APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1978, p. 720-723
0099-2240/78/0036-0720$02.00/ Copyright © 1978 American Society for Microbiology
Vol. 36, No. 5 Printed in U.S.A.
Bioproduction of ['4C]Ochratoxin A in Submerged Culture E. B. LILLEHOJ,I*
AALUND,2 AND B. HALD2 Northern Regional Research Center, Feder.al Research, Science and Education Administration, U.S. 0.
Department of Agriculture, Peoria, Illinois 61604,' and Royal Veterinary and Agricultural University, DK1870 Copenhagen, Denmark2
Received for publication 5 September 1978
A number of Aspergillus and Penicillium species were tested for production of ochratoxin A (OA) in several media. After 8 days of static incubations of submerged cultures at 28°C, toxin yields of 25 and 30 ,ig/ml were obtained with Aspergillus alliaceus NRRL 4181 in Ferreiras and 2% yeast extract-4% sucrose media, respectively. However, the largest production observed in the preliminary screening was 54 jig/ml; this highest level was produced by A. sulphureus NRRL 4077 in a modified Czapek solution. The medium contained the basal salts and sucrose of Czapek plus urea (3%) and corn steep liquor (0.5% solids). A time study of toxin production demonstrated maximum yield of 350 ,ug/ml by the A. sulphureus isolate in the modified Czapek medium after 11 days of static incubation at 280C. The optimal production conditions were employed in additional tests designed to measure the efficiency of 14C incorporation from sodium [1_I4C]_ acetate into OA. Samples (20 ,uCi) of sodium acetate were added to separate culture flasks at 24-h intervals during the initial 9 days of the fermentation. Addition of [14C]acetate on day 4 of incubation provided the maximum yield of labeled OA. The highest specific activity of labeled toxin obtained was 0.07 ,uCi/ mg of OA and the maximum incorporation rate of labeled acetate was 5.3%.
Ochratoxin A (OA), 7-carboxyl-5-chloro-5-hy- on solid substrates (3, 4, 10). Steyn et al. (11) droxyl 3,4-dihydro-3-R-methyl isocoumarin and Yamazaki et al. (13) demonstrated that the linked to L-phenylalanine, is a toxic metabolite formation of the dihydroisocoumarin moiety of of several Aspergillus and Penicillium species OA is derived from a straight polyketide chain (3,4, 10). Although several symptoms have been of acetate units. Wei et al. (12) studied the associated with OA poisoning in animals, the incorporation of 36Cl into the toxin; they obprinciple target of the toxin is the kidney (7, 10). served that time of addition of chlorine to the A spontaneous occurrence of renal disease in fermentation medium was an important factor Scandinavian swine has been related to ochra- in the rate of synthesis of halogenated OA. toxin ingestion (7). Investigation of feed samples High specific activity of radioactively labeled from areas with a high incidence of the disease OA is a requisite for effective study of tissue showed occurrence rates exceeding 50% (7). OA distribution and the mode of action of the toxin. has also been implicated in kidney disease of Chang and Chu (1) prepared [3H]OA with spechickens (7, 10). Doerr et al. (5) have provided cific activities of 2.6 to 3.0 Ci/mmol by an exevidence that the toxin is the most potent change procedure with tritiated water. Although growth inhibitory mycotoxin studied in chick- the specific activity of the [3H]OA was high, ens. tritium exchange in tissue could provide ambigA kidney disease of humans identified in cer- uous results in some test systems. Therefore, the tain areas of Bulgaria, Rumania, and Yugoslavia current study was carried out to identify optialso has been linked to OA (7). Recent studies mum conditions for incorporation of ['4C]acetate of food in areas experiencing a frequent occur- into the ring structure of OA. rence of the disease demonstrated a high incidence of the toxin (8). In