556
BIOCHIMICA ET BIOPHYSICA ACTA
VOL. 2 9
(1958)
A METHOD FOR THE DETERMINATION OF D(--)-LACTIC ACID
C. J. A. VAN D E N H A M E R * AND R. W. E L I A S
Bacteriological Department, Laboratory o] Hygiene, University o] Utrecht (The Netherlands)
INTRODUCTION
Accounts of several good methods for the determination of the total amount of lactic
acid have been published1, 2. Reliable methods for the separate determination of
D(--)- or L(+)-lactic acid are less plentiful. The usual method is the determination
of the water content and the optical rotation of the zinc salt s . Furthermore, oxidation
with D P N and lactic acid dehydrogenase 4, 5, oxidation with resting cells e and oxidation
with cell-free preparations of microorganisms ~ are often used. A separation of optically
active and racemic lactic acid by paper chromatography is also possible 8. Each of
these methods has its own advantages and specific applications. Isolation of zinc
lactate, for instance, is time-consuming and only possible with large quantities of
lactic acid. The spectrophotometric determination with D P N and lactic acid deh y d r o g e n a s e - - c o m m o n l y used in hospitals--is very sensitive, but expensive.
The method described here is based on the observation of HAUGAARD9 that a
cell-free preparation of E. coli B, grown in a medium with DL-lactate as the only
carbon source, oxidizes both D(--)- and L(+)-lactate to pyruvate, whereas an acetone
powder of this preparation attacks only D(--)-lactate; in the latter case methylene
blue has to be added. We observed that an acetone powder of whole cells of E. coli B
has the same properties. When a lactic acid-containing sample is incubated with the
acetone powder, methylene blue and phosphate buffer in a Warburg apparatus, the
oxygen consumption is a measure for the amount of D(--)-lactic acid in the sample.
The method was specially useful in the study of bacterial fermentation products.
METHODS AND MATERIALS
Preparation o~ acetone powder
S t o c k c u l t u r e s of a strain of E. coli B were kept on n u t r i e n t agar. I n order to prepare t h e
a c e t o n e p o w d e r t h e o r g a n i s m w a s i n o c u l a t e d f r o m s t o c k cultures into a s m a l l v o l u m e of a liquid
m e d i u m w i t h DL-lactate as t h e sole c a r b o n source. This m e d i u m w a s p r e p a r e d a c c o r d i n g to
MclLWAIN TM. I t c o n t a i n s per liter: 4.5 g KH2PO4, 0.5 g (NH4)zSO4, 0.5 g NH4C1, 2.8 g Na-DLlactate, a n d t r a c e s of F e S O 4 a n d MgSO 4. I t w a s a d j u s t e d to p H 7.6 w i t h s o d i u m h y d r o x i d e ,
filtered, a n d sterilized b y boiling for s o m e m i n u t e s . This c u l t u r e w a s s h a k e n or a e r a t e d w i t h
sterile air a t 3 °o C for 24 h a n d t h e w h o l e c u l t u r e w a s used as a n i n o c u l u m for a IS-fold v o l u m e
of t h e s a m e m e d i u m . In this w a y larger q u a n t i t i e s of cells w e r e obtained. T h e final c u l t u r e w a s
a g a i n a e r a t e d at 3o~C for 24 hours. W h e n a e r a t i o n had been sufficient t h e c u l t u r e w a s very
turbid, showing 8o % light a b s o r p t i o n (white light).
Cells w e r e h a r v e s t e d b y c e n t r i f u g a t i o n , w a s h e d t w i c e w i t h saline, a n d s u s p e n d e d in a v o l u m e
of saline ( a b o u t 0.25 % of t h e c u l t u r e v o l u m e ) . W h i l e stirring v i g o r o u s l y a n d cooling w i t h w a t e r
of o ° C a 5-fold v o l u m e of cold a c e t o n e w a s s l o w l y added. T h e p r e c i p i t a t e d cells w e r e r e s u s p e n d e d
b y m e c h a n i c a l stirring in a b o u t one v o l u m e of cold a c e t o n e and centrifuged. This p r o c e d u r e w a s
* This i n v e s t i g a t i o n w a s s u p p o r t e d b y a g r a n t from t h e N e t h e r l a n d s O r g a n i z a t i o n for Pure
R e s e a r c h (Z.W.O.).
Re/erences p. 561/562.
VOL. 29 (1958)
DETERMINATION
O F D(--)-LACTIC ACID
557
r e p e a t e d three times, water-free acetone being used for t h e last t r e a t m e n t . The wet acetone
p o w d e r w a s t h e n s p r e a d in a t h i n layer in a Petri dish a n d dried at r o o m t e m p e r a t u r e in vacuo.
