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Colorimetric Estimation of Lactose and
Its H y d r o l y t i c Products
T. A. NICKERSON, I. F. VUJICIC 1, and A. Y. LIN
Department of Food Science and Technology
University of California
Davis 95616
ABSTRACT reacts with methylamine in h o t alkaline solu-
Current interest in enzymic and acid tion to forln a red compound. The reaction,
hydrolysis of lactose creates a need for first described by Fearon (2), is the basis for
sensitive methods for routine measure- quantitative procedures developed by Malpress
ment of decreases in lactose and con- and Morrison (6), Horowitz et al. (4), and
comitant increases in glucose and galac- Karasz et al. (5). Our paper describes conditions
rose. To differentiate among these sugars, in which this reaction can be used to measure
two color reactions were used. Lactose lactose in whey or milk products.
reacted with methylamine in h o t alkaline Benham and Despaul (1) developed a quanti-
solution to form a red compound with tative colorimetric m e t h o d based on the blue
maximum absorbance at 540 nm; glucose color produced by heating sugar in the presence
and galactose do not interfere under these of ammonium molybdate and potassium dihy-
conditions. In the other reaction, glucose drogen phosphate. This was modified by Potter
and galactose were measured by the (7) to determine the degree of lactose hydroly-
development of a blue color with maxi- sis. Although glucose and galactose produce
mum absorbance at 710 nm after heating different amounts of color, the reaction has the
in an ammonium molybdate solution advantage that lactose gives essentially no color.
buffered with phosphate-phthalate at pH Hence, the quantity of lactose has no effect on
5.3, a condition under which lactose does the color developed.
not interfere. Proper color development This study describes modifications of these
in both reactions requires careful control two methods for determining lactose and its
of conditions. Both methods have been hydrolytic products.
used to follow the acid hydrolysis of a
lactose solution and also have been EXPERIMENTAL METHODS
adapted to milk and whey by use o f zinc
Determination of Lactose by Methylamine
acetate-phosphotungstic acid to remove
the proteins prior to color development. Reagents.
(a) Zinc acetate-phosphotungstic acid
INTRODUCTION (ZAPT) reagent.-Dissolve 25.0 g
Current interest in improving whey utiliza- zinc acetate and 12.5 g phospho-
tion is the occasion for research on lactose tungstic acid in water. Add 20 ml
which makes up some 70% of the solids in glacial acetic acid and dilute to 100
whey. None of the methods suggested for ml.
(b) Glycine-NaOH b u f f e r . - M i x 150 ml
analysis of lactose is completely satisfactory as
o f glycine solution containing
a convenient, routine procedure.
2.4768 g glycine and 1.9359 g NaC1
In studies of lactose hydrolysis by acid and
with 850 ml .385 N NaOH to pH
enzyme, methods were needed to measure the
decrease in lactose and concomitant increase in 12.8.
(c) Methylamine s o l u t i o n . - 5.0%
glucose and galactose. F e w reactions distinguish
(wt/vol) methylamine-HC1 in dis-
among these reducing sugars. In one, lactose
tilled water. Store in refrigerator.
(d) Sodium sulfite solution.-1.0%
(wt/vol). Dissolve 1.0 g Na2SO3 in
Received August 1, 1975.
l Fulbright Scholar on leave from Faculty of distilled water and dilute to 100 ml.
Agriculture, Novi Sad, Yugoslavia. Prepare daily.
386
COLORIMETRY OF LACTOSE 387
(e) L a c t o se standard s o l u t i o n s . - 1) (b) Ph osphate-phthalate b u f f e r . - p H
Stock solution. Dissolve 2.6315 g 5.3. Dissolve .3 g KH2PO4 and 5.1
lactose monohydrate, USP grade, g potassium acid phthalate in 158
and dilute to 200 ml with .1% .1 N NaOH and dilute to 250 ml.
(wt/vol) benzoic acid. Store in re- Add a few drops of toluene.
frigerator. 2) Working solutions. (c) Glucose-galactose standard solu-
.50, .75, 1.00, 1.25, and 1.50 nag t i o n s . - 1 ) Stock solution. 1.000 g
lactose/ml. Dilute 10, 15, 20, 25, glucose and 1.000 g galactose are
and 30 ml stock solution to 250 ml. dissolved and diluted in .1%
(wt/vol) benzoic acid to 200 ml to
Preparation of sample. prevent fermentation. Store in re-
frigerator. 2) Working solutions pre-
(a) To 8 ml of milk or whey add 1 ml
pared with .1% benzoic acid solu-
ZAPT reagent. Dilute to 10 ml,
tion: .5, 1.0, 1.5, 2.0, 2.5, and 3.0
mix, and after 10 rain filter (with
mg glucose-galactose/ml.
Whatman No. 1). Correct for vol-
ume of fat and protein by method
Procedure.
of Grimbleby (3).
(b) To .5 ml filtrate add .5 ml 1 N (a) Pipet 5 ml phosphate-phthalate buf-
NaOH, dilute to 10 ml, and filter fer in a 25-ml test tube.
(with Whatman No. 1). (b) Add 1.0 ml standard, unknown, or
(c) Dilute 5 ml of filtrate to 10 ml, water (blank).
which becomes the sample. (c) Add 5 ml ammonium molybdate
solution and mix thoroughly.
