THE LIPOLYTIC ENZYME SYSTEMS

OF MILK

Dissertation

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

By

THEODORE FERER IRMITER, B. S., M. Sc.

The Ohio State University

1951

Approved by: TABUS OP CONTENTS

page

Acknowledgments ...... lii Introduction ...... 1 Review of literature ...... 4 Part I. Lipase Activity in M i l k ...... 4 Introduction...... 4 Nature of Lipase Present in M i l k ...... 4 Specificity of Lipase Action ...... 8 Occurrence in Milk ...... 8 Distribution in M i l k ...... 11 Some Pactors Affecting Lipase Action 12 Lipase Activity in Cheese Flavor Development 23 Lipase Action on Other Characteristics of Dairy Products ...... 211- Conclusion ...... 23 Part II. Determination of Patty Acids ...... 26 Introduction...... 26 General Methods ...... 26 Methods Employed to Measure Patty Acids in Dairy Products ...... $8 Analysis for Other Products of Lipolysis ... 44 Conclusion...... 44 Part III. Lipase Activity in Other Biological Systems 45 Introduction...... 43 Other Lipases Used vith Dairy Products 45 Bacterial Lipases ...... 47 Pancreatic Lipases and Liver Esterase ...... 30 Methods of Separation and Concentration of Lipases ...... 52 General Notes of Enzymes ...... 54 Conclusion...... » 56 Experimental Data ...... 5& Discussion ...... 103 Summary ...... 104 Bibliography ...... 106 Autobiography ...... 112

ii

592525 AC Kw OWLEDGMENTS

The author wishes -bo egress his grateful appreciation to t h o s e who gave assistance and aid during the preparation of this d i s s ertation.

Special -fclnanks should he given to Dr. E. F. Almy, Professor

in. -the Department of Agricultural Biochemistry, for judicious

c01.3n.sel and a d v i c e throughout the problem; to the Department of

Agricultural Biochemistry for the use of its facilities; and to

t h e Department; of Dairy Technology for providing funds for a

fellowship d u r i n g the course of experimental work.

The author is also indebted to William A. Bulen, fellow class-

m a t e in the Department o f Agricultural Biochemistry, for permission

to u.se certain techniques developed by him and as yet unpublished.

Thanks m u s t also be given to Dr. H. P. Brown of the Goodrich

R e s e a r c h Center, Brecksville, Ohio, who supplied the samples of.

G e o n used in tliis research.

The author wishes t o thank Dr. W. James Harper, Department of

D a i r y Technology, for supplying samples used in this investigation

a n d for technical advice given during the course of the problem. INTRODUCTION

"And so these men of Indostan

Disputed loud and long,

Each in his own opinion

Exceeding stiff and strong;

Though each was partly in the right,

And all were in the wrong."

"The Blind Men and the Elephant"

by John Saxe.

One of the major problems encountered in the field of Dairy

Technology has to do with the development of fatty acid flavors in certain products as a result of hydrolytic decomposition of the fat.

In its early stages the taste of the product may be only slightly af­ fected, and is variously described as bitter, cocoanut, wintry, etc.

As the degree of deterioration increases, however, rancid flavors attributed largely to release of significant amounts of strong-flavored, volatile fatty acids of lower molecular weights become increasingly ap­ parent. This development of rancidity has been found to be an important problem in the storage of dried mills products, and particularly in the keeping quality of homogenized milk which is being increasingly adopted by the consumer.

On the other hand, the proper development of flavor in cheese

during ripening is thought to be dependent to a certain extent upon the amount and kind of fatty acids liberated from the milk fat present during

the ripening of the cheese. Thus the liberation of fatty acids may lead

to undesirable quality in some dairy products, while in others the result 2 is regarded as a desirable flavor change. In either case it is important for the dairy technologist to learn as much as possible about the factors responsible for fat decomposition and the condi­ tions governing such changes.

While the subject has been widely investigated there are still a great many unanswered questions as to just what is involved in the varying degrees of hydrolytic change which butterfat may undergo. It is, of course, known that the essential chemical change involves the hydrolysis of the complex mixture of mixed triglycerides found in milk fat into its component fatty acids and glycerol. Organic chemists state that this reaction may be promoted by high temperature, by alkalies with the formation of soaps, or by acids. Biochemists add a fourth type of factor - the fat-splitting enzymes known as lipases - which enable fat decomposition to go on rapidly at much lower temperatures than is the case with other catalysts. Since most of the hydrolytic changes in dairy products are of this latter type the work reported in the main body of this paper has to do entirely with the problem of fat changes resulting from the action of the fat-splitting enzymes or the lipases in milk products.

Maass (8l) la 1909 was the first to show that milk contained a true lipase. In the strict sense of the term, lipase should be confined to those enzymes which split the fatty acids from mixed triglycerides. Some authors misuse the term lipase when they use it to refer to such enzymes as esterases which can cleave fatty-acid esters. These esterases are incapable of hydrolyzing a mixture of mixed triglycerides such as milk 3 fat (123). This confusion in terminology has led to many erroneous conclusions in connection vith investigations of lipase systems in dairy products.

In order to draw any valid conclusions as to the nature and pro­ perties of milk lipase two criteria must "be fulfilled:

1. The lipase must act on milk fat in its natural medium.

2. The method used to analyze for the liberated fatty acids must

be accurate and give good recoveries of all fatty acids.

Failure to meet either one of these criteria leads to a state of confusion with respect to the nature of lipase activity resembling that of the

"honorable men of Indostan" referred to in the quotation above. It is the objective of the work to be described here to develop methods which will allow these criteria to be met. 4 REVIEW OF LITERATURE

Part I. Lipase Activity in Milk

Introduction

The off flavors of milk which have been characterized as bitter,

"wintry", and sometimes as resembling cocoanut, result from the presence of very small amount of free fatty acids from to (123) Lipase, which is a normal constituent of all milks, brings about this breakdown of the fat. (22, 32, 4l, 43, 68, 70, 84, 91, 92, 105) Milk lipase is usually regarded as a nonspecific fat-splitting enzyme capable of producing lipolysis upon a wide variety of fatty substrates such as tributyrin, ethyl laurate, etc. under favorable conditions. (33) It would be well to point out that any enzyme which brings about lipolysis in dairy products must function under the conditions which are found in the particular product.

For example, many workers have reported an enzyme in milk which is capable of hydrolyzing fats and esters at an optimum pH of 8.0 to 8.8. (62, 84,

112, 113, 115) It is of interest to ask of just what importance is this enzyme in milk, where the pH is in the range of 6.6 to 6.8, or in cheese where the pH is in the range 4 to 5, unless it can be shown that its activity is still significant in these more usual pH ranges for dairy products.

Nature of Lipases Present in Milk

There are some general conclusions which appear to be fairly well

accepted concerning milk lipase. First, it seems that there is more than 5 one type of lipase in milk. Tarassuk and Jack (123) have divided the causes of lipolysis into two broad groups:

1. Activated lipolysis which results from homogenization,

violent shaking of warm milk, and warming of the precooled milk

to about 30° C. and cooling again below 10° C. These treatments

lead to disruption, partial displacement or distortion of the

natural adsorption layer of the fat globules, and therein lies

the clue to the mechanism of activation.

2. Spontaneous lipolysis where the only condition necessary

for the initiation of lipolysis is the cooling of the milk. Lipase

of naturally lipolytically-active milk is present in milk plasma

prior to cooling. By cooling the milk, the lipase is irreversibly

adsorbed on the fat globules, and the lipolysis begins immediately

upon the adsorption.

Dunkley (13) has very recently shown a positive correlation between

tributyrinase activity and lipase activity. He further showed a coincidence

of the pH optima of both tributyrinase and lipase at 10° C. For some

research purposes the tributyrinase determination may be useful as a measure

of lipase in milk. It may well be that these two determinations measure

the same enzyme or enzymes, but the data are insufficient to justify a

definite conclusion on this point.

There is other evidence which also leads to the conclusion that there

may be more than one milk lipase. Gould (32) has shown that the lipase

action which occurs in homogenized raw milk is usually affected by various

external factors differently than is lipase in unhomogenized milk. Thus in 6 unhomogenized milk lipase activity was inhibited by copper while in homogenized milk no such inhibition occurred. Moreover, it was shown that in unhomogenized milk oxidative changes occur simultaneously with or precede normal lipase action while in homogenized milk no oxidative changes could be detected by means of the peroxide determination although large amounts of fat splitting had taken place. The addition of formaldehyde to homogenized milk had no effect on lipase activity while in normal milk lipase activity was inhibited by the addition of formalin. These data may indicate the presence of more than one lipase. Other workers have also observed the effect of formalin on milk lipase.

Herrington (4l, ^3) contends that milk contains at least two lipases. One is inhibited completely by very small amounts of formalin.

The other is apparently not sensitive to moderate amounts of formalin.

In some milk samples the formaldehyde tolerant lipase predominates; in others, the formaldehyde sensitive enzyme was more important. Most of the samples contained both enzymes. However, data did not reveal any difference in the abilities of the formaldehyde-tolerant and formalde­ hyde-sensitive enzymes to act in the temperature range from 32° F. to

”15° F.(77) It has been observed that the addition of formaldehyde and aging will increase the rate of lipolysis of milk without prior cooling. The addition of formaldehyde to well-cooled milk has no effect on the lipolysis. (12^) It has also been noted that in cream containing formaldehyde the degree of hydrolysis increased as the holding tempera­ ture of the cream increased within the range of 5° to 21° F. (22) Tributyrin has often been used as a substrate to measure lipase activity. (88, 89, 112, 113, 13) The significance of this is also questionable since there is no tributyrin in milk fat. In fact one of the workers states, "There is a possibility that milk lipase, which hydrolyzes milk fat, and the enzyme that splits tributyrin may not be

identical. Therefore, some authors use the designation tributyrinase for the agent responsible for the latter reaction. The present authors prefer the term lipase until evidence substantiates the double enzyme theory." (112) This approach to the problem does nothing to help solve

the question as to whether tributyrinase with an optimum pH of 8 or 9 has any aisnigicance in lipolytic changes observed at the normal pH of milk.

The data suggest the presence in cows milk of at least 2 lipases,

one more stable than the other. The ratio of the 2 enzymes seem to vary

from cow to cow since varying proportions of the lipolytic activity are

lost during the first few hours. Assuming the presence of several li­ pases, it is of course obvious that the rate of hydrolysis of tributyrin

at pH 8.5 is not necessarily directly related to the rate of hydrolysis

of the various butterfat glycerides at other pH values. (88)

Dunkley (113) very recently has done some work in this direction.

He is the first worker to attempt to show positively a direct relation­

ship between tributyrinase and milk lipase. In his work he used the

term "tributyrinase" and "lipase" to indicate the type of substrate

hydrolyzed, without intending to prejudice a conclusion as to whether

the enzymes involved are separte entities. He described a determina­

tion of tributyrinase based on measurement of tributyrin hydrolysis, and 8 an estimation of lipase by measurement of milk fat hydrolysis. His results illustrated a correlation between tributyrinase and lipase determinations and further a similarity of the pH activity curves for the enzymes measured by the two methods. His results provide greater justification for the use of the tributyrinase determination as a measure of lipase in milk. He found the optimum pH for both tributyri­ nase and lipase was 9»5 to 10° C.

A milk esterase has been reported which has optimum activity at

37° G. and pH 8.0.

Specificity of Lipase Action

It has not been conclusively demonstrated, but it has been thought that there is a certain degree of specificity of milk lipase. (123) Milk lipase appeared to show some selectivity in its hydrolysis, acting more rapidly on the volatile than on the non-volatile fatty acid containing substrates. (70)

Occurrence in Milk

Many workers have reported lipase to be present in all samples of milk, (kl, l+7» 73j 100) Herrington (4l) has shown that there is only a slight variation in the acidity of the fat in the milk of different cows when it is drawn from the udder, but after a few hours there may be wide variations in the acidity. The rate of lipase action is influenced by the rate of cooling of the milk, being retarded by sufficiently rapid cooling. 9 The amount of lipase which is present in milk is influenced by- several factors. Some lipase activity can be attributed to the secre­ tory tissue of the mammary glands if the glands have undergone the development of pregnancy. (68) There is a seasonal variation in which the amount of lipase reaches a minimum in early summer and a maximum in early winter. (k7, 2k) However, Dunkley (lk) did not observe this seasonal variation when the tributyrinase activity was measured. Opinion is divided as to the effect of the stage of lactation on the amount of lipase present. Some workers claim that the amount of lipase increases as the lactation is prolonged. (k7, 122, 2k) On the other hand, some workers deny that the stage of lactation has any effect on the quantity of lipase present, (lk, k3, 91) The effect of the estrus cycle on lipase activity is also open to debate. Advanced gestation may increase lipase activity (122, 7k) or according to some authors the estrus cycle may have no effect. (k3, 2k) There is also some evidence that different

quarters of the udder of the same cow produce milk of varying lipolytic

activity. (92)

It was confirmed (122) that the advanced stage of lactation is a

contributing or major factor in the secretion of milk high in the con­

centration of naturally active lipase. In this experiment every cow in

a late stage of lactation and gestation on the diet of bleached alfalfa

hay and concentrates secreted milk that spontaneously developed strong

rancid flavor. The ingestion of green feed causes a drop in lipase and

generally improves the flavor of milk; however, the principle responsible

for the beneficial effect of green feed, in preventing spontaneous

lipolysis of the milk fat, is not its high carotene content, furthermore the hydrolytic rancidity occurring in the milk of cows near the end of gestation on a dry-feed diet cannot he attributed to a low carotene content of the ration.

Ruth Reder (98) has attempted to correlate changes in other,,, milk constituents with lipase activity in an effort to determine which factors regulate lipase activity. First she noted that milk from cows frequently producing rancid samples has a higher chloride and a lower lactose content than normal milk produced in the same period of lactation. The chloride- lactose ratio of rancid milk is high. The high chloride and low lactose content appears to he characteristic of all milk produced hy those animals whose milk is frequently rancid. Occasional rancidity occurring in the milk of animals producing normal milk, or conversely, the production of normal milk hy animals frequently producing rancid samples, cannot he explained on the hasis of changes in the chloride and lactose content of milk.

It was also found (99) that, in general, rancid milk has a higher content of total solids, fat and protein than does normal milk of the same period of lactation. The increased protein content of rancid milk is attributed to an increase in the amounts of both the casein and lact- albumin fractions. The high content of these constituents appears to he characteristic of all milk produced hy those animals whose milk is fre­ quently rancid.

Further, rancid milk was found usually to have a higher titratable acidity and hydrogen-ion concentration than normal milk of the same period of lactation. The mean titratable acidity and pH of all rancid samples 11 were significantly higher than the mean values for all normal samples.

(100) This information is not startling in view of the acids produced

by lipolysis. It does, however, show that considerable quantities of

acids are produced. When one notes these changes in view of the

buffer capacity of milk, considerable quantities of fatty acids are

liberated. In fact, Kelly (68) has noted that lipolytic activity can

affect as much as 25$ of the fat ordinarily synthesized in milk in 2k

hours.

Reder (101) has also found that the fatty acid and cholesterol

content of the blood serum of cows producing rancid milk follows the

same trend as does that of cows producing normal milk during corres­

ponding periods of lactation. There is no increased lipolytic activity

in the blood serum of cows producing rancid milk, although such milk

has a greater lipase content than normal milk. The production of rancid

milk cannot be explained on the basis of a change in any one of the above

blood constituents.

