JOURNAL OF DAIRY SCIENCE

VOLUME X X X V I I July, 1954 NUMBER 7

LIPASE: A REVIEW

B. L. HERRINGTON

Department of Dairy Industry

Cornell University, Ithaca, New Yortc

These pages were written with the hope that they might prove useful to
those who know little about lipase, and to those who know nmch. The references
cited m a y not be the first paper on a given topic nor the last. The first publica-
tion may be indecisive, incomplete. The last m a y be less informative than an
earlier publication. Some may feel that too much has been omitted; an equal
number may feel that too much has been included. So be it.
I f these pages help some to understand what is already known; if they point
the way for further research; if they stinmlate some to start new investigations,
then the time spent in writing this has been well spent.

I. T H E R A N C I D I T Y 1 P R O B L E M

At one time, some chemists doubted that normal milk contained lipase. I t
is now accepted that the great m a j o r i t y of cows secrete milk containing" fat-
splitting enzynles but, in most cases, these enzymes are inactive. Under certain
conditions they may become active and cause serious loss. I n view of this fact,
it is more important to know the activity of lipase in raw milk than to know the
q u a n t i t y ; and handlers of milk must p a y particular attention to those factors
which increase or reduce the activity of the enzyme. 2
Several times in the history of the dairy industry, new methods of handling
or processing milk have activated the lipase system and produced rancid flavors.
ttomogenization is the best known example of this. Raw nfilk becomes rancid
so quickly after homogenization that the sale of homogenized milk would be
impossible if lipase could not be inactivated by simple means.
As a second example of changes in practice which led to trouble with lipase,
we may cite the change in temperature of separation which occurred in the mar-
ket cream industry some years ago. At one time, milk was warmed to a fairly

Received for publication February 8, 1954.

1In this paper, rancidity refers only to hydrolysis with liberation of fatty acids. Oxidation
is not included.
~'"Activation of lipase" should not be interpreted in a narrow sense. The phrase is used in
place of the more accurate statement: '~acceleration of the process of enzymatic hydrolysis."
775,
776 g.L. HERRINGTON

high t e m p e r a t u r e (110-120 ° F.) before separation to reduce fat losses in the

skimmilk. W h e n it was realized that a more viscous product could be produced
by s e p a r a t i n g at a lower t e m p e r a t u r e (80-90 ° F.) and improvements ill sepa-
rators made this possible, the t e m p e r a t u r e of separation was g r a d u a l l y lowered.
At the same time, there was an increase in the incidence of rancidity. The
relation between these two events was not understood until the phenomenon of
" t e m p e r a t u r e a c t i v a t i o n " ~ was reported in 1939 (51).
Recently, there has been an increase in the n u m b e r of reports reaching this
d e p a r t m e n t concerning rancid milk. This time, the trouble seems associated with
the introduction of pipeline milkers and f a r m tanks into the New York area.
Because the use of such equipment offers m a n y advantages and is likely to be
adopted widely, r a n c i d i t y m a y become an i m p o r t a n t problem once more.
I t is known that several investigations of the effect of pipeline milkers and
bulk handling upon the quality of milk are now under way, but little or nothing
has yet been published. Until the results of others appear, comment must be
based largely upon our own investigations. Full details of these will be pub-
lished later.
According to reports reaching us, the milk handIed b y the newer methods
f r e q u e n t l y contains b u t t e r particles and in some cases it is rancid. Churning
is evidence of agitation, and the agitation of r a w milk is known to accelerate
lipolysis (52). I t seemed probable that the two defects could be traced to the
same source.
I n a n u m b e r of cases, the trouble was traced to " r i s e r s " in the pipe lines,
vertical sections connecting one pipe line to another at a higher level. The milk
is carried up by a stream of air which produces considerable agitation a n d foam.
A n y given portion of milk m a y be thrown up and fall back a n u m b e r of times
before it passes over.
I n New York State, where milk is collected each day, the average acid
degree 4 of milk reaching pasteurizing plants is a p p r o x i m a t e l y 0.5 unless it has
been handled in a pipeline milker (32). I n t h a t case, the acid degree m a y be
much higher. To show clearly the effect of a pipeline milker upon lipolysis, a
sample of the entire night milking was taken on three successive days at a f a r m
where there were five risers in the pipe line. On the second d a y of the sequence,
pail milkers were used and the milk was carried b y h a n d to the milkhouse ; on
the first and third days, the milk passed through the pipe line. The acid degree
values a f t e r holding for 36 hours were 2.87, 0.92, and 2.59, respectively.
I f risers could be eliminated, much of the trouble would disappear. Ulti-
mately, it m a y be possible to design risers which cause less damage to the milk.
The mechanism by which agitation activates lipolysis is not understood in detail.
I t is difficult to u n d e r s t a n d why agitating w a r m raw milk with air in a vertical