addition, an association MATERIALS AND METHODS between the kidney disease and enhanced rate Media. A number of media were tested as screening of urinary tract tumors has been reported (2). for elaboration of fluorescence by ochratoxinThe fungi that produce OA are widely distrib- agents producing fungi. The basal medium was Czapek soluuted and have been identified in many foods and tion agar containing: 1.0 g; MgSO4 7H20, 0.5 feeds (3, 10). Although several of the toxin-pro- g; HCI, 0.5 g; FeSO4. K2HP04, 7H20, 0.01 g; sucrose, 30 g; agar, ducing isolates elaborate the toxin in submerged 20 g; and distilled water, 1 liter. Amendments included: culture, the highest yields have been obtained (NH4)H2PO4, 3 g; Casamino Acids (Difco), 3 g; urea, 3 720
VOL. 36, 1978
['4C]OCHRATOXIN A IN SUBMERGED CULTURE
and corn steep liquor (CS) (Clinton Corn Processing Co.), 0.5 g of solids (dry weight). Submerged media for OA production included: (i) modified Czapek solution (MC) (basal medium plus urea, 3 g; CS, 0.5 g/liter); (ii) yeast extract-sucrose (YES) (yeast extract [Difco], 20 g; sucrose, 40 g; distilled water, 1 liter); (iii) Ferreira medium (FM) (6) (KH2PO4, 1 g; KCl, 0.5 g; MgSO4.7H20, 0.5 g; FeSO4, 18 mg; ZnSO4, 22 mg; MnSO4 H20, 8 mg; CuSO4.5H20, 4 mg; ammonium molybdate, 2.5 mg; glutamic acid, 10 g; sucrose, 30 g; distilled water, 1 liter); (iv) Raulin-Thom medium (RT) [tartaric acid, 2.6 g; diammonium tartrate, 2.6 g; (NH4)H2PO4, 0.4 g; K2CO3, 0.4 g; MgCO3, 0.25 g; (NH4)2SO4, 0.16 g; ZnSO4* 7H20, 0.6 g; FeSO4. 7H20, 0.06 g; glucose, 50 g; distilled water, 1 liter]. Culture. Petri plates containing sterile media were incoulated at the center of the agar surface with test fungi conidia. Plates were incubated at 28°C for 10 days and subsequently examined under UV light (366 nm) for characteristic bluish fluorescence of ochratoxin. Liquid media were dispensed into either 300-ml Erlenmeyer flasks (50 ml) or 2.8-liter Fernbach flasks (200 ml), autoclaved, inoculated with fungal conidia, and incubated statically at varied temperatures and different times. [1-'4C]sodium acetate (specific activity, 5 mCi/mmol) was dissolved in distilled water, and appropriate quantities were added to the fermentation medium at designated times. Liquid media were inoculated by adding a 1-ml conidial suspension to 300-ml Erlenmeyer flasks and a 10-ml conidial suspension to 2.8-liter Fernbach flasks. Conidial suspensions were prepared by scraping spores from the surface of a 10to 14-day-old potato-dextrose-agar slant into an aqueous, sterile solution of Triton X (0.01%). Extraction and isolation of OA. OA was extracted from the fermentation broth by adjusting the pH to 2.5, adding 2x the volume of CHC13, stirring the g;
721
two phases gently for 8 h, and reducing the CHCl3 solution to 1 to 5 ml. The toxin-containing solution was applied to thin-layer chromatographic glass plates 20 by 20 cm layered with 0.75 mm of Adsorbosil-1
(Applied Sciences Laboratories). Plates were developed in benzene:acetic acid (90:10, vol/vol) in unlined, unequilibrated tanks. The OA band was identified under UV light and scraped from the plate; the toxin was eluted with CHCl3:methanol (9:1). Concentrations of the toxin were determined by visual comparison of fluorescent intensities of specific volumes of test solutions with standard quantities of OA after development on thin-layer chromatographic plates (0.25 mm of Adsorbosil-1) in benzene:acetic acid (90:10). Labeled OA. Radioactivity of OA was measured in a Beckman LS-250 liquid scintillation spectrometer. The liquid scintillation fluor contained 4 g of PPO (2,5-diphenyloxazole) plus 40 mg dimethyl-POPOP [1,4-bis-2-(4-methyl-5-phenyloxazolyl)-benzene] in 1 liter of toluene. Specific activities of labeled OA were determined by the number of disintegrations per minute per weight of the toxin.
