biochemj00914-0136
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5 2 7 The Colorimetric Determination o f Lactic Acid in Silage B Y A . J . G . BARNETT Division o f Agricultural Biochemistry, Department of Biological Chemistry, University o f Aberdeen (Received February 1951) The fundamental reaction which takes place i n th e process o f ensilage i s the conversion of a proportion o f t h e soluble carbohydrates present t o lactic acid. This reaction i s o f such importance that when material o f high protein content i s being ensiled, i t i s desirable t o add molasses t o provide a readily available substrate fo r t h e lactobacilli which occur along with coliform organisms i n th e original grass crop. When lactic acid is formed i n suitable amount i t acts a s a preservative f o r t h e ensiled material a n d serves, o n account o f t h e lowering o f p H which accompanies i t s formation, t o inhibit t h e activity of coliform bacteria which produce undesirable b y - products. Despite t h e importance o f this reaction i t h a s never been fully investigated, partly o n account o f t h e pre-occupation o f many investigators with t h e question o f quality i n silage, f o r t h e determination o f which t h e p H is a reasonable guide, a n d partly because th e methods used fo r i t s estimation a r e tedious a nd i n some cases inaccurate. F o r example, t h e method o f Foreman (1920) depends upon a difference calculation which involves t h e experi- mental error o f t h e other acid determinations, a n d gives a result which represents t h e total non-volatile acid content, of which b u t a part i s lactic acid, a fact which h a s been stressed b y Watson & Ferguson (1937) i n their study o f th e method. The method o f Karstr6m & Virtanen (1937), modified from t h e original techniques o f Hirsch-Kauffmnann (1924) and Lieb & Zacherl (1932), involves the u s e o f large amounts o f material a n d complicated procedures fo r t h e elimination o f proteins a n d carbohydrates. Miller & Muntz (1938) developed a method, later modified by Barker & Summerson (1941), f o r t h e colorimetric estimation o f lactic acid which depends upon t h e fact that lactic acid i s oxidized by sulphuric acid t o acetaldehyde which gives a purple-red colour with p-hydroxydiphenyl i n t h e presence o f metallic ions, o f which copper has been found t o b e t h e most suitable (Barker & Summerson, 1941). T h e reaction is similar t o that o f Mendel & Goldscheider (1925) who, however, used o-dimethoxybenzene a s t h e colour-producing reagent. Davidson (1949) hasused th e Barker & Summerson technique f o r t h e determination o f lactic acid i n milk a n d milk products, demonstrating i t s applica- bility to amounts o f 20-400 m g . lactic acid. I n the case o f these latter, a n d blood or minced tissue, steps have to b e taken t o remove th e protein matter involved. Silage, however, may b e looked upon a s solid matter covered with, or impregnated with, lactic acid t o a greater o r lesser extent a n d t h e amounts o f protein extractable b y water from silage a r e relatively small. O f t h e 5 g . portions o f fresh silage which a re used i n t h e method o f analysis described below, less than 0 4 g. i s protein an d o f this only a part is soluble in water. This small pro- portion m a y b e removed from t h e prepared extract a t t h e same time a s th e soluble carbohydrates. T h e technique o f Barker & Summerson, a s used fo r th e determination o f lactic acid in silage, i s described below. REAGENTS Standard lactic acid solution. Pure zinc lactate (0-0135 g.) is dissolved i n 1 0 m l . o f distilled water contained i a 1 1 . standard flask. Conc. H2SO4 ( 0 5 ml.) i s added a n d t h e volume made u p t o t h e mark with distilled water. This solution i s stable but should b e kept in a refrigerator. p-Hydroxydiphenyl reagent. p-Hydroxydiphenyl (1-5 g . ) i s dissolved i n 1 0 m l . 