"~The r a t e of l i p o l y s i s in cold r a w m i l k is i n c r e a s e d g r e a t l y i f cold m i l k is w a n n e d momen-

t a r i l y to 85 ° F. and recooled. W a r m i n g to h i g h e r or l ow e r t e m p e r a t u r e s prodllees less " a c t i v a -
tion. ' '
' A c i d d egre e is the n u m b e r of m i l l i l i t e r s of n o r m a l a l k a l i r e q u i r e d to n e u t r a l i z e t he f r e e
acid i n 100 g. of f a t .
 LIPASE : A ~EVIEW 777

pipe should produce m u c h more activation t h a n agitation for the same time in
a W a r i n g Blendor. T h a t is the case if the W a r i n g Blendor is filled completely
so t h a t no space is left for air.
The effectiveness of agitation is not determined by its violence alone. Other
factors must be considered. The role of t e m p e r a t u r e is important. As fresh
milk cools, it passes t h r o u g h a critical t e m p e r a t u r e zone where it is most suscep-
tible to churning (43). I f pipe lines in barns were heated to p r e v e n t cooling
of the milk, churning in the pipe could be prevented, but this would not reduce
trouble f r o m rancidity. The sensitivity of milk 'to activation b y agitation is
related to the fluidity of the fat, and it increases r a p i d l y as the t e m p e r a t u r e is
increased up to 75 or 85 ° F.
W i t h the introduction of f a r m tanks and t a n k t r u c k transportation, there
is a grow{'ng interest in the possibility of alternate d a y pick-up f r o m the farms.
I f this practice is adopted, an increased effort will be needed to control lipase.
Milk supplies which do not develop organoleptic r a n c i d i t y u n d e r present methods
of handling m a y do so if the milk is held r a w for an additional 24 hours. U n d e r
such conditions, a n y factor which m a y accelerate or delay the appearance of
rancidity must be given careful consideration.
The effect of agitating fresh w a r m milk has been mentioned. F o r t u n a t e l y ,
the agitation of cold milk has much less effect ~pon lipase activity. The most
critical periods are when the w a r m milk is in the pipeline and when w a r m milk
enters an e m p t y t a n k and does not completely cover a propeller-type agitator.
Crowe (12) compared milks cooled in cans with milks cooled in a vat. The vat-
cooled milk contained more water-insoluble acid, and the value increased with
increasing amounts of agitation. The agitation of milk in t a n k trucks d u r i n g
shipment is of lesser consequence because the t e m p e r a t u r e is usually v e r y low
and the subsequent holding.period is relatively short.
The possibility of t e m p e r a t u r e activation when fresh w a r m milk is added
to cold milk in the t a n k should not be overlooked. I n the worst possible case,
all of the second milking would be added at once to the cold milk in the tank.
Under such conditions, the t e m p e r a t u r e might exceed 68 ° F. This is high enough
to produce a definite increase in the rate of lipolysis (51). I n practice, however,
the w a r m milk is added over a period of time while the refrigeration unit is in
continuous operation, and the cold milk is not w a r m e d enough to produce such
a great effect. I n some cases, however, this small effect m i g h t be just enough "~o
c a r r y the milk across the threshold of organoleptic rancidity, especially if alter-
nate d a y pick-up is practiced.
On the positive side, only one means of controlling r a n c i d i t y has been dis-
covered that might be p u t into practice on the d a i r y farm. I t is instantaneous
cooling:. I f the milk is cooled in a few seconds by means of a t u b u l a r or surface
cooler, lipolysis during subsequent storage is reduced greatly (34). F u r t h e r m o r e ,
this avoids all risk of t e m p e r a t u r e activation and reduces activation b y agitation
in the vat. I n s t a n t cooling m a y become an i m p o r t a n t step in a p r o g r a m of
alternate day hauling.
I t should be emphasized that low t e m p e r a t u r e alone does not check lipolysis.
778 B.L, HERRINGTON

