ENEYMATTC PROTEOLYSIS 0F SELECTED MILK f
PROTEINS AS AFFECTED BY HEAT
TREATMENT AND OTHER AGENTS

Thesis for the Degree of Ph. D.
MICHIGAN STATE UNIVERSITY
GEORGE A. PURVIS .,
1969

LIBRARY

Michigan State
University

 

This is to certify that the

thesis entitled

Enzymatic Proteolysis of Selected Milk
Proteins as Affected by Heat Treatment
and Other Agents.

presented by
George A. Purvis

has been accepted towards fulfillment
of the requirements for

Ph.D. degree in Food Science

  

Major professor

   

Date January 23. 1969

 

0-169

 

 

ABSTRACT

ENZYMATIC PROTEOLYSIS OF SELECTED MILK
PROTEINS AS AFFECTED BY HEAT
TREATMENT AND OTHER AGENTS

By

George A. Purvis

Milk proteins were studied during enzymatic proteoly-
sis as model protein systems with and without heat treat—
ment. The effect of selected components on enzymatic
proteolysis of model systems was evaluated. Heat-
induced protein interactions were considered as they
influence enzymatic proteolysis.

This study was divided into three parts: pepsin-
pancreatin hydrolysis of model proteins and assessment of
amino acids and peptides liberated; evaluation of model
proteins enzymatically hydrolyzed during gel filtration;
and rate of proteolytic proton release monitored with a pH-
stat.

Milk protein treatments included heat treatment (121
C for 30 min), sugars, chemical denaturing agents and addi-
tion of calcium and sodium salts.

An essential amino acid index was applied for inter-
pretation of enzyme released amino acids and correlated

with published biological data. Casein enzyme-released

George A. Purvis

amino acids were lowered 13% by heat treatment and 36% by
heat treatment with added lactose. Enzyme liberated amino
acids from aS—casein were lowered 16% by heat treatment and
12% by heat with added lactose. k-Casein enzyme-freed
amino acids were reduced 23% by heat treatment with added
lactose. Amino acids freed in enzymatic digests of B-
lactoglobulin were not changed by heat treatment.

Enzymatic hydrolysis of k-casein and B-lactoglobulin
during gel filtration was 2U% more extensive after heat
treatment than before. The proteins were assessed for
contents of e-amino lysine before and after gel filtra-
tion proteolysis, and the data were ineffective indicators
of susceptibility to proteolysis, possibly due to inter-
ference by terminal amino groups.

Rates of enzymatic proteolysis were estimated by
pH—stat monitored proton release for proteins treated with
chemical denaturing agents. Urea treatment of casein and
B-lactoglobulin resulted in increased proteolysis rates;
however heat treatment reduced proteolysis rates. Protein
treatment with mercaptoethanol resulted in slightly
reduced proteolysis rates for all proteins studied except
as-casein. Performic acid oxidation of milk proteins
increased rates of proteolysis, and rates were not sig-
nificantly influenced by heat treatment.

Heat treatment at pH 5 reduced digestion rates of

aS-casein to immeasurable levels but increased k-casein

George A. Purvis

(26%) and B-lactoglobulin (20%). Heat treatment at pH 9
did not alter the rates of proteolysis for k-casein and
B-lactoglobulin but reduced the rate for ds-casein to 28%
of the original level.

The model protein systems were used to provide an
indication of enzymatic proteolysis of food proteins as
affected by heat treatment and chemical denaturing agents.
The observations derived can be used to describe similar
treatment to other food protein systems. The amino acid
release rate upon enzymatic proteolysis provides the basis
for an index of protein digestibility. Measurement of
rate of enzymatic proteolysis provides a potential method

for rapid estimation of protein digestibility.

ENZYMATIC PROTEOLYSIS OF SELECTED MILK
PROTEINS AS AFFECTED BY HEAT

TREATMENT AND OTHER AGENTS

By

George A. Purvis

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Department of Food Science

1969

ACKNOWLEDGMENTS

The author expresses his sincere appreciation to
Dr. J. R. Brunner, Professor of Food Science, for his
inspiration, counsel and patience during this study and
for his assistance in the preparation of this manuscript.

Grateful acknowledgment is due Dr. B. S. Schweigert,
Chairman of the Department of Food Science, for making
arrangements so that this study could be made and for
making the research facilities available. Acknowledgment
is also due Miss Ursula Koch for analyses on the amino
acid analyzer.

The author thanks Dr. R. A. Stewart, Director of
Research, Gerber Products Company, for his encouragement.
Grateful acknowledgment is due Gerber Products Company for
financial support during this study.

The writer is grateful to his wife, Norma, for
typing the drafts and for her understanding encouragement.
The author also thanks his children: Amy, Beth, Tom and

Meg for waiting.

11

TABLE OF CONTENTS

Page
ACKNOWLEDGMENTS o o o o o o ' o a o o o 0 11

LIST OF TABLES . . . . . . . . . . . . . vi
LIST OF FIGURES . . . . . . . . . . . . . viii
INTRODUCTION . . . . . . . . . . . . . 1
LITERATURE REVIEW . . . . . . . . . . . . 3
Commentary and Objectives . . . . . . . . 17
EXPERIMENTAL . . . . . . . . . . . . . 19
Apparatus and Equipment . . . . . . . . . 19
Chemicals and Materials . . . . . . . . . 2O
Preparative Procedures for Milk Protein Fractions . 20
Preparation of Whole Casein . . . . . . . 21
Preparation of as-Casein . . . . . . . . 21
Preparation of k- Casein . . . . . . . . 22
Preparation of B- Lactoglobulin. . . . . . . 22
Acrylamide Gel Electrophoresis . . . . 23
Preparative Procedures for Treated Milk Protein
Fractions . . . . . . . 2A
Heat Treatment of Milk Proteins . . . . . . 25
Heat Treatment of Milk Proteins with Added
Lactose . . . . . . . . . . . . . 25
Pepsin Treatment . . . . . . . . . 26
Treatment with Isotonic Saline . . . . . 27
Treatment with Calcium Ions . . . . . . 27
pH Adjustment . . . . . . . . . . . . 27
Equal Cleavage Sites . . . . . . . . . . 27
Treatment with Sugars . . . . . . . 28
Treatment with Denaturing Agents . . . . . . 29
Performic Acid . . . . . . . . . . . 29
2- -Mercaptoethanol . . . . . . . . . . 29
Eight Molar Urea . . . . . . . . . . 30
Chemical Analyses . . . . . . . . . . 3O
Nitrogen . . . . . . . . . . . . . 30
Total Protein . . . . . . . 31
Trichloroacetic Acid- Soluble Protein . . . . 32
e-Amino Lysine . . . . . . . . . . . . 33

iii

Amino Acids--Acid Hydrolysates . .
Amino Acids and Peptides--Enzymatic

Hydrolysates . . . . . . . . . .
Measurement of Enzymatic Hydrolysis . .
Gel Filtration . . . . . . . . . .
Pepsin and Pancreatin . . . . . . . .
Proton Release by pH—Stat . . . . . . .
RESULTS . . . . . . . . . . . . .
Milk Protein Preparations . . . . . . .
Amino Acids . . . . . . . . .
Characteristics of Protein Preparations Upon
Heat Treatment . . . . . . . . . .
Pepsin-Pancreatin Digest of Proteins and Heat-
Treated Proteins . . . . . .~ . . . .
Amino Acids and Peptides . . .
Trichloroacetic Acid- Soluble Proteolysis
Products . . . . . . .
Enzymatic Proteolysis During Gel Filtration .
Total Protein Distribution . . . .
Trichloroacetic Acid- Soluble Nitrogenous
Material . . . . . .
Reaction with 1- Fluoro- 2, A- Dinitrobenzene .

Rates of Proteolysis by pH- -Stat. . . . . .
Equal Cleavage Sites . . . . . . .
Chemical Denaturing Agents

Sugars . . . . . . . . . . . . .
Control Measurements . . . . .
DISCUSSION . . . . . . . . . . . .

Milk Protein Preparations . . . . . . .

Amino Acids . .
Characteristics of Protein Preparations Upon

Heat Treatment . . . . . . . . .
Pepsin-Pancreatin Digest of Proteins and Heat-
Treated Proteins . . . . . . . .
Amino Acids and Peptides . . . . .

Proteolysis During Pepsin—Pancreatin
Digestion . . . . . . .
Enzymatic Proteolysis During Gel Filtration .
Total Protein Distribution . . . . .

Trichloroacetic Acid Supernatant Protein . .
Reaction with 1-F1uoro-2,A-Dinitrobenzene .

iv

Page

3“

35
37
37
38
39

AA

AA
AA

“A

96
M6

50
51
51

52
53
5A
55
56
59
6O

88

88
88

88

9O
90

99
102

103
103
10“

Page

Rate of Proteolysis by pH-Stat . . . . . . . 107
Equal Cleavage Sites . . . . . . . . . 107
Chemical Denaturing Agents . . . . . . . 108

SUMMARY 0 C O O O O O O O l 0 fl 0 6 0 ll 8
BIBLIOGRAPHY . . . . . . . . . . . . . 123

LIST OF TABLES

Table Page

1. Summary of the thin-layer chromatographic
procedure for amino acids . . . . . . . A2

2. A comparison of the amino acid compositions
of the milk proteins utilized in this
study with those reported by others . . . 61

3. Free amino acids in pepsin-pancreatin digest
of casein and heat-treated casein .. . . . 62

A. Free amino acids in pepsin-pancreatin digest
of as—casein and heat-treated aS-casein . . 6A

5. Free amino acids in pepsin-pancreatin digest
of k-casein and heat-treated k-casein . . . 66

6. Free amino acids in pepsin-pancreatin digest
of B-lactoglobulin and heat-treated
B-lactoglobulin . . . . . . . . . . 68

7. Peptides in pepsin-pancreatin digest of milk
proteins and heat—treated milk proteins . . 7O

8. Trichloroacetic acid soluble protein measured
at intervals during pepsin-pancreatin digest
of milk proteins and heat-treated milk
proteins . . . . . . . . . . . . 71

9. Measurement of the protein content in fractions
of proteins and heat- treated proteins sub-
jected to tryptic proteolysis during gel
filtration . . . . . . . . . . . 72

10. Trichloroacetic acid soluble nitrogen con-
taining material from proteins and heat-
treated proteins subjected to tryptic
proteolysis during gel filtration . . . . 73

ll. Terminal amino groups of proteins and heat-

treated proteins subjected to tryptic pro-
teolysis during gel filtration . . . . . 74

vi

Table

Page
12. Rates of proteolysis of protein fractions at
equal cleavage site concentrations measured
by proton release . . . . . . . . . 75
13. Rates of tryptic proteolysis of casein and
heat-treated casein after treatment with
sugars, chemical denaturing agents and
pepsin digest

. . . 76
Rates of tryptic proteolysis of as-casein and
heat-treated as-casein after treatment with
chemical denaturing agents and pepsin digest

1“.

77
15.

Rates of tryptic proteolysis of k-casein and
heat-treated k-casein after treatment with
chemical denaturing agents and pepsin
digest

o o o 78
Rates of tryptic proteolysis of B-lactoglobulin
and heat-treated B-lactoglobulin after treat-

ment with sugars, chemical denaturing agents
and pepsin digest . . . 79

Pepsin-pancreatin indices of milk proteins and
heat-treated milk proteins

l6.

17.

117

vii

LIST OF FIGURES

Figure Page

1. Procedure for the preparation of milk protein
fractions . . . . . . . . . . . . 43

2. Free essential amino acid and peptide profiles
in pepsin-pancreatin digest of casein and
heat—treated casein . . . . . . . . . 8O

3. Free essential amino acid and peptide profiles
in pepsin-pancreatin digest of ds-casein and
heat-treated ds-casein . . . . . . . . 81

A. Free essential amino acid and peptide profiles
in pepsin-pancreatin digest of k-casein and
heat-treated k-Casein o o o o o o o o 82

5. Free essential amino acid and peptide profiles
in pepsin-pancreatin digest of B-
lactoglobulin and heat-treated B-
lactoglobulin . . . . . . . . . . . 83

6. Proteolysis during pepsin—pancreatin digest of
casein measured by trichloroacetic acid-
soluble protein . . . .- . . . . . . 8A

7. Proteolysis during pepsin-pancreatin digest
of d -casein measured by trichloroacetic
acid-soluble protein . . . . . . . . 85

8. Proteolysis during pepsin-pancreatin digest of
k-casein measured by trichloroacetic acid-
soluble protein . . . .7 . . . .' . . 86

9. Proteolysis during pepsin-pancreatin digest of

B-lactoglobulin measured by trichloroacetic
acid-soluble protein . . . . . . . . 87

viii

INTRODUCTION

Milk proteins exist in nature as a complex mixture
of micellar casein and whey proteins. Individual milk
proteins have characteristics which have been established
by physical and chemical studies. The individual milk
proteins derived have been used as model protein systems
for the study of enzymatic proteolysis with and without
heat treatment in this study. An_effort has been made to
evaluate some of the components of foods on the model
protein systems as they affect enzymatic hydrolysis.
Protein interactions induced by heat have been reported as
they are measured by physical behavior. The interactions
thus described have been further examined relative to the
influence they may have on proteolytic behavior.

The enzymatic breakdown of proteins has been related
to digestion of proteins during the digestive process. The
use of model protein systems to further describe the break-
doWn of proteins may provide a further insight into the
behavior of proteins upon digestion.

Measurement of protein quality as the ability to
support growth has been related to amino acid content,
specific amino acid availability and amino acid release

upon exposure to proteolytic enzymes. The rate of

l

proteolysis of a protein may provide another means to
attain an indication of protein quality.

This study was conducted to examine the behavior of
model milk protein systems during enzymatic proteolysis.
The observations thus derived can be related to the treat-
ments encountered in food processing operations. The
behavior of model proteins upon hydrolysis can also be

associated with the behavior during digestive processes.

LITERATURE REVIEW

The primary importance of enzymatic proteolysis is
the specific set of functions as performed in the digestive
process. The breakdown of dietary proteins involves de-
gradation by enzymatic cleavage to yield residues to be
absorbed by the intestinal mucosa. The proteins ingested
are subjected to proteolysis by successive attack by first
pepsin at a relatively low pH, then by the proteolytic
enzymes secreted by the pancreas at higher pH. The condi-
tions for proteolysis are optimal for each enzyme. The
optimal pH for pepsin is 1.8 compared to 7-8 for trypsin
and pancreatin. The temperature Optimum for each of the
proteolytic enzymes considered is 37 C.

The order of hydrolysis must be taken into account.
Initially pepsin acts in the gastric juice at pH 1.8-2,
followed by pancreatic enzymes in the intestine at pH 7-8.
The systems must be considered in the order of their place
in the digestive process.

The principle enzyme of gastric juice is pepsin
(3.4.9.1) which has a pH optimum of 1.8 as described by
Bovey and Yanari (1960). Further proteolysis is accom-
plished after passage to the intestine and adjustment of

the intestinal pH to between 7 and 8, by trypsin (3.4.9.4)

and chymotrypsin (3.4.4.5). The enzymes trypsin and chymo—
trypsin are so closely related in their behavior that they
may be discussed together as Desnuelle (1960) has suggested.
The characteristic action of trypsin and chymotrypsin
includes not only cleavage of peptide linkages, but ester
linkages as well, probably by attack on the nucleophilic
centers and serine moieties as described by Cunningham
(1957). The mechanism of action for trypsin has been sug-
gested by Graae and Rasmussen (1961) to be identical for
proteins of different structures. Smith and Hill (1960)
proposed that leucine aminopeptidase (3.4.1.1), sometimes
referred to by its trivial name, erepsin, should be given
more active consideration in physiologic proteolysis.

The specificity of each of the enzymes involved in
physiologic digestion is unique and has been summarized by
Hill (1965). Pepsin is least specific of the proteolytic
enzymes involved, cleaving amino or carboxyl groups of
phenylalanine, tryosine, glutamic acid, cystine and
cysteine. The specificity of rennin (3.4.4.3) and cathep-
sin (3.4.4.9) is similar to pepsin. Trypsin peptide
specificity is restricted to peptide bonds formed by
carboxyl groups of lysine and arginine. Chymotrypsin
cleaves at the carboxyl bonds of tyrosine, phenylalanine
and tryptophan. Leucine aminopeptidase cleaves peptide
bonds adjacent to the a—amino group but shows preferential

treatment to leucine residues. Specificity for each of

the enzymes is also influenced by the isomeric form, with
the L—form favorably attacked as described by Rapp gt_al.
(1966).

Proteolysis during digestion is specific not only
to individual proteins but specificity is changed by the

condition of the individual proteins. Structural dif-

p:
ferences in native proteins, specifically plasma albumin g
and y—globulin, have been observed by Epstein and Possick I
(1961) to influence their susceptibility to enzymatic

cleavage. They attributed these differences to stresses l

 

involved in tertiary linkages between polypeptide chains.
Allison (1964) pointed out that each native protein is
unique in its proteolysis in much the same way that
behavioral reactions are unique and that proteolysis is
as dependent on structure as is behavior.

Donoso et_§1. (1962) proposed that changes occurred
in structural characteristics of pork protein as a result
of extensive heating. These included formation of
aldehyde condensation products and reduction of disulfide
cross-linkage reaction products to sulfhydryl groups.
Neither of the protein treatments exhibiting these types
of interactions were available to enzymatic cleavage.

The products of proteolysis, amino acids and
"oligopeptides" (i.e., peptides with 2-4 amino acids) have
been studied as important determinants of rate and extent

of intestinal absorption by a group of British researchers;

Matthews, Crampton and L13 (1968), Matthews, Craft and
Crampton (1968), and Hamilton et_§l. (1968). They postu-
late that protein degradation products may be most
favorably absorbed as "oligopeptides" although hydrolysis
to amino acids must occur prior to entry into the blood.
The transport of dipeptides (methionyl-methionine,
methionyl-glycine, glycyl-methionine) was more rapid than
component amino acids. The transport rate for each
dipeptide studied was unique. This may partially explain
the distinctly greater utilization of pathways alternate
to the glycolytic scheme observed by Ahrens and Wilson
(1966) in rats fed casein as opposed to rats fed an amino
acid mixture simulating casein.

The release rate and total content of amino acids
was comprehensively examined by Block and Mitchell (1946).
They observed that the value for growth support is depen-
dent not only on total content of amino acids but on
relative proportion of these amino acids.

