Occurrence of Mycotoxins in Foods and Feeds - Advances in

2

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Occurrence of Mycotoxins in Foods and Feeds LEONARD STOLOFF Division of Food Technology, Food and Drug Administration, Washington, D. C. 20204 The year 1960 divides the descriptive phase of mycotoxin investigations from the analytical one. The descriptive episodes are related in this review to current knowledge of specific toxins. The analytical information covers commodities susceptible to aflatoxin contamination (oil-seeds, tree nuts, grains, dried fruits, legumes) and mycotoxins other than aflatoxins (ochratoxin, zearalenone, citrinin, penicillic acid, patulin, sterigmatocystin) for which analytical methods have been devised and applied in surveys. Incidence and level data are given in each case. The current concern about the possible occurrence of toxins in moldy foods and feeds is a far cry from the conclusion of an eminent pro fessor of bacteriology (1) published in 1932 that "there is very little evidence that moldy food causes illness." Mold-Related

Human

Incidents

In fact when this statement was published, the role of a saprophytic fungus (Claviceps purpurea) i n the formation of the poisonous ergot grains on rye had been established; the first conjecture to this effect was made i n 1711 ( 2 ) . Reviews of cause and effect relationships between human consumption of moldy food and illness (3,4) cite numerous cases during the period 1826-1888 of poisoning from the consumption of moldy bread, an 1843 incident caused by moldy army rations i n Paris, an 1878 poisoning attributed to moldy pudding, and numerous deaths between 1906 and 1909 from eating moldy corn meal. Scabby grain has a long history of causing illness i n those areas such as Eastern Europe, China, and Japan where some form of grain is a dietary staple (5, 6, 7). Scab refers to that type of kernel blight caused by species of Fusarium. 23 In Mycotoxins and Other Fungal Related Food Problems; Rodricks, J.; Advances in Chemistry; American Chemical Society: Washington, DC, 1976.

24

MYCOTOXINS

M o r e r e c e n t l y , c o n s i d e r a b l e effort w a s e x p e n d e d i n R u s s i a to i n v e s t i gate r e c u r r i n g e n d e m i c instances of a s y n d r o m e d e s c r i b e d as a l i m e n t a r y t o x i c a l e u k i a ( A T A ) (4)

t r a c e d to t h e c o n s u m p t i o n of foods m a d e f r o m

cereals w h i c h h a d o v e r w i n t e r e d i n fields u n d e r snow. T h e e t i o l o g y of the disease was e s t a b l i s h e d i n 1948 as b e i n g r e l a t e d t o i n v a s i o n of t h e g r a i n b y Fusarium

sporotrichiella

(F.

trincinctum,

Synder and Hansen).

The

disease w a s n o t t r a n s m i s s i b l e , a n d a r a b b i t s k i n test f o r t o x i c i t y w a s d e Downloaded by UNIV OF TENNESSEE KNOXVILLE on December 21, 2014 | http://pubs.acs.org Publication Date: June 1, 1976 | doi: 10.1021/ba-1976-0149.ch002

v i s e d a n d u s e d to d i v e r t t o x i c g r a i n to a l c o h o l i c f e r m e n t a t i o n ( t h e t o x i n does n o t d i s t i l l ) . T h i s is the s e c o n d instance of a c o r r e l a t i o n b e t w e e n a specific m o l d a n d a specific s y n d r o m e . F r o m the s y m p t o m s a n d the m o l d s i n v o l v e d , investigators i n the U n i t e d States (8)

a n d i n J a p a n (7)

conjec

t u r e d , b a s e d o n c u r r e n t k n o w l e d g e , t h a t t h e toxins b e l o n g to the t r i c h t h e cene g r o u p of m o l d metabolites. T h i s conjecture has b e e n s u b s t a n t i a t e d b y d i r e c t t e s t i n g of a n extract f r o m a c u l t u r e of one of the R u s s i a n m o l d isolates ( 9 ) . R e p o r t s of illness i n J a p a n f r o m e a t i n g m o l d y r i c e w e r e a p p a r e n t l y r a r e u n t i l i m p o r t s of r i c e f r o m other countries i n c r e a s e d d u r i n g a n d after W o r l d W a r I I ( J O ) . A y e l l o w d i s c o l o r a t i o n of kernels w a s associated w i t h some toxic lots. T o x i n - p r o d u c i n g m o l d s i s o l a t e d f r o m lots of y e l l o w r i c e w e r e Pénicillium sum.

citreo-viride,

P. islandicum,

P. citrinum,

a n d P.

rugulo-

T o x i n s p r o d u c e d b y these m o l d s i n l a b o r a t o r y c u l t u r e h a v e b e e n

i s o l a t e d a n d c h a r a c t e r i z e d (11)

a n d t h e i r effects o n a n i m a l s d e s c r i b e d ,

b u t n o d e s c r i p t i o n of the h u m a n s y m p t o m s f r o m e a t i n g m o l d y r i c e is a v a i l a b l e for c o m p a r i s o n . Mold-Related

Animal

Incidents

M o l d y f e e d has r e c e i v e d c o n s i d e r a b l y m o r e a t t e n t i o n t h a n has m o l d y f o o d , p r o b a b l y because of the greater f r e q u e n c y of a n i m a l exposure a n d t h e d r a m a t i c manifestations of the m a s s i v e c h a l l e n g e t h a t a n i m a l s u s u a l l y r e c e i v e . T h e e a r l y l i t e r a t u r e has b e e n t h o r o u g h l y r e v i e w e d ( 3 ,

4,12,13).

S p e c i a l note is t a k e n here o n l y of those situations w h e r e a specific m o l d has b e e n i n c r i m i n a t e d i n a n u m b e r of s i m i l a r i n c i d e n t s . I f a source is n o t s p e c i f i c a l l y c i t e d , reference to one of the c i t e d r e v i e w s is i m p l i e d . H e m o r r h a g i c sweet c l o v e r disease is a t y p i c a l of o t h e r m y c o t o x i c o s e s e n c o u n t e r e d i n t h a t a specific p r e c u r s o r , c o u m a r i n , r a t h e r t h a n a specific m o l d , is r e q u i r e d i n the substrate p l a n t to m a k e the t o x i n . T h e disease is m e n t i o n e d h e r e b e c a u s e i t is the first i n s t a n c e since the l o n g f o r g o t t e n i s o l a t i o n of the ergot a l k a l o i d s t h a t a specific t o x i n w a s i d e n t i f i e d . T h e c o m p o u n d is d i c o u m a r o l .

T h i s h e m o r r h a g i c disease affected

c a t t l e i n the

N o r t h C e n t r a l States a n d C a n a d a d u r i n g the 1920s w h e n sweet c l o v e r w a s i n t r o d u c e d as a f o r a g e c r o p . S t a c h y b o t r y o t o x i c o s i s w a s o r i g i n a l l y d e s c r i b e d as a u s u a l l y f a t a l d i s -

In Mycotoxins and Other Fungal Related Food Problems; Rodricks, J.; Advances in Chemistry; American Chemical Society: Washington, DC, 1976.

2.

Occurrence

STOLOFF

in Foods and

25

Feeds

ease of horses a n d less f r e q u e n t l y of sheep a n d goats.

T y p i c a l case h i s

tories d u r i n g t h e 1 9 3 0 s i n d i c a t e d t h a t the p r o b l e m w a s w i d e s p r e a d i n E a s t e r n E u r o p e . R u s s i a n investigators i d e n t i f i e d the causative o r g a n i s m as Stachybotrys

( S . atra, B i s b y ) . T h e y also d e m o n s t r a t e d t h a t t h e

alternans

typical symptoms could be p r o d u c e d b y feeding grain invaded b y F . sporotnchieUa

(see

p r e c e d i n g d i s c u s s i o n of A T A ) . T h e s e observations are

n o t s u r p r i s i n g i n t h e l i g h t of c u r r e n t k n o w l e d g e

that both

organisms


p r o d u c e r e l a t e d t o x i c c o m p o u n d s of t h e t r i c h o t h e c e n e g r o u p (8,

14).

S c a b b y g r a i n has b e e n associated w i t h b o t h a n i m a l a n d h u m a n p r o b lems.

S c a b b y g r a i n f e d to s w i n e is often rejected, b u t i f eaten, causes

v o m i t i n g ( 1 5 , 1 6 ) . T h e same effect was o b t a i n e d w i t h c o r n i n t e n t i o n a l l y m o l d e d b y Fusarium

graminearum

f e c t f o r m Gibberella

zeae)

( F . roseum,

Snyder and Hansen, per

w h i l e g r o w i n g . T h e s e observations are r e m i

n i s c e n t of recent experiences of f a r m e r s i n t h e n o r t h c e n t r a l U n i t e d States w i t h Gibberella-iniected w h a t w e k n o w today. m a n y Fusaria,

c o r n (17).

T h e pieces of i n f o r m a t i o n fit w i t h

A n u m b e r of trichothecenes, toxins p r o d u c e d

are d e m o n s t r a t e d emetics (4,18,19,

by

at a r e l a t i v e l y

20, 21)

l o w dosage. A t h i g h e r levels the s y m p t o m s a r e those of s t a c h y b o t r y o t o x i cosis o r of A T A , r e f e r r e d to e a r l i e r . A disease i n s w i n e a n d cattle, c a u s e d b y e a t i n g m o l d y c o r n , w a s r e p o r t e d as e n d e m i c i n F l o r i d a , A l a b a m a , a n d G e o r g i a . I t w a s also r e l a t e d to feed-associated h e p a t i t i s of dogs ( 2 2 ) . 1 9 5 0 s i m p l i c a t e d Aspergillus

flavus

tive molds.

of the

T h e symptoms

Increased research attention i n the

a n d Penicilium field

as the c a u s a

rubrum

and experimentally

developed

m o l d y c o r n diseases, as d e s c r i b e d i n the 1950 l i t e r a t u r e , are r e m a r k a b l y s i m i l a r to the s y m p t o m s of acute aflatoxicosis d e v e l o p e d e x p e r i m e n t a l l y i n s w i n e w i t h p u r e aflatoxins

(23).

M o l d y f e e d toxicosis, also c a l l e d h e m o r r h a g i c s y n d r o m e , of p o u l t r y has b e e n a consistent p r o b l e m i n the U n i t e d States; i t e x h i b i t s extreme v a r i a b i l i t y i n c l i n i c a l signs, course, m o r t a l i t y , a n d gross a n d h i s t o p a t h o l o g i c a l m a n i f e s t a t i o n b e t w e e n b i r d s a n d b e t w e e n flocks (12).

A m o n g the f u n g i

t h a t h a v e b e e n i s o l a t e d f r o m s u s p e c t e d f e e d are s e v e r a l n o w k n o w n to p r o d u c e specific t o x i n s — v i z . Aspergillus

clavatus,

Pénicillium

a n d P. rubrum.

citrinum,

P. purpurogenum,

A flavus,

A.

fumigatus,

F e e d on w h i c h

e a c h of these m o l d s h a d b e e n c u l t u r e d p r o d u c e d the t y p i c a l s y n d r o m e . F a c i a l e c z e m a , a m a j o r p h o t o s e n i t i z a t i o n disease of r u m i n a n t s

(12),

w a s first r e p o r t e d f r o m N e w Z e a l a n d i n 1897 a n d w a s t h o u g h t p e c u l i a r t o t h a t area. A s i m i l a r disease has since b e e n r e p o r t e d i n the southeastern U n i t e d States. I n 1958 t h e cause of the N e w Z e a l a n d p r o b l e m w a s t r a c e d to the g r o w t h of Pithomyces

chartarum

o n d e a d p a s t u r e grass a n d later to

the e l a b o r a t i o n b y t h a t m o l d of a n y of a series of l i v e r toxins m i n s ) c h a r a c t e r i z e d as e p i p o l y t h i o d i o x o p i p e r a z i n e s (24). associated w i t h t h e p r o b l e m i n t h e U n i t e d States is Perconia

(sporides-

T h e organism minutissima,


26

MYCOTOXINS

also s a p r o p h y t i c o n d e a d h e r b a g e , b u t the s t u d y has n o t b e e n c a r r i e d b e y o n d that point. Era of

Mycotoxins

A n i n c i d e n t i n 1960 m a r k s a c h a n g e i n the g e n e r a l a t t i t u d e to m y c o toxins a n d a n awareness of the scope of the p r o b l e m . I t w a s n o t t h e loss Downloaded by UNIV OF TENNESSEE KNOXVILLE on December 21, 2014 | http://pubs.acs.org Publication Date: June 1, 1976 | doi: 10.1021/ba-1976-0149.ch002

of 100,000 t u r k e y p o u l t s ( 2 5 ) i n this E n g l i s h i n c i d e n t , n o r the severe losses of d u c k l i n g s i n K e n y a f r o m a s i m i l a r , l e s s - p u b l i c i z e d , c o n c o m i t a n t d e n t (26)

inci

t h a t l e d to the change. T h e s e w e r e b u t t h e i n i t i a l i n c i d e n t s i n

a series of events t h a t e s t a b l i s h e d the g e n e r a l t o x i c i t y of p e a n u t m e a l s t o p o u l t r y a n d l i v e s t o c k , t h e r e l a t i o n of the t o x i c i t y to a g r o u p of

fluorescent

c o m p o u n d s t h a t c o u l d b e e x t r a c t e d f r o m t o x i c m e a l s , a n d t h e a b i l i t y of a f u n g u s , Aspergillus compounds

(26).

flavus, i s o l a t e d f r o m a t o x i c m e a l to p r o d u c e t h e same This w o u l d still have been only a veterinary problem,

i f the toxic m e a l h a d not b e e n s h o w n c a p a b l e of p r o d u c i n g c a n c e r of t h e l i v e r (27, 2 8 ) , s t i m u l a t i n g a s t u d y of the i s o l a t e d c o m p o u n d s n o w c a l l e d aflatoxins. T h i s s t u d y e s t a b l i s h e d the extreme o n c o g e n i c i t y of the c o m p o u n d s to the r a t . C o i n c i d e n t a l studies i n F r a n c e ( 2 9 ) l i s h e d t h e r o l e of F. flavus as a p r o d u c e r of c o m p o u n d s

f u r t h e r estab

hepatocarcinogenic

to the rat. T h e s e observations a p p e a r e d at the same t i m e t h a t the W o r l d H e a l t h O r g a n i z a t i o n ( F A O / U N I C E F ) w a s c o n s i d e r i n g p e a n u t m e a l as a p r o t e i n s u p p l e m e n t i n foods for u n d e r n o u r i s h e d c h i l d r e n (30).

