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A P P L I E D AND ENVIRONMENTAL M I C R O B I O L O G Y , J u n e 1 9 8 5 , p . 1 4 0 7 - 1 4 1 10 0 9 9 - 2 2 4 0 / 8 5 / 0 6 1 4 0 7 - 0 5 0 2 . 0 0 / 0C o p y r i g h t 1 9 8 5 , A m e r i c a n S o c i e t y f o r M i c r o b i o l o g y

I n h i b i t i o n b y A n t i m i c r o b i a l F o o d A d d i t i v e s o f O c h r a t o x i n AP r o d u c t i o n b y A s p e r g i l l u s s u l p h u r e u s a n d P e n i c i l l i u m v i r i d i c a t u mCHAO HSIANG TONG AND FRANCES A . DRAUGHON

D e p a r t m e n t o f Food T e c h n o l o g y and S c i e n c e , U n i v e r s i t y o f T e n n e s s e e , K n o x v i l l e , T e n n e s s e e 3 7 9 0 1 - 1 0 7 1R e c e i v e d 1 3 November 1 9 8 4 / A c c e p t e d 1 3 M a r c h 1 9 8 5

T h e e f f e c t s o f a n t i m i cr o b i al f o o d a d d i t i v e s on g r o w t h a n d o c h r a t o x i n A p r o d u c t i o n b y A s p e r g i l l u s s u l p h u r e u sN R R L 4 0 7 7 i n d P e n i c i U u m v i r i d i c a t u m N R R L 3 7 1 1 were i n v e s t i g a t e d . A t pH 4 . 5 , g r o w t h a n d t o x i n p r o d u c t i o nb y b o t h A . s u l p h u r e u s a n d P . v i r i d i c a t u m were c o m p l e t e l y i n h i b i t e d b y 0 . 0 2 p o t a s s i u m s o r b a t e , 0 . 0 6 7m e t h y l p a r a b e n , 0 . 0 6 6 7 m e t h y l p a r a b e n , a n d 0.2 s o d i u m p r o p i o n a t e . A t pH 5 . 5 , 0.134 p o t a s s i u ms o r b a t e a n d 0 . 0 6 7 m e t h y l p a r a b e n c o m p l e t e l y i n h i b i t e d g r o w t h a n d o c h r a t o x i n A p r o d u c t i o n b y b o t h f u n g i .S o d i u m b i s u l f i t e a t 0 . 1 , t h e m aximum l e v e l t e s t e d , was f o u n d t o i n h i b i t g r o w t h o f A . s u l p h u r e u s a n d P .v i r i d i c a t u m b y 4 5 a n d 89 , r e s p e c t i v e l y . T o x i n p r o d u c t i o n was i n h i b i t e d b y 9 7 a n d 99 , r e s p e c t i v e l y . Sodiump r o p i o n a t e ( 0 . 6 4 ) a t pH 5 . 5 i n h i b i t e d g r o w t h o f A . s u l p h u r e u s a n d P . v i r i d i c a t u m b y 7 6 a n d 90 ,r e s p e c t i v e l y . T o x i n p r o d u c t i o n was i n h i b i t e d b y >9 9 f o r e a c h f u n g u s . A n t i m i c r o b i a l a g e n t s were r a n k e d a st o e f f e c t i v e n e s s b y c o m p a r i n g t h e l e v e l r e q u i r e d f o r c o m p l e t e i n h i b i t i o n o f o c h r a t o x i n A p r o d u c t i o n t o t h eh i g h e s t a n t i m i c r o b i a l a g e n t l e v e l n o r m a l l y u s e d i n f o o d . A t pH 4 . 5 , t h e most e f f e c t i v e i n h i b i t o r o f g r o w t h a n dt o x i n p r o d u c t i o n was p o t a s s i u m s o r b a t e , f o l l o w e d b y s o d i u m p r o p i o n a t e , m e t h y l p a r a b e n , a n d s o d i u m b i s u f i t e ,r e s p e c t i v e l y , f o r b o t h f u n g i . H o w e v e r , a t pH 5 . 5 , t h e m o s t e f f e c t i v e a n t i m i c r o b i a l a g e n t s f o r i n h i b i t i n go c h r a t o x i n p r o d u c t i o n were m e t h y l p a r a b e n a n d p o t a s s i u m s o r b a t e , f o l l o w e d b y s o d i u m p r o p i o n a t e . S o d i u mb i s u l f i t e wa s n o t h i g h l y i n h i b i t o r y t o t h e s e t o x i g e n i c f u n g i a t t h e h i g h e r pH v a l u e t e s t e d .

T h e o c h r a t o x i n s c o n s t i t u t e a g r o u p o f s t r u c t u r a l l y r e l a t e dm e t a b o l i t e s p r o d u c e d b y s e v e r a l s p e c i e s o f A s p e r g i l l u s a n dP e n i c i l l i u m . O c h r a t o x i n A ( 7 - c a r b o x y l - 5 - c h l o r o - 8 - h y d r o x y l -3 - 4 - d i h y d r o - 3 - m e t h y l i s o c o u m a r i n l i n k e d t o L - , 3 - p h e n y l a l a -n i n e ) was t h e f i r s t t o x i n d i s c o v e r e d i n t h i s group ( 2 4 ) . I na d d i t i o n t o b e i n g a s s o c i a t e d w i t h n e p h r o p a t h y i n c h i c k e n s( 5 ) a n d s w i n e ( 1 1 , 1 2 ) , o c h r a t o x i n A h a s b e e n f o u n d t o b et o x i c t o s p e c i e s s u c h a s p o u l t r y , r a t s , m i c e , d o g s , c a t t l e ,b r i n e s h r i m p , a n d b a c t e r i a ( 1 0 , 1 6 ) . O c h r a t o x i n A i s t e r a t o -g e n i c i n m i c e ( 8 ) , r a t s ( 2 ) , c h i c k e m b r y o s ( 7 ) , a n d h a m s t e r s( 9 ) . I n t h e U n i t e d S t a t e s , t h e o c h r a t o x i n s h a v e n o t b e e ns t u d i e d a s i n t e n s i v e l y a s t h e a f l a t o x i n s b e c a u s e most r e p o r t so f i t s occurrence h a v e b e e n u n s u b s t a n t i a t e d . H o w e v e r ,o c h r a t o x i n h a s b e e n w i d e l y s t u d i e d i n S c a n d i n a v i a n cou n-t r i e s b e c a u s e o f i t s occurrence i n s m a l l g r a i n s s u c h a s b a r l e ya n d b e c a u s e o f i t s t o x i c i t y t o s w i n e ( 1 0 ) . I n a surv ey o f af a r m a t w h i c h p i g s a re s l a u g h t e r e d , o c h r a t o x i n A was f o u n di n t i s s u e s o f 1 8 o f 1 9 p i g s f e d o c h r a t o x i n - c o n t a m i n a t e db a r l e y , a n d r e s i d u e l e v e l s i n t i s s u e were a s h i g h a t 6 7 , u g / k g( 1 0 ) . R e s i d u e s o f o c h r a t o x i n A have a l s o b e e n f o u n d i nk i d n e y s , l i v e r s , a n d m u s c u l a r t i s s u e s o f s l a u g h t e r e d p o u l t r ya t c o n c e n t r a t i o n s b e t w e e n 4 . 3 a n d 5 0 , u g / k g ( 5 ) . T h e o n l yo b s e r v a b l e l e s i o n i n a f f l i c t e d a n i m a l s i s o f t e n e v i d e n c e d a sk i d n e y d a m a g e ; t h e r e f o r e , t h e r e m a i n i n g p a r t s o f t h e carcassmay pass meat i n s p e c t i o n , a n d t h e t r a n s f e r o f o c h r a t o x i n Af r o m a n i m a l f e e d t o human f o o d i s p o s s i b l e ,T h e r e h a v e b e e n f e w s t u d i e s o n t h e c h e m i c a l c o n t r o l o fo c h r a t o x i n A p r o d u c t i o n . V a n d e g r a f t e t a l . ( 2 2 , 2 3 ) h a v ei n v e s t i g a t e d t h e e f f e c t s o f f u m i g a n t s on o c h r a t o x i n p r o d u c -t i o n i n s t o r e d g r a i n a n d h a v e f o u n d t h a t n o n e r e d u c e do c h r a t o x i n p r o d u c t i o n . T he i n s e c t i c i d e d i c h l o r v o s , a t 3 0m g / 1 0 0 m l o f c u l t u r e m e d i u m , i n h i b i t e d o c h r a t o x i n p r o d u c -t i o n b y 8 0 ( 2 5 ) . I n a l a t e r s t u d y , V a n d e g r a f t a n d c o - w o r k -e rs ( 2 1 ) e x a m i n e d g r a i n p e r s e r v a t i v e s s u c h a s p r o p i o n i c a c i da n d ammonia i n cor n t o d e t e r m i n e t h e i r e f f e c t s on o c h r a -