The rate of proteolysis of several proteins from
animal and plant sources was studied in white rats by
Zebrowski (1968). Casein emptied from the stomach much
more rapidly than did heated soya protein. The intes-
tines contained more soluble nitrogen when the rats were
fed casein diets as compared to heated soya protein diets.
The intestine contained extremely large amounts of protein
nitrogen when rats were fed raw soya protein relative to

either heated soya or casein.

 

The influence of heat treatment of protein on rate
and extent of hydrolysis has been related to a number of
considerations including structure of the protein and
reactions of individual groups within the protein mole-
cule. Kakade and Evans (1966a) credited denaturation of
navy bean protein for improved proteolysis as measured by 1r-
lysine availability. They separated the effect of a heat
labile trypsin inhibitor in a further study (Kakade and
Evans, 1966b) as an independent consideration.

The extent of heating and protein concentration

 

were related to the rate of disappearance of food from

the digestive tract by Rice and Beuk (1953). They noted
that heating of casein for 8 hr at 100 0 did not reduce
the growth-promoting properties of casein, however toast-
ing did cause an impairment. Heat treatment of evaporated
milk for a period of 3 hr was required at 120 C to result
in measurable depression of rat growth.

The loss of "nutritive efficiency" after heating
milk was reported by McCullom and Davis in 1915. The loss
was attributed to "sulfur" due to the characteristic
sulfur odor upon heating. The observation that destruc-
tive changes take place has been developed from several
aspects by numerous studies.

The most striking results for milk protein alone
have been accomplished with milk powder after autoclaving.

Kraft and Morgan (1951) fed rats milk powder heated for

15 to 25 min at 120 C. Rat growth was depressed by heat

treatment to 50% of the rate when rats were fed control

diets. The lack of rat growth could be reversed by addi-
tion of lysine. In an experiment with weanling dogs for
a 120 day period with the same diets there was no observ-
able difference in growth rate or nitrogen balance. ,—
Eldred and Rodney (1946) performed a study igDXitgg_with A
heat treatment of dry casein at 150 C for 70 min. Step- ;

wise hydrolyses were performed with pepsin, trypsin and ;

 

pancreatin; and lysine was determined manometrically with g
lysine decarboxylase. Digestion rates measured by Van
Slyke a-amino nitrogen did not differ significantly,
however lysine contents were significantly lower than
observed with unheated casein. Acid hydrolysates of
heated and unheated samples were not measurably different
in their amino—acid content. Hankes e£_§1, (1948) made

a similar observation that autoclave heating of casein for
as long as 15 hr did not reduce the content of total amino
acids but did reduce in_yi££g digestibility by 25 to 47%.
Losses in lysine, methionine and tryptophan in commer-
cially processed products were reported by Mauron e£_al.
(1951). The only products they examined which reflected
significant losses were roller-dried milk powder (23 to
62%) and evaporated milk (11%). Spray—dried milk powder,
sweetened condensed milk and boiled milk losses were mini-

mal. In contrast, Fricker (1964) fed rats through five

generations raw milk and "Uperized" (flash heated) milk
heated to 150 C for 2.4 sec. He was unable to observe any
differences in rate of growth, histological development or
reproductive ability.

Heat treatment of milk proteins with sugars has been
examined extensively from the standpoint of susceptibility

to hydrolysis. Patton et a1. (1948) were able to demon-

EI_. l—_ E III.
IT
'5 t
’1

strate decreases of 15 to 27% in lysine, arginine, and

tryptophan in 5% casein-glucose solutions heated for 24 hr

 

at 95.5 C. McInroy gt_a1. (1948) demonstrated the rela-
tive effects of added dextrose and protein concentration
after heat treatment at 121 C for 2 hr. They reported that
rats lost weight with casein and dextrose autoclaved;
however growth rates were 79% of the control when casein
was autoclaved alone. Mixtures of casein with dextrose at
50% moisture were examined by Lowry and Thiessen (1950) with
in XEEEQ enzymatic digestion. Pepsin was effective in
hydrolysis of the casein-dextrose complexes while trypsin
was almost totally ineffective as measured by a-amino
nitrogen release. They suggested the formation of ester
linkages by casein molecules which were susceptible to
pepsin cleavage but not to trypsin. Lowe g£_al. (1964)
demonstrated significant growth advantage in infants fed

a formula sterilized at 146 C for 6 sec when compared to
conventional autoclave processes. The pertinent measure-

ments included weight gain per energy intake as well as

10

total growth measurement. Differences in nitrogen balance
and serum proteins were not discerned by Fomon and Owen
(1962) for infant formulas fed with and without autoclav—
ing.

Milk proteins must be distinguished as to their
presence in milk or as derived from milk. The native
state of the individual proteins is in the form of a com-
plex as a stable colloidal suspension. Casein, as'
described by Morr (1967), consists of loosely packed cal-
clum caseinate complex units joined by calcium and calcium
phosphate—citrate linkages. Casein has been defined by
Thompson e£_§l, (1965) as a heterogeneous group of
phospho-proteins precipitated from skim milk at pH 4.6
and 20 C. The complex units or micelles in casein are
stabilized by interaction of the casein fractions as
described by Waugh (1961). The k-casein fraction is
believed to function as a "protective colloid" for micellar
casein (Swaisgood et_al., 1964). Thompson gt_al. (1965)
have defined k-casein as a phospho-glyco-protein capable
of stabilizing dS-casein against precipitation by calcium.
The proteins remaining after casein has been removed from
skim milk comprise whey proteins or milk serum proteins as
designated by Gordon and Whittier (1965). The protein
fractionated from milk designated as ds—casein is a calcium
sensitive, disordered protein without intermolecular

disulfide bonds as described by McKenzie (1967). The

ll

behavior of dS-casein is advantageous in that it is sus-
ceptible to digestion particularly by the newborn because
of its disordered configuration. In contrast, k-casein
has been described by Swaisgood g£_al. (1964) as an
ordered glyco—protein with intramolecular disulfide bonds
determining a specific tertiary configuration. The carbo- L»
hydrate component of k-casein is sialic acid. Waugh (1961)
concluded that all the cystine in casein existed in the

k-casein fraction. The k-casein fraction is sensitive to

 

specific attack by rennet. The "caseino-glycopeptide"
formed by heat treatment was reported by Alais e£_a1.
(1967) to be similar to the rennet hydrolysis product.
The milk whey protein B-lactoglobulin is a globular
protein containing disulfide linkages and sulfhydryl
groups susceptible to reversible disulfide interchange
as reported by Morr (1967). B—lactoglobulin was considered
by early workers to have external secondary structure due
to its ability to form crystals. More recent information
compiled by Tanford et_a1. (1962) indicates that the
native structure is compact and that refolding after de-
naturation results in a large number of d-helices.
Tanford e£_al. (1967) have further observed that specific
and different denatured states are produced by explicit
denaturing agents. McKenzie (1967) has concluded that
B-lactoglobulin exhibits a high degree of change in

secondary structure depending on environmental conditions.

12

A proteolytic enzyme was reported in casein by Warner
and Polis in 1945 which decreased casein solubility and
increased trichloroacetic acid soluble nitrogenous pro—
ducts. Harper et_al, (1960) reported that raw milk
usually but not always contains a small amount of a pro-
tease which releases tyrosine. Zittle (1963) observed the r»
association of milk protease with the k—casein fraction.
Trypsin inhibitor was reported by Kiermeer and Semper =
(1960) in cow's milk. The presence of both a protease and

inhibitor has been suggested by Shahani (1966) as a reason

 

for inability to establish quantities or activities for
either component.

The ordered form of a native protein may be disrupted
as described by Kauzman (1956) to modify the primary,
secondary, or tertiary structure. The properties of a
native protein may be sensitive to change by heat, light,
pressure or chemical reagents. Chemical denaturing agents
have been examined and specific changes attributed to them.
Performic acid has been utilized by Hirs (1956) for oxida-
tion of the sulfur moieties of sulfur—containing amino
acids. Proteins oxidized with performic acid were approxi-
mately one—tenth as susceptible to tryptic digest as were
the original proteins. Mercaptoethanol has been utilized
for reversible disulfide interchange. Acid and base bind-
ing groups were released by denaturation of ovalbumin

with guanadine by Harrington (1955).

l3

Electrophoretic changes in casein were observed by
Tamauchi and Tsugo (1961) as a result of heating skim
milk. Heat treatment at 120 C for 30 min resulted in
severely decreased contents of as- and B-caseins. The
.changes were probably due to complexes of the type
described by Trautman and Swanson (1959). The complexes Fr“
they observed between <i-casein components and B—lacto-
globulin could be prevented by sulfhydryl blocking agents.
Morr and Josephson (1968) reported that whey protein

aggregation was influenced by thiol-disulfide group

 

reactions and calcium concentration.

The degree and severity of heat denaturation can be
altered by a number of factors; pH, ionic strength, ultra-
violet treatment and concentration; as described by Joly
(1965). The stabilization against heat denaturation by
anions of fatty acids was reported by Boyer g£_al. in
1946. Epstein and Possick (1961) suggested that fatty
acid anions reduce denaturation to cause a reduction in
susceptibility to enzymatic proteolysis. Protein denatura-
tion may occur by enzymatic action when hydrolysis does
not occur as observed by Lundgren (1941) with thyroglobu-
1in. Allosteric effects may in part account for reversible
denaturation observed in proteolytic enzymes (Gerhart and
Pardee, 1962). ~

The influence of denaturation on susceptibility to

proteolysis is unique to the substrate protein.

l4

Linderstrsm-Lang gt_§1. (1938) cited an example of

enhanced tryptic proteolysis of B—lactoglobulin when the
temperature was adjusted to induce the denatured form of
the protein. Ledford gt_§1. (1968) reported that as-casein
was hydrolyzed before whole casein or B-casein by rennin.
In studies with k-casein, Zittle (1961) noted that in com-

bination with heat, chymotrypsin destroyed the stabilizing

_ _......| '3 In]

property of k-casein for aS-casein in the presence of

calcium ions.

 

Measurement of protein quality has traditionally been I
made by growth of animals, usually rats, under carefully
defined conditions. Expression of protein quality has been
as Protein Efficiency Ratio (PER) or gain in body weight
divided by amount of protein consumed (Campbell, 1963).
Another method for expression described by Miller (1963)
is Net Protein Utilization (NPU) or carcass nitrogen with
test protein compared to control protein. Many refinements
and modifications of these basic methods have been used
for specific purposes: AOAC, 1965; Braham gt_al., 1959;
Summers and Fisher, 1961; Derse, 1962.

The relative amino acid contents have also been
used as measurements of protein quality, since the nutri-
tive value of dietary protein depends on the pattern and
quantity of essential amino acids. The two most common
methods for expression of amino acid values have been the

Chemical Score suggested by Mitchell and Block (1946) and

15

the Essential Amino Acid Index proposed by Oser (1951).
Both techniques establish an index based on the amino

acid composition of protein relative to the requirements
of the rat. Comparison may also be to a reference pat—
tern such as that suggested by the FAO (1961). Advantages
of speed can be attained with chemical methods, however
losses during hydrolysis as reported by Patton (1950) and
variations in digestibility and availability as noted by
Akeson and Stahmann (1964) diminish their value.

A number of enzymatic digest methods for protein
evaluation have been developed to overcome the disadvan-
tages of animal studies and chemical methods; time,
expense and lack of accuracy. An early method was proposed
by Sheffner e£_§1, (1956) involving a pepsin digest followed
by microbiological examination of residual protein and
released essential amino acids. Mauron e£_§1. (1955)
utilized sequential digestion with pepsin and pancreatin
in a dialysis sac. Measurements of the amino acids
tryptophan, tyrosine, methionine and lysine were accom-
plished in the dialysate using manometric techniques.

Ford and Salter (1966) used a similar hydrolysis, removing
the amino acids and small peptides on Sephadex gel (G-10).
The retained residues were quantitated microbiologically.
A stepwise hydrolysis method was reported by Frangne and
Adrian (1967) utilizing pepsin, pancreatin and erypsin,

sequentially applied. Unhydrolyzed proteins were removed

-l-

 

 

16

by alcohol and heat treatment. Lysine and methionine

were estimated in the filtrate by microbiological methods.
The technique suggested by Akeson and Stahmann (1964)
involved hydrolysis with pepsin then pancreatin followed
by analysis for amino acids of the picric acid supernatant
by means of an automatic amino acid analyzer. Each of

the enzymatic degradation techniques described here pro—
vided an accurate index of protein quality as related to
animal growth measurements.

Chemical indices of protein quality have been
directed to lysine studies. Selection of lysine was based
on observations by Lea and Hannan (1950) and Henry and Kon
(1950) that availability of lysine was paramount to chick
growth on the protein under consideration. Donoso g£_al.
(1962) suggested that cross linkage reactions as well as
condensation reactions were responsible for loss of lysine
in heated systems. The reaction of the Sanger reagent,
l-fluoro-2, 4-dinitrobenzene (FDNB), with protein to form
epsilon—2, 4-dinitropheny1 lysine was applied by Carpenter
(1960) to measure the availability of lysine in aminal
proteins. Evaluations of the technique have been per-
formed independently by several groups. Roach et a1.
(1967) noted that differences in heat treatment could be
measured quantitatively. Bujard gt_§1. (1967) observed
values for the FDNB method that fell between the total

lysine contents as determined enzymatically and those

l7

determined by hydrolysis. From their studies with animals,
Boctor and Harper (1968) concluded that the FDNB method

was not suitable for estimation of lysine in heat—treated
foods. Some of the lysine reported as available with

FDNB was a part of the undigestible residue in the feces

of rats. Either a combination of measurements or a more
precise measurement of amino acid availability is therefore

indicated.

Commentary and Objectives

 

Protein systems and food protein mixtures have been
examined in many studies relative to their susceptibility
to enzymatic proteolysis. The characteristics of many
individual proteins have been reported in terms of physical
and chemical properties. The functions of proteolytic
enzymes have been separately defined, usually with single
substrates. Specific relationships between protein systems,
component protein fractions and protein treatments are,
however, not well defined in terms of enzymatic hydrolysis.
Animal studies have been used to indicate total protein
digestion; however in yitgg techniques are more definitive
because stepwise observations can be made.

The objectives of this study include an evaluation
of enzymatic proteolysis of milk proteins treated as
model protein systems. The model protein systems were
selected to be representative of food proteins. Treatments

chosen are commonly encountered in processing of food

18

proteins. Additional selected chemical treatments were
applied to evaluate the effect on enzymatic proteolysis.
One of the purposes of this study is to apply the amino
acid release and proteolysis rate data to a method for
accurate assessment of protein digestibility. Another
purpose is to describe differences in behavior upon proteo-
lysis of model milk protein systems and to relate them to
previously described physical and chemical protein dif-
ferences. A third purpose is to examine existing methods

for protein evaluation as applied to model protein systems.

EXPERIMENTAL

Apparatus and Equipment

The milk used for preparation of milk protein
fractions was collected in five-gallon stainless steel
cans and separated with a DeLaval Model 9 disc-type sepa-
rator. A Beckman expanded scale pH meter with glass
electrode was used to measure pH. Low speed room tempera-
ture centrifugations were performed with an International
Model U centrifuge. Intermediate speed and refrigerated
centrifugations were performed with a Sorvall RC-2B
refrigerated centrifuge.

The eluate from gel filtration columns was monitored
at 280 nm with an ISCO UA-5 recording monitor manufactured
by Instrument Specialties Company.

Laboratory weightings were made utilizing a tOp-
loading, direct-reading Mettler Type K-7 balance.
‘Analytical weighings were made with a Mettler H-15 analyti-
cal balance.

Amino acid analyses were performed with a Beckman-
Spinco Model 1200 amino acid analyzer.

A Coleman Model 14 Universal SpectrOphotometer was,

used for all spectrophotometric examinations.

19

20

Proton release during enzymatic proteolysis was

measured with a Sargent Recording pH-Stat.

Chemicals and Materials

 

The principle chemicals used and their suppliers
are given below. Glucose and lactose were Baker Analyzed
and obtained from J. T. Baker Chemical Company. Galactose
was Mann Assayed from Mann Research Laboratories, Inc.
Bio—Gel P-2 and a 1% solution of dimethyldichlorosilane
in benzene were obtained from Bio-Rad Laboratories. The
anion exchange resin, Dowex 2-X 8, was purchased from J. T.
Baker Chemical Co., as were 2-mercaptoethanol and l-fluoro-
2,.4-dinitrobenzene. The N—dinitrophenyl-epsilon-L-
lysine monohydrochloride used as a standard was obtained
from Mann Research Laboratories.

The trypsin was 2X recrystallized purchased from
Worthington Biochemical Corporation. Pepsin utilized was
3X recrystallized from General Biochemicals. Pancreatin
was "B" Grade purchased from Calbiochem.

The other chemicals used in this study were of
reagent grade.

Preparative Procedures for Milk
Protein Fractions

 

Milk used in this study was obtained from the
Michigan State University dairy herd which consisted of

Holstein, Jersey and Brown Swiss cows. A11 milk was

21

obtained immediately after milking and separated while

still warm.

Preparation of Whole Casein

Whole casein was prepared from fresh skim milk by
precipitation at pH of 4.5 at 25 C. The precipitated
whole casein was filtered from the supernatant with
double cheesecloth and drained for 15 minutes. The casein
was redissolved at 3% in deionized water by adjustment
of pH to 7 with 0.1 N NaOH. The sodium caseinate is
referred to as casein. The casein solution was stored at

-20 C in approximately 100 g quantities for subsequent uSe.

Preparation of aS-casein

 

The method of preparation for dS—casein was suggested
by Zittle and Custer (1963). Approximately 350 g of frozen
whole casein were dissolved in 1 liter of 6.6 M urea. The
solution was acidified with 200 ml of 7 N sulfuric acid
and 2 liters deionized water added. The resulting pH was
1.4 to 1.5. A flocculent precipitate which formed during
a 2 hr period was removed by centrifugation. The centri-
fugal supernatant was retained for preparation of k-casein.
The aforementioned precipitate was dissolved by adjustment
of the pH to 7.2 with 0.1 N NaOH. The resulting clear
solution was added to an equal volume of 95% ethanol until

maximum precipitation was achieved. The precipitate was

removed by centrifugation and discarded. The supernatant

22

was acidified to pH 5.0 with 3.0 N HCl. The precipitate
formed in this operation was separated by centrifugation
and dissolved at pH 7.5 with 0.1 N sodium hydroxide. The
resulting as-casein solution was dialyzed against deionized
water and pervaporated to a concentration of approximately

10 mg per ml.