In n a t u r a l milk, the reverse is t r u e ; lipolysis is accelerated as the t e m p e r a t u r e

is reduced (53). I f the holding period is prolonged, microorganisms m a y attack
the f a t at higher t e m p e r a t u r e s and, u n d e r these conditions, investigators m a y
observe less lipolysis at intermediate t e m p e r a t u r e s (60 ° F.) than at higher or
lower t e m p e r a t u r e s (12, 70).
I t seems probable t h a t there will be increased interest in the s t u d y of lipase
in the near future. Those nlaking such studies, or reading about them, should
u n d e r s t a n d some of the differences between the methods used to s t u d y lipase.
Failure to do this has been the cause of much confusion.
Some methods measure the deterioration of milk fat itself, but they do not
measure the same kind of deterioration. F o r that reason, their results n m y not
agree closely with each other. Other methods do not measure the deterioration
of milk fat. They yield results which m a y or m a y not be significant in the actual
handling of milk. Such data must be i n t e r p r e t e d with caution. The most widely
used methods which measure changes in milk f a t are :
1. Orga~oleptic examinatiom This is p r i m a r i l y a measure of free, short-chain,
water-soluble, f a t t y acids. Liberation of long-chain acids lms little effect
on the flavor or odor of the milk. This method yields results of most sig-
nificance in terms of consumer response but it does not yield numerical
data, and the method is of low sensitivity. I t can not detect changes before
they become commercially significant.
2. Surface tension measurements. I f the change in surface tension is due to
the liberated f a t t y acids, as some suppose, then it should be most sensitive
to the longer water-soluble acids (eaprie, eaprylie). Butyric acid, the
most i m p o r t a n t factor in the organoleptie measurement, has little effect on
the surface tension of nlilk (10). On the other hand, the possible role of
monoglycerides and of diglycerides in reducb~g surface tension nmst not
be overlooked. I f they are the m a j o r factors causing surface tension de-
pression, then the depression of surface tension should be nearly inde-
pendent of the nature of the acid liberated.
Unfortunately, some of the published measurements of surface tension
are difficult to interpret for several reasons. First, merely cooling fresh
milk and immediately r e w a r m i n g it to room t e m p e r a t u r e will produce a
change in surface tension of several dynes which is not due to lipase
action (50.). I f workers using surface tension measurements have taken
this into account in p l a n n i n g their experiments, they have not always
made the fact clear, nor explained what provision was made to eliminate it.
Second, there is some doubt about the correctness of the values reported
in the literature. F e w details have been published concerning the tech-
niques that have been employed so that others might duplicate the work.
F o r example, the surface tension of a fresh surface decreases r a p i d l y at
first, then more slowly. The first change is so r a p i d that it can scarcely be
observed unless the milk is diluted (83). Subsequently, the surface tension
falls more slowly, probably because fat globules are accumulating in the
surface. (The surfaee tension of cream is lower t h a n that of whole milk;
 LIPASE : A I~EVIEW 779