C o r r e s p o n d i n g a u t h o r .

t o x i n p r o d u c t i o n . B o t h 1 p r o p i o n i c a c i d a n d 2 ammonias i g n i f i c a n t l y r e d u c e d m o l d g r o w t h a n d o c h r a t o x i n p r o d u c -t i o n .T h e p r e s e n t s t u d y was d e s i g n e d t o e v a l u a t e t h e e f f e c t i v e -ness o f f o u r a n t i m i c r o b i a l f o o d a d d i t i v e s , s o d i u m p r o p i o -n a t e , m e t h y l p a r a b e n , s o d i u m b i s u l f i t e , a n d p o t a s s i u m s o r -b a t e , a t pH 4 . 5 a n d 5 . 5 , f o r i n h i b i t i o n o f o c h r a t o x i n Ap r o d u c t i o n a n d m y c e l i u m f o r m a t i o n b y A s p e r g i l l u s s u l p h u -reus a n d P e n ic i l l i u m v i r i di c a t u m .

M A T E R I A L S ANDMETHODST e s t o r g a n i s m s a n d g r o w t h m e d i u m . A . s u l p h u r e u s NRRL4 0 7 7 a n d P . v i r i d i c a t u m NRRL 3 7 1 1 , known o c h r a t o x i n - p r o -d u c i n g f u n g i , were u s e d t h r o u g h o u t t h i s s t u d y . C u l t u r e s werei n o c u l a t e d o n t o 2 y e a s t e x t r a c t - 4 sucrose ( Y E S ) agars l a n t s , i n c u b a t e d f o r 2 weeks a t 2 8 C t o p r o d u c e s p o r e s , ands t o r e d a t 4 C u n t i l n e e d e d . C o n i d i a l s u s p e n s i o n s were p r e -p a r e d i n a s t e r i l e aq u e o u s s o l u t i o n o f T r i t o n X - 1 0 0 ( 0 . 0 0 5[ v o l / v o l ] ) i m m e d i a t e l y b e f o r e i n o c u l a t i o n .F o r e v a l u a t i o n o f o c h r a t o x i n p r o d u c t i o n and g r o w t h , 1 0 7f u n g a l spores were a d d e d t o 1 0 0 m l o f YES b r o t h i n a 5 0 0 - m lE r l e n m e y e r f l a s k . T he b r o t h c u l t u r e s were i n c u b a t e d f o r 1 4d a y s a t 2 8 0 C . R e s u l t s o f p r e l i m i n a r y e x p e r i m e n t s c o n f i r m e dt h a t o p t i m u m o c h r a t o x i n p r o d u c t i o n o c c u r r e d i n a 2 ( w t / v o l )

y e a s t e x t r a c t - 4 ( w t / v o l ) sucrose b r o t h ( 4 ) w i t h t h e f u n g a ls t r a i n s c h o s e n f o r e x p e r i m e n t a t i o n .A n t i m i c r o b i a l a g e n t s . Sodium p r o p i o n a t e and sodium b i -s u l f i t e ( F i s h e r S c i e n t i f i c C o . , A t l a n t a , G a .) , f o o d g r a d em e t h y l p a r a b e n ( U e n o F i n e C h e m i c a l I n d u s t r i e s , I n c . ,O s a k a , J a p a n ) , a n d p o t a s s i u m s o r b a t e ( T r i - K I n d u s t r i e s ,I n c . , W e s t w o o d , N . J . ) were e v a l u a t e d a s a n t i m i c r o b i a l a g e n t sa g a i n s t o c h r a t o x i n p r o d u c e r s . M e t h y l p a r a b e n was d i s s o l v e di n 75 e t h a n o l b e c a u s e o f i t s low s o l u b i l i t y i n w a t e r . Othera n t i m i c r o b i a l a g e n t s were d i s s o l v e d i n w a t e r . S o l u t i o n s o fa n t i m i c r o b i a l a g e n t s were f i l t e r s t e r i l i z e d an d s t o r e d a t 4 Cu n t i l n e e d e d .YE S medium was a u t o c l a v e d a t 1 2 1 C f o r 1 5 m i n . When1 4 0 7