Preparation of k-casein
The method proposed by Zittle and Custer (1963) also

was used for k-casein. The supernatant from the acid-

 

urea fractionation of aS-casein was treated with 132 g
ammonium sulfate per liter. The precipitate formed was
removed by centrifugation, suspended in deionized water
and dissolved by adjustment of the pH to 7.5 with 1.0 N
sodium hydroxide. The solution, containing principally
k-casein, was dialyzed against deionized water and per-
vaporated to a concentration of approximately 10 mg

protein per m1.

Preparation of B—lactoglobulin

 

The preparation method for B-lactoglobulin was that
suggested by Fox e£_§1. (1967). The acid whey was
recovered from casein preparation at pH 4.5. Trichloro-
acetic acid was added to the solution at a rate of 34.2 g
per liter. The precipitate was removed by centrifugation
at room temperature and discarded. The clear supernatant

was concentrated by pervaporation to one-tenth the

23

original volume. Saturated ammonium sulfate was added

in an amount to yield an 0.4 saturated solution with
reference to ammonium sulfate. The mixture was allowed

to stand for 12 hr at room temperature, and the precipi-
tate was removed by centrifugation. The precipitate was
suspended in saturated ammonium sulfate and dialyzed
against deionized water at 4 C. The protein concentration
was adjusted by pervaporation to approximately 10 mg
protein per ml. A summary of milk protein preparation

procedures is presented in Figure 1.

 

Acrylamide Gel ElectrOphoresis

 

The electrophoretic homogeneity of milk protein
fractions was established with the continuous acrylamide.
gel electrophoresis system used by Peterson (1963). The
buffer system was a tris—borate buffer with disodium
ethylenediamine-tetraacetic acid (disodium EDTA). Stock
buffer was diluted 1:9 for addition to buffer tanks. The
acrylamide gel for one 250 ml gel bed was prepared by
adding 27.5 ml stock buffer, 17.5 g Cyanogum 41 and 62.5
g urea to 150 ml glass distilled water. Immediately
before pouring the liquid gel into the gel bed, 0.5 m1
B-amino propionitrile and 0.3 g ammonium persulfate were
added to the solution. After pouring, the gel was poly-
merized in a nitrogen atmosphere for 20-30 minutes and
covered with saran wrap to minimize evaporation. The

diluted tris—borate buffer was poured into the buffer tanks

24

to complete the system. Samples were introduced into
preformed slots in amounts equivalent to 1-2 mg protein.
One drop of a bromo phenol blue solution was introduced
into one of the slots to act as an indicator since
bromo phenol blue'migrates with the most rapidly moving
proteins. Platinum electrodes were inserted in the
buffer tanks. Current was applied with a Heathkit JP-32
power supply, with an initial voltage of 100 volts,
gradually increased to 250 volts after 15 minutes with a
current of 75 ma. Electrophoresis was continued for 5 hr,
at which time the dyd marker had moved within 2 cm of the
end of the gel bed. When electorphoresis was completed,
the gel was removed from the gel bed and stained with a
dye solution containing 250 ml water, 250 m1 methanol,
50 m1 glacial acetic acid, and 5 g Amido Black. After a
staining period of 30-40 min, the gel was electrolytically
destained in a 7% acetic acid solution. The destained gel
was then wrapped in saran wrap to prevent evaporation. A
single aS-casein band moved near the dye marker, 8-
lactoglobulin moved in a single band directly behind, and
k-casein moved in a diffuse single band approximately 5
cm from the origin.
Preparative Procedures for Treated
Milk Protein Fractions
The milk protein fractions were treated with heat in

a manner consistent with normal processing practice. The

F...

 

25

proteins were heated alone as well as with reducing
sugars. Chemical denaturing agents intended to modify
the configuration of the proteins were employed in other

experiments.

Heat Treatment of Milk Proteins

The milk protein fractions were prepared, as pre—
viously described in concentrations adjusted to 10 mg
protein per ml. The solutions were placed in 250 ml

Erlenmeyer flasks and covered with aluminum foil. The

 

flasks were subjected to autoclave treatment at 15 lb
pressure for a period of 30 min in an Amsco Cyclomatic
Sterilizer. After sterilization, the samples were imme-
diately cooled and stored at 4 C until use.

Heat Treatment of Milk Proteins

with Added Lactose

Milk protein fractions adjusted to protein concen-
trations of 10 mg per ml were made to 0.06 M lactose.
Heat treatment was identical to that for heated proteins,
in the same autoclave whenever possible. The pH of
samples was adjusted to 7 prior to heating in all cases
unless otherwise indicated.

Samples designated as heated milk proteins and milk
proteins heated in the presence of lactose were the basic
samples used in all evaluations of enzymatic hydrolysis;
pepsin and pancreatin, gel filtration and measurement of

proton release. All other variables were evaluated only

26

by monitoring of proton release during proteolysis by pH-
stat. Observations were made with control samples,
samples after heating and after heat treatment in the
presence of lactose.

Variable samples were prepared from milk protein
fractions for enzymatic proteolysis. Treatments included
denaturation with selective denaturing agents; 2-
mercaptoethanol, performic acid, urea, pH adjustment, and
pepsin digest prior to tryptic proteolysis. The samples
were treated with salts of the cations of sodium and cal-
cium. Protein concentrations were adjusted according to
the number of cleavage sites in the individual protein
preparations to provide equal concentrations with respect
to number of trypsin cleavage sites (lysine and arginine
residues). Preparations were made of proteins with
selected sugars for measurement of proton release upon

hydrolysis.

Pepsin Treatment

Milk protein fractions, adjusted to a concentration
of 10 mg protein per ml, were subjected to pepsin treat-
ment according to the procedure suggested by Rick (1963).
Pepsin was added in quantities equivalent to 1 mg pepsin
per 100 mg protein. Proteolysis was conducted in a water
bath at 37 C for 3 hr. The pH was then adjusted to 8.0
and tryptic proteolysis evaluated by monitoring the

protons released with a pH-stat-

27

Treatment with Isotonic Saline
Milk protein fractions were treated by the addition
of sodium chloride to yield a solution 0.15 M in sodium

chloride.

Treatment with Calcium Ions
Milk protein fractions were treated by the addition

of calcium chloride to yield a solution 0.3 M in calcium.

pH Adjustment

’ “a. 7"?! ‘1'!

Milk protein fractions were heated at pH 5 and pH 9.

 

The other samples were adjusted to pH 7 prior to heating.
The pH adjusted samples were divided after heating and one
'portion treated with pepsin prior to evaluation of their

susceptibility to proteolysis by pH-stat monitoring.

Equal Cleavage Sites

Concentrations of milk protein fractions were
adjusted to establish an equal number of lysine and
arginine residues per unit volume for each protein. The
amino acid content of casein assumed for these calcula-
tions was that of Block and Weiss (1956), with the number
of lysine and arginine residues 26.2 per molecular weight.
The molecular weight of dS-casein used was 28,000 although
the range for genetic variants may be from 28-30,000, as
discussed by Gordon et_al. (1965). The number of lysine
and arginine residues per monomer (24.3) was also reported

by Gordon et a1. (1965). The molecular weight for k-casein

28

(28,000) and number of lysine and arginine residues

(18.0) of Swaisgood gt_al. (1964) were utilized. The
values reported by Kalan gt_al, (1964) for B-lactoglobulin
as monomer molecular weight (18,000) and number of lysine

and arginine residues per monomer (18.0) were used.

Treatment with Sugars

Milk protein fractions adjusted to a concentration
of'lOmg per ml were treated with selected sugars. Lactose
was added at a rate of 20 mg per ml to yield a concentra-
tion of 0.06 M in lactose. Glucose and galactose were
added to separate solutions at a rate of 10 mg per ml to
yield a concentration of 0.06 M in glucose and galactose
respectively. Glucose and galactose were added together
to the milk protein fractions at rates of 10 mg per ml
each to yield a concentration 0.06 M in each glucose and
galactose.

Lactose represents a reducing disaccharide native to
milk. Glucose and galactose are reducing monosaccharides.
Glucose and galactose are the monosaccharide components of
lactose and were included for evaluation of the effect of
molar concentration.

The protein fractions prepared with sugars were
divided with one portion heated and the other retained as
a control. Protein-sugar preparations thus treated were
examined for behavior upon proteolysis by monitoring of

proton release using the pH-stat.

29

Treatment with Denaturing Agents

 

Performic Acid

The protein fractions were oxidized with performic
acid using the procedure suggested by Hirs (1956). Per-
formic acid was prepared by addition of 0.5 m1 of 30%
hydrogen peroxide to 4.5 ml 88% formic acid at 50 C. The
performic acid so prepared was added to protein fractions
in a volume of 0.8 ml performic acid to 10 ml sample

solution. The reaction mixture was held at 50 C for 15 g

 

min and the performic acid was removed by passage over a '.
Bio-Gel P-2 desalting gel. Protein recovery was monitored

by measuring absorption at 280 nm. Aliquots of performic

acid oxidized samples were further treated with heat and

heat in the presence of lactose as previously described.

2—Mercaptoethanol

The disulfide linkages of protein fractions pre-
viously described were disrupted with 2-mercaptoethanol
as suggested by Anfinsen and Haber (1961). 2-Mercapto-
ethanol was added to the protein fractions in amounts of
0.1 ml per 10 ml sample at 1 mg protein per ml in 0.1 N
acetic acid. The reaction was allowed to proceed for 30
min in a nitrogen atmosphere. Freshly boiled deionized
water was used in preparation of all reagents and for
dilutions. Removal of 2-mercapthethanol was accomplished

by passage over a Bio-Gel P-2 desalting gel with protein

30

recovery monitored at 280 nm. Aliquots of 2-
mercaptoethanol-treated samples were further treated with
heat and heat in the presence of lactose as previously

described.

Eight Molar Urea

 

The milk protein preparations of casein and B-
lactoglobulin were treated with 8 M urea to dissociate the
secondary structural characteristics. Urea in amounts of
4.8 g per 10 ml was added to the preparations of casein and
B-lactoglobulin adjusted to 1 mg protein per m1 and dis-
solved by stirring with a magnetic stirrer. The solution
was allowed to standau3room temperature for 30 min. The
urea was removed by passage over a Bio-Gel P-2 desalting

gel with monitoring of protein elution at 280 nm.

Chemical Analyses

 

Nitrogen

 

Micro-Kjeldahl nitrogen analyses were performed in
duplicate. An apparatus with round-bottom flasks and
ground glass joints was used. The digestion mixture was
concentrated sulfuric acid (500 ml) to which 5.0 g SeO2
and 5.0 g CuSou~5H2O were added. The mixture was vigor—
ously shaken before each usage. Ten to fifty mg of
protein were digested with 4 m1 of the digestion mixture
for 1 hr or more. After cooling, 1 ml of 30% H202 was

added and digestion continued for 1 hr. The digestion

31

flasks were rinsed with 10 ml of deionized water after
cooling and connected to the distillation apparatus.
Approximately 25 ml of a 40% sodium hydroxide solution
was added to neutralize the digest. The released ammonia
was distilled into a flask containing 15 ml of a 4% boric
acid solution to which 4 to 5 drops of indicator had been 1
added. The indicator contained 400 mg bromcresol green
and 40 ml methyl red in 100 m1 of 95% ethanol. Distilla-
tion was terminated when a total volume of at least 60 m1 g

was present in the receiving flask. The ammonia was

 

 

titrated with 0.1036 N hydrochloric acid. Tris(hydroxy-
methyl)aminomethane (Sigma 121) was used as the primary
standard for determination of the normality of the hydro-

chloric acid.

Total Protein

Total protein was determined using the Folin reac-
tion as described by Lowry §t_al. (1951). A reagent
solution was prepared containing 50 ml 0.1 N sodium
hydroxide with 2% Na2CO3 and 1 ml of a sodium tartrate
solution with 0.5% CuSou'5H2O. One ml of the reagent
was added to a protein solution containing 10 to 100 mg
protein. The mixture was thoroughly mixed and allowed
to stand for 10 min at room temperature. Folin-Ciocalteu
reagent, diluted to 1 N in acid, was added in an amount
of 0.1 ml. The reaction mixture was allowed to stand 30

min at room temperature. Optical Density (OD) was

32

measured at 700 nm. The relationship between protein
concentration and OD was linear within the range 100-400
ug per ml. The linear equations were the same for casein,
dS-casein and k-casein, however a different relationship
existed for B—lactoglobulin. The equations were as

follows:

F“

Caseins--Optical Density = 0.45 (ug Prot/ml)

+ 0.101

B-Lactoglobulin--Optical Density = 0.59 1

 

(ug Prot/ml) + 0.0876

(Kjeldahl nitrogen x 6.38 was used as a reference standard
for all milk protein cnalyses.)
Trichloroacetic Acid
soluble Protein

The protein in trichloroacetic acid supernatants was
determined by the McDonald and Chen (1965) modification of
the method of Lowry et_31. (1951) previously described.
The modification involved preparation of a reaction mixture
containing sufficient NaOH to neutralize the trichloro-
acetic acid and acid in the Folin-Ciocalteu phenol reagent.
Standard curves were prepared and were identical to those

for total protein.

33

e—Amino Lysine

The method suggested by Carpenter (1960) for .
available lysine (e-amino lysine) was adapted for heated
samples and selected hydrolysates. The principle of the
procedure was to convert lysine residues with reactive
c-NH2 groups to yellow e-DNP lysine by treatment with l-
fluoro-2, 4—dinitrobenzene (FDNB), followed by acid,
hydrolysis. Duplicate samples were prepared containing
30-50 mg protein in flat-bottom boiling flasks with ground
glass fittings. To each of the flasks was added 8 ml of

8% (w/v) NaHCO followed by 12 ml ethanol containing 0.3

3
ml FDNB. The flasks were covered and shaken gently on a
mechanical shaker for 2 hr. The flasks were held in a
boiling water bath until there was no further effervescence
upon swirling. The flasks were cooled, 24 ml 8.1 N
hydrochloric acid added, and gently refluxed for 16 hr on

a hot plate with water cooled condensors. The flasks were
cooled and placed in an ice bath for 1 hr, then filtered
through Whatman No. 41_filter paper for removal of
unreacted FDNB. The filtrate was made to 100 ml with l

N hydrochloric acid. Interferring substances to colori—
metric determination were removed by extraction with 50

ml ethyl ether. Optical density of the solution was deter-
mined at 435 nm with a Coleman Model 14 Spectrophotometer.

The standard curve was prepared using N-dinitrophenyl—

epsilon—L-lysine monohydrochloride (e—DNP-Lysine) in the

34

range 20 to 200 pg per 10 m1 aliquot. The equation for

the standard curve was:

Optical Density - 0.58 (ug e—DNP-Lysine/lO m1)
+ 0.012

(Results were expressed as mg e-DNP—Lysine per 100 g Fe

protein.) ;

Amino Acids--Acid Hydrolysates

 

Amino acid analyses were performed on hydrochloric

 

acid hydrolysates of the proteins using a Beckman Amino
Acid Analyzer, Model 1200 (Moore, Speckman, and Stein,
1958). Protein samples were placed in 20 m1 ampoules.
Four mg of protein was used weighed within 0.1 mg. Eight
m1 of 6 N hydrochloric acid were added to the ampoules.
The contents of the ampoules were frozen in a dry ice-
ethanol bath. The ampoules were evacuated with a high
vacuum pump, removed from the dry ice-ethanol bath and
allowed to melt slowly to remove any gases in the frozen
samples. The freezing and thawing procedure was repeated
2 times, after which they were sealed, while frozen, with
an air-propane flame. The sealed ampoules were placed in
an oil bath in a forced draft, recirculating oven regu-
lated at 100 i 2 C. The proteins were hydrolyzed for 20
and 70 hr. The ampoules were removed from the oven,

cooled and stored in a deep freeze until used.

35

The hydrolysate was transferred quantitatively to a
25 m1 pear-shaped flask fitted with a ground glass joint.
The hydrolysate was evaporated to dryness on a rotary
evaporator, redissolved in a small amount of deionized
water and redried. The dried hydrolysate was dissolved
in citrate HCl buffer (pH 2.2) and the final volume 11
adjusted to 5 ml. An aliquot was-removed for application

to the amino acid analyzer. Standard amino acid mixtures

were analyzed using the same ninhydrin solution within the

same four-day period.

 

Amino Acids and Peptides--
Enzymatic Hydrolysates

The trichloroacetic acid supernatant from pepsin-
pancreatin hydrolysates of protein preparations were
evaluated for amino acid and peptide content after removal
of trichloroacetic acid.

Amino acids and peptides were measured in the three-
solvent single dimension thin-layer chromatography (TLC)
system described by Pataki (1968). The solvent systems
were those utilized by Wollenweber (1962) and by Bujard
and Mauron (1966). The procedures and solvents for TLC
of amino acids are summarized in Table l. Solvents were
redistilled, water was glass redistilled and the glacial
acetic acid and ammonium hydroxide were fresh reagent
grade. Separation was accomplished on a stationary phase

of Machery and Nagel 300 cellulose powder (MN-300

36

cellulose). Glass plates of dimensions 20 x 20 cm were
Spread with a 0.25 mm thickness of MN—300 cellulose slurry.
The slurry was prepared for five plates by homogenization
of 15 g of MN-300 cellulose powder with 90 ml deionized
water in a Waring Blender. After spreading, the plates
were air dried overnight and activated for 10 min at 100 C Fm-
in a dry air oven. The plates were developed in solvent
tanks with a saturated atmosphere at room temperature
until the solvent front had proceeded 18 cm.

Visualization was accomplished by spraying with 0.2%

 

IF..." I

ninhydrin solution in n-butanol prepared fresh each day.
The color was developed in an oven at 100 i 2 C for 10 min.
Quantization of the plates was accomplished by use of a
recording densitometer with the technique described by
Squibb (1963a, 1963b). The equipment used consisted of a
Photovolt Thin-Layer plate scanner (Model 520) attached
to‘a Photovolt Recording ElectrOphoresis Densitometer
(Model 542) with an Integraph Automatic Integrator (Model
49). Standard curves were prepared from thin-layer

plates developed in the same fashion with incremental
known amounts of amino acids to be measured. Quantitative
relationships were established by relative areas as
measured by integration. Peptides were noted, unique to
each sample, and identified by Rf value for the solvent
involved. Relative quantities were established by measure-

ment of areas according to the lysine standard curve.

37

Measurement of Enzymatic Hydrolysis

Gel Filtration

Proteins were digested during gel filtration to
remove the digestion products from the site of action of
the digesting enzyme. The method proposed by Ford and
Salter (1966) was used with modifications.