the surface tension of skimmilk is higher.) Because of this slower change,

one who works r a p i d l y will obtain higher readings t h a n one who works
more slowly. More i m p o r t a n t still, measurements made with a du Noiiy
tensiometer (the i n s t r u m e n t most widely used today) must be corrected
as explained by the makers of the instrument. Personal correspondence
has revealed that some workers have not nlade this correction. There is a
possibility that others, also, have failed to correct their data. This does
not necessarily invalidate the conclusions of an individual investigator,
but it does make it difficult to compare the values of one worker with those
of another unless there is assurance that both have applied p r o p e r cor-
rections to their measurenlents. ( W i t h our own instrmnent, the correction
is a p p r o x i m a t e l y --9%.)
3. Meas~trement of fat solvable acids ((wid degree val~tes). Most of these acids
have little effect on surface tension or upon the flavor of milk. Several
procedures are in use for their measurement. Some p r e f e r to recover the
f a t by extraction, some b y churning (46). ( I t m a y be possible to use some
of the emulsion breakers investigated b y Stiue and Patton, 78.) Some
have titrated the acids in alcohol, and sonle have used ether and other
solvents (80). These modifications all yield essentially the same results
though there are small differences2 Measurements of f a t soluble acids
have the advantage that they yield numerical results which are repro-
ducible and which reveal v e r y small changes in the fat. Before any change
can be detected organoleptically, the titration value will usually increase
f r o m an initial value of 0.20 to 0.30 ml. the normal range for fresh milk
fat (32, 33)] up to 1.5 to 2.0 ml. (17). Consequently, the effect of minor
changes in handling milk can be detected easily b y this method.
I t should be emphasized that each of these three methods measures deteriora-
tion of f a t b y hydrolysis. However, it is a common observation that a n y one of
the three measures of deterioration (orga~oleptic rancidity, surface tension de-
pression, or water-insoluble acids) m a y change independently of the others.
F o r t h a t reason, there are some who believe that several different enzymes are
present in milk and t h a t they do not always occur in the same proportions.
Other methods have been used to s t u d y lipase which do not measure the
breakdown of milk fat directly. F o r example, some have measured the breakdown
of substrates foreign to milk, t r i b u t y r i n (18, 55), p-nitrophenol stearate (45),
naphthol acetate (62, 74, 75), etc. W i t h these methods, it is possible t o show
differences in milks, but the significance of such data is not clear. The limitation
of these methods is a p p a r e n t when you consider t h a t the effects of homogeniza-
tion and of t e m p e r a t u r e activation upon lipolytic activity in milk can not be
shown by such techniques. These methods m a y be useful in estimating the quanti-
ties of enzyme but, in practice, the activity of the enzyme system is of m u c h m o r e
importance t h a n the quantity when handling r a w milk.

5 H i l l i g ' s method f o r water-insoluble acids is relatively specific f o r acids of 16 carbons or

more. W I A values are not strictly comparable with measuremellts of total f a t soluble acid
(acid degree).
780 B.L. HERRINGTON

II. SOME R E C E N T W O R K ON L I P A S E

Several reviews dealing with lipase have a p p e a r e d in recent years. That of

H e r r i n g t o n (31) dealt p r i m a r i l y with lipase in milk and its products. A m m o n
(1) did not restrict his review to a n y p a r t i c u l a r field. B r a d s h a w (5) was con-
cerned with lipase in cereal products, and Desneulle (1~) with lipase in digestive
processes. Frisell's (23) review of nonoxidative, nonproteolytic enzymes lists
only 12 references to lipase. I n contrast, D u n k l e y ' s p a p e r (17) was not intended
p r i m a r i l y as a review, but it does cite m a n y references to the literature of the
subject. Those interested in extensive bibliographies should consult these papers
for early references. This p a p e r is intended to supplement, not duplicate, these
earlier reviews.
IJIPASE I N M I L K

Dunkley and his associates published a series of articles of much interest.

I n the first of these (17), D u n k l e y compared the acid degree values of 92
samples of cream with organoleptic ratings. Samples classified as slightly rancid
ranged in acid degree f r o m 1.47 to 4.88, with an average of 2.59. Since fresh
milk f a t has an acid degree of a p p r o x i m a t e l y 0.3 (32, 33), it is evident that
extensive hydrolysis m a y ocur in some samples before it can be detected organo-
leptically. He found t h a t surface tension values were a better index of organo-
leptic rancidity t h a n acid degrees. I n general, samples having a surface tension
below 45 dynes at 20 ° C. were rancid, and those above 46 were seldom rancid.
However, the f a t content of the milk should be taken into consideration when
making predictions. Some samples containing only 2% of f a t showed no organo~
leptic r a n c i d i t y even though the surface tension fell below 42 dynes. He made
the curious observation that samples stored in test tubes differed in surface ten-
sion f r o m those held in 1/~-pint bottles by a p p r o x i m a t e l y 1.4 dynes. He also
measured tile surface tension of milk f r o m individual quarters of the same u d d e r
and occasionally found large differences.
I n a second paper, Fredeen et al. (22) reported t h a t lipolytie activity, as
revealed b y surface tension measurements, varied with season and stage of lacta-
tion but not with gestation or estrous cycle. This last observation should be
contrasted with the results of another s t u d y at M a r y l a n d on the relation beween
lipolytie activity and both n a t u r a l and induced estrus (2). I n the M a r y l a n d
investigation, they measured changes in the acidity of the f a t and found a maxi-
m u m concentration of lipase on the d a y of estrus, and a m a x i m u m acidity in the
f a t on the day following estrus. W h e n estrus was induced by injections of
diethyl stilbestrol, the milk became v e r y rancid. P e r h a p s the difference between
these conflicting reports is due to differences in the cows, perhaps to differences
in the methods used to detect lipolysis.
F r e d e e n et al. reported that injections of pituitrin and stilbestrol sometimes
caused a decrease of almost 15 dynes in the surface tension of the milk secreted,
but five cows did not show this response to the injection. No infomnation about
organoleptie r a n c i d i t y was given.
I n the t h i r d p a p e r of the series, Dunkley and Smith (18) compared the
 L I P A S E : A Y,'EV1E'W 781