V o l . 4 9 , N o . 6

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1 4 0 8 TONG AND DRAUGHONt h e c u l t u r e medium r e a c h e d a m b i e n t t e m p e r a t u r e , a p p r o p r i -a t e a m o un ts o f t h e a n t i m i c r o b i a l a g e n t s were a d d e d , a n d t h epH was t h e n a s e p t i c a l l y a d j u s t e d t o pH 4 . 5 or 5 . 5 b y u s i n gv a r y i n g n o r m a l i t i e s o f s t e r i l e HC I a n d Na OH.A n t i m i c r o b i a l a g e n t s were a d d e d t o YE S b r o t h a t t h ef o l l o w i n g c o n c e n t r a t i o n s : s o d i u m p r o p i o n a t e ( 0 , 0 . 1 ,0 . 2 , 0 . 3 2 , 0 . 4 8 , 0 . 6 4 ) , m e t h y l p a r a b e n ( 0 , 0 . 0 1 1 ,0 . 0 2 2 , 0 . 0 3 3 , 0 . 0 67 ) , s o d i u m b i s u l f i t e ( 0 , 0 . 0 1 6 7 ,0 . 0 3 3 4 , 0 . 0 5 0 0 , 0 . 0 6 6 7 , 0 . 0 8 3 4 , 0 . 1 0 0 0 , 0 . 1 2 0 0 ) ,a n d p o t a s s i u m s o r b a t e ( 0 , 0 . 0 1 1 , 0 .0 22 , 0 .0 4 4 ,0 . 0 6 7 , 0 . 1 3 4 ) . L e v e l s o f a n t i m i c r o b i a l a g e n t s p e r m i t t e dor g e n e r a l l y u s e d ( 6 , 1 4 ) i n f o o d s f o r human c o n s u m p t i o nwere c o n s i d e r e d when l e v e l s o f a n t im i c r o bi a l a g e n t s weres e l e c t e d .E x t r a c t i o n a n d a n a l y s i s o f o c h r a t o x i n . O c h r a t o x i n A wase x t r a c t e d f r o m t h e b r o t h b y t h e p r o c e d u r e s o f S c o t t e t a l .( 1 7 ) . The c h l o r o f o r m e x t r a c t s were c o m b i n e d a n d d r i e d b yf i l t e r i n g t h r o u g h a n h y d r o u s s o d i u m s u l f a t e t o remove w a t e r .The c h l o r o f o r m was e v a p o r a t e d t o d r y n e s s a t 6 0 C w i t h af l a s h e v a p o r a t o r ( R o t o v a p ) . T he r e s i d u e was d i s s o l v e d i nc h l o r o f o r m ( 5 m l ) . A f t e r t h e s a m p l e s we r e f i l t e r e d t h r o u g h a0 . 4 5 - p . m M i l l i p o r e f i l t e r , t h e y were s e a l e d i n v i a l s a n dq u a n t i t a t e d b y h i g h - p r e s s u r e l i q u i d c h r o m a t o g r a p h y HPLC)f o r o c h r a t o x i n A .The h i g h - p r e s s u r e l i q u i d c h r o m a t o g r a p h ( W a t e r s A s s o c i -a t e s , M i l f o r d , M a s s . ) was e q u i p p e d w i t h d u a l m o d e l M6000pu mps , a m o d e l 4 4 0 UV d e t e c t o r ( 3 4 0 n m ) , a m o d e l 4 2 0f l u o r e s c e n c e d e t e c t o r ( 3 4 0 - n m e x c i t a t i o n f i l t e r a n d a 4 4 0 - n me m i s s i o n f i l t e r , a n d a U6 K s e p t u m l e s s i n j e c t o r . A p L - P o r a s i lc o l u m n ( W a t e r s A s s o c i a t e s ) was u s e d a s t h e s t a t i o n a r yp h a s e w i t h a s o l v e n t system o f b e n z e n e - a c e t i c a c i d - m e t h a n o l( 9 0 : 1 0 : 5 [ v o l / v o l / v o l ] ) . The average r e t e n t i o n t i m e f o r o c h r a -t o x i n A was 3 m i n a n d 4 0 s a t a f l o w r a t e o f 1 m l / m i n . Ane x p e r i m e n t was p e r f o r m e d t o d e t e r m i n e t h e r e p r o d u c i b i l i t yo f t h e H P L C a n a l y s i s when t h e i n j e c t i o n v o l u m e , t h e s e n s i -t i v i t y o f t h e d e t e c t o r , a n d t h e c o n c e n t r a t i o n were v a r i e d .Each o f f i v e s t a n d a r d s o l u t i o n s r a n g i n g i n c o n c e n t r a t i o n sf r o m 0 . 1 t o 0 . 0 0 1 m g / m l was i n j e c t e d 1 0 t i m e s i n t o t h ec h r o m a t o g r a p h . The s t a n d a r d c u r v e was f o u n d t o b e l i n e a r .The c o e f f i c i e n t o f v a r i a t i o n f o r e a c h s e r i e s o f i n j e c t i o n s wash i g h l y s a t i s f a c t o r y ( T a b l e 1 ) .S a m p l e s were s p o t t e d o n t h i n - l a y e r c h r o m a t o g r a p h i c p l a t e s( R e d i ; F i s h e r S c i e n t i f i c C o . ) w h i c h were p r e c o a t e d w i t hs i l i c a g e l G a n d d e v e l o p e d w i t h b e n z e n e - a c e t i c a c i d ( 9 0 : 1 0[ v o l / v o l ] . T h e p r ese nc e o f o c h r a t o x i n A was c o n f i r m e d b yb o r o n t r i f l u o r i d e d e r i v a t i z a t i o n ( 1 ) . T h e r ec ov e r y o f o c h r a -t o x i n f r o m YE S b r o t h was d e t e r m i n e d b y s p i k i n g e x p e r i -ments.

A f t e r e x t r a c t i o n , m y c e l i a were s e p a r a t e d f r o m t h e b r o t hc u l t u r e b y f i l t e r i n g w i t h a B u c h n e r f u n n e l w i t h a vacuum a n dWhatman n o. 1 f i l t e r paper. T h e m y c e l i a l ma t was t r a n s -f e r r e d t o p r e w e i g h e d f i l t e r paper, d r i e d a t 6 0 C f o r 2 4 h , a n dw e i g h e d on a M e t t l e r a n a l y t i c a l b a l a n c e t o d e t e r m i n e t h ea m o un t o f g r o w t h ( d r y w e i g h t ) .

A n a l y s i s a n d e x p e r i m e n t a l d e s i g n . T h e e n t i r e e x p e r i m e n twas r e p l i c a t e d t w i c e ( t h r e e s e p a r a t e r u n s ) . T h r e e f l a s k s werep r e p a r e d f o r e a c h t r e a t m e n t f o r e a c h r e p l i c a t e . An a n a l y s i so f v a r i a n c e was p e r f o r m e d t o a n a l y z e t h e d a t a . Whens i g n i f i c a n t d i f f e r e n c e s were f o u n d , t h e Duncan m u l t i p l er a nge t e s t was u s e d t o d e t e r m i n e t h e s i g n i f i c a n t d i f f e r e n c e samong mean v a l u e s .

R E S U L T S AND D I S C U S S I O NM y c e l i u m i n h i b i t o r s . F i g u r e 1 s h o w s t h e e f f e c t s o f p o t a s -

sium s o r b a t e , m e t h y l p a r a b e n , s o d i u m b i s u l f i t e , a n d s o d i u m

T A B L E 1 . R e p r o d u c i b i l i t y o f t h e H P L C s y s t e mC o n c n o f C o e f f i c i e n ts t a n d a r d Mean p e a k a r e a o fs o l u t i o n + S D h v a r i a t i o n( m g / m l ) ( )

0 . 1 2 1 9 . 5 6 . 4 5 1 2 . 9 40 . 0 5 1 7 8 . 3 + 4 . 2 3 4 2 . 3 80 . 0 1 1 2 4 . 2 7 . 4 3 5 5 . 9 90 . 0 0 5 1 3 0 . 3 + 6 . 8 8 5 5 . 2 80 . 0 0 1 1 2 4 . 2 6 . 8 8 8 5 . 5 5 R e p r o d u c i b i l i t y w a s d e t e r m in e d w it h a , u - P o r a s i l c o l u m n a n d a s o l v e n ts y s t e m o f b e nz e n e - ac e t ic a c i d - m e t h a n o l a t a f l o w r a t e o f 1 m l p e r m i n u s i n gt h e f l u o r e s c e n c e d e t e c t o r .b Mean o f 1 0 i n j e c t i o n s . T h e p e a k a r e a i s s u b j e c t t o c h a n g e w i t h a c h a n g e i nt h e v o l u m e o f i n j e c t i o n a n d t h e g a i n o f t h e f l u o r e s c e n c e d e t e c t o r . P e a k a r e a sw e r e h e l d b e t w e e n 1 0 0 a n d 2 5 0 f o r o p t i m a l v i s u a l i z a t i o n .