Chromatographic columns were prepared of 2.25 cm
inside diameter and approximately 40 cm length. The
columns were jacketed by wrapping with l/4—inch tygon
tubing in circuit with a circulating water bath. The
columns were coated with dimethyldichlorosilane to insure
a uniform flow rate at the glass-gel interface. Gel
beads (Bio-Gel P-2) were hydrated in phosphate buffer
(pH 7.6, 0.02 M) for 24 hr at 37 C. The columns were
poured to a gel height of 21 cm to yield a void volume of
28.0 ml. Flow rate was adjusted by adjustment of head
pressure with a 5 liter aspirator bottle fitted with a
Mariotte constant pressure tube. The elution volumes
for proteins and amino acids were established by adding
known quantities of protein (B-lactoglobulin) and an
amino acid (tyrosine). The effluent was monitored at 280
nm and the fraction volume established to include the
desired component.

Continuous absorption at 280 nm resulted in a lack
of resolution as hydrolysis allowed continuous release of

amino acids. Volumetric fractions were established for a

 

38

protein without hydrolysis and for an amino acid. The
volumetric fractions were based on elution of B—
lactoglobulin in the 12—18 m1 volume following the void
volume and elution of tyrosine at 74—80 ml following the
void volume. Fraction 1 represents the 28 ml quantity
following the void volume to contain intact protein. The
second 28 ml quantity following the void volume was desig-
nated as Fraction 2 and contained peptides and slowly
hydrolyzed amino acids. Volume designated Fraction 3 was-
the last 50 ml volume collected and contained the com-
pounds (amino acids and peptides) included in the gel, of
molecular weight less than 2600. Temperature was main-
tained at 37 C i 0.2 C in the jacketed columns.

Sample addition was performed by addition of protein
solution containing 100 mg protein mixed immediately before
application with 2 mg trypsin. Flow rate was adjusted
with 0.02 M phOSphate buffer, pH 7.6, to enable an elution
time of 4 hr t 15 min. Fractions were collected manually,
monitored for both time and volume. Enzyme blanks were
prepared in a manner analogous to the samples, including

collection and measurement of volumetric fractions.

Pepsin and Pancreatin

 

The degree and form of protein hydrolysis was
measured using the pepsin—Pancreatin Digest of Akeson and
Stahmann (1964). Pepsin followed by pancreatin digests

were prepared by incubating samples containing 100 mg

39

protein with 1.5 mg pepsin in 15 m1 of 0.1 N hydrochloric
acid at 27 C for 3 hr. The samples were adjusted to pH 8
with 0.2 N sodium hydroxide. After addition of 4 mg
pancreatin in 7.5 m1 phosphate buffer (pH 8, 0.1 M), the
samples were incubated for an additional 24 hr at 37 C.
Enzyme blanks were prepared under the same conditions

with the protein sample omitted. Upon completion of the
digestion, 25 ml of a 10% trichloroacetic acid (TCA)
solution were added to the digest, and the final volume
adjusted to 50 ml. Undigested protein was removed by cen-
trifugation, and the supernatant was passed over an anion
exchange resin (AG 2 x 8) in the chloride form to remove
TCA. Samples were immediately dried in a rotary vacuum
evaporator. The residue was dissolved in a small amount
of citrate-HCl buffer (pH 2.2), and the volume was adjusted
to 5 ml for measurement of amino acids and peptides.

The pepsin-pancreatin digestion was measured in
duplicate preparations by measurement of TCA supernatant
protein. One milliliter aliquots of the digest samples
were added to tubes containing 1 ml of 10% trichloroacetic
acid at incremental times. The supernatant protein con-
tent was determined after centrifugation using the method

of McDonald (1965) described previously.

Proton Release byng-Stat

 

The Sargent Recording pH-Stat was employed to

measure proton release as the uptake of standardized

40

sodium hydroxide to maintain constant pH. The procedures
described by Jacobsen g£_a1. (1947) with conditions stipu-
lated by Fasold and Gundlach (1963) were used.

The pH-stat was standardized at pH 8 with the
Sorenson buffer described in Kolthoff and Laitenin (1948).
At temperature 37 C, pH 8.00 was attained with a mixture
of 5.75 ml of 0.05 M borax with 4.25 ml 0.10 N hydrochloric
acid. Samples were prepared with a known protein content
within the range 2-50 mg. The pH of samples was adjusted
to 8.0 prior to storage at 4 C.

Enzyme solutions were prepared containing 0.083 mg
trypsin or 0.240 mg pancreatin per ml. The enzyme prepara—
tions were stored at 4 C after adjustment of the pH to
8.0. Storage times of no longer than 2 hr were necessary
to prevent enzyme autolysis.

Immediately prior to measurement of each preparation,
an aliquot of the sample was placed in a 37 C water bath
to increase the temperature to that for Optimum enzyme,
activity. In a separate beaker 2-3 ml of enzyme solution
were held under the same conditions. The substrate pro-
tein concentration was varied by quantitative dilution of
the preparations of known protein content. The final
volume applied to the pH-stat was 5 m1. Each 5 mlesub-
strate sample was allowed to equilibrate to 37 C and pH 8
in the pH-stat during baseline settings. Electrode control

was activated after equilibration, and 1 m1 of pH adjusted

41

enzyme solution at 37 C was added. During the reaction

in the pH-stat reaction vessel, 0.0502 N sodium hydroxide
was added to maintain the pH at 8.00. The volume of sodium
hydroxide required to maintain the desired pH was auto-
matically recorded relative to time of the reaction.

Initial velocity for each reaction was estimated
as the slope of the recorded curve. The line slope was
assessed as that normal to the plane of a mirror when the
curve was continuous in the mirror (Bergmeyer, 1963).
Substrate concentrations were derived as molar quantities
using the molecular weights for milk proteins compiled
by McKenzie (1967). (Casein = 33,600, ds-casein-s28,000,
k-casein = 28,000, B-lactoglobulin = 18,000).

Reaction calculations were made using the double
reciprocal plot technique essentially as treated by
Lineweaver and Burk (1934) for pure enzyme—substrate
systems. In these applications, the velocity and substrate
terms represent emperical values since Dixon and Webb
(1964) explain that only pure systems can be characterized

in absolute terms.

42

TABLE l.--Summary of the thin-layer chromatographic pro-

cedure for amino acids.

 

 

Amino Acid Solvent Rf
Lysine . . . Butanol/Glacial Acetic Acid/Water . 0.42~
4:1:5 (v/v) (Wollenweber, 1962)
Aspartic Acid 0.46
Serine 0.50
Glutamic Acid 0.35
Glycine 0.48
Alanine 0.38
Cystine 0.14
Valine 0.58
Isoleucine 0.67
Leucine 0.70
Tryptophan 0.62
Histidine . . . Methanol/Water/Pyridine . . 0.27
20:5:1 (v/v) (Wollenweber, 1962)
Arginine 0.04
Threonine 0.53
Proline . . Chloroform/Methanol/17% Ammonia 0.75
2: 2: 1 (v/v) (Bujard and Mauron, 1966)
Methionine 0.81
Tyrosine 0.62
Phenylalanine 0.89

 

43

Fresh Milk

Separated at 25 C

 

|
Residue

Skim Milk
lAdjust pH to 4.5
r—r
Supernatant
TCA
32.4 g/l

 

Suspend at pH 7

CASEIN

‘—

Residue (discard)

Supernatant
concentrate
10:1
0.4 saturated (NH4)2SO4

Allow to stand 12 hr.

 

 

at 25 C
[11
Supernatant
(discard)
Residue

Suspend in
Sat. (NH ) SO
Dialyze 4 2 4

 

8-LACTOGLOBULIN

Dissolve in 1
- liter 6.6 M urea
Acidity w/7NH280u

 

 

 

 

 

 

 

 

 

Dilute to
3 liters
(pH 1.4)
I
Residue
Supernatant
Adjust pH
132 g(NHu)2SOu to 7.2, Add
per liter equal vol.
95% Ethanol
“‘1 Add 1.0 M
Residue NH“ Acetate
[ T
Supernatant Dissolve Supernatant Residue
(discard) at (discard)
pH 7.5 Adjust
Dialyze pH to
Pervaporate 5.0
Supernatant Residue
(discard)
Adjust to
k-CASEIN pH 7.5
Pervaporate
«Salaam

Figure l.--Procedure for the preparation of milk
protein fractions.

RESULTS

Milk Protein Preparations

 

Amino Acids

 

Amino acid analysis of the protein preparations was
conducted after acid hydrolysis to verify the composition
of the proteins studied.‘ The amino acid compositions are
compared to published amino acid contents of similar pro-
teins in Table 2. Comparison of the amino acid contents
to published results indicates that the preparations are
essentially the same. The descrepancies noted can be
associated with differences in preparative technique,
analytical methods and inherent variation in milk proteins.
Characteristics of Protein

Preparations upon Heat
Treatment

 

 

The milk protein preparations each demonstrated a
unique set of characteristics upon heat treatment. Changes
included color development and pH differences after heat
treatment, both alone and with lactose added. Before heat
treatment each of the protein preparations was white. The
pH was adjusted to 7 when necessary.

Casein demonstrated no observable color change asso-

ciated with heat treatment alone, but when heated in the

44

45

presence of lactose, a uniform light tan color was
observed. The pH of casein after heat treatment was 6.2
to 6.3 for both heated samples and those heated in the
presence of lactose.

The color of dS-casein was not perceptibly changed
after heating; however heat treatment with added lactose a.
resulted in a light tan color similar in intensity to
casein. The pH of dS—casein was 6.3 to 6.4 in samples
heated alone and heated in the presence of lactose.

The color observed in heated k-casein preparations

 

was a very faint tan. There was no discernable color
difference between heated k-casein and that heated in the
presence of lactose. The pH of k-casein after heat treat-
ment was 5.7 to 5.8 in preparations heated alone and heated
with added lactose.

The color observed in heated B-lactoglobulin prepara—
tions was more pronounced than in any of the heated casein
samples. The color was gray-brown, compared to the tan in
heated casein preparations. The pH of heated B-
lactoglobulin preparations was 6.5 to 6.6 which represented
less change from the original pH 7 than any other proteins
observed.

B—lactoglobulin showed distinct coagulation after
heating in the presence of added sodium chloride, added

calcium and pH adjustment.

46

Pepsin-Pancreatin Digest of Proteins
and Heat-Treated Proteins

 

 

Amino Acids and Peptides

The amino acids liberated by pepsin-pancreatin pro-
teolysis of milk protein preparations are reported in
Tables 3 to 6. Table 3 contains amino acid results for
casein, 4 for aS-casein, 5 for k-casein and 6 for B-
lactoglobulin. The results for peptides estimated for the
same milk protein preparations are presented in Table 7,
designated by Rf values and expressed as lysine.

Amino acids and peptides in the unheated protein
digests were assessed by analysis on an amino acid analyzer.
The amino acid analyzer could not be used for the enzymatic
hydrolysates of heated proteins and protein—lactose pre-
parations. The ion-exchange resins evidently were unable
to accept the digest residues other than amino acids
(peptides and complexes) contained in hydrolysates of
heated protein preparations. One digest of a heated
sample could be satisfactorily eluted; however application
of a second digest resulted in excessive volumn pressure
buildup (200-300 lb). The residual material responsible
for excessive pressure could notvbe removed by normal re-
generation procedures. The contamination necessitated
removal of resin from the column, washing and repacking.
An ion-exchange resin designed for peptide analysis may

have made use of the analyzer possible for digests of

47

heated preparations. An alternate method for amino acid
assessment utilized quantitative thin-layer chromatography.
The amino acid results for individual protein digests
reported in Tables 3 to 6 represent analyses conducted
with quantitative thin-layer chromatography. The amino
acid results for control (unheated) protein preparations
represent analyses conducted by thin-layer chromatography
and essentially substantiated by the automatic amino acid
analyzer (within i 10 per cent). 3

The free amino acids from enzymatic digests of

 

casein preparations are reported in Table 3 as quantities
and as per cent of the total amino acid composition. The
amino acid release by proteolysis was less from heated
casein than from unheated casein and further reduced from
casein heated in the presence of lactose for 10 of the 19
amino acids. Lysine, arginine and isoleucine were, on the
contrary, released in greater amounts in enzymatic hydro-
1ysates of casein heated in the presence of lactose than
of unheated preparations.

An essential amino acid and peptide profile for the
digest of casein preparations is Figure 2. The relative
differences in the amounts of enzymatically hydrolyzed
amino acids are pronounced, particularly decreases in
threonine, valine, methionine and leucine released from
casein heated in the presence of lactose. The six peptides

measured were all present in greater amounts in

48

hydrolysates of heated than of unheated casein. The
peptide designated 31 was present in the greatest rela-
tive amount for the digest of heated casein; while pep-
tide 86 was predominant in the hydrolysate of casein
heated in the presence of lactose.

The free amino acid contents of ds-casein hydro-
lysates are reported in Table 4 as total amounts and as
per cent of casein amino acid composition. The amino
acids in dS-casein hydrolysates of heated preparations
were less than the unheated preparation for eight of the
amino acids, and no significant change was noted for five
amino acids.

Figure 3 represents the essential amino acids and
peptides as profiles for enzymatic digests of dS-casein
preparations. The essential amino acid pattern formed
for aS—casein digests of heated and control preparations
further indicates relatively smaller changes associated
with heating in dS-casein than in casein. The hydrolysate
of dS-casein heated in the presence of lactose contained
smaller amounts than the control hydrolysate of histidine,
threonine and leucine; however larger amounts were noted
for valine and phenylalanine. The peptide contents of
ds—casein preparation digests were, as in the casein
preparations, larger for heated than for unheated protein

preparations.

49

The pepsin-pancreatin digest free amino acids from
k-casein are reported in Table 5 as total amounts and as
per cent of k-casein amino acid composition. The relative
stability of k-casein to heat is indicated by the lack of
significant difference in nine of the amino acids
between heated and unheated preparation digests.

k—Casein essential amino acids and peptides from
enzymatic digests for unheated and heated protein prepara-
tions are described in Figure 4. The relative amounts of
histidine and threonine in digests of k-casein heated in
the presence of lactose are notable as they represent
slightly more than half the amounts in corresponding
samples from control preparations. The peptide designated.
31 was present in much greater quantity in the digest of
heated k-casein than in digests of the unehated protein or
k—casein heated in the presence of lactose. Peptide 68
was present in increased quantity in the digest of k-
casein heated in the presence of lactose relative to the
unheated preparations.

The free amino acids and peptides from digests of
B—lactoglobulin preparations are in Table 6, presented
as total quantities and as percentages of amino acid
composition. The amino acids in enzymatic digests from
heated B-lactoglobulin preparations were present in
greater amounts in eight cases and lesser amounts in six

amino acids than in the control (unheated) preparations.

50

The essential amino acids and peptides from digests
of B-lactoglobulin (Figure 5) illustrate inconsistent
amino acid release upon proteolysis associated with heat
treatment. The amino acids valine and histidine were
released in considerably smaller amounts in the heated
preparation digests than in unheated counterparts. The
peptides from pepsin-pancreatin digests of B-lactoglobulin
were unique among the proteins examined as peptides were
released in greater quantities from heated protein than
from protein heated in the presence of lactose, except
peptide 86.

Trichloroacetic Acid-Soluble
‘Proteolysis Products

 

 

The enzymatic cleavage of milk protein preparations
during pepsin-pancreatin proteolysis was monitored by
assessment of the trichloroacetic acid—soluble hydrolysis
products. The soluble proteins released by stepwise
hydrolysis during first pepsin, then pancreatin pro—
teolysis are reported for milk protein preparations in
Table 8. The graphic descriptions of enzymatic pro-
teolysis products from individual milk proteins for
unheated and heated preparations are in Figures 6 through
9. The order of appearance of milk protein preparations
in both tabular and graphic presentation is: casein,

dS—casein, k—casein and B—lactoglobulin. All measurements

51

were made on the same sample for the individual protein
preparations. The transition from pepsin to pancreatin
represents adjustment of the pH from 2 to 8 in the protein
solution and the addition of pancreatin.

The changes in appearance of the protein preparations
during hydrolysis were conspicuous and consistent. Ini-
tially all preparations were turbid and individually
colored as previously described. Within 1 hr after initia-
tion of proteolysis turbidity had disappeared from all
samples. The heated samples which were tan-colored upon
initiation of proteolysis became a clear brown. The
unheated ds-casein preparation had a distinct blue cast
which was not observed in the heated dS-casein sample or
in other unheated protein preparations.

Enzymatic Proteolysis During
Gel Filtration

 

Total Protein Distribution

 

Milk protein preparations; unheated, heated and
heated in the presence of lactose were fractionated
during tryptic proteolysis on an acrylamide desalting gel
(Bio—Gel P-2). The protein contents of the fractions
derived are reported in Table 9; including Fraction 1--
intact protein, Fraction 2-—large peptides and slowly
hydrolyzed amino acids and Fraction 3--small (MW less than

2600) peptides and amino acids.

52

The protein quantities contained in the intact
protein portion of the enzymatic hydrolysates were 50-
100% greater from heated casein and 22-41% greater from
heated ds-casein than from the corresponding unheated
protein preparations. The protein contents of the intact
protein fractions were less than 10% different from heated
and unheated k-casein preparations. The intact protein
after tryptic proteolysis of B-lactoglobulin was less than
half in heated preparations of that in the unheated
protein. The amino acid and peptide-containing fractions
(Fractions 2 and 3) were complementary to the intact
protein, as the sums of the protein contents were similar
for all protein preparations examined.

Trichloroacetic Acid-Soluble
Nitrogenous Material

 

 

The extent of enzymatic proteolysis for each of
the model proteins during gel filtration hydrolysis was
estimated by measurement of the Lowry-Folin reacting
material in trichloroacetic acid supernatants of intact
protein, peptide and amino acid fractions. These data
are reported in Table 10. The trichloroacetic acid-
soluble protein contents of Fraction 1 (intact protein)
are considerably greater in heated preparations of casein
(15 go 20 times) and of aS-casein (3 to 4 times) than the
corresponding unheated (control) proteins. The tri-

chloroacetic acid supernatant protein of the k-casein

53

intact protein fraction was influenced much less by heat
treatment, with a 25% increase noted only after heat
treatment in the preSence of lactose. The trichloro-
acetic acid-soluble protein from the intact protein
fraction after gel-filtration proteolysis of B-
lactoglobulin was half the amount from heated protein as
it was from the unheated control. There was, therefore,
a correlation between trichloroacetic acid—soluble protein
and total protein which was also prevalent in Fraction 2
and Fraction 3.
Reaction with l-Fluoro-2,
4-dinitrobenzene

The quantities of materials reactive with l-fluoro-
2, 4-dinitrobenzene (FDNB) are reported in Table 11 for
protein preparations before gel-filtration proteolysis and
for the fractions obtained after gel-filtration hydrolysis.
The unit for expression of FDNB reactive materials in
proteins and hydrolysis fractions was adapted to terminal
amino rather than the usual e—amino connotation, even

though eNH -DNP-lysine was used as the basic standard.