Distribution in Milk

One point on which all workers seem to agree is that lipase is

found in the skim milk fraction. (32, 7k, 87, 92, 91) Rao (97) has

raised the question as to whether lipase might be one of the euglobu-

lins. Naturally it must become associated with the milk fat before it

can act and this mechanism will be discussed in more detail later on in

the section on the state of the fat globule, (lk, 97, 123) 12

Some Factors Affecting Lipase Action

A. Concentration of Enzyme

It should be pointed out that the degree of off flavor which develops is thought to be proportional to the amount of lipase present.

(92, 100) Dunkley (l4) has shown that the amount of lipase present usually is not the principal factor determining whether milk will become rancid. Because rancidity can be induced in most milks by treatments such as homogenization, agitation and controlled temperature fluctuations, it is a reasonable assumption that sufficient lipase is present to pro­ mote the lipolysis. It was his opinion that the condition of the fat surface appears to be the most important factor determining whether milk develops rancidity. (lb)

B. Influence of pH

The problem of the proper pH for lipase activity was mentioned earlier and the question was raised at that time of the significance of an enzyme active at a pH of 8 to 9 when the pH of milk averages 6.6 to

6 .8 . As mentioned before, Dunkley (13) found the optimum pH for both tributyrinase and lipase to be 9.5 at 10° C.

Using sugar saturated cream as the substrate Roahen and Sommer (103,

10b) found the optimum pH for lipase activity to be 8 .b to 8 .6 . They also found some activity at pH 6.6 and at pH 6.25J the activity was only one- third as much as at pH 6 .6 .

Gould (33) found the optimum pH for lipase action in homogenized rennet whey-fat emulsions was within the range of pH 8 to pH 9* bow pH 13 values adversely and permanently affect lipase activity. Acidified whey failed to exhibit appreciable lipolytic activity even though the pH was subsequently raised to the optimum range.

Proks (92) has also shown that the production of lactic acid slowed down the rate of development of certain flavor defects due to the lipoly­ sis of milk fat.

In the curing of raw milk cheddar cheese off flavors developed due to lipolysis. The addition of HC1 as a means of acidification was only partly successful. Larger amounts of starter and longer ripening period were beneficial. These results are explained on the basis of lipase inhibition by contact with acid over a period of time, as well as by influence of acid which affects lipase action in such a way as to produce mainly non-volatile acids. (55)

Since the optimum pH of tributyrinase is in the range of pH 8 to 9> this may indicate that there may be other enzymes which preferably hydro­ lyzes the longer chain fatty acids as indicated in the reference above.

While these longer chain, non-volatile fatty acids do not affect the flavor this observation is very interesting from the theoretical point of view.

C. Temperature

Temperature also plays a vital role ±1 lipolysis. In a comparison of $ to 4° C., 27° C. and 37° C. lipolysis was most extensive at 37° C., slightly less at 27° C. and decidedly less at ? to 4° C. (104)

Samples of unsalted sweds-cream butter were stored at a series of temperatures ranging from 32° F. to -15° F. The extent of lipolysis was 14 measured at intervals by titration of the free acids in the fat and the data indicates that lipolysis by natural lipases of milk is o ° inhibited at 5 F. or lower, though they are active at 30 to 32° F.

Further the data did not reveal any difference in the abilities of the

HCHO -tolerant and HCHO -sensitive enzymes to act at low temperature.

(77)

Butter samples were churned from fresh cream held at 110, 125,

140, 155, 170 and l80° F. for periods of time ranging from 1 to 150 minutes. The rate of lipolysis during storage of the butter prepared from the cream was measured by titration of free acids in the fat. At

110° F. lipolysis was first activated, then reduced as the holding time was increased. The rate was reduced by 2/3 when held for 150 minutes. At 125° F. the rate of lipolysis was reduced about l/2 after about 20 minutes but it was still measurable after 150 minutes. At l40° F. the rate of lipolysis was reduced more than l/2 at zero holding time. The rate was measurable with a holding period of 15 minutes but not after 35 minutes. For the butter prepared from cream heated to 155°

F. the rate of lipolysis was scarcely measurable after zero minutes of holding. (76)

Gould (30) has obtained about the same results in raw milk. Raw milk was heated momentarily and homogenized at 70, 105, 115, 125, 135, and 145° F. underwent lipolysis in every case. The maximum fat split­ ting occurred at 105° to 125° F. At 135° to 145° F. the acceleration of lipolysis by homogenization was slowed down but not entirely prevented. 15

Cream separated at 110° F. showed less lipolysis than cream separated at 75° F. both as observed by holding the samples at f to

1° C. and by using the cream to inoculate sugar-saturated cream and incubating at 37° C. Similarly- the 110° F. separated skimmilk showed less lipolysis than the 75° F. skim milk in sugar-saturated cream.

This can not be explained by the assumption that under this condition more lipase goes into the skim milk since the skim milk also exhibited less lipolytic activity than was found with the lower temperature of separation. (103, 104)

An increase in temperature of the milk during separation caused a decrease of lipase activity of both the skim milk and cream. A marked decrease occurs when the milk is separated at 120° F. The slime of the separator bowl is an excellent source of lipase and possesses about three times the lipolytic activity of the original milk. Decreased activity from high temperature of separation is believed due to inhibition of enzyme by heat. Since the inhibition is about the same in the cream, skim milk and separator slime, it appears that the effect of higher tem­ perature of separation is due to inactivation rather than fractionation of the enzyme. (91)

The time required at any temperature to inactivate the lipase was found to vary with the rate of heating to and cooling from the holding temperature. When milk was heated to lk2° F. at the rate of 5° F.increase per minute, instantaneous exposure at 1^2° F. was sufficient to inacti­ vate the enzyme. When heating by means of the Mallory unit to the re­ quired temperature within 5 seconds, a temperature of 185° F. with instantaneous exposure was necessary to result in inactivation. (h6) 16

Cream containing lipase pasteurized at 150° F. for 30 minutes o made good butter. If cream was held for 3 days at 40 F. before pas­ teurizing inferior butter resulted. (63)

From the foregoing discussion it can be seen that lipase can act over a wide temperature range, even at freezing. It can also withstand considerable heat. However, lipase can be inactivated by proper pas­ teurization.

D. State of Fat Globule

Some mention was made earlier of the activation of milk lipase.

Temperature also plays an important role in this activation process.

Krukovsky and Herrington (42) have stated that the rate of lipolysis seems to depend upon the crystalline state of the fat and consequently upon the previous temperature history of the milk. It also seems prob­ able that this unrecognized phenomena of activation has been responsible,

in part at least, for many experimental results which have been attri­ buted to other factors.

The rate of lipolysis in milk stored at low termperature depends upon the rate at which the milk was cooled before the storage period.

To secure a minimum rate of lipolysis, the cooling time should be reduced

to a few seconds. There is a critical temperature range in which the rate of cooling is most important. The upper limit of this range is 20 to 25° C. The lower limit is approximately 0° C. with natural milk and approximately 10° C. with temperature activated milk. (45)

Crystalline fractions separated from milk fat by stepwise cooling

show a broken trend toward higher iodine, Reicher-Meissl and saponifica­

tion numbers. The lower the temperature required for crystallization of 17 the fat fractions, the greater the increase in acidity when they are used as a substrate for milk lipase; this fact indicates that the rate of lipolysis is dependent upon the melting point of the fat or upon the degree of solidification of fat at a given temperature a

The degree of solidification is determined by the mutual solubility properties of the individual glycerides of which milk fat is composed.

(7*)

E. State of Fat Globule Surface o Milk lipase is activated by cooling the milk to 20 C. or below.

It is suggested that the cooling affects the permeability of the adsorp­ tion membrane around the fat globules to lipase, for agitation of milk, without cooling, also activates the enzyme. If milk is held at to 37°

C. for 1 to 3 hours after drawing subsequent lipolysis is much retarded. (124)

Lipolysis of milk fat is accelerated by resurfacing the fat globules.

Resurfaced fat globules, show no further increase in lipolysis due to cooking, warming, and cooling. The rate of lipolysis of resurfaced fat globules increases with increasing temperature (showing a normal tempera­ ture coefficient); whereas the rate of lipolysis of fat globules with the original normal surface increases as the temperature is lowered (showing a negative temperature coefficient). These experiments demonstrated the influence of the conditions of the fat-plasma interface on lipolytic activity in milk and cream. (74)

Rao (97) has very recently shown that the presence of phospholipids accelerates lipase action. Phospholipids assume an important role at the fat globule surface and probably control the activity of lipase.

However, the data is insufficient to conclude that they are necessary for the typical temperature activation phenomenon to be shown in an artificial emulsion. He raises the question as to whether milk lipase is one of the euglobulins or closely associated with the euglobulin fractions. The euglobulins are adsorbed nn the solid fat globules and are released into the serum when the fat globule is in a liquid condi­ tion.

Agitation, violent shaking or homogenization are other methods by which cettain types of lipase may be activated. Gould (33) has shown that homogenization creates a condition which very greatly enhances lipolysis as produced by pancreatic extract.

Shaking of raw, whole cow's milk while the fat is in the liquid or partially liquified state induces lipolysis and such induced lipolysis will continue after the milk has been cooled to low temperatures. The effect of shaking is attributed to an alteration in the surface charac­ teristics of the fat globules which creates a condition more favorable for lipolysis.

The increase in the rate of lipolysis due to homogenization or shaking may be due to the increase of surface area of the fat brought about by such treatment. It is well known in colloid chemistry that such increases in surface area can increase the rate of a surface re­ action. 19

Larsen (79) found that rancidity developed rapidly in mixtures of milk composed of:

a) unhomogenized raw milk and homogenized pasteurized milk

b) homogenized raw milk and homogenized pasteurized milk

c) unhomogenized raw milk and homogenized raw milk

The development of rancidity seemed to be equally dependent upon the amount of lipase present and upon the amount of acceleration afforded by the newly created surfaces. Gould and Trout (35) also obtained similar results earlier.

Cheese made from agitated milk possessed objectionable flavors due to increased lipase action. Cheese milk was submitted to vigorous agita­ tion by churning at temperatures from ^5 to 86° F. for periods of from 5 to 15 minutes. The effect of this treatment, particularly at the higher temperatures, was to activate the milk lipase and produce a rancid flavor in the resulting cheese. When the activation of lipase was less, flavors normally termed "unclean" were produced. The more prolonged the agita­ tion of the milk, and the higher the temperature of agitation, the lower was the flavor score of the resulting cheese. The increase in the fat- aqueous interface due to fat dispersion may explain in part the mechanism of increased lipase action observed in agitated milk. It is recommended that all cheese milk should be cooled at the farm, and unnecessary agita­ tion of the milk at the farm, in transit, and at the factory prior to setting the vat should be avoided.(53*5*0

F. Factors Inhibiting Lipase Activity

Not much appears in the literature on spontaneous lipolysis. The development of hydrolytic rancidity in raw milk by a naturally occurring 20 lipase present in high concentration can he successfully prevented hy mixing with normal milk within an hour after milking. The amounts to he mixed depend upon the concentration of the lipase. The mixing of lipase milk with normal milk in a proportion of 1 to 4 or higher always prevented rancidity. To insure the effectiveness of this method

of preventing rancidity, the mixing must he made within an hour after milking, before cooling, or immediately after cooling. If a milk contain­

ing a naturally active lipase is allowed, after cooling, to age separately

and thus become rancid, then the addition of a very small amount to a

normal supply will impart the rancid flavor to the whole mixture. (121).

Lipase in normal milk may he inactivated hy a reaction with dis­

solved oxygen in which dissolved copper acts as a catalyst. Normal milk

contains enough copper or other catalyst to cause the inactivation of

lipase hy dissolved oxygen at relatively low temperature. The relatively

low temperature of destruction of lipase in normal milk is the resultant

of two processes: the effect of increased temperature in accelerating the

inactivation of lipase hy dissolved oxygen, and the general destructive

effect of heat on enzymes. Dissolved copper causes no inactivation of

lipase in normal whole milk in the absence of dissolved oxygen.(75)

The acidity produced hy lipase was accompanied hy fat-perioxide

formation in amounts varying directly with the acidity. This was attri­

buted to the free oleic acid formed. The depression of lipase activity

hy the various metals varied directly with their catalytic power in

inducing fat oxidation later, and it is suggested that destruction of

lipase was catalyzed hy the traces of heavy metals according to their

varying powers of activating oxygen. Copper was the most potent inhi- 21

"bitor, while iron, nickel, cohalt, manganese and chromium were less active and tin and aluminum had no effect. (10)

Although copper inhibits lipase activity in milk under certain conditions it was ineffective as a lipase inhibitor in Cheddar cheese.

This is attributed to the strongly reducing potential of the cheese. The potential of the cheese remains at a sufficiently negative value to be able to reduce lipase to its active condition. It is, therefore, unlikely that slight variations of the oxidation-reduction potential or copper contamination would have any effect on the spontaneous development of rancid flavor under commercial conditions. (52)

The reversibility of oxidative inactivation of milk lipase was studied with three oxidation-reduction systems of interest in Cheddar cheese. It appears that cysteine can reverse the inactivation of milk lipase brought about by aeration, or by aeration and copper. Some augmen­ tation of lipase activity was also noted. Anaerobic environment may bring about some reversibility of oxidatively inactivated milk lipase but not in the presence of copper. The results with ascorbic acid were inconclusive. (50)

Exposure of fresh milk in glass vessels to bright summer sunshine for ten minutes destroys kQFjo of the lipase (tributyrinase). Exposure for thirty minutes destroys about 80$ of the enzyme. Exposure to an

800 watt, quartz mercury vapor lamp at a distance of 15 centimeters destroys 75$ of the lipolytic power of milk. If oxygen is removed from the milk before exposure to sunlight, the destructive effect is greatly diminished. (67) 22

The addition of 0.2 to 0.3$ electrolytic 39$ hydrogen peroxide solution to cow milk both at freezing temperature and at 20 to 30° C. did not affect lipase, amylase, tryptase and phosphatase but nearly destroyed peroxidase, catalase and reductase. The multiplication of the surviving microorganisms restores the catalase and reductase power to the milk some hours after the treatment. (8 )

Other substances have be&n shown to exert an effect on lipase activity. Some substances have an inhibiting effect while others pro­ mote lipolysis. Thus, salt has an inhibiting effect on lipase (91) and at the 5 to 8$ level it prevents lipolysis in homogenized milk. (32)

The addition of pitocin to samples of normal milk and those which developed rancidity quite consistently activated the tributyrinase found in both. When the longer-chain fats or butter oil were used, the pitocin caused unpredictable results with some series activated and some inhibited. (69)

The esterase which is found* in various milks exists in an inactive state in cow or goat milk and can be activated with a very small amount of ammonium hydroxide. The milk esterase also has lipase activity which

is strongly inhibited by atoxyl but not by quinine. (62)

Since all enzymes are protein in nature, it is to be expected that proteolytic enzymes might exert an effect on the enzymes and indeed such has been the case. Typical rancid cheese was reproduced experimentally with the addition of commercial lipase to cheese milk. Such rancid

cheese was also reproduced experimentally with the addition of homogenized milk to the regular cheese milk. Milk lipase was at least partially 23 inactivated by the use of varying amounts and proportions of rennet and

■pepsin. While similar flavor scores were found between vats activated and inactivated at the first grading, there was a tendency for the numerical grade of the cheese made from inactivated milk to fall in grade after a period of six weeks storage, (49, 6l)

Following the observations above, Gould (36) performed a few experi­ ments on lipolysis in homogenized mixtures of whey and cream or butterfat to which 3 to 20 ounces of rennet extracts per 1000 pounds had been added.