5% (w/v) aqueous NaOH a n d made u p t o a litre with water. This reagent i s also stable b u t should be stored in a n amber bottle. Calcium hydroxide. T h e brand used is Judex. Copper sulphate solutions. T w o CUSO4 solutions a r e required, o n e 0-80M a n d t h e other 0 16M. These a r e prepared from A.R. CuS04.5H20. Concentrated sulphuric acid. I t h a s been shown by Russell (1944) that nitrates and nitrites interfere with th e reaction, a n d f o r this reason t h e N-free H,SO4 essential for Kjeldahl determinations i s used. METHOD O f th e representative sample o f silage (about 3 kg.) brought t o t h e laboratory f o r examination, about 1 0 0 g . taken from t h e centre o f t h e mass i s finely chopped an d treated after t h e fashion described below. A further 1 0 0 g . portion i s mixed with 100 l . o f distilled water i n a beaker a n d s e t aside f o r 2 4 h r . pending a p H determination o n t h e extract. A dry- matter determination is performed on approxi- mately 1 k g . o f t h e material b y drying this a t 8 0 0 f o r 24 h r. O f th e chopped material t w o 5 g . portions a r e placed i n t w o 2 5 0 m l. beakers 1 0 0 ml. o f boiling distilled water added a n d th e whole allowed t o cool
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Transcript of biochemj00914-0136
8/2/2019 biochemj00914-0136
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5 2 7
The C o l o r i m e t r i c D e t e r m i n a t i o n o f L a c t i c A c i d i n S i l a g e
BY A . J . G . BARNETT
D i v i s i o n o f A g r i c u l t u r a l B i o c h e m i s t r y , D e p a r t m e n t o f B i o l og i c al C h e m i s t r y , U n i v e r s i t y o f A b e r d e e n
( R e c e i v e d 1 4 F e b r u a r y 1 9 5 1 )
he f u n d a m e n t a l r e a c t i o n w h i c h t a k e s p l a c e i n t h e
o f e n s i l a g e i s t h e c o n v e r s i o n o f a p r o p o r t i o n
f t h e s o l u b l e c a r b o h y d r a t e s p r e s e n t t o l a c t i c a c i d .r e a c t i o n i s o f s u c h i m p o r t a n c e t h a t when
o f h i g h p r o t e i n c o n t e n t i s b e i n g e n s i l e d , i ts d e s i r a b l e t o add m o l a s s e s t o p r o v i d e a r e a d i l y
s u b s t r a t e f o r t h e l a c t o b a c i l l i w h i c h o c c u r
w i t h c o l i f o r m o r g a n i s m s i n t h e o r i g i n a l g r a s s
When l a c t i c a c i d i s f o r m e d i n s u i t a b l e amount
t a c t s a s a p r e s e r v a t i v e f o r t h e e n s i l e d m a t e r i a l a n don a c c o u n t o f t h e l o w e r i n g o f pH w h i c hi t s f o r m a t i o n , t o i n h i b i t t h e a c t i v i t y
c o l i f o r m b a c t e r i a w h i c h p r o d u c e u n d e s i r a b l e b y -
D e s p i t e t h e i m p o r t a n c e o f t h i s r e a c t i o n i t h a s
b e e n f u l l y i n v e s t i g a t e d , p a r t l y on a c c o u n t o f
h e p r e - o c c u p a t i o n o f m a n y i n v e s t i g a t o r s w i t h t h e
u e s t i o n o f q u a l i t y i n s i l a g e , f o r t h e d e t e r m i n a t i o n
f w h i c h t h e pH i s a r e a s o n a b l e g u i d e , a n d p a r t l y
t h e m e t h o d s u s e d f o r i t s e s t i m a t i o n a r e
a n d i n some c a s e s i n a c c u r a t e . F o r e x a m p l e ,
h e method o f Foreman ( 1 9 2 0 ) d e p e n d s upon a
i f f e r e n c e c a l c u l a t i o n w h i c h i n v o l v e s t h e e x p e r i -
e n t a l e r r o r o f t h e o t h e r a c id d e t e r m i n a t i o n s , a nd
i v e s a r e s u l t w h i c h r e p r e s e n t s t h e t o t a l n o n - v o l a t i l e
a c i d c o n t e n t , o f w h i c h b u t a p a r t i s l a c t i c a c i d , a f a c t
w h i c h h a s b e e n s t r e s s e d by Watson & F e r g u s o n
( 1 9 3 7 ) i n t h e i r s t u d y o f t h e m e t h o d . T he m et ho d o f
K a r s t r 6 m & V i r t a n e n ( 1 9 3 7 ) , m o d i f i e d f r o m t h e
o r i g i n a l t e c h n i q u e s o f H i r s c h - K a u f f m n a n n ( 1 9 2 4 )
a n d L i e b & Z a c h e r l ( 1 9 3 2 ) , i n v o l v e s t h e u s e o f l a r g eamounts o f m a t e r i a l and c o m p l i c a t e d p r o c e d u r e s f o r
t h e e l i m i n a t i o n o f p r o t e i n s and c a r b o h y d r a t e s .