RESULTS AND DISCUSSION To expedite broad screening of fungi for OA production, a number of agar media were tested for their ability to support production of fluorescent material that could be associated with toxin synthesis (Table 1). The presence of a bluishwhite fluorescence was observed in the agar of some of the isolates; this fluorescence was associated with OA because CHC13 extraction of agar of test plates with and without the fluorescent material demonstrated the qualitative occurrence of OA exclusively in extracts of the positive plates. The medium providing the broadest detection of toxin-producing isolates was MC with
TABLE 1. Detection of ochratoxin-producing species by elaboration offluorescence in a nutrient agar medium Intensity of blue fluorescencea with amendments to basal mediumb: CA CA + CS U + CS (NIL)H2P04 U (NH4)H2P04 + A. alliaceus NRRL 4181 + + A. ochraceus NRRL 410 + A. ochraceus NRRL 5175 + + + + + A. ochraceus NRRL 5222 + + A. ochraceus NRRL 5223 + A. melleus NRRL 5227 A. scierotiorum NRRL 5170 A. scierotiorum NRRL 3793 + + + ++ A. sulphureus NRRL 4077 P. viridicatum NRRL 963 P. viridicatum NRRL 1161 P. viridicatum NRRL 3586 P. viridicatum NRRL 3710 P. viridicatum NRRL 3711 P. viridicatum NRRL 3712 a Plates were examined under UV (366 nm) light after 10 days of incubation at 28°C. b Basal medium was Czapek nutrient agar. Amendments included: (NH4)HPO4, 0.3%; CS, weight); CA, Casaniino Acids, 0.3%; U, urea, 0.3%.
+ + _ -
++ + + + + ++ + -
++
+++
-
-
+
-
-
+ +
-
0.05% solids (dry
722
LILLEHOJ, AALUND, AND HALD
APPL. ENVIRON. MICROBIOL.
urea and CS. In addition, the most intense fluorescent zones around the developing fungal colony were observed in MC medium, particularly with the A. sulphureus NRRL 4077 isolate. Daily examination of test plates after day 5 of incubation indicated that maximum fluorescence occurred in the agar at 9 to 12 days of incubation. Production of OA by A. sulphureus in submerged culture of MC was compared with yields in other media (28°C, 8 days) that had been utilized in earlier studies (3, 4, 6) (Table 2). Low OA levels were observed in all media inoculated with some isolates of A. ochraceus, A. sclerotiorum, and Penicillium viridicatum. Middlerange yields of 23 to 30 jig of OA per ml of medium were observed by A. alliaceus in MC, FM, and YES media. However, the highest toxin production (54 jig/ml) was obtained by A. sulphureus culture in MC medium. Examination of the kinetics of toxin accumulation in submerged culture of A. sulphureus in MC medium demonstrated that the highest OA
levels occurred day 11 of incubation at 28°C (Fig. 1). Parallel fermentations at 20°C with the 28°C incubation showed that about three times more OA accumulated at 28°C than at 20°C. Maximum toxin yields at 20°C occurred 1 day after the highest OA levels were determined in the culture media incubated at 28°C. A marked reduction in toxin levels at both incubation temperatures occurred during the period after maximum OA accumulation. The relatively high yields of OA in submerged culture of A. sulphureus in MC medium at 28°C provided an opportunity for efficient ring labeling of OA with 4C of substrate acetate. The rate of incorporation of acetate during the fermentation was studied by addition of labeled substrate each day during the first 8 days of an 11day incubation (Table 3). Maximum incorporation of '4C occurred in media that had been amended with labeled acetate on day 4 of incubation; these conditions provided a 5.3% rate of incorporation of ['4C]acetate into OA. TABLE 3. A. sulphureus NRRL 4077 incorporation of ['4Cacetate into OA
TABLE 2. Production of OA in submerged culture by several species of Aspergillus and Penicillium
Addition of ['"C]acetate' (day)
OA (,g/ml)' in medium:
Organism YES
FM 25
RT
MC
1 30 alliaceus NRRL 4181 23 27 ochraceus NRRL 3174 ND tr 1 tr ochraceus NRRL 410 ND ND tr ochraceus NRRL 5223 tr ND 12 ND tr ND ND tr ochraceus NRRL 5175 melleus NRRL 5227 4 tr 1 5 tr tr sclerotiorum NRRL 5170 tr ND 21 1 suphureus NRRL 4077 22 54 viridicatum NRRL 1161 tr ND tr ND viridicatum NRRL 3711 tr ND tr ND viridicatum NRRL 3710 ND tr ND ND "Means of duplicate 300-ml Erlenmeyer flasks containing 50 ml of medium (28°C, 8 days). ND, None detected. tr, Trace, less than 1 g/ml.
A. A. A. A. A. A. A. A. P. P. P.
0 1 2 3 4 5 6 7 8
OA Sp act
(,ICi/lLg)h
3.2 5.7 8.4 18.1 70.5 55.1 21.8 7.3 0.9
0.1 'A 20-,uCi amount of ['4C]acetate added at designated periods. ' Means of duplicate 300-ml Erlenmeyer flasks containing 50 ml of MC incubated for 11 days at 28°C.
400 E' E 300 0
W
200
._
x-
100
C.