p r o p i o n a t e o n g r o w t h o f A . s u l p h u r e u s NRRL 4 0 7 7 a n d P .v i r i d i c a t u m NRRL 3 7 1 1 i n YE S b r o t h a t pH v a l u e s o f 4 . 5a n d 5 . 5 . T h e a b i l i t y o f t h e f o o d a d d i t i v e s t o i n h i b i t m y c e l i u mp r o d u c t i o n v a r i e d w i d e l y . H o w e v e r , a s i g n i f i c a n t d e c r e a s e( P < 0 . 0 5 ) i n m y c e l i a l w e i g h t w a s o b t a i n e d w i t h t h e a d d i t i o no f e a c h o f t h e f o u r a n t i m i c r o b i a l a g e n t s . P o t a s s i u m s o r b a t ew a s h i g h l y e f f e c t i v e f o r r e d u c i n g f u n g a l g r o w t h a t pH 4 . 5 .A l t h o u g h p o t a s s i u m s o r b a t e i s l i s t e d a s a g e n e r a l l y r e c o g -n i z e d a s s a f e ( G R A S ) s u b s t a n c e , i t s l e v e l o f u s e i n f o o d d o e sn o t g e n e r a l l y e x c e e d 0 . 2 ( 6 , 1 4 ) . P o t a s s i u m s o r b a t e w a sf o u n d t o c o m p l e t e l y i n h i b i t f u n g a l g r o w t h a t 0 . 1 3 4 , t w o -t h i r d s o f t h e n o r m a l u s e l e v e l , when t h e pH o f t h e b r o t h w a s5 . 5 . O r i g i n a l l y , p o t a s s i u m s o r b a t e l e v e l s o f 0 , 0 . 0 2 2 ,0 . 0 4 4 , 0 . 0 6 7 , a n d 0 . 1 3 4 w e r e s t u d i e d . H o w e v e r , m y c e -l i u m p r o d u c t i o n w a s n o t d e t e c t e d e v e n a t 0 . 0 2 2 a t pH 4 . 5 .T h e e f f e c t i v e n e s s o f p o t a s s i u m s o r b a t e a t pH 4 . 5 w a sa p p r o x i m a t e l y s e v e n t i m e s h i g h e r t h a n t h a t a t pH 5 . 5 b a s e do n t h e a m o u n t u s e d a n d t h e p e r c e n t i n h i b i t i o n .M e t h yl p a r a b e n w a s 1 0 0 e f f e c t i v e i n t h e i n h i b i t i o n o fm y c e l i a l g r o w t h a t l o w l e v e l s . T h e g r o w t h i n h i b i t i o n p a t t e r n sw e r e s i m i l a r w i t h m e t h y l p a r a b e n , r e g a r d l e s s o f t h e pHv a l u e s ( F i g . 1 . A s i g n i f i c a n t d e c r e a s e i n w e i g h t o f m yc e l i a lm a t o c c u r r e d w i t h 0 . 0 3 3 m e t h y l p a r a b e n .S o d i u m b i s u l f i t e i s n o t g e n e r a l l y u s e d i n f o o d a t l e v e l s i ne x c e s s o f 5 0 0 ppm b e c a u s e o f a n o t i c e a b l e t a s t e a b o v e t h i sl e v e l ( 6 ) . B e c a u s e i n h i b i t i o n a t 5 0 0 ppm w a s v e r y p o o r a t pH5 . 5 , h i g h e r l e v e l s w e r e t e s t e d . A t 1 , 0 0 0 p p m , s o d i u m b i s u l f i t ei n h i b i t e d g r o w t h o f A . s u l i p h u r e u s a n d P . i ' r i d i c a t u m b y 8 5a n d 9 1 , r e s p e c t i v e l y . A t 5 0 0 p p m , s o d i u m b i s u l f i t e i n h i b -i t e d g r o w t h o f A . s u l p h i u i e i s a n d P . x ' i r i d i c a t u m b y 9 a n d1 1 , r e s p e c t i v e l y , a t pH 5 . 5 ( F i g . 1 ) . To o b t a i n 1 0 0i n h i b i t i o n o f g r o w t h f o r b o t h f u n g i , 6 6 7 ppm s o d i u m b i s u l f i t ew a s r e q u i r e d a t pH 4 . 5 . S i g n i f i c a n t d e c r e a s e s i n m y c e l i a lw e i g h t o c c u r r e d f r o m 3 3 4 t o 5 0 0 ppm a t pH 4 . 5 a n d 8 3 4 t o1 , 0 0 0 ppm a t pH 5 . 5 , r e s p e c t i v e l y ( F i g . 1 . T h e r e d u c e da n t i f u n g a l a c t i v i t y o f s o d i u m b i s u l f i t e a t pH 5 . 5 v e r s u s 4 . 5 i st y p i c a l o f a n t i m i c r o b i a l a g e n t s t h a t a r e m o s t a c t i v e i n t h eu n d i s s o c i a t e d f o r m .T h e p r a c t i c a l u s e o f s o d i u m p r o p i o n a t e a s a f u n g a l i n h i b -i t o r w a s s i g n i f i c a n t l y r e l a t e d t o p H . W i t h 0 . 2 0 s o d i u mp r o p i o n a t e , g r o w t h o f A . s i i l p h i u r - e u s w a s i n h i b i t e d b y 1 3 a tpH 5 . 5 c o m p a r e d w i t h 1 0 0 a t pH 4 . 5 a n d , s i m i l a r l y , 1 6c o m p a r e d w i t h 1 0 0 f o r P . v i r i d i c a u i n N R R L 3 7 1 1 . A t0 . 6 4 s o d i u m p r o p i o n a t e , 8 0 a n d 8 8 i n h i b i t i o n o f g r o w t ho c c u r r e d a t pH 5 . 5 f o r A . s u i l p h u r ) e i u s a n d P . O i r i d i c a t u m ,r e s p e c t i v e l y .P . v i r i d i c a t u m w a s f o u n d t o p r o d u c e m o r e m y c e l i u m t h a nA . s u i l p h i u r e i u s . I t w a s f o u n d t h a t b o t h s t r a i n s p r o d u c e d m o r em y c e l i a l m a t a t pH 4 . 5 t h a n a t pH 5 . 5 . S i m i l a r r e s u l t s h a v e

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F I G . 1 . E f f e c t o f a n t i m i c r o b i a l a g e n t s on t h e g r o w t h o f A . s u l p h u r e u s a n d P . v i r id ic a t u m when i n c u b a t e d a t 2 8 C f o r 2 w e e k s .