2
The expression terminal amino groups more precisely
describes the reaction measured than seamino lysine.
There was up to 36 times as much_FDNB reacting material in
gel-filtration hydrolyzed proteins as in the same protein

preparations before hydrolysis.

54

The terminal amino groups measured in milk protein
preparations before hydrolysis were essentially the same,
irrespective of heat treatment, with the exception of
heat—treated B-lactoglobulin which was 17% greater than
the unheated protein. The terminal amino groups of the
intact protein fraction (Fraction 1) derived after gel-
filtration hydrolysis of proteins varied in the same
way as the total protein contents of corresponding protein
preparations. The terminal amino groups for peptides and
amino acids (Fractions 2 and 3) from gel filtration pro-
teolysis reflected the amino groups reactive with FDNB,
and were erratic but in all cases amounted to more than
the lysine content. The amounts of FDNB reacting material
in excess of the lysine content indicated the reaction

with d— and e—amino groups.

Rate of Proteolysis byng-Stat

 

Evaluation of protein preparations during proteoly-
sis by monitoring proton release provides a means for
examination of the substrate differences by the kinetics
of hydrolysis. A more extensive examination of protein
treatments influencing proteolysis was possible with use
of the pH-stat than by the more involved techniques pre-
viously reported.

The expression terms for results of proteolysis-
rate measurements are substrate concentration and specific

activity. The more familiar counterparts for homogeneous

55

systems are Km for substrate concentration and V for

Specific activity.

Equal Cleavage Sites

The influence of trypsin cleavage site concentra-
tion was evaluated at equal lysine and arginine concen-
tration for each model protein by enzymatic hydrolysis
rate measurement. Expressions of results for rate measure—
ments in Table 12 are in terms of number of trypsin
cleavage sites rather than the conventional molar con-
centration.

The differences between rate measurements for model
protein preparations reflect individual protein behavior
other than that attributable to the number of cleavage
sites. The specific activity, or reaction velocity, for
B-lactoglobulin control (unheated) was less than one-third
that for dS—casein control and half that for casein and
k-casein controls. The unheated proteins also demonstrated
individual cleavage rate characteristics independent of the
number of cleavage sites, with a range of specific activity
from 1.0 to 26.6. Protein preparations heated in the
presence of lactose reflected behavior similar to heated
preparations with a range in specific activity from 7.0
to 30.9. The lack of similarity of rate measurements for
individual protein preparations indicated that numbers of
cleavage sites did not govern differences in rates of

proteolysis.

56

'Chemical Denaturing Agents

The influences of chemical denaturing agents; urea,
2—mercaptoethanol and performic acid on rates of prote-
olysis are reported in Table 13 to 16. Reaction rate
measurements of casein are in Table 13, aS-casein in
Table 14, k-casein in Table 15 and B—lactoglobulin in
Table 16. The rates of proteolysis after heat treatment
at pH 5 and 9 are also reported for each protein.
Enzymatic proteolysis rate for each protein fraction sub-
jected to a chemical denaturing agent was assessed unheated,
after heat treatment and after heat treatment in the
presence of lactose. Urea treatment was evaluated for
only casein and B-lactoglobulin, as dS—casein and k-casein
were treated with urea as a part of their fractionation.

Casein demonstrated no difference in specific
activity between urea-treated and untreated preparations.
The specific activity for urea-treated casein was twice
as great, however, after heat treatment and was five times
as great after heat treatment in the presence of lactose.

B-lactoglobulin treated with urea demonstrated an
activity twice that observed in the untreated preparation.
The urea-treated B-lactoglobulin also reflected more rapid
hydrolysis after heat treatment, double the specific
activity for the corresponding untreated protein. A

slight reduction in hydrolysis rate was noted for

57

urea-treated B-lactoglobulin after heat treatment in the
presence of lactose.

Casein treated with 2—mercaptoethanol demonstrated
a reduced velocity of enzymatic proteolysis, one-tenth
the specific activity of the untreated casein preparation.
Casein treated with 2-mercaptoethanol and then heated
demonstrated a five-fold increase in activity and when
heated in the presence of lactose resulted in an increase
in specific activity of 25%.

B-Lactoglobulin treated with 2-mercaptoethanol was
enzymatically hydrolyzed less rapidly than the untreated
protein with a Specific activity one-third as great for
treated as untreated proteins. The specific activity for
2—mercaptoethanol-treated, then heated B—lactoglobulin
was two-thirds that of the untreated, but an increase of
10% was observed in the specific activity for the 2-
mercaptoethanol—treated protein heated in the presence of
lactose from the corresponding untreated preparation.

Performic acid treatment of casein resulted in
increased rates of proteolysis, measured as Specific
activity for all protein preparations; unheated, heated
and heated in the presence of lactose.

ds-Casein treated with performic acid was hydrolyzed
25% more rapidly than the untreated protein, indicated by
Specific activity. The proteolysis velocity of performic

acid—treated dS-casein after heat treatment was reduced to

58

one—third the control protein, but heat treatment in the
presence of lactose resulted in a slight increase in the
rate of enzymatic hydrolysis.

k-Casein proteolysis velocity was reduced in all
cases by performic acid treatment for unheated and heat—
treated preparations.

Performic acid-treated B-lactoglobulin was hydrolyzed
more rapidly by approximately 30% in all cases, indicated
by specific activity assessment compared to control pro-
tein preparations.

Heat treatment at pH 5 resulted in reduction of
enzymatic proteolysis to immeasurable quantities for
casein and as-casein. k-Casein and B-lactoglobulin were
hydrolyzed more rapidly after heat treatment at pH 5 as
evidenced by a 50% increase in Specific activity for k-
casein and a slight increase for B-lactoglobulin, com-
pared to corresponding untreated proteins.

Heat treatment at pH 9 resulted in decreases in rate
of proteolysis for each of the proteins compared to their
untreated controls.

The treatment of protein preparations with 0.3 M
calcium is reported as a treatment for individual proteins
in Tables 13 through 16. The presence of calcium ions
in casein and B-lactoglobulin preparations resulted in
greater enzymatic proteolysis velocity in all cases. The

rate of proteolysis indicated by specific activity for

59

calcium—treated as-casein preparations was less than half
the velocity for preparations not containing calcium.
k-Casein preparations containing calcium were hydrolyzed
less rapidly in each preparation, but the reduction was
not pronounced.

Protein treatments with 0.15 M sodium chloride
(isotonic saline) as they influenced rates of proteolysis
are reported as one of the treatments for each protein in
Tables 13 through 16. Sodium chloride treatment apparently
stabilized the protein preparations to heat treatment,
but the specific activity for isotonic saline-treated
protein preparations was less than the specific activity

for the untreated control proteins.

Sugars

The sugars; glucose, lactose, galactose and glucose
plus galactose are reported as treatments to evaluate
their influence on enzymatic proteolysis rate of casein
in Table 13 and of B—lactoglobulin in Table 16. The
enzymatic hydrolysis rate for casein was greater for each
sugar—treated casein preparation than it was for untreated
casein. The specific activity for sugar—casein prepara-
tions was less after heat treatment than before, but only
after heat treatment in the presence of lactose was the
rate less than heated untreated casein.

B-lactoglobulin solutions containing added sugars

reflected rates of proteolysis 5-20% higher than untreated

60

protein. B-lactoglobulin heat treatment in the presence
of sugars, however, resulted in specific activity measure-
ments less than or equal to velocities observed with no

added sugar.

Control Measurements

Control measurements (blanks) were made for each of
the variables, and in several cases, the observations are
pertinent to interpretation of results.

Each of the protein preparations; casein, dS-casein,
k-casein and B-lactoglobulin was examined for proteolytic
enzymes inherent to the proteins by monitoring proton
release without added enzyme. Proteolysis was not measure-
able by pH—stat in any protein preparation examined.

Lactose concentration was evaluated within the range
of solubility for possible changes in enzymatic proteoly-
sis rate. Within the range of lactose concentration
(0.06 M to 0.24 M) there was no observed difference in
enzymatic proteolysis rate monitored by proton release.

Enzyme autolysis was considered in each experiment,
and corrections were made when indicated. Amino acid
analyses were corrected to compensate for the enzyme con-
tribution of each amino acid established by separate
enzyme blank analysis. The activity of prepared enzyme
solutions was evaluated during storage and autolysis by
the enzymes made it necessary to prepare fresh solutions

after no more than 2 hr storage at 4 C.

61

TABLE 2.--A comparison of the amino acid compositions of the milk proteins
utilized in this study with those reported by others.a

 

 

 

 

 

 

B-Lacto-

Amino Acid Casein dS—Casein k-Casein globulin

“.233. £232. E233. m 32:2. m

(mb

Lys 8.9 7.1 8 3 8.2 5 8 5 6 28.1 28.5
His 3.4 2 6 1.8 2.9 2 1 2.0 1 1 1.4
Arg 4.1 3.7 4 o 3.9 3 6 3.5 2 1 2.6
Asp 7.0 5. 6.3 6.8 6 2 6.6 9 8 9.7
Thr 3.9 3.8 2 5 2.5 5 7 5 7 4 5 4.4
Ser 4.6 5.2 4.8 5.3 4 2 4.1 3 1 3 1
Glu 23.0 20.2 22.8 21.4 19 4 17.3 17 0 17 1
Pro 10.6 10.3 8.4 6.9 8.9 9.1 4.4 4.5
Gly 1.6 1.6 2.1 2.1 0.8 0.9 1 2 0.9
Ala 2.8 2 4 2 8 2.6 4 5 4.3 5.4 5.3
1/2 Cys 0.0 0.3 0 1 0 0 0 8 0.9 8 1.8
Val 6.3 6.3 5 2 4.7 5 2 5.3 5.0 5
Met 2.0 2.9 1 8 2.2 1 1 1.1 1.9 2.2
Ile 5.4 5.7 5.5 5.5 5.9 6 0 5.6 5 9
Leu 9.0 8.7 7.0 6.9 5.1 5 2 11.7 12.8
Tyr 5.6 5.7 7.0 7.0 6.2 6.8 5
Phe 5.2 5.2 5.2 3 9 3.3 3.4 3 4 3 2
Trp 2.5 7 3 5 2.0
Phosphorus I 1.01 0.22
Carbohydrate 1.4

TOTAL N 15.4 15.3 15.3 8.8

 

aAnalytical data compared with data reported in the following sources:
(1) Block and Weiss (1956); (2) Gordon et a1. (1965); (3) Swaisgood et a1.
(1964); (4) Kalan et a1. (1965).

bAmino acids expressed as anhydrous residues.

 

62

 

 

 

om :.m om :.m om H.Hm m.az use
om H.om om m.ma m: m.ms m.sm qu
mm s.om w: m.sm mm m.ma H.mm mHH
m: :.m Son s.om moa m.om m.mH 662
:m m.ma m: m.mm H: H.2m S.mw Hm>
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m m.H ma m.m SH 3.: H.Hm mam
mm m.om mm m.mm we o.mm :.mw was
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ofimwmmouMMMmWon 1V ongv Am\moaoe\1v OARV Am\moaoe\1v Awmwmwmsmmv
monomonm CH oopmom homomom Hompsoo mQOHpHmoosoo msoflmom
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.CHommo

ompmmnplpmon one cfiommo mo pmowfip capmonocmalcfimdmd CH memos ocasm commit.m mqm¢e

63

f

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n

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mm
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Hmfipcommmlcoz

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64

 

 

 

 

as s.om mm m.sm mm m.mm m.m: has
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65

 

ms
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m.ss a: m.ss z: m.ss m.msa Sam
m.mm mm m.mm mm H.3m 2.3m pom
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H.SH mm m.mm mm 8.3m m.mm mes

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S.H Tu A.H Tu A.H I. ate
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66

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mm o.wm mm 0.0m am m.om m.so has
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monomonm CH ooumom nompmom Honpsoo mCOHpHmOQEoo ozpfimom
psmEpmonB mpfio< ocas¢
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powwoSplpmon one cflmmmotx mo pmowap cfipmonocmatcfimdod CH mofiom ocflsm commit.m mqm<e

 

67

.COHpamOQEoo HmpOp no ommpcoonod mm commondxmo

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Q

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mm
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pCoEpmoCB mpfio< OCHEC

 

.CHHCCOHwOpomHIm condoms

lemon pCm CHHCnoawopomHTm mo pmowfio CapmoCoCmalCHond CH moflom OCHEm ooCmTI.m mumCB

 

69

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m.om
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mm
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70

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t)
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p
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9.0 u.ms o.o m.ma m.sm 0.0 C.MH 0.3C 0.0 m.ma m.sm 0.0 OHM am
. . . . . . . . . . . . . . . . . 6Aw\msn SHOE av . . . . . . . . . . . . . . . . .
ow0pomq do swepomq do om0pom; do mmoSomq do
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msfisgma

CfiasnonOSomqlm Cfiommotx CfimmmUde Cfiommo

 

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71

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m.mm m.ms m.m0 0.0a m.Hm m.00 0.sm o.mm s.mm m.wn H.m0 m.0m CC :m
0.00 0.05 m.am 0.00 0.00 s.:0 :.Ns H.ms 0.00 m.ms m.:0 0.00 CC 0H
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n.0m 0.0m a. m w.mm m.:m 0.0m m.m: 0.0m 0.Hm 0.0m 0.00 0.m: UCC H
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72

 

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mom 0m: 0: mmH HSH 0m HMH NMH OHH Hm m0 am mm coHCosCC
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mocmwmkm mocmmwhm mocmmwhm mocmwmhm
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a

 

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uCoEopsmmCZIl.m mqm<e

73

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.CHE om Com 0 omH um Umumozm

 

 

s.©m H.mH o.m N.mH N.NH o.m o.mH m.© 0.:H ©.m 0.0 v.0 mm COHHQMLm
3.0m. 0.0H O.NH 0.5H m.HH 0.0m w.ma m.m 3.0m m.mm m.om 0.0: Um COHCUMCQ
m.mm H.mm 0.0: 0.»: m.©m m.mm m.mm H.©© m.©H w.©m 5.0: m.m -oH COHpomCm
m.m m.OH m.H . w.NH H.mH 0.m m.HH >.m 0.: 3.0 H.w 0.0 HCHCHCH
. . . . . . .. . . . . . . . . . .CAwE 00H\How Cue wEV . . . . . . . . . . . . . . . .
mmoComC Co mwoComC Co mmouomq mo omoComC Co
moCmmmCm mocmmoCa ooCommCm ooComoCm

mCH copmo: assume: HOCCCoo mCH Coumom mumnmo: HOCCCoo mCH Cmpmoz sponsor HOCCCoo mCH nonmo: spouse: HOCCCOQ

III ||l.l CoEHooqm

 

 

CHHzConoComCT0 CHommotx CHmmCOTms CHowmo

 

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74

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How 0mm HHH mas 0H0 0mm 0: Ham om om 00H.H 000.m 0m :oHsosCC
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00m 0zs 00H mmH 00m 1H0 0m 0HH m0: 0mm mxm 00a CH :oHsomCC
03H 00H mmH 03H qu 03H mmH HmH mmH HHH m:H mzH HmHCHcH
o a o o o o o o o o o o o o - AWOOH\\W\HWH rmmnfiq N.HHH|W wficv o o o o o o o o o o o o o o o
omoComH omoComH mmopumq «monomq
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mocwmwch vncmmmmc. mocvnmhg wocwmmpnw
CH CH CH CH
mcoumoz mCmCmo: HOCCCOU ammusom spouse: HOCCCOQ msmnmm: Spouse: HQCCCOQ wwonmo: spouse: HOCCCoo
CHHzConoComCT0 CHomnolx CHommotmo CHommo coEHomdm

 

.CoHCmCCHHC How wCHC30

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memHomCOCQ oHCQmCC

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.CHE om Com 0 omH Cm Umummzo

.CHE\mmCHm 0Hx “HHH>HCom CHCHooqm

 

75

 

 

 

 

ml C
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Co ooComoCd
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CHSHCo. COHCCCCCooCoo mCH>HCo< COHCmCCCooCOQ HCH>HC0< CoHCmCCCooCoo HCH>Hpo¢ COHCmeCoocoo
HCHomdm moCmCCmCsm CoHCHoodm mmaCCijm COHCHoodm moCmCCmCdm ConHooqm moCmCCmCCm
CCoECmoCs
CHHsConOComCT0 CHmmCOIx CHmmmoIms CHommo

 

 

 

.ommoHoC COCOCQ mC CoCCmmoE mCoHmeCCooCoo mpHm omm>mmHo Hmzvo Cm mCoHComCm CHoCOCQ Co mHmmHooCOCQ mo mopmmll.mH mHm<B

76

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m.0 T ssHoH600
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H.m 0.0 m0.0 00.0 666606H60
0.H 0.0 m0.0 mm.H 6660666H
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m.H 0m.0 0 m 060660
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smouomq mo omouomq no
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66H 660660 6666660 HOCmcoo 66H 660660 6066660 H066600 066606669

 

 

HCH>Hpo< OHCHomam COHmepCmoCoo mmemesm

 

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77

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0.0 0.0 0.0 00.0 00.0 00.0 00000 0000060
0.0 0.0 0.0 00.0 00.0 00.0 0000 000000060
0.0 0.0 0.0 00.0 00.0 00.0 00060060006006:
0.0 00.0 0 00 060660
000 0 00 060660
0.0 0.0 0.0 00.0 00.0 00.0 0000000
. . 0000\00 m:000 . . . 00 0.03 .
6000000 00 6000000 00
60060600 60060600
000 060060 0060060 0000000 000 060060 0060060 0000000 006800609

 

.006000 000060 000 000600 0000000060 00008600 0003 006000600 06000
006000100 0600600|0060 000 006000|00 00 00000060000 0000000 00 0600mln.00 00009

78

omwfihoano Ezfiuom z ma.o I mafiamm oaQOpomHo

.2 moo I Edfloamon

.cHe om MOM o oma pm cmpmmmm

 

 

 

 

w.: :m.m m ma cmpmmm
m.o movm m ma Umpmmm
N.© m.m m.: o~.m Hmea omoa “pmmwfim cawamm
ocm m.m m.m mmea mmco mmoo omcfipoano szfioom
oom ©.m m.m mm.o mmuo mmoo meOH ssfioamo
H.H mom m.o mm.o mmuo mm.o ©H0< OflEQOQme
o.oH oom m.H omuo aqoo mmflo Hocmnmepamopmz
m.m move ‘ a ma ompmmm
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o ACHE\ZE M'OHV o o o .- c .1 o A: MIDI—”V o u o 9
mmOpomq mo mmOQowq mo
mocmmmpm mocmmmpm
mcfi vmpmmm mwmpmmm Hompcoo mcfi Umpmmm mumpmmm Hoppcoo
pcmspmmpe
sz>Hpo¢ oamaomam coapmmpcmonoo mpmhpmnsm

 

.pmmeU cfimmma 6cm mpsmww wCHmSpmcmv HwOHEmgo £pfi3 pamEQanp
mmpmm QfimmMolx Umuwmmplummn cum camwmolx mo mammaompoma OHQanu mo mmpmmll.ma mqm<e

79

.wcfipmmn com: coaumHsmmoo Umpmppmcoamam

0: ma.o I mcHHMm cacouomHm
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m.m om.m m mm cmpmmm

m.m om.o m ma umpmmm
w.m mum m.m 3mg: mm.m mm.o "pmmmfiu camamm
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mo.oa mm.m m.m mmm.m moo.m omta nchH asfioamo
m.m m.m m.m Hm.o Nmuo omIo UHo¢ anpompmm
m.m m.H ”.0 :H.o :HIO mmuo Hocmcmepamopmz
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oIH H.m pa.m om.o ammOQUmHmo
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80

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86

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DISCUSSION

Milk Protein Preparations

Amino Acids

The amino acid contents contained in Table 2 for
milk protein preparations indicate that basically the
amino acid contents verify those published for protein
fractions. Differences between amino acid contents.from
this study and those previously published represent varia-
tions in preparative techniques, analytical methods and
inherent variation in milk. Particular amino acid dif—
ferences are obvious for casein. The results reported by
Block and Weiss (1956) represent analyses performed
microbiologically, and the results for this study were
analyzed chemically.
Characteristics of Protein

Preparations Upon Heat
Treatment

 

The formation of color during heat treatment of pro-
teins can be associated with chemical characteristics of
the individual proteins. The lack of color change with
heat treatment of dS-casein may be a ramification of the
disordered configuration, lack of internal disulfide link-

ages for interchange and minimal aggregation tendencies

88

89

at the treatment temperature and pH. Swaisgood and
Timasheff (1968) reported that at pH values greater than
7, low ionic strength (to 0.02) and protein concentration
less than 10 g per liter, aS-casein aggregation was
favored at room temperature.