0
2
4
6
['"C]acetate in-
corporation(% 0.3 0.5 0.8 1.8 5.3 4.4 2.0 0.7
8
Days
10
12
14
16
FIG. 1. Production of OA by A. sulphureus in CM incubated at 20 and 28°C.
['4C]OCHRATOXIN A IN SUBMERGED CULTURE
VOL. 36, 1978
TABLE 4. Variation in specific activity of['4C]OA synthesized by A. sulphureus NRRL 4077 by addition of varied levels of P4Clacetate
OAa
['4C]acetate (uCi/
mi)b 40 80 160 320
Total (mg) 22
26 24 24
Sp act
(uLCi/ug)
60 130 210 570
a Means of duplicate 2.8-liter Fernbach flasks containing 200 ml of MC. b Labeled acetate added at day 4 of an 11-day incubation at 28°C.
An OA production study in 2.8-liter Fernbach flasks was carried out to determine yields in larger fermentation of A. sulphureus in MC media (Table 4). Yields in the 200-ml fermentation broths after 11 days of incubation at 280C ranged from 22 to 24 mg of OA per flask. Addition of 40 to 320 ,iCi of ['4C]acetate per flask on day 4 of incubation provided specific radioactivity in the OA of 0.06 to 0.57 ,tCi/mg of OA. Yields of toxin obtained with A. sulphureus in the MC medium provide quantities of ring-labeled OA at levels of specific activity required for sensitive investigations of tissue distribution in test animals. LITERATURE CITED 1. Chang, F. C., and F. S. Chu. 1976. Preparation of 'H-
labeled ochratoxins. J. Labelled Compd. Radiopharm. 12:231-238. 2. Chernozimsky, I. N., I. S. Stoyanov, T. K. PetkovaBocharova, I. G. Nicolov, I. V. Draganov, I. I.
723
Stoichev, Y. Tanchev, and N. D. Kalcheva. 1977. Geographic correlation between the occurrence of endemic nephropathy and urinary tract tumours in Vratza district, Bulgaria. Int. J. Cancer 19:1-11. 3. Chu, F. S. 1974. Studies on ochratoxin. CRC Crit. Rev. Toxicol. 3:499-524. 4. Ciegler, A. 1972. Bioproduction of ochratoxin A and penicillic acid by members of the Aspergillus ochraceus group. Can. J. Microbiol. 18:631-634. 5. Doerr, J. A., W. E. Huff, H. T. Tung, R. D. Wyatt, and P. B. Hamilton. 1974. A survey of T-2 toxin, ochratoxin, and aflatoxin for their effects on the coagulation of blood in young broiler chickens. Poult. Sci. 53:1728-1734. 6. Ferreira, N. P. 1967. Recent advances in research on ochratoxin. Part 2, Microbiological aspects, p. 157-168. In R. I. Mateles and G. N. Wogan (ed.), Biochemistry of some food-borne microbial toxins. MIT Press, Cambridge, Mass. 7. Krogh, P. 1976. Mycotoxic nephropathy. Adv. Vet. Sci. Comp. Med. 20:147-170. 8. Krogh, P., B. Hald, R. Plestina, and S. Ceovic. 1977. Balkan (endemic) nephropathy and foodborne ochratoxin A: preliminary results of a survey of foodstuffs. Acta Pathol. Microbiol. Scand. Sect. B 85:238-240. 9. Scott, P. M., W. van Walbeek, B. Kennedy, and D. Anyeti. 1972. Mycotoxins (ochratoxin A, citrinin, and sterigmatocystin) and toxigenic fungi in grains and other agricultural products. J. Agric. Food Chem. 20:1103-1109. 10. Steyn, P. S. 1971. Ochratoxin and other dihydroisocoumarins, p. 179-205. In A. Ciegler, S. Kadis, and S. J. Ajl (ed.), Microbial toxins, vol. 6. Fungal toxins. Academic Press Inc., New York. 11. Steyn, P. S., C. W. Holzapfel, and N. P. Ferreira. 1970. The biosynthesis of the ochratoxins, metabolites of Aspergillus ochraceus. Phytochemistry 9:1977-1983. 12. Wei, R.-D., F. M. Strong, and E. B. Smalley. 1971. Incorporation of chlorine-36 into ochratoxin A. Appl. Microbiol. 22:276-277. 13. Yamazaki, M. Y., Maebayashi, and K. Miyaki. 1971. Biosynthesis of ochratoxin A. Tetrahedron Lett.
