BAER ET AL.: J. ASSOC. OFF. ANAL. CHEM. (VOL. 63, NO.

3, 1980) 587

DAIRY PRODUCTS

Freezing Point Measurement of Lactose Hydrolysis in Acid Whey

and Lactose Solutions
ROBERT J. BAER, JOSEPH F. FRANK and MORRISON LOEWENSTEIN
University of Georgia, Animal and Dairy Science Department, Athens, GA 30602

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A method for determining lactose hydrolysis by ported that the true freezing points of these
freezing point determinations is described. There solutions were —0.408 and —0.600°C, respec-
is a direct relationship between freezing point and tively. Thus, there is a measurable difference
lactose hydrolysis in neutralized acid whey (r =
0.985) and lactose solutions (r = 0.998). In both between the Hortvet scale and the centigrade
the acid whey and the lactose solutions that con- scale. Most cryoscopes are currently calibrated
tained 4.6 to 5.0% lactose, the freezing point was to operate on the Hortvet (°H) scale.
depressed approximately 0.050°H for each 1% The freezing point method is commonly
lactose hydrolyzed. used to determine the presence of added water
in milk (10). The freezing point of a liquid is
Milk and whey can be treated with lactase based on the number of molecules in solution,
(/?-D-galactoside/galactohydrolase; EC 3.2.1.23) not the weight of the dispersed substances
to hydrolyze lactose to glucose and galactose (11), and therefore the hydrolysis of lactose
(1, 2, 3). This is advantageous in producing should have a direct effect on freezing point.
low lactose dairy products for individuals who Several methods are available for determin-
are lactose intolerant or lactase deficient (2, 3). ing degree of lactose hydrolysis (1, 12), but
The treatment of whey with lactase may offer these methods are complicated and lengthy.
a partial solution to the whey disposal problem Because freezing point measurement would
(1, 3). Benefits of hydrolyzing lactose are pre- offer a simple, quick, and accurate measure-
vention of lactose crystallization, increased ment of lactose hydrolysis, this study was de-
solubility, higher osmotic pressure, lower vis- signed to determine the relationship between
cosity, increased sweetness, and increased level freezing point depression and lactose hydroly-
of reducing sugars (1). A great potential exists sis.
for the use of lactase-treated whey in many
products. Lactase-treated whey has been used Experimental
in the manufacture of whey wines (4); Lacto- Materials
fruit, a prototype snack-type soft drink (5); Maxilact® T. G. (20,000 ONP units/g) lactase
ice cream (6); candies, bakery products, sirups, from Saccharomyces lactis was purchased from
and confections (1, 3). Gist-Brocades N.V., Delft, The Netherlands,
through GB Fermentation Industries, Inc., Des
Maxilact lactase is produced from the yeast Plaines, IL. Acid whey from cottage cheese was
Saccharomyces lactis and has optimum activity obtained from the University of Georgia Dairy
at pH 6.6-7.0 (7). The optimum temperature Manufacturing Plant. The 5.0% (w/v) lactose
for hydrolysis of lactose in milk and whey is solutions were prepared with C. P. lactose mono-
between 30 and 35°C (1). hydrate in deionized water.
Freezing point of cow's milk has long been
recognized as one of its most constant values, Methods
normally varying between —0.530 and The acid whey was analyzed, in duplicate or
—0.566cH (reported by investigators as triplicate, for the following components using the
-0.530 and -0.566°C) for all herd milk and methods indicated: Fat, Babcock test (13); total
most individual cow's milk (8, 9). In early solids, sec. 16.032 (10); ash, sec. 16.035 (10);
literature on cryoscopy it was reported that the nitrogen, micro-Kjeldahl method, sees. 34.021,
47.021 (10); titratable acidity with 0.1N NaOH
freezing point of a 7% sucrose solution was and phenolphthalein expressed as per cent lactic
—0.422°C and for a 10% sucrose solution the
value was — 0.621 °C; the Hortvet scale was This research was supported by State and Hatch funds
based on these measurements. It was later re- allocated to the Georgia Agricultural Experiment Stations.
0004-5756/80/6303-0587-04$01 .00
© Association of Official Analytical Chemists, Inc.
588 BAER ET At.: J. ASSOC. OFF. ANAL. CHEM. (VOL. 63, NO. 3, 1980)

