Colorimetric Estimation of Lactose and
Its H y d r o l y t i c Products
ABSTRACT
Current interest in enzymic and acid
hydrolysis of lactose creates a need for
sensitive methods for routine measure-
ment of decreases in lactose and con-
comitant increases in glucose and galac-
rose. To differentiate among these sugars,
two color reactions were used. Lactose
reacted with methylamine in h o t alkaline
solution to form a red compound with
maximum absorbance at 540 nm; glucose
and galactose do not interfere under these
conditions. In the other reaction, glucose
and galactose were measured by the
development of a blue color with maxi-
mum absorbance at 710 nm after heating
in an ammonium molybdate solution
buffered with phosphate-phthalate at pH
5.3, a condition under which lactose does
not interfere. Proper color development
in both reactions requires careful control
of conditions. Both methods have been
used to follow the acid hydrolysis of a
lactose solution and also have been
adapted to milk and whey by use o f zinc
acetate-phosphotungstic acid to remove
the proteins prior to color development.
INTRODUCTION
Current interest in improving whey utiliza-
tion is the occasion for research on lactose
which makes up some 70% of the solids in
whey. None of the methods suggested for
analysis of lactose is completely satisfactory as
a convenient, routine procedure.
In studies of lactose hydrolysis by acid and
enzyme, methods were needed to measure the
decrease in lactose and concomitant increase in
glucose and galactose. F e w reactions distinguish
among these reducing sugars. In one, lactose
Received August 1, 1975.
l Fulbright Scholar on leave from Faculty of
Agriculture, Novi Sad, Yugoslavia.
386
T. A. NICKERSON, I. F. VUJICIC 1, and A. Y. LIN
Department of Food Science and Technology
University of California
Davis 95616
reacts with methylamine in h o t alkaline solu-
tion to forln a red compound. The reaction,
first described by Fearon (2), is the basis for
quantitative procedures developed by Malpress
and Morrison (6), Horowitz et al. (4), and
Karasz et al. (5). Our paper describes conditions
in which this reaction can be used to measure
lactose in whey or milk products.
Benham and Despaul (1) developed a quanti-
tative colorimetric m e t h o d based on the blue
color produced by heating sugar in the presence
of ammonium molybdate and potassium dihy-
drogen phosphate. This was modified by Potter
(7) to determine the degree of lactose hydroly-
sis. Although glucose and galactose produce
different amounts of color, the reaction has the
advantage that lactose gives essentially no color.
Hence, the quantity of lactose has no effect on
the color developed.
This study describes modifications of these
two methods for determining lactose and its
hydrolytic products.
EXPERIMENTAL METHODS
Determination of Lactose by Methylamine
Reagents.
(a) Zinc acetate-phosphotungstic acid
(ZAPT) reagent.-Dissolve 25.0 g
zinc acetate and 12.5 g phospho-
tungstic acid in water. Add 20 ml
glacial acetic acid and dilute to 100
ml.
(b) Glycine-NaOH b u f f e r . - M i x 150 ml
o f glycine solution containing
2.4768 g glycine and 1.9359 g NaC1
with 850 ml .385 N NaOH to pH
12.8.
(c) Methylamine s o l u t i o n . - 5.0%
(wt/vol) methylamine-HC1 in dis-
tilled water. Store in refrigerator.
(d) Sodium sulfite solution.-1.0%
(wt/vol). Dissolve 1.0 g Na2SO3 in