While

t h a t o r g a n i z a t i o n w a s p o n d e r i n g the q u e s t i o n of p r o t e i n s t a r v a t i o n vs. cancer, a r e p o r t (31)

of a n a p p a r e n t l y u n r e l a t e d s t u d y f r o m A u b u r n

U n i v e r s i t y d i s p e l l e d the " i t can't h a p p e n h e r e " a t t i t u d e i n t h e U . S . T h e A u b u r n report related U.S.-produced peanut m e a l i n laboratory a n i m a l f e e d to the o c c u r r e n c e of l i v e r cancer i n rats. T h e s i m i l a r i t y to t h e E n g l i s h observations ( 2 7 , 2 8 ) , n o t e d i n the p a p e r d e s c r i b i n g t h e s t u d y , w a s also o b s e r v e d b y those i n g o v e r n m e n t a n d i n d u s t r y c o n c e r n e d w i t h t h e safety of the f o o d s u p p l y a n d the p r o d u c t i o n of peanuts a n d p e a n u t p r o d u c t s . T h e r e s u l t w a s a spate of a c t i v i t y t h a t p r o d u c e d i s o l a t e d c o m p o u n d s w o r k w i t h a n d s i m p l e , sensitive, a n a l y t i c a l m e t h o d o l o g y

for

to

research,

s u r v e y , a n d c o n t r o l . I t is this d e v e l o p m e n t t h a t c h a r a c t e r i z e s t h e difference b e t w e e n the o l d a n d n e w m y c o t o x i n i n v e s t i g a t i o n s . T h e n e w c o n c e r n w a s f o r the p o s s i b i l i t y of cancer o r o r g a n d a m a g e f r o m c h r o n i c i n g e s t i o n of i n i t i a l l y s u b c l i n i c a l a m o u n t s of m o l d toxins. T h e extent of exposure to a t o x i n w h o s e effects m i g h t b e e v i d e n t o n l y years after i n g e s t i o n a n d the safety of the f o o d s u p p l y c o u l d b e d e t e r m i n e d o n l y b y d i r e c t a n a l y s i s f o r t h e toxic c o m p o u n d .

W e e n t e r e d a n e w e r a , a n e r a of a n a l y t i c a l d e t e r m i

n a t i o n of the i n c i d e n c e a n d l e v e l of m y c o t o x i n s i n s u s c e p t i b l e foods a n d a s e a r c h for specific c h e m i c a l entities p r o d u c e d b y m o l d s t h a t m i g h t i n s o m e w a y b e l i n k e d to i d i o p a t h i c disease.


2.

STOLOFF

Occurrence

in Foods and

27

Feeds

I n c i d e n c e , as u s e d i n this p a p e r , refers to t h e n u m b e r o f s a m p l e s w i t h d e t e c t a b l e m y c o t o x i n , u s u a l l y as a p e r c e n t o f the n u m b e r of

samples

e x a m i n e d a n d sometimes as a r a t i o . T h e l e v e l w i l l b e e x p r e s s e d as a n a v e r a g e of a l l samples w i t h detectable m y c o t o x i n a n d as a r a n g e o f t h e levels f o u n d . T h e l o w e r figure i n the r a n g e w i l l u s u a l l y b e t h e l o w e r l i m i t of d e t e c t i o n for the m e t h o d u s e d . O f the b i o l o g i c a l l y a c t i v e m o l d m e t a b o l i t e s that h a v e c o m e t o o u r Downloaded by UNIV OF TENNESSEE KNOXVILLE on December 21, 2014 | http://pubs.acs.org Publication Date: June 1, 1976 | doi: 10.1021/ba-1976-0149.ch002

a t t e n t i o n , significant o c c u r r e n c e i n f o o d or f e e d has b e e n e s t a b l i s h e d f o r aflatoxins, c i t r i n i n , o c h r a t o x i n A , p a t u l i n , p e n i c i l l i c a c i d , t r i c h o t h e c e n e s , a n d zearalenone. A d e q u a t e l y sensitive a n a l y t i c a l m e t h o d s h a v e b e e n d e v e l o p e d a n d v a l i d a t e d for aflatoxins ( 3 2 ) , o c h r a t o x i n A ( 3 3 ) , a n d p a t u l i n (34),

a n d u n v a l i d a t e d b u t u s e f u l m e t h o d s for c i t r i n i n ( 3 5 ) , p e n i c i l l i c a c i d

(36)

a n d zearalenone (37)

are b e i n g u s e d . T h e r e has b e e n s o m e c o n

c e r n a b o u t s t e r i g m a t o c y s t i n b e c a u s e of its d e m o n s t r a t e d c a r c i n o g e n i c i t y (38)

a n d s t r u c t u r a l r e l a t i o n to aflatoxins ( 3 9 ) .

c y s t i n has b e e n v a l i d a t e d (40)

A method for sterigmato

a n d u s e d i n F D A surveys o f suspect foods

a n d feeds, b u t n o o c c u r r e n c e has b e e n d e t e c t e d , n o r w a s s t e r i g m a t o c y s t i n f o u n d i n a n y of 173 samples of v a r i o u s foods s u s c e p t i b l e to m o l d a t t a c k i n a s t u d y of

fiver

M o z a m b i q u e (41),

cancer e p i d e m i o l o g y

i n the I n h a m b a n e r e g i o n

of

a l t h o u g h aflatoxin w a s d e t e c t e d i n 5 % o f t h e s a m p l e s .

S t e r i g m a t o c y s t i n has b e e n i d e n t i f i e d i n a b a d l y m o l d e d s a m p l e of w h e a t (42)

a n d i n m o l d y g r e e n coffee

(43).

T h e d e d u c e d presence of trichothecenes i n grains has b e e n n o t e d , b u t its d e t e c t i o n as a n a t u r a l c o n t a m i n a n t has b e e n r e c o r d e d f o r o n l y one s a m p l e of severely m o l d e d c o r n (44).

A d e q u a t e l y sensitive a n a l y t i c a l

m e t h o d s h a v e n o t y e t b e e n d e v e l o p e d for trichothecenes. Aflatoxins A f l a t o x i n c o n t a m i n a t i o n appears to b e a p r o b l e m w i t h

commodities

s u c h as nuts a n d g r a i n s w h i c h are p r e s e r v e d b y r e d u c t i o n of w a t e r a c t i v i t y , a n d is m o r e l i k e l y to o c c u r at h i g h p r e v a i l i n g t e m p e r a t u r e s . d u c i n g m o l d s , Aspergillus

flavus

a n d A . parasiticus,

The pro

compete best w i t h

o t h e r m i c r o f l o r a w h e n the w a t e r a c t i v i t y is m a r g i n a l f o r p r e s e r v a t i o n (Ow — 0.84-0.86) a n d w h e n t h e t e m p e r a t u r e is r e l a t i v e l y h i g h ( 2 5 ° - 4 5 ° C ) (45).

I n a d e q u a t e d r y i n g at harvest a n d p o o r storage p r a c t i c e s w e r e o r i g i

n a l l y t h o u g h t to b e the m a j o r r e a s o n for c o n t a m i n a t i o n a n d p r o b a b l y are i n m a n y instances, b u t e v i d e n c e is a c c u m u l a t i n g t h a t s i g n i f i c a n t c o n t a m i n a t i o n c a n o c c u r p r i o r to harvest. I n some instances t h e r e is a d e m o n s t r a b l e insect v e c t o r ; i n others n o cause f o r t h e m o l d i n v a s i o n is i m m e d i a t e l y apparent. T h o s e c o m m o d i t i e s c o n s u m e d i n t h e U n i t e d States i n w h i c h s o m e aflatoxin c o n t a m i n a t i o n o f m a r k e t p l a c e samples has b e e n f o u n d


are

28

MYCOTOXINS

Table I.

Farmers' Stock Peanuts Rejected for Visible Aspergillus by Year and by A r e a , as Percent of Total Crop

flavus

Year


Area Southeast Southwest Virginia-Carolina A l l Areas

1968 6.3 1.2 0.5 3.7

1969 1.4 2.4 0.07 1.4

1970 3.4 1.4 0.4 2.1

1971 2.6 2.5 0.3 2.1

1972 13.5 1.9 0.2 8.1

1978 2.6 0.8 0.1 1.6

p e a n u t s , B r a z i l a n d p i s t a c h i o n u t s , a l m o n d s , w a l n u t s , pecans, filberts, cot tonseed, c o p r a , c o r n , g r a i n s o r g h u m , r i c e , a n d figs. Peanuts.

T h e p e a n u t is t h e m o s t t h o r o u g h l y s t u d i e d of t h e

com

m o d i t i e s s u s c e p t i b l e to aflatoxin c o n t a m i n a t i o n , for a n u m b e r of unassoc i a t e d reasons. I t w a s t h e o r i g i n a l p r o b l e m source; i t is a m a j o r f o o d c r o p ; a n d m a r k e t i n g i n the U n i t e d States is u n d e r t h e c o n t r o l of the D e p a r t m e n t o f A g r i c u l t u r e ( U S D A ) . F o r this last r e a s o n a n d also b e c a u s e t h e r e s p o n s i b l e u n i t s of g o v e r n m e n t

(FDA, USDA)

and industry (National

P e a n u t C o u n c i l ) h a d a g r e e d to a system of certification for s h e l l e d peanuts w h e n the p r o b l e m w a s first r e c o g n i z e d , there is c o m p r e h e n s i v e y e a r - t o -

20

40

60

80

TOTAL AFLATOXINS, PPB Figure I . Cumulative percent of lots with aflatoxin levels below the given values for each crop year—1967 to 1972, southwest, all varieties


2.

Occurrence

STOLOFF

in Foods and

29

Feeds


100

40

60

80

100

TOTAL AFLATOXINS, PPB Figure 2. Cumulative percent of lots with aflatoxin levels below the given values for each crop year—1967 to 1972, southeast, all varieties y e a r d a t a for p r a c t i c a l l y the entire c r o p as f a r m e r s ' stock ( T a b l e I ) o r as s h e l l e d nuts ( F i g u r e s 1 - 4 ) .

These data show considerable

year-to-year

a n d area-to-area differences i n t h e i n c i d e n c e a n d levels e n c o u n t e r e d .

The

r e l a t i v e l y s m a l l p o r t i o n of the c r o p c o n s u m e d as r o a s t e d i n - s h e l l p e a n u t s has b e e n free of aflatoxins b e c a u s e of t h e v a r i e t y , r e g i o n , a n d q u a l i t y i n volved i n their production.