Lipolysis was slightly, but not significantly retarded by the rennet.

Lipase Activity in Cheese Flavor Development

It was mentioned in the beginning of this paper that fatty acids lead to off flavors in milk. In some cases, however, the acids are desirable and improve the flavor of dairy products.

Harper (39) has very recently undertaken studies of the lipase system of various rennet pastes and related enzyme preparations in order to explain differences in flavor of Provolone and Romano cheese made with different rennet preparations. Several commercial rennet pastes that produced cheese of little flavor were found to possess only slight lipase activity.

Babel (3 ) has found the addition of 13 to 40 grams of rennet pastes to 1000 pounds of cheese milk yielded a cheese of superior flavor. The fat acidities of this cheese were very similar to cheese made from milk without added lipase. Addition of 50 to 225 grams of rennet paste per

1000 pounds of milk resulted in a significant increase in fat acidity. 2h

All of the cheese showed rancidity at some time during the ripening and some lots were rancid at six months.

Lipase Action on Other Characteristics of Dairy Products

Certain other deleterious effects may arise from the liberation of fatty acids in milk. The difficulty in churning and the abnormal foam formation found in cream obtained from the milk of cows in advanced lac­

tation is thought to be due largely to lipolytic activity and the con­

centration of the resultant soaps and fatty acids in the air-plasma

interface. Less difficulty in churning and less lipolytic activity are

encountered if the cream is separated while the fat globules are in a

liquid state as contrasted to the solid state. Pasteurization of the

milk or cream as soon as drawn largely prevents the difficulty in

churning. (72)

Laurjc acid and oleic acid in milk resulting from enzyme activity

or added directly to the milk will, under proper conditions, seriously

interfere with and even inhibit entirely the clotting of the milk by

rennet. Cauric acid is the only other common acid in milk fat which

has this effect. The adverse effects of the saturated fat acids are

shown only after a period of aging at reduced temperature and addition

of rennet at 35° C. (or lower) and may be completely reversed by heat-

treatment for 30 minutes or more at 40° C. Some reversal occurs during

several hours heating at 35° C.The adverse effects of oleic acid occur

without aging and are not overcome completely by the same heat-treatment.

The clot-preventing effects of lauric acid are not evident at 35° C. if 25

CaCl2 is added to the milk in the curd tension test. This is probably due to chemical reaction between salt and acid and/or its compounds but may also be due in part to the lower pH of the CaClg-treated milk. When lipoly­ sis is induced in raw whole milk by agitation at proper temperature and subsequent holding at low temperature, some interference with normal rennet clotting results. Similar lipolytic effects are not necessarily induced in raw cream by churning although the curd tension of the butter­ milk will be low. Protein denaturation involving the fat-globule protein may be concerned in this pehnomena. (87)

Conclusion

The results discussed in the preceding section have been reported per se but it will be brought out in the section to follow that many of the analytical methods are not very satisfactory and this must be remem­ bered when interpreting the data.

It would seem that a great deal of the confusion in determining the extent of lipolysis arises because of the different analytical methods used and the lack of a sensitive analytical method which can be used for such measurements under all conditions. It is doubtful whether the same thing is being measured in all cases. There must be some sort of standardi­ zation as to techniques and methods before the true nature of lipase activity can be unraveled. 26

Part II. Methods for Determination of Fatty Acids

Introduction

The isolation and identification of the component acids comprising a natural fat is generally a difficult process, especially if it is to be carried out in a quantitative rather than a qualitative manner. The available techniques may be divided into three general types, involving separation by (l) distillation, (2 ) differences in solubility, and (3) adsorption, of fatty acids per se, or after conversion into esters, salts, or halogenated derivatives. Generally, the mixture of acids will be too complex to render direct separation of the individual components possible by a single process, for example, distillation. Therefore, it is usually necessary to separate the mixture into two or more gross frac­ tions containing a smaller number of components of relatively similar nature. (82, 93)

In this section the available methods of analysis will be discussed from the point of view of accuracy, sensitivity and limitations imposed by the substrate and the nature of the problem. Only enough of the actual procedure will be presented to permit an evaluation of the method.

General Methods

A. Distillation

The distillation method can be further subdivided into (l) regular distillation, (2) vacuum distillation, (3) steam distillation, and (^) molecular distillation. 27

The first two of these procedures have "been used very extensively in the study of natural fats. The first step in these procedures is to saponify the fat and then liberate the fatty acids with mineral acid and distill the free fatty acids or form the methyl or ethyl esters and distill the esters. The saponification step eliminates the procedure for use in this particular problem which is concerned only with the measure­ ment of the fatty acids already liberated by lipase activity. Other limitations of this method are the amount of fat required (100 to 300 grams), the time required for a single analysis, and the elaborate equip­ ment required. Examination of results of these methods on butterfat show wide variance in the fatty acid composition of butterfat. (106)

The Duclaux method for the determination of volatile fatty acids is an empirical technique based on distillation procedures. This method has been applied to butterfat. (610 It involves distilling a dilute solution of free volatile acids made up to a definite volume, collecting the dis­ tillate in ten equal fractions and titrating the acid in each fraction.

The procedure is based on the fact that each of the acids of the series

CnH2n+i C00H has a constant rate of vaporization when distilled under given conditions. Duclaux assumed that each acid in a mixture behaves as if it were alone and follows its own law of distillation. The chief difficulty with the Duclaux method lies in separating the total volatile acids originally present in the unknown without contamination with fixed and relatively fixed acids. (66) This difficulty along with the unwarranted basic assumption makes this method unsatisfactory.

Steam distillation has been used extensively in the examination of food fats. The Reichert-Meissl and Polenske determinations are well known to food and dairy chemists. Such methods are necessarily empirical because most of the fatty acids found in common fats are volatile with steam in varying degrees depending on the temperature, pressure, through­ put of steam and other factors developing during distillation. (82)

The Reichert-Meissl value varies considerably from season to season, with butterfat produced in various countries, and from other conditions.

These variations coupled with the empirical nature of the method make steam distillation unsuitable for use in this problem.

Mattick and Kay (8^) have employed steam distillation of a tributyrin substrate to determine the occurrence of lipase and its relationship to the lactation cycle.

Molecular distillation is used chiefly for removal of very high boiling fractions auch as high boiling alcohols and vitamins. Very elaborate equipment is required and the method can not do anything that can not be done easier by one of the simpler distillation techniques.

B. Solubility

Various methods are available for the separation of fatty acid mixtures which depend on the relative solubilities of individual acids, their salts, or bromo derivatives in different solvents, or in the same

solvent at different temperatures. These methods make it possible to

separate a mixture of fatty acids into saturated and unsaturated fractions, and to separate the unsaturated acids into sub-fractions of different degrees of unsaturation or into individual components. None of these methods are strictly quantitative owing to the mutual solubility effects 29 exerted by one component of the mixture on the solubility of the other components. However, they afford a means of resolving relatively complex mixtures into simpler ones which can in turn be separated further by other means.

Most of the methods for determination of fatty acids in dairy products are based on solubility techniques and will be discussed in detdil in the next section.

It has been suggested that the resonating character of benzene molecules exerts a specific solubilizing influence upon fatty acids.

The high solubilities of fatty acids in the relatively low polar 1,^- dioxane is evidence either of a dipole-dipole attraction of the 1 ,^- dioxane molecules for those of the acid, or of the occurrence of hydro­ gen bonding between those molecules. The relatively low solubilities of fatty acids in furfural result from the extensively "associated" nature of this highly polar solvent. The solubilities of fatty acids in dichloroehhane are between those in chloroform and carbon tetra­ chloride, indicating that there is evidently some hydrogen bonding of the fatty acids with the solvent molecules. (56)

The separation of mixtures by partition between two imiscible solvents, such as ether and water, is a well known chemical procedure.

The separation depends upon differences in partition coefficient which is defined as follows:

Partition coefficient . , (t OC. ; s _* *gm.solute/ml.of------=—non-mobile — --- — phase =--- gm.solute/ml. of mobile phase

Barry (5) has applied this principle to the separation of fatty acids by a technique known as counter-current distribution. The proce- 30 dure gives good separation of all fatty acids from formic to stearic.

It is necessary to use four different pairs of solvents to accomplish the separation. One pair of solvents is used for acids from to C^, a second pair for acids from C5 to Cg, a third pair for acids from Cg to and the fourth pair for acids from C ^ to C^g. However, this technique has not been used to analyze a mixture which contains the fatty acids from butyric acid through stearic acid in a single mixture.

The apparatus required for counter-current distribution is very elaborate and expensive. The procedure is also very time consuming.

C. Adsorption

Another general technique which is available is based on adsorption phenomena. These methods, in general, comprise solution of the products

to be separated in a suitable solvent and passing the solution through a

column of adsorbent. As a result of the difference in the degree to which

each molecular species is adsorbed, the various components of the solution

will be separated into a series of bands. Separation into bands results

from the fact that the weakly adsorbed substances will move through the

column more rapidly than those components which are more strongly ad­

sorbed. (82)

Chromatographic techniques are classified as follows:

1. Adsorption chromatography

2. Partition chromatography

3. Ion-exchange 31 Of these three techniques, only the second has been applied to the separation of fatty acids, so no further mention will be made of adsorption chromatography or ion-exchange.

In partition chromatography the equilibrium is established between two liquid phases, one of which is held stationary by a solid material rather than between a liquid and a solid as in adsorp­ tion chromatography or ion-exchange. In this case the separation de­ pends on the partition coefficient which was defined on the preceding page.

Many different adsorbents have been used in partition chromatog­

raphy of fatty acids with varying degrees of success. Among these are

silica gel, charcoal and various elastomers. Filter paper chromatography

is classed by some as a type of partition chromatography but from the

theoretical standpoint this classification is erroneous. Filter paper

chromatography has been developed only in a qualitative manner for the

fatty acids so will not be discussed in more detail.

In order to discuss partition chromatography there are several terms

peculiar to this technique which should be defined:

Column - vertical glass tube, which is packed with the adsorbent

carrier.

Carrier - the solid adsorbent material of which the column is

constructed.

Immobile phase - the solvent which is adsorbed intimatply on the

carrier. Mobile phase - the solvent which percolates down through

the column. This is collected in fractions and

titrated with standard base. This phase must be

imiscible in the immobile phase.

Elsden (15) was one of the first to extend the idea of partition chromatography to fatty acids. He employed a silica gel column but was not able to separate fatty acids containing more than five carbon atoms.

Ramsey and Patterson (9k-, 99, 96) have worked out a system for the

separation of fatty acids from one carbon atom to nineteen carbon atoms.

In order to bring about the separation they found it necessary to

separate the acids into three groups.

The first group consisted of the saturated volatile fatty acids

from formic acid through butyric acid. The acids were separated on a

silica gel column with water as the immobile phase and butanol-

chloroform mixtures as the mobile phase. Formic, acetic, and propionic

acids are separated completely from one another, but n-butyric and iso- butyric acids are obtained together, free from their homologues, however.

Positive identification of all the acids except isobutyric is based

upon the microscopic examination of a characteristic salt. Identifica­

tion of isobutyric acid is based upon its oxidation to acetone by acid potassium permanganate.

The second group consists of the saturated fatty acids from

valeric acid through capric acid. The acids were separated on a

silica gel column with methyl alcohol as the immobile phase and iso­

octane as the mobile phase. The separated acids were titrated with standard sodium ethylate and tentatively identified by their threshold volumes; and the identification in each case was confirmed by adding an approximately equal amount of an authentic sample of the suspected acid and testing the chromatographic homogeneity of the mixture on a

fresh column.

The last group consists of the straight chain saturated fatty acids from undecylic acid through nondecylic acid. The separation of the

even-numbered carbon atom acids from each other and of the odd-numbered

carbon atom acids from each other is fairly complete in a single fraction­ ation. However, an odd-numbered acid can not be separated from an adjacent

even-numbered acid. Recoveries of added acids are essentially quantitative

The fatty acids are separated on a column of silica gel using a mixture of

furfuryl alcohol and 2-aminopyridine as the immoble solvent and n-hexane

as the mobile solvent. Each acid is tentatively identified by the thresh­

old volume, and the identification is confirmed either by a melting point

determination or by adding an approximately equal amount of an authentic

sample of the suspected acid to the unknown and testing the chromatographic

homogeneity of the mixture on a fresh column.

The authors have not used their scheme on a mixture of all the

naturally occurring fatty acids. It is not known, for example, what

would happen if a mixture of C-j_ to fatty acids contained non-volatile

acids such as palmitic or stearic acid.

McRoberts (85) has adapted the method of Ramsey and Patterson to

the determination of acetic and propionic acids in bread. The sample is

subjected to a rapid distillation to give complete recovery of the two

acids which are then separated. 34

Moyle (86) and his co-workers have developed a chromatographic method of separation of the low molecular weight fatty acids occurring

in rumen digests. Silica gel is the carrier with various phosphate

"buffers as the immobile phase and mixtures of n-butanol-chloroform as

the mobile phase. Reported recoveries of fatty acids in varous mix­

tures range from 97 to 103%.

The buffers were prepared from 2M stock solutions of the various

phosphates as follows:

Buffer 1 - 2 vol. K^HPO^ + 1 vol KHgPO^

Buffer II - 2.5 vol. KgHPOi,. + 3-5 vol. KgPO^

Buffer III - K^POjj. alone

Buffer IV - KHgPO^ alone

The columns were prepared by mixing 5 gms. silica gel with 3 cc.

of the proper buffer in a small mortar. This mixture is then slurried

with 40 cc. of 1% n-butanol in chloroform and poured into a glass tube

18 mm. in diameter. The silica gel settles and is packed by allowing

the mobile solvent to percolate down through it. The acids in solution

were added to the top of the column. The proper solvent was added to

the top of the column., allowed to percolate down through the column,

and collected in fractions at the bottom of the column. The fractions

were titrated with 0.005 N KOH in methanol using 0.05% cresol red in metha­

nol as the indicator. When the volume of base used is plotted against the

number of the fraction a series of maxima or peaks occur. Each acid will 35 exhibit a peak and hy proper selection of buffer and solvent the various acids were separated. The solvents used were 1$>, 10fo, and

30$ mixtures of n-butanol in chloroform.

Their analytical scheme may be summarized as follows:

Table I

Buffer pH* BuOH i Eluted By-passed** Retained

I 6.6 1 Ci, and higher 10 C3 30 C2

II 8.1* 1 c6 Cj and higher C3-C2 10 c5 30

III 9.5 1 C7 and higher — Cj, and lower 10 c6 30 c5

*Glass electrode at l6° G. 5 gm. silica gel + 3 cc. buffer suspended in 10 cc. H2O. **In first 5 to 6 samples.

Some of their typical results are summarized in the following

table: Table II

Column Acids taken io Recovered I n-butyric 102 I propionic 96 I acetic 10k II n-caproic 100 II n-valeric 100 II n-butyric 97 III n-capric ) III n-caprylic) 100 III n-caproic 98 36

It will be noted in Table I that the Type II column is supposed to by-pass and higher fatty acids. The authors do not give any data to support this. In fact they found it necessary to effect a prelimi­ nary separation of the lower acids from lauric acid and higher members of the series by a method of steam distillation. The separated volatile acids can then be chromatographed.