distilled water and dilute to 100 ml.
Prepare daily.
 COLORIMETRY OF LACTOSE
(e) L a c t o se standard s o l u t i o n s . - 1)
Stock solution. Dissolve 2.6315 g
lactose monohydrate, USP grade,
and dilute to 200 ml with .1%
(wt/vol) benzoic acid. Store in re-
frigerator. 2) Working solutions.
.50, .75, 1.00, 1.25, and 1.50 nag
lactose/ml. Dilute 10, 15, 20, 25,
and 30 ml stock solution to 250 ml.
Preparation of sample.
(a) To 8 ml of milk or whey add 1 ml
ZAPT reagent. Dilute to 10 ml,
mix, and after 10 rain filter (with
Whatman No. 1). Correct for vol-
ume of fat and protein by method
of Grimbleby (3).
(b) To .5 ml filtrate add .5 ml 1 N
NaOH, dilute to 10 ml, and filter
(with Whatman No. 1).
(c) Dilute 5 ml of filtrate to 10 ml,
which becomes the sample.
Procedure.
(a) Pipet 5 ml of standard, unknown,
or water (blank) into 25 ml (18 mm
OD) test tube.
(b) Add 5 ml glycine-NaOH buffer.
(c) Add .5 ml methylamine solution.
(d) Add .5 ml sodium sulfite solution
and mix thoroughly.
(e) Heat tubes in a thermostatically
controlled water bath at 65 C for
exactly 25 min, and cool immedi-
ately in an ice-water bath for 2 rain
to stop reaction. We recommend
including standard solutions for
comparison.
(f) Read absorbance against blank
(using water in place of sample) at
540 nm in a Beckman Model DB
spectrophotometer.
(g) Make standard curve by plotting
absorbance against lactose concen-
tration in standard solutions.
Determination of Glucose and Galactose
Reagents.
(a) A m m o n i u m molybdate s o l u t i o n . -
6% (wt/vol). Dissolve 15.0 g
(NH4)6 Mo7024 " 4 H 2 0 analytical
grade in water and dilute to 250 ml.
387
(b) Ph osphate-phthalate b u f f e r . - p H
5.3. Dissolve .3 g KH2PO4 and 5.1
g potassium acid phthalate in 158
.1 N NaOH and dilute to 250 ml.
Add a few drops of toluene.
(c) Glucose-galactose standard solu-
t i o n s . - 1 ) Stock solution. 1.000 g
glucose and 1.000 g galactose are
dissolved and diluted in .1%
(wt/vol) benzoic acid to 200 ml to
prevent fermentation. Store in re-
frigerator. 2) Working solutions pre-
pared with .1% benzoic acid solu-
tion: .5, 1.0, 1.5, 2.0, 2.5, and 3.0
mg glucose-galactose/ml.
Procedure.
(a) Pipet 5 ml phosphate-phthalate buf-
fer in a 25-ml test tube.
(b) Add 1.0 ml standard, unknown, or
water (blank).
(c) Add 5 ml ammonium molybdate
solution and mix thoroughly.
(d) Heat tubes in a boiling-water bath
for exactly 15 min, and cool imme-
diately in tap water for 5 min to
stop reaction. We recommend in-
cluding standard solutions for com-
parison.
(e) Read absorbance against blank
(using water in place of sample) at
710 nm in a Beckman Model DB
spectrophotometer.
(f) Make standard curve by plotting
absorbance against concentrations
in standard solutions.
RESULTS A N D DISCUSSION
The methylamine color method of Fearon
(2) was modified to increase sensitivity and
reliability. The modification involves use of
buffer for pH control and a change in reaction
temperature.
Effect of pH
The literature indicated that a highly alka-
line solution is needed for development of the
red color reaction but did not specify further.
Table 1 shows the effect of pH on color
development. Color did not develop until pH
was over 12. As pH was increased, the red color
became more intense, then lighter, and above
Journal of Dairy Science Vol. 59, No. 3
388 NICKERSON ET AL.

