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A m m o n i a K i l l s S p o i l a g e M o l d s i n C o r nR . J . B O T H A S T , E . B . L A N C AS T E R , a n d C . W . H E S S E L T I N EN o r t h e r n R e g io n a l R e s e ar ch L a b o r a t o r y , A R S , U S D A

P e o r i a , I l l i n o i s 6 1 6 0 4

A b s t r a c tCorn con ta in ing 26% and 12% mo is tu rewe re t rea t ed wi th amm onia a t 2?o and .5?oo f c o r n d r y w e i g h t , r e s p e c t i v e l y . A m -m o n i a t i o n a t b o t h c o n c e n t r a t i o n s e l i m i -n a t e d e x t e r n a l a n d i n f e c t i n g m o l d s a n dy e a s t s a n d t e n d e d t o r e d u c e b a c t e r i a lcounts . Molds k i l l ed were spec ies o f

AspergiUus, Penicillium, Fusarium, Trich-oderma, and Rhizopus. In feed ing t r i a l s ,m i c e s h o w e d n o p r e f e r e n c e b e t w e e n t e m -p e r e d a n d 2 ~ a m m o n i a t e d c o r n t h a t i n i -t i a l l y h a d a p u n g e n t o d o r , b u t m i c e c o n -s u m e d m o r e c o n t r o l c o r n t h a n c o r n a m -monia ted a t .5%.I n t r o d u c t i o n

S t u d i e s a t t h e N o r t h e r n R e g i o n a l R e s e a r c hL a b o r a t o r y ar e b e i n g c o n d u c t e d t o d e s t r o ya f l a t o x i n i n n a t u r a l l y c o n t a m i n a t e d c o r n u s i n ga q u e o u s o r g a s e o us a m m o n i a . P r e s e r v a t io n a n ds t o r a g e o f h i g h - m o i s t u r e c o r n h a v e c o n c e r n e dt h e f a r m e r a n d e l e v a t o r o p e r a t o r . C o n c e r n h a si n t e n s i f i e d w i t h a d v e n t o f t h e p i c k e r - s h e l l e rand a r t i f i c ia l d ry ing fac i l i t i e s , which haves t i m u l a t e d h a r v e s t i n g h i g h - m o i s t u r e c o r n ( 2 0to 35% mois tu re ) to reduce f ie ld losses . Corna t these mois tu res i s sub jec t to mold spo i lagea n d m y c o t o x i n fo r m a t i o n u n l e ss i t is r a p i d l ydr ied . (An es t imated 8 .8 b i l l ion l i t e r s o f corni s l o s t i n t h e U n i t e d S t a t e s e v e r y y e a r ( 5 ) . )M e t h o d s u s e d t o p r e v e n t m o l d g r o w t h i n c l ud ed r y i n g , l o w - t e m p e r a t u r e s t o r a g e , r e d u c e d o x y -g e n c o n t e n t o f s t o r a g e a t m o s p h e r e , a n d a p p l y -ing o rgan ic ac ids (ace t ic , p rop ion ie , fo rmic ,a n d s o r b i c ) . H o w e v e r , t h e f u n g i c i d a l p r o p e r -t i es o f a m m o n i a o n c o r n h a v e n e v e r b e e n i n -ves t iga ted .B o t t i n i ( 2 ) r e p o r t e d t h a t a m m o n i a g a s p r e -v e n t e d d e c a y o f c i tr u s f r u it s . S i n ce , t h e r e h a sb e e n m u c h r e s e a r c h o n f u m i g a t i o n o f c i t r u sf r u i t s w i t h a m m o n i a t o c o n t r o l s u c h m o l d s a sPen ic~llium funiculosum , P. italicum , P.digi ta tum, Rhizopus n igr icans , a n d Diplodianatalensis (6, 10, 11, 12, 14, 17, 18, 21, a n d2 2 ) . Despi te e f fe c t iv e n e s s o f a m m o n i a f u m i g a -

Received for publication September 6, 1972.

t i o n t h e t r e a t m e n t h a s n e v e r b e e n p o p u l a r b e -cause i t i s d i f f i cu lt to con t ro l am m onia gen-e ra t ion , and sens i t ive c i t rus f ru i t s show dark-e n e d s k i n a n d i n j u r e d s t e m e n d s ( 7 ) . E n o e ta l. ( 8 ) r e d u c e d p o p u l a t i o n s o f f u n g i , b a c t e r i a ,a n d n e m a t o d e s i n F l o r i d a s a n d w i t h a n h y d ro u sammonia . Nea l e t a l . (16) s ta ted tha t NH~rap id ly k i l l ed sc le ro t ia o f Phymatotrichumomnivorum, M c C a l l a n a n d S e t t e r s t r o m ( 1 5 )d e m o n s t r a t e d t h a t N H 3 i s t o x i c t o m a n y f u n g i ,and o thers repor ted s imi la r resu l t s (3 , 4 , 9 , 19 ,2 3 ) . T h e r e f o r e , o u r r e s e a r c h w a s t o d e t e r m i n ethe feas ib i l i ty o f us ing ammonia as a fung ic ideon she l led corn .M a t e r i a l s a n d M e t h o d s