activity of individual samples of skimmilk upon t r i b u t y r i n a n d upon a special

homogenized milk fat substrate. I n both cases, they observed the same p H opti-
mum, 9.5 at 10 ° C. A t 37 ° C., the o p t i n m m fell to p H 8.8 with t r i b u t y r i n ; no
value was given for milk fat. The results with different milks on the two sub-
strates showed v e r y high correlation: 0.984, 0.923, and 0.941 in three sets of
samples. They concluded that measurements of tributyrinase are a useful meas-
ure of lipasc. I t should be noted that they did not compare either m e a s u r e m e n t
with changes occurring in the original milk.
In the f o u r t h paper, t r i b u t y r i n a s e activity was compared with changes in
surface tension in n a t u r a l and in t e m p e r a t u r e - a c t i v a t e d milk. D u r i n g the first
3-4 months of lactation, the two showed considerable correlation but there was
none d u r i n g the latter half of the lactation period. They make the curious state-
ment that the amount of tributyrinase present is not a limiting factor in produc-
ing r a n c i d i t y in the latter p a r t of the lactation period (when the amount of
tributyrinase is low and lipase activity as measured by surface tension changes
is high), but it is a limiting factor in the latter p a r t of the lactation cycle when
the tributyrinase values are high and lipolysis (measured by surface tension) is
low. This can be i n t e r p r e t e d in several ways. W h a t they m e a n t is not clear.
I n their experiments, they found that the tributyrinase activity of s k i m m i l k was
independent of the separation t e m p e r a t u r e and concluded t h a t cooling milk did
not cause an increase in adsorption of lipase upon the f a t globules. This is in
agreement with the conclusions of K r u k o v s k y (50), who f o u n d no difference
between skimmilks p r e p a r e d at 10 ° C. and at 45 ° C. when tested against tri-
b u t y r i n and against heated homogenized cream. K r u k o v s k y went f u r t h e r and
found that there was no difference between the creams if the lipase was activated
b y homogenization. W i t h o u t activation, the 45 ° cream showed no lipolysis but
the 10 ° cream became rancid v e r y quickly.
These findings are in conflict with the view of Tarassuk and J a c k (79) that
milk lipase is adsorbed upon the f a t when it is cooled. I t is true t h a t their
experimental conditions were v e r y different. Their conclusion was based upon
the observation that some samples of milk will become rancid if cooled but do
not become rancid if k e p t warm. I t is possible to explain their observations by
the fact that lipolysis in n a t u r a l milk is more r a p i d at low t e m p e r a t u r e , a
phenomenon which can not be explained by adsorption alone.
Weinstein and T r o u t (82.) compared the susceptibility of milk of different
breeds to activation b y homogenization. No definite conclusions can be drawn
since they studied only 18 cows distributed among five breeds.

LIPASE IN MILK PRODUCTS

Shotwell et al. (7"7) made a s t u d y of rancidity in ice cream. They employed

a solvent extraction method to recover the fat for m e a s u r e m e n t of acid degree
values. Samples of vanilla ice cream collected f r o m ten m a n u f a c t u r e r s at four
periods during the year had a mean acid degree value of 2.91 and a range of
1.33 to 6.24. I n a separate s t u d y of organoleptic rancidity of ice cream, they pre-
p a r e d rancid samples by using homogenized raw cream. Samples having acid
782 B.L. HERRINGTON

degree values of 4-5 were judged slightly rancid. Those above five were judged
rancid, or worse. They made similar studies of chocolate ice cream. The mean
values for acid degree were higher but the organoleptic threshold was higher also.
Hood (44) has published statistics on the incidence of rancidity in 718,0.96
Canadian Cheddar cheeses, and others (47) have reported on rancidity in the
butter of Quebec.
Christensen et al. (8) found that spray powders made from milks which lind
not been preheated were rancid. They attributed this to the agitation in the
vacuum pan during condensing. The acidity of the fat continued to increase
slowly during storage even though the moisture content was less than 3%. Pre-
heating for 20 minutes at 140 ° F., or higher, was sufficient to prevent rancidity.