B e c a u s e o n l y s h e l l e d nuts w i t h aflatoxins

b e l o w a specified g u i d l i n e are used for edible products a n d because the n o r m a l s o r t i n g a n d r o a s t i n g processes s u b s t a n t i a l l y r e d u c e t h e aflatoxin l e v e l , c o n s u m e r p e a n u t p r o d u c t s i n the U n i t e d States s h o u l d b e r e l a t i v e l y free o f aflatoxins. R o u t i n e F D A m o n i t o r i n g has v e r i f i e d this a s s u m p t i o n . A more comprehensive F D A survey, carried out i n the spring of (46),

1973

has p r o v i d e d d a t a i n g e n e r a l a g r e e m e n t w i t h t h a t f r o m the F D A

m o n i t o r i n g a n d w i t h i n d e p e n d e n t s u r v e y d a t a f r o m C a n a d a o n aflatoxin



30

MYCOTOXINS

20

40

60

80

100

TOTAL AFLATOXINS, PPB Figure 3. Cumulative percent of lots with aflatoxin levels below the given values for each crop year—1967 to 1972, Virginia and Carolinas, all vaneties i n peanut butter (Table I I ) .

S o m e samples of

finished

products

with

levels t h a t e x c e e d t h e r a w m a t e r i a l c e r t i f i c a t i o n g u i d e l i n e c a n b e a t t r i b u t e d to the great v a r i a n c e i n h e r e n t i n s a m p l i n g a n d a n a l y z i n g peanuts f o r aflatoxin c o n t a m i n a t i o n (47).

T h e s e figures are i n m a r k e d contrast to t h e

c o n t a m i n a t i o n of peanuts i n T h a i l a n d (48) samples tested c o n t a i n e d

where 4 9 %

aflatoxin w i t h a n average

Mg/kg> ° r to p e a n u t b u t t e r i n t h e P h i l i p p i n e s (49,

of the m a r k e t

B i l e v e l of

50)

1530

where 9 7 %

of

t h e samples c o n t a i n e d aflatoxins w i t h a n average B i l e v e l of 213 f t g / k g , o r to peanuts i n U g a n d a w h e r e 1 7 % of t h e samples tested h a d a n average aflatoxin B i l e v e l of 363 f t g / k g Brazil N u t s .

(51).

T h e p r e s e n c e of aflatoxins i n some samples of B r a z i l

nuts offered f o r e n t r y w a s first o b s e r v e d i n 1965 b y the V a n c o u v e r

Re

g i o n a l L a b o r a t o r y of the C a n a d i a n F o o d a n d D r u g D i r e c t o r a t e f o l l o w i n g a g e n e r a l p r o g r a m of i n v e s t i g a t i o n of a l l nuts f o r aflatoxins. T h e U . S . F D A w a s a l e r t e d a n d , after c o n f i r m i n g the o b s e r v a t i o n , i n s t i t u t e d a


100%

2.

Occurrence

STOLOFF

31

in Foods and Feeds

s a m p l i n g of B r a z i l n u t i m p o r t s i n 1967. A s a r e s u l t , 2 7 % o f t h e lots offered f o r entry w e r e d e t a i n e d f o r r e c o n d i t i o n i n g o r r e t u r n .

Subsequently

a v o l u n t a r y c o n t r o l agreement w a s w o r k e d o u t b e t w e e n t h e i m p o r t e r s a n d the U S D A w i t h the assistance a n d c o n c u r r e n c e o f t h e F D A . W h e n t h e a g r e e m e n t w a s i m p l e m e n t e d i n 1968, 3 2 % o f t h e lots w e r e

detained.

T h r o u g h t h e influence o f i m p o r t e r s i n e d u c a t i n g t h e i r sources o f s u p p l y , t h e d e t e n t i o n rate has d e c l i n e d so that i n t h e past three years o n l y 1 % Downloaded by UNIV OF TENNESSEE KNOXVILLE on December 21, 2014 | http://pubs.acs.org Publication Date: June 1, 1976 | doi: 10.1021/ba-1976-0149.ch002

of t h e lots offered f o r e n t r y h a s b e e n d e t a i n e d . Pistachio Nuts. P i s t a c h i o nuts n o w i m p o r t e d i n t o t h e U n i t e d States are p r o d u c e d i n T u r k e y a n d I r a n . M o s t p i s t a c h i o nuts enter t h r o u g h t h e port of N e w York. O n the initiative of personnel of t h e F D A N e w Y o r k D i s t r i c t Office, a n e x a m i n a t i o n o f p i s t a c h i o nuts f o r aflatoxins w a s c o n d u c t e d as p a r t of t h e g e n e r a l s u r v e y f o r c o m m o d i t i e s aflatoxin c o n t a m i n a t i o n .

Since positive

findings

susceptible

to

o f v i o l a t i v e levels of

aflatoxins w e r e e n c o u n t e r e d , t h e c o v e r a g e o f p i s t a c h i o nuts w a s i n c r e a s e d u n t i l i n e a r l y 1972 m o r e t h a n 8 0 % o f t h e lots offered f o r e n t r y w e r e 100

86

20

40

60

80

100

TOTAL AFLATOXINS, PPB


32

MYCOTOXINS

Table II. Consumer Peanut Products—Cumulative Percent of Samples Examined in Various Aflatoxin Contamination Categories United States, Shelled Products Surveillance FY

a

N o . Samples E x a m i n e d

1973 98


R a n g e , T o t a l A f l a t o x i n s , μg/kg 20-50 15-19 10-14 5- 9 - 4

Canada, Peanut Butter

Survey

Surveillance

Swing 1973 361

FY

1973 428

C u m u l a t i v e % of S a m p l e s 2 4 5 11 25

3 4 6 8 15

5 7 9 12 16

% of S a m p l e s None Detectable

84

85

75

° F Y — fiscal year.

detained.

T h e b o t t l e n e c k w a s b r o k e n w h e n the p i s t a c h i o n u t i m p o r t e r s

o r g a n i z e d a v o l u n t a r y c e r t i f i c a t i o n a g r e e m e n t s i m i l a r to the o n e

worked

o u t w i t h t h e B r a z i l n u t i m p o r t e r s . A t the same t i m e a t e a m of U . S . g o v e r n m e n t a f l a t o x i n experts v i s i t e d T u r k e y a n d I r a n at t h e i r request to d e t e r m i n e h o w a f l a t o x i n c o n t a m i n a t i o n m i g h t b e p r e v e n t e d a n d to i n s t r u c t k e y technical personnel i n analytical control methods.

F o l l o w i n g the visit,

detentions of p i s t a c h i o nuts f o r aflatoxin c o n t a m i n a t i o n d r o p p e d to v i r t u a l l y z e r o a n d h a v e r e m a i n e d t h a t w a y for T u r k i s h s h i p m e n t s ;

however

i n t h e first h a l f of 1974, 1 3 % of 246 lots s h i p p e d f r o m I r a n w e r e d e t a i n e d . T h i s r e v e r s i o n w a s a t t r i b u t e d i n p a r t to l a x c o n t r o l at t h e p o i n t of s h i p m e n t a n d i n p a r t to a n i n c r e a s e d i n c i d e n c e of c o n t a m i n a t i o n o n the tree (52). Almonds, Walnuts, Pecans, Filberts. T h e s e n u t s w e r e e x a m i n e d as p a r t o f t h e g e n e r a l F D A s u r v e y of aflatoxin-susceptible c o m m o d i t i e s .

Al

m o s t a l l t h e a l m o n d s , w a l n u t s , pecans, a n d i n - s h e l l filberts c o n s u m e d i n t h e U n i t e d States are p r o d u c e d shelled

filberts

consumed

A m a j o r p o r t i o n of

the

i n t h e U n i t e d States c o m e f r o m T u r k e y .

domestically.

No

aflatoxin w a s d e t e c t e d i n a n y of the d o m e s t i c

filberts

sampled over two

c r o p y e a r s , b u t a p p r o x i m a t e l y 8 % of the 142 samples of i m p o r t e d tested w e r e p o s i t i v e for aflatoxins ( t o t a l aflatoxins a v e r a g e d n u t m e a t s ; r a n g e , 2 - 1 0 0 μg/kg). s a m p l e s of d o m e s t i c

filberts

33 / * g / k g

Aflatoxins have been found i n market

a l m o n d s , pecans, a n d w a l n u t s ( T a b l e

III)

at a n

i n c i d e n c e of a b o u t 6 % of the samples a n d a n average t o t a l aflatoxin l e v e l of 2 3 / A g / k g n u t meats. W h e n a l e r t e d to t h e p r o b l e m , the r e s p o n s i b l e i n dustries i n v e s t i g a t e d t h e cause a n d i n s t i t u t e d p r o c e d u r e s to p r e v e n t

con

t a m i n a t e d n u t s f r o m r e a c h i n g the m a r k e t p l a c e . T h e m a j o r aflatoxin c o n -


2.

STOLOFF

Occurrence

33

in Foods and Feeds

t a m i n a t i o n of a l m o n d s was t r a c e d to d a m a g e b y n a v e l o r a n g e w o r m s S o r t i n g of the s h e l l e d a l m o n d s f o r specific types of d a m a g e w a s

(53).

effective

i n s e g r e g a t i n g those nuts c o n t a i n i n g aflatoxins. T h e m a j o r c o n t a m i n a t i o n of w a l n u t s w a s t r a c e d to those varieties p r o n e to s u n b u r n (54),

a n d as

w i t h a l m o n d s , s o r t i n g o r i e n t e d to the specific d a m a g e effectively r e m o v e d c o n t a m i n a t e d kernels. A f l a t o x i n c o n t a m i n a t i o n of p e c a n k e r n e l s does n o t correlate w i t h a n y t y p e of d a m a g e t h a t c a n b e o b s e r v e d ; a f l a t o x i n c o n t a m i Downloaded by UNIV OF TENNESSEE KNOXVILLE on December 21, 2014 | http://pubs.acs.org Publication Date: June 1, 1976 | doi: 10.1021/ba-1976-0149.ch002

n a t i o n has b e e n f o u n d i n p e c a n lots w i t h n o v i s i b l e k e r n e l d a m a g e .

Re

s e a r c h is i n progress to d e t e r m i n e the cause a n d s u b s e q u e n t m e t h o d f o r control. Cottonseed.

I n the same y e a r t h a t t u r k e y X disease d e c i m a t e d t h e

n e w l y h a t c h e d b i r d s i n the E n g l i s h

flocks,

an epizootic

of

hepatoma

o c c u r r e d i n t r o u t hatcheries i n t h e U n i t e d States. T h e cause w a s t r a c e d to the cottonseed m e a l c o m p o n e n t of the t r o u t r a t i o n a n d e v e n t u a l l y to aflatoxin i n the m e a l ( 5 5 ) .

E v e n b e f o r e the association w a s firmly estab

l i s h e d , surveys of cottonseed a n d cottonseed m e a l f o r i n c i d e n c e of afla toxins h a d b e e n started. T h e first surveys (56)

c o v e r e d t h r e e c r o p years

a n d i n v o l v e d samples of cottonseed a n d cottonseed m e a l c o l l e c t e d o n a w e e k l y basis d u r i n g t h e c r u s h i n g season f r o m p l a n t s s e l e c t e d as a r e p r e sentative cross-section of process a n d p r o d u c t i o n ( 5 7 ) .

T h e survey data

for the three-year p e r i o d ( T a b l e I V ) s h o w e d a n 8 % i n c i d e n c e of detect a b l e aflatoxin ( > 3

f i g / k g , aflatoxin B i ) i n cottonseed

and a 19%

inci

d e n c e i n m e a l d e r i v e d f r o m t h a t seed, w i t h average aflatoxin B i levels of 143 a n d 99 f t g / k g .

T h e r e was a m a r k e d year-to-year v a r i a t i o n i n i n c i

d e n c e a n d l e v e l of c o n t a m i n a t i o n . T h e h i g h e r i n c i d e n c e of aflatoxin i n t h e m e a l c o m p a r e d w i t h the cottonseed

is p r o b a b l y a n a r t i f a c t of

the

d i f f i c u l t y i n o b t a i n i n g a representative s a m p l e of seed. T h e h i g h e r a v e r a g e l e v e l i n the seed c o m p a r e d to the m e a l i n d i c a t e s a p r e f e r e n t i a l p a r t i t i o n of aflatoxin into the o i l . T h e s u r v e y results also s h o w e d a m a r k e d l y h i g h e r i n c i d e n c e a n d l e v e l of aflatoxin c o n t a m i n a t i o n i n samples f r o m the s o u t h e r n v a l l e y s of C a l i f o r n i a a n d the l o w e r a l t i t u d e areas of A r i z o n a c o m p a r e d w i t h f r o m the r e m a i n d e r of the cotton b e l t ( 5 8 ) . firmed

samples

L a t e r surveys (59, 60)

con

the c o n t a m i n a t i o n p r o b l e m i n these areas. T h e surveys w e r e a i d e d

Table III. Almonds, Pecans, Walnuts—Incidence and Level of Aflatoxin Contamination in Domestic Production Sampled Fiscal Years 1970-1974 Detectable Aflatoxins, Total TVo. o/ μρ/kg Nut Meats Nut Almond Pecan Walnut

of Samples 345 406 205

of Samples 8 6.5 3.5

Average 20 26 27

Range 2 - 94 2-172 2 - 70


34

MYCOTOXINS

Table I V . Cottonseed and Cottonseed Meal—Incidence and Level of Aflatoxin Contamination in Three Successive Crop Years Detectable Aflatoxin Crop

Year

of

% of Samples

No. Samples

Average

B

x

Highest (ng/kg)

Range


Cottonseed 1964- 5 1965- 6 1966- 7

928 1319 943

>1500 1 5 1 - 500 500-1500

220 44 228

6.5 8.2 8.8

Cottonseed M e a l 1964- 5 1965- 6 1966- 7

964 1293 961

21.1 21.5 12.8

500-1500 1 5 1 - 500 500-1500

136 33 150

b y t h e o b s e r v a t i o n of a field b o l l r o t c a u s e d b y Aspergillus

flavus,

its asso

c i a t i o n w i t h b r i g h t g r e e n i s h - y e l l o w ( B G Y ) fluorescing spots o n t h e a n d s e e d (61),

fiber

a n d t h e association of the B G Y fluorescence w i t h t h e p r e s

e n c e of aflatoxins (62,

63, 64).