TABLE 1. Effect of pH on color development•

Io / LACTOSE
pH As 40 Color
0
10.75 .015 •. . 8 / (WITHOUT
11.70 .015
12.50 .475 orange-red
12.70 .650 red
12.80 .670 red
12.90 .585 red
13.00 .505 light red
13.15 .300 pink
13.25 .190 yellow GLU-GAL
13.28 .160 yellow 0 5 I0 15 20
CONCENTRATION (mg/ml)

FIG. 1. Calibration curve for standard lactose

pH 13 c h a n g e d to yellow. Our p r e l i m i n a r y
o b s e r v a t i o n s during the e v o l u t i o n of a suitable
p r o c e d u r e suggested t h a t closer pH c o n t r o l was
necessary f o r r e p r o d u c i b l e results since t h e
r e a c t i o n s h o w s a sharp o p t i m u m near pH 12.7.
A g l y c i n e - N a O H b u f f e r p r o v e d s a t i s f a c t o r y in
stabilizing p H u n d e r the c o n d i t i o n s of this test.
Effect of Reaction Temperature
Various investigators have used d i f f e r e n t
t e m p e r a t u r e t r e a t m e n t s to develop the color.
F o r e x a m p l e , H o r o w i t z et al. (4) used 55 C for
30 rain w h e r e a s Karasz e t al. (5) used 95 C for
5 rain. T h e color t h a t d e v e l o p e d in a q u e o u s
lactose s o l u t i o n s at pH 12.7 was u n s t a b l e in a
b o i l i n g - w a t e r bath. It d e v e l o p e d quickly b u t
also d e c o m p o s e d rapidly, m a k i n g o p t i m u m
color difficult to achieve. On the o t h e r h a n d , at
55 C color d e v e l o p m e n t r e q u i r e d an excessive
solutions, effect of eliminating Na 2 SO 3 from reagents,
and color development by glucose and galactose with
the methylamine (lactose) reaction•
reaction time (Table 2). Finally, 65 C was
selected because a deep-red c o l o r developed in a
reasonable time. Both time a n d t e m p e r a t u r e of
h e a t i n g m u s t be c o n t r o l l e d carefully a n d the
optical density read w i t h i n 10 m i n for consis-
t e n t results.
Red color d e v e l o p e d slowly overnight at
r o o m t e m p e r a t u r e in the p r e s e n c e o f glycine
b u f f e r b u t in t h e a b s e n c e o f m e t h y l a m i n e .
Therefore, the test s o l u t i o n a n d buffer m u s t
n o t be m i x e d u n t i l analysis.
Effect of Hydrolysis Products in the
Assay Mixture
To aid the procedure to measure changes in
lactose c o n c e n t r a t i o n during acid or e n z y m i c

TABLE 2. Effect of heating at 55 and 65 C.

7 GALACTOSE

As4o
Time 55 C 65 C
_~ 5
(min)
~ 4
10 .245
15 10;2 .640 3
18 . . . . 79O
(.
20 .O40 . . .
21 . . . . 875 "~ 2

25 .125 .925
I
30 .270 .90O
35 .435 .850 LACTOSE
40 .580 ...
45 . . . . 825 CONCENTRATION ( g / l O 0 ml)
55 .850 .720
90 .850 ... F1G. 2. Calibration curves for standard sugar
solutions with the molybdate reaction.

Journal of Dairy Science Vol. 59, No. 3

 COLORIMETRY OF LACTOSE 389

TABLE 3. Summary of recovery experiments from skim milk with added lactose using the methylamine lactose
test a .

Av. lactose Theoretical

Sample found value Recovery

(mg/ml) (%)
1) Skim milk 47.63 +- .97 47.63-+.97
2) + 12.5 mg lactose b 58.89 -+ 2.03 60.13 + .97 97[96 -+4.30
3) + 25.0 mg lactose b 72.21 -+ 1 . 6 6 72.63 -+ .97 9 9 . 4 4 +- 3.03
4) + 37.5 mg lactose b 84.34 +- 1.65 85.13 -+ .97 99.08 -+ 2.58

asix replications for each concentration.

bAnhydrous lactose.

hydrolysis, the effects of glucose a n d galactose in p r o d u c i n g a clear f i l t r a t e s u i t a b l e f o r lactose

were negligible (Fig. 1). Glucose a n d galactose
p r o d u c e a y e l l o w color u n d e r c o n d i t i o n s o f t h e
test w i t h a m a x i m u m a b s o r b a n c e at a p p r o x i -
m a t e l y 370 n m b u t little a b s o r b a n c e at 540 nm,
the o p t i m u m w a v e l e n g t h for m e a s u r i n g light
a b s o r b a n c e b y the r e d color. This was t h e
w a v e l e n g t h u s e d also by Karasz e t al. (5).
analysis. His zinc a c e t a t e - p h o s p h o t u n g s t i c acid
(ZAPT) reagent removed the protein without
i n t e r f e r i n g w i t h the c o l o r r e a c t i o n . T r i c h l o r o -
acetic acid was u n s u i t a b l e since it d e c r e a s e d the
a m o u n t of red color p r o d u c e d . Table 3 s u m m a -
rizes t h e recovery e x p e r i m e n t s w i t h various
q u a n t i t i e s o f lactose a d d e d t o skim milk.

