Lactase A c t i v i t y o f C u l t u r e d a n d A c i d i f i e d D a i r y P r o d u c t s 1

A. KILARA and K. M. SHAHANI

Department of Food Science and Technology
University of Nebraska
Lincoln 68583

ABSTRACT was also lactose tolerant. Those villagers that

could not digest milk were able to digest a
A m e t h o d was standardized for deter-
mining lactase activity in cultured and partially fermented milk product with reduced
acidified dairy products such as cottage lactose content. In the manufacture of fer-
mented milk products, some of the lactose is
cheese, sour cream, and yogurt. Cottage
converted to lactic acid by microorganisms.
cheese and sour cream prepared by both
Vakil and Shahani ( 1 4 ) d e m o n s t r a t e d lactase in
the cultured and acidified processes and
yogurt prepared by the direct acidifica-
Streptococcus lactis. Citti et al. (3) reported
that the lactase of S. lactis was inducible. More
tion process did not possess lactase activ-
recently, a survey of 15 microorganisms re-
ity. However, cultured yogurt possessed
vealed that among others, Streptococcus tber-
considerable enzyme activity mainly due
mopbilus and Lactobacillus bulgaricus were
to lactase as an endoenzyme in the yogurt
able to hydrolyze 50 to 60% of lactose in
culture (Lactobacillus bulgaricus and
acidified cheese whey within 5 h at 50 C (15).
Streptococcus thermophilus). Enzyme in
yogurt increased with time of incubation,
S. lactis, S. thermophilus, and L. bulgaricus
commonly are used as lactic cultures in the
reaching a m a x i m u m of 8 orthonitro-
phenol /3-d-galactopyranoside units per manufacture of yogurt, sour cream, and cottage
gram of yogurt in 4 h. S. thermophilus cheese. Recent technological advances have
contained approximately three times evolved processes to obtain these products by
more lactase than did L. bulgaricus. Also, direct acidification. Instead of relying on bacte-
an in vitro digestion process appeared to ria to produce acid, acidulants such as glucono-
enhance the release of lactase from the delta-lactone, hydrochloric acid, and citric acid
composite yogurt culture. It was felt that are employed.
cultured yogurt would be beneficial to The purpose of this investigation was to
individuals suffering from lactose intoler- study lactase in cultured and acidified dairy
products, namely, yogurt, cottage cheese, and
ance not only because of reduced lactose
sour cream. The behavior of the composite
but also because of lactase.
yogurt culture, L. bulgaricus and S. thermo-
philus, in skim milk and broth systems was
INTRODUCTION determined. Also, the effect of an in vitro
gastric digestion of yogurt on lactase was
Considerable attention has been focused on
determined.
the problem of lactose intolerance, a condition
that is believed to arise from the deficiency of
lactase (t3-galactosidase) in the intestinal micro- MATERIALS AND METHODS
villi, particularly in individuals of certain ethnic
groups (6, 9, 13). Kretchmer (9) in a study of Manufacture of Products
various Nigerian tribes discovered that a tribe Cultured yogurt, cottage cheese, and sour
involved in milk production and consumption cream were prepared by conventional methods
as described b y Kosikowski (8) and Emmons
and Tuckey (4). Acidified yogurt was prepared
Received December 3, 1975. as described b y Reddy and Shahani (12). The
1Published as paper No. 5020, Journal Series, acidified cottage cheese samples were prepared
Agricultural Experiment Station, Lincoln, NE. Re- by a patented process (5) and acidified sour
search was conducted on project 16-026. This work
was supported in part by grants from Dairy Research cream was produced by a technique described
Inc. (DRINC) and Dannon Products Inc. by Little (10).