N i n e k i l o g r a m s o f U . S . G r a d e 5 w h i t e c o r n( 1 2% m o i s t u r e ) f r o m t h e b o o t h e e l a r ea o fMissouri were sealed in a 38-l i ter , s ter i le , s ta in-l e s s s t e e l d r u m . C o r n w a s t e m p e r e d t o 2 6 ~mois tu re wi th s te r i l e wate r a t 26 C fo r 24 h r ,a n d 2 . 0 % N H s ( w / w ) w a s a d d e d a s a m -m o n i u m h y d r o x i d e t o b r i n g f in a l m o i s t u r e c o n -t e n t o f g r a i n t o 3 0% . A m m o n i a t e d c o r n w a sh e l d 1 4 d a y s a t t h e s a m e t e m p e r a t u r e . S a m -p l e s o f o r i g in a l c a m , c o r n a f t e r 2 4 h r o f t e m p e r -ing , I h r a f te r ammor t ia f ion , and 14 days a f te ra m m o n i a t io n w e r e r e m o v e d f o r m i cr o b io l o g i-ca l examina t ion . Ana lyses fo r to ta l ae rob ic bac-te r ia , molds and yeas t s , and in fec t ing o r in -

l O , O O O , O 0 01 , 0 0 0 , 0 0 0E Bacteria

lO 0 , O 0 0 ~ I I' ~ 1 0 . 0 0 0. _ ~ 1 0 0 0

1 0 0 ~ M e l d s & Yeasts" , , T r i a l s l ~ Olo " , ,

0 L

Fzc. 1. T he Srdluence of applying 2~ ammonia t ohigh-moisture co rn on m icrobial growth a~ter 24 h rof tempering. Fungi (dotted line) were eliminated;bacteria (solid line) did not proliferate.241

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242 BOTHAST ET ALt e rna l fung i were the p rocedures o f Bo thas t e taI . (1) . In a secon d experiment , repl icatet rea tments were made wi th 2 .3 kg o f o r ig ina lcorn t r ea ted w i th .5% NH~ ( w /w ) as am-m o n i u m h y d r o x i d e a n d s a m p l e d a f t er 1 h rand af ter 14 days. The f i rs t exper iment wasrepeated on this smaller scale .A m m o n i a t e d c o r n w a s f e d t o m i c e in t w o1-wk pre fe rence t ri al s . Twe lve m ice (4 to 8 wkold , n ine males an d th ree females ) were pu tin four cages ( th ree mice pe r cage , same sexin each cag e) and g iven a da i ly cho ice o f 50kerne l s o f ammonia ted corn f rom the f i r s t ex -per im ent an d 50 ke rnel s o f un t re a ted t emp ered

corn in separa te con ta ine rs . N um ber o f ke rne l sc o n s u m e d p e r d a y f r o m e a c h c o n t a in e r w a s r e -corded . Ana lys i s o f va r iance and ch i squaretest s (20) de te rmin ed t re a tmen t d i f fe rences .Th e seco nd t r ia l was ident ica l to the f i rst ex-cep t fo r the mice (4 to 8 wk o ld , s ix males ands ix females ) an d the feed ( .5~ amm onia te dand or iginal 12% moisture corn ) .Results

Effec t o f t emp er ing and subsequ en t e f fec tof 2 .0~ NH 3 on the microf lora of corn are inFig. 1 . The or iginal corn contained 2 .0 X 10~

FIG. 2. Mold growth from surface-sterilized corn kernels after 5 to 7 days of incubation at 28 C.A, corn conta ining 12~ moisture an d n ot treate d w ith amm onia; B, corn tempe red to 26~ moisture; a nd(3, application of .5% amm onia which destroyed fungi and preven ted germination. Same results with2~ ammonia.JOURNAL OF DAI RY SCIENCE VOL. 56, No . 2