M i l l e r & Muntz ( 1 9 3 8 ) d e v e l o p e d a m e t h o d , l a t e r
m o d i f i e d by B a r k e r & Summerson ( 1 9 4 1 ) , f o r t h ec o l o r i m e t r i c e s t i m a t i o n o f l a c t i c a c i d w h i c h d e p e n d supon t h e f a c t t h a t l a c t i c a c i d i s o x i d i z e d by s u l p h u r i c
a c i d t o a c e t a l d e h y d e w h i c h g i v e s a p u r p l e - r e d c o l o u r
w i t h p - h y d r o x y d i p h e n y l i n t h e p r e s e n c e o f m e t a l l i c
i o n s , o f w h i c h c o p p e r h a s b e e n f o u n d t o b e t h e m o s ts u i t a b l e ( B a r k e r & S u m m e r s o n , 1 9 4 1 ) . The r e a c t i o n
i s s i m i l a r t o t h a t o f M e n d e l & G o l d s c h e i d e r ( 1 9 2 5 )
w h o , h o w e v e r , u s e d o - d i m e t h o x y b e n z e n e a s t h e
c o l o u r - p r o d u c i n g r e a g e n t .
D a v i d s o n ( 1 9 4 9 ) h a s u s e d t h e B a r k e r & Summerson
t e c h n i q u e f o r t h e d e t e r m i n a t i o n o f l a c t i c a c i d i n
m i l k a nd m i l k p r o d u c t s , d e m o n s t r a t i n g i t s a p p l i c a -b i l i t y t o amounts o f 2 0 - 4 0 0 m g . l a c t i c a c i d . I n t h e
c a s e o f t h e s e l a t t e r , and b l o o d o r m i n c e d t i s s u e , s t e p s
h a v e t o b e t a k e n t o remove t h e p r o t e i n m a t t e r
i n v o l v e d . S i l a g e , h o w e v e r , m a y b e l o o k e d upon a s
s o l i d m a t t e r c o v e r e d w i t h , o r i m p r e g n a t e d w i t h ,
l a c t i c a c i d t o a g r e a t e r o r l e s s e r e x t e n t and t h e
a m o u n t s o f p r o t e i n e x t r a c t a b l e by w a t e r f r o m s i l a g ea r e r e l a t i v e l y s m a l l . Of t h e 5 g . p o r t i o n s o f f r e s hs i l a g e w h i c h a r e u s e d i n t h e method o f a n a l y s i s
d e s c r i b e d b e l o w , l e s s t h a n 0 4 g . i s p r o t e i n and o f
t h i s o n l y a p a r t i s s o l u b l e i n w at e r . T h i s s m a l l p r o -p o r t i o n m a y b e r e m o v e d f r o m t h e p r e p a r e d e x t r a c t
a t t h e same t i m e a s t h e s o l u b l e c a r b o h y d r a t e s .
The t e c h n i q u e o f B a r k e r & S u m m e r s o n , a s u s e d
f o r t h e d e t e r m i n a t i o n o f l a c t i c a c i d i n s i l a g e , i sd e s c r i b e d b e l o w .
REAGENTS
S t a n d a r d l a c t i c a c i d s o l u t i o n . P u r e z i n c l a c t a t e ( 0 - 0 1 3 5 g . )i s d i s s o l v e d i n 1 0 m l . o f d i s t i l l e d w a t e r c o n t a i n e d i n a 1 1 .
s t a n d a r d f l a s k . C o n c . H 2 S O 4 ( 0 5 m l . ) i s a d d e d a n d t h e
v o l u m e made u p t o t h e mark w i t h d i s t i l l e d w a t e r . T h i s
s o l u t i o n i s s t a b l e b u t s h o u l d b e k e p t i n a r e f r i g e r a t o r .
p - H y d r o x y d i p h e n y l r e a g e n t . p - H y d r o x y d i p h e n y l ( 1 - 5 g . )i s d i s s o l v e d i n 1 0 m l . 5% ( w / v ) a q u e o u s Na OH a n d made
u p t o a l i t r e w i t h w a t e r . T h i s r e a g e n t i s a l s o s t a b l e b u t
s h ou l d b e s t o r e d i n a n a m b e r b o t t l e .C a l c i u m h y d r o x i d e . T h e b r a n d u s e d i s J u d e x .