2031
2032 KILARA AND SHAHANI

Preparation of Samples for Lactase Assay bulgaricus and S. tbermophilus was grown in
APT broth, S. tbermophilus appeared to domi-
Ten grams of the product were macerated
nate the flora. Therefore, these studies dealt
with water in a Waring blender and the final
with single organisms only. One hundred milli-
volume was made to 100 ml in a volumetric
liters of broth were inoculated with a 1% active
flask. Aliquots of the diluted sample were
culture and incubated at 43 C for 16 h. At the
subjected to sonic disintegration by a Biosonik
end of the incubation period, cells were har-
III cell disruptor (Bronwill Scientific Co., Roch-
vested by passing the broth through a Millipore
ester, NY) at a frequency of 12 KHz. One-milli-
filter. The resulting cell paste was diluted to 10
liter portions of the sonicated solution were
ml with water. A 5-ml aliquot of the cell
used to determine enzyme activity.
suspension was divided into two portions. One
To study the effect of incubation time on
portion of the sonicated suspension was used
the enzyme activity of yogurt, 100-g samples of
directly for the assay of lactase and the other
milk were each inoculated at 4% with an active
portion was centrifuged at 10,000 x g to obtain
yogurt culture. This composite inoculated milk
a cell free supernatant which also was assayed
was apportioned in 10-g batches and incubated
for lactase. The lactase activity of the former
at 43 C for periods up to 8 h. Samples were
fraction indicated the total enzyme while the
taken at hourly intervals for determining titrat-
latter reflected the free enzyme. The difference
able acidity, pH, and lactase activity. Titratable
between the total and free enzyme was taken as
acidity was determined by titrating a 9 g sample
the enzyme bound to the cell wall. Dry weights
with .1 N sodium hydroxide to a phenolphtha-
of cell suspensions were determined by drying
lein end point. The lactase assay is described
samples at 110 C f o r 3 h.
later in this section.
Assay of Lactase
Culture Studies
The release of o-nitrophenol from the hy-
Pure cultures of L. bulgaricus and S. tbermo-
drolysis of o-nitrophenol-fl-D-galacto-
pbilus were isolated from a commercial sample
pyranosidase (ONPG) was used as the param-
of yogurt. The isolates were examined for
eter for measuring lactase activity (3). Solutions
purity by conventional methods (1) and then
of ONPG at 1.66 x 10-3 M were prepared in .1
maintained in sterile skim milk and Difco
M phosphate buffer, pH 7.0, and used as the
All-Purpose Tween (APT) broth. Cultures were
substrate. To 5 ml o f the substrate was added 1
transferred successively three times before use.
ml of the test solution and the reaction carried
Batches of sterile skim milk (100 ml) were
out at 30 C for 10 min. The reaction was
inoculated with 1% active cultures of L. bulgari-
stopped by the addition of 2 ml of 1.0 M
cus and S. tbermopbilus and incubated at 43 C
sodium carbonate. The absorbancy at 420 nm
for 16 h. Also, the composite yogurt culture (L.
was measured in a Beckman Model 25 spectro-
bulgaricus plus S. tbermopbilus) was inoculated
photometer. A standard curve was prepared
at 4% into sterile skim milk and incubated at 43
from known activities of pure E. coli lactase
C for 4 h. At the end of the incubation period,
(Worthington Biochemicals, Freehold, NJ). One
a 10-g portion of the cultured skim milk was
unit of enzyme activity was defined as the
removed and assayed for lactase. A 50-g portion
amount needed to hydrolyze 1 pmole of
of the same cultured milk was subjected to an
ONPG/min at 30 C and pH 7.0.
in vitro gastric digestion process described by
Breslaw and Kleyn (2). Aliquots of the digest
RESULTS A N D DISCUSSION
were withdrawn at O, 1, 2, and 3 h intervals and
analyzed for thier lactase content. Initial qualitative examination of several
The isolates maintained in broth were grown dairy products revealed that only cultured
separately in APT broth (Difco) containing yogurt contained detectable amounts of lactase.
glucose or lactose as the carbohydrate source. The cottage cheese, sour cream, and acidified
Preliminary trials revealed that it was not yogurt samples, and the milk from which these
possible to attain a one to one ratio between L. products were made failed to yield the charac-
bulgaricus and S. tbermopbilus in a broth teristic yellow color with ONPG even after
system. When a composite yogurt culture of L. prolonged reaction times of 2 h, indicating that