25:2301-2304.
0099-2240/78/0036-0720$02.00/ Copyright © 1978 American Society for Microbiology
Vol. 36, No. 5 Printed in U.S.A.
Bioproduction of ['4C]Ochratoxin A in Submerged Culture E. B. LILLEHOJ,I*
AALUND,2 AND B. HALD2 Northern Regional Research Center, Feder.al Research, Science and Education Administration, U.S. 0.
Department of Agriculture, Peoria, Illinois 61604,' and Royal Veterinary and Agricultural University, DK1870 Copenhagen, Denmark2
Received for publication 5 September 1978
A number of Aspergillus and Penicillium species were tested for production of ochratoxin A (OA) in several media. After 8 days of static incubations of submerged cultures at 28°C, toxin yields of 25 and 30 ,ig/ml were obtained with Aspergillus alliaceus NRRL 4181 in Ferreiras and 2% yeast extract-4% sucrose media, respectively. However, the largest production observed in the preliminary screening was 54 jig/ml; this highest level was produced by A. sulphureus NRRL 4077 in a modified Czapek solution. The medium contained the basal salts and sucrose of Czapek plus urea (3%) and corn steep liquor (0.5% solids). A time study of toxin production demonstrated maximum yield of 350 ,ug/ml by the A. sulphureus isolate in the modified Czapek medium after 11 days of static incubation at 280C. The optimal production conditions were employed in additional tests designed to measure the efficiency of 14C incorporation from sodium [1_I4C]_ acetate into OA. Samples (20 ,uCi) of sodium acetate were added to separate culture flasks at 24-h intervals during the initial 9 days of the fermentation. Addition of [14C]acetate on day 4 of incubation provided the maximum yield of labeled OA. The highest specific activity of labeled toxin obtained was 0.07 ,uCi/ mg of OA and the maximum incorporation rate of labeled acetate was 5.3%.
Ochratoxin A (OA), 7-carboxyl-5-chloro-5-hy- on solid substrates (3, 4, 10). Steyn et al. (11) droxyl 3,4-dihydro-3-R-methyl isocoumarin and Yamazaki et al. (13) demonstrated that the linked to L-phenylalanine, is a toxic metabolite formation of the dihydroisocoumarin moiety of of several Aspergillus and Penicillium species OA is derived from a straight polyketide chain (3,4, 10). Although several symptoms have been of acetate units. Wei et al. (12) studied the associated with OA poisoning in animals, the incorporation of 36Cl into the toxin; they obprinciple target of the toxin is the kidney (7, 10). served that time of addition of chlorine to the A spontaneous occurrence of renal disease in fermentation medium was an important factor Scandinavian swine has been related to ochra- in the rate of synthesis of halogenated OA. toxin ingestion (7). Investigation of feed samples High specific activity of radioactively labeled from areas with a high incidence of the disease OA is a requisite for effective study of tissue showed occurrence rates exceeding 50% (7). OA distribution and the mode of action of the toxin. has also been implicated in kidney disease of Chang and Chu (1) prepared [3H]OA with spechickens (7, 10). Doerr et al. (5) have provided cific activities of 2.6 to 3.0 Ci/mmol by an exevidence that the toxin is the most potent change procedure with tritiated water. Although growth inhibitory mycotoxin studied in chick- the specific activity of the [3H]OA was high, ens. tritium exchange in tissue could provide ambigA kidney disease of humans identified in cer- uous results in some test systems. Therefore, the tain areas of Bulgaria, Rumania, and Yugoslavia current study was carried out to identify optialso has been linked to OA (7). Recent studies mum conditions for incorporation of ['4C]acetate of food in areas experiencing a frequent occur- into the ring structure of OA. rence of the disease demonstrated a high incidence of the toxin (8). In addition, an association