T h e B G Y fluorescence, w h i c h w a s l a t e r

r e l a t e d to t h e a c t i o n of p e r o x i d a s e i n the i n v a d e d tissue o n k o j i c a c i d p r o d u c e d i n c o p i o u s q u a n t i t y b y Aspergillus

flavus

(65),

became a valuable

s c r e e n i n g t o o l . T h e m e c h a n i c a l d a m a g e c a u s e d b y the p i n k b o l l w o r m has b e e n i d e n t i f i e d as one r o u t e of e n t r y to t h e b o l l for A . flavus P r o l o n g e d h i g h t e m p e r a t u r e a n d h i g h h u m i d i t y caused p r a c t i c e s also c o n t r i b u t e to A . flavus

i n v a s i o n (67,

68).

(66).

by irrigation Intensive l o c a l

s a m p l i n g i n one of t h e h i g h r i s k areas has s h o w n 1 0 0 %

incidence

of

aflatoxin c o n t a m i n a t i o n of lots of seed g o i n g i n t o storage w i t h t h e m e a n t o t a l aflatoxins l e v e l of i n c o m i n g seed u p to 2600 j u g / k g

(69).

A f l a t o x i n c o n t a m i n a t i o n of cottonseed is n o t confined to t h e U n i t e d States a l t h o u g h the w o r l d w i d e p r o b l e m appears to b e less serious t h a n i n t h e U . S . S o u t h w e s t . A D a n i s h s u r v e y (70) ucts f r o m e i g h t m a j o r e x p o r t i n g countries

of i m p o r t e d cottonseed p r o d (not

i n c l u d i n g the U n i t e d

States) f o u n d aflatoxins i n 1 / 3 of the 120 samples tested ( t o t a l aflatoxins a v e r a g e 30 f t g / k g ; r a n g e , 5 - 1 2 0 / A g / k g ) . T h e i n c i d e n c e b y c o u n t r y r a n g e d f r o m 21 to 1 0 0 % of t h e samples tested. A r e p o r t (71)

of a s u r v e y i n I n d i a , a c o u n t r y not i n c l u d e d i n t h e

D a n i s h survey, confirmed the correlation between fluorescence

i n cottonseed.

aflatoxin a n d B G Y

A f l a t o x i n w a s f o u n d i n 5 4 % of 388

samples

e x a m i n e d ; 1 2 % of t h e samples h a d aflatoxin B i levels greater t h a n 500 / A g / k g . T h e r e w a s a s t r o n g r e l a t i o n b e t w e e n the area h u m i d i t y a n d t h e l e v e l a n d i n c i d e n c e of aflatoxin c o n t a m i n a t i o n . Copra. C r u d e c o c o n u t o i l has r e p l a c e d c o p r a i m p o r t s to t h e U n i t e d States since F D A s u r v e i l l a n c e a c t i v i t y f o u n d

aflatoxin i n 8 8 %


of

72

2.

STOLOFF

Occurrence

in Foods and

35

Feeds

samples of c o p r a a n d c o p r a m e a l tested ( t o t a l aflatoxins average 46

pg/

k g ; r a n g e , t r a c e - 2 0 0 / x g / k g ) . A s t u d y of F i n n i s h c o p r a i m p o r t s ( 7 2 )

pro

v i d e d m u c h the same p i c t u r e . A f l a t o x i n s w e r e f o u n d i n 6 3 % of t h e 16 samples tested ( a v e r a g e 37 / x g / k g ; r a n g e 1 0 - 1 0 0 / x g / k g ) . N o cause for t h e c o n t a m i n a t i o n has b e e n e s t a b l i s h e d a l t h o u g h i n a d e q u a t e d r y i n g a n d i m p r o p e r h a n d l i n g i n t r a n s p o r t are suspected. Seed O i l . P e a n u t s , cottonseed, a n d c o p r a are m a j o r sources of e d i b l e


oils. A c o n s i d e r a b l e p o r t i o n of t h e aflatoxin i n the s e e d is f o u n d i n t h e expressed or e x t r a c t e d o i l .

S u c h oils, i f u s e d i n t h e c r u d e state, c a n

c o n t r i b u t e to h u m a n aflatoxin exposure

(73, 74).

N o r m a l r e f i n i n g of

e d i b l e oils effectively r e m o v e s aflatoxins ( 7 3 , 7 5 ) . C o r n . I n the pre-1960 p e r i o d , m o l d y c o r n w a s f r e q u e n t l y i m p l i c a t e d i n a n i m a l toxicosis; Aspergillus

was a c o m m o n

flavus

t o x i c c o r n isolate,

a n d the s y m p t o m s of m o l d y c o r n toxicosis are m u c h l i k e those d e s c r i b e d for aflatoxicosis. C o r n is the o n l y g r a i n n o r m a l l y h a r v e s t e d at a m o i s t u r e level that can support m o l d growth.

T h e r e q u i r e m e n t s of

mechanical

h a r v e s t i n g h a v e f u r t h e r i n c r e a s e d t h a t l e v e l , p u t t i n g greater d e m a n d s

on

the d r y i n g operations n o r m a l l y o v e r b u r d e n e d at h a r v e s t t i m e . T h e p o s s i b i l i t y of aflatoxin c o n t a m i n a t i o n of c o r n w a s a n a t u r a l conjecture p a r t i c u l a r l y w h e n a r e l a t i o n to e p i z o o t i c s of t o x i c hepatitis h a d b e e n strated ( 2 3 ) .

demon

T o determine the possibility a n d incidence, the A g r i c u l t u r a l

R e s e a r c h S e r v i c e ( A R S ) of U S D A u n d e r t o o k surveys of c o r n f r o m c r o p years 1 9 6 4 , 1 9 6 5 , a n d 1967 (76, 7 7 ) . T h e surveys c o v e r e d 1594 samples of a l l grades a n d f r o m a l l g r o w i n g areas. A f l a t o x i n w a s d e t e c t e d i n 2 . 5 %

of

t h e samples ( t o t a l aflatoxins average 9 / x g / k g ; r a n g e 3 - 3 7 / x g / k g ) , m o s t of t h e m i n the S a m p l e G r a d e category.

A subsequent s u r v e y (78)

of

293

samples of c o r n d e s t i n e d f o r e x p o r t d u r i n g the 1 9 6 8 - 1 9 6 9 p e r i o d f o u n d 2.7%

of the samples w i t h d e t e c t a b l e aflatoxins ( t o t a l aflatoxins

average

18 / x g / k g ; r a n g e 2 - 3 1 / x g / k g ) , a p i c t u r e s i m i l a r to t h a t d e v e l o p e d i n t h e p r e v i o u s w i d e r s u r v e y except that t h e c o n t a m i n a t i o n of the e x p o r t c o r n i n v o l v e d a l l grades a l m o s t e q u a l l y . C o n c e n t r a t i n g o n c o r n f r o m the s o u t h eastern states, e v e n t h o u g h t h a t a r e a accounts for o n l y a b o u t 6 % U . S . p r o d u c t i o n , t h e next U S D A s u r v e y ( 7 9 ) aflatoxin i n c o r n i n t o better focus.

of t o t a l

b r o u g h t the p r o b l e m

Aflatoxin was f o u n d i n 3 5 %

of

of 60

samples of c o r n m a r k e t e d i n the 1 9 6 9 - 1 9 7 0 p e r i o d ( t o t a l aflatoxins average 66 / x g / k g ; r a n g e 6 - 3 4 8 / x g / k g ) . A h i g h i n c i d e n c e of c o n t a m i n a t i o n w a s f o u n d i n a l l g r a d e categories. F u r t h e r s u r v e y w a s f a c i l i t a t e d b y the o b s e r v a t i o n t h a t a B G Y rescence, analogous

to t h a t seen

i n cottonseed,

was

associated

fluo with

aflatoxin c o n t a m i n a t e d kernels ( S O ) . T h e U S D A ' s A g r i c u l t u r a l M a r k e t i n g S e r v i c e ( A M S ) u s e d this s i m p l e o b s e r v a t i o n i n c o n j u n c t i o n w i t h a s e m i q u a n t i t a t i v e s c r e e n i n g m e t h o d f o r aflatoxin assay (81)

at 15 selected s t a

tions to e x a m i n e 2866 samples of 1973 c r o p c o r n s u b m i t t e d f o r g r a d e d e -


36

MYCOTOXINS

termination (82).

T h e assay m e t h o d has a s e n s i t i v i t y l i m i t for t o t a l afla

toxins of a b o u t 5 / x g / k g ; i t w a s u s e d w i t h a 15 / x g / k g reference s t a n d a r d to d e t e r m i n e d e t e c t a b l e

aflatoxins greater or less t h a n the

s t a n d a r d i n a l l samples of c o r n i n w h i c h some B G Y

reference

fluorescence

had been

o b s e r v e d . A f l a t o x i n was d e t e c t e d i n 8 . 2 % of the s a m p l e s e x a m i n e d ; 2 . 5 % of the samples h a d t o t a l aflatoxin levels greater t h a n 15 / x g / k g . T h e i n c i d e n c e v a r i e d f r o m s t a t i o n to s t a t i o n ; the highest was i n c o r n r e c e i v e d at Downloaded by UNIV OF TENNESSEE KNOXVILLE on December 21, 2014 | http://pubs.acs.org Publication Date: June 1, 1976 | doi: 10.1021/ba-1976-0149.ch002

M o b i l e , C h i c a g o , Norfolk, O m a h a , Peoria, a n d Sacramento, i n that order. T h e average i n c i d e n c e of detectable aflatoxins f r o m these stations w a s 1 5 . 6 % of the 1128 samples e x a m i n e d ; 5 . 6 % of the samples f r o m these six stations h a d levels greater t h a n 15 / x g / k g . B e c a u s e of the s m a l l s a m p l e size ( 2 l b ) , these figures p r o b a b l y u n d e r e s t i m a t e the r e a l i n c i d e n c e . T h e a p p e a r a n c e of B G Y

fluorescence

i n c o r n has a h i g h c o r r e l a t i o n

w i t h the presence of aflatoxins (82, 83, 84),

but the observation should

a c c o m p a n y the k n o w l e d g e t h a t t h e f o r m a t i o n of the

fluorescent

compound

d e p e n d s o n a peroxidase i n t h e v i a b l e k e r n e l . S i n c e c u r r e n t e v i d e n c e i n d i cates t h a t aflatoxin c o n t a m i n a t i o n of c o r n is p r i m a r i l y a field a n d f a r m p r o b l e m (85, 86, 87, 88),

m o s t c o n t a m i n a t i o n c a n p r o b a b l y be p i c k e d u p

b y this m e t h o d . A f l a t o x i n i n c o r n seems to b e a w o r l d w i d e p r o b l e m .

L i m i t e d sur

veys s h o w a 4 0 % i n c i d e n c e of aflatoxin i n c o r n samples t a k e n i n U g a n d a (aflatoxin B

x

average 133 / x g / k g ) (51),

( a f l a t o x i n B i average 400 / x g / k g ) (48), p i n e i s l a n d of C e b u (aflatoxin B

x

a 35%

incidence i n T h a i l a n d

a n d a 9 7 % i n c i d e n c e i n the P h i l i p

average 213 / x g / k g ) (49, 50).

I n some

areas c o n v e n t i o n a l m e t h o d s f o r u s i n g c o r n r e s u l t i n d e s t r u c t i o n or d i v e r s i o n of the aflatoxin. A l k a l i p r o c e s s i n g of c o r n destroys m u c h of t h e afla t o x i n (89).

B o t h w e t m i l l i n g a n d d r y m i l l i n g of c o r n concentrate

aflatoxins i n those fractions u s e d for f e e d or o i l r e c o v e r y (90,

the

91).