Journal of Dairy Science Vol. 59, No. 12

 LACTASE IN CULTURED PRODUCTS 203 3

these products did not comain any detectable lC 2.0

amounts of lactase. Cultured yogurt, on the
other hand, gave a positive reaction with
ONPG. Therefore, subsequent studies were fo- Lactase
cused only on cultured yogurt. The choice of
ONPG as a substrate was necessitated by the Acidity 1.6
fact that glucose and galactose resulting from
lactose hydrolysis are metabolized rapidly to
lactic acid and other intermediates of the
Embden-Myerhoff-Parnas pathway (14). Hence,
.2
measuring the glucose or galactose in the t)
product would not be an accurate index of the g o
lactase activity.
Since the milk used in the manufacture of
cultured yogurt did not show any lactase 4 .8
activity, the enzyme activity of this product
f
was attributed to the yogurt culture. Studies m - - e ~ i re
were made to determine the effect of sonica-
tion on the release of lactase. Yogurt samples
J 1 A
were sonicated at 12 KHz for periods up to 10
min. The sonicated samples were reacted with
!
ONPG and the resultant color recorded at 420
nm. Maximum color was noticed after 7 min of
sonication (Fig. 1). Sonication for periods
I I
2 4
I I
6 10
I
longer than 7 rain resulted in less color. In all
TIME (Hr)
subsequent studies involving milk cultures, a
sonication time of 7 min at 12 KHz was used. FIG. 2. Effect of time of incubation on titratable
acidity, pH, and lactase content of cultured yogurt.
Effect of Incubation Time on Enzyme Production
in Yogurt incubation during the manufacture of yogurt
The production of lactic acid and other (Fig. 2). Lactic acid reached a m a x i m u m of
metabolites was affected by the length of 1.5% after 6-h incubation and remained at that
upon prolonged incubation. The pH, on the
other hand, declined gradually to 4.5 in 7 h.
1.0
The acidity of yogurt after 6-h incubation may
be considered excessive; however, after only 4-h
incubation, it was in the optimal acidity range
e,-
o
- °
/./ \. of 1.0 to 1.2%. Lactase activity increased
steadily up to 4 h of incubation, reaching a
maximal value of 8 units/g. Further incubation
u __ °~o__o/ resulted in a marked lowering of the enzyme
activity to about 3 units/g before leveling off.
i .4 The decrease in enzyme activity between the
4th and 6th h of incubation was believed due to
0
m the increase in titratable acidity or the decrease
< .2-- in pH. Since 4-h incubation periods are com-
monly employed in the manufacture of yogurt,
the lactase content would be near maximum in
I I I I 1 such a product.
0 2 4 6 8 10
TIME (Min) Culture Studies

FIG. 1. Effect of sonication time at 12 KHz on the From the above experiments it was uncer-
release of lactase from cultured yogurt. tain whether a single species of bacterium was

Journal of Dairy Science Vol. 59, No. 12

2034 KILARA AND SHAHANI

TABLE 1. Lactase content of L. butgaricus, S. tbermopbitus, and yogurt culture in milk.

Composite
L. S. yogurt
Treatment bulgaricus t bermopbilus culture

(Units of lactase per gram culture)

Unsonicated (control) .40 .45 .81
Sonicated 1.52 2.41 3.84

responsible for the lactase activity or whether these cultures in a broth system. The cultures
the two organisms, L. bulgaricus and S. tberrno- differed from one another in such a system.
pbilus, exerted a synergistic effect. Pette and The lactase of S. tbermopbilus was an inducible
Lolkema (11) reported that a composite yogurt enzyme since cells grown in lactose containing
culture exhibited a synergistic effect upon APT broth (LAPT) showed an activity of
growth and acid production. Rods, cocci, and 12,000 units of lactase whereas ceils grown in
the composite yogurt culture were grown sepa- glucose containing APT broth (APT) showed
rately and their enzymic activities determined. only 4900 units/g (Table 2). However, L.
Table 1 shows the effect of sonication on the bulgaricus grown in LAPT broth did not show
release of lactase from individual cultures. The any increase in activity. A substantial part of
unsonicated cultures of L. bulgaricus and S. the lactase was bound to the cell-debris. In
tbermopbilus contained nearly the same either case the enzyme was an endoenzyme
amounts of lactase activity. However, on soni- since the cell-free medium did not possess any
cation S. tbermopbilus had 1.59 times more enzyme activity. A decrease in total lactase
lactase activity than L. bulgaricus. One gram of activity of L. bulgaricus grown in LAPT broth
the unsonicated composite yogurt culture con- compared with the activity of cells grown in
tained .81 u of lactase. Since one gram of the APT broth could have been due to inhibition of
composite yogurt sample consisted of .5 g each the enzyme by galactose (16).
of L. bulfaricus and S. tbermopbilus, the total
activity should have been (.40 + .45) + 2 = .425
u/g of composite sample. However, the ob- Effect of In Vitro Digestion on Release of
served value was .81 u/g which seemed to sug- Lactase
gest a synergistic relationship between the cul- Since substantial amounts of lactase were
tures grown together. b o u n d to bacterial ceils, gastric digestion may
Since lactase is an inducible enzyme in aid in the release of this enzyme. Trials per-
certain microorganisms, experiments were de- formed by subjecting 16-h-old cultures of L.
signed to study the production of lactase by bulgaricus and S. tbermopbilus and 4-h-old

TABLE 2. Lactase activity of cultures growth in broth.