It will be shown later in the experimental data that acids above Cq can not be eluted from the alkaline columns (Type II and III). This blockage comes about because as the length of the fatty chain is in­ creased, the solubility of the soaps decreases rapidly in the alkaline medium.

Boldingh (6) has achieved the separation of fatty acids from caproic acid through stearic acid. He has used various elastomers such as weakly vulcanized Hevea rubber, neoprene, Dow Silastic and Geon as the adsorbent carrier and benzene as the immobile phase. The mobile phase consists of various mixtures of methanol, acetone and water. A solution (M) of 3 parts methanol and 1 part acetone was prepared. This solution was then mixed with distilled water in the following proportions:

Solution Parts M Parts HgO

M ko ko 60 M 60 6o ko M 6 5 65 35 M 70 70 30 M 7k 7k 26

His results are summarized in Table III on page 37* ■ 37 Table III

Ac id Mobile Phase Retention Vol. (ml)*

C£ M 40 20 C8 M 60 80 C10 M 60 140 Cl2 M 60 230 Cl^ M 65 3I+O Cl6 + Cpg unsat'd M 70 5^0-600 Cl8 M 7^ 7^0 *[The volume of mobile solvent where the titration value for the particular

acid is at a maximum.)

He has used from 2 to 10 mg. samples when single acids were used

and kO mg. samples when mixtures were to be analyzed. It must be

pointed out that temperature is critical in this method and must be

maintained at 20° to 22° C. throughout the analysis.

More will be said of this method in the experimental data since it

was found necessary to change some of the details because of differences

in the rubber samples used.

Masuyama (83) has prepared colored esters of the normal saturated

fatty acids from C-j_ to Cp8 with 4-phenylazophenacyl bromide. These esters

were then separated by chromatographic techniques.

Holman (58, 59) has demonstrated that saturated fatty acids can

displace their lower homologs from charcoal, making the application of

displacement possible in the fatty acid series. However, in its present

state of development, displacement analysis is not suitable for analysis

of unknown samples. 38 Methods Employed to Measure Fatty Acids in

Dairy Products

Balls (4) has discussed the difficulties from the twofold stand­ point of chemical analysis and regulatory enforcement of the determina­ tion of the activity of the fat splitting enzymes.

As stated previously in the general discussion the measurement of

fatty acids in dairy products usually depends on some solubility tech­ nique. With this in mind the methods which have been used to study

dairy products will be outlined.

A. Titration of entire mixture

The hydrolysis of equimolar amounts of a series of triglycerides

containing saturated fatty acids with 2, 3> k, 5> 6, 8, 10, 12, 14, and

18 carbon atoms by a series of lipases including human milk lipase was

investigated in a system composed of an ammonium buffer, calcium chloride*

and ox bile in glycerol, the activity being measured either by titration

of the liberated acid or by measuring the liberated carbon dioxide in a

Warburg Apparatus (109). The latter method will be discussed in more

detail in the section on miscellaneous methods.

Peterson, Johnson and Price (88, 89) studied lipase activity in

milk and cheese by measuring the extent of hydrolysis of tributyrin in

buffered solutions. They measured the amount of acids liberated by

titrating the whole mixtures before and after hydrolysis. However, they

could only get their results to check within 10$. The lack of sensitivity

of this method is readily apparent. In any buffered mhfcure the unex­

hausted buffer would have to be titrated in addition to the liberated acids. If a milk product is to t>e used as a substrate the production of lactic acid would seriously interfere and it would also be neces­ sary to consider the buffer index of the particular product being used.

It is very probable that only a very small part of the titration would be due to fatty acids.

B. Titration of the fat phase only

The direct titration of milk fat to measure free fatty acid content

is a far more sensitive means of detecting lipolysis than by titration

of the milk or by pH measurements (31).

The problem immediately arises as to how to remove the fat phase

from the milk. One of the earliest techniques was to churn the fat out and then clarify it by melting, centrifuging and filtering. However,

several difficulties have been encountered with this technique. In high

acid samples it was difficult to get the cream to churn. Difficulty in

churning was also noted when preservatives such as formaldehyde were used.

The chief objection to this technique is that the more water-soluble

volatile fatty acids are lost in the buttermilk leading to low yields of

free fatty acids (6*0.

The other possibility for removal offet would involve an extraction

with some good fat solvent. Various alcohols have been tried under

different conditions. A 12-fold increase in the acid number of rancid

fat over normal fat was found when the fat was extracted with ethyl

alcohol. However, the extraction was not considered complete (34, 64).

Hollender (57) presented a paper at the 1948 American Dairy Science

Association Weeting in which he reported an alcohol extraction process 40 for powdered milk, cream and other high fat dairy products. The product was extracted twice with boiling 95$ ethyl alcohol* cooled and centrifuged.

The extract was titrated with alcoholic alkali to the phenophthalein end point. They reported quantitative recoveries of oleic, palmitic and butyric acid and various mixtures of these three acids. The question of the extraction of non-fatty acid acidity (lactic) was raised but not answered. Later reports on this work could not be found.

Johnson and Gould (64) tried various mixtures of ethyl ether and

Skellysolve F, but the best combination gave only 6 .65$ recovery of butyric acid and 87 .05$ recovery of oleic acid. They also tried various treatments of the milk prior to extraction with these solvents in an effort to improve the recoveries. Saturation of the milk with salts such as MgSOif. and NaCl prior to extraction did not give any significant in­ creases in recoveries. However, acidification of the milk to pH 2 with sulfuric acid prior to extraction did result in 30$ recovery of butyric acid. They also tried continuous extraction of milk which had been pre­ viously dried in plaster of Paris but did not get any improvement in results (65).

Fatty acids were titrated in an ether solution of the fat, using an underlying layer of 5$ barium or crlcium chloride to observe the end point with phenophthalein. The method prevented products of proteolysis from interfering with the titration values and minimized hydrolysis of the soaps which were formed (10). No figures were given for recoveries but from the conclusion drawn it is doubtful if good recoveries of butyric acid were obtained. 41

The literature dealing with the relation between acidity of milk fat

(usually expressed as acid degree, i.e., milliliters of IN NaOH required to neutralize lOOg. of fat) and the rancid flavor in the product from which it was obtained shows considerable lack of agreement. On the basis of acid degree determinations applied to fat obtained from cream samples, it has been concluded that fat acidity determinations are helpful in classifying cream as rancid or not rancid, but they are of little value as a measure of the intensity of rancidity when compared with organoleptic evaluations as the standard.(12).

Dunkley (13) has worked out a method for determination of lipase

in which a buffered milk fat emulsion was used as the substrate. After the incubation period the fat was extracted with ether and titrated. He has also used a similar procedure for the determination of tributyrinase activity. He did not make any trials in which he added known amounts of acids to measure the recovery. He found good correlation between tribu­ tyrinase activity and lipase activity.

Hillig (48) has developed an extraction procedure for measurement of the water-insoluble fatty acids only in cream and butter.

A procedure has been outlined for the estimation of milk lipase which is based on the titration of an ether extract of butyric acid

derived from the enzymatic hydrolysis of tributyrin under standardized

conditions. Approximately 90$ of added butyric acid was recovered. How­

ever, this method measures only tributyrinase activity. The procedure

has been extended to measure bacterial lipase and lipase activity in

cheese.(112, 113, 114) Another mixed ether extraction procedure has been worked out in which Tween 20 (polyoxyethylene sorbitan mono“laurate) is used as the substrate. The great advantage of this method is that the substrate is completely water soluble (l). However, it has not been proved that the enzyme which hydrolyzes this ester is a true lipase.

C. Surface tension

It has been found possible to detect the development of rancidity in milk by following its progress through surface tension measurements.

(120) The relation between lipase activity as indicated by surface tension measurements and flavor as judged by 144 milk samples including not more than 4 samples from one cow and representing 51 individual cows gave a correlation coefficient of 0 .23. (51)

Dunkley (12) found that rancidity in milk could be estimated more conveniently by changes in surface tension than by changes in acid degree. His results can be summarized in the following tables:

Table IV Acid Degree Class No. of Samples Average Range

Not rancid 45 O.98 0.44 - 2.10 Slightly rancid 15 2.59 1.47 - 4.88 Rancid 32 3.46 1.09 - 5-9b

Table V

No. of Surface Tension (dynes/cm.) 20° C. Class Samples Average Std. Deviation Range

Not rancid 421 46.61 0.95 44.4-50.4 Slightly rancid 193 44.23 1.02 41.8-47.1 Rancid 238 40.74 2.52 32.3-46.1 There are several other factors which can influence the surface tension of milk besides the liberated fatty acids. Of these the percent fat has perhaps the greatest effect. Surface tension dropped from about

50 dynes/cm. at 0$> fat to about kG dynes/cm at 6$ fat. Above Gfjo there was little decrease in surface tension with increased fat percentage.

The percentage of fat also influenced the surface tension at which rancidity was detected organoleptically. Rancidity was not detected in three recombined milk samples containing approximately 2$> fat, even though their surflace tensions had dropped below k2 dynes/cm. Results also indicate that the type of sample bottle may be a factor influencing lipolysis. Temperature profoundly influences surface tension and must be controlled in order to obtain reproducible results. Since surface tension measurements show only gross aspects of lipolysis, they are of little value in fundamental research.

D. Miscellaneous Methods

Colorimetric Methods - Several compounds which yield colored products upon hydrolysis have been proposed as substrates in which to measure lipolytic activity. The optical density cf the solution will depend thus upon the amount of substrate split. In these procedures it is debatable whether they measure activity of true lipase or of an esterase. Gad (25) has proposed p-nitrophenyl-butyrate as a substrate for such a procedure. Greenbank (37) proposed a similar scheme at the

1950 American Dairy Science Association. His scheme is based on the splitting of 0(.-naphthyl esters of the fatty acids, chiefly OC-naphthyl acetate. Upon hydrolysis these esters react with 2,6-dibromoquinone- chloromide to yield purple colored compounds. The method detects 1 pound of raw milk in 2000 pounds of heated milk.

Manometric Techniques - Lipase can he measured indirectly in the

Warburg apparatus. If lipolysis is carried out in a Warburg flask with

some sodium bicarbonate present, a quantity of CO2 will be generated

which is proportional to the amount of fatty acid liberated ty lipolysis.

(109)

Analysis for Other Products of Lipolysis

It may be possible that some of the other products of lipolysis

such as mono- or di-glycerides contribute to the overall flavor effect of

lipase. Desnuelle (ll) has developed methods for the analysis of mono-

and di-glycerides. He has also shown that these products can determine

the ultimate course of lipolysis.

Conclusion

From the preceding discussion of the methods available it is seen

that they fail to be of any considerable value for the purpose of deter­

mining the lipase activity in dairy products because of one or more of the

following reasons:

1. Only the gross results of lipolysis are measured.

2. Esterase rather than lipase activity is measured.

3* A synthetic medium is used in an unnatural environment.

4. Incomplete recovery of all of the liberated fatty adids.

5. No means of separating the fatty acids formed by lipolysis is

offered. 45

III. Lipase Activity in Other Biological Systems

Introduction

If one surveys the biochemical literature on lipase activity there is very little information to be found on milk lipase. This is especially true if one seeks information on the mechanism and rate of lipolysis.

It is possible that such enzymes as milk lipase, pancreatic lipase and liver esterase may ultimately be traced to the same source.

When one analyzes the data in regard to pancreatic lipase, for example, many similarities are noted between its activity and that of milk lipase.

With these similarities in mind it will be profitable to examine the role of lipases in other biological systems. This could well give some clues to the mechanism of the hydrolysis of milk fat by lipase.

Other Lipases Used with Dairy Products

Raw skim milk and whey have been shown to contain an esterase which accelerates the hydrolysis of diglycol laurate, diglycol oleate and other esters at temperatures below 10° C. This enzyme is heat labile and can be removed by ultrafiltration. (87)

The role of lipase in the ripening of cheese was discussed at

some length in the first section on the general discussion of lipase

in dairy products. Peterson (90) however contends that milk lipase

disappears during the making of cheese and is completely absent in the

young cheese. The addition of rennet extract during Cheddar cheese- making causes an increase in lipolytic activity. This increase disappears within a period of about 30 minutes. After 5 to 20 days, lipases which are considered bacterial begin to make their appearance in the young Cheddar cheese.

Mulberry juice is a source of lipase which has been used in the production of Cheddar cheese. Addition of mulberry juice to pasteurized milk did not improve the flavor of the resulting cheese. Small amounts had little effect while larger amounts resulted in rancid and unclean

flavors. The rancidity developed in cheese made with rennet pastes was not as objectionable as the rancidity produced by mulberry juice. The

results suggest some selective hydrolysis. (3) These results would also

seem to indicate that the effects of rennet extract last longer than

indicated by Peterson.

Harper (39) has studied the lipase system of rennet pastes and

related enzyme preparations in order to explain differences in flavor of

Provolone and Romano cheese made with different rennet preparations.

Rennet pastes made from kids produced a more typical Romano and Provolone

cheese than did calf rennet pastes. The differences in flavor were

attributed to possible differences in the lipase systems.

Hydrolysis of a series of triglycerides by human milk lipase

followed the equation for unimolecular reactions up to 20$> hydrolysis

for the lower molecular weight triglycerides, and up to 60 to 80$ hydroly­

sis for those triglycerides of highest molecular weight. Human milk lipase

failed to catalyze the hydrolysis of trilaurin and trimyristin at an ap­

preciable rate. The rate of decrease of hydrolysis with increasing kl enzyme concentration was as much as 5 times as great for human milk lipase as for pancreatic lipase. (109)

Bacterial Lipases

Many different types of microorganisms have been shown to possess lipolytic enzyme systems. Richards (102) has studied rancidity in butter as produced by the action of microorganisms and thinks it prob­ able that many different lipases would be required to complete the hydrolysis of a mixture of mixed triglycerides such as butter fat. He has found that liquid acids make up the greatest proportion of the

fatty acids recovered. From 77 to 85$ of the total acids recovered were liquid, the chief ones being oleic and linoleic. He used an ether

extraction procedure to separate the fat and titration with sodium hydroxide to measure the fatty acids.

Fouts (23) has studied the effect of lactic acid on the hydrolysis

of fat in cream by pure cultures of lipolytic microorganisms. Oospora

lactis and Achromobacter llpolyticum were inhibited somewhat by the

growth of butter culture organisms in cream as judged by changes in the

acid value of the fat. Mycobacterium lipolytica showed increased growth

in the presence of the butter culture organisms. Lipolysis, even in

high acid cream, was extensive enough with all organisms investigated to

be of importance in cream quality, lactis, Myc. lipolytica, and Ach.

lipolyticum were definitely inhibited by the addition to the cream of

excessive amounts of lactic acid. However, they all grew well in cream containing sufficient added lactic acid to give a titratable acidity of about 1$. The first two species caused lipolysis in cream with an acidity of 2.08$. The addition of lactic acid to sterilized cream

in amounts sufficient to increase titratable acidity up to 2 .08$ did not cause changes in the acid numbers of the fat after holding for six

days at 21° C.