The color formation of k—casein, in contrast, was
not changed significantly by the presence of lactose
during heat treatment. Color formation with heat treatment
of k—casein suggests reaction of the carbohydrate moiety
of the molecule as well as complex formation resulting
from the interactions of the internal disulfide linkages.
The color formation observed after B-lactoglobulin was
heated both with and without lactose was more pronounced
than casein preparations and of a particular gray—tan
color. The heated B-lactoglobulin color change may be
partially attributable to complex formation of the type
described by Trautman and Swanson (1959) or by the disul-
fide interactions suggested by Morr and Josephson (1968).
The B-lactoglobulin susceptibility to coagulation, par-
ticularly during heat treatment in the presence of ionic
calcium, has been reported by Rose (1962) as well as by
Morr and Josephson (1968). The heat induced changes in
B-lactoglobulin indicated by color change can be associated
with unique characteristics upon enzymatic hydrolysis, par-
ticularly the rate of proteolysis observations reported

in Table 16.

 

9O

Pepsin-Pancreatin Digest of Proteins
and Heat-Treated Proteins

 

 

Amino Acids and Peptides

 

The free amino acid contents of individual protein
digests were indicative of enzymatic release character-
istic for each protein, amino acid and treatment. The Pm
complexity of possible combinations and their signifi-
cance makes examination of the amino acid data by several
methods desirable.

The amino acids lysine and arginine are significant

 

‘ Mann
1

in that they represent the cleavage sites for trypsin
(Hill, 1965). The lysine and arginine liberated by
enzymatic proteolysis are expressed as percentages of the
original quantities for each milk protein in Tables 3
through 6, and no consistent reduction in release rates
can be associated with heat treatment. The lysine-
arginine amounts released from casein were greater after
heat treatment in the presence of lactose than unheated
casein, but no change was associated with heat treatment
without lactose. ds-Casein demonstrated a decrease in
freed lysine—arginine quantities when heated, consistent
with the observations by Bujard gt_§l, (1967) for milk
proteins.

The free lysine-arginine contents from k-casein and
B-lactoglobulin hydrolysates_increased markedly after

heat treatment. Heat treatment of k-casein and

91

B-lactoglobulin in the presence of lactose resulted in
free lysine-arginine contents similar to unheated prepara-
tions. This suggests that heat denaturation may have made
the cleavage sites more exposed and susceptible to com—
plexes of this type suggested by Donoso g£_al.(l962) in
the presence of lactose. The complexes they described
formed a stable condensation bond between the reducing
sugar components and amino groups. The carbon-nitrogen
linkage formed was not hydrolyzable by digestive enzymes.

The enzymatic release of amino acids other than

 

lysine and arginine was likewise individually different
with preparation and treatment variation to make a single
basis for expression desirable. Interpretive calculations
have been proposed by several authors (Oser, 1959;
Sheffner gt_al., 1956; Block and Mitchell, 1950) which
provide correlation with animal studies. The manipulation
used by Sheffner gt_al. (1956) as adapted by Akeson and
Stahmann (196“) was used. The pepsin—pancreatin indices
are logarithmic-geometric averages of free essential

amino acid contents described as a ratio to egg protein
under similar conditions. The pepsin-pancreatin indices
for milk protein preparations are contained in Table 17.
The pepsin-pancreatin essential amino acid index for
casein (78) was in agreement with that reported by

Akeson and Stahmann (1964) for casein. These results

were greater than the value of 69 originally reported by

92

Sheffner gg_§l. (1956), but their results were for a
commercially dried preparation. A study conducted using
humans as subjects by Hawley g£_al,(l948) led to an egg
protein index of 73 for casein. The pepsin—pancreatin
amino acid index therefore provides a correlary with
animal studies.

Animal studies were not possible with the protein

preparations in this study. The primary deterrent was

 

the necessity for concentration of the protein prepara-

tions to provide a diet containing 8 to 10% protein.

 

Protein concentration provides conditions favorable to,
for example, aggregation for dS-casein (Swaisgood and
Timasheff, 1968); loss of free amino nitrogen in stored
high—moisture milk powder (Lea, 1948); and B-lactoglobulin
complex formation in evaporated milk (Trautman and Swanson,
1959). Another reason that animal studies were not con-
ducted was that the heat treatment, 120 C for 30 min,

was mild in comparison to many common processing condi—
tions. It is therefore questionable that the differences
would be significant, if measurable. Fricker (1964) was
unable to demonstrate differences related to high tem-
perature short time heat-treated milk in extensive feeding
trials. The quantity of milk that would be required to
prepare sufficient quantities of proteins for animal feed—
ing was also a limitation. Zittle and Custer (1963)
reported a yield of 12% for k-casein; however the yields

in this study were approximately 3%.

93

The pepsin-pancreatin essential amino acid indices
reported in Table 17 represent composites of free amino
acid contents for each protein. The component amino
acids can best be discussed relative to the essential
amino acid index for individual model systems.

Casein.--The essential amino acid pepsin-pancreatin
index for casein was lower after heat treatment (68)
than unheated (78) and was considerably less (46) after
heat treatment in the presence of lactose. Three essen-
tial amino acids were associated with the changes observed;
histidine, threonine and leucine. Even though isoleucine
and lysine were liberated in larger amounts from heat-
treated casein, their contents are not reflected in the
index since their quantities were greater than those in
the reference protein.

The peptides from the pepsin-pancreatin digest of
casein reported in Table 7 have a relationship to the
pepsin-pancreatin index. The peptide quantities vary
inversely with the pepsin—pancreatin index. Peptides
designated 31 and 86 quantities are greater from the
digest of casein heated in the presence of lactose cor-
responding to decreases in the amino acids; histidine,
threonine and leucine. The association between peptides
31 and 86 and the essential amino acids indicates that
these peptides may contain histidine, threonine or

leucine.

94

Non-essential amino acids are not considered in the
pepsin-pancreatin index; however their release by enzyme
hydrolysis is pertinent. The amino acids aspartic acid,
glutamic acid, glycine and alanine were released in
smaller amounts after heat treatment with added lactose.
The enzymatic liberation of non-essential amino acids
corresponds to the pepsin-pancreatin index and reflects
less complete proteolysis after heat treatment, especially
with added lactose, than for unheated casein.

Casein, as a mixed protein, was unique among the
proteins studied, and the inconsistent liberation of amino
acids by enzymatic proteolysis can be noted in the amino
acid profiles in Figure 2. In contrast, the amino acid

profiles for more homogeneous preparations; a -casein,

s
Figure 3; k—casein, Figure u; and B-lactoglobulin, Figure
5; are more consistent in free amino acid contents of
digests associated with heat treatments.

qs-Casein.—-The pepsin-pancreatin indes for dS-casein
was analogous to casein in magnitude and reduction associ-
ated with heat treatment, from 80 for unheated to 67 for
heated protein. The similarity for heated dS-casein to
casein is carried through to amino acid differences.
Histidine and threonine were less in the heated than un-
heated aS-casein digest in amounts similar to comparable

measurements for casein digests. Likewise, the peptides

31 and 86 were present in larger amounts in the heated

 

95

aS-casein digest than the unheated, to suggest contents of
histidine and/or threonine.

aS-Casein heated in the presence of lactose resulted
in a pepsin-pancreatin index slightly higher (70 to 67)
than that for the heated protein. The amino acid index
increase associated with heat treatment in the presence
of lactose was observed only for as—casein. Also signifi-
cant was the outstanding amount of peptide 68 in the digest
of as-casein heated in the presence of lactose. Comparison
of essential amino acids from casein and aS-casein in
pepsin-pancreatin digests indicates that the amino acids
that can be associated with the striking differences are
threonine and histidine. The outstanding and unique
increase in peptide 68 in the digest of dS-casein heated
in the presence of lactose was associated with a smaller
amount of arginine, suggesting that arginine may be a
component of peptide 68.

k—Casein.--A significant increase in the pepsin-
pancreatin index from 6H for unheated to 79 for heated was
derived for k-casein. The pepsin-pancreatin index of k-
casein heated in the presence of lactose (73) was also
greater that that for the unheated preparation and only
slightly less than that for k—casein heated alone. The
more extensive k—casein proteolysis associated with heat
treatment is possibly attributable to configuration

characteristics after heat denaturation. A similar

 

96

phenomenon was reported by Kakade and Evans (1966b) for
navy bean protein. Mitchell and Block (1946) prOposed

that heat rendered the amino acids of ordered proteins

more available without changing actual contents of the

amino acids.

The amino acids released from heated k-casein pre—
parations in greater quantities than from unheated prepara-
tions were phenylalanine, valine and lysine, as reported
in Table 5. The amounts of histidine and threonine were
less in digests of heated k-casein than unheated, in magni-
tudes similar to the other casein hydrolysates. The
quantities of histidine and threonine present in the
hydrolysates of k-casein preparations were greater than
amounts in the index protein and, as a result, the quan-
tities were not limiting and did not influence the pepsin-
pancreatin index. The compensation for non-limiting amino
acids represents an inherent difficulty in index evalua-
tion of amino acid quantities. The validity of the index
technique has been established, however, as a proper com-
pensation (Block and Mitchell, 1946; Oser, 1959;

Sheffner, et al., 1956).

The peptide distribution for digests of k-casein
preparations was unique among proteins examined, as peptide
31 was outstanding particularly from heated protein. The
contents of histidine and threonine associated with the
peptide 31 suggest that histidine and/or threonine may be

components as has been noted for casein and dS-casein.

97

The free arginine in digests of k-casein proteolysis
preparation was 30% greater from the heated than the un-
heated preparations. Since arginine is one of the tryptic
cleavage sites, the increased quantity may implicate heat-
induced availability associated with denaturation.

B-Lactoglobulin.--The pepsin—pancreatin indices for
B-lactoglobulin were not significantly different for
unheated or heated preparations. The inability fo dis-
criminate between heated and unheated B-lactoglobulin can
be attributed to the amounts of essential amino acids in
excess of the index protein. The same observation was
noted for heated k-casein hydrolysates. The amounts of
lysine and arginine freed by hydrolysis in Table 6 suggest
that heat treatment may cause denaturation to make the
tryptic cleavage sites more readily available. The quan-
tities of lysine and arginine are 5 to 15% greater from
heated preparations, with and without lactose, than from
control preparations.

The amounts of free tyrosine in the pepsin—pancreatin
hydrolysates for B—lactoglobulin in Table 6 are all
greater than the total composition released by acid
hydrolysis. The descrepancy may represent an analytical
inconsistency and a trait of hydrolysis. The losses of
tyrosine upon acid hydrolysis may be as great as 10%
suggested by Block and Weiss (l9h6). Pepsin-pancreatin

digests were not subjected to this loss. Tyrosine is one

98

of the cleavage sites for pepsin (Hill, 1965). The con—
formation of B-lactoglobulin may make many of the tyrosine
sites available for pepsin cleavage. The efficient pepsin
cleavage of B—lactoglobulin may be associated with
increased degree of tryptic digest after pepsin treatment
as observed by Linderstrbm-Lang, e£_al. (1938).

The free non—essential amino acids from B-
lactoglobulin digests were present in larger amounts after

heat treatment than in unheated protein. An important

 

exception is the content of cysteine, which was freed in :E
much smaller quantities from heated B-lactoglobulin. The
decrease in cysteine content associated with heat treatment
indicates the lability of the sulhydryl-disulfide group
and the reactivity described by Morr and Josephson (1968).
A similar decrease was not noted from digests of heated
k—casein, although k-casein also contains internal disul-
fide linkages. This may indicate that the same secondary
structural characteristics suggested by Tanford g£_al.
(1962) for B-lactoglobulin do not exist for k—casein.
Peptides.-—The peptides observed in the pepsin—
pancreatin digests of each protein preparation (Table 7)
represent relative quantities since peptide standards are
not available. The disappearance of histidine and threo-
nine was associated with increased amounts of peptide 31,
implying that one or both of these amino acids may be

constituents. Mobility in the methanol/water/pyridine

 

99

solvent system indicated behavior similar to that of
histidine and threonine, as peptide 31 moved between the
two amino acids. Peptide 86 was also increased in
digests of heated proteins. The mobility of peptide 86
was indicative of an aliphatic amino acid.

Proteolysis During Pepsin-
Pancreatin Digestion

 

The pepsin-pancreatin proteolysis of milk protein
preparations, monitored by trichloroacetic acid-soluble
Lowry-Folin reactive material, resulted in a number of
observations true for all preparations.- The trichloro-
acetic acid supernatant protein was greater in heated
protein preparations than for unheated samples before
enzymatic proteolysis. Similar observations were reported
by Ghadimi and Pecora (1963) for picric acid supernatants.
The initial trichloroacetic acid-soluble protein may sug-
gest disruption of the protein molecular configuration to
either make a portion of each molecule soluble or a por-
tion of the total number of molecules soluble.

Casein.——The pepsin—pancreatin proteolysis of casein
monitored by trichloroacetic acid-soluble protein is
described graphically in Figure 6. Pepsin proteolysis of
casein reflects distinct differences between heated and
unheated proteins. Peptic proteolysis of heated prepara-
tions proceeded to completion within a l-hr period, while

the unheated protein was more slowly hydrolyzed throughout

 

lulu, |.ll|' IlllIJ

100

the 3-hr period. Pancreatin hydrolysis of casein,
unheated and heated, proceeded at essentially the same
rate. The casein preparation heated in the presence of
lactose was hydrolyzed by pancreatin considerably less
rapidly with the final amount of hydrolyzed protein 20%
less than preparations otherwise treated. The observa-
tions for casein are essentially consistent with pepsin-
pancreatin index measurements.

gs-Casein.--Peptic proteolysis of ds-casein (in

Figure 7) was analogous to that for casein. Heated pro-

 

tein preparations had attained the final level of tri-
chloroacetic acid-soluble protein within the first hour.
A larger portion of the protein was solubilized by pepsin
in the case of aS-casein (28 to 31%) than in the case of
casein (23 to 24%). The pancreatin hydrolysis rate for
unheated aS-casein was similar to casein; however a much
more pronounced heat treatment influence was obvious
with heated ds-casein which was less extensively hydrolyzed.
The reSponse to heat treatment may be associated with the
disorganized structure of as-casein reported by Herskovits
(1966). The amounts of ds-casein solubilized by pepsin-
pancreatin hydrolysis were consistent with the pepsin-
pancreatin indices for aS-casein.

k-Casein.--The k-casein pepsin—pancreatin proteolysis
monitored by trichloroacetic acid—soluble protein is

described graphically ianigure 6. The peptic proteolysis

101

for k-casein preparations monitored by trichloroacetic
acid-soluble protein was not as extensively influenced by
heat treatment as were casein and ds-casein. The degree
of hydrolysis by pepsin had attained the final level for
all preparations after a l-hr exposure. The extent of
pancreatic proteolysis of k-casein by heat treatment and a“
heat treatment in the presence of lactose was reduced by
19 to 20% from unheated k-casein. The reduction in pan-

creatic proteolysis associated with heat treatment for

k—casein was not in agreement with the pepsin-pancreatin

 

amino acid index which was higher for heated than unheated
proteins. The difference between the amino acid index

and trichloroacetic acid-soluble protein is due to the
released amounts of threonine and histidine from k-casein
which are not limiting when compared to the index egg
protein.

B-Lactoglobulin.--The pepsin—pancreatin proteolysis
of B—lactoglobulin monitored by trichloroacetic acid—
soluble protein is indicated in Figure 9. Peptic digest
of B—lactoglobulin did not proceed after the first hour
in any case. Proteolysis of heated protein was only half
as extensive as was peptic hydrolysis of unheated B-
lactoglobulin. Heat-induced complex formation of the
type suggested by Trautman‘and Swanson (1959) or the
molecular associations described by Tanford gt_al. (1962)

may have been partially responsible for the less extensive

102

proteolysis of B-lactoglobulin. Pancreatic hydrolysis

of B-lactoglobulin, on the contrary, was more extensive
than unheated protein for heated preparations and still
more extensive for the protein heated with added lactose.
The pepsin-pancreatin amino acid index for B-lactoglobulin
did not indicate similar amounts of amino acid release

as the trichloroacetic acid-soluble protein. The amino
acids threonine and histidine were not limiting, as was
the case with k-casein.

