconds on a Vortex Jr. mixer (Scientific Products), and quantitatively determined by use of a DU-2 spectrophotometer at 358 nm. (Rodricks and Stoloff, 1970).
CHIPLEY
Exposure of this organism to 25, 50, 100, 150, 200, 300, 400 or 600 krad. did not induce the production of aflatoxin Bi by this organism on either wheat or in the synthetic medium. However, the quantities of aflatoxin Bi produced from the irradiated toxinogenic strain (NRRL3145) were greater in both wheat and synthetic media following a 10-day incubation period at 28°C. than were nonirradiated toxinogenic controls (Fig. 1). Aflatoxin Bi production on wheat medium was increased by exposing spores from this strain to 150 or 200 krad. Of the six doses of ionizing irradiation studied (25, 50, 100, 150, 200 and 300 krad.), only the 150 krad. doses induced higher Bi production by the toxinogenic strain than by nonirradiated toxinogenic controls following a 10-day incubation in synthetic medium (Fig. 2). DISCUSSION
These results contradict those by Jemmali and Guilbot (1969). These investigators reported that exposure of seven strains of non-toxinogenic A. flavus to 100
RESULTS
The growth and sporulation of Aspergillus flavus NRRL-A-12268 were greatly reduced in both the wheat and synthetic media by exposure of the fungus to 300 krad., with complete growth inhibition resulting from exposures to 400 krad. of irradiation. These results are similar to those reported by Bridges et al. (1956) who observed that spores of A. flavus were inactivated by an ionizing radiation dose of 300 krad.
Time (days)
60
FIG. 1. Effects of Co irradiation on aflatoxin Bi production by Aspergillus flavus on 25 grams of wheat.
A F L A T O X I N PRODUCTION
FIG. 2. Effects of 60Co irradiation on aflatoxin Bi production by Aspergillusfiavusin 25 ml. of synthetic medium.
1495
were conducted since the Rf values of these spots were significantly different from those of the Bi standards. Although results of this investigation indicate t h a t exposure to gamma irradiation does not induce aflatoxin production by non-toxinogenic aspergilli, toxinogenic aspergilli appear to be induced to produce significantly larger amounts of aflatoxin Bi when exposed to irradiation levels of 150 or 200 krad. Since cobalt irradiation is presently sanctioned by the United States D e p a r t m e n t of Agriculture for irradiation of wheat, the induced production of anatoxins by irradiated toxinogenic strains of fungi could be of public health significance. ACKNOWLEDGMENT
krad. induced the individual strains to produce varying amounts of aflatoxin Bi. I t is possible t h a t this difference in results between the present study and those of Jemmali and Guilbot (1969) m a y be due to difference in test organisms since the strains of A. flavus which they used were not available for comparison with A.flavus NRRL-A-12268. I t is also possible t h a t the strains of A. flavus which they used were, in fact, aflatoxin-producing organisms b u t produced too little aflatoxin for detection by the techniques employed. Irradiation then m a y have increased the level of aflatoxin production to a detectable level rather t h a n induced anatoxin production by their strains. I t is also possible t h a t substances which exhibit the same blue fluorescent properties of aflatoxin on thin-layer chromatography (TLC) plates m a y have been mistaken b y Jemmali and Guilbot (1969) as anatoxins (Mislivec et al., 1968). I n the present study, similar faint blue spots were observed periodically on T L C plates after growth of irradiated N R R L A-12268 spores on wheat. No bioassays