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AMMONIA AND CORN MOLD 2 4 3b a c t e r i a p e r g r a m a n d 7 .9 X 1 04 m o l d s a n dy e a s t s p e r g r a m . F r o m t e n t a t iv e i d e n ti f ic a t io no f e a c h m o l d c o l o n y o n d i l u t i o n p l a t e sc o u n t e d , s p e c i e s o f F u s a r i u m c o m p o s e d 7 5 7 oo f e x t e rn a l m o l d p o p u l a t i o n ; t h e r e m a i n d e rw a s p r e d o m i n a n t l y A s p e r g i U u s f l a v u s a n ds p e c i e s o f Penic i l l i um. A f t e r 2 4 h r o f te m p e r -i n g t o 2 6 % m o i s t u r e , b a c t e r i a i n c r e a s e d t o 1 . 4X 1 0 r p e r g r a m a n d m o l d s a n d y e a s t s i n c r e a s e dt o 5 . 1 X 1 0 5 p e r g r a m . B u t 1 h r a f t e r 2 $ N H sw a s a d d e d , m o l d s a n d y e a s t s w e r e e l i m i n a t e da n d b a c t e r i a l c o u n t s r e d u c e d t o 1 . 8 X 1 04 p e rg r a m . A f t e r 2 w k o f a m m o n i a t e d s t o r ag e , n ov i a b l e m o l d s a n d y e a s t s w e r e e v i d e n t , a n db a c t e r i a l c o u n t s w e r e f u r t h e r r e d u c e d t o 9. 7X 1 0 a p e r g r a m . T e n t a t i v e i d e n ti f i ca t i o n i n d i -c a t e d t h a t s p e c i e s o f Baci l lus s u r v i v e d a m -m o n i a t r e a t m e n t .S u r f a c e - s t e r i l i z e d o r i g i n a l , t e m p e r e d , a n dt r e a t e d c o r n s a m p l e s p l a t e d o n m a l t e x t r a c ta g a r ( 3 . 0 g m a l t e x t r a c t a n d 1 .5 % a g a r ) a r es h o w n in F i g . 2 . F u n g i t h a t g r e w f r o m t h e sek e r n e l s a r e v i a b l e i n t e r n a l m o l d s . T h e n u m b e ra n d p e r c e n t o f m o l d s i n f e c t i n g t h e s e s a m p l e sa r e i n T a b l e 1 . A . f l avus , s p e c i e s o f F u s a r i u m ,a n d A . n i g e r i n fe c t ed 4 3 .1 , 2 3 .5 , an d 1 9.07 oo f t h e o r i g in a l c o rn . H o w e v e r , d u r i n g t e m p e r -i n g t h e f a s t e r g r o w i n g m o l d s w e r e f a v o r e d ;c o n s e q u e n t l y , s p e c i e s o f T r i c h o d e r m a a n dR h i z o p u s w e r e i n a s i g n i f ic a n t p o r t i o n o fk e r n e l s . A t e i t h e r 1 h r o r 2 w k a f t e r a m m o n i at r e a t m e n t , n o i n t e r n a l m o l d s w e r e vi a b le .T h e r e w a s e v i d e n c e o f b a c t e r i a l g r o w t h o n af e w a m m o n i a t e d k e r n e l s , b u t z o n e s o n t h ep e r i p h e r y o f k e r n e ls ( F i g . 2 C ) a r e n o t b a c -t e ri a l g r o w t h b u t a p r e c ip i t a t e in t h e m e d i u m .P e r h a p s a m m o n i a d i f f u s e d i n t o t h e m e d i u m

100,000E~ lO ,OOO.~ 1000

lO 0

z0 IJ

\- \ BacteriaTr ia ls A & B\

\ Molds & Yeasts T r i a l s A & B

FIG. 3. The influence on microbial growth oft rea t ing 12% -mois ture corn wi th .5% ammonia .Trea tmen t e l imin a t ed fu n g i (d o t t ed l i n e ) wi th o u ts t imula t ing bacter ia (so l id l ine) .a n d a n a m m o n i u m s a l t f o r m e d . A l s o , a g a r i nt h es e p l a te s b e c a m e b r o w n b e c a u s e o f p i g m e n td i ff u s io n f r o m t r e a t e d c o r n . H o w e v e r , t h ism e d i u m w a s s til l c a p a b l e o f s u p p o r t i n g g r o w t ha s d e m o n s t r a t e d b y A . f l a v u s f o l lo w i n g i n o c u -l a ti o n. S u c h g r o w t h w a s e x p e c t e d s i n ce m a n ym o l d s , i n c l u d i n g m o s t P e n i e i l l i a a n d A s p e r g i l l i ,c a n r e a d i l y u s e a m m o n i u m s a lt s a s a n i t r o g e ns o u r c e .T h e e f f e e t o f .5% N H 8 o n e x t e r n a l m i c r o -f l o ra o f n o rma l 1 2 %-mo i s tu re eo rn i s p lo t t edi n F i g . 3 . T h i s c o r n h a d t h e s a m e f l o r a a s t h eo r i g i n a l c o r n i n t h e f i r s t e x p e r i m e n t . H o w e v e r ,i n t h e s e c o n d e x p e r i m e n t t h e c o r n w a s n o tt e m p e r e d . O n e h o u r a f t e r a m m o n i a t r e a t m e n t ,b a c t e r i a i n c r e a s e d ( 2 . 0 X 1 0 a t o 6 . 8 X 1 0 s p e rg r a m ) a n d m o l d s a n d y e a s t s w e r e n e a r l y d e -s t r o y e d . L e s s t h a n 1 0 m o l d s p e r g r a m ( s p e c i e s