F r o m i 1 of m e d i u m , o.5-o.6 5 g d r y p o w d e r w a s obtained. After storage during one y e a r at 4 ° C
or one m o n t h at r o o m t e m p e r a t u r e no reduction in activity was found. N o t all strains gave
equally satisfactory results.
Standard assaT;
I n order to determine D(--)-lactic acid in a f e r m e n t a t i o n s u p e r n a t a n t , 35 ° mg acetone
p o w d e r was suspended into io ml p h o s p h a t e buffer 0.2 M, p H 7.5, as h o m o g e n e o u s l y as possible.
Elimination of c l u m p s b y decantation a n d filtration over a Willstiitter nail w i t h o u t filter p a p e r
can produce a m o r e h o m o g e n e o u s suspension.
F o r the determination, conventional W'arburg vessels were used. The centre well contained
o.I ml lO% K O H , the sidearm(s) 0.2-0.6 ml sample, and the m a i n c o m p a r t m e n t i ml suspension
of acetone p o w d e r in M/5 p h o s p h a t e buffer, p H 7.5, 0.3 ml 2. 5 % methylene blue and water,
so t h a t the final v o l u m e in the whole flask was 2. 4 ml. The gas p h a s e was air, the incubation
t e m p e r a t u r e 37 ° , and t h e shaking frequency a b o u t 7° per minute. The o x y g e n c o n s u m p t i o n was
c o m p a r e d in the usual m a n n e r with t h a t in a vessel w i t h o u t s a m p l O I.
Fig. i s h o w s a typical experiment. All values were corrected for the value of the vessel
w i t h o u t sample. The oxygen c o n s u m p t i o n was f e u n d after the m a n o m e t e r readings had become
constant. I n those cases where the curve did n o t become completely horizontal b u t continued
to rise at a low slope the oxygen c o n s u m p t i o n was found b y e x t r a p o l a t i o n to zero time (Fig. I).
Total and L( +)-lactic acid
The total a m o u n t of lactic acid in the sample was generally determined according to a
modification 12 of the procedure of BARKER AND SUMMERSON2. The a m o u n t of L( +)-lactic acid
was found as the difference b e t w e e n total and n ( - - ) - l a c t i c acid.
Lactate preparations used
I n order to test the m e t h o d the following lactate p r e p a r a t i o n s were used:
(a) L( +)-lactic acid, obtained commercially as the free acid. Before use it was neutralized
with K O H .
(b) n(--)-lactic acid, prepared with LactobaciUus leichmannii ATCC 4797 and isolated as the
zinc salt, according to BRIN 13, after it had been extracted from the acidified f e r m e n t a t i o n m e d i u m
with ethyl ether 14. Before use it was converted to the free acid with Dowex-5o and neutralized
with KOH.
(c) Li-oL-lactate was a gift of the Central I n s t i t u t e for Food Research (Mr. WILLEMSE). I t
WaS used in the state in which it was received.
RESULTS AND DISCUSSION
Our acetone powder, prepared from whole cells of E. coli B, acted in the presence
of methylene blue in the same way as the acetone powder from a cell-free preparation
of E. coli B described by HAUGAARD9 (Fig. I). 0. 5/~mole oxygen was consumed per
/~mole D(--)-lactic acid, whereas L( +)-lactic acid was scarcely affected. After omission
of K O H from the centre well essentially the same manometer readings resulted, so
no CO 2 was produced. After oxygen consumption was complete, pyruvic acid was
found 15 in the vessels in quantities equimolecular with the amount of D(--)-lactic
acid added.
Optimal conditions
As the method appeared s a t i s f a c t o r y - - a n d , in fact, after it had been used for
some time with good r e s u l t s - - w e checked whether the conditions of assay were the
optimal conditions (Fig. 2-6). In these figures the conditions of assay are indicated
by an arrow. It was found that the concentration of methylene blue and of acetone
powder were not limiting factors (Fig. 2 and 3). The amount of D(--)-lactic acid in
the vessels had no influence on the reaction velocity within a range of 0.25-3.0 mg
(Fig. 4). Variation of the buffer concentration from 0.05-0.28 M did not influence
the results. The p H optimum was studied in phosphate and glycine-sodium hydroxide
References p. 561/562.
558
C.J.A.
VAN DEN HAMER, R. W. ELIAS
VOL. 2 9
(i958)
buffers (Fig. 5). The pH of the buffer (Fig. 5) used in the assay was not quite optimal.