The lipolytic activity in skim milk cultures of microorganisms

representing a number of species and ganera were studied by the extrac­

tion- titration method described on page 4l. (112, 113) Wo evidence was

found of a bacterial lipase having an activity optimum on the acid side

of neutrality. No lipase active at a pH of about 5-0 was demonstrated in

20 samples of commercial Cheddar cheese of varying age or one sample of

blue-veined cheese on measurement by the extraction-titration method or

by the method of Peterson on page 37- (88, 89). Weak lipolytic activity

was found in one sample of blue-veined cheese by the extraction-titration

method. No lipolytic activity at pH 8.5 was demonstrated in one sample

of cheese by the extraction-titration method. (114)

The lipolytic activity of a strain of Pseudomonas fluorescens was

investigated. Activity was greatest in the pH range 8 to 9 at about 42° C.

Calcium chloride inhibited rather than enhanced lipolytic activity. The

lipase is not specific for tributyrin but hydrolyzes tricaproin and

tricaprylin as well although with decreasing ease. This confirms the

observations of Collins and Hammer that bacterial lipase is not charac­

terized by narrow substrate specificity. (115)

The lipases obtained from the broth of Aspergillus niger and

Penicillium roqueforti exhibit distinct differences in relative specifi­ ^9

city. Tributyrin, tricaproin, tricaprylin, and tripropionin were hydrolyzed by the Penicillium. lipase in this order in decreasing

rates. The order of decreasing rates for the Aspergillus lipase was:

tricaprylin, tributyrin, tricaproin, and tripropionin. Triacetin was

not hydrolyzed by either lipase.

Hydrolysis of an equimolar mixture of tributyrin and tricaprylin

by the Aspergillus lipase liberated butyric and caprylic acids in the

ratio of about 1 : whereas hydrolysis of tributyrin and tricaprylin

separately gave a ratio of approximately 1:1.7. In both cases the

Penicillium lipase liberated butyric and caprylic acids in the ratio

of about 3:1-

The effect of butyric, oleic, and caprylic acids on the hydrolysis

of tributyrin by the two lipases was studied. The Penicillium lipase

was affected less than the Aspergillus lipase. The different acids

showed different effects.

The optimum pH for the two lipases was found to be between 5*0 and

5*5. The optimum temperature was found to be between 30° to 35° C. for

the Penicillium lipase, and between 35° to 40° C. of the Aspergillus

lipase.

Calcium chloride accelerated the action of both lipases. Acetone,

ethyl alcohol, formaldehyde, dioxane, toluene, and ethyl ether inhibited

both enzymes although not to the same degree.

The lipase in the culture broth was concentrated by slowly freezing

out some of the water, followed by precipitation with ammonium sulfate.

(110) 5°

Rosenfeld (107) has confirmed the existence of bacteria capable of anaerobic lipolysis. Among these organisms are the important group of sulfate reducers. Hydrolyzable substrates, included esters of mono- hydric alcohols, glycerides of both soluble and insoluble fatty acids and more complex fats and oils.

Pancreatic Lipase and Liver Esterase

The pancreatic juice of dogs 0 to 12 days of age does not contain

any lipase. Lipase makes its appearance at 1.5 months of age and rises

rapidly to quantities characteristic of the adult animals. (78)

The pH activity curves are essentially the same for liver and pancreatic lipases but vary with the nature of the buffer, the degree

of enzymatic purity, and the constitution of the substrate. Observa­

tions of this nature suggest the possibility of a genetic relationship

between the lipases of the pancreas, stomach, intestinal mucosa, serum,

milk, etc. The properties of an enzyme elaborated in one organ may be

changed by transition into another organ or body fluid. Pancreatic

lipase has been shown tote associated with globulin while liver esterase

is associated with albumin. Pancreatic lipase and liver esterase differ

from each other in the following ways:

1. by their physiological function

2. by the nature of the protein fraction in which they are found

3. by their substrate specificity

4. by the type of their kinetics

5. by the difference of affinity for a given substrate

6. by the influence of a foreign substance on their action. The authors have postulated that the substrate is bound to inactive areas of colloidal pancreas globulin particles in such a manner that it is not accessible to the active groups of the pancreatic enzyme itself, but remains available to added liver esterase. If this hypothesis is correct, the combination oflarge amounts of substrate with inactive areas or "dead spots" on the lipase particles or with altogether inactive globulin particles evidently would diminish the actual concen­ tration of substrate in the aqueous phase. This, in turn, would increase the apparent solubility of the substrate.

The existence of inactive areas on the enzyme gives a basis for the understanding of the differences in kinetics between pancreas and liver enzymes, offers an explanation of non-competitive and excess substrate inhibition, and makes clearer the mechanism of acceleration. (ll6)

The evidence obtained strongly suggests the coexistence in pan­ creatic extracts of at least two enzymes concerned with ester hydrolysis.

One enzyme hydrolyzes the triglycerides beginning with tripropionin with a high initial rate of hydrolysis, which, however, slows down very soon and never under the conditions used brings about more than kQPjo hydrolysis.

This enzyme possesses a relatively high resistance toward inactivation by heat, crystalline trypsin, and alkali. The inactivation seems rather to be concerned with a decrease in the capacity to hydrolyze the sub­ strates at their former high initial velocity, than to fail to bring about hydrolysis at all. The other, more labile enzyme hydrolyzes monovalent alcohol esters, the glycol esters and triacetin with a lower initial rate of velocity which is maintained until the hydrolysis has reached 30$) thereafter, the time curves of the hydrolysis ceases to remain linear, but continues at least to 90fo. Treatment of this enzyme with heat, alkali, or crystalline trypsin results in an almost complete destruction of the hydrolytic capacity. (19, 21)

It is probable that both cholesterol esterase and the esterases which hydrolyze methyl butyrate and its homologs are either very similar to each other or else identical. (20)

Inhibition of enzymes has been shown to be due to the formation of inactive complexes while activation results when the interfacial tension between the enzyme and substrate is lowered. (27)

In the course of an investigation on the nature of phospholipid- splitting enzymes, an apparently new phenomenon was observed: That of the probable formation of an ether soluble phospholipid-enzyme complex, and the subsequent degradation of this complex in an ether solution.

The products were free fatty acids and lysophospholipids. It was proved that the phenomena was not an interface reaction. (3 6 ).

Christiansen (7) has discussed a method to derive the mechanism of an enzymatic reaction the kinetics of which are not known.

Methods of Separation and Concentration of Lipases

Glick (28) has pointed out the protein nature of the lipases. Thus, many of the techniques used to isolate and concentrate proteins in general may profitably be applied to the separation of enzymes.

Tabuer (119) has listed the following techniques which have been applied to the separation of enzymes: (l) adsorption methods; (2) extraction methods, (3) salting out, (4) electrophoresis, and (5) frac- 53 tional iso-electric precipitation. All these techniques may not he adaptable to a given enzyme and it may be necessary to use combinations

of these methods to achieve the desired results.

Adsorption by cotton may prove useful in the selective concentra­

tion of enzymes and in testing the purity of crystalline enzymes.

Pepsin, rennin, and catalase can be removed from their solutions by a

single filtration through cotton or to a certain extent by filtration

through filter paper, whereas, peroxidase is only slightly adsorbed.

(118)

A method has been described whereby proteins, enzymes and similar

substances of high molecular weight are adsorbed quite strongly in the

presence of salt solutions in low concentration. On some salt free

adsorbers the solutions show no affinity or only slight affinity for

the adsorbers. Five cc. of 1$ egg albumin was adsorbed on 0.5 gm.

silica gel at concentrations of 0 to 2.0 M ammonium sulfate. The adsorp­

tion was measured by changes in the ultraviolet extinction at 280 mu on

a Beckman spectrophotometer.(125)

The fungi Fusarium lini var. Boli has been shown to possess a true

lipase. This enzyme has been isolated from the mold and can be concen­

trated and purified about fifteen-fold over the original starting ma­

terial. The method involves essentially the use of acid and alcohol.

The lipase is intra-cellular, ionstable in water but stable in the dry

state, soluble in glycerol, glucose, and salt solutions, and does not

possess a dissociable prosthetic group. (17, l8) 54

Conners (9) has purified an esterase from horse liver acetone powder by a combination of ammonium sulfate and acetone fractiona­ tions, heat and heavy metal denaturations and dialysis procedures.

However, the procedure was not applicable to beef liver as it ap­ peared to have a lower esterase content than horse liver.

General Notes on Enzymes

In conclusion it would be well to summarize briefly a few points which can not be overlooked in the study of enzymes.

An exothermic reaction (such as adsorption) on the surface of a large molecule (such as a protein) or a colloidal particle may cause local temperature increases before the heat evolved spreads over the whole molecule or particle. By use of probable values for the heat conductivity of the particle and for the heat of adsorption, it is shown that the surface temperature may be 70° above the mean tempera­ ture. This may be important for enzymatic reactions at the surface of protein molecules, (ill)

Studies have indicated that the action of a lipase on any one substrate is of limited value as a basis for predicting its action on any other substrate. In attempts to overcome procedural difficulties in lipase studies, numerous simple synthetic substrates have been sub­ stituted for natural fats. However, one should not assume that any correlation necessarily exists between the lipolysis of synthetic sub­ strates and the lipolysis of natural fats. (110) 55 Falk (l6) has presented evidence which is best interpreted in the sense that the enzyme responsible for ester-hydrolyzing or lipase actions possesses a definite composition or is present as a definite grouping. The different actions observed when proteins or tissue extracts are present or added are due to their composition and properties. Their actions can not be considered solely or even essentially protective because of the striking specificities observed, although a certain amount of stabilizing or protective action is cer­ tainly present. The following general principle may be stated:

Actions on

Substrate A Substrate B

Enzyme E + + Proteins P 8s Q 0 0 E + P +-H- + E + Q + (p e ) ■ 1-1-1- + (PQ) +

It is apparent that in discussing enzyme actions, the system as a whole must be considered. Taking one constituent and determining its action or reaction may give an incomplete and misleading picture of the changes which occur in the complete system. At the present time, the various actions and interactions of the constituents of biological systems are not sufficiently well defined to make possible the predic­ tion of the behavior of the whole from a knowledge of the parts. 56

Conclusion

General conclusions from observation of lipase activities in other systems indicate:

1 . lipases may act more readily on some triglycerides than on

others. (109, 110)

2. different lipases acting on same fat appear to give different

products as judged by flavor. (3)

3. possibility that many different lipases would be required to

complete hydrolysis of complex mixture such as butterfat has

been suggested. (102)

Such observations would suggest that in the lipolysis of milk fat similar findings may. be expected. Thus one might expect to find differ­ ing activity of the lipases present depending upon their source, whether present in milk as secreted, or from bacterial or mold contamination, from rennet, etc. Likewise varying conditions as to acidity, tempera­ ture, presence of inhibiting agents, etc., might be expected.to alter the change and the products liberated either by repressing one lipase and favoring another, or by shifting the point of attack on the trigly­

cerides to cause the release of different acids under varying conditions

To enable one to follow such changes accurately it becomes apparent

that the investigator should be able to accurately determine both the kinds of fatty acids liberated and the amounts. Up to the present no

very satisfactory methods have been available for this purpose, so the

knowledge of the exact results of lipolysis is based largely on estima­

tion of total fatty acid liberation. This gives little basis for dis­

tinguishing between the characteristics of individual lipases which may 57 be concerned. It has been possible to measure the relative degree of total lipolysis, but not the value of the contribution of each separate lipase when two or more are present in the same system for the most part. Neither has it been possible to determine with much accuracy the individual acids released by any given enzyme to compare action with another type of lipase. 58 EXPERIMENTAL DATA

Standard Procedures

Certain equipment and procedures used throughout the work are

standard methods of quantitative analysis and so will he described

only briefly. Various standard alkali solutions were used in the

course of the work. These were prepared from chemically pure re­

agents and the procedure used in making them up will be treated as

they come up in the discussion. However, in all cases the alkalies

were standardized against potassium acid phthalate to the phenophtha-

lein end point. The potassium acid phthalate was dried for two hours

at 110° C. and stored in a desiccator until required. New standard

solutions of potassium acid phthalate (approx. 0.025 N) were prepared

from time to time to insure their accuracy. The alkalies were stan­

dardized each day they were used. The alkalies used were approximately

0.01 N.

A 10 ml. self filling semimicro burette was used throughout. It

was graduated in 0.02 ml. units but could be read fairly accurately to

0.01 ml. A 1 liter dropping funnel was used as a reservoir and was

connected permanently to the burette. All air entering this system was

passed through drying tubes containing calcium chloride and soda lime.

One drying tube was attached to the top of the burette and another to

the inlet of the separatory funnel.

Samples of the following fatty acids were obtained to use in the

work: butyric acid, caproic acid, caprylic acid, capric acid, lauric

acid, palmitic acid, oleic acid and stearic acid. The stearic acid was 59 found to be impure and was not used. The oleic acid was re-distilled and the fraction boiling at 19^° C. at 2.2 mm. was collected. This gave a refractive iidex of 1.^575 at 25° C. The literature gives a value of 1.U6 at 17.7° C. for pure oleic acid. The oleic acid had a light yellow color and was placed in sealed tubes to be removed as re­ quired. The other fatty acids were of the chemically pure grade and were not purified further.

Titration of fatty acids

One of the first problems encountered was to find the most satis­ factory procedure for titrating the fatty acids. In the first trials an aqueous solution of standardized NaOH was used. A sample of 20 mg. palmitic acid was added to 20 gm. of the solvent indicated and titrated.

The titration was carried out by adding 10 cc. of water and 5 drops of

2$ Dfeft and titrating to the cresol red end point.

Table VI

Titration of Palmitic Acid in Various Solvents

i of BuOH/CHCLo Vol. 0.01 H NaOH

0 0.22 5 0.19 10 0.18 15 0.16 50 0.17

This volume of base amounts to less than 2 of the theoretical volume

required, so the aqueous system was discarded in favor of a titration

in alcohol. 60

The next step was to try standard sodium ethylate in a non- aqueous medium. The sodium ethylate was prepared by adding freshly cut sodium to absolute alcohol and filtering to obtain a solution approximately 0.02 H. Two mg. of palmitic were added to 20gm. of 5$

BuOH/CHCl^ and titrated to the phenol red end point. The blank value on 20 gm. of solvent was 0.10 cc. of base. The results of five trials required from 103 to 110$ of the theoretical base.

Oleic acid and mixtures of butyric acid, palmitic acid and oleic acid were titrated with sodium ethylate to the phenol red end point.

Quantitative recoveries were obtained. However, when the eluate con­ taining fat was titrated a satisfactory end point could not be obtained.

The end point was not sharp and it faded very rapidly. It would seem that the sodium ethylate reacted slowly with the fat.

Accordingly, sodium hydroxide was dissolved in absolute alcohol.

When butyric acid, palmitic acid and oleic acid were titrated quantita­ tive recoveries were obtained. It was also found that phenophthalein gave a sharper end point than phenol red.