Wheat, G r a i n , Sorghum, Oats, Rice. O t h e r c e r e a l grains i n c l u d e d i n U S D A - A R S surveys for aflatoxins are w h e a t , g r a i n s o r g h u m , a n d oats (92,93).

S a m p l e s w e r e o b t a i n e d f r o m U S D A - A M S g r a d e r stations r e p r e

s e n t i n g a l l grades a n d g r o w i n g areas. N o aflatoxins w e r e d e t e c t e d i n a n y of t h e 1379 samples of w h e a t , 533 samples of g r a i n s o r g h u m , or 304 s a m ples of oats. F D A s u r v e i l l a n c e a c t i v i t y f o u n d n o aflatoxins i n 106 samples of w h e a t , b u t aflatoxin w a s d e t e c t e d

i n t w o of 66 samples of

grain

s o r g h u m ( 13 a n d 50 / x g / k g ) a n d i n one of 157 samples of r i c e at 5 / x g / k g . G r a i n s o r g h u m w a s s u s c e p t i b l e to aflatoxin c o n t a m i n a t i o n i n U g a n d a w h e r e 2 3 % of 69 samples e x a m i n e d h a d a n average aflatoxin B i l e v e l of 152 / x g / k g (51).

R i c e i n the m a r k e t s i n U g a n d a , T h a i l a n d , a n d t h e

P h i l i p p i n e s w a s r e m a r k a b l y c l e a n (48,

49, 50, 51);

aflatoxin w a s f o u n d

i n o n l y eight of 447 samples at a n average u n d e r 10 / x g / k g , b u t c o o k e d r i c e k e p t u n d e r p r i m i t i v e c o n d i t i o n s p r o v e d to b e a m a j o r source of h u m a n e x p o s u r e to aflatoxins ( 9 4 ) .

T h e s m a l l grains i n g e n e r a l d o n o t a p p e a r to


2.

STOLOFF

Occurrence

in Foods and

37

Feeds

b e a n i m p o r t a n t source of aflatoxin exposure unless a b u s e d i n storage or after p r e p a r a t i o n . Legumes. L e g u m e s i n the U n i t e d States a p p e a r to b e r e l a t i v e l y free of aflatoxin c o n t a m i n a t i o n . A U S D A - A R S s u r v e y of 866 s o y b e a n samples (76)

f o u n d t w o samples c o n t a m i n a t e d at t o t a l aflatoxins levels of 10 a n d

11 /xg/kg. A n F D A s u r v e y of 117 samples of d r i e d beans ( b l a c k e y e , b l a c k t u r t l e , g a r b a n z o , great n o r t h e r n , l i m a , n a v y , p i n k , p i n t o , a n d r e d ) f o u n d n o Downloaded by UNIV OF TENNESSEE KNOXVILLE on December 21, 2014 | http://pubs.acs.org Publication Date: June 1, 1976 | doi: 10.1021/ba-1976-0149.ch002

d e t e c t a b l e aflatoxins i n a n y s a m p l e . A s t u d y o f the s u s c e p t i b i l i t y of soy beans to i n v a s i o n b y Aspergillus

flavus

(95)

i n d i c a t e d t h e presence of a n

A . flavus g r o w t h i n h i b i t o r . H o w e v e r i n U g a n d a 23% a s s a y e d h a d a n average aflatoxin B T h a i l a n d 3% /xg/kg (48).

x

of 64 b e a n samples

l e v e l of 500 /xg/kg (51),

and i n

of 322 samples h a d a n average aflatoxin B i l e v e l of

106

T h e factors c r e a t i n g these differences i n o b s e r v a t i o n h a v e

not been determined. Peppers. P u b l i s h e d reports of a h i g h i n c i d e n c e of Aspergillus

flavus

isolates f r o m v a r i o u s types of p e p p e r s p r o v i d e d t h e i m p e t u s for a C a n a d i a n H e a l t h P r o t e c t i o n B r a n c h s u r v e y of b l a c k , w h i t e , a n d c a p s i c u m p e p p e r s (96). pepper.

N o aflatoxins w e r e f o u n d i n 24 samples of b l a c k or w h i t e

O f the Capsicum

b a s e d p e p p e r s , 14 of 33 samples of

cayenne

p e p p e r a n d a l l six I n d i a n c h i l i p o w d e r samples c o n t a i n e d aflatoxins at levels f r o m ca. 2 to 8 /xg/kg. A n o t h e r 30 samples of Capsicum (paprika, chili powder,

Singapore

chili powder)

h a d no

pepper detectable

aflatoxins. Dried Fruits, Wine.

S o m e f r u i t s s u c h as dates, figs, a n d raisins

are p r e s e r v e d b y d r y i n g , a n d u n p u b l i s h e d m o l d profiles of t h e d r i e d c o m m o d i t i e s l i s t e d Aspergillus species.

flavus

among

the commonly

encountered

A l i m i t e d F D A s u r v e y f o u n d n o aflatoxins i n 108 samples

raisins a n d 62 samples of dates, b u t aflatoxins w e r e f o u n d i n six of samples of figs ( t o t a l aflatoxins average 13 /xg/kg; r a n g e , 2-29 T h e c o n s i d e r a t i o n of Aspergillus

of 165

/xg/kg).

flavus as a n i n v a d e r of grapes also l e d to

t h e e x a m i n a t i o n of w i n e s . I n one s t u d y of 33 G e r m a n w i n e s f r o m the S o u t h B a d e n v i n e y a r d c o u n t r y (97), aflatoxin w a s f o u n d i n t w o samples at levels less t h a n 1 /xg/1. I n another s t u d y (98)

of 17 G e r m a n R h i n e

c o u n t r y w i n e s f r o m the years w h e n g r a p e r o t w a s a p r o b l e m , n o aflatoxins w e r e detected; n o r w e r e aflatoxins d e t e c t e d i n 13 samples of v a r i o u s w i n e s i m p o r t e d i n t o t h e U n i t e d States (99)

u s i n g a m e t h o d sensitive to

0.3

D a i r y Products. A f l a t o x i n M i , a m a m m a l i a n h y d r o x y l a t i o n p r o d u c t of aflatoxin B i , is f o u n d i n the m i l k of l a c t a t i n g a n i m a l s e x p o s e d to afla toxin B i i n their feed.

A n F D A s u r v e y (100)

of m i l k products (cottage

cheese d r y c u r d , n o n f a t d r y m n l k , e v a p o r a t e d m i l k ) p r o d u c e d i n the U n i t e d States i n the first t h r e e m o n t h s of 1973 f o u n d M i i n samples t a k e n f r o m areas w h e r e aflatoxin c o n t a m i n a t i o n of f e e d i n g r e d i e n t s h a d b e e n


38

MYCOTOXINS

suspected.

I n one m i l k s h e d area, aflatoxin M i was d e t e c t e d i n a l l of 16

s a m p l e s t a k e n . A lesser i n c i d e n c e w a s f o u n d i n t w o other areas. A f l a t o x i n M i was found i n 8 %

of t h e 320 samples assayed f r o m 0.05 to 0.5 /xg/1

( a l l c a l c u l a t i o n s w e r e b a s e d o n t h e o r i g i n a l m i l k f o r easy i n t e r c o m p a r i son).

T h e first r e p o r t of aflatoxin M i i n m a r k e t m i l k w a s i n 1968 f r o m

S o u t h A f r i c a (101)

w h e r e d e t e c t a b l e aflatoxin w a s f o u n d i n five of 21

s a m p l e s of fluid m i l k f r o m r e t a i l outlets at a trace ( < 0 . 0 2 to 0.2 /xg/1). Downloaded by UNIV OF TENNESSEE KNOXVILLE on December 21, 2014 | http://pubs.acs.org Publication Date: June 1, 1976 | doi: 10.1021/ba-1976-0149.ch002

A G e r m a n s t u d y of c o m m e r c i a l l y d r i e d m i l k p r o d u c t s (102) toxin M i i n 5 %

f o u n d afla

of 166 samples at 0.07-0.2 /xg/1 ( r e c a l c u l a t e d to

fluid

m i l k basis ). T w o years later a s u r v e y of d r i e d m i l k p r o d u c t s i n G e r m a n y b y a n o t h e r t e a m of investigators (103)

f o u n d aflatoxin M i i n 6 2 % of 120

s a m p l e s p i c k e d u p at m o n t h l y i n t e r v a l s over a p e r i o d of IV2 years.

The

l e v e l s ( f l u i d m i l k b a s i s ) r a n g e d f r o m 0.02 to 0.4 /xg/1 w i t h a d i s t i n c t s e a s o n a l t r e n d to a h i g h e r i n c i d e n c e w h e n the c o w s w o u l d n o r m a l l y b e o n s t o r e d f e e d . T h e s e d a t a are s u p p o r t e d f u r t h e r b y a n F D A e x a m i n a t i o n of 3 5 s a m p l e s of v a r i o u s cheeses i m p o r t e d f r o m E u r o p e . A f l a t o x i n M i w a s f o u n d i n t w o of e i g h t samples f r o m G e r m a n y , t w o of f o u r samples f r o m S w i t z e r l a n d , a n d one of 11 samples f r o m F r a n c e . L e v e l s r a n g e d f r o m 0.1 to 0.6 ttg/kg cheese. N o aflatoxin w a s f o u n d i n the 12 samples f r o m I t a l y and Greece.

F r o m c o n v e r s i o n d a t a (104, 105, i 0 6 , 107, 108),

it m a y be

e s t i m a t e d t h a t aflatoxin M i i n the m i l k at 0.1 /xg/1 c a m e f r o m aflatoxin B i i n t h e f e e d at a b o u t 25 /xg/kg d r y f e e d w e i g h t .

Other

Mycotoxins

A l t h o u g h i t appears t h a t most of the m y c o t o x i n s u r v e y w o r k has b e e n c o n f i n e d to aflatoxins, t h e s e a r c h has i n c l u d e d other m y c o t o x i n s f o r w h i c h a n a l y t i c a l m e t h o d o l o g y of some sort w a s a v a i l a b l e a n d for w h i c h s o m e r e a s o n existed to suspect a p o t e n t i a l for h a r m . E a c h d i s t u r b i n g i n c i d e n c e o f a m y c o t o x i n i n a f o o d c o m m o d i t y has t r i g g e r e d a t o x i c o l o g i c a l effort to d e t e r m i n e t h e r e a l p o t e n t i a l for h a r m f r o m c h r o n i c i n g e s t i o n . Zearalenone.

T h e o c c u r r e n c e of z e a r a l e n o n e is r e l a t e d to t h e i n v a

s i o n o f g r a i n b y v a r i o u s species of Fusarium cinctum

a n d F . moniliforne

nearium

(=Gibberella

(109,110,

111 ) s u c h as F . tri-

b u t p a r t i c u l a r l y b y F . roseum

var.

grami-

zeae) c a l l e d G i b b i n f a r m v e r n a c u l a r . T h e s e o r g a

n i s m s i n v a d e d e v e l o p i n g c o r n at the s i l k i n g stage i n p e r i o d s of

heavy

r a i n f a l l a n d p r o l i f e r a t e o n m a t u r e grains t h a t h a v e n o t d r i e d b e c a u s e of w e t w e a t h e r at h a r v e s t or o n grains t h a t are s t o r e d w e t (17, I n s t o r e d c o r n t h e Fusaria

a n d b a c t e r i a (114).

Penicillia

112,

113).

o c c u p y the w a t e r a c t i v i t y n i c h e b e t w e e n the F i e l d o b s e r v a t i o n of a p i n k d i s c o l o r a t i o n of

k e r n e l s signals the p r e s e n c e of F. roseum i n c o r n ; s c a b is associated w i t h F . roseum

i n s m a l l grains. S i n c e l o w t e m p e r a t u r e is n e e d e d to i n i t i a t e


2.

Occurrence

STOLOFF

in Foods and

39

Feeds

a n d m a i n t a i n the p r o d u c t i o n of z e a r a l e n o n e ( 1 1 5 )

from m o l d , the pres

ence of m o l d is insufficient e v i d e n c e for the presence of the m e t a b o l i t e . I n the U S D A s u r v e y s of c o r n f r o m the 1967 c r o p ( 7 7 ) a n d of c o r n f o r export d u r i n g 1 9 6 8 - 1 9 6 9

( 7 8 ) , z e a r a l e n o n e w a s f o u n d i n six of the 576

samples at a n a v e r a g e l e v e l of 625 /xg/kg ( r a n g e 4 5 0 - 8 0 0 /xg/kg). I n s o m e years c o n d i t i o n s are c o n d u c i v e to Fusarium (17).

ear rot i n e p i d e m i c p r o p o r t i o n s

O n e s u c h y e a r w a s 1972. I n the s p r i n g of the f o l l o w i n g y e a r , the


F D A c o l l e c t e d s a m p l e s of c o r n at t e r m i n a l elevators s e r v i c i n g areas w h e r e t h e r e h a d b e e n s o m e e v i d e n c e of F . roseum d a m a g e (116). was found i n 1 7 %

Zearalenone

of t h e 223 samples assayed, at a n average l e v e l of

0.9 m g / k g ( r a n g e 0 . 1 - 5 . 0 m g / k g ) w i t h n o r e l a t i o n to grade or i n t e n d e d use i n c l u d i n g f o o d use. W i t h i n t h e a r e a s u r v e y e d there was a g e o g r a p h i c a l c o n c e n t r a t i o n of t h e c o n t a m i n a t i o n i n s i d e a 150 m i l e r a d i u s of t h e s o u t h e r n t i p of L a k e M i c h i g a n . Z e a r a l e n o n e c o n t a m i n a t i o n of f e e d grains is not c o n f i n e d to t h e U n i t e d States; instances of f e e d c o n t a m i n a t i o n h a v e b e e n r e p o r t e d f r o m F i n l a n d ( 1 1 7 ) , D e n m a r k (118), England

F r a n c e (119),

and

(120).