ANALYTICAL ~IETtIODS
Papers dealing with analytical procedures may be divided into two groups:
those measuring changes produced by lipolysis and those attempting to measure
the enzyme itself. Obviously, the two are closely related.
Hillig's (35) method for the determination of water-insoluble acids ( W I A )
in b u t t e r has been adopted by the A.O.A.C. I t enables the analyst to judge the
quality of the original cream, since the W I A are not lost during churning (39).
Most of the butyric acid is lost when butter is made in conventional churns (42),
though one-third may remain in the butter made by the continuous process (40).
This fact has been t u r n e d to advantage in judging whether deterioration in
quality has .occurred before or after churning. Microbial activity in butter
usually results in a sharp increase in the butyric a c i d / W I A ratio (49).
Because of the importance of such measurements, the Hillig test has been the
subject of many papers. Several have presented evidence concerning its relia-
bility (4, 28, 36, 39, 41). It has been found to correlate reasonably well with the
alpha naphtholphthalein (ANP) test when the W1A value did not exceed 400 rag/
100 g. (3, 30). When compared with organoleptie scores, there were m a n y incon-
sistencies (4). This is to be expected since the water-insoluble acids, themselves,
are practically tasteless and odorless. It has been reported (69) that the results
of the Hillig test were sometimes much higher when 2.0 ml. of excess alkali
(beyond that required for neutralization) was used. Hillig (37), however, has
explained this by showing that excess alkali is needed to insure complete extrac-
tion of the free f a t t y acids. The official method recognizes this by prescribing
0.5 ml. of excess alkali.
The original Hillig test is time-consuming. A quicker procedure has been
described (38), which measures the W I A by volumetric titration (instead of
isolation and weighing) and the assumption that the mean equivalent weight
of the acids is 270. B y actual test, the value felt between 260 and 280 in 71 out
of 73 samples. Ramsey and Hess (72) reported good results on a collaborative
study of the butyric acid test, but they used aqueous solutions of short chain
acids, not milk fat. It should be noted that lipolysis in butter and in sour cream
is due chiefly to enzymes of microorganisms, not of the milk (42).
Armstrong and I~arper (3) described an improved form of the A N P test
 LIPASE: A REVIEW 783

and adapted it for the examination of cream (30). This test can not measure
small differences in W I A values but they recommend it as a sorting test. Goiffon
(28) described another colorimetrie test based upon the fact that Nile blue, which
ordinarily turns red in the presence of excess sodium carbonate, does not do so
in the presence of free u n s a t u r a t e d f a t t y acid. He describes both colorimetric and
titrimetric procedures based upon ,this fact. Such a test might permit the r a p i d
estimation of oleic acid in the W I A of butter. Tlle value of such information is
not yet known.
Tucker and B i r d (80) substituted methanol for ethanol in p r e p a r i n g their
s t a n d a r d alkali solutions because it was cheaper and easier to obtain. This
produced t u r b i d i t y during the titration, but that trouble was eliminated by
dissolving the fat in a mixture of Skellysolve B and propanol, 4 + 1;
The chemical methods whieh have been used to s t u d y the breakdown of milk
fat are all measures of the acid products of hydrolysis. Desnuelle (14, 15) and
Mattson et al. (57) have nieasured the other products (diglyceride, monoglyeer-
ide, and glycerine) formed d u r i n g the digestion of fats. The first step in the
hydrolysis Seemed niost easy; the last, most difficult. The yield of glycerine was
increased b y the addition of calcium and bile salts (15). P e r h a p s sonic One will
be able to make similar studies on rancid miik. Because of the great surface
activity of the mono- and diglycerides, such information might be useful.

] g S T I M A T I O N OF L I P A S E

Interest in methods of measuring lipase is not limited to the dairy industry.