MATERIALS AND METHODS between the kidney disease and enhanced rate Media. A number of media were tested as screening of urinary tract tumors has been reported (2). for elaboration of fluorescence by ochratoxinThe fungi that produce OA are widely distrib- agents producing fungi. The basal medium was Czapek soluuted and have been identified in many foods and tion agar containing: 1.0 g; MgSO4 7H20, 0.5 feeds (3, 10). Although several of the toxin-pro- g; HCI, 0.5 g; FeSO4. K2HP04, 7H20, 0.01 g; sucrose, 30 g; agar, ducing isolates elaborate the toxin in submerged 20 g; and distilled water, 1 liter. Amendments included: culture, the highest yields have been obtained (NH4)H2PO4, 3 g; Casamino Acids (Difco), 3 g; urea, 3 720
VOL. 36, 1978
['4C]OCHRATOXIN A IN SUBMERGED CULTURE
and corn steep liquor (CS) (Clinton Corn Processing Co.), 0.5 g of solids (dry weight). Submerged media for OA production included: (i) modified Czapek solution (MC) (basal medium plus urea, 3 g; CS, 0.5 g/liter); (ii) yeast extract-sucrose (YES) (yeast extract [Difco], 20 g; sucrose, 40 g; distilled water, 1 liter); (iii) Ferreira medium (FM) (6) (KH2PO4, 1 g; KCl, 0.5 g; MgSO4.7H20, 0.5 g; FeSO4, 18 mg; ZnSO4, 22 mg; MnSO4 H20, 8 mg; CuSO4.5H20, 4 mg; ammonium molybdate, 2.5 mg; glutamic acid, 10 g; sucrose, 30 g; distilled water, 1 liter); (iv) Raulin-Thom medium (RT) [tartaric acid, 2.6 g; diammonium tartrate, 2.6 g; (NH4)H2PO4, 0.4 g; K2CO3, 0.4 g; MgCO3, 0.25 g; (NH4)2SO4, 0.16 g; ZnSO4* 7H20, 0.6 g; FeSO4. 7H20, 0.06 g; glucose, 50 g; distilled water, 1 liter]. Culture. Petri plates containing sterile media were incoulated at the center of the agar surface with test fungi conidia. Plates were incubated at 28°C for 10 days and subsequently examined under UV light (366 nm) for characteristic bluish fluorescence of ochratoxin. Liquid media were dispensed into either 300-ml Erlenmeyer flasks (50 ml) or 2.8-liter Fernbach flasks (200 ml), autoclaved, inoculated with fungal conidia, and incubated statically at varied temperatures and different times. [1-'4C]sodium acetate (specific activity, 5 mCi/mmol) was dissolved in distilled water, and appropriate quantities were added to the fermentation medium at designated times. Liquid media were inoculated by adding a 1-ml conidial suspension to 300-ml Erlenmeyer flasks and a 10-ml conidial suspension to 2.8-liter Fernbach flasks. Conidial suspensions were prepared by scraping spores from the surface of a 10to 14-day-old potato-dextrose-agar slant into an aqueous, sterile solution of Triton X (0.01%). Extraction and isolation of OA. OA was extracted from the fermentation broth by adjusting the pH to 2.5, adding 2x the volume of CHC13, stirring the g;
721
two phases gently for 8 h, and reducing the CHCl3 solution to 1 to 5 ml. The toxin-containing solution was applied to thin-layer chromatographic glass plates 20 by 20 cm layered with 0.75 mm of Adsorbosil-1
(Applied Sciences Laboratories). Plates were developed in benzene:acetic acid (90:10, vol/vol) in unlined, unequilibrated tanks. The OA band was identified under UV light and scraped from the plate; the toxin was eluted with CHCl3:methanol (9:1). Concentrations of the toxin were determined by visual comparison of fluorescent intensities of specific volumes of test solutions with standard quantities of OA after development on thin-layer chromatographic plates (0.25 mm of Adsorbosil-1) in benzene:acetic acid (90:10). Labeled OA. Radioactivity of OA was measured in a Beckman LS-250 liquid scintillation spectrometer. The liquid scintillation fluor contained 4 g of PPO (2,5-diphenyloxazole) plus 40 mg dimethyl-POPOP [1,4-bis-2-(4-methyl-5-phenyloxazolyl)-benzene] in 1 liter of toluene. Specific activities of labeled OA were determined by the number of disintegrations per minute per weight of the toxin.