The senior author wishes to acknowledge the assistance of Dr. J. F. Stephens in this study. REFERENCES Adyes, J., and R. I. Mateles, 1964. Incorporation of labeled compounds into anatoxins. Biochem. Biophys. Acta, 86: 418-420. AUcroft, R., B. A. Roberts and W. H. Butler, 1967. Aflatoxin in milk. Food Cosmet. Toxicol. 5: 597. Archibald, R. McG., H. J. Smith and J. D. Smith, 1962. Brazilian ground-nut toxicosis in Canadian broiler chickens. Can. Vet. J. 3: 322-325. Arseculeratne, S. N., L. M. Desilva, S. Wijesundera and C. H. S. R. Banduntha, 1969. Coconut as a medium for experimental production of aflatoxin. Appl. Microbiol. 18: 88-94. Asplin, F. D., and R. B. A. Carnaghan, 1961. The toxicity of certain groundnut meals for poultry with special reference to their effect on ducklings and chickens. Vet. Res. 73: 1215-1218. Blount, W. P., 1971. Turkey "X" disease. Turkeys (J. Brit. Turkey Federation) 9 (2): 52, 55-58, 61, 77. Bridges, A. E., J. P. Olive and V. L. Chandler, 1965. Relative resistance of microorganisms to cathode rays. Appl. Microbiol. 4: 147-152. Christensen, C. M., and B. W. Kennedy, 1971. Filamentous fungi and bacteria in macaroni and spaghetti products. Appl. Microbiol. 21:144-146. Epstein, E., M. P. Steinberg, A. I. Nelson and L. S. Wei, 1970. Aflatoxin production as affected by
1496
K. L. APPLEGATE AND J. R. CHIPLEY
environmental conditions. J. Food Sci. 35: 389390. Halver, J. F., 1965. In: Mycotoxins in Foodstuffs G. N. Wogan, ed., M.I.T. Press, Cambridge, Mass. pp. 204-234. Jemmali, M., and A. Guilbot, 1969. Influence de l'irradiation des spores d'A. flavus sur la production d'aflatoxin Bi. C. R. Acad. Sc. Paris, T. 269, series D : 2271-2273. Mislivec, P. B., J. H. Hunter, and J. Tuile, 1968. Assay for anatoxin production by the genera Aspergillus and Penicillium. Appl. Microbiol. 16: 1053-1055. Oettle, A. G., 1964. Cancer in Africa, especially in regions south of the Sahara. J. Natl. Cancer Inst. 33:383-439. Oettle, A. G., 1965. The etiology of primary carcinoma of the liver in Africa: A critical appraisal of previous ideas with an outline of the mycotoxin hypothesis. South Africa Med. J. 39: 817-925. Pons, W. A., Jr., 1969. Collaborative study on the determination of anatoxins in cottonseed products. J. Assoc. Ofiic. Anal. Chemists, 52: 61-72.
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Moisture and Microwave Effects on Selected Characteristics of Turkey Pectoral Muscles GEORGIA W. CREWS 1 AND GRAYCE E. GOERTZ Department of Food Science and Institution Administration, College of Home Economics and Agricultural Experiment Station, The University of Tennessee, Knoxville, Tennessee 37916 (Received for publication November 10, 1972) ABSTRACT The effect of added water and microwave heating on the water holding capacity as indicated by expressible moisture index (EMI), total moisture, cooking losses and p H for U.S.D.A. Grade A turkey toms was studied. Samples (200 g.) of ground composites of pectoral muscles containing 0,15, or 30 ml. added water were heated in a microwave oven for 0, 70 or 130 sec. The 15 and 30 ml. samples represented 7 and 13% added water and end point temperatures for 70 and 130 sec. were 44 to 47°C. and 61 to 64°C. Expressible moisture index was the best measure of treatment effects. Percent variation was greater for the E M I than for measures of cooking losses and total moisture. Total moisture values indicated water loss during cooking but some added water was retained. Cooking time caused the greatest percent variation; added water also caused significant effects on all characteristics studied. POULTRY SCIENCE 52: 1496-1500, 1973
INTRODUCTION
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EATING alters the chemical and physical characteristics of muscle protein, as evidenced by denaturation and :
Present Address: Department of Human Nutrition and Food, Virginia Polytechnic Institute and State University, Blacksburg, Virginia.
decreased ability of muscle to retain water. Factors that affect the chemical and physical composition of protein also affect water in ways that alter the juiciness, tenderness, and overall acceptability of meat. In previous studies, heat denaturation of tube-cooked beef began at 40°C. and was