Organism and
growth medium Bound Free Total

[Units of lactase per gram cells (dry wt)]

S. tbermopbilus
Glucose broth (APT) 2927 1962 4889
Lactose broth (LAPT) 8427 3655 I2,082

L. bulgaricus
Glucose broth (APT) 646 144 790
Lactose broth (LAPT) 641 3 644

Journal of Dairy Science Vol. 59, No. 12

 LACTASE IN CULTURED PRODUCTS 2035

TABLE 3. Effect of in vitro gastric digestion on the release of lactase from cultures.

Composite
L. S. yogurt
Treatment bulgaricus thermophilus culture

(Units of lactase per gram culture)

Control a .40 .45 .85

Digestion control b 3.4 3.9 4.0

After 1-h digestion 2.3 2.5 4.1
After 2-h digestion 2.2 1.9 4.5
After 3-h digestion 2.0 1.1 4.9

aNo treatments.
bzero time sample, i.e., culture + acid + enzymes.

c o m p o s i t e y o g u r t c u l t u r e to an in v i t r o gastric 3 Citti, J. E., W. E. Sandine, and P. R. Elliker. 1965.

digestion p r o c e s s revealed t h a t lactase was 3-galactosidase of Streptococcus lactis. J. Bacteriol.
89:937.
a f f e c t e d b y digestion. While t h e e n z y m e in
4 Emmons, D. B., and J. L. Tuckey. 1967. Cottage
individual c u l t u r e s d e c l i n e d w i t h t i m e o f diges- cheese and other cultured milk products. Pub.
t i o n , lactase in t h e c o m p o s i t e y o g u r t c u l t u r e (3has. Pfizer Co., Inc., New York, NY.
s e e m e d t o s h o w a n increase ( T a b l e 3). T h i s 5 Ernstrom, C. A. 1967. Process for preparing
e v i d e n c e can b e c o r r o b o r a t e d w i t h t h e observa- Cottage cheese curd. U.S. Pat. 3,298,836.
6 Flatz, G., and Ch. Saengudom. 1969. Lactose
t i o n s o f G o o d e n o u g h a n d K l e y n (7), w h o s t a t e d intolerance in Asians. A family study. Nature
that yogurt microflora may contribute to the 224:915.
successful m e t a b o l i s m o f lactose. 7 Goodenough, E. R., and D. H. Kleyn. 1976.
A c u l t u r e d milk p r o d u c t , s u c h as y o g u r t , Influence of viable yogurt microflora on digestion
m a y be m o r e digestible b y l a c t o s e i n t o l e r a n t of lactose by the rat. J. Dairy Sci. 59:601.
8 Kosikowski, F. V. 1970. Cheese and fermented
individuals b e c a u s e o f a r e d u c e d lactose c o n t e n t milk foods. Pub. F. V. Kosikowski, Ithaca, NY.
a n d t h e p r e s e n c e o f t h e e n z y m e lactase. I n a 9 Kretchmer, N. 1972. Lactose and lactase. Sci.
m i l k s y s t e m a synergistic e f f e c t b e t w e e n r o d s Amer. 227:70.
a n d cocci o n lactase a c t i v i t y was o b s e r v e d . In 10 Little, L. 1963. The direct aid process for manufac-
turing sour cream. Milk Dealer 52: 28.
b r o t h c u l t u r e s t h e lactase o f S. tberrnopbilus
11 Pette, J. W., and H. Lolkema. 1950. Yogurt: I.
was i n d u c i b l e . In v i t r o d i g e s t i o n also was Symbiosis and antibiosis in mixed cultures of
h e l p f u l in releasing lactase f r o m t h e c o m p o s i t e Lactobacillus bulgaricus and Streptococcus tber-
yogurt culture. mopbilus. Neth. Milk Dairy J. 4:197.
12 Reddy, K. P., and K. M. Shahani. 1972. B-Complex
ACKNOWLEDGMENT vitamins in yogurt. J. Dairy Sci. 55:660. (Abstr.)
13 Rosenweig, N. S. 1969. Adult human milk intoler-
Skillful t e c h n i c a l assistance o f Clara Zoz in ance and intestinal lactase deficiency. A review. J.
p e r f o r m i n g s o m e o f t h e analyses is a p p r e c i a t e d . Dairy Sci. 52:585.
14 Valdl, J. R., and K. M. Shahani. 1962. Different
pathways of lactose metabolism of Streptococcus
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