TABLE 1. Effe ct of NH3 on internal m olds of surface-steri lized wh ole corn.Co rn t emp ered t o 26% H2 0

Microorganism Original Af ter 24 hr

12g-moisture cornAfter 2% amm onia af te r .5% amm oniat rea tmen t t r ea tmen t1 h r 2 wk 1 h r 2 wk

(no.)~ (%)b (no .) (%)AsperglUus f lavus 66 43.1 30 19.7Aspergillus niger 29 19.0 7 4.6Fusarium 36 23.5 51 33.6PeniciUium 15 9.8 8 5.3Trichoderma 4 2.6 23 15.1Rhizopus 28 18.4Othe r 3 2 .0 5 3 .3Tota l 153 100 152 100

(no . (no . (no . (no .and %) and %) and %) and %)0 0 0 0O 0 0 00 0 8 (100 ) 00 0 0 00 0 0 0O 0 0 0O 0 0 O

o o -(lOO) oNumber of kernels f rom which mold grew (100 kernelsb Per cen t o f to ta l m old popula t ion . were ex amin ed ) .

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2 4 4 B O T H A S T E T A L

T A B L E 2. Mouse preference for ammonia ted c o m .Exper iment Trea tment

No. 1, corn tem pere d to 26~ moisture 2.0% NH~ add ed Tem pere dKernels remaining 477 522Consumed 923 878Tota l 1,400 1,400Perce nt consumed 65.9 62.7x 2 = 3 .Ol (P> .0~)

No. 2, corn with 12% moisture 0.5% NH~ add ed No neKernels remaining 640 550Consumed 960 1,050Tota l 1,600 1,600Perce nt consumed 60.0 65.6

x : 10.6 ( P < . 0 1 )

o f Fusarium o n l y ) s u r v i v e d a m m o n i a tr e a t -m e n t a n d a f t e r a 2 - w k s t o ra g e w i t h a m m o n i a ,t h e y w e r e a l s o e l im in a t e d . B a c t e r i a l c o u n t s( 4 .2 X l 0 s ) r e m a i n e d a b o u t t h e s a m e a f t e r 2wk s to r a g e . T h e e f f e c t o f .5 ?o NH3 o n i n t e r n a lmo ld s o f s u r f a c e - s te r i l i z e d c o r n i s i n T a b le 1 .As w i th e x t e r n a l mo ld s , a f e w s p e c i e s o fFusarium i n i t i a l l y w i t h s t o o d a m m o n i a t r e a t -me n t . Ho we v e r , a f t e r a 2 - wk s to r a g e t h e y a l s ow e r e d e s t r o y e d .P r e l i m i n a r y o b s e r v a t i o n s o f a m m o n i a t e dc o r n s t o r e d 2 t o 3 m o i n d i c a t e d t h a t b a c t e r i a lf l or a d id n o t c h a n g e w i th .5% N H 3 t r e a tm e n ta n d t h e 2% t r e a t m e n t d e s t r o y e d b a c t e r i a d u r -i n g s to r a g e . Ap p a r e n t ly , a m m o n ia w i ll n o tc r e a t e a h e a l t h h a z a r d b y a l l o w i ng p a t h o g e n i co r g a n i s m s t o p r o p a g a t e .B a s e d o n a n a ly s i s o f v a r i a n c e a n d c h i s q u a r et e s t s f o r E x p e r ime n t 1 , t h e r e wa s n o e v id e n c eo f t r e a tm e n t d i f f e r en c e s ( T a b le 2 ) ; e . g . , t h e2~o a m m o n i a t e d a n d t e m p e r e d c o r n w e r e c o n -s u m e d e q u a l l y w e l l b y m i c e . H o w e v e r , r es u l tsw e r e e x t r e m e l y i nc o n s i s te n t a m o n g f o u r c a g e s .I n E x p e r i m e n t 2 , d i f f e r e n c e s a m o n g c a g e sw e r e m o r e c o n si st e nt . E f f e c t o f t r e a t m e n t w a sno t s ign if ican t by ana lys is o f va r ianc e , bu t o nt h e e h i s q u a r e t e s t u n t r e a t e d c o n t r o l c o r n w a sc o n s u m e d s l i g h tl y b e t t e r t h a n .5% a m m o n i -a t e d c o r n .Discussion