RESULTS AND DISCUSSION To expedite broad screening of fungi for OA production, a number of agar media were tested for their ability to support production of fluorescent material that could be associated with toxin synthesis (Table 1). The presence of a bluishwhite fluorescence was observed in the agar of some of the isolates; this fluorescence was associated with OA because CHC13 extraction of agar of test plates with and without the fluorescent material demonstrated the qualitative occurrence of OA exclusively in extracts of the positive plates. The medium providing the broadest detection of toxin-producing isolates was MC with
TABLE 1. Detection of ochratoxin-producing species by elaboration offluorescence in a nutrient agar medium Intensity of blue fluorescencea with amendments to basal mediumb: CA CA + CS U + CS (NIL)H2P04 U (NH4)H2P04 + A. alliaceus NRRL 4181 + + A. ochraceus NRRL 410 + A. ochraceus NRRL 5175 + + + + + A. ochraceus NRRL 5222 + + A. ochraceus NRRL 5223 + A. melleus NRRL 5227 A. scierotiorum NRRL 5170 A. scierotiorum NRRL 3793 + + + ++ A. sulphureus NRRL 4077 P. viridicatum NRRL 963 P. viridicatum NRRL 1161 P. viridicatum NRRL 3586 P. viridicatum NRRL 3710 P. viridicatum NRRL 3711 P. viridicatum NRRL 3712 a Plates were examined under UV (366 nm) light after 10 days of incubation at 28°C. b Basal medium was Czapek nutrient agar. Amendments included: (NH4)HPO4, 0.3%; CS, weight); CA, Casaniino Acids, 0.3%; U, urea, 0.3%.
+ + _ -
++ + + + + ++ + -
++
+++
-
-
+
-
-
+ +
-
0.05% solids (dry
722
LILLEHOJ, AALUND, AND HALD
APPL. ENVIRON. MICROBIOL.
urea and CS. In addition, the most intense fluorescent zones around the developing fungal colony were observed in MC medium, particularly with the A. sulphureus NRRL 4077 isolate. Daily examination of test plates after day 5 of incubation indicated that maximum fluorescence occurred in the agar at 9 to 12 days of incubation. Production of OA by A. sulphureus in submerged culture of MC was compared with yields in other media (28°C, 8 days) that had been utilized in earlier studies (3, 4, 6) (Table 2). Low OA levels were observed in all media inoculated with some isolates of A. ochraceus, A. sclerotiorum, and Penicillium viridicatum. Middlerange yields of 23 to 30 jig of OA per ml of medium were observed by A. alliaceus in MC, FM, and YES media. However, the highest toxin production (54 jig/ml) was obtained by A. sulphureus culture in MC medium. Examination of the kinetics of toxin accumulation in submerged culture of A. sulphureus in MC medium demonstrated that the highest OA
levels occurred day 11 of incubation at 28°C (Fig. 1). Parallel fermentations at 20°C with the 28°C incubation showed that about three times more OA accumulated at 28°C than at 20°C. Maximum toxin yields at 20°C occurred 1 day after the highest OA levels were determined in the culture media incubated at 28°C. A marked reduction in toxin levels at both incubation temperatures occurred during the period after maximum OA accumulation. The relatively high yields of OA in submerged culture of A. sulphureus in MC medium at 28°C provided an opportunity for efficient ring labeling of OA with 4C of substrate acetate. The rate of incorporation of acetate during the fermentation was studied by addition of labeled substrate each day during the first 8 days of an 11day incubation (Table 3). Maximum incorporation of '4C occurred in media that had been amended with labeled acetate on day 4 of incubation; these conditions provided a 5.3% rate of incorporation of ['4C]acetate into OA. TABLE 3. A. sulphureus NRRL 4077 incorporation of ['4Cacetate into OA
TABLE 2. Production of OA in submerged culture by several species of Aspergillus and Penicillium
Addition of ['"C]acetate' (day)
OA (,g/ml)' in medium:
Organism YES
FM 25
RT
MC
1 30 alliaceus NRRL 4181 23 27 ochraceus NRRL 3174 ND tr 1 tr ochraceus NRRL 410 ND ND tr ochraceus NRRL 5223 tr ND 12 ND tr ND ND tr ochraceus NRRL 5175 melleus NRRL 5227 4 tr 1 5 tr tr sclerotiorum NRRL 5170 tr ND 21 1 suphureus NRRL 4077 22 54 viridicatum NRRL 1161 tr ND tr ND viridicatum NRRL 3711 tr ND tr ND viridicatum NRRL 3710 ND tr ND ND "Means of duplicate 300-ml Erlenmeyer flasks containing 50 ml of medium (28°C, 8 days). ND, None detected. tr, Trace, less than 1 g/ml.
A. A. A. A. A. A. A. A. P. P. P.
0 1 2 3 4 5 6 7 8
OA Sp act
(,ICi/lLg)h
3.2 5.7 8.4 18.1 70.5 55.1 21.8 7.3 0.9
0.1 'A 20-,uCi amount of ['4C]acetate added at designated periods. ' Means of duplicate 300-ml Erlenmeyer flasks containing 50 ml of MC incubated for 11 days at 28°C.
400 E' E 300 0
W
200
._
x-
100
C.