As the use of phosphate buffer was more convenient for our purposes and the buffer
capacity of phosphates at pH 8 is small we continued to use pH 7.5. The oxygen
supply is the limiting factor of the reaction velocity (Fig. 6). An increase of shaking
jl
mote 0 2 consumed
8
6
,.,I=----.--..e
~ctate
/
•
4
2/
L(+)-t0ctete
J u
0
'
x
,
0
40
20
60
T i m e in
80
minutes
Fig. x. D e t e r m i n a t i o n of D ( - - ) - l a c t i c acid. The W a r b u r g vessels c o n t a i n e d o.I ml K O H (in t h e
c e n t r e wells), 35 m g a c e t o n e p o w d e r of E. cell B s u s p e n d e d in i ml 0.2 M p h o s p h a t e buffer,
p H 7.5, 0.3 ml 2 . 5 % m e t h y l e n e blue, i i . I p m o l e s u b s t r a t e as i n d i c a t e d (in t h e "side arm) a n d
w a t e r to a final v o l u m e of 2. 4 ml. O t h e r c o n d i t i o n s see t e x t .
~1 02 consumed perlO rain.
50
40
_.~e
30
I
~ - -
20
f "
10
, o
0
0.I
o2
O.3
I~
,
~4
0.5
I~
m[ 2.50/o methytene bl.ue per ve~se[
Fig. 2. Effect of t h e m e t h y l e n e blue c o n c e n t r a t i o n on t h e r a t e of o x i d a t i o n . C o n d i t i o n s a re as
d e s c r i b e d in Fig. i. The a r r o w i n d i c a t e s t h e a m o u n t of m e t h y l e n e blue as used in our s t a n d a r d
assay. N o t e d i s c o n t i n u a t i o n in t h e scale.
ReJerences p. 561]56z.
VOL. 29 (1958)
DETERMINATION
OF D(--)-LACTIC ACID
559
frequency of the Warburg vessels from 4 ° to about 9o per minute augmented the
reaction rate 2. 4 times. The substitution of the air in the gas phase by pure oxygen
augmented the reaction velocity 2.o times at the low frequency and 2.6 times at
the higher frequency of shaking.
Fig. 2-6 show that the chosen conditions are nearly optimal. The reaction rate
j,t 0 2 consumed per
50
10 m i n'Jte$
40-
]
30
=
=
•
1(1
0
0
10
20
30
40
50
I~0
mg acetone powder per vessel
F i g . 3. E f f e c t of t h e a m o u n t o f a c e t o n e p o w d e r o n t h e r a t e o f o x i d a t i o n . C o n d i t i o n s a r e a s d e s c r i b e d
in Fig. i The ~rrow indicates the amount of acetone powder as used in our standard assay.
~l 02
consumed
2oo
i
I
6mg OL- Lactic acid
I
160
]
.°
/~mL
D1-)- Lactic
I
.;r
,o
I
.~ ' ~ * ~ 0 . 2 5 ~
0
10
20
mg O(-)- Lactic acid
30
~0
50
Time in rn~nutes
F i g . 4- E f f e c t o f t h e s u b s t r a t e c o n c e n t r a t i o n o n t h e r a t e o f o x i d a t i o n .
O t h e r c o n d i t i o n s a r e a s d e s c r i b e d i n F i g . I.
Re/erences p. 56z/562.
Substrates
as indicated.
560
c.J.A.
VAN D E N
H A M E R , R. W . E L I A S
VOL. 29
(z958)
could be increased by a higher pH or by increased oxygen supply. As the determination of I)(--)-lactic acid did not depend on tlle reaction rate but on the total amount
of oxygen consumed, we continued to use the original conditions.
~umote 0 2 consumed
14
ja[ 0 2 c o n s u m e d
80
~
I
40 per mln. I
per
90-100 /
per m i n . /
10 m i n u t e s
.....
10
6o
2°!
.
;
6
I/
/
I
5
/I
"
'~11./ ' ~
6
I
?
8
I
9
10
[~H of
I
11
12
tho b u f f e r
0
S
I
10
15
Time in
F i g . 5. E f f e c t o f t h e p H o n t h e r a t e o f o x i d a t i o n .
C o n d i t i o n s a r e a s d e s c r i b e d i n F i g . I. T h e a r r o w
indicates the pH of our standard assay.
.
20
25
minutes
F i g . 6. E f f e c t of o x y g e n o n t h e r a t e o f o x i d a t i o n .