Table VII

Titration of Fatty Acids with Alcoholic NaOH Solvent Benzene Acid Mg. Acid Used Mg.acid by titration 1o Recovery Palmitic 3-75 3-87 103.2 Palmitic 3-75 3.72 99.3 Palmitic 7-50 7 -Mt- 99-3 Palmitic 7.50 7.52 100.2 Palmitic 9.00 8.93 99.3 Palmitic 15.00 1^.7^ 98.3 Butyric 1.20 1.23 102.5 Butyric 1.20 1.18 98. k Oleic k.oB it-. 02 98.5 Oleic k.oB it-.10 100.5 Preliminary trials on separation of fatty acids on silica gel

Early it appeared that some sort of chromatographic technique would best meet the requirements of this problem. The first attempt

to develop such a procedure was made with silica gel. In all the work on silica gel columns Mallinkrodt's 200 mesh silica gel which has been prepared especially for chromatographic work was used. Sufficient

silica gel was obtained at the start so that the same lot was used

throughout the work. The silica gel was not treated in any manner o prior to use except to dry it at 100 C. for at least 12 hours. On weighed samples of silica gel no changes in weight were noticed after

12 hours at this temperature.

The solvents used were various mixtures of butanol and chloroform,

and in some trials benzene. The chloroform was washed twice with dis­

tilled water and the chloroform layer passed through dry filter paper.

It was not purified further. The n-butanol used was chemically pure

grade and was not purified further. It was found necessary to wash the

benzene twice with dilute sulfuric acid and then three times with dis­

tilled water. The benzene layer was then passed through a dry filter

paper. After this treatment, 10 gm. of benzene required 0.03 cc. of

0.01 N base to become alkaline to phenol red. The solvents were mixed

and equilibrated against water before use.

For the first trials a 2" x 12" Pyrex glass tube was used. The

bottom of the tube was constricted slightly to support a Witte plate

which was covered with a moist filter paper. The bottom of the tube was

placed in a short stemmed funnel which was equipped with a stopcock. The

fractions were collected in 20- cc. Erlenmeyer flasks which were placed 62 on a triple "beam "balance. Twenty gm. fractions were collected and titrated.

In the first trials the solutions to he chromatographed were pre­ pared as follows:

7 ml. borate buffer

2 gm. double cream

15 gm. silica gel

These mixtures were used because of the nature of the work planned

later where this method would be used. It was planned to incubate buffered cream samples under various conditions and use the technique

developed here to separate and measure the liberated fatty acids.

The cream and buffer were mixed and the silica gel added. The whole was ground until a free flowing powder was obtained. This powder

was slurried with chloroform and poured into the tube. The column was

allowed to pack by the down flow of solution through it. The column was

then washed with chloroform and the fat thus removed while the fatty

acids were retained on the column with this solvent. The fatty acids

could then be eluted with 15$ butanol in chloroform.

The first trials gave variable results with recoveries of palmitic

and oleic acids varying from 65 to 200$.

Several variations were tried such as using a smaller diameter

column (38 mm.) to slow down the rate of flow through the column and

making the buffered aream acid with dilute sulfuric acid before adding

the silica gel. However, no separation of the acids was obtained.

These results are summarized in Tables 8 and 9 . 63 Table VIII

Recovery of Palmitic Acid

Run # Mg. acid used Mg. recovered jo recovery

11 U-.8 6.93 li+5 14 4.6 7.86 171 20 0.9 1.07 119

Table IX

Tecoveries of Mixtures of Palmitic, Oleic and Butyric Acids

Total Mg. Vol. base Vol. Run£. Acid acid used theor. reqd. base used Recovery

12 Palmitic 4.8 Oleic 37.7 26.17 24.33 93-1 Butyric 18.0

15 Palmitic 5.0 Oleic 17.3 16.84 16.60 98.5 Butyric 13.3

17 Palmitic 6.6 Oleic 17.4 20.54 21.90 107 Butyric 15.2

18 Palmitic 5-9 Oleic 13.4 72.82 27.90 100 Butyric 24.8

21 Palmitic 1.7 Oleic 2.6 3.86 1.12 29 Butyric 4.2

On the runs reported in Tables VIII and IX an extra quantity of silica gel was put on the column before the sample was added. Eighteen gm. of silica gel was mixed with 9 cc. of water, ground to a free flowing powder and slurried with chloroform. The slurry was poured into the column and allowed to pack. The sample was added on top of silica gel layer as described previously. 6b

It should ho pointed out that the Immobile phase of this lover part: of the column was water. The fact that the higher fatty acids are so insoluble In this solvent may account for some of the varia­ tions. Perhaps there was a mutual solubility effect here, or it may be because the pH of the column was too near neutrality. It will be shown in the discussion of the phosphate buffered columns that the

column must be acid for the higher fatty acids to be eluted.

Further runs were made by modifying the procedures used but no

Improvement was obtained In the results.

Separation of fatty acids on phosphate buffered silica gel columns

Since the procedure just described did not give the desired

results it was decided to try the technique of Moyle et al employing

buffered silica gel chromatographic columns (86).

For this work new chromatographic columns were prepared. Sigh-

teen mm. Pyrex tubing was cut into 30 cm. lengths and a stopcock

sealed at one end. Automatic siphons which delivered b cc. were also

made from Pyrex glass. In use a plug of glass wool was placed in the

bottom of the tube to support the silica gel. A reservoir for solvent

was made by sealing a stopcock on the bottom of a 123 cc. Srlenmyer

flask. The reservoir was oonnected to the chromatographic column with

a rubber stopper. It was found necessary to use air pressure to force

the solvent through the packed column at the desired rate. The top of

the reservoir was connected to the air line and a manometer was placed

in the line in order to regulate accurately the air pressure. The appa­

ratus is diagramed in Figure 1. Manometer

Figure I - Apparatus 66

Stock solutions of 2-molar monopotassium phosphate and dipotas­

sium phosphate were prepared from c.p. reagents. Since tripotassium phosphate of satisfactory purity could not he obtained this reagent was prepared according to the following reaction:

KByPO^ + 2 KOH K^POj,. + 2 HgO

1%, 10%, and 30% solutions of n-butanol in chloroform were prepared

as before.

The following buffers were prepared from these stock solutions:

Buffer 1 - 2 vol. KyHPO^ + 1 vol. KHpPO^ Buffer II - 2.5 vol. KgHPO^ + 3-5 vol. KgPO^ Buffer III - K^PO^alone Buffer IV - KH^PO^ alone

The pH of the various columns was checked by mixing 5 g®. of

silica gel, 3 cc. of the appropriate buffer, grinding in a mortar until

a well mixed, free flowing powder was obtained. This powder was then

"slurried with 10 cc. of water and the pH determined on a Beckman Model

H pH meter.

Table X

pH of buffered silica gel

______Ei______Type of buffer Immediately After 2k hours

I 6.62 6.58 II 8.01 8.40 III 8.68 8.88 IV 3-99 ^.00

It will be noticed that the pH of the type II and III columns

vary on standing due to a shift in equilibrium of the phosphate salts.

4 67

The pH of these types of buffer was followed over a period of time and

is reported in Figure 2. In order to overcome any discrepancies due to such shifts of pH, all columns were prepared, packed and allowed to

stand at least 18 hours before use.

The columns were prepared by mixing 5 gm. of silica gel and 3 cc.

of the proper buffer, mixing until a free flowing powder in a mortar,

slurrying with Vj> BuOH in CHCl^ and packing into the tube. After pack­

ing the column measured Ti- to 8-g- cm. in length. After standing, Vfo

BuOH in CHCl^ solvent was percolated down through the column and col­

lected in 5 cc. fractions. These blanks on the solvent required 0.01

cc. of 0.01 N alcoholic NaOH with phenophthalein as the indicator used.

The first runs on these columns were on standard fatty acids in

benzene solution. The column was prepared as indicated and an aliquot

of the standard acid added to the top of the column and the column de­

veloped with proper solvent.

It was pointed out in the discussion of Moyle's article (86) in

the review of literature that the fatty acids above should be eluted

with 1$ BuOH in CHCl^ in the first 5 or 6 fractions on a Type II or

Type III column. However, this was not found to be the case. When

caprylic acid (Cq) was placed on a Type II column it could not be washed

off even though three times the required volume of solvent was used. In

view of this failure to wash out the higher fatty acids on the alkaline

columns (Type II, Type III) they were not used further. 9.0 Type M

8.8

8.6 Type IT

5CL 8.4

8 2 -

8.0

78 20 24 28 Time (hours)

Figure 2 - Effect of standing on pH of phosphate buffered silica gel columns. o\ 00 The results on the Type I (pH 6.6) column are summarized in

Table XI and graphed in Figure 3. The results on the Type IV (pH 4.0) column are summarized in Table XII and graphed in Figure 4.

It will be seen from examination of Figure 3 that separation of butyric acid from the higher fatty acids would have been accomplished if the acids had come out sharply and not "tailed out". From figure 4 it can be seen that although the separation of butyric acid from its higher homologues was not achieved the "tailing" was eliminated. A rate of flow of the solvent of 1 cc./minute was adopted from here on.

It was thought that a column with properties intermediate between the Type I (pH 6.6) and Type IV (pH 4.0) column could be constructed which would give the desired separation but eliminate the tailing out.

A new buffer was made up by mixing equal volumes of 2 M K^HPO^ and

2 M KH2PO4 . When a column was prepared using this buffer a pHof 6.3 was obtained. This column was designated Type la (pH 6 .3 ) and results obtained with various fatty acids are summarized in Table XIII and graphed in Figure 5.

Good separation was obtained and the tailing of caproic acid and caprylic acid was eliminated, however, the butyric acid still tailed out. It will be shown later that the tailing of butyric acid can be eliminated by washing it from the column with 5$ BuOH in CHCl^ after the higher homologues are removed with the 1$ solvent mixture.

Attempts were made to separate caproic and caprylic acids using a Type la (pH 6.3) column and benzene as the movile solvent. However, no separation was obtained as can be seen in Table XIV. TO

Table XI

Recovery of Various Acids on Type I (pH 6.6) Column

Motile Solvent - 1$ BuOH in CHC1- o

Vol. Fractions Mg. Mg. acid (cc) containing Acid Used Acid Recovered Recovery solvent acid

Butyric 9-^ 9-02 96.0 1^0 10-30

Butyric* ^.7 5-13 109.1 100 1^-25

Caproic 5-92 5.85 98.8 60 3 t 11

Caproic 5-92 5-99 101.0 60 3-12

Palmitic* 12.82 12.52 97.6 60 3-10

*Reduced flow rate of solvent through column to 1 cc/min. / 71

Table XII

Recovery of Various Acid in Type IV (pH 4.0) Column

Mobile solvent - 1$ BuOH in CHC1 Rate of flow - 1 ec/min. ^

Fractions Mg. Mg. acid io Vol. containing Acid used acidRecovered Recovery Solvent acid

Butyric 4.70 4.12 87.7 50 4 - 7

Butyric 4.70 4.79 101.9 50 4 - 5

Caproic 5.92 5.89 99-5 60 3 - 8

Caproic 5.92 6.47 109-3 50 3 - 4

Caprylic 7.12 7.28 102.2 50 3 - 4 Volume (cc.) O.OI alcoholic NaOH per fraction 2.6 2.4 0.4 0.8 2.0 2.2 0.2 0.6 n ye (pH I Type on eoey f at ais on acids fatty of Recovery 5 l tc acid itic alm P 0 5 0 25 20 15 10 ari acid Caproic iue 3 Figure 6 6 column ) rcin Number Fraction Butyric acid

Recovery of fatty acidsfatty of Recovery n ye E p 4.0) (pH EE Type on colum n colum 1 15 10 5 K iue 4 Figure - - Butyricacid - Coprofcacid 72

73

Table XIII

Recovery of Various Fatty Acids in Type la (pH 6 .3) Column

Vol. Fractions Mg. Mg. Acid 1° (cc) containing Acid Used Acid Recovered Recovery Solvent Solvent acid

Butyric 5.05 4.54 89.9 lfoBuOH 80 9-24 11 Caproic 5-97 5.79 97-0 50 1)-- 6 Caproic 5.97 5.90 98.8 1st BuOH 60 5- 8 t! Caprylic 7.12 6.82 95-9 60 4- 7 *Caproic 5.66 5.55 93-0 CHClo 70 8-15 11*2 *Caprylic 6.85 6.68 93.8 60 6-11 * These two columns were l6 cm. long instead of the usual 8 cm. in an attempt to separate caproic acid from caprylic acid.

Table XIV

Recovery of Various Fatty Acids in Type la (pH 6 .3) Column

Vol. Fractions Mg. Mg. Acid * (cc) containing Acid Used Acid Recovered Recovery Solvent Solvent Acid

Caproic 5-97 Strung out Benzene 60 8 - ? Caprylic 6.88 6.97 101.3 60 4-11 Caproic 101.2 %f> BuOH/ 50 4-9 5.97 6.04 ( ) Caprylic 6.88 6.82 99.1 Benzene 60 4-7 Volume (cc.) O.OI N alcoholic NaOH per fraction 0 iue - Sprto o fty cd o Tp 1a (H .) column. 6.3) (pH 1(a) Type on acids fatty of Separation - 5 Figure . Solvent: 1 Length = ofcolumn BO i CHCI in BuOH % ari acid Caproic 0 5 0 25 20 15 10 \ acid \ / I Butyric — V 3 8 cm.

Solvent; •j!r% BuOH in GHCI Lengthcolumn of = 8 cm. rcin Number Fraction I acid 'I I Caprylic •Caproic acid 0 5 0 15 10 3

-- Solvent: 1 CHCI egh f oun 16cm. = column Length of 1 1 2 25 20 15 10 5 3 J *«Cpoc acid *V«-Caproic / fir 1 V . fboo . 1 V -arlc acid *-Caprylic 1 ___ 1

The results which can he achieved with this type column can he seen in figure 6 . For this run a mixture of fatty acids were made up as follows: Theor. Vol. Base Wt. Acid (0.01^68 N) Reqd. a Butyric acid 8.7 mg. 6.73 Caproic acid 5-1 mg- 2.99 Caprylic acid 5*9 mg* 2.79 Caproic acid 6.2 mg. 2.k5 Laurie acid 9*7 “g* 3-30 Palmitic acid 9-5 mg> 2.52

This mixture was placed on top of a Type la (pH 6 .3) column using the unpublished technique of Bulen. To the mixture of fatty acids was added 0.3 cc. of 0.5 N HgSO^, 0.5 gm. of silica gel and the whole ground to a free flowing powder in a mortar. The powder was slurried with a small amount (3 go k cc) of 1$ BuOH in CHCI3 and poured on top of the column. This silica gel was allowed to pack and then 50 cc. of 1$

BuOH was added to develop the column. After 10 fractions were collected, k0 cc. of 5/o BuOH in CHCl^ were added to elude the butyric acid.

Caprylic and the higher acids were taken off in fractions 3 through 8 with a 98.5$ recovery. Butyric acid; was obtained in fractions 9 through

16 with a recovery of 96.656.

It is to be noted in Figure 6 that sharp separation was obtained with no tailing of the acids. The rounded hump which appears in the butyric acid curve arises from the fact that the shift from 1$ BuOH in

CHCI2 to 5/o BuOH in CHCl^ was not made soon enough. This was eliminated in following runs by changing solvents sooner. * Private communication. 76

The recoveries were calculated in the following manner:

Higher Fatty Acids

13.90 cc Vol. vase used to titrate fractions 3 through 8 -0.06 cc Vol. "base equivalent to solvent (Blank)

13.8k cc Vol. "base required for fatty acids

15.05 cc Theor. vol. of "base required.

x 100 - 98.5$

Butyric Acid

6.59 cc Vol. hase to titrate fractions 9 through l6 -0.09 cc Vol. base equivalent to solvent (Blank)

6.50 cc Vol. base required for fatty acids

6.73 Theor. vol. of base required

| ‘5° x 100 - 96.6$ 6.73

Separation of Fatty Acids on Elastomers

The method of Boldingh employing elastomers (6 ) offers a scheme to separate the fatty acids from Cg through C^q. This coupled with the separations already obtained on silica gel would give the desired re­ sults for the purpose of the study of fatty acids liberated by lipolysis.