b e e n r e p o r t e d b y L a i e t a l . ( 1 3 ) . G e n e r a l l y , t h e i n h i b i t i o np a t t e r n s f o r a n t i m i c r o b i a l f o o d a d d i t i v e s we r e a l m o s t i d e n -t i c a l f o r A . s u l p h u r e u s NRRL 4 0 7 7 a n d P . v i r i d i c a t u m N R R 13 7 1 1 a t pH 4 . 5 . H o w e v e r , a t pH 5 . 5 a n d a t h i g h e r concen-t r a t i o n s , t h e f o r m e r was g e n e r a l l y f o u n d t o b e more r e s i s t a n tt o t h e a n t i m i c r o b i a l a g e n t s t h a n was t h e l a t t e r .T o x i n i n h i b i t i o n . T a b l e s 2 a n d 3 show t h e e f f e c t s o fa n t i m i c r o b i a l f o o d a d d i t i v e s on o c h r a t o x i n A p r o d u c t i o n b yA . s u l p h u r e u s NRRL 4 0 7 7 a n d P . v i r i d i c a t u m NRRL 3 7 1 1 i nYE S b r o t h a t two p H s . The a d d i t i o n o f t h e f o o d a d d i t i v e sr e s u l t e d i n a s i g n i f i c a n t d e c r e a s e i n o c h r a t o x i n A p r o d u c t i o n .P o t a s s i u m s o r b a t e ( 0 . 1 3 4 ) a n d m e t h y l p a r a b e n ( 0 . 0 6 7 )c o m p l e t e l y i n h i b i t e d t o x i n p r o d u c t i o n b y b o t h f u n g i a t t h etwo pH l e v e l s t e s t e d w i t h o u t e x c e e d i n g l e v e l s n o r m a l l y u s e di n f o o d f o r human c o n s u m p t i o n . H o w e v e r , two t i m e s t h eamou nt o f s o d i u m b i s u l f i t e a n d t h r e e t i m e s t h e amou nt o fs o d i u m p r o p i o n a t e n o r m a l l y u s e d d i d n o t c o m p l e t e l y i n h i b i tt o x i n p r o d u c t i o n a t pH 5 . 5 . By i n c r e a s i n g t h e c o n c e n t r a t i o n so f s o d i u m b i s u l f i t e a n d s o d i u m p r o p i o n a t e t o 0 . 1 a n d 0 . 6 4 ,more t h a n 90 i n h i b i t i o n i n o c h r a t o x i n A p r o d u c t i o n wasa c h i e v e d f o r b o t h s t r a i n s a t b o t h pH v a l u e s ( T a b l e s 2 a n d 3 ) .G e n e r a l l y , t o x i n p r o d u c t i o n d e c r e a s e d as m y c e l i u m f o r -m a t i o n d e c r e a s e d . H o w e v e r , t h e a n t i mi c r o b i al a g e n t i n h i b i -t i o n o f t o x i n was more s i g n i f i c a n t t h a n m y c e l i a l i n h i b i t i o n .A l t h o u g h t h e r e ason f o r t h i s h a s n o t b e e n shown e x p e r i m e n -t a l l y , i t i s a l o g i c a l f i n d i n g b e c a u s e i n t o x i n b i o s y n t h e s i s t h eexcess a c e t a t e a n d m a l o n a t e f o r m e d b y p r i m a r y b i o s y n t h e s i s

ar e u s e d . T h u s , e v e n s m a l l c h a n g e s i n p r i m a r y b i o s y n t h e s i sca n h a v e m a j o r e f f e c t s o n s e c o n d a r y b i o s y n t h e s i s .I n t h e c o n t r o l s , A . s u l p h u r e u s p r o d u c e d a b o u t 1 0 0 t i m e sm n o r e o c h r a t o x i n A t h a n di d P . v i r id i c a t u m. P e n i c i l l i a ns p e c i e s f r e q u e n t l y p r o d u c e m a x i m u m t o x i n a t l o w e r t e m p e r -a t u r e s t h a n d o a s p e r g i l l i . T h i s i s t r u e o f P . v i r i d i c a t u m ,w h i c h h a s o p t i m a l o c h r a t o x i n p r o d u c t i o n a t 2 0 C ( 1 8 , 1 9 ) .H o w e v e r , P . v i r i d i c a t u m p r o d u c e d more m y c e l i a l w e i g h tt h a n d i d A . s u l p h u r e u s i n YE S b r o t h a t 2 8 C . L a i e t a l . ( 1 3 )h a v e r e p o r t e d t h a t pH 5 . 5 i s b e t t e r f o r o c h r a t o x i n A p r o d u c -t i o n t h a n pH 4 . 5 . N e v e r t h e l e s s , i n t h i s s t u d y , no s i g n i f i c a n td i f f e r e n c e i n o c h r a t o x i n A f o r m a t i o n was o b s e r v e d a s ar e s u l t o f pH d i f f e r e n c e s i n t h e c o n t r o l f l a s k s . I t was a l s of o u n d t h a t t h e s t a n d a r d d e v i a t i o n s o f mean v a l u e s f o r t o x i nwere much h i g h e r t h a n t h e d e v i a t i o n s o f mean v a l u e s f o rm y c e l i u m . T he l a r g e v a r i a ti o n f o r t o x i n c o u l d b e du e t o t h ef l u c t u a t i o n o f i n c u b a t i o n t e m p e r a t u r e , e x t r a c t i o n proce-d u r e s , d e g r a d a t i o n d u r i n g h e a t i n g and e v a p o r a t i o n , HPLCq u a n t i t a t i o n , a n d b i o l o g i c a l d i f f e r e n c e s . R e s u l t s o f o u r ex-p e r i m e n t s showed t h a t o n l y m i n o r v a r i a t i o n s were due t o t h eHPLC a n a l y s i s . R e c o v e r i e s o f o c h r a t o x i n i n s p i k i n g e x p e r -i m e n t s r a n g e d f r o m 8 3 t o 9 5 . D e s p i t e a r e p o r t b y C hu andB u t z ( 3 ) t h a t o c h r a t o x i n A i s v e ry s t a b l e when s t o r e d underr e f r i g e r a t i o n , t h e d e g r a d a t i o n o f s t a n da r d s o l u t i o n f o r HPLCd e t e r m i n a t i o n s c o u l d a l s o b e a f a c t o r t h a t c o n t r i b u t e s t o t h ev a r i a t i o n .T r e n k e t a l . ( 2 0 ) h a v e f o u n d t h a t o c h r a t o x i n A ca n p e r s i s t