Table 1. Original acid whey composition"

Lactose Total Titra table Freezing

Ash Protein, 6 % Fat, % (w/v) solids acidity 0 pH point, °H

0.860 0.975 0.01 4.82 7.102 0.56 4.62 -0.751

0.873 0.755 0.01 4.96 7.351 0.54 4.62 -0.774
0.847 0.822 0.02 4.98 7.195 0.57 4.65 -0.766
0.826 0.767 0.04 4.92 7.293 0.55 4.68 -0.759
0.819 0.790 0.04 4.89 7.321 0.57 4.65 -0.776

" Means of duplicate or triplicate determinations for each of 5 trials.

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6
Nitrogen X 6.38 = % protein.
r
Expressed as % lactic acid.

acid (13); and pH, determined with a glass elec- ple to be analyzed for lactose stopped the lactase
trode potentiometer (Model 701 digital pH meter, activity. ZAPT reagent was not added to samples
Orion Research Inc., 380 Putnam Ave, Cambridge, used for freezing point analysis. For each of the
MA 02139). 5 trials, duplicate freezing points and lactose de-
Lactose content was determined by a modifica- terminations were carried out. Fifteen freezing
tion of the method of Nickerson et al. (14) in that points and 10 lactose analyses were performed on
(under Preparation of Sample, part c) the 5 mL the original acid wheys. Forty-five freezing points
of filtrate was not diluted to 10 mL with water and 38 lactose analyses were performed on the
before addition of reagents. A Coleman 111- neutralized acid wheys.
Perkin-Elmer UV-Vis spectrophotometer was used
to read absorbances. Freezing points were ob- Preparation of 5.0% (w/v) Lactose
tained with a Fiske MS™ cryoscope calibrated A 5.0% (w/v) lactose solution (500 mL) was
with the following aqueous NaCl standards from prepared. Freezing point and lactose content were
Fiske Associates (Wheeler Rd. Burlington, MA determined. Lactase (0.4 g) was added to and
01803): -0.422°H (-0.408°C), -0.540°H thoroughly mixed with 400 mL lactose solution.
(—0.522°C), —0.621°H (—0.600°C), and distilled The remaining unhydrolyzed lactose solution was
water (0.000°C). Cryoscope readout was in milli- the control. Solutions were incubated at 33°C. At
degrees Hortvet (°H). various intervals (between 15 and 240 min after
inoculation) samples were removed for freezing
Preparation of Neutralized Acid Whey point determination and lactose analysis. A
greater distribution of freezing points and lactose
Acid whey (500 mL) was neutralized to pH 6.90
contents was obtained by using different inter-
with 5.0% (w/v) NaOH, and freezing point and
vals of time for hydrolysis of lactose. This was re-
lactose content were determined. Neutralized acid
peated for 5 trials with duplicate analyses for
whey (100 mL) not treated with lactase served as
freezing point and lactose on each sample. Forty
a control. Lactase (0.4 g) was added to 400 mL
freezing points and 36 lactose analyses were per-
neutralized whey with thorough mixing. The
formed on the lactose solutions.
mixture was incubated at 33°C. At 30, 60, and 150
min intervals after inoculation, samples were re-
moved for freezing point determination and lac- Results
tose analysis. The addition of 1 mL zinc acetate- Composition of acid whey is shown in Table
phosphotungstic acid (ZAPT) reagent to the sam- 1. There was no change in titratable acidity of

Table 2. Freezing point (°H) and lactose concentration (%, w/v) of neutralized
acid whey before and during lactose hydrolysis9

Before hydrolysis During hydrolysis

Freezing Freezing Freezing Freezing
Trial No. point Lactose point Lactose point Lactose point Lactose

1 -0.756 4.60 -0.825 3.12 -0.880 2.12 -0.953 1.02

2 -0.779 4.65 -0.840 3.46 -0.891 2.44 -0.959 1.20
3 -0.773 4.65 -0.845 3.22 -0.894 2.24 -0.961 1.08
4 -0.764 4.74 -0.839 3.11 -0.889 2.09 -0.954 1.08
5 -0.782 4.69 -0.851 3.18 -0.901 2.22 -0.964 1.17
Mean 6 -0.766 4.67 -0.829 3.22 -0.880 2.22 -0.947 1.11