The first sample of rubber used was a butadiene-styrene copolymer prepared by low temperature polymerization. The polymer had a molecular weight of about 8000 so in this respect should have been quite similar to the Hevea rubber used by Boldingh. The polymer had been milled and some placticizer had been added. The polymer was in soft shreds when ihr ooous Tp l (H clm. Solvent: column. ) 3 6 (pH la Type homologues. higher Figure uao i chloroform. in butanol

6 Volume (cc.) O.OI N NaOH per fraction eaain f uyi ai fo a itr of mixture a from acid butyric of Separation - 7.0 5 6 5.5 5 7 4.0 4.5 6.0 3.5 2.5 3.0 5.0 2.0 0.5 Fraction Number itr o higher of Mixture homologues 5% uyi acid Butyric

78 obtained and handled very nicely. A sample of unmilled natural rubber was also obtained but it could not be worked up.

The GRS copolymer shreds were washed twice with acetone and most of the placticizer removed. It was allowed to stand overnight in benzene and next morning the benzene was drained off. The rubber was pressed dry with paper towels and ground in a Wiley mill to pass a 20 mesh screen while in the swollen state. The ground rubber was washed with methanol and then with acetone to remove the benzene. It was extracted with acetone for 2h hours.

The rubber was then washed with dilute E^SO^ at pH 2.0 by agitating violently for 30 minutes to remove any carbonates which might have been used in the milling. It was next washed repeatedly with distilled water until the wash water was neutral to litmus. It was drained dry, then washed twice with methanol and finally extracted continuously for 9 hours with methanol. The treated sample was stored under methanol until used.

In the runs using elastomers a 12 mm. column was used. This tube was surrounded by a water jacket which was equipped so that the tempera­

ture of the col wan could be maintained at 20 to 22° C. Otherwise the

setup of apparatus was the same as illustrated in Figure 1.

A mixture of 3 parts methanol to 1 part acetone (v/v) was prepared

and designated as "M" solution. The "M" solution was then mixed (v/v)

with water in varying proportions as follows:

Solution Parts M Parts Water M h-0 kO 60 M 60 60 kO M 65 65 35 M 70 70 30 M 7^ 75 26 79

The methanol and acetone were redistilled using a 2k" frac­ tionating column packed with glass helices. The distillation was carried out in all glass apparatus with the receiver protected from the air by a drying tube containing CaClg and soda lime.

Baker's c.p. methanol was used and the fraction boiling at 6k. 5° C. collected. Baker’s c.p. acetone was distilled and the fraction boiling at 56° C. was collected.

Two and one-half gm. of the prepared rubber was washed with

50 ml. of M lj-0 and the solvent was drained off. The rubber was suspended in 750 cc. of M 40 which had been saturated with benzene and to which an additional 6 cc. of benzene was added. This was stirred violently for 5 minutes after which the solvent was poured off and the column packed. This gave a packed column of 20 cm. and was compressed to 18 cm.

Several runs were made using caproic acid and caprylic acid.

However, in all cases the recovery was exactly zero. None of the fatty acids could be washed off the column. The reason for this is not immediately apparent. It is possible that the polarity of the rubber is such that the fatty acids are adsorbed too strongly to be eluted. All the elastomers used by Boldingh were polymers of ali­ phatic compounds and it is possible that the styrene in the polymer used made the rubber more polar than the samples used by Boldingh.

It would be well if this point could be investigated further but it was beyond the scope of the problem at hand, so other types of elasto mers were obtained for trials. A sample of Geon (polyvinylchloride) of low molecular weight

(estimated 8,000 to 10,000) was obtained and was washed three times with methanol before use. It was not further purified at this time.

A column was prepared and packed as indicated in the discussion of the GRS copolymer. However, 2.5 gm. of Geon gave a column only

8 cm. long as compared to a column length of 18 cm. reported by the above authors. This first trial on this column with caproic acid gave a recovery of 95«7When a mixture of caproic acid and caprylic acid was placed on an 8 cm. column 98.0$ recovery was obtained, however, no separation was effected, probably because the column was too short.

After several trials, it was found that 6.0 gm. of Geon and 15 cc. of benzene when stirred with 750 cc. of M 50 solvent saturated with benzene gave a column of about 20 cm. when packed. These quantities were used in all the trials which follow. The 750 cc. of benzene- saturated M 40 solvent were placed in a 1 liter beaker and stirred violently by means of a magnetic stirrer. The Geon was poured in and the 15 cc. of benzene added slowly from a dropping funnel while the stirring was continued. The mixture was agitated for a total of 45 minutes. Most of the liquid was poured off and the remainder used to aid in packing the column. Care must be taken when packing the Geon

column to keep air bubbles out. To do this, the tube was removed

from the water jacket for packing. The tube was rotated constantly

and tapped sharply with a heavy rubber policeman while the column was being packed. 81

For the first trial on the long Geon column the following mixture was weighed out:

Caproic acid 18.0 mg. Caprylic acid 20.2 " Capric acid 6.1 " Laurie acid 13-3 " Palmitic acid 3.3 "

Total 60.9 mg-

The results of this trial are shown in Figure 7 .

Consideration of the results above showed that several errors were made in this trial. The total quantity of acids was too great.

It would seem that the total quantity of acids should not exceed

^0 mg. as suggested by Boldingh. The amounts of caproic and caprylic acids were much too large and these two acids did not separate coim-; pletely. Further the amount of caprylic acid used was so great in comparison with the weight of capric acid that the capric acid was almost totally obscured;, by the caprylic acid. Because of the poor separations no recoveries were calculated in this rim.

For the second trial, on a freshly prepared column, the following fatty acids were weighed out and gave more satisfactory results. The weights of acids used were as follows:

Caproic acid 10.0 mg. Caprylic acid 6.2 mg. Capric acid 6.9 " Laurie acid 8.6 " Palmitic acid 7.9 "

Total 39.6 mg.

The results of this trial are shown in Figure 8 .

The separation obtained here was much better than that obtained

in the previous run. By extrapolation (dotted lines) the actual titra Volume (cc) OOI N NaOH per fraction - 5 . 2 20 3.0 0 4 0.5 3.5 0 iue - eaain f at ais n Go column. Geon on a acids fatty of Separation - 7 Figure 1 1 2 2 3 3 4 4 5 5 60 55 50 45 40 35 30 25 20 15 10 5 M40 ari acid Caproic arlc acid Caprylic rcin Number Fraction . acid . * c c 4 fractions Capric 0 6 M

fractions ^ftacid /-Laurie 8 cc Palmitic acid Volume O.OI NoOH per fraction 2.0 2.5 0.5 0 Figure 1 1 2 2 3 3 4 45 0 5 0 6 55 50 4 5 40 35 30 25 20 15 10 5 Caproic 8 acid M40 Sprto o fty cd n Go column. Geon aon acids fatty of Separation - Caprylic acid rcin Number Fraction Kacid ► — 0 6 M Capric M65 Laurie acid ►-.acid Palmitic 84 tion values may be determined and the recoveries of the various acids calculated. The percentage recoveries were as follows:

Caproic acid 95*6 Caprylic acid 93-4 Capric acid 9^-2 Lauiic acid 87.5 Palmitic acid 6l.O

Good recoveries were obtained for all but palmitic acid. It may be that the palmitic acid used in these experiments contains an impurity. Ramsey and Patterson (98) found that the sample of palmitic acid used in their work contained 0.5$ stearic acid.

The sample of palmitic acid used here had a melting point of

61.5 to 62.5° C. compared to a value of 64° C. given in the litera­ ture. A sample of palmitic acid was chromatographed and the results are shown in Figure 9* A sample of oleic acid was then chromatographed on the same column and is also shown in Figure 9*

Both palmitic and oleic acid come out in the same fractions

(2 through 14) and in both cases the recovery obtained was 80$.

It would thus seem that the sample of palmitic acid used here con­ tained some stearic acid.

Removal and separation of fatty acids from milk samples

From the preceding discussion it is noted that butyric acid can be separated from the higher fatty acids on buffered silica gel

columns, and the acids from caproic through caprylic can be separated

on Geon columns. It was also shown earlier that mixtures of fatty

acids could be recovered quantitatively from buffered cream samples

employing silica gel. 2.5

c 2.0 o o o Palmitic acid

0) CL X o o z o o a> E 3 | 0.5 Stearic acid ? Oleic acid

0 5 10 15 20 25 0 5 10 15 20 25 Fraction Number

Figure 9 - Chromatographs of palmitic acid and oleic acid on a Geon column. 86 The three techniques were combined to separate and recover mixtures of fatty acids from buffered cream samples. The removal of the fatty acids from the buffered cream and the separation of butyric acid from its higher homologues was achieved on silica gel columns. For this phase of the separation a 38 ram* tube was employed. The silica gel column was packed in two parts. The lower layer was made up of 10 gm. dried silica gel and 6 cc. of phosphate buffer la (pH 6 .3). The top part of the column contained the sample and was mixed as follows:

5 cc. borate buffer (pH 8 .5) 2.5 gm. bQPp cream 15 gm. silica gel 4 to 10 drops 4N H^O^ Mixtures of fatty acids

Sufficient ^ S O ^ was added hereto give a pH of about 2 as indicated by "Hydrion" paper.

The mixture for the lower layer was ground well, slurried with kO cc. of chloroform and poured into the tube. After this portion of the tube had been packed the top layer was slurried with chloro­ form and poured into the tube. It should be noted that the level of the chloroform should not be allowed to go below the top level of the

silica gel or channeling of the silica gel will result. The collection of samples was started as soon as the top layer was poured into the tube. After the top layer had packed additional solvent was added to

complete development of the chromatograph.

A preliminary trial was made in which 4.72 mg. of butyric acid and 5*06 mg. of caproic acid were added to the buffered cream. A tdal Volume O.OI N NaOH per fraction 2.0 0.8 0.2 Q4 Q6 iue 0 Sprto o btrc cd from acid butyric of Separation 10- Figure ari ai i a ra sample. cream a in acid caproic 0 CHCl 1 1 2 2 3 3 40 35 30 25 20 15 10 5 Caproic acid rcin Number Fraction uH n CHCI in BuOH % 5 Butyric acid 3

87 88 of 100 cc. of chloroform and 150 cc. of 5$ BuOH in CHCl^ was used to develop the chromatogram. The caproic acid was recovered 100$ and the "butyric acid was recovered 99*5$. The results are summarized in Figure 10. The first hump in the caproic acid part of the curve is due to the fact that the switch from chloroform to the more polar 5$

BuOH in CHCI3 was not made soon enough. This was eliminated in sub­ sequent experiments by switching solvents sooner.

For the next run a mixture of six fatty acids was prepared as follows:

Butyric acid Ik 72 mg. Caproic acid 5.07 mg. Caprylic acid 6.k 1mg. Capric acid U.83 mg. Laurie acid 3.03 mg. Palmitic acid 3-kk mg.

To this mixture of fatty acids was added 5 cc. of borate buffer

(pH 8 .5), 2.5 gm. of 1+0$ cream, 7 drops 4N H2S02). and 15 gm. silica gel. The lower part of the column was prepared as above using 10 gm. of silica gel and 6 cc. of phosphate buffer I (a) (pH 6 .3). The

column was packed and developed using a total of 50 cc. of chloroform

and lJ+0 cc. of 5$ BuOH in chloroform. The first four samples were blank while the higher fatty acids were contained in fractions 5

through 2k and the butyric acid in fractions 25 through 32. The

higher fatty acids were recovered to the extent of 102.2$ and the buty

ric acid: recovered to the extenbof 102$. This phase of the separation

is shown in Figure 11.

After titration fractions 5 through 2k were pooled and the flasks

rinsed with 2 to 3 cc. distilled water, this mixture of the salts of

the fatty acids in the butanol-chloroform were extracted with Volume (cc) O.OI N NaOH per fraction 2.2 2.0 0.8 0.2 0.6 0.4 iue I eaain f uyi ai from acid butyric of Separation - II Figure ihr at ais n cem sample. cream a in acids fatty higher rcin Number Fraction ihr acids Higher %BO i CHCI in BuOH 5% uyi acid Butyric

89 90 distilled water. The solvent was extracted three times and a total of 250 cc. of water was used. The water extract was evaporated to dryness on a water "bath. Sufficient 0.5N HgSOj,. (0.55 cc.) was added to neutralize the base used to titrate these fractions. Care must be taken not to add excess mineral acid because it will pass through the Geon column with the caproic acid. However, sufficient mineral acid must be added to assure that all the salts are converted back to the free acids.

Five cc. of the M-ltO solvent were used to wash this fatty acid mixture onto a freshly prepared Geon column. This chromatogram was

then developed. Caproic acid was eluted in fractions h through 15 using a total of 60 cc. of the VL-bQ solvent. A total of 150 cc. of

M-60 solvent was used to elute caprylic acid and capric acid.

Caprylic acid came off in fractions 15 through 26 and capric acid in fractions 25 through 36 . Separation was not complete, but the acids

could be resolved graphically. laurlc acid was eluted with 75 cc.

of the M-65 solvent in fractions 39 through 50 and palmitic acid was

eluted with 90 cc. of the M-70 solvent in fractions 55 through 66.

This phase of the separation is shown in Figure 12 and Table XV.

Good separation was obtained and good recoveries were achieved

for all but the palmitic acid. The poor recovery of the palmitic

acid was first thought to be due to an impurity in the palmitic acid

used which caused it to tail out. From later observations it was

concluded that It was more probably due to incomplete elution under

the conditions used. Volume (cc) O.OI N NaOH per fraction 0.4 0.8 Q2 0 1.0 . 6 iue1-Sprto o fty cd o a en column. Geon a on acids fatty of Separation 12- Figure - C 0 -6 C -8 M60M40 rcin Number Fraction C-IO C-12 M65 C-16 70 M Table XV

Separation of Fatty Acids on a Geon Column

Length of column - 22 cm.

Temperature - 19° C.

Fraction Acid Solvent containing acid Recovery

Caproic M-ltO i^ -13 102.7

Caprylic m-6o 15-26 99.2

Capric M-60 25-36 97-0

Laurie M-65 39*50 92.1

Palmitic M-70 55-66 72.6 95

Runs were made on fresh pasteurized cream samples to which no fatty acids had been added. Upon development it was found that the cream contained higher fatty acids (Cg and above) equivalent to 0.2 to 0.3 cc. of 0.01 N base and no measurable butyric acid.

It was also determined that lactic acid is not eluted from silica gel with % BuOH in chloroform but can be eluted with 1 %

BuOH in chloroform. This may offer a new method for the determina­ tion of lactic acid in milk and might be investigated further.

Conclusions

As a result of the investigation reported in the preceding

section the following procedure was evolved for the quantitative

separation of mixtures of fatty acids which might be expected to

result from lipolytic changes in natural fats.

1. Preparation of sample

The sample should be of such a size so as to contain a total

of 30 to 35 mg. of fatty acids. On unknown samples a preliminary

trial may be necessary to determine the proper size of the sample.