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1 4 1 0 TONG AND DRAUGHONi n f o o d s e v e n a f t e r 3 h o f a u t o c l a v i n g . T h e r e m o v a l o fo c h r a t o x i n A w o u l d b e d i f f i c u l t o n c e f o o d s a re c o n t a m i -n a t e d , a n d t h e b e s t p r o t e c t i o n w o u l d b e t o p r e v e n t t o x i nf o r m a t i o n t h r o u g h proper d r y i n g a n d t h e a dd it i o n o f a n t i m i -c r o b i a l a g e n t s . T h e e f f e c t i v e n e s s o f t h e f o u r a n t i m i c r o b i a lf o o d a d d i t i v e s a n d t h e e f f e c t s o f pH v a l u e s were e v a l u a t e d i nYE S b r o t h t o p r o v i d e i n f o r m a t i o n f o r f u t u r e a p p l i c a t i o n s i ng r a i n s .I t i s w e l l known t h a t t h e a c t i v i t y o f a n t i m i c r o b i a l f o o da d d i t i v e s , e x c e p t f o r t h e p a r a b e n s , i s d e p e n d e n t o n p H .U s u a l l y , t h e l o w e r t h e p H , t h e h i g h e r t h e e f f e c t i v e n e s s . T h eo p t i m u m pH r a nge f o r s o d i u m p r o p i o n a t e a n d s o d i u m b i -s u l f i t e i s b e t w e e n 2 . 5 a n d 5 . 0 a n d b e t w e e n 3 . 0 a n d 6 . 5 f o rp o t a s s i u m s o r b a t e . T h e p a r a b e n s a re e f f e c t i v e a t a b o v e pH 7 6 . I n t h i s s t u d y , p o t a s s i u m s o r b a t e a n d m e t h yl p a r a b e nwere e f f e c t i v e f o r i n h i b i t i n g f u n g a l g r o w t h a t l o w c o n c e n t r a -t i o n s a t b o t h pH 4 . 5 a n d 5 . 5 . A l t h o u g h t h e l e v e l r e q u i r e d t oc o m p l e t e l y i n h i b i t f u n g a l g r o w t h a n d o c h r a t o x i n A p r o d u c -t i o n was t h e same f o r m e t h y l p a r a b e n a t pH 4 . 5 a n d 5 . 5 , t h ep e r c e n t a g e s o f i n h i b i t i o n w e r e s t i l l a f f e c t e d b y p H . V a n -d e r g r a f t e t a l . ( 2 1 ) h a v e r e p o r t e d t h a t n o o c h r a t o x i n A wasd e t e c t e d i n corn when t h e corn was t r e a t e d w i t h 1 p r o p i -o n i c a c i d . T h e pH o f corn i s a b o u t pH 6 . 0 ( 1 5 ) . B e c a u s e t h ea d d i t i o n o f 0 . 6 4 s o d i u m p r o p i o n a t e r e s u l t e d i n 99 i n h i b i -t i o n i n t o x i n p r o d u c t i o n , a l e v e l b e t w e e n 0 . 6 4 a n d 1 i se x p e c t e d t o i n h i b i t o c h r a t o x i n A p r o d u c t i o n b y 1 0 0 . B e -cause o f i t s poor i n h i b i t i o n o f t h e f u n g i a t pH 5 . 5 , s o d i u mb i s u l f i t e may n o t f e a s i b l y b e a d d e d i n t o most f o o d s b e c a u s ei t s s t r o n g o d o r i s d e t e c t a b l e when t h e l e v e l e x c e e d s 5 0 0 pp m .

T ABL E 2 . E f f e c t o f a n t i m i c r o b i a l a g e n t s o n o c h r a t o x i n Ap r o d u c t i o n b y A . s u l p h u r e u s NRRL 4 0 7 7 i n YE S b r o t h i n c u b a t e da t 2 8 ' C f o r 2 w e e k s

Me an t o x i n l e v e l S D ( , u g / 1 0 0 m l o fA n t i m i c r o b i a l L e v e l b r o t h ) a t t h e f o l l o w i n g p H s a :a g e n t ( )

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P o t a s s i u m s o r b a t e 0 2 , 2 9 7 4 4 b 1 , 9 1 4 t 3 70 . 0 1 1 6 9 6 8 C ( 7 0 ) 1 , 6 2 1 3 0 b ( 1 5 )0 . 0 2 2 o d ( 1 0 0 ) 4 7 2 1 3 C ( 7 5 )0 . 0 4 4 0 ( 1 0 0 ) 1 9 6 4 d ( 9 0 )0 . 0 6 7 0 ( 1 0 0 ) 4 o d ( 9 9 )0 . 1 3 4 0 ( 1 0 0 ) 0d ( 1 0 0 )M e t h y l p a r a b e n 0 2 , 7 3 3 4 5 b 1 , 5 9 1 3 30 . 0 1 1 1 1 1 4 ' ( 9 6 ) 4 0 7 2 2 c ( 7 4 )0 . 0 2 2 4 0 c ( 9 9 ) 4 9 2 d ( 9 7 )0 . 0 3 3 3 Oc ( 9 9 ) 2 2 0 d ( 9 9 )0 . 0 6 7 OC ( 1 0 0 ) od ( 1 0 0 )S o d i u m p r o p i o n a t e 0 1 , 6 1 1 3 1 b 1 , 7 7 0 4 6 b0 . 1 0 1 7 Oc ( 9 9 ) 1 , 4 3 0 3 3 c ( 1 9 )0 . 2 0 o d ( 1 0 0 ) 6 3 5 2 6 d ( 6 4 )0 . 3 2 0 ( 1 0 0 ) 2 2 7 9 ( 8 7 )

0 . 4 8 0 ( 1 0 0 ) 2 0 1 2 4 e ( 8 9 )0 . 6 4 0 ( 1 0 0 ) 1 0 + e( 9 9 )Sodium b i s u l f i t e 0 2 , 1 8 8 2 7 2 , 5 1 6 2 9 b0 . 0 1 6 7 4 1 6 2 1 ' ( 8 1 ) 2 , 1 2 9 2 4 c ( 1 5 )0 . 0 3 3 4 2 6 4 1 4 d ( 8 8 ) 1 , 9 5 0 1 6 c ( 2 2 )0 . 0 5 0 0 4 5 ( 9 9 ) 9 9 7 1 7 d ( 6 0 )0 . 0 6 6 7 o e ( 1 0 0 ) 4 6 0 1 3 e ( 8 2 )0 . 0 8 3 4 0 ( 1 0 0 ) 1 8 6 9 f ( 9 3 )0 . 1 0 0 0 0 ( 1 0 0 ) 7 1 4 f ( 9 7 )

a V a l u e s w i t h i n t r e a t m e n t s f o l l o w e d b y t h e s a m e l e t t e r ( b t h r o u g h j ) ar e n o ts i g n i f i c a n t l y d i f f e r e n t ( P < 0 . 0 5 ) b y t h e D u n c a n m u l t i p l e r a ng e t e s t . V a l u e s i np a r e n t h e s e s a re p r e c e n t i n h i b i t i o n .

T ABL E 3 . E f f e c t o f a n t i m i c r o b i a l a g e n t s o n o c h r a t o x i n Ap r o d u c t i o n b y P . v i r i d i c a t u m N R R L 3 7 7 1 i n YE S b r o t h i n c u b a t e da t 2 8 C f o r 2 w e e k sMean t o x i n l e v e l S D t i g / 1 0 0 m l o fA n t i m i c r o b i a l L e v e l b r o t h ) a t t h e f o l l o w i n g p H s a :a g e n t ( % ) pH 4 . 5 pH 5 . 5

P o t a s s i u m s o r b a t e 0 2 1 4 . 0 b 1 8 3 . 0 b0 . 0 1 1 4 1 . 0 ( 8 1 ) 1 7 3 . 0 b ( 6 )0 . 0 2 2 o d ( 1 0 0 ) 1 4 1 . 0 C ( 2 2 )0 . 0 4 4 0 ( 1 0 0 ) 1 2 1 . 0 C ( 3 3 )0 . 0 6 7 0 ( 1 0 0 ) 7 d ( 6 1 )0 . 1 3 4 0 ( 1 0 0 ) o e ( 1 0 0 )