A f t e r l o m i n u t e s t h e s h a k i n g f r e q u e n c y is i n creased from 4 ° per minute to 9O-lOO per
minute. Solid line indicates pure oxygen as gas
p h a s e . S u b s t r a t e w a s 66. 7 / 1 m o l e L i - D L - l a c t a t e .
O t h e r c o n d i t i o n s a r e a s d e s c r i b e d i n F i g . I.
Specificits,
For a number of compounds other than lactic acid we estimated the oxidation
by the acetone-powder under the conditions of our assay. Especially those compounds
were tested that might be present as substrate or product in fermentation fluids of
lactic acid bacteria.Table I gives some ot these results.The rate of oxygen consumption
rather than total oxygen consumption is given here, since another substance broken
TABLE
I
OXIDATION RATES OF VARIOUS SUBSTANCES
t,moles
20
2o
i i. i
20
13.3
i 1.2
i I.I
13. 3
13-3
ReIerences p. 56I]562.
Compound
K-o(--)-lactate
Li-DL-lactate
L( + ) - l a c t a t e
K-D(--)-lactate
Arabinose
Gluconolactone
Glucose
Ribose
Xylose
Id 0 t i~ Io rain
+ 20 t~mole A s 2 0 3
3o-32
3o-32
i
30-32
o
i
o
o
o
VOl.. 29
(1958)
DETERMINATIONOF D(--)-LACTIC ACID
561
down at a very low rate would n o t affect specificity v e r y much. I t should be remembered t h a t u n d e r our conditions the rate is not optimal. To date no s u b s t a n c e has
been found which was oxidized at a n appreciable rate. A possible d e c a r b o x y l a t i o n
of some substances has not been excluded since in all cases the centre well c o n t a i n e d
KOH. W i t h pure lactic acid preparations the K O H can be omitted.
W i t h i n the limits of e x p e r i m e n t a l accuracy no oxygen c o n s u m p t i o n was o b t a i n e d
with the following substances: acetate, acetylmethylcarbinol, K-citrate, ethanol,
glycerol, glycine, glycylglycine, K-DL-malate, K-oxalacetate, K - p r o p i o n a t e a n d Napyruvate.
Sensitivity
The sensitivity depends m a i n l y on the shape of the b e n d of tile curve (Fig. I),
i.e., in fact, on the velocity of the reaction. U n d e r our conditions O.l-O.2 mg lactic
acid h a d to be present for reasonable accuracy. The limiting factor is the scattering
of the m a n o m e t e r readings in the horizontal part of the curve, which determine the
total oxygen c o n s u m p t i o n .
A c c u r a c y was not studied in detail. I n IO observations derived from three series
of experiments with IO/~moles D(--)-lactate the q u a n t i t i e s of oxygen c o n s u m e d varied
from 9.73 to IO.I tzatoms of oxygen. The m e a n was 9 . 9 2 / , a t o m s a n d the s t a n d a r d
error of each observation was o.182 tzatoms. W i t h smaller a m o u n t s of lactic acid the
error would be larger. U n d e r our conditions, with a m o u n t s of lactic acid in the region
of o.1-2 mg lactic acid the error of a single e x p e r i m e n t would be in the order of 5-1o %.
The described m e t h o d permits the e s t i m a t i o n of D(--)-lactic acid in a n u m b e r
of samples (depending on the apparatus) in a r a t h e r short time. The m e t h o d requires
a W a r b u r g apparatus. A t t e m p t s to use T h u n b e r g technique showed less accurate
results.
ACKNOWLEDGEMENT
We are very grateful to Professor Dr. K. C. WINKLER for his c o n s t a n t interest a n d
to Mr. K. C. STRASTERS for preparing D(--)-lactic acid a n d carrying out some prelimin a r y experiments.
SUMMARY
A method for the determination of D(--)-lactic acid is described. An acetone powder from Escherichia coli B in the presence of methylene blue oxidizes n(--)-lactic specifically. Oxygen consumption in a Warburg apparatus was used as a measure of the n(--)-lactic acid.
REFERENCES
1 T. E. FRIEDEMANNAND J. B. GRAESER, J. Biol. Chem., ioo (1933) 291.
2 j. B. BARKERAND W. H. SUMMERSON,ft. Biol. Chem., 138 (I94 I) 535.
3 C. S. PEDERSON, W. H. PETERSON AND E. B. FRED, J. Biol. Chem., 68 (1926) 151.
4 G. PFLEIDERERAND K. DOSE, Biochem. Z., 326 (I955) 436.
5 H. D. HORN AND F. H. BRUNS, Biochim. Biophys. Acta, 21 (1956) 378.
e K. KITAHARAAND S. FUKUI, Symposia on Enzyme Chem., 8 (1953) lO8.