Effect of Reducing Agent, Sodium Sulfite Analysis of Glucose and Galactose

H o r o w i t z a n d c o w o r k e r s (4) b u b b l e d nitro-
gen t h r o u g h t h e i r biological samples for 10 to
15 m i n p r i o r to heating, b u t Karasz et al. (5)
f o u n d t h a t a r e d u c i n g agent, such as s o d i u m
sulfite, stabilized the color. Figure 1 s h o w s t h e
i m p r o v e d c o l o r d e v e l o p m e n t with s o d i u m sul-
fite in t h e r e a c t i o n m i x t u r e .
F o r m i l k a n d w h e y samples, the p r o t e i n
m u s t be r e m o v e d p r i o r to color d e v e l o p m e n t .
Many p r o t e i n p r e c i p i t a n t s have b e e n used in
different procedures for lactose analysis.
G r i m b l e b y (3) c o m p a r e d several reagents useful
T h e p r o c e d u r e of P o t t e r (7) was s e l e c t e d to
measure glucose a n d galactose. This is based o n
t h e d e v e l o p m e n t of m o l y b d e n u m blue. The
i n t e n s i t y of color varies w i t h t h e t y p e of sugar,
as in Fig. 2. A l t h o u g h glucose a n d galactose
differ in t h e a m o u n t of color p r o d u c e d , this
p r o c e d u r e is useful in f o l l o w i n g h y d r o l y s i s as
long as t h e 1:1 ratio is n o t a l t e r e d a p p r e c i a b l y
d u r i n g h y d r o l y s i s . F o r t u n a t e l y , lactose pro-
duces little color u n d e r t h e c o n d i t i o n s o f this
test so t h a t changes in lactose c o n c e n t r a t i o n are
of little c o n s e q u e n c e in f o l l o w i n g hydrolysis.

"FABLE 4. Summary of recovery experiments from skim milk with added lactose using the molybdate (glucose-
galactose) reaction a.

Theoretical
Sample Av. lactose value Recovery

(g/lO0 ml) (%)

1) Skim milk 0 0 ...
2) + 5.00 g (;-GI. h 4.79 +- .27 5.O0 95.85 -+ 5.33
3) + 10.00 g G-GI. 9.51 -+ .32 10.00 95.12 -+ 3.15
4) + 15.00 g G-GI. 14.00 + .22 15.00 93.30 ± 1.44

asix replications for each concentration.

bEqual weights of anhydrous glucose and galactosc.

Journal of Dairy Science Vol. 59, No. 3

390 NICKERSON ET AL.
C o n d i t i o n s for d e v e l o p m e n t o f t h e color are
e x t r e m e l y critical, r e q u i r i n g careful c o n t r o l for
c o n s i s t e n t results. T h e c o l o r r e a c t i o n is n o t
c o m p l e t e u n d e r test c o n d i t i o n s , n o r is it com-
p l e t e even a f t e r 45 m i n in a b o i l i n g - w a t e r b a t h .
T h e r e f o r e , h e a t i n g m u s t be well c o n t r o l l e d .
B e n h a m a n d Despaul (1) r e p o r t e d t h a t t h e
o p t i m u m p H f o r the r e a c t i o n was a b o u t 5.3. We
f o u n d t h a t a p h o s p h a t e - p h t h a l a t e b u f f e r im-
p r o v e d reliability a n d r e p r o d u c i b i l i t y . R e d u c i n g
t h e r e a c t i o n t i m e e l i m i n a t e d occasional p r o b -
lems w i t h cloudiness d u r i n g h e a t i n g t h a t gave
erratic readings. T h e s h o r t e r r e a c t i o n p e r i o d
also allowed a b r o a d e r r a n g e o f sugar c o n c e n t r a -
t i o n t o b e c o v e r e d in the t e s t solution.
Table 4 s u m m a r i z e s t h e r e c o v e r y experi-
m e n t s w h e r e various a m o u n t s o f an e q u a l
m i x t u r e o f glucose a n d galactose were a d d e d to
skim milk.
B o t h of these tests have b e e n useful in o u r
research o n lactose a n d s h o u l d - b e w e l c o m e to
s u p p l e m e n t t h e available p o l a r i m e t r i c a n d en-
z y m i c p r o c e d u r e s . Their use in s t u d y i n g acid
and e n z y m a t i c h y d r o l y s i s o f lactose will be
r e p o r t e d in a s u b s e q u e n t paper.
Journal of Dairy Science Vol. 59, No. 3
ACKNOWLEDGMENT
This research was s u p p o r t e d in part b y a
g r a n t f r o m t h e Dairy Council o f California.
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colorimetric method for determining carbohy-
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2 Fearon, W. R. 1942. The detection of lactose and
maltose by means of methylamine. Analyst
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3 Grimbleby, F. H. 1956. The determination of
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4 Horowitz, M. G., H. M. Davidson, F. D. Howard,
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1971. Determination of added lactose (nonfat dry
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semi-micro estimation of lactose alone and in the
presence of other sugars. Biochem. J. 45:455.
7 Potter, F. E. 1950. A colorimetric method for the
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