C o p p e r s u l p h a t e s o l u t i o n s . Two C U S O 4 s o l u t i o n s a r e
r e q u i r e d , o n e 0 - 8 0 M a n d t h e o t h e r 0 1 6 M . T h e s e a r e p r e p a r e d
f r o m A . R . C u S 0 4 . 5 H 2 0 .C o n c e n t r a t e d s u l p h u r i c a c i d . I t h a s b e e n s h o w n b y R u s s e l l
( 1 9 4 4 ) t h a t n i t r a t e s a n d n i t r i t e s i n t e r f e r e w i t h t h e r e a c t i o n ,a n d f o r t h i s r e a s o n t h e N - f r e e H , S O 4 e s s e n t i a l f o r K j e l d a h l
d e t e r m i n a t i o n s i s u s e d .
METHOD
O f t h e r e p r e s e n t a t i v e s a m p l e o f s i l a g e ( a b o u t 3 k g . )b r o u g h t t o t h e l a b o r a t o r y f o r e x a m i n a t i o n , a b o u t
1 0 0 g . t a k e n f r o m t h e c e n t r e o f t h e mass i s f i n e l yc h o p p e d and t r e a t e d a f t e r t h e f a s h i o n d e s c r i b e d
b e l o w . A f u r t h e r 1 0 0 g . p o r t i o n i s m i x e d w i t h 1 00 m l .
o f d i s t i l l e d w a t e r i n a b e a k e r and s e t a s i d e f o r 2 4 h r .p e n d i n g a pH d e t e r m i n a t i o n on t h e e x t r a c t . A d r y -
m a t t e r d e t e r m i n a t i o n i s p e r f o r m e d on a p p r o x i -
m a t e l y 1 k g . o f t h e m a t e r i a l b y d r y i n g t h i s a t 8 0 0 f o r2 4 h r .
O f t h e c h o p p e d m a t e r i a l two 5 g . p o r t i o n s a r e
p l a c e d i n two 2 5 0 m l . b e a k e r s a n d 1 0 0 m l . o f b o i l i n gd i s t i l l e d w a t e r a d d e d and t h e w h o l e a l l o w e d t o c o o l
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A . J . G . BARNETT
f o r 5 m i n . Each p r e p a r a t i o n i s t r e a t e d i n t h e f o l l o w -
i n g w a y . The m i x t u r e i s s t i r r e d m e c h a n i c a l l y v e r y
v i g o r o u s l y f o r 5 m i n . , d u r i n g w h i c h t i m e a b o u t 0 5 g .o f s o l i d C a ( O H ) 2 i s a d d e d t o a s s i s t t h e s u b s e q u e n t
f i l t r a t i o n . The p r o d u c t i s f i l t e r e d t h r o u g h a h a r df i l t e r p a p e r and t h e f i r s t 2 0 m l . o f f i l t r a t e a r e d i s -c a r d e d . Of t h e f i l t r a t e 2 m l . a r e t a k e n and d i l u t e d
t o 1 0 m l . w i t h d i s t i l l e d w a t e r , and o f t h i s s o l u t i o n1 m l . i s p l a c e d i n a s m a l l g l a s s - s t o p p e r e d b o t t l e t o
w h i c h a r e a d d e d , i n t h e f o l l o w i n g o r d e r , 8 m l . o f
d i s t i l l e d w a t e r f r o m a b u r e t t e , 1 m l . o f 0 - 8 0 M -
CuSO4 and 1 g . o f C a ( O H ) , . A s t a n d a r d f o r c o m -
p a r i s o n i s p r e p a r e d by u s i ng 5 m l . o f s t a n d a r d l a c t i ca c i d s o l u t i o n a nd m a k i n g t h i s up a s d e s c r i b e d f o r t h e
t e s t p o r t i o n , u s i n g o n l y 4 m l . o f d i s t i l l e d w a t e r .
S i m i l a r l y , a b l a n k i s p r e p a r e d by u s i n g 1 m l . o f
w a t e r i n s t e a d o f t h e u n k n o w n . The f o u r b o t t l e s a r e
t h e n s t o p p e r e d a nd s h a k e n f o r 2 0 m i n . i n a s m a l l
m e c h a n i c a l s h a k e r , a f t e r w h i c h t i m e t h e c o n t e n t s a r e
f i l t e r e d t h r o u g h s m a l lf i l t e r p a p e r s d i s c a r d i n g t h ef i r s t 2 m l . o f f i l t r a t e i n e a c h c a s e . The r e m a i n d e r o f
e a c h f i l t r a t e i s c o l l e c t e d i n s m a l l t e s t t u b e s . Each
f i l t r a t e i s t r e at e d a s f o l l o w s : The f i l t r a t e ( 1 m l . ) i sp l a c e d i n a P y r e x t u b e ( 2 0 0 x 3 0 m m . ) a n d m i x e dw i t h 0 . 0 5 m l . o f 0 - 1 6 M - C u S O 4 a n d 6 m l . o f N 2 - f r e eH 2 S O 4 added f r o m a b u r e t t e w i t h g r e a s e - f r e e t a p .