O u r f i n d i n g s i n d i c a t e t h a t a m m o n i a h a s e x -c e l l e n t f u n g ic id a l p r o p e r t i e s a s l o w a s . 5 % o ft h e d r y w e i g h t o f c or n . I m p o r t a n c e to t h ef a r m e r o r e l e v a t o r o p e r a t o r h a s y e t t o b ea s s e s se d . C o n s id e r in g s imi l a r i t y o f a m m o n iat o u r e a , n o w u s e d a s a p r o t e i n s u p p l e m e n t f o rr u min a n t s , p r o s p e c t s f o r s a t i s f a c to r y o r b e n e -f i c i a l f e e d in g o f a mmo n ia t e d g r a in a r e e n -c o u r a g in g . Ho we v e r , i t s u t i l i t y d e p e n d s o n a d -JOURNAL OF DAIRY SCIENCE VOL. 56, NO. 2

v e r s e e ff e c t f r o m d i s c o lo r a t io n o f g r a in ,r e s i d u a l a m m o n i a o d o r , o r c h e m i c a l c h a n g e sin c o r n c o n s t i t u e n t s .U r e a h a s a p r o t e i n e q u i v a l e n t f o r t h er u m i n a n t a n i m a l o f 6 . 2 5 t i m e s t h e n i t r o g e nc o n t e n t a n d i s r a p i d l y d e c o m p o s e d t o a m m o n i aa n d c a r b o n d i o x i d e b y r u m e n m i c r o o r g a n i s m s .A m m o n i a i n c o r n s h o u l d b e e q u a l l y u s e f u l a sa p r o t e i n s u p p l e m e n t . H u b e r a n d S a n t a n a( 1 3 ) r e p o r t e d t h a t h e i fe r s a t e m o r e a m -mo n ia t e d c o r n s i l a g e t h a n c o n t r o l s i l a g e a n dt h a t l a c t a t i n g c o w s s h o w e d h i g h e r m i l k y i e ld so n a m m o n i a - a n d u r e a - t r e a t e d s i la g e s t h a n o nn e g a t i v e c o n t r o l r a t i o n s w i t h n o a d d e d n i t r o -g e n . No s ig n if i c an t d i f f e re n c e s i n p r o d u c t io nw e r e n o t e d f o r c o ws f e d c o n t r o l , u r e a - t r e a t e d ,o r a m m o n i a t e d s i l a g e s a t e q u a l d i e t a r y n i t r o -g e n . S in c e a mmo n ia t e d c o r n h a s s o me r e s id u a la m m o n i a o d o r , a c c e p t a b i l i t y o f t r e a t e d c o r nm u s t b e d e t er m i n e d .A m m o n i a , b e i n g v o l at il e , w o u l d b e e x p e c t e dto p e r me a te r a p id ly s t o r e d c o r n , k i l l i n g a l l i n -t e r n a l f u n g i , a n d e l im in a t i n g h o t s p o t s . Am-mo n ia ' s d i f f u s ib i l i t y s h o u ld ma k e a p p l i c a t i o nt e c h n ic a l l y s imp le .A l o n g w i t h l a r g e - s c a l e t r e a t m e n t a n d f e e d -h a g s tu d i e s , f u r th e r l a b o r a to r y e x p e r ime n t s t od e t e r m i n e s e p a r a t e a n d c o m b i n e d e f f e c t s o fm o i s t u re , t e m p e r a t u r e , a n d a m m o n i a c o n t e n two u ld b e u s e f u l . M ic r o b i a l a n d b io lo g i c a l e f -f e c t s o f a m m o n i a f o r l o n g e r t i m e s t h a n a r e i n -e l u d e d i n t h is s t u d y n e e d t o b e in v e s t ig a t e d .T h e u l t i m a t e t e s t w o u l d b e t o t r e a t a n d f e e dh ig h - mo i s tu r e c o r n f r o m th e f i e ld .Acknowledgments

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