Enzymatic Proteolysis During
Gel Filtration

 

 

Enzymatic proteolysis of heated proteins during gel
filtration provides a rapid method for estimation of the
degree of protein hydrolysis. Broad interpretation of
the results derived must be approached with caution, as.
the specificity of the enzyme may be measured rather than
treatment differences (Ford and Salter, 1964).

Zebrowska (1968) confirmed the precaution suggesting that
only "broad similarity" exists between the in vitrg
measurements from gel filtration proteolysis and those
obtained with rats. Several advantages are ascribed to
the technique of enzymatic proteolysis during gel filtra-
tion, including: minimal transamination, stepwise
proteolysis and proteolysis under realistic conditions

insofar as absorption of amino acids is concerned.

103

Total Protein Distribution

The protein content of fractions separated after
enzymatic proteolysis during gel filtration of milk pro-
teins are reported in Table 9. The variable protein
amounts eluted as intact protein indicate that a general
statement to describe influence of heat treatment on
susceptibility to tryptic cleavage is not possible.

Casein and aS-casein heat-treated preparations
contained less protein in the combined peptide and amino
acid fractions (Fractions 2 and 3) than the unheated pro-
tein. The protein preparations containing disulfide
linkages, k-casein and B-lactoglobulin, demonstrated a
dissimilar tendency, as the amount of protein in the
amino acid and peptide fractions from heated proteins was
greater than or equal to the amount from unheated pro-
teins. The stimulation of proteolysis by heat treatment
of k-casein and B-lactoglobulin was previously noted in
pepsin-pancreatin proteolysis and may be associated with
heat denaturation.

Trichloroacetic Acid
Supernatant Protein

 

Trichloroacetic acid-soluble Lowry-Folin reactive
material in fractions collected after gel filtration
proteolysis provides an indication of the degree of
tryptic hydrolysis. The trichloroacetic acid—soluble

protein contents reported in Table 10 demonstrated the

104

same characteristics described for the total protein
content of the gel-filtration proteolysis fractions.

The trichloroacetic acid-soluble portion of the intact
protein (Fraction 1) showed increases attributable to
heat treatment of 20 times for casein and 3 to 4 times
for as-casein. On the contrary, the intact protein
after gel filtration proteolysis of heated k-casein was
lower than the unheated protein, and an increase of only
2M% was observed after heat treatment with added lactose.
The trichloroacetic acid-soluble.portion of the intact
protein fraction from B-lactoglobulin gel filtration
proteolysis was approximately half the amount from control
protein in the heated preparations.

The trichloroacetic acid-soluble protein from heated
preparations before hydrolysis was.higher than from
unheated preparations suggesting that dissociation of
complexes and some heat-induced peptide—linkage cleavages
may occur.

Reaction with 1-Fluoro-2,
fl-Dinitrobenzene

The reaction of protein with FDNB to measure avail-
able lysine has gained acceptance since the method was
published by Carpenter (1960). The milk proteins in this
study were evaluated by reaction with FDNB before and
after heat treatment. The protein fractions from gel

filtration proteolysis were also reacted with FDNB. The

105

initial quantities of e-amino-DNP-lysine for milk pro-
teins in Table 10 are slightly greater (15 to 23%) than
the results for milk protein reported by Bujard eg_al.
(1961), but similar to those reported by Lea and Hannan
(1950). Lysine inactivation of 20% was described in a
report by Bujard §t_al. (1967) for evaporated milk (heated
in condensed state at 113 C for 15 min). Casein results
showed a A; decrease in lysine associated with heat treat—
ment in the presence of lactose in this study. Pepsin-
pancreatin digest of the evaporated milk described by
Bujard g£_a13 (1967) led to a lysine availability of 7A%
compared to 59% for casein in this study. The descrepancy
in lysine contents is probably associated with three dif-
ferences in sample preparation. The evaporated milk of
Bujard g£_al. (1967) contained the entire milk protein at
a concentration of 8.9% protein. Their conditions were
more advantageous to protein-carbohydrate complex forma-
tion than the relatively low concentration and homogeneous
systems in this study. Heat treatment at 113 C for 15 min
by Bujard gt_al. (1967) was considerably less than for
proteins in this study (120 C, 30 min). The comparative
results indicate greater apparent lysine loss with
evaporated milk but fewer linkages resistant to hydrolysis.
The third difference involves liberation of c-amino
linkages during heat treatment. The trichloroacetic acid-

soluble protein fractions have indicated that release of

106

soluble protein may be attributable partially to heat-
induced peptide cleavage. The reaction of a-amino groups
freed by heat-induced cleavage would tend to offset the
loss of availability suggested by Bujard gt_al. (1967).
Boctor and Harper (1968) have evaluated the FDNB method
for protein evaluation in rats and contend that it is not
suitable for lysine estimation in heat-treated proteins.
Their contention was based on the large amounts of FDNB
reactive compounds in the feces of rats.

Milk protein fractions collected after proteolysis
during gel filtration (Table 10) contained in all cases
more FDNB reactive material estimated as lysine than did
the original protein. The observation of Boctor and
Harper (1967) is substantiated by the contents of the gel-
filtration hydrolysis fractions. Fecal material would
reflect any FDNB reactive material not absorbed and would
include a-amino groups in addition to e-amino lysine.

Quantities of terminal amino groups noted in the
fractions from proteolysis during gel filtration of
unheated casein and k-casein were much larger than other
protein preparations. The initial proteolysis during
pepsin-pancreatin digest was rapid and was possibly a
measurement of the same phenomenon. The FDNB reactive
material in the three fractions derived from gel hydrolysis
of B-lactoglobulin indicates more extensive hydrolysis

after heat treatment, as was observed in the free amino

107

acids released by pepsin-pancreatin proteolysis. The
reduced amounts of FDNB reactive materials observed in
the fractionated effluents derived from ds-casein heated
in the presence of lactose suggest the formation of

complexes not susceptible to tryptic cleavage.

Rate of Proteolysis byng-Stat

 

The rates of proteolysis estimated by pH-stat
monitored proton release provide a rapid and sensitive
method for evaluation of enzymatic proteolysis. The
methods of expression, substrate concentration and specific
activity as an expression of reaction velocity, provide
duplicate bases for discussion of the observation. Rapid
and efficient proteolysis is described by a maximum rate

at minimum concentration.

Equal Cleavage Sites

 

Preliminary examination of milk proteins resulted
in kinetic differences between individual protein prepara-
tions equated on the basis of molar concentration. The
hypothesis that differences in proteolysis rate was
governed by the number of cleavage sites rather than
molar concentration was evaluated. The substrate con-
centrations and specific activities for milk protein pre-
parations with equal lysine and arginine concentrations
are reported in Table 12. The dissimilarity of rates of

proteolysis suggests that chemical and physical composition

108

are more responsible for enzymatic proteolysis rate than

number of cleavage sites.

Chemical Denaturing Agents

The basis for discussion of treatments evaluated by
rates of enzymatic proteolysis must be established by
description of fundamental milk protein measurements.
The rates of proteolysis are enumerated for milk protein
systems in Tables 13 through 16.

Casein specific activity results were less after
heat treatment (2.2 x 10.3 mM/min) than untreated (2.8 x
10’3 mM/min) and reduced to 1.6 x 10’3 mM/min after heat
treatment with added lactose. The casein proteolysis
rate results can be related to pepsin-pancreatin indices
for corresponding casein preparations; control - 78,
heated - 68, and heated in the presence of lactose - A6.

The aS-casein rate measurements are similar to the
corresponding treatment values observed for the pepsin-
pancreatin amino acid index. The as-casein relationship
is not as straightforward as was casein because the
substrate concentration optima were reduced for dS-casein.

The Specific activity for k-casein was the same for
heated as unheated preparations. The k-casein proteolysis
rate after heat treatment with added lactose was greater
than the unheated protein corresponding to the pepsin-

pancreatin indices for k-casein preparations.

 

109

The specific activity measurements for B-
lactoglobulin were the same for control, heated, and
heated in the presence of lactose. The uniformity in
specific activity for B-lactoglobulin corresponds to
similar uniformity in pepsin-pancreatin indices for B-
lactoglobulin preparations. Substrate concentration
optima suggest previously noted heat denaturation and
complex formation since a decrease in concentration from
4.08 to 1.11 x 10"3 M resulted from heat treatment dena-
turation. A concentration Optimum of 2.50 x 10-3 M for B-
lactoglobulin heated with lactose suggested possible
lactose-protein complex formation.

grga.--Casein and B-lactoglobulin treated with 8M
urea were enzymatically hydrolyzed at a more rapid-rate
than the corresponding untreated proteins. Casein and
B—lactoglobulin only were treated with urea as dS—casein
and k-casein were so treated in fractionation. Casein
demonstrated a two-fold increase in specific activity
associated with heat treatment and a four-fold increase
when heat treated with added lactose. B—Lactoglobulin,
on the other hand, showed an increase of 25% in specific
activity due to heat treatment. B-Lactoglobulin heated
in the presence of lactose was hydrolyzed at a rate equi-
valent to 35% of the unheated protein. Concentration
optima for B-lactoglobulin after urea treatment were less

tha 1/10 of the untreated proteins, suggesting denaturation.

 

110

Since the same difference in concentration optima were
not obvious for casein, the results suggest that dis-
sociation of casein by urea was reversible but was not
for B-lactoglobulin.

Mercaptoethanol.--2-Mercaptoethanol is a denaturing

 

agent used for disruption of disulfide linkages as

reported by Anfinsen and Haker (1961). The rate of pro-

 

teolysis measurements of 2-mercaptoethanol-treated

aS-casein were the same as for untreated preparations.

 

dS-Casein contains no disulfide linkages (McKenzie, 1967)
which probably can be associated with the lack of change
in proteolysis rate attributable to 2—mercaptoethanol
treatment. The specific activities for casein, k-casein
and B-lactoglobulin unheated preparations were lower for
2-mercaptoethanol-treated than for untreated proteins.

The reduced proteolysis rates after heat treatment suggest
that the rearranged protein forms after denaturation led
to less readily available cleavage sites. Heat treatment
of 2-mercaptoethanol-treated casein and k—casein resulted
in Specific activities greater than untreated preparations;
however the reverse relationship existed for B—
lactoglobulin. The reduced rate of proteolysis for heated
B-lactoglobulin suggested an occlusion of cleavage sites
in the denatured protein. The rates of proteolysis for
casein, k-casein and B-lactoglobulin after heat treatment

with added lactose were considerably greater than untreated

lll

preparations. Reassociation was apparently kept to a
minimum by the presence of lactose, resulting in a
readily hydrolyzable configuration.

Performic acid.--Hirs (1956) described the tryptic

 

hydrolysis of ribonuclease after performic acid oxidation
as approximately l/10 its original quantity. Rate
measurements monitored by pH-stat provide a somewhat
different observation although not in conflict. The rates
of proteolysis, described as specific activity, were
greater after performic acid oxidation than for corres-
ponding untreated preparations. The concentration Optima
were approximately one-fourth the original values for cor-
responding protein preparations. The combined phenomena
suggest that irreversible denaturation occurred in a
uniform amount not significantly influenced by heat. The
irreversible denaturation may render a large protion of
the original protein molecule available for hydrolysis,
to conform with the observation by Hirs (1956). The
influence of performic acid treatment on aS-casein in a
manner analogous to proteins containing disulfide link-
ages suggests that methionine may be involved in site
availability since dS-casein contains no disulfide link—
ages.

Calcium.--The presence of calcium ions resulted in
the most pronounced change in rate of proteolysis for

ds-casein. The calcium sensitivity for dS-casein described

 

112

by McKenzie (1967) resulted in reduction of Specific
activity for dS-casein by 45% in unheated and heated Sam-
ples and 25% in samples heated with lactose. Aggregation
tendencies were indicated in dS-casein preparations by
higher concentration Optima for calcium-treated proteins,
particularly in combination with heat treatment. The spe—
cific activity for k—casein was not changed by calcium treat-
ment, indicating the relative calcium stability reported by
Zittle and Custer (1963). B-Lactoglobulin preparations
demonstrated up to fivefold increases in specific activity
associated with calcium addition. The most pronounced
increases in B-lactoglobulin specific activity, three times
control for heated and five times control when heated in the
presence of lactose, were accompanied by visible coagulation
of the protein. The coagulation of B-lactoglobulin may pro—
vide denatured conditions favorable to proteolysis.

Stabilization of trypsin by calcium has been de-
scribed by Bergmeyer (1963). Trypsin stability brought
about by the presence of calcium may have been responsible
for a part of the increased proteolysis rates.

Sodium chloride.--Treatment of milk protein prepara-

 

tions with isotonic saline resulted in minimal changes in
specific activity associated with heat treatment or heat
treatment with added lactose. The milk protein mOlecules
may have been stabilized or "salted in" by the presence of

isotonic saline.

113

Pepsin pretreatment.--The influence of partial
selective proteolysis before tryptic proteolysis rate
evaluation was examined by pepsin pretreatment of milk
protein preparations. Linderstrom—Lang (1938) observed
that B-lactoglobulin was more susceptible to tryptic
proteolysis after pepsin digest, which was verified by
pH-stat measurement in this study. The action of pepsin
has been compared to rennin by Fish (1957) making an

analogy possible for pepsin predigestion. Porter (1964)

 

reported that the action of rennin did not modify the .
behavior of casein upon tryptic hydrolysis. The change ”7
in specific activity of k-casein preparations was not

large due to pepsin pretreatment to correspond with the

contention set forth by Porter (1964). Proteolysis rate
measurements for casein preparations reflected disruption

by peptic digest, as rate increases of as much as four

times the untreated preparations were observed. The

specific activity measruements for ds-casein preparations

were substantially reduced for unheated and heated

samples after pepsin predigestion. The reduction in

tryptic digest rates may be indicative of extensive

peptic proteolysis of as-casein resulting in insufficient

amounts of the protein remaining for precise measurement.

pH treatment during heating.--Milk protein prepara-

 

tions were heated at pH 5 and at pH 9 before proteolysis

rate measurement. The pH values represent a logical

114

pH range encountered in protein containing, heat—treated
foods.

Heat treatment of casein and dS-casein preparations
at pH 5 resulted in reduction of proteolysis to levels
less than could be measured. Heat treatment of casein at
pH 5 followed by pepsin pretreatment led to specific
activity slightly greater than untreated casein but at an a
insignificant concentration optimum. as-Casein after heat

treatment at pH 5 had a specific activity one-tenth as.

 

great as unheated protein. Proteolysis of k-casein heated
at pH 5 was enhanced, as reflected by a 26% increase in
specific activity at a 15 times higher substrate optimum
than unheated k-casein. B—Lactoglobulin heated at pH 5

was more rapidly hydrolyzed than the unheated protein,
Similar to the response by k-casein. The Specific activity
for B—lactoglobulin heated at pH 5 increased by 20%, and
the concentration optimum was slightly more than double

the unheated protein.

Casein preparations heated at pH 9 reflected a 10%
reduction from unheated proteins in both specific activity
and substrate concentration optima. The same casein pre-
parations after pepsin pretreatment were not measurably
hydrolyzed. The observations-suggest change in micellar
structure of casein. The hydrolysis sites were evidently
available only after pepsin predigestion. ds-Casein

heated at pH 9 was also less rapidly hydrolyzed, with

115

reduction of specific activity to 25% of the control value
and reduction of substrate concentration optimum to 32% of
the unheated protein. k-Casein preparations heated at pH
9 did not demonstrate marked reduction in specific activity.
The k-casein heated at pH 9 and then subjected to pepsin
pretreatment was hydrolyzed more rapidly by trypsin. Heat
treatment of B-lactoglobulin at pH 9 resulted in no sig—
nificant change in rate of tryptic hydrolysis. B-
Lactoglobulin heated at pH 9 was much more readily
hydrolyzed after pepsin pretreatment, as was the case
after heat treatment at pH 7.

Sugars.--The mono- and di-saccharide components of
lactose were evaluated to establish their influence upon
rates of proteolysis with and without application of heat.
The sugars exerted effects on an individual basis not
associated with chemical reactions of the sugars. Specific
activities and substrate concentration optima for casein
were greater in each preparation with sugar added. The
proteolysis rates suggested dissociation of the micelle
followed by reversible complexing as proposed by Lea and
Hannan (1950). Treatment of B-lactoglobulin with sugars
without heat treatment resulted in small increases in
specific activity, accompanied by reduced substrate con—
centration optima. Heat treatment in the presence of
glucose and of galactose resulted in reduction of specific

activity by 50% and in doubled substrate concentration

116

optimum. Heat treatment of B-lactoglobulin with added
lactose and with added glucose plus galactose brought

about no change in rate of proteolysis.

117

TABLE l7.—-Pepsin-pancreatin indicesa of milk proteins
and heat-treated milk proteins.

 

Pepsin—Pancreatin Index
Milk Protein

 

 

Heated in Presence

 

 

Control Heated of Lactose
Casein 78a (78)b 68 46 7
as-Casein 80 67 70
k-Casein 64 79 76
B-Lactoglobulin 63 63 62

aAnimal Study Indices: 78 (Rippon, 1959); 73
(Mitchell and Block, 1946).

bAkeson and Stahmann (1964).

SUMMARY

The milk proteins were studied during enzymatic
proteolysis as model protein systems with and without
heat treatment. The milk proteins used were: casein,
as-casein, k-casein and B-lactoglobulin.

This research was divided into three parts. The
first was pepsin-pancreatin proteolysis with examination
of proteins during hydrolysis and assessment of amino
acids and peptides freed in the digest. The second part
was evaluation of milk proteins enzymatically hydrolyzed
during gel filtration. And, the third part was rate of
proteolysis examination by pH-stat monitoring of proton
release.

Milk protein treatments included heat treatment
(121 C for 30 min), sugars, chemical denaturing agents,
pH adjustment and addition of calcium and sodium salts.