M e t h y l p a r a b e n 0 1 9 6 . 0 b 2 2 8 . 0 b0 . 0 1 1 2 1 5 . 0 b 1 3 4 . 0 c ( 4 1 )0 . 0 2 2 2 2 . 0 ' ( 8 9 ) 7 3 . 0 d ( 6 9 )0 . 0 3 3 2 0 . 1 c ( 8 9 ) 1 0 . l e ( 9 5 )0 . 0 6 7 0 C ( 1 0 0 ) o e ( 1 0 0 )S o d i u m p r o p i o n a t e 0 1 9 3 . 0 b 2 1 + 6 . 0 b c0 . 1 0 5 2 . 0 c ( 6 9 ) 3 4 2 4 . 00 . 2 0 o d ( 1 0 0 ) 1 2 + 4 . 0 ' ( 4 3 )0 . 3 2 0 ( 1 0 0 ) 1 0 . 1 C ( 9 5 )0 . 4 8 0 ( 1 0 0 ) 2 0 . 1 c ( 9 9 )

0 . 6 4 0 ( 1 0 0 ) O c ( l 0 0 )S o d i u m b i s u l f i t e 0 2 1 3 . 0 b 2 2 7 . 0 b0 . 0 1 6 7 4 1 . 0 c ( 8 1 ) 1 9 2 . 0 ( 1 3 )0 . 0 3 3 4 2 1 . 0 d ( 9 0 ) 1 2 2 . 0 c ( 4 6 )0 . 0 5 0 0 2 O . l e ( 9 9 ) 9 1 . 0 c ( 5 8 )0 . 0 6 6 7 o e ( 1 0 0 ) 4 2 . 0 d ( 8 0 )0 . 0 8 3 4 0 ( 1 0 0 ) 0 . 8 0 . 3 e ( 9 6 )0 . 1 0 0 0 0 ( 1 0 0 ) 0 . 1 0 . l e ( 9 9 )

a V a l u e s w i t h i n t r e a t m e n t s b y t h e s a m e l e t t e r ( b t h r o u g h e ) a r e n o ts i g n i f i c a n t l y d i f f e r e n t ( P < 0 . 0 5 ) b y t h e D u n c a n m u l t i p l e r a n g e t e s t . V a l u e s i np a r e n t h e s e s a r e p e r c e n t i n h i b i t i o n .

A n t i m i c r o b i a l a g e n t s w e r e r a n k e d a s t o t h e i r e f f e c t i v e n e s sb y c o m p a r i n g t h e l e v e l r e q u i r e d f o r c o m p l e t e i n h i b i t i o n o fo c h r a t o x i n A p r o d u c t i o n w i t h t h e h i g h e s t l e v e l o f a n t i m i c r o -b i a l a g e n t s n o r m a l l y u s e d i n f o o d . A t pH 4 . 5 , t h e m o s te f f e c t i v e i n h i b i t o r o f g r o w t h a n d t o x i n p r o d u c t i o n w a s p o -t a s s i u m s o r b a t e . I n d e s c e n d i n g o r d e r , i t w a s f o l l o w e d b ys o d i u m n p r o p i o n a t e , m e t h y l p a r a b e n , a n d s o d i u m b i s u l f i t e .H o w e v e r , a t pH 5 . 5 , t h e m o s t i n h i b i t o r y a n t i m i c r o b i a la g e n t s w e r e p o t a s s i u m s o r b a t e a n d m e t h y l p a r a b e n , f o l -l o w e d b y s o d i u m p r o p i o n a t e a n d s o d i u m b i s u l f i t e . B o t h A .s u l p h u r e u s a n d P . v i r i d i c a t u m f o l l o w e d s i m i l a r p a t t e r n s o fi n h i b i t i o n w h e n e x p o s e d t o a n t i m i c r o b i a l a g e n t s a t pH 4 . 5a n d 5 . 5 .F o r f o o d s w i t h pHs b e t w e e n S a n d 6 , s u c h a s r y e a n ds o r g h u m , t h e a n t i m i c r o b i a l a g e n t o f c h o i c e f o r f u t u r e s t u d i e sw o u l d b e m e t h yl p a r a b e n o r p o t a s s i u m s o r b a t e . T h e a p p l i -c a t i o n o f t h e s e a n t i m i c r o b i a l a g e n t s i s f e a s i b l e , a l t h o u g ht h e r e a r e p r o b l e m s t o b e o v e r c o m e i n t h e a p p l i c a t i o n a n dd i s t r i b u t i o n o f t h e a n t i m i c r o b i a l a g e n t s i n g r a i n . C o r n s i l a g ea n d s i m i l a r p r o d u c t s w i t h p H s o f a b o u t 4 . 5 s h o u l d b e t e s t e dw i t h m e t h y l p a r a b e n , p o t a s s i u m s o r b a t e , o r s o d i u m p r o p i -o n a t e .

L I T E R A T U R E C I T E D1 . A s s o c i a t i o n o f O f f i c i a l A n a l y t i c a l C h e m i s t s . 1 9 8 0 . N a t u r a l p o i -s o n s , p . 4 2 6 - 4 4 0 . I n O f f i c i a l M e t h o d s o f A n a l y s i s , 1 3 t h e d .A s so c ia t i o n o f O f f i c i a l A n a l y t i t c a l C h e m i s t s , W a s h i n g t o n , D . C .2 . B r o w n , M . H . , G . M . S z c z e c h , a n d B . P . P u r m a l i s . 1 9 7 6 .T e r a t o g e n i c a n d t o x i c e f f e c t s o f o c h r a t o x i n A i n r a t s . T o x i c o l .A p p l . P h a r m a c o l . 3 7 : 3 3 1 - 3 3 8 .

A P P L . E N V I R O N . M I C R O B I O L .

