appl. environ. microbiol. 1985 tong 1407 11
TRANSCRIPT
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8/16/2019 Appl. Environ. Microbiol. 1985 Tong 1407 11
1/5
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 1
0 0 9 9 - 2 2 4 0 / 8 5 / 0 6 1 4 0 7 - 0 5 0 2 . 0 0 / 0
C 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 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 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 m
CHAO 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 1
R 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 s
N 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 n
b 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 7
m 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 m
s 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 .
Sodium
p 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 s
t 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 e
h 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 d
t 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 g
o 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 m
b 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 d
m 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 d
P 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 n
a 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 e
t 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 n
s 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 s
o 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 y
a 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
a
f 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 d
i 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 d
b 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 n
k 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 y
a 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 y
o 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 s
k 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
carcass
may 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
A
f 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 f
o 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 e
i 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 d
o 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 0
m 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 d
a 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
ammonia
s 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
A
p 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
AND
METHODS
T 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
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 ,
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
were
i 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 )
agar
s 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 ,
and
s 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 7
f 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 l
E 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 4
d 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 d
t 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 l
s 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 e
m 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 s
a 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 d
i 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 .
Other
a 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 f
a 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 ° C
u 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 .
When
1 4 0 7
V o l . 4 9 , N o . 6
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8/16/2019 Appl. Environ. Microbiol. 1985 Tong 1407 11
2/5
1 4 0 8 TONG AND DRAUGHON
t 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 e
pH
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 g
v 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 e
f 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 d
or
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 n
were 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
were
s 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
was
e 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 y
f 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 a
f 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 n
c 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
a
0 . 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 d
q 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
M6000
pu 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 0
f 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 m
e 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 l
c 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 y
p 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 .
An
e 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 y
o 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 s
f 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 e
c 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 was
h 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 h
s 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 y
b 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 h
c 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 d
Whatman 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 d
w 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 e
a 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 t
was
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
were
p 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 s
o 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 .
When
s 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 e
r 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 s
among
mean
v a l u e s .
R E S U L T S AND
D I S C U S S I O N
M 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 m
C o n c n
o f
C o e f f i c i e n t
s t a n d a r d Mean
p e a k
a r e a o f
s 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 4
0 . 0 5 1 7 8 . 3 + 4 . 2 3 4 2 . 3 8
0 . 0 1
1 2 4 . 2 ± 7 . 4 3 5 5 . 9 9
0 . 0 0 5
1 3 0 . 3
+
6 . 8 8 5
5 . 2 8
0 . 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 t
s 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 g
t 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 n
t 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 s
w 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 . 5
a 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 m
p 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 n
o 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 e
w 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 s
n 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 s
f 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 s
5 . 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 s
a 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 d
o 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 f
m 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 s
w 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
pH
v 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 l
m 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 n
e 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 s
l 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
pH
5 . 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 e
i 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 5
a 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 d
1 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 0
i 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 e
w 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 l
w 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 o
1 , 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 d
a 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 s
t 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 e
u 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 m
p 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 t
pH
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 6
c 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 t
0 . 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 h
o 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 n
A .
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 e
m 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
A P P L .
E N V I R O N . M I C R O BI O L .
-
8/16/2019 Appl. Environ. Microbiol. 1985 Tong 1407 11
3/5
AN T I M I C R OBIA L INHIBITION O F OCHRATOXIN 1 4 0 9
o P .
v i r i d . , pH 5.5
P .
V i r i d . ,
p H
4 . 5
A .
s u l p h . ,
pH
5 . 5
O A .
s u l p h . ,
pH 4.5
z
I.
4 c
-i
4 c
j
LA J
u
. u z . 0 4 .0 6
. 0 8
. 1 0
S OD IUM
BISULFITE
CONCENTRATION
( % )
2 . 0
1 . 8
1 . 6
1.4
E
1 . 2
1.0
Q
0 . 8
0 . 6
0 . 4
0 . 2
0
0
1 2
.2 4 . 3 6 . 4 8 . 6 0
S OD IUM
P R O P I O N A T E C O N C E N T R A T I O N ( S )
0 . 0 2 . 0 4
. 0 6 .0 8
P OTA SS IUM S O RBA T E C O N
0
0
o
P .
v i r i d . ,
p H 5 . 5
P .
v i r i d . ,
p H
4 . 5
A .
s u l p h. , p H
5 . 5
O A .
s u l p h . , p H
4 . 5
. 1 0 . 1 2 . 1 4
CENTRATION
5)
P .
v i r i d . ,
pH
5 . 5
P . v i r i d . , pH 4 . 5
A .
s u l p h . .
pH 5 . 5
A .
s u l p h . ,
pH
4 . 5
. 0 1
. 0 2 . 0 3 . 0 4 . 0 5 . 0 6 . 0 7
M E T HYL P AR A BE N
C O NC E NT R AT ION
( S )
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 n
p 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 1
3 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 t
t 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 f
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 on 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 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 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 s
r 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 e
two
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 d
i 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 e
amou 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 f
s 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 t
t 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 s
o 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
was
a 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 e
excess 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 s
ca 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 s
m 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 n
s 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 t
t 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 t
d 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
a
r 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 o
f 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 n
were
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 r
m 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 e
f 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 ,
HPLC
q 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 e
HPLC
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 and
B 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
under
r 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 HPLC
d 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 e
v 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
2 . 7 5
2 . 5 0
2 . 2 5
2 . 0 0
E
1 . 7 5
< 1 . 5 0
I 1 . 2 5
X 1 . 0 0
0 . 7 5
0 . 5 0 -
0 . 2 5 -
0
2 . 0
1 . 8
1 . 6
1 . 4 -
1 . 2
1 . 0 -
- J
0 8 '
, 0 . 6
E
0 . 4
V O L .
4 9 ,
1 9 8 5
-
8/16/2019 Appl. Environ. Microbiol. 1985 Tong 1407 11
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1 4 1 0
TONG
AND DRAUGHON
i 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 f
o 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 n
f 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 l
f 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 n
YE 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 n
g 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 d
a 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 e
o 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 r
p 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 n
were
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 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 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 e
p 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
was
d 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 e
a 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 s
e 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 m
b 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 e
i 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
A
p 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 d
a 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 f
A 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 ( )
4.5 5.5
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 7
0 . 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 3
0 . 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 b
0 . 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 b
0 . 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 t
s 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 n
p 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
A
p 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 d
a t
2 8 ° C f o r 2
w e e k s
Mean
t o x i n
l e v e l
±
S D
t i g / 1 0 0
m l
o f
A 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 b
0 . 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 b
0 . 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 c
0 . 1 0
5
±
2 . 0 c ( 6 9 )
3 4
±
2 4 . 0
0 . 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 b
0 . 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 t
s 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 n
p 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 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
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 t
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 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 y
s 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 l
a 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 f
i 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 . 5
a 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 d
s 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 s
w 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 h
t 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 d
d 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 e
a 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 d
w 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 D
1 .
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 1
3 .
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 n
o 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 c
m 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 in
A .
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 f
o 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 c
e 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 l
e 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 l
f 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 n
A . 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 s
f 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 a
s 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 y
G . 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 y
o 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 d
p 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 f
P 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 d
i 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 f
P 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 d
t 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 f
o 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 f
w 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 e
t 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 d
J . 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 d
b 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 n
o 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 d
C h e m . 2 2 : 5 3 6 - 5 3 7 .
V O L .
4 9 ,
1 9 8 5