T h e c o n c e n t r a t i o n of z e a r a l e n o n e w i t h i n the c o r n k e r n e l is d i s t r i b u t e d , as e x p e c t e d , i n t h e s a m e f a s h i o n as aflatoxin (121).

O n d r y m i l l i n g of

c o n t a m i n a t e d c o r n , t h e h i g h e s t concentrations of zearalenone w e r e i n the h i g h fat fractions u s u a l l y u s e d f o r o i l a n d f e e d .

A n F D A f o l l o w - u p to

a finding of z e a r a l e n o n e - c o n t a m i n a t e d c o r n (120 /xg/kg) u s e d for p r o d u c t i o n of s t a r c h f o u n d z e a r a l e n o n e at 15 /xg/kg i n t h e starch. Ochratoxin. A l t h o u g h o c h r a t o x i n s A a n d Β a n d the e t h y l ester of ochratoxin A (ochratoxin C ) have been isolated from laboratory cultures, o n l y o c h r a t o x i n A has b e e n d e t e c t e d i n m o s t cases of n a t u r a l occurrence. T h e toxins w e r e o r i g i n a l l y i s o l a t e d f r o m strains of Aspergillus

ochraceus

f o u n d to b e t o x i n p r o d u c e r s , as p a r t of a s c r e e n i n g p r o g r a m for t o x i g e n i c m o l d s (122,

123).

Pénicillium

p r o d u c e r of o c h r a t o x i n (124,

viridicatum 125)

has also b e e n i d e n t i f i e d as a

a n d has b e e n associated w i t h the n a

t u r a l o c c u r r e n c e o f o c h r a t o x i n A i n m o s t situations w h e r e a n association could be made.

B o t h species are w i d e l y d i s t r i b u t e d a n d f r e q u e n t l y e n

c o u n t e r e d o n g r a i n s , l e g u m e s , a n d other c o m m o d i t i e s u s u a l l y p r o t e c t e d b y a r e d u c t i o n of w a t e r a c t i v i t y . A w a t e r a c t i v i t y t h a t favors the g r o w t h of P. viridicatum

o n w h e a t a n d b a r l e y (126)

is ca. 0.90, a l e v e l far h i g h e r

t h a n t h a t r e c o m m e n d e d f o r safe storage of grains a n d a h i g h e r r u n g i n the w a t e r a c t i v i t y l a d d e r t h a n t h a t f a v o r a b l e for the g r o w t h of A .

flavus

(0.84-0.86). T h e first d e t e c t i o n o f o c h r a t o x i n A as a n a t u r a l c o n t a m i n a n t w a s i n a U S D A - A R S s u r v e y o f c o r n f o r aflatoxins, ochratoxins, a n d zearalenone (77, 1 2 7 ) .

O c h r a t o x i n w a s f o u n d at 130 /xg/kg i n one S a m p l e G r a d e

s a m p l e of 283 s a m p l e s of v a r i o u s grades of c o r n r e c e i v e d f r o m

com

m e r c i a l m a r k e t s i n 1967. T h r e e m o r e samples w i t h o c h r a t o x i n (83, 119,


40

MYCOTOXINS

a n d 166 /xg/kg) w e r e e n c o u n t e r e d i n a l a t e r s u r v e y of 293 s a m p l e s of c o r n i n t e n d e d for export (78).

I n a three-year U S D A - A R S survey for

aflatoxin a n d o c h r a t o x i n i n w h e a t (291 h a r d r e d w i n t e r , 286 h a r d r e d s p r i n g , 271 soft r e d w i n t e r ) f r o m g r a d e r s ' samples ( 9 3 ) , o c h r a t o x i n w a s f o u n d i n t w o samples of h a r d r e d w i n t e r w h e a t at 25 a n d 35 /xg/kg a n d i n seven samples of h a r d r e d s p r i n g w h e a t at 2 0 - 1 1 4 /xg/kg. A l l c o n t a m i n a t e d samples w e r e i n the p o o r e r grades. Pénicillium Downloaded by UNIV OF TENNESSEE KNOXVILLE on December 21, 2014 | http://pubs.acs.org Publication Date: June 1, 1976 | doi: 10.1021/ba-1976-0149.ch002

w a s the

viridicatum

associated m o l d . B e c a u s e of reports f r o m D e n m a r k (128)

of o c h r a t o x i n i n

b a r l e y associated w i t h p o r c i n e n e p h r o p a t h y , the F D A u n d e r t o o k a s u r v e y of d o m e s t i c b a r l e y u s i n g samples s u b m i t t e d to U S D A - A M S for g r a d i n g . O c h r a t o x i n w a s f o u n d i n 1 4 % of 159 samples w i t h n o r e l a t i o n to g r a d e . H a l f the detections w e r e trace a m o u n t s ( < 1 0 / x g / k g ) ; the average l e v e l of o c h r a t o x i n i n the m e a s u r a b l e detections /xg/kg).

w a s 18 / * g / k g

(range

10-29

B e c a u s e o c h r a t o x i n h a d b e e n d e t e c t e d i n 2 2 % of 37 samples of

m a l t b a r l e y , a f o l l o w - u p s u r v e y w a s m a d e of m a l t b a r l e y a n d beer p i c k e d u p at 138 U . S . b r e w e r i e s . N o o c h r a t o x i n w a s d e t e c t e d i n either b a r l e y or beer.

E x p e r i m e n t a l D a n i s h beers m a d e f r o m o c h r a t o x i n A c o n t a m i n a t e d

barley retained approximately 4 %

of the o r i g i n a l o c h r a t o x i n ( 1 2 9 ) .

At

this rate of o c h r a t o x i n loss, b a r l e y s w i t h sufficient o c h r a t o x i n A t o r e s u l t i n d e t e c t a b l e o c h r a t o x i n i n the b e e r m a d e f r o m t h e m w o u l d b e rejected f o r m a l t i n g because of i n a d e q u a t e g e r m i n a t i o n . A D a n i s h s u r v e y of b a r l e y a n d oats (125)

provided a m u c h dif

f e r e n t p i c t u r e . O c h r a t o x i n w a s f o u n d i n 5 8 % of 3 3 samples of f e e d g r a i n s , m o s t l y b a r l e y , t a k e n i n districts e x p e r i e n c i n g a h i g h i n c i d e n c e of s w i n e n e p h r o p a t h y ; the average l e v e l w a s 3 m g / k g

(range 0.03-28

mg/kg).

O c h r a t o x i n Β i n a d d i t i o n to o c h r a t o x i n A w a s d e t e c t e d i n t w o of t h e b a r l e y samples (130).

O c h r a t o x i n was also f o u n d i n 6 % of 50 samples of

b a r l e y selected as h i g h q u a l i t y g r a i n at 9, 44, a n d 189 /xg/kg.

Subsequent

to these findings, a f a r m w a s l o c a t e d at w h i c h the g r a i n b e i n g f e d to p i g s w a s c o n t a m i n a t e d w i t h o c h r a t o x i n . R e s i d u e s of o c h r a t o x i n w e r e f o u n d at slaughter i n the k i d n e y s of 18 of 19 p i g s e x a m i n e d (131).

N o explanation

has b e e n offered for the h i g h i n c i d e n c e of o c h r a t o x i n c o n t a m i n a t i o n of D a n i s h barley. I n a C a n a d i a n s u r v e y of m o l d y feedstuffs (42)

ochratoxin was found

i n 18 of 29 samples of h e a t e d g r a i n ( w h e a t , oats, a n d r y e ) at 0 . 0 3 - 2 7 m g / k g a n d i n three of f o u r samples of d r i e d w h i t e beans at 0.02, 0.03, a n d 1.9 mg/kg.

Pénicillium

viridicatum

was consistently f o u n d to b e the m o l d

r e l a t e d to the presence of o c h r a t o x i n , as w a s also the case i n the D a n i s h studies. I n a n F D A s u r v e y of d r i e d beans for aflatoxins a n d m o l d p o p u l a t i o n , a l l samples w i t h a h i g h i n c i d e n c e of A . ochraceus ochratoxins; none were detected.

w e r e a n a l y z e d for

T h e e v i d e n c e p o i n t s to P.

viridicatum

as the u s u a l case of o c h r a t o x i n c o n t a m i n a t i o n . O c h r a t o x i n has also b e e n f o u n d i n g r e e n coffee beans ( 1 3 2 ) , i n i t i a l l y


2.

Occurrence

STOLOFF

in Foods and

41

Feeds

i n f o u r of five samples of h e a v i l y m o l d e d beans at < 2 0 - 4 0 0 xtg/kg.

The

s t u d y c o n t i n u e d w i t h a n e x a m i n a t i o n of samples f r o m 267 b a g s of beans o r i g i n a t i n g i n six countries. S a m p l e s w e r e t a k e n after o b v i o u s l y s p o i l e d beans h a d b e e n r e m o v e d .

O c h r a t o x i n w a s f o u n d i n 19 samples at a n

average of 47 /xg/kg ( r a n g e 2 0 - 3 6 0 / x g / k g ) . analyzed.

H a l f of the samples h a d b e e n

A n o t h e r 68 samples w e r e

flown

i n f r o m the c o u n t r y of

o r i g i n to r e m o v e the factor of o c e a n t r a n s p o r t ; the other h a l f w e r e l a b o r a Downloaded by UNIV OF TENNESSEE KNOXVILLE on December 21, 2014 | http://pubs.acs.org Publication Date: June 1, 1976 | doi: 10.1021/ba-1976-0149.ch002

t o r y samples of c l e a n g r e e n coffee. O c h r a t o x i n w a s f o u n d i n t w o samples at a b o u t 20 /xg/kg a n d i n one s a m p l e at 80 xtg/kg. A s u p e r f i c i a l s t u d y of the m o l d flora s h o w e d A . ochraceus

a n d u n i d e n t i f i e d species of

Penicillia.

N o a t t e m p t w a s m a d e to d e t e r m i n e the o c h r a t o x i n - p r o d u c i n g c a p a b i l i t y of a n y of t h e isolates. L a t e r at the same l a b o r a t o r y a m o r e c o m p r e h e n s i v e s t u d y of the m o l d flora of g r e e n coffee (133) P. viridicatum. A . ochraceus

f o u n d b o t h A . ochraceus

and

O c h r a t o x i n w a s p r o d u c e d i n c u l t u r e b y m o s t isolates of b u t b y n o n e of t h e isolates of P . viridicatum.

coffee destroys m u c h of the o c h r a t o x i n i n the b e a n

R o a s t i n g of

(132).

Citrinin. C i t r i n i n w a s o r i g i n a l l y i s o l a t e d f r o m Pénicillium

citrinum

as p a r t of the m o n u m e n t a l effort b y R a i s t r i c k a n d h i s c o - w o r k e r s at

(134)

the L o n d o n S c h o o l of H y g i e n e a n d T r o p i c a l M e d i c i n e to isolate a n d c h a r a c t e r i z e the m e t a b o l i c p r o d u c t s of m o l d s . P . citrinum

C i t r i n i n - p r o d u c i n g isolates of

h a v e b e e n o b t a i n e d f r o m y e l l o w - c o l o r e d r i c e of the

associated i n J a p a n w i t h toxic s y m p t o m s (11).

type

H o w e v e r , the o c c u r r e n c e

of c i t r i n i n as a c o n t a m i n a n t of feedstuffs has b e e n associated w i t h P . viridicatum

a n d a l w a y s as a c o - c o n t a m i n a n t w i t h o c h r a t o x i n .

I n the

C a n a d i a n s u r v e y of m o l d y feedstuffs p r e v i o u s l y r e f e r r e d to ( 4 2 ) , c i t r i n i n w a s f o u n d at 0 . 0 7 - 8 0 m g / k g levels i n 13 of t h e 18 samples i n w h i c h ochratoxin had been found.