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ANT I M I C R OBI AL INHIBITION O F O C HR A TO XI N 1 4 1 13 . C h u , F . S . , a n d M. E . B u t z . 1 9 7 0 . S p e c t r o p h o t o f l u o r o d e n s i t o m e -t r i c m e a s u r e m e n t o f o c h r a t o x i n A i n c e r e a l p r o d u c t s . J . A s s o c .O f f . A n a l . C h e m . 5 3 : 1 2 5 3 - 1 2 5 7 .4 . D a v i s , N . D . , J . W. S e a r c y , a n d U . L . D i e n e r . 1 9 6 9 . P r o d u c t i o no f o c hr a t o x in A b y A s p e r g i l l u s o c h r a c e u s i n a s e mi s yn t he t i cm e d i u m . A p p l . M i c r o b i o l . 1 7 : 7 4 2 - 7 4 4 .5 . E l l i n g , F . , B . H al d, C . J a c o b s e n , a n d P . K r o g h . 1 9 7 5 . S p o n t a -n e o u s t o x i c n e p h r o p a t h y i n p o u l t r y a s s o c i a t e d w i t h o c hr a t o x inA . A c t a P a t h o l . M i c r o b i o l . S c a n d . S e c t . A 8 3 : 7 3 9 - 7 4 1 .6 . F u r i a , T . E . 1 9 7 2 . H a n d b o o k o f f o o d a d d i t i v e s , 2 n d e d . D . F .C h i c h e s t e r a n d F . W. T a n n e r ( e d . ) . C R C P r e s s , C l e v e l a n d ,O h i o .7 . G i l a n i , S . H . , J . B a n c r o f t , a n d M. R e i l y . 1 9 7 8 . T e r a t o g e n i c i t y o fo c h r a t o x i n A i n c h i c k e m b r y o s . T o x i c o l . A p p l . P h a r m a c o l .4 6 : 5 4 3 - 5 4 6 .8 . H a y e s , A . W . , R . S . H o o d , a n d H . L . L e e. 1 9 7 4 . T e r a t o g e n i ce f f e c t s o f o c h r a t o x i n A i n m i c e . T e r a t o l o g y 9 : 9 3 - 9 8 .9 . H o o d , R . D . , M. J . N a u g h t o n , a n d A . W. H a y e s . 1 9 7 6 . P r e n a t a le f f e c t s o f o c h r a t o x i n A i n h a m s t e r s . T e r a t o l o g y 1 3 : 1 1 - 1 4 .1 0 . K r o g h , P . 1 9 7 8 . C a u s a l a s s o c i a t i o n s o f m y c o t o x i c n e p h r o p a t h y .A c t a P a t h o l . M i c r o b i o l . S c a n d . S e c t . A . 2 6 9 ( S u p p l . ) : 6 - 2 8 .1 1 . K r o g h , P . , F . E l l i n g , N . G y r d - H a n s e n , B . H a l d, A . E . L a r s e n ,E . B . L i l l e h o j , A . M a d s e n , H . P . M o r t e n s e n , a n d U . R a v n s k o v .1 9 7 6 . E x p e r i m e n t a l p o r c i n e n e p h r o p a t h y : c h a n g e s o f r e n a lf u n c t i o n a n d s t r u c t u r e o r a l l y i n d u c e d b y c r y s t a l l i n e o c h r a t o x i nA . A c t a P a t h o l . M i c r o b i o l . S c a n d . S e c t . A 8 4 : 4 2 9 - 4 3 4 .1 2 . K r o g h , P . , F . E l l i n g , B . H a l d , A . E . L a r s e n , E . B . L i l l e h o j , A .M a d s e n , a n d H . P . M o r t e n s e n . 1 9 7 6 . T i m e - d e p e n d e n t d i s a p p e a r -a n c e o f o c h ra t o x i n A r e s i d u e s i n t i s s u e s o f b a c o n p i g s . T o x i c o l -o g y 6 : 2 3 5 - 2 4 2 .1 3 . L a i , M . , G . S e m e n i u k , a n d C . W. H e s s e l t i n e . 1 9 7 0 . C o n d i t i o n sf o r p r o d u c t i o n o f o c h r a t o x i n A b y A s p e r g i l l u s s p e c i e s i n as y n t h e t i c m e d i u m . A p p l . M i c r o b i o l . 1 9 : 5 4 2 - 5 4 4 .1 4 . L u e c k , E . 1 9 8 0 . A n t i m i c r o b i a l f o o d a d d i t i v e s . T r a n s l a t e d b yG . F . E d w a r d s . S p r i n g e r - V e r l a g , N e w Y o r k .

1 5 . O c k e r m a n , H . W. 1 9 7 8 . S o u r c e b o o k f o r f o o d s c i e n t i s t s , p . 7 5 .AV I P u bl i s hi n g C o ., I n c . , W e s t p o r t , C o n n .1 6 . R i b e l i n , W. E . , K . F u k u s h i m a , a n d P . E . S t i l l . 1 9 7 8 . T h e t o x i c i t yo f o c h r at o x i n t o r u m i n a n t s . C a n . J . C o m p . M e d . 4 2 : 1 7 2 - 1 7 6 .1 7 . S c o t t , P . M . , B . K e n n e d y , a n d W. v a n W a l b e e k . 1 9 7 1 . S i m p l i f i e dp r o c e d u r e f o r t h e p u r i f i c a t i o n o f o c h r a t o x i n A f r o m e x t r a c t s o fP e n i c i l l i u m v i r i d i c a t u m . J . A s s o c . O f f . A n a l . C h e m . 5 4 : 1 4 4 5 -1 4 4 7 .1 8 . S c o t t , P . M . , W. v a n W a l b e e k , J . H a r w i g , a n d D . I . F e n n e l l .1 9 7 0 . O c c u r r e n c e o f a m y c o t o x i n , o c h r a t o x i n A , i n w h e a t a n di s o l a t i o n o f o c h r a t o x i n A a n d c i t r i n i n p r o d u c i n g s t r a i n s o fP e n i c i l l i u m v i r i d i c a t u m . C a n . J . P l a n t S c i . 5 0 : 5 8 3 - 5 8 5 .1 9 . S c o t t , P . M . , W. v a n W a l b e e k , B . K e n n e dy , a n d D . A n y e t i . 1 9 7 2 .M y c o t o x i n s ( o c h r a t o x i n A , c i t r i n i n , a n d s t e r i g m a t o c y s t i n ) a n dt o x i g e n i c f u n g i i n g r a i n s a n d o t h e r a g r i c u l t u r a l p r o d u c t s . J .A g r i c . F o o d C h e m . 2 0 : 1 1 0 3 - 1 1 0 8 .2 0 . T r e n k , H . L . , M. E . B u t z a n d F . S . C h u . 1 9 7 1 . P r o d u c t i o n o fo c h r a t o x i n s i n d i f f e r e n t c e r e a l p r o d u c t s b y A s p e r g i l l u s o c h r a -c e u s . A p p l . M i c r o b i o l . 2 1 : 1 0 3 2 - 1 0 3 5 .2 1 . V a n d e g r a f t , E . E . , C . W. H e s s e l t i n e , a n d 0 . L . S h o t w e l l . 1 9 7 5 .G r a i n p r e s e r v a t i v e s : e f f e c t o n a f l a t o x i n a n d o c h r a t o x i n p r o d u c -t i o n . C e r e a l C h e m . 5 2 : 7 9 - 8 4 .2 2 . V a n d e g r a f t , E . E . , 0 . L . S h o t w e l l , M. L . S m i t h , a n d C . W.H e s s e l t i n e . 1 9 7 3 . M y c o t o x i n f o r m a t i o n a f f e c t e d b y f u m i g a t i o n o fw he a t . C er e al S c i . T o d a y 1 8 : 4 1 2 - 4 1 4 .2 3 . V a n d e g r a f t , E . E . , 0 . L . S h o t w e l l , M. L . S m i t h , a n d C . W.H e s s e l t i n e . 1 9 7 3 . M y c o t o x i n p r o d u c t i o n a f f e c t e d b y i n s e c t i c i d et r e a t m e n t o f w h e a t . C e r e a l C h e m . 5 0 : 2 6 4 - 2 7 0 .2 4 . Van D e r M e r w e , K . J . , P . S . S t e y n , L . F o u r i e , D . B . S c o t t , a n dJ . J . T h e r o n . 1 5 . O c h r a t o x i n A , a t o x i c m e t a b o l i t e p r o d u c e db y A s p e r g i l l u s o c h r a c e u s W i l h . N a t u r e ( L o n d o n ) 2 0 5 : 1 1 1 2 -1 1 1 3 .2 5 . Wu , M. T . , a n d J . C . A y r e s . 1 9 7 4 . E f f e c t s o f d i c hl o r v o s o no c h r a t o x i n p r o d u c t i o n b y A s p e r g i l l u s o c h r a c e u s . J . A g r i c . F o o dC h e m . 2 2 : 5 3 6 - 5 3 7 .

V O L . 4 9 , 1 9 8 5