Procedure. (d) Heat tubes in a boiling-water bath
for exactly 15 min, and cool imme-
(a) Pipet 5 ml of standard, unknown,
diately in tap water for 5 min to
or water (blank) into 25 ml (18 mm
stop reaction. We recommend in-
OD) test tube.
cluding standard solutions for com-
(b) Add 5 ml glycine-NaOH buffer.
parison.
(c) Add .5 ml methylamine solution.
(e) Read absorbance against blank
(d) Add .5 ml sodium sulfite solution
(using water in place of sample) at
and mix thoroughly.
710 nm in a Beckman Model DB
(e) Heat tubes in a thermostatically
spectrophotometer.
controlled water bath at 65 C for
(f) Make standard curve by plotting
exactly 25 min, and cool immedi-
absorbance against concentrations
ately in an ice-water bath for 2 rain
in standard solutions.
to stop reaction. We recommend
including standard solutions for
RESULTS A N D DISCUSSION
comparison.
(f) Read absorbance against blank The methylamine color method of Fearon
(using water in place of sample) at (2) was modified to increase sensitivity and
540 nm in a Beckman Model DB reliability. The modification involves use of
spectrophotometer. buffer for pH control and a change in reaction
(g) Make standard curve by plotting temperature.
absorbance against lactose concen-
tration in standard solutions. Effect of pH
The literature indicated that a highly alka-
Determination of Glucose and Galactose
line solution is needed for development of the
Reagents. red color reaction but did not specify further.
(a) A m m o n i u m molybdate s o l u t i o n . - Table 1 shows the effect of pH on color
6% (wt/vol). Dissolve 15.0 g development. Color did not develop until pH
(NH4)6 Mo7024 " 4 H 2 0 analytical was over 12. As pH was increased, the red color
grade in water and dilute to 250 ml. 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•
o
I / 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 L
GLU-GA
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 solutions, effect of eliminating Na 2 SO 3 from reagents,
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 and color development by glucose and galactose with
p r o c e d u r e suggested t h a t closer pH c o n t r o l was the methylamine (lactose) reaction•
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. reaction time (Table 2). Finally, 65 C was
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 selected because a deep-red c o l o r developed in a
stabilizing p H u n d e r the c o n d i t i o n s of this test. 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
Effect of Reaction Temperature optical density read w i t h i n 10 m i n for consis-
Various investigators have used d i f f e r e n t t e n t results.
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. Red color d e v e l o p e d slowly overnight at
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 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
30 rain w h e r e a s Karasz e t al. (5) used 95 C for 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 .
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 Therefore, the test s o l u t i o n a n d buffer m u s t
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 n o t be m i x e d u n t i l analysis.
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 Effect of Hydrolysis Products in the
Assay Mixture
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 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 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
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 (ZAPT) reagent removed the protein without
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 - 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 -
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, 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
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 m o u n t of red color p r o d u c e d . Table 3 s u m m a -
a b s o r b a n c e b y the r e d color. This was t h e rizes t h e recovery e x p e r i m e n t s w i t h various
w a v e l e n g t h u s e d also by Karasz e t al. (5). 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- 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
gen t h r o u g h t h e i r biological samples for 10 to measure glucose a n d galactose. This is based o n
15 m i n p r i o r to heating, b u t Karasz et al. (5) 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
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 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,
sulfite, stabilized the color. Figure 1 s h o w s t h e as in Fig. 2. A l t h o u g h glucose a n d galactose
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- differ in t h e a m o u n t of color p r o d u c e d , this
fite in t h e r e a c t i o n m i x t u r e . 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
F o r m i l k a n d w h e y samples, the p r o t e i n 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
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 . 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-
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 duces little color u n d e r t h e c o n d i t i o n s o f this
different procedures for lactose analysis. test so t h a t changes in lactose c o n c e n t r a t i o n are
G r i m b l e b y (3) c o m p a r e d several reagents useful 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 ACKNOWLEDGMENT
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 This research was s u p p o r t e d in part b y a
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 g r a n t f r o m t h e Dairy Council o f California.
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
REFERENCES
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colorimetric method for determining carbohy-
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 drates. Anal. Chem. 20:933.
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 - 2 Fearon, W. R. 1942. The detection of lactose and
lems w i t h cloudiness d u r i n g h e a t i n g t h a t gave maltose by means of methylamine. Analyst
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 67:130.
3 Grimbleby, F. H. 1956. The determination of
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 - lactose in milk. J. Dairy Res. 23:229.
t i o n t o b e c o v e r e d in the t e s t solution. 4 Horowitz, M. G., H. M. Davidson, F. D. Howard,
Table 4 s u m m a r i z e s t h e r e c o v e r y experi- and F. J. Reithel. 1951. Determination of lactose
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 in biological materials. Anal. Chem. 23 : 375.
5 Karasz, A. B., W. M. Gantenbein, and L. Bokus.
m i x t u r e o f glucose a n d galactose were a d d e d to
1971. Determination of added lactose (nonfat dry
skim milk. milk) in meat products. I. Colorimetric method. J.
B o t h of these tests have b e e n useful in o u r Ass. Off. Agr. (hem. 54:1436.
research o n lactose a n d s h o u l d - b e w e l c o m e to 6 Malpress, F. H., and A. B. Morrison. 1949. The
semi-micro estimation of lactose alone and in the
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-
presence of other sugars. Biochem. J. 45:455.
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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 quantitative determination of the degree of lactose
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Journal of Dairy Science Vol. 59, No. 3