T h e r e w a s n o consistent r a t i o of t h e t w o

toxins. I n the D a n i s h s u r v e y of grains associated w i t h s w i n e n e p h r o p a t h y (125),

c i t r i n i n w a s d e t e r m i n e d at 0.16, 1.0, a n d 2.0 m g / k g i n three of t h e

22 samples of b a r l e y i n w h i c h o c h r a t o x i n h a d b e e n detected. E x p e r i m e n t a l beers m a d e f r o m m a l t e d b a r l e y w i t h c i t r i n i n a d d e d at 1 m g / k g m a l t h a d n o d e t e c t a b l e c i t r i n i n ( 129). B a r l e y s n a t u r a l l y c o n t a m i n a t e d w i t h c i t r i n i n h a d i n a d e q u a t e g e r m i n a t i o n for m a l t i n g . P e n i c i l l i c A c i d . P e n i c i l l i c a c i d w a s first i s o l a t e d f r o m a m o l d c u l t u r e i n 1913 as p a r t of a s t u d y of c o r n d e t e r i o r a t i o n (135). p l e t e d 60 years l a t e r w h e n a n e w l y d e v e l o p e d

T h e cycle was com

analytical method

was

tested o n samples of c o r n c o l l e c t e d as p a r t of a n F D A s u r v e y of t h e 1972 c r o p (136).

P e n i c i l l i c a c i d w a s f o u n d i n seven of 20 r a n d o m samples

assayed at 5 - 2 3 1 /xg/kg ( a v e r a g e 59 /xg/kg).

S i n c e the f u n g i associated

w i t h the b l u e - g r e e n d i s c o l o r a t i o n of c o r n k n o w n as b l u e eye w e r e d e m o n strated p r o d u c e r s of p e n i c i l l i c a c i d (137),

the m e t h o d w a s also tested o n

48 samples of c o r n selected b y U S D A s A g r i c u l t u r a l M a r k e t i n g S e r v i c e graders as h a v i n g this d i s c o l o r a t i o n . P e n i c i l l i c a c i d w a s d e t e c t e d i n a l l


42

MYCOTOXINS

samples at 5 - 1 8 4 f i g / k g ( average 4 6 /xg/kg ) . D r i e d b e a n s a m p l e s , s e l e c t e d f r o m a n a t i o n w i d e s u r v e y b e c a u s e a h i g h p r o p o r t i o n o f seeds c o n t a i n e d v i a b l e Pénicillium

cyclopium,

w e r e also a s s a y e d ( P . cyclopium

is o n e o f

t h e b l u e eye m o l d s ) . P e n i c i l l i c a c i d w a s f o u n d i n five o f 2 0 samples tested at 1 1 - 1 7 9 /xg/kg ( a v e r a g e 82 / x g / k g ) . I n a s t u d y of m o l d species i s o l a t e d f r o m f e r m e n t e d sausage

(138),

p e n i c i l l i c a c i d w a s p r o d u c e d i n s y n t h e t i c c u l t u r e b y 4 4 o f 421 isolates a n d Downloaded by UNIV OF TENNESSEE KNOXVILLE on December 21, 2014 | http://pubs.acs.org Publication Date: June 1, 1976 | doi: 10.1021/ba-1976-0149.ch002

b y a t least one isolate i n seven o f 18 i d e n t i f i e d species i n c l u d i n g some n o t p r e v i o u s l y r e c o r d e d as p r o d u c e r s o f p e n i c i l l i c a c i d .

However,

when

d e m o n s t r a t e d p e n i c i l l i c a c i d p r o d u c e r s w e r e u s e d to f e r m e n t sausage, n o penicillic acid could be found (139).

P e n i c i l l i c a c i d a d d e d to sausage

meat could be totally recovered i f extracted i m m e d i a t e l y f o l l o w i n g a d d i t i o n , b u t after three days o n l y 5 % c o u l d b e r e c o v e r e d .

T h e loss w a s

a t t r i b u t e d to the f o r m a t i o n o f a d d u c t s w i t h c y s t e i n e o r g l u t a t h i o n e i n t h e meat. S i m i l a r observations h a v e b e e n m a d e o n t h e d i s a p p e a r a n c e of p e n i c i l l i c a c i d a d d e d to w h e a t flour (140)

w i t h the same proposed mecha

n i s m of r e a c t i o n . I n t e n t i o n a l l y f o r m e d a d d u c t s o f p e n i c i l l i c a c i d w i t h these c o m p o u n d s w e r e m a r k e d l y less t o x i c t h a n t h e p e n i c i l l i c a c i d alone. P a t u l i n . T h e n a t u r a l o c c u r r e n c e of p a t u l i n h a s b e e n associated w i t h Pénicillium

expansum

r o t i n a p p l e s (141, 142, 143)

soils a n d p l a n t s t u b b l e b y Pénicillium

urticae

a n d t h e i n v a s i o n of

(P. patulum)

Both

(144).

m o l d s are c o m m o n a n d h i g h l y c o m p e t i t i v e i n t h e i r n o r m a l e n v i r o n m e n t s . C o n c e n t r a t i o n s of p a t u l i n f o u n d i n n a t u r a l a p p l e rots h a v e b e e n as h i g h as 136 m g / k g of f r u i t (143).

T h e first o b s e r v a t i o n f o r p a t u l i n i n c o m m e r

c i a l p r o d u c t s w a s i n a l i m i t e d C a n a d i a n s u r v e y (145).

Patulin was found

at 1.0 m g / 1 . i n o n e of 12 samples o f a p p l e j u i c e . A 1971 s u r v e y of f o u r c i d e r m i l l s i n u p p e r N e w Y o r k State s h o w e d t h a t t h e presence of p a t u l i n i n t h e a p p l e juice w a s r e l a t e d to t h e i n c l u s i o n o f d e c a y e d a p p l e s w i t h t h e f r u i t g o i n g i n t o the press. N i n e of 40 samples o f j u i c e f r o m t w o m i l l s t h a t u s e d u p to 5 0 % d e c a y e d a p p l e s h a d p a t u l i n l e v e l s of 2 0 - 4 5 m g / 1 . ( 146). P a t u l i n has also b e e n i s o l a t e d f r o m five o f 21 s a m p l e s o f b a k e d goods c o v e r e d w i t h a green m o l d i d e n t i f i e d as a Pénicillium

species

(147).

B a s e d o n these observations, t h e U . S . F D A c o n d u c t e d a s u r v e y d u r i n g fiscal 1973 of a p p l e j u i c e o n t h e U . S . m a r k e t . P a t u l i n w a s d e t e c t e d i n 3 7 % of 136 samples at a n average c o n t a m i n a t i o n l e v e l o f 69 /xg/1. ( r a n g e 4 0 - 4 4 0 /xg/1.). T h e p o s s i b i l i t y of p a t u l i n o c c u r r e n c e has b e e n

demon

strated i n other fruits ( p e a c h e s , pears, a p r i c o t s , c h e r r i e s ) f o r w h i c h P . expansum

is a c o m m o n storage r o t o r g a n i s m

(148).

T h e r e are o n l y a f e w n a t u r a l substrates s u c h as a p p l e j u i c e i n w h i c h p a t u l i n has s h o w n a reasonable s t a b i l i t y (140, 149). p r o d u c i n g strains of P . expansum

Although patulin-

h a v e b e e n u s e d f o r r i p e n i n g s o m e types

of f e r m e n t e d sausage, p a t u l i n is n o t d e t e c t a b l e i n t h e (150)

finished

product

n o r is there a n y d e t e c t a b l e b i o l o g i c a l a c t i v i t y t h a t c o u l d b e r e l a t e d


2.

43

Occurrence in Foods and Feeds

STOLOFF

to p a t u l i n (151).

T h e i n a c t i v a t i o n of p a t u l i n has b e e n r e l a t e d t o its

a b i l i t y to r e a c t w i t h s u l f h y d r y l c o m p o u n d s s u c h as cysteine a n d g l u t a t h i o n e (141,

152),

s i m i l a r l y to p e n i c i l l i c a c i d .

Alcoholic fermentation

of a c o n t a m i n a t e d j u i c e w i l l also e l i m i n a t e p a t u l i n ( 1 5 2 ) . Conclusion Information on mycotoxin incidence should be interpreted cautiously. Downloaded by UNIV OF TENNESSEE KNOXVILLE on December 21, 2014 | http://pubs.acs.org Publication Date: June 1, 1976 | doi: 10.1021/ba-1976-0149.ch002

I n c i d e n c e d a t a c a n t e l l o n l y w h a t has h a p p e n e d i n t h e past a n d a p p l i e s o n l y to t h a t cross-section of t h e c o m m o d i t y r e p r e s e n t e d b y t h e s a m p l e . U n l e s s i n f o r m a t i o n is i n c l u d e d o n h o w t h e c o n t a m i n a t i o n o c c u r r e d , t h e r e is n o basis f o r f u t u r e projections. H o w e v e r , some i d e a of t h e p a s t is n e e d e d to d e t e r m i n e h o w m u c h effort to p u t i n t o t h e f u t u r e . O f t e n this effort c o n sists i n d e t e r m i n i n g t h e m a g n i t u d e o f t h e t o x i c o l o g i c a l r i s k f r o m t h e levels of t o x i n e n c o u n t e r e d since r e l i a b l e i n f o r m a t i o n o n t h e effects o f c h r o n i c h u m a n exposure to these toxins is p r a c t i c a l l y nonexistent. Literature

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77. Shotwell, Ο. L., Hesseltine, C. W., Goulden, M . L., Vandegraft, Ε. E., "Survey of Corn for Aflatoxin, Zearalenone, and Ochratoxin," Cereal Chem. (1970) 47, 700-707. 78. Shotwell, O. L., Hesseltine, C. W., Vandegraft, Ε. E., Goulden, M . L., "Survey of Corn from Different Regions for Aflatoxin, Ochratoxin, and Zearalenone," Cereal Sci. Today (1971) 16, 266-273. 79. Shotwell, O. L., Hesseltine, C. W., Goulden, M . L., "Incidence of Afla toxin in Southern Corn, 1969-1970," Cereal Sci. Today (1973) 18, 192-195. 80. Shotwell, O. L., Goulden, M. L., Hesseltine, C. W., "Aflatoxin Contamina tion: Association with Foreign Material and Characteristic Fluorescence in Damaged Corn Kernels," Cereal Chem. (1972) 49, 458-465. 81. Velasco, J., "Detection of Aflatoxin Using Small Columns of Florisil," J. Amer. Oil Chem. Soc. (1973) 49, 141-142. 82. Hunt, W. H., Liebe, E. B., Velasco, J., "Incidence of Aflatoxin in Corn and a Field Method for Aflatoxin Analysis," Cereal Chem., submitted for publication, 1975. 83. Shotwell, O. L., Goulden, M. L., Hesseltine, C. W., "Aflatoxin: Distribu tion in Contaminated Corn," Cereal Chem. (1974) 51, 492-499. 84. Shotwell, O. L., Goulden, M. L., Jepson, A. M., Kwolek, W. F., Hesseltine, C. W., "Aflatoxin Occurrence in White Corn Under Loan, 1971. III. Association with Bright Greenish-Yellow Fluorescence in Corn," Cereal Chem., in press, 1975. 85. Lillehoj, Ε. B., Kwolek, W. F., Fennell, D. I., Milburn, M . S., "Bright Greenish-Yellow Fluorescence and Aflatoxin in Freshly Harvested HighMoisture Corn from Southern Illinois and Southeastern Missouri," Cereal Chem. (1975) 52, 403-412. 86. Lillehoj, Ε. B., Kwolek, W. F., Shannon, G. M., Shotwell, O. L., Hessel tine, C. W., "Aflatoxin Occurrence in 1973 Field Corn: A Limited Survey in the Southeastern U.S.," Cereal Chem., in press, 1975. 87. Knake, R. P., Deyoe, C. W., "Production of Aflatoxin by Aspergillus parasiticus in Maturing White Corn," Poultry Sci. (1973) 52, 2049. 88. Rambo, G., Tuite, J., Crane, P., "Preharvest Inoculation and Infection of Dent Corn Ears with Aspergillusflavusand A. parasiticus," Phyto pathology (1974) 64, 797-800. 89. Ullola-Sosa, M., Schroeder, H . W., "Note on Aflatoxin Decomposition in the Process of Making Tortillas from Corn," Cereal Chem. (1969) 46, 397-400. 90. Yahl, K. R., Watson, S. Α., Smith, R. J., Barabolok, R., "Laboratory Wet Milling of Corn Containing High Levels of Aflatoxin and a Survey of Commercial Wet-Milling Products," Cereal Chem. (1971) 48, 385-391. 91. Brekke, O. L., Peplinski, A. J., Nelson, G. Ε. N., Griffin, E. L., Jr., "Pilotplant Dry Milling of Corn Containing Aflatoxin," Cereal Chem. (1975) 52, 205-211. 92. Shotwell, O. L., Hesseltine, C. W., Burmeister, H . R., Kwolek, W. F., Shannon, G. M., Hall, Η. H., "Survey of Cereal Grains and Soybeans for the Presence of Aflatoxin: I. Wheat, Grain Sorghum, and Oats," Cereal Chem. (1969) 46, 446-454. 93. Shotwell, O. L., Goulden, M. L., Fennell, D. I., Hesseltine, C. W., "Survey of Wheat for Aflatoxin and Ochratoxin," J. Agr. Food Chem., sub mitted for publication, 1975. 94. Shank, R. C., Gordon, J. E., Wogan, C. N., Nondasuta, Α., Subhamani, B., "Dietary Aflatoxins and Human Liver Cancer. III. Field Survey of Rural Thai Families for Ingested Aflatoxins," Food Cosmet. Toxicol. (1972) 10, 71-84.