0
2
4
6
['"C]acetate in-
corporation(% 0.3 0.5 0.8 1.8 5.3 4.4 2.0 0.7
8
Days
10
12
14
16
FIG. 1. Production of OA by A. sulphureus in CM incubated at 20 and 28°C.
['4C]OCHRATOXIN A IN SUBMERGED CULTURE
VOL. 36, 1978
TABLE 4. Variation in specific activity of['4C]OA synthesized by A. sulphureus NRRL 4077 by addition of varied levels of P4Clacetate
OAa
['4C]acetate (uCi/
mi)b 40 80 160 320
Total (mg) 22
26 24 24
Sp act
(uLCi/ug)
60 130 210 570
a Means of duplicate 2.8-liter Fernbach flasks containing 200 ml of MC. b Labeled acetate added at day 4 of an 11-day incubation at 28°C.
An OA production study in 2.8-liter Fernbach flasks was carried out to determine yields in larger fermentation of A. sulphureus in MC media (Table 4). Yields in the 200-ml fermentation broths after 11 days of incubation at 280C ranged from 22 to 24 mg of OA per flask. Addition of 40 to 320 ,iCi of ['4C]acetate per flask on day 4 of incubation provided specific radioactivity in the OA of 0.06 to 0.57 ,tCi/mg of OA. Yields of toxin obtained with A. sulphureus in the MC medium provide quantities of ring-labeled OA at levels of specific activity required for sensitive investigations of tissue distribution in test animals. LITERATURE CITED 1. Chang, F. C., and F. S. Chu. 1976. Preparation of 'H-
labeled ochratoxins. J. Labelled Compd. Radiopharm. 12:231-238. 2. Chernozimsky, I. N., I. S. Stoyanov, T. K. PetkovaBocharova, I. G. Nicolov, I. V. Draganov, I. I.
723
Stoichev, Y. Tanchev, and N. D. Kalcheva. 1977. Geographic correlation between the occurrence of endemic nephropathy and urinary tract tumours in Vratza district, Bulgaria. Int. J. Cancer 19:1-11. 3. Chu, F. S. 1974. Studies on ochratoxin. CRC Crit. Rev. Toxicol. 3:499-524. 4. Ciegler, A. 1972. Bioproduction of ochratoxin A and penicillic acid by members of the Aspergillus ochraceus group. Can. J. Microbiol. 18:631-634. 5. Doerr, J. A., W. E. Huff, H. T. Tung, R. D. Wyatt, and P. B. Hamilton. 1974. A survey of T-2 toxin, ochratoxin, and aflatoxin for their effects on the coagulation of blood in young broiler chickens. Poult. Sci. 53:1728-1734. 6. Ferreira, N. P. 1967. Recent advances in research on ochratoxin. Part 2, Microbiological aspects, p. 157-168. In R. I. Mateles and G. N. Wogan (ed.), Biochemistry of some food-borne microbial toxins. MIT Press, Cambridge, Mass. 7. Krogh, P. 1976. Mycotoxic nephropathy. Adv. Vet. Sci. Comp. Med. 20:147-170. 8. Krogh, P., B. Hald, R. Plestina, and S. Ceovic. 1977. Balkan (endemic) nephropathy and foodborne ochratoxin A: preliminary results of a survey of foodstuffs. Acta Pathol. Microbiol. Scand. Sect. B 85:238-240. 9. Scott, P. M., W. van Walbeek, B. Kennedy, and D. Anyeti. 1972. Mycotoxins (ochratoxin A, citrinin, and sterigmatocystin) and toxigenic fungi in grains and other agricultural products. J. Agric. Food Chem. 20:1103-1109. 10. Steyn, P. S. 1971. Ochratoxin and other dihydroisocoumarins, p. 179-205. In A. Ciegler, S. Kadis, and S. J. Ajl (ed.), Microbial toxins, vol. 6. Fungal toxins. Academic Press Inc., New York. 11. Steyn, P. S., C. W. Holzapfel, and N. P. Ferreira. 1970. The biosynthesis of the ochratoxins, metabolites of Aspergillus ochraceus. Phytochemistry 9:1977-1983. 12. Wei, R.-D., F. M. Strong, and E. B. Smalley. 1971. Incorporation of chlorine-36 into ochratoxin A. Appl. Microbiol. 22:276-277. 13. Yamazaki, M. Y., Maebayashi, and K. Miyaki. 1971. Biosynthesis of ochratoxin A. Tetrahedron Lett.
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