The t u b e s a r e a l l o w e d t o s t a n d i n b o i l i n g w a t e r f o r
5 m i n . and a r e t h e n c o o l e d by s t a n d i n g i n a s t r e a m o f
r u n n i n g w a t e r . To e a c h t u b e i s a d d e d 0 1 m l . o f t h e
p - h y d r o x y d i p h e n y l r e a g e n t and t h e c o n t e n t s s h a k e n
l a t e r a l l y t o e n s u r e e v e n d i s t r i b u t i o n o f t h e i n s o l u b l er e a g e n t . The t u b e s a r e t h e n p l a c e d i n a w a t e r b a t h
f o r 3 0 m i n . a t 3 0 - 3 3 ° , b e i n g s h a k e n a t 1 0 m i n .i n t e r v a l s . F i n a l l y , t h e t u b e s and t h e i r c o nt e nt s a re
h e a t e d f o r 2 m i n . i n a b a t h o f b o i l i n g w a t e r a nd c o o l e d
t o r o o m t e m p e r a t u r e . R e a d i n g s o f t h e c o l o u r
d e v e l o p m e n t s a r e m a d e o n a n EEL ( E v a n s E l e c t r o -
s e l e n i u m L t d . , H a r l o w , E s s e x ) p h o t o e l e c t r i c c o l o r i -meter u s i n g t h e s t a n d a r d g r e e n f i l t e r s u p p l i e d byt h e m a k e r s and w a t e r a s a z e r o . As t h e 1 m l . c o p p e r -
c o n t a i n i n g p o r t i o n o f s t a n d a r d l a c t i c a c i d c o n t a i n s
0 - 0 0 5 m g . / l m l . p o r t i o n u s e d , t h e c o n c e n t r a t i o n o f
l a c t i c a c i d i n t h e unknown i s g i v e n by t h e f o r m u l a :
% L a c t i c a ci d i n f r e s h s i l a g eR e a d i n g f o r unknown ( - b l a n k ) x O i
R e a d i n g f o r s t a n d a r d ( - b l a n k )
and f o r t h e amount o f l a c t i c a c i d b a s e d on t h e d r ym a t t e r ( D M ) c o n t e n t o f t h e s i l a g e :
% L a c t ic a ci d
R e a d i n g f o r unknown ( - b l a n k ) 5 0
R e a d i n g f o r s t a n d a r d ( - b l a n k ) %DM c o n t e n t
The r e s u l t s o f a c o n t r o l e x p e r i m e n t t o t e s t t h e
a c c u r a c y o f t h e method a r e i n d i c a t e d i n F i g . 1 , w h i l e
r e c o v e r y o f l a c t i c a c i d f r o m m i x t u r e s o f known
s t a n d a r d and unknown v a r i e s from 9 6 t o 1 0 2 %, andt h e o n l y p r e c a u t i o n r e q u i r e d i s a b s o l u t e c l e a n l i n e s s
a s , o w i n g t o t h e s e n s i t i v e n e s s o f t h e r e a c t i o n ,i m p u r i t i e s w h i c h g i v e r i s e t o l a c t i c a c i d on h e a t i n g
w i t h H 2 S O 4 n u l l i f y t h e r e s u l t s . A l l g l a s s w a r e u s e d
' 6 0
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E
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0 0 9 0 8 0 7 0 6 0 . 5 0 4 0 3 0 2 0 1 0 0 L a c t i c a c i d ( % )0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 0 0 Unknown ( ° , )
F i g . 1 . S p e c t r o p h o t o m e t e r r e a d i n g s f o r m i x t u r e s o f s t a n d a r d
l a c t i c a c i d s o l u t i o n a n d s i l a g e e x t r a c t a s p r e p a r e d f o ra n a l y s i s i n p e r c e n t a g e s s h o w n ( b y v o l . ) . F o r d e t a i l s o f
p r o c e d u r e s e e T e x t .
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p H
F i g . 2 . R e l a ti o n o f l a c t i c a c i d c o n t e n t
o f w e t s i l a g e t o p H .
5 - 6
i s s u c c e s s i v e l y w a s h e d w i t h d i l . H C I , warm water
w i t h a l i t t l e d e t e r g e n t , c o l d w a t e r and f i n a l l y d r i e di n an o v e n .
I 9 5 I2 8
II III IIII I Lc
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