On fluid milk samples of average fat content an 8 cc. sample would be

about right. Add kN ^SOi,. dropwise until the sample has a pH of 2.

Add 15 gm. of dried silica gel and grind to a free flowing powder in

a mortar. The ratio of silica gel to liquid should be kept in the

order of 10: k in order to give the proper rates of flow of solvent

through the column. If it is found that it is necessary to change the sample size the amount of silica gel used must be changed accordingly.

2. Preparation of buffered silica gel column

A phosphate buffer is prepared by mixing equal parts of 2 M

KgHPO^ and 2 M KHgPO^. The column is prepared by grinding 10 gm.

of dried silica gel and 6 cc. of the above buffer to a dry free-

flowing powder in a mortar. This powder is slurried with kO cc.

of washed chloroform, which has been equilibrated against the

buffer, and poured into a 38 mm. tube. An additional 10 cc. of

chloroform is used to rinse the beaker in which the slurry was made

and this rinse is also poured into the tube. The column is packed

by the solvent flowing through. It may be necessary to rap the tube

sharply with a heavy policeman to aid in removing air bubbles. The

columfl should be evenly packed and free of air bubbles and channels.

3. Elution of fatty acids

The sample which has been prepared in step 1 is slurried with

kO cc. of chloroform and poured on top of the column prepared in

the preceding step. An additional 10 cc. of chloroform is used to

rinse the beaker in which the slurry is prepared and this is also

poured into the column. The collection of fractions is started

when the sample is added to the column. Each 5 cc. fraction is ti­

trated immediately after it is collected with 0.01N alcoholic NaOH

to the phenophthalein end point. When the level of the chloroform

reaches the top of the silica gel, lkO cc. of 5$ butanol in chloro­ 95 form is added. The first 4 fractions should not contain any fatty acids. The higher fatty acids (caproic and higher) will come out in fractions 5 through about 2k depending on the amount present. The butyric acid will come off in fractions 25 through

32 again depending on the amount present. If the titration values are plotted against the fraction numbers as the titrations are made, the separation can be followed very closely.

4. Collection of samples containing the higher fatty acids

After the titration has been completed the fractions contain­ ing the higher fatty acids are pooled and the flasks which contained them are rinsed three times with small portions of distilled water and the rinse water added to the pooled solvent. Enough water is added to make a total of about 150 cc. of water. The water layer is removed and saved and the solvent layer is extracted twice more using

50 cc. of water each time. The aqueous layers are collected together and evaporated to dryness on a water bath. A gentle stream of air may be used to aid in the evaporation. The temperature of the water solution of the salts should be kept below 6o°C.

5. Preparation of Geon column

Seven hundred and fifty cc. of benzene saturated M-kO solvent are stirred in a 1 liter beaker by means of a magnetic stirrer, (p. 78).

Six gm. of Geon are added slowly,~and an additional 15 cc. of ben­

zene is added dropwise with continuous stirring. The stirring should be continued for k5 minutes. After this time about 600 cc. of the 96 solvent is poured off and the remainder used to pack the column. A tube 12 mm. in diameter is used here. The swollen Geon is added at the top and the column packed by allowing the solvent to flow down through it. It is necessary to rotate the tube continuously and rap

it sharply with the policeman to keep out air bubbles. When the column is packed it should be about 22 cm. long. A small Witte plate

is placed on top of the column and it is compressed to 20 cm. with a

glass rod. After the column is completed it is placed in a water

jacket and brought to 20 to 22° C. This temperature must be maintained

throughout the run.

6 . Separation of higher fatty acids.

To the dried residue prepared in step k is added a quantity of

0.5 N HgSO^ which is equivalent to the base used to titrate these

fractions. About 0.5 cc. will be required and an excess of mineral

acid is to be avoided. The salts are dissolved in the acid by stir­

ring and washed onto the freshly prepared Geon column with 2 to 3 cc.

of M-^0 solvent which has been saturated with benzene. The dish is

rinsed twice with 1 cc. of M-40 and these rinses are poured into the

tube. The solvent used to pack the column should be drained to the

level of the Geon before the sample is added. The 5 cc. of M-kO con­

taining the sample is then allowed to flow into the bed and 25 cc.

of M-kO added above the column. Five-tenths cc. of benzene is added

on top of this solvent to be sure that the solvent is kept saturated

with benzene. A total of about 60 cc. of M-40 will be required to 97 elute caproic acid and the remaining 35 cc. is poured into the sol­ vent reservoir. The "bottom stopcock is opened all the way and a rate of flow of 1 cc. per minute maintained by adjusting the top stopcock.

The first 3 fractions will be blank and caproic acid will come off in fractions ^ through 13. One hundred, thirty cc. of benzene saturated M-6o are then added to elute caprylic acid and capric acid. One hudrired, fifty cc. of benzene saturated M-65 are then added to elute lauric and myristic acid. Ninety cc. of M-70 will elute palmitic acid and the unsaturated C-^0 acids as a single group.

Finally 90 cc. of M-7^ will elute stearic acid.

The progress of the separation should be followed by plotting the titration values against the fraction number. By so doing the proper place to change solvents becomes readily apparent. This is especially true if one of the acids would not be present. The quan­ tities of solvent given above are approximately correct when 0.1 millimole of each acid is present.

It was found best to use a silica gel column once and discard it after the run was completed. The Geon columns should only be used for one run also but the Geon can be reclaimed by extracting it continuously with ether for at least four hours. The reclaimed Geon can then be used to prepare a fresh column. Experience with this method seems to indicate, however, that better results would be obtained if fresh Geon was used for each run. 98 Application of Proposed Method of Pried Milk Preparations

Tiro samples of dry whole mills: were examined following the procedure outlined above. Both samples were rancid powders which had been prepared especially for use in chocolate candy manufac­ ture.

One sample was called Teknican and was produced by the Golden

State Creameries of California. In this product raw milk is allowed to become rancid by natural lypolysis, it is then pasteurized and spray dried. The final product contains 28.5$ fat. The sample used had an oxidized odor with only a faint trace of butyric acid odor. According to the usual methods of analysis, this product has an acid degree of 17*

The other sample examined was Mil-La it, produced by the Dairy land

Food Labs. This powder is prepared by adding a lipolytic enzyme preparation obtained according to the patent No. 2,531,329 of

M. G. Farnham. This extract is added at thejrate of 4 gms. per 1000 pounds of milk and lipolysis allowed to proceed. The milk is then pasteurized and spray dried. The final product contains 28.5$ fat and according to the usual methods of analysis has an acid degree of

33. This powder has a very strong butyric acid odor.

Preliminary trials showed that 1 gm. sample of Mil-Lait and a

2 gm. sample of Teknican would give satisfactory results. The separa­

tion on silica gel showed that the Teknican contained only small

amounts of the volatile fatty acids in comparison to the amounts of 99 non-valatlle adds. Mil-Iait, on the other hand, contained large quantities of the l w molecular weight, volatile fatty acids and relatively small quantities of the high molecular weight, non­ volatile fatty acids.

The data were also used to calculate the acid degree of each sample. The results of the analysis are shown in Table 171.

Table XVI

Fatty Acid Content of Dried Milk Powders Expressed as mg. acid/gm. powder

On Silica Gel

Acid _____Teknican Mil-Ialt mg. acid Fraction mg. acid fraction Caproic and higher 11.26* 6-20 11.24* 6 - 21

Butyric 0.28 21 - 36 2.07 22 - 29

On Geon

Acid Teknican Mil-Iait mg. acid fraction mg. acid fraction

Caproic 0.27 8-10 2.84 6-11

Caprylic 0.18 15 - 18 3.58 13 - 19

Capric 0.27 18 - 21 3.25 20-26 lauric 0.29 28-29 O.71 31 - 3 6

**Eigh®r fatty acids (3.58) high (0.97) low

Acid degree 17.3 40.2

♦Calculated as caproic acid. ** Myrietic acid and higher.

More exact calculations can not be made for these acids because they were not eluted quantitatively from the Geon column. It would appear that these acids are adsorbed directly on the Geon and could not be washed off quantitatively.

The data from Table XVI has been used to calculate the ratio

of each acid liberated as compared to the amount present In milk.

For these calculations the fatty acid composition of a sample milk

fat as given by Eogers (106) was used. The calculations are only

as accurate as the values assumed for the fatty acid composition of

the fat, however, they should show any considerable differences in

the relative amounts of each acid liberated. The results of these

calculations are shown in Table XVII.

It is noted first that the glandular enzyme preparation is much

more active than natural milk lipase. The glandular enzyme prepara­

tion liberates a mufih higher ratio of the low molecular-weight,

volatile fatty acids than high molecular-weight, non-volatile fatty

acids. The activity appears to follow a definite pattern, maximum

activity occurring with caprylic acid. This may indicate a certain

degree of specificity, or if the enzyme preparation is a mixture of

different specific lipases, that the lipase which cleaves caprylic

acid occurs in the greatest amount.

On the other hand, natural milk lipase shows no definite trend,

but it is about ^.5 times as reactive toward the high molecular-

weight, non-volatile acids than toward the low-molecular weight,

volatile acids.

It must be again noted, however, that the fat used in comparison

is not the same as the fat present in the sample analyzed, so too much Table XVII

Acid $ in milk Millimoles present Millimoles liberated .Mole, jo Liberated fat (106) Teknican Mil-lait Teknican Mil-lait______Teknican Mil-lait

Butyric 3.^ 0.220 0.110 0.0032 0.023 l.k-5 20.9

Caproic 3-3 0.168 0.081 0.0023 0 . 02k 1.37 29.6

Caprylic 1.9 0.075 O.O38 0.0012 0.025 1.60 65.8

Capric 3-0 0.099 0.050 0.0016 0.019 1.62 38.8

Laurie 3-7 0.105 0.053 0.001k 0.0035 1.33 6.6

*Higker acids 20.8 0 .k63 O .231 0.032 0.0086 6.91 3 . 7

Calculated as palmitic acid.

H o 101 weight cannot be given to such conclusions in this case. The data merely serve to indicate the possible manner in which application of this method of fatty acid analysis may be useful in future studies of lipase action.

The Geon used here was reclaimed several times and it may be

that the reclaimed material had different properties than fresh

Geon. There was insufficient Geon available to have a fresh sample

for each run. Although Boldingh (6) reported separation and quanti­

tative recovery of palmitic acid on natural rubber this could not

be done under the present experimental conditions. The type of

elastomer used, particle size, concentration of solvent, temperature,

rate of flow and number of times the elastomer has been used are some

of the factors which may be concerned. More work must be done in order

to realize the quantitative recovery of and higher acids.

It should be pointed out that the method described offers a

better means for determining acid degree than the usual extraction

procedure. From the results obtained on the Mil-lait powder it is

noted that the acid degree determined by the usual method is low.

This is due to the large amount of water soluble butyric acid in this

powder which is not extracted with ether to any great extent. If the

acid degree is calculated only on the basis of the higher fatty acids

(Cg and above), which were separated out on the silica gel, it agrees

with that determined by the usual method. The acid degree of the

Teknican powder, which is low in water soluble acids, determined by 102 the usual method, agrees with the acid degree calculated from the present data. Thus, it is shown that the usual method for deter­ mining acid degree is not reliable, especially when high concentra­

tions of low-molecular weight, water soluble acids are present.

The silica gel separation also may be used to estimate the

relative amounts of butyric acid as compared to the high-molecular

weight acids (caproic and above). The silica gel separation offers

another advantage in that it is less time consuming than the usual

ether extraction procedure.

By combining the silica gel technique with the elastomer

technique further quantitative separation of the acids from caproic

through laurlc can be achieved. This should be of considerable assls

tance in future studies related to the distribution of fatty acids

released by lipase activity under varying conditions, or by lipases

of different possible types.

I 103 DISCUSSION

The method described has been shown to be accurate, however, some precautions must be taken. The quantity of each fatty acid used was of the order of 0.1 millimole. In certain cases where one fatty acid occurs in a higher ratio than others,separation may not be achieved. Attention is called to Figure 7 where a small quantity of capric acid was almost totally obscured by a relatively large quantity of caprylic acid.

Mention should also be made of the rate of flow of the solvent

through the column. In the 38 nnn. tubes a rate of flow of 2 cc. per minute was found to be satisfactory while in the 12 mm. and

18 mm. tubes a rate of 1 cc. per minute was found best. The solvent

must percolate slow enough to allow equilibrium to be reached but

nothing is to be gained by running the solvent too slowly. With the

Geon columns any delay causes the benzene to be washed off the

column. The Geon column should be developed as rapidly as possible.

The control of temperature with the Geon column must be

stressed. It must be held at 20 to 22° C. in order to obtain

reproducible results. The solubility of the fatty acids in benzene

increases rapidly with increases in temperature. The partition co­

efficient of the fatty acid between benzene and the aqueous solvent

would thus be changed.

If It becomes necessary to change batches of Geon, samples of

known fatty acids should be run on the new Geon before any unknowns

are run because each batch will have slightly different properties

which might affect the results. 10^ SUMMARY

1. A technique for the titration of fatty acids in non-

aqueous solvents has been presented. Alcoholic sodium hydroxide

■was used to titrate fatty acids dissolved in benzene, chloroform,

and butanol-chloroform mixtures. Phenophthalein was found to be the

most satisfactory indicator for these titrations.

2. A method has been presented for the removal and separation

of fatty acids from milk which has undergone lipolysis. The proce­

dure is based on chromatographic techniques and is carried out in

two stages:

A. Separation of fatty acids from milk and separation of

butyric acid from higher fatty acids has been achieved on a

two phase silica gel column. The top of the column contains

the milk sample to be tested and under the conditions used,

all the fatty acids were eluted and the other components of

milk remain adsorbed to the silica gel. The lower part of the

column is buffered at pH 6.3 and serves to separate butyric

acid from the higher fatty acids.

B. Separation of the higher fatty acids (Cg to C^g) on a

benzene swollen Geon chromatographic column. These higher

fatty acids were eluted as a group in the first step and were

quantitatively separated on a Geon (polyvinyl chloride) which

had been swollen with benzene. Acids of Cih and higher were

not quantitatively recoverable by this process under the condi­

tions used here. 105

C. Based on the two phase silica gel separation procedure a method has heen presented for the determina­ tion of acid degree which is more accurate and less time consuming than the methods which are in use at present.

X>. By a combination of the procedures using the silica gel and the rubber chromatographic processes the separate fatty acids in a mixture may be quantitatively determined up to laurlc acid, thus making available a method of comparing the distribution of these more highly flavored volatile acids resulting from lipolysis of milk fat. 10 6 BIBLIOGRAPHY

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AUTOBIOGRAPHY

I, Theodore Ferer Irmiter, was born in Meadville,

Pennsylvania, July 9, 1922. I received my secondary school education in the public schools of the City of Kent, Ohio.

My undergraduate training was obtained at Kent State University, from which I received the degree Bachelor of Science in 1943.

From 19^3 to 19^6, I served in the Army of the United States.

After working as a research chemist at the B.F. Goodrich Co.,

Akron, Ohio, for one year, I entered the Graduate School at

The Ohio State University in 19^7. I held an appointment as a

Graduate Assistant in the Department of Agricultural Biochemistry for three years and in 19^9 received the degree Master of Science in Dairy Chemistry. From October 1950 to December 1951, I held a research fellowship in die Department of Dairy Technology, while completing the requirements for the degree Doctor of Philosophy.