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95. Hesseltine, C. W., Shotwell, O. L., Ellis, J. J., Stubblefield, R. D., "Afla toxin Formation by Aspergillusflavus,"Bacteriol. Rev. (1966) 30, 795-805. 96. Scott, P. M., Kennedy, B. P. C., "Analysis and Survey of Ground Black, White, and Capsicum Peppers for Aflatoxins," J. Ass. Off. Anal. Chem. (1973) 56, 1452-1457. 97. Schuller, P. L., Ockuizen, Th., Werringloer, J., Marquardt, P., "Aflatoxin B und Histamin in Wein," Arzneim. Forsch. (1967) 17, 888-890. 98. Drawert, F., Barton, H., "Zun Nachweis von Aflatoxinen in Wein," Z. Lebensm. Unters. Forsch. (1974) 154, 223-224. 99. Takahashi, D. M., "Thin Layer Chromatographic Determination of Afla toxins in Wine," J. Ass. Offic. Anal. Chem. (1974) 57, 875-879. 100. "Report on a Surveillance Program—Aflatoxins in Milk Products," FDA Bureau of Foods, 1973. 101. Purchase, I. F. H., Vorster, L. J., "Aflatoxin in Commercial Milk Samples," S. Afr. Med. J. (1968) 42, 219. 102. Neumann-Kleinpaul, Α., Terplan, G., "Zum Vorkommen von Aflatoxin M in Trockenmilchprodukten," Arch. Lebensmittelhyg. (1972) 23, 128131. 103. Jung, M., Hanssen, E., "Uber das Vorkommen von Aflatoxin M in Trockenmilcherzeugnissen," Food Cosmet. Toxicol. (1974) 12, 131138. 104. van der Linde, J. Α., Frens, A. M., van Esch, C. J., "Experiments with Cows Fed Groundnut Meal Containing Aflatoxin," in "Mycotoxins in Foodstuffs," G. Wogan, Ed., pp. 247-249, MIT Press, Cambridge, Mass., 1965. 105. Polan, C. E., Hayes, J. R., Campbell, T. C., "Consumption and Fate of Aflatoxin B by Lactating Cows," J. Agr. Food Chem. (1974) 22, 635638. 106. McKinney, J. D., Cavanagh, G. C., Bell, J. T., Hoversland, A. S., Nelson, D. M., Pearson, J., Selkirk, R. J., "Effects of Ammoniation on Aflatoxins in Rations Fed Lactating Cows," J. Amer. Oil Chem. Soc. (1973) 50, 79-84. 107. Masri, M . S., Garcia, V. C., Page, J. R., "The Aflatoxin M Content of Milk from Cows Fed Known Amounts of Aflatoxin," Vet. Rec. (1969) 84, 146-147. 108. Allcroft, R., Roberts, Β. Α., "Toxic Groundnut Meal: The Relationship Between Aflatoxin B Intake by Cows and Excretion of Aflatoxin M in Milk," Vet. Rec. (1968) 82, 116-118. 109. Mirocha, C. J., Christensen, C. M., Nelson, G. H., "Estrogenic Metabolite Produced by Fusarium graminearum in Stored Corn," Appl. Microbiol. (1967) 15, 497-503. 110. Caldwell, R. W., Tuite, J., Stob, M., Baldwin, R., "Zearalenone Produc tion by Fusarium Species," Appl. Microbiol. (1970) 20, 31-34. 111. Mirocha, C. J., Christensen, C. M., Nelson, G. H., "Biosynthesis of the Fungal Estrogen F-2 and a Naturally Occurring Derivative (F-3) by Fusarium moniliforme," Appl. Microbiol. (1969) 17, 482-483. 112. Caldwell, R. W., Tuite, J., "Zearalenone Production in Field Corn in Indiana," Phytopathology (1970) 60, 1696-1697. 113. Caldwell, R. W., Tuite, J., "Zearalenone in Freshly Harvested Corn," Phytopathology (1974) 64, 752-753. 114. Koehler, B., "Fungus Growth in Shelled Corn as Affected by Moisture," J. Agr. Res. (1938) 56, 291-307. 115. Mirocha, C. J., Christensen, C. M., Nelson, G. H., "F-2 (Zearalenone) Estrogenic Mycotoxin from Fusarium," in "Microbial Toxins," Vol. VII, S. Kadis, A. Ciegler, S. A. Ajl, Eds., pp. 107-138, Academic Press, New York, 1971.


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116. Eppley, R. M., Stoloff, L., Trucksess, M . W., Chung, C. W., "Survey of Corn for Fusarium Toxins," J. Ass. Offic. Anal. Chem. (1974) 57, 632635. 117. Roine, K., Korpinen, E-L., Kallela, K., "Mycotoxicosis as a Probable Cause of Infertility in Dairy Cows," Nord. Vet. Med. (1971) 23, 628633. 118. Erikson, E., "Estrogenic Factors in Moldy Grain," Nord. Vet. Med. (1968) 20, 396-401. 119. Jemmali, M., "Presence dun Facteur Oestrogenique d'Origine Fongique la Zearalenone ou F-2, comme Contaminant Naturel, dans du Mais," Ann. Microbiol. (Inst. Pasteur) (1973) 124B, 109-114. 120. Mirocha, C. J., Harrison, J., Nichols, Α. Α., McClintock, M., "Detection of a Fungal Estrogen (F-2) in Hay Associated with Infertility in Dairy Cattle," Appl. Microbiol. (1968) 16, 797-798. 121. Bennett, G. Α., Peplinski, A. J., Brekke, O. L., Jackson, L., Wischer, R., "Distribution of Zearalenone in Dry Milled Fractions of Contaminated Corn," Cereal Chem., submitted for publication, 1975. 122. Scott, De Β., "Toxigenic Fungi Isolated from Cereal and Legume Prod ucts," Mycopath. Mycolog. (1965) 25, 213-222. 123. van der Merwe, K. J., Steyn, P. S., Fourie, L., Scott, De Β., Theron, J. J., "Ochratoxin A, a Toxic Metabolite Produced by Aspergillus ochraceus Wilh," Nature (1965) 205, 1112-1113. 124. van Walbeek, W., Scott, P. M., Harwig, J., Lawrence, J. W., "Penicillium viridicatum Westling: a New Source of Ochratoxin A," Can. J. Micro biol. (1969) 15, 1281-1285. 125. Krogh, P., Hald, B., Pedersen, J., "Occurrence of Ochratoxin A and Citrinin in Cereals Associated with Mycotoxic Porcine Mephropathy," Acta. Path. Microbiol. Scand. (1973) B81, 689-695. 126. Harwig, J., Chen, Y.-K., "Some Conditions Favoring Production of Ochra toxin A and Citrinin by Penicillium viridicatum in Wheat and Barley," Can. J. Plant Sci. (1974) 54, 17-22. 127. Shotwell, O. L., Hesseltine, C. W., Goulden, M. L., "Ochratoxin A: Occur rence as a Natural Contaminant of a Corn Sample," Appl. Microbiol. (1969) 17, 765-766. 128. Krogh, P., Royal Veterinary and Agricultural University, Denmark, 1971, personal communication. 129. Krogh, P., Hald, B., Gjertsen, P., Myken, F., "Fate of Ochratoxin A and Citrinin During Malting and Brewing Experiments," Appl. Microbiol. (1974) 28, 31-34. 130. Krogh, P., Hald, B., 1974, personal communication. 131. Hald, B., Krogh, P., Ochratoxin Residues in Bacon Pigs. Abstr. IUPACsponsored symp. "Control of Mycotoxins," Goeteborg, Sweden (1972) 18. 132. Levi, C. P., Trenk, H. L., Mohr, H. K., "Study of the Occurrence of Ochratoxin A in Green Coffee Beans," J. Ass. Offic. Anal. Chem. (1974) 57, 866-870. 133. Trenk, H., General Foods Technical Center, 1974, personal communica tion. 134. Hetherington, A. C., Raistrick, H., "Biochemistry of Microorganisms XIV. Production and Chemical Constitution of a New Yellow Coloring Matter, Citrinin, Produced from Dextrose by Penicillium citrinum Thom," Trans. Royal Soc. (London) (1931) B220, 269-296. 135. Alsberg, C. L., Black, O. F., "Contributions to the Study of Maize De terioration," U.S. Dept. Agr., Bur. Plant Ind. Bull. (1913) 270, 7-48. 136. Thorpe, C. W., Johnson, R. L., "Analysis of Penicillic Acid by Gas-Liquid Chromatography," J. Ass. Offic. Anal. Chem. (1974) 57, 861-865.



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137. Ciegler, Α., Kurtzman, C. P., "Penicillic Acid Production by Blue-Eye Fungi on Various Agricultural Commodities," Appl. Microbiol (1970) 20, 761-764. 138. Ciegler, Α., Mintzlaff, H-J, Machnik, W., Leistner, L., "Untersuchungen uber das Toxinbidungsvermogen von Rohwursten Isolierter Schimmelpilze der Gattung Penicillium," Fleischwirtschaft (1972) 52, 13111318. 139. Ciegler, Α., Mintzlaff, H-J., Weisleder, D., Leistner, L., "Potential Pro duction and Detoxification of Penicillic Acid in Mold-Fermented Sau sage (Salami)," Appl. Microbiol. (1972) 24, 114-119. 140. Scott, P. M., Somers, E., "Stability of Patulin and Penicillic Acid in Fruit Juices and Flour," J. Agr. Food Chem. (1968) 16, 483-485. 141. Brian, P. W., Elson, G. W., Lowe, D., "Production of Patulin in Apple Fruits by Penicillium expansum," Nature (1956) 178, 263-264. 142. Walker, J. R. L., "Inhibition of the Apple Phenolase System through In fection by Penicillium expansum" Photochemistry (1969) 8, 561-566. 143. Harwig, J., Chen, Y-K., Kennedy, B. P. C., Scott, P. M., "Occurrence of Patulin and Patulin-Producing Strains of Penicillium expansum in Nat ural Rots of Apple in Canada," Can. Inst. Food Sci. Technol. J. (1973) 6, 22-25. 144. Norstadt, F. Α., McCalla, T. M., "Microbially Induced Phytotoxicity in Stubble-Mulched Soil," Soil Sci. Soc. Amer. Proc. (1968 ) 32, 241-245. 145. Scott, P. M., Miles, W. F., Toft, P., Dubé, J., "Occurrence of Patulin in Apple Juice," J. Agr. Food Chem. (1972) 20, 450-451. 146. Wilson, D. M., 1974, private communication. 147. Reiss, J., "Nachweis von Patulin in Spontan Verschimmeltem Brot und Geback," Naturwissenschaften (1972) 59, 37. 148. Buchanan, J. R., Sommer, N . F., Fortlage, R. J., Maxie, Ε. C., Mitchell, F. G., Hsieh, D. P. H., "Patulin from Penicillium expansum in Stone Fruits and Pears," J. Amer. Soc. Hort. Sci. (1974) 99, 262-265. 149. Pohland, A. E., Allen, R., "Stability Studies with Patulin," J. Ass. Offic. Anal Chem. (1970) 53, 688-691. 150. Alperden, L, Mintzlaff, H-J., Tauchmann, F., Leistner, L., "Untersuch ungen uber die Bildung des Mykotoxins Patulin in Rohwurst," Fleisch wirtschaft (1973) 53, 566-568. 151. Hofmann, K., Mintzlaff, H-J., Alperden, I., Leistner, L., "Untersuchung uber die Inaktivierung des Mykotoxins Patulin durch Sulfhydrylgruppen," Fleischwirtschaft (1971) 51, 1534-1536. 152. Harwig, J., Scott, P. M., Kennedy, B. P. C., Chen, Y-K., "Disappearance of Patulin from Apple Juice Fermented by Saccharomyces spp," J. Inst. Can. Sci. Technol. Aliment. (1973) 6, 45-46. RECEIVED November 8,

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