7 j. G. DEWANAND D. E. GREEN, Biochem. ,[., 31 (1937) lO74.
s H. KATAGIRI,K. [MAI AND T. SUGIMORI,Symposia on Enzyme Chem., IO (1954) 203-
562
c . J . ix. VAN DEN HAMER, R. W. ELIAS
VOL. 2 9
(1958)
9 N. HAUGAARD,Federation Proc., 9 (195o) 182.
10 H. MCILWAIM,Biochem. J., 35 (1941) 13 TM
11 W. VV. UMBREIT, R. H. BURRIS AND J . F . STAUFFER, Manometric techniques, 2nd ed., Burgess
Publ. Co., Minneapolis, 1949.
is A. C. NEISH, Analytical Methods for Bacteriol. Fermentation, 2nd rev. ed., 1952.
13 M. BRIM, Biochem. Prep., 3 (I953) 6I.
14 M. BRIM, 1R. E. OLSEMAND F. J. STARE, Arch. Biochem. Biophys., 39 (1952) 214.
1~ TH. E. FRIEDEMAMNAND G. E. HAUGEM.J. Biol. Chem., 147 (1943) 415 •
Received March 6th, 1958
QUANTITATIVE
BY MEANS
AMINO
ACID ANALYSIS
OF A SINGLE
OF FOOD
ION-EXCHANGE
PROTEINS
COLUMN
D. S. BIDMEAD AND F. J. LEY
Unilever Ltd., Food Research Department, Sharnbrook, Bed[ordshire (Great Britain)
T h e MOORE AND STEIN technique of ion-exchange c h r o m a t o g r a p h y for q u a n t i t a t i v e
s e p a r a t i o n a n d d e t e r m i n a t i o n of the i n d i v i d u a l amino acids in a m i x t u r e has been
w i d e l y used in one or o t h e r of its modifications, and has been a p p l i e d to the analysis
of food-protein h y d r o l y s a t e s b y SCHRAM et al. 1. T h e y discussed the p r e c a u t i o n s to be
t a k e n in the acid h y d r o l y s i s of proteins in different m a t e r i a l s especially those with
a high c a r b o h y d r a t e content, a n d referred p a r t i c u l a r l y to the difficulty of a c c u r a t e l y
d e t e r m i n i n g cystine a n d methionine in an acid h y d r o l y s a t e . Cystine a n d t r y p t o p h a n
were d e s t r o y e d to a large e x t e n t during the h y d r o l y s i s so t h a t s e p a r a t e d e t e r m i n a t i o n s
were required for these a m i n o acids. Methionine also was p a r t i a l l y oxidized, forming
isomeric sulphoxides which a p p e a r e d as two small peaks in the c h r o m a t o g r a m , a n d
where the m e t h i o n i n e content of a p a r t i c u l a r sample was low, the a m o u n t present
h a d to be c a l c u l a t e d from the s u m m a t i o n of three v e r y small peaks; consequently
e s t i m a t i o n s were often inaccurate.
SCHRAM, MOORE AND BIGWOOD2 devised a m e t h o d for the s e p a r a t e d e t e r m i n a t i o n
of the cystine c o n t e n t of a protein b y oxidizing the protein m a t e r i a l with performic
acid prior to hydrolysis. This c o n v e r t e d the cystine residues to cysteic acid which
was stable to acid hydrolysis. The h y d r o l y s a t e was then c h r o m a t o g r a p h e d on Dowex-2
in the c h l o r o - a c e t a t e form, the cysteic acid being eluted as a s e p a r a t e p e a k following
all the o t h e r a m i n o acids. PARTRIDGE AND DAVIS3 also used performic acid for the
o x i d a t i o n of cystine b u t carried out the c h r o m a t o g r a p h y on Dowex-5o. This gave
quicker results, as the cysteic acid a p p e a r e d in the first few fractions. The same
reagent was used b y us to convert methionine q u a n t i t a t i v e l y to the sulphone which
was also stable to acid hydrolysis, a n d the technique was modified to p e r m i t simultaneous d e t e r m i n a t i o n of cystine as cysteic acid a n d methionine as methionine
sulphone. Thus, two s e p a r a t e analyses were required on each sample, one on the
oxidized m a t e r i a l for cystine a n d methionine, a n d one on the unoxidized m a t e r i a l
for all the o t h e r a m i n o acids.
Re#rences p. 567.