Copenhauer (9) has described a W a r b u r g method b y which he could measure
the esterase activity of liver at p H 8.4, using as little as 0.5 rag. of fresh tissue.
L u b e r t et al. (.55) developed an extraction-titration method which could be used
over a wide range of p H values. Fiore and Nord (20) recommended polyvinyt
alcohol as an emulsifying agent in lipase assays. Para-nitrophenol esters have
been used by some investigators to measure lipase activity (45). This colorless
substrate yields a yellow color upon hydrolysis. IIowever, Dirks and Boyer (16)
found that m a n y substances which were not enzymes wo.uld catalyze this reaction.
Glutathione was p a r t i c u l a r l y active, and crystalline bovine serum albumin,
crystalline egg albumin and fi-lactoglobulin possessed activity, even a f t e r heat
treatment. Similar studies should be made to determine whether other nonfat
substrates such as the beta naphthol esters are specific reagents for enzymes.

ISOLATION OF LIPASES
A few have a t t e m p t e d to concentrate and p u r i f y lipase. Zechmeister (85)
and Giri (27) have described chronlatographic techniques, and Wallenfels (81)
used electrophoresis in a p a p e r sheet to separate the enzymes of a fungus. He
was able to identify zones of amylase, proteinase, lipase, and phosphatase. How-
ever, lipase was identified b y the hydrolysis of p a r a - n i t r o p h e n y l stearate and,
in view of D i r k s ' results, the identification of lipase is not conclusive.
Morton (59) reported that m a n y enzymes could not be extracted f r o m n a t u r a l
materials because they were bound to solid particles of lipid material. I n some
784 B . L . HERRINGTON

cases they could be released by extracting the lipids with butanol. This may be
helpful in the study of lipase. [It is of interest to dairymen that he was able
to concentrate milk phosphatase 5,600-fold by this technique (60).] Nasllif and
Nelson (63) were able to precipitate the lipase of Pse~tdomonas fragi from solw
tion with (NH~)~SO~, but the temperature must be kept v e r y low to avoid
inactivation. Shipe (76), also, used ammonium sulfate to precipitate the enzyme
from cultures of Penicilli~m roq~teforti and Aspergillus niger, but the broth was
concentrated first by removing approximately three-fourth,~ of the water as ice.

SOME E F F E C T S OF L ] P O L Y S [ S

The growth of S. lactis is inhibited in strongly rancid milk. Costilow and

Speck (10) studied this phenomenon by adding small amounts of purified f a t t y
acids to milk inoculated with test organisms. Butyric acid, caproie, oleic, linoleic.
]inolenic, arachidonic, and palmitic acids bad no appreciable effects upon growth.
Capric acid was most toxic, followed by caprylic and laurie. Toxicity did not
correlate with the depression of surface tension. In a second paper (11) they
reported that the growth of S. zymoge~es, of S. boy is, and of E. colt Was retarded
in rancid milk, but less than that of S. lactis. They confirmed the fact that the
surface tension of rancid milk tends to rise during incubation with S. lactis.
This parallels the observations of Cherrington and H a m m e r (7), who found'
that rancidity in young Cheddar cheese was removed by the action of an organism
thought to be a variant of S. lactis.
As little as 0.] % rancid milk fat proved to be a very effective foam breaker
during the condensing of skimmilk and whey ('6). The effect was attributed t o
the mono- and diglyeerides present. Sagar (73) found that rancid milk fat was
assimilated by young children even more rapidly than homogenized milk. This,
also. was attributed to the surface activity of the mono- and diglycerides. H e
measured the interfacial tension between a 1% solution of olive oil iu mineral
oil and aqueous solutions of alpha mono-stearyl glyceride, alpha, beta di-stearyI
glyeeride, and bile. The interracial tensions were 2, 4, and 12 dynes, respec-.
tively. With no addition,, the interfacial tension was 22 dynes.
Mukherjee (61) has repeated the observation of Greenbank and Hohn (29)
that fat containing free f a t t y acid is more susceptible to oxidation. Mukherjee
held milk fat under various conditions at 37 ° C. and examined it periodically
for 85 days. The peroxide value and the Kreis test increased more rapidly in the
fats having higher acid values. The possible relation between lipase activity
a~d oxidation in market milk deserves more attention than it has received.