Enzyme released amino acid and peptide contents
from unheated proteins and those heated in the presence
and absence of lactose showed results necessitating the
use of an amino acid index for interpretation. Amino
acid indices for milk proteins reflected the amino acid
release quantities and correlated with published bio—

logical data. Casein amino acids enzymatically released

118

119

were 13% lower after heat treatment than when unheated,
and a 36% reduction in freed amino acids was noted after
heat treatment in the presence of lactose. The enzyme
liberated amino acids from aS-casein were 16% lower in
heated than unheated digests and 12% lower when heated
in the presence of lactose than unheated. The k—casein
amino acids enzymatically released after heat treatment
were 23% lower than unheated, and heating in the presence
of lactose resulted in 19% less free amino acids than
when the same protein was not heated. The enzymatic
release of amino acids from B-lactoglobulin was not mea-
surably different as a result of heat treatment with or
without the presence of lactose.

Six peptides were observed in trichloroacetic acid
soluble portion of the digests of heat—treated protein
preparations, and they were measured concurrently with
amino acid analyses. The relative amounts of the peptides
and amino acid quantities from protein digests indicated
that two of the peptides contained predominately histi—
dine, threonine, and leucine.

The proteins were examined by enzymatic hydrolysis
during gel filtration. The milk protein with internal
disulfide linkages (k-casein and B-lactoglobulin) were
24% more completely hydrolyzed during gel filtration than
casein and aS-casein, with 2 to 10 times as much intact

protein remaining after proteolysis of the proteins

 

120

containing internal disulfide linkages. The proteins
hydrolyzed during gel filtration were assessed for e—amino
lysine before and after proteolysis. The e—amino lysine
data were ineffective indicators of susceptibility to
proteolysis during gel filtration for milk proteins after
heat treatment, possibly due to interference by terminal
amino groups.

The rates of enzymatic hydrolysis for milk protein
preparations were assessed by pH-stat monitored proton
release. The cleavage site concentration (lysine and
arginine residues) was not the predominant factor influ-
encing rate of proteolysis for milk protein systems.

The protein systems were treated with chemical
denaturing agents, and the rates of proteolysis for the
altered proteins were evaluated by measurement of proton
release. Urea treatment of casein and B-lactoglobulin
resulted in increased proteolysis rates; however upon
heat treatment the rates of proteolysis were reduced which
suggested formation of complexes. The treatment of milk
proteins with mercaptoethanol to disrupt internal disul-
fide linkages resulted in slightly decreased rates of
proteolysis for casein, k-casein and B-lactoglobulin;
however proteolysis rates were considerably increased
after heat treatment in the presence of lactose. Perfor-
mic acid oxidation of milk proteins resulted in enhanced

rates of proteolysis for all proteins studied, and

121

hydrolysis rates were not significantly influenced by
heat treatment.

Calcium (0.3 M) was added to the milk protein
systems which reduced the hydrolysis rate for aS-casein
by 45% and caused a fivefold increase in the B-
lactoglobulin rate of proteolysis.

The milk protein systems were heated at pH 5 and pH
9 followed by assessment of enzymatic proteolysis rates
by pH—stat proton release evaluation. The rates of
proteolysis of casein and aS—casein after heat treatment
at pH 5 were reduced to immeasurable levels. Enzymatic
hydrolysis rates were increased for k-casein (26%) and B-
lactoglobulin (20%) upon heat treatment at pH 5. The rate
of enzymatic proteolysis for casein reflected a 10%
reduction attributable to heat treatment at pH 9, and the
proteolysis rate for dS-casein was 25% of the untreated
protein.

Rates of enzymatic proteolysis of casein and B-
lactoglobulin were evaluated with added glucose, galac-
tose and lactose. The rates of proteolysis for casein
were increased by glucose and galactose addition and
reduced by 50% after heat treatment. B-Lactoglobulin
rates of proteolysis were not extensively changed by
sugar addition, with or without heat treatment.

The milk proteins studied as model protein systems

reflected the individual characteristics of each protein

122

by behavior unique to each upon enzymatic proteolysis.
The amino acid quantities released by pepsin-pancreatin
digest provided the basis for an index which could be
correlated with other evaluation techniques studied,
proteolysis during gel filtration and hydrolysis rate
measurements. The rate of protein hydrolysis assessed

by proton release provides a potential method for

rapid estimation of protein digestibility, as correlation
was observed between rate of proteolysis and digest

amino acid index.

BIBLIOGRAPHY

123

BIBLIOGRAPHY*

Ahrens, R. A. and Wilson, J. E. 1966. Carbohydrate
Metabolism and Physical Activity in Rats Fed Diets
Containing Purified Casein Versus a Mixture of
Amino Acids Simulating Casein. J. Nutr. 90g63.

Akeson, W. R. and Stahmann, M. A. 1964. A Pepsin
Pancreatin Digest Index of Protein Quality Evalua-
tion. J. Nutr. 83;257.

Alais, C.; Kiger, N.; Jolles, P. 1967. Action of Heat
on Cow k-Casein. Heat Caseino—GlyCOpeptide. J.
Dairy Sci. 5251738.

Allison, J. B. 1964. The Nutritive Value of Dietary
Proteins. In Munro, H. N. and Allison, J. B. (ed.)
Mammalian Protein Metabolism, Vol. 11. Academic
Press, new York. p. 41.

 

Anfinsen, C. B. and Haker, E. 1961. Studies on the Reduc-
tion and Re—formation of Protein Disulfide Bonds.
J. Biol. Chem. 236:1361.

Association of Official Agricultural Chemists. 1965.
Methods of Analysis, Ed. 10. Washington, D.C.,

p, 785.

 

Bailur, A. 1967. Studies on Availability of Amino Acids
from Selected Proteins. Ph.D. Thesis. Cornell
University, Ithica, New York.

Bahadur, K. and Atreya, B. D. 1959. A Study of the
Influence of Casein Concentrations on the Papain-
Casein Reaction at Different Hydrogen Ion Concentra-
tions Employing Boric Acid/Borate and Citric Acid
Phosphate Buffers. Enzymologia 155327.

Bergmeyer, H. U. 1963. Methods of Enzymatic Analysis.
Academic Press, New York. 1064 pp.

 

Block, R. J. and Weiss, K. W. 1956. Amino Acid Handbook.
Charles C. Thomas, Springfield, Ill. 3867pp.

 

 

*

Cited according to Conference of Biological Editors.
1964. Style Manual for Biological Journals. 2nd Ed. Am.
Inst. Biol. Sci., Washington. 117 pp.

 

124

125

Block, R. J. and Mitchell, H. M. 1946. The Correlation
of the Amino Acid Composition of Proteins with
their Nutritive Value. Nutr. Abstr. Revs. 16:249.

Boctor, A. M. and Harper, A. E. 1968. Measurement of
Available Lysine in Heated and Unheated Foodstuffs
by Chemical and Biological Methods. J. Nutr. 24:289.

Bovey, F. A. and Yanari, S. S. 1960. Pepsin.ln Boyer,
P. D. Lardy, H and Myrbach, K. (ed.) The Enzymes.
Vol 4. Academic Press, New York, p. 63.

 

Boyer, P. D.; Lum, . G.; Ballou, G. A.; Luch, J. M.; and
Rice, R. G. 1946. The Combination of Fatty Acids
and Related Compounds with Serum Albumin. 1.
Stabilization Against Heat Denaturation. J. Biol.
Chem. 162:181.

Braham, J. E.; Bressani, R.; and Guzman, M. A. 1959. A
Rapid Procedure for the Determination of Net Protein
Utilization (NPU) with New Hampshire Chicks.‘ Fed.
Proc. 18:518.

Bujard, E., Handwerck, V. and Mauron, J. 1967. The Dif-
ferential Determination of Lysine in Heated Milk. I.
In vitro Methods. J. Sci. Food Agric. 18:52.

Bujard, E. and Mauron, J. 1966. A Two-Dimensional Separa-
tion of Acid, Neutral and Basic Amino Acids by Thin-
Layer Chromatography on Cellulose. J. Chromatog.
21:19.

Campbell, J. A. 1963. Method for Determination of PER and
NPR. In Evaluation of Protein Quality, National
Academy of Sciences-:NatiOnalmResearch Council Pub—
lication 1100. p. 31.

 

 

Carpenter, K. J. 1960. The Estimation of the Available
Lysine in Animal-Protein Foods. Biochem. J. 11:
604.

Cunningham, L. W. 1957. Proposed Mechanism of Action of
Hydrolytic Enzymes. Science 125:1145.

Derse, P. H. 1962. Evaluation of Protein Quality (Bio-
logical Method). J. Am. Assoc. Agr. Chem. 45:418.

Desnuelle, P. 1960. Chymotrypsin. In Boyer, P. D.;
Lardy, H.; and Myrbach, K. (ed.7‘ The Enzymes, Vol.
4. Academic Press, New York. p. 631.

 

126

Dixon, M. and Webb, E. C. 1964. The Enzymes. Academic
Press, New York. 950 pp.

 

Donoso, G.; Lewis, 0. A. M.; Miller, D. S.; and Payne, P. R.
1962. Effect of Heat Treatment on the Nutritive
Value of Proteins; Chemical and Balance Studies. J.
Sci. Food Agric. 13:192.

Eldred, N. R. and Rodney, G. 1946. The Effect of Proteo—
lytic Enzymes on Raw and Heated Casein. J. Biol.
Chem. 162:261.

Epstein, S. I. and Possick, P. A. 1961. Inhibition of
Enzymic Hydrolysis of Plasma Albumin by Detergents.
Arch. Biochem. Biophys..93:538.

Fasold, H. and Gundlach, G. 1963. Characterization of
Peptides and Proteins with Enzymes. In Bergmeyer,
H. U. (ed.) Methods 6: Enzymatic Analysis. Academic
Press, New York. p. 350.

Fish, J. C. 1957. Activity and Specificity of Rennin.
Nature 180:345.

Fomon, S. J. and Owen, G. M. 1962. Retention of Nitrogen
by Normal Full-Term Infants Receiving an Autoclaved
Formula. Pediatrics 29:1005.

Ford, J. E. and Salter, D. N. 1966. Analysis of
Enzymically Digested Food Proteins by Sephadex-gel
Filtration. Brit. J. Nutr. 26:843.

Fox, K. K.; Holsinger, V. H.; Posati, L. P.; Pallansch,
M. J. 1967. Separation of B-Lactoglobulin from
Other Milk Serum Proteins by Trichloroacetic Acid.
J. Dairy Sci. 66:1363.

Frangne, R. and Adrian, J. 1967. La Reaction de Maillard.
V. Proteolyse Enzymatique des Aliments Grilles et
Signification des Resultats. Ann. Nutrit. Alim.
21:163.

Fricker, A. 1964. Nutritive Properties of Uperized Milk.
Kiel Milschwirtsch. Forschungsber. 16:315.
(Original not seen. Cited in Chem. Abstr. 66:763.)

Gerhard, J. C. and Pardee, A. B. 1962. The Enzymology of
Control by Feedback Inhibition. J. Biol. Chem.
237:891.

127

Ghadimi, H. and Pecora, P. 1963. Free Amino Acids of
Different Kinds of Milk. Am. J. Clin. Nutr. 13:75.

Gordon, W. G. and Whittier, E. O. 1965. Proteins in
Milk. £2 Webb, B, H, and Johnson, A. H. (ed.)
Fundamentals gprairy Chemistry. AVI, Westport,
Conn. p. 54.

Gordon, W. G.; Basch, J. J. and Thompson, M. P. 1965.
Genetic Polymorphism in Caseins of Cow's Milk. VI.
Amino Acid Composition of a -Caseins A, B, and C.
J. Dairy Sci. 36:1010. S

Graae, J. and Rasmussen, E. S. 1961. The Action of
Trypsin on Casein. Acta. Chem. Scand. 16:703.

Hankes, L. V.; Reisen, W. H.; Henderson, L. M.; and
Elvehjem, C. A. 1948. Liberation of Amino Acids
from Raw and Heated Casein by Acid and Enzyme
Hydrolysis. J. Biol. Chem. 116:467.

Hamilton, J. D. and McMichael, H. B. 1968. Role of the
Microvillus in the Absorption of Disaccharides.
Lancet II:154.

Harrington, W. F. 1955. The Liberation of Acid and Base
Binding Groups on Denaturation of Ovalbumin.
Biochim. Biophys. Acta. 16:450.

Harper, W. J.; Robertson, J. A. and Gould, I. A. 1960.
Observations on Milk Protease. J. Dairy Sci.
43:1850.

Hawley, E. E.; Murlin, J. R.; Nasset, E. S.; and Szymanski,
T. A. 1948. Biological Values for Six Partially
Purified Proteins. J. Nutr. 365153.

Henry, K. M. and Kon, S. K. 1950. Effect of Reaction
with Glucose on Nutritive Value of Casein. Biochim.
Biophys. Acta. 6:455.

Herskovits, T. T. 1966. On the Conformation of Caseins.
Optical Rotary Properties. Biochemistry 6:1018.

Hill, R. L. 1965. Hydrolysis of Protein. lg Anfinsen,
C. B., Anson, M. L., Edsall, J. T. and Richards,
F. M.- (ed.) Advances i6 Protein Chemistry. 161.
20. Academic Press, New York. p. 37.

 

 

Hirs, C. H. W. 1956. The Oxidation of Ribonuclease with
Performic Acid. J. Biol. Chem. 219:611.

128

Jacobsen, C. F.; Leonis, J.; LinderstrOm—Lang, K.; Offeson,
M. 1957. The pH—STAT and its Use in Biochemistry.
In Glick, E. (ed.) Methods of Biochemical Analysis.
26}, IX, Interscience PuEIiSHers, New York. p. 171.

 

Joly, M. 1965. A Physico-Chemical Approach to the Denatura-
tion gf Proteins. Academic Press, New YErk. 350 pp.

 

 

 

 

Kakade, M. L. and Evans, R. J. 1966a. Chemical and
Enzymatic Determination of Available Lysine in Raw
and Heated Navy Beans. (Phaseolus vulgaris). Can. J.
Biochem. 44:648. ‘

 

Kakade, M. L. and Evans, R. J. 1966b. Growth Inhibition
of Rats Fed Raw Navy Beans (Phaseolus vulgaris).
J. Nutr. 265191.

 

Kalan, E. B.; Greenberg, R., and Walter, M. 1965. Studies
on B-lactoglobulins A, B, and C. I. Comparison of
Chemical Properties. Biochem. 45991.

Kauzmann, W. 1956. Structural Factors in Protein Denatura-
tion. J. Cellular and Comp. Physiol. 41:113.

Kiermeir, F. and Semper, G. 1960. Uber das Vorkommen
eines Proteolytischen Enzymes und eines Trypsin—
Inhibitors in Kuhmilch. Z. Lebensm.-Untersuch.
u.-Forsch. 11;:282.

Kolthoff, I. M. and Laitenin, H. A. 1941. E5 and Electro
Titrations. 2nd Ed. Wiley, New York. 190 pp.

 

Kraft, R. A. and Morgan, A. F. 1951. Effect of Heat
Treatment on the Nutritive Value of Milk Proteins.
IV. The Biological Value of Unheated and Autoclaved
Dried Skim Milk. J. Nutr. 46:567.

Lea, C. H. 1948. The Reaction Between Milk Protein and
Reducing Sugar in the "Dry" State. J. Dairy Res.
15:369.

Lea, C. H. and Hannan, R. S. 1950. Studies of the Reaction
Between Proteins and Reducing Sugars in the Dry
State. 11. Further Observations on the Formation
of the Casein-Glucose Complex. Biochem. Biophys.
Acta. 4:518.

Ledford, R. A.; Chen, J. H. and Nath, K. R. 1968.
Degradation of Casein Fractions by Rennet Extract.
J. Dairy Sci. 61:792.

LinderstrOm-Lang, K.; Hotchkiss, R. D.; and Johansen, G.
1938. Peptide Bonds in Globular Proteins. Nature
142:996.

129

Lineweaver, H. and Burk, D. 1934. Determination of Enzyme
Dissociation Constants. J. Am. Chem. Soc. 66:658.

Lowe, C. U., Mosovich, L. L. and Pessin, V. 1964. Effects
of Protein Level and Type of Heat Treatment of Milk
Formulas on Growth and Maturation of Infants. J.
Pediatrics 64:666.

Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; and Randall,
R. J. 1951. Protein Measurement with the Folin
Phenol Reagent. J. Biol. Chem. 193:265.

Lowry, J. R. and Thiessen, R. 1950. Studies of the
Digestive Impariment of Proteins Heated with
Carbohydrates. II. In vitro Digestion Studies.
Arch. Biochem. 66:148.

Lundgren, H. D. 1941. The Catalytic Effect of Active
Crystalline Papain on the Denaturation of Thyro—
globulin. J. Biol. Chem. 138:293.

Matthews, D. M.; Crampton, R. F.; and Lis, M. T. 1968.
Intestinal Absorption of Peptides. Lancet 11:639.

Matthews, D. M.; Craft, 1. L.; and Crampton, R. F. 1968.
Intestinal Absorption of Saccharides and Peptides.
Lancet 66:49.

Mauron, J.; Mottu, F.; Bujard, E.; and Egli, R. H. 1951.
Availability of Lysine, Methionine, and Tryptophan
in Condensed Milk and Milk Powder. In vitro
Digestion Studies. Arch. Biochem. Biophys. 66:433.

McCullom, E. V. and Davis, M. 1915. The Cause of the
Loss of Nutritive Efficiency of Heated Milk. J.
Biol. Chem. 66:247.

McDonald, C. E. and Chen, L. L. 1965. The Lowry Modifica-
tion of the Folin Reagent for Determination of
Proteinase Activity. Anal. Biochem. 66:175.

McInroy, E. E.; Murer, H. K.; and Thiessen, R. 1948.
Effect of Autoclaving with Dextrose on the
Nutritive Value of Casein. Arch. Biochem. 66:256.
McKenzie, H. A. 1967. Milk Proteins. 66 Anfinsen, C. B.;
Anson, M. L.; Edsall, J. T.; Richards, F. M. (ed.)
Advances 12 Protein Chemistry, 266. 66. Academic
Press, New York. p. 55.

 

 

130

Miller, D. S. 1963. A Procedure for Determination of NPU
Using Rats Body N Technique. 12 Evaluation 66
Protein Quality. National Academy of Science—-
National Research Council. Publication 1100. p. 34.

 

 

Mitchell, H. H. and Block, R. J. 1946. Some Relationships
Between the Amino Acid Contents of Proteins and Their
Nutritive Values for the Rat. J. Biol. Chem. 163:

599.

Moore, 8.; Spackman, D. H.; and Stein, W. H. 1958.
Chromatography of Amino Acids on Sulfonated Poly-
styrene Resins. Anal. Chem. 66