" Means of duplicate analyses.

h
Mean of 5 trials with correction factors for lactase and NaOH applied.
 BAER ET AL.: J. ASSOC. OFF. ANAL. CHEM. (VOL. 63, NO. 3, 1980) 589

Table 3. Freezing point (°H) and lactose concentration of lactose solutions (5.0%, w/v)
before and during lactose hydrolysis"
Before hydrolysis During hydrolysis
Freezing Freezing Freezing Freezing
Trial No. point Lactose point Lactose point Lactose point Lactose

1 -0.298 5.04 -0.368 3.54 -0.458 1.60 -0.496 1.05

2 -0.298 5.01 -0.344 4.09 -0.433 2.18 -0.507 0.90
3 -J> _ t -0.332 4.40 -0.377 3.33 - 0 507 1.00
4 -0.297 5.01 -0.327 4.63 _b b
1.22
-0.486
5 -0.297 4.98 -0.349 3.97 -0.410 2.73 -0.440 2.12

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Mean d -0.298 5.01 -0.344 4.13 -0.420 2.46 -0.487 . 1.26

'• Means of duplicate analyses.

b
No data.
c
Mean of 5 trials with correction factors for lactase applied.

the whey samples during incubation. The neu- 5.0% lactose solutions, respectively. Points on
tralization of the acid whey with NaOH de- Figures 1 and 2 indicate measured changes be-
pressed the freezing point approximately tween freezing point depression and per cent
0.005 °H and also diluted the lactose content lactose hydrolyzed, which were calculated from
in the neutralized whey (Table 2). data in Tables 2 and 3, using correction factors
In both the acid whey and the 5.0% lactose where applicable. The lines of best fit as cal-
samples, the freezing point was depressed ap- culated from linear regression analysis are
proximately 0.006 °H by the addition of the shown. A linear relationship between freezing
lactase. point depression and per cent lactose hydro-
The effect of lactose hydrolysis on freezing lyzed for both the acid whey and the pure
point depression in neutralized acid whey and 5.0% lactose solution was obtained.
in the 5.0% lactose solution during various
stages of lactose hydrolysis is shown in Tables Discussion
2 and 3, respectively. The hydrolysis of lactose by lactase pro-
Figures 1 and 2 demonstrate the relationship duces not only the simple breakdown of lac-
between freezing point depression and per cent tose to glucose and galactose, but also the for-
lactose hydrolyzed in the acid whey and the mation of various oligosaccharides (15-21). A

90 90

80
?708 °
IO
*- 60
•o

* 50

4 0
|
u

" 30

* 20

10
0 .02 .04 .06 .08 .10 .12 .14 .16 .18 0 .02 .04 .06 .08 .10 .12 .14 .16 .18 .20
F r e e z i n g Point Depression H Freezing Point D e p r e s s ion H

FIG. 1—Effect of lactose hydrolysis on freezing point FIG. 2—Effect of lactose hydrolysis on freezing point
depression in neutralized acid whey with an initial lac- depression in 5.0% (w/v) lactose solutions.
tose concentration of 4.60-4.74%.
590 BAER ET At.: J. ASSOC. OFF. ANAL. CHEM. (VOL. 63, NO. 3, 1980)

small amount of galactose is always trans- can provide a fast, reliable method for estimat-
ferred to another sugar molecule during hy- ing the amount of lactose hydrolysis in whey
drolysis, thus there is slightly more free glu- and similar products.
cose than galactose present after the reaction
(1). Oligosaccharide formation probably causes Acknowledgments
the freezing point depression during lactose The authors wish to thank Diane Little for
hydrolysis to be less than it would be if there the statistical analysis and William R. McLean
was just a simple breakdown of lactose to glu- of Fiske Associates, Uxbridge, MA for furnish-
cose and galactose. ing the cryoscope.