T H E N A T U R E OF L I P A S E

Relatively little is actually known about the lipases. There is evidence that
some can be split into a thermostable coenzyme and a thermolabile apoenzyme
(,~'~). Perhaps this is true of all lipases. When lipases from different sources are
compared with respect to p H range, temperature of inactivation, activity against
different substrates, and response to different activating or inactivating agents,
it-seems that scarcely any two enzymes are exactly alike. Do we have as many-
 LIPASE: A REVIEW 785

lipases as we have sources, or are we dealing with mixtures of a much smaller

n u m b e r of enzymes ? I f we are dealing with mixtures, how can we separate them ?
How specific are individual components in their action ? These questions need
answers, and some investigators are a t t e m p t i n g to find them.
P a u l J. F o d o r has published m a n y papers on the properties of enzymes f r o m
insects and other sources2 Others (21, 58, 63, 64, 65, 66, 67, 68, 71, 76) have
studied the lipases of organisms i m p o r t a n t in the d a i r y industry. F r o m the
published data, it is clear that these enzymes are different and not interchange-
able.
Shipe (76) described a new technique for the s t u d y of relative specificity. He
used an equimolecular mixture of t r i b u t y r i n and t r i c a p r y l i n as a substrate and
determined the relative amounts of butyric and caprylic acids set free using a
chromatographic separation. The lipase f r o m Aspergillus niger liberated 4 mols
of caprylic acid per tool of butyric, that f r o m Penicillit~m roqueforti liberated
only 1/..~ tool per mol of butyric acid. W i t h equal activity on t r i b u t y r i n , there is
a twelvefold difference in activity on tricaprylin. Clearly, we must dismiss the
idea that "lipases are of low specificity."
An even more striking case of specificity was reported by Martin and Peers
(56), who found that oat lipase would split only one molecule of butyric acid
f r o m tributyrin. I t had no action on either alpha, alpha dibutyrill or alpha,
beta dibutyrin, nor would it attack either alpha or beta monobutyrin. I t did
attack triacetin (at one-third the rate on t r i b u t y r i n ) . D u r i n g purification, the
relative activities against t r i b u t y r i n and triolein remained constant. This m a y
mean that the same enzyme is involved in both cases, but the evidence is not
conclusive.
F u j i m u r a and H a m a g u c h i (2-1, 25, 26) have published several papers which
seem of unusual interest though the original nmnuscripts are not yet available
to the reviewer. They found that a complex of casein and ascorbic acid possessed
esterase activity which was lost by destruction of the ascorbic acid. I t was
restored by the subsequent addition of ascorbic acid. This should open a new
field of inquiry regarding the possible relation of ascorbie acid to lipase activity
in milk. I t m a y be significant that both lipase and ascorbic acid are destroyed
b y exposure of milk to sunlight (48), and both are sensitive to oxygen (54).
F r o m the practical viewpoint, an u n d e r s t a n d i n g of the phenomena of activa-
tion m a y be of greater value to the dairy i n d u s t r y t h a n information r e g a r d i n g
the enzyme itself. I n the original report (32) describing t e m p e r a t u r e activation
it was stated t h a t : " I t seems probable that activation is more dependent upon
changes in the state of the.fat t h a n upon changes in the l i p a s e . " This view has
been expressed more recently b y Dunkley and Smith (19), who wrote: " A c t i v a -
tion t r e a t m e n t s such as temperature-fluctuation, agitation and homogenization
generally are eousidered to depend on changes in the substrate for their effec-
tiveness. ' '
The writer would go one step f u r t h e r and postulate that activation by
6 T h e s e p a p e r s a r e n o t l i s t e d h e r e b u t m a y be l o c a t e d t h r o u g h t h e a u t h o r i n d e x t o C h e m i c a l
Abstracts.
786 B.L HERRINGTON

temperature changes, by agitation, or by homogenization is nonselective with

respect to the kind of acids subsequently liberated. The relative amounts of the
different acids set free in any given sample will depend upon the nature of the
enzymes present. If this view is correct, then the choice of method for studying
the effect of processing upon lipolytic activity in milk need be based only on
sensitivity and convenience.
The exact nature of the changes in the fat surface during activation needs
further study. It seems likely to the writer that at least two kinds of activation
must exist. The original surface material may be removed irreversibly by
mechanical forces, or the adsorptive and reactive properties of the fat may be
changed by the phase transformations which follow cooling. The completion of
a thesis dealing with the relation between lipolysis and the properties of the
surface of the fat globule has been announced (13). No further information is
available at present, but the title suggests that the results may be of unusual
interest.
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 LIPASE : A REVIEW 787

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~788 B.L. HERRINGTON

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 LIPASE : A ]¢EVIEW 789

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