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In milk the lactose and chlorides have the REFERENCES
greatest influence on freezing point (13), so this (1) Bouvy, F. A. M. Food Prod. Dev. (1975) 9(2),
should also be true for acid whey. Butterfat 10-14
has no effect on freezing point (13). The lac- (2) Guy, E. J., & Bingham, E. W. (1978) 7. Dairy
tose and chloride contents of whey will vary, Sci. 61,147-151
thus affecting the initial freezing point. Thus, (3) Holsinger, V. H. (1978) Food Technol. 32(3),
35-40
to use the freezing point method to determine (4) Roland, J. F., & Aim, W. L. (1975) Biotechnol.
proportionate lactose hydrolysis it is necessary Bioeng. 17,1443-1453
to determine the initial freezing point of the (5) Food Eng. (1978) 50(3), 116-117
sample. (6) Guy, E. J., Tamsma, A., Kontson, A. & Hol-
A freezing point correction factor account- singer, V. H. (1974) Food Prod. Dev. 8(8), 50
(7) Maxilact: Introduction and Applications of
ing for the effect of lactase addition and neu- Maxilact (1978) GB Fermentation Industries
tralization was used in conducting statistical Inc., Des Plaines, IL, Technical Bulletin pp.
analysis. For the neutralized acid whey with 1-4
no lactase added, 0.005cH was added to the (8) Keeney, P. G., & Kroger, M. (1974) in Funda-
freezing point depression to adjust for the mentals of Dairy Chemistry, 2nd Ed., B. H.
NaOH. In the neutralized acid whey with lac- Webb, A. H. lohnson, & J. A. Alford (Eds.),
AVI Publishing Co., Inc., Westport, CT, pp.
tase added 0.011°H was added to the freezing 876-877
point depression. For the pure lactose solu- (9) Shipe, W. F. (1959) J. Dairy Sci. 42, 1745-
tions, 0.006°H was added to the freezing point 1762
depression to adjust for the lactase effect on (10) Official Methods of Analysis (1980) 13th Ed.,
the freezing point. AOAC, Arlington, VA
(11) Nielsen, V. H. (1976) Am. Dairy Rev. 38(6),
Statistical analysis demonstrated a direct re- 20
lationship between freezing point and lactose (12) Maxilact: Optimal process conditions for
hydrolysis in neutralized acid whey and lac- treatment. (1978) GB Fermentation Industries
tose solutions. A correlation coefficient of Inc., Des Plaines, IL, Technical Bulletin, pp.
0.985 was obtained with the whey. In the acid 1-4
whey for each 1% lactose hydrolyzed the (13) Atherton, H. V., & Newlander, J. A. (1977) in
Chemistry and Testing of Dairy Products,
freezing point was depressed 0.0510°H with a 4th Ed., AVI Publishing Co., Inc., Westport,
standard error of 3.0%. In the lactose solu- CT, pp. 38, 71, 250
tions a correlation coefficient of 0.998 was ob- (14) Nickerson, T. A., Vujicic, I. F., & Lin, A. Y.
tained. In the lactose solutions for each 1% (1976) J. Dairy Sci. 59, 386-390
lactose hydrolyzed the freezing point was de- (15) Aronson, M. (1952) Arch. Biochem. Biophys.
pressed 0.0486°H with a standard error of 39, 370-378
(16) Pazur, J. H. (1953) Science 117, 355-356
1.1%. (17) Pazur, I. H., Tipton, C. L., Budovich, T., &
By determining the initial lactose concentra- Marsh, J. M. (1958) J. Am. Chem. Soc. 80,
tion and freezing point of a sample the per 119-121
cent lactose hydrolyzed can be calculated from (18) Roberts, H. R., & McFarren, E. F. (1953) J.
the subsequent freezing point, since the freez- Dairy Sci. 36, 620-632
(19) Roberts, H. R., & Pettinati, J. D. (1957) J.
ing point will be depressed 0.050°H for each Agric. Food Chem. 5, 130-134
1% reduction in lactose concentration. In solu- (20) Toba, T., & Adachi, S. (1978) J. Dairy Sci. 61,
tions containing approximately 5.0% lactose, 33-38
the freezing point method can be used to de- (21) Wierzbicki, L. E., & Kosikowski, F. V. (1973)
termine up to 80% lactose hydrolysis. Our re- 7. Dairy Sci. 56,1400-1404
sults show that freezing point measurements Received October 25. 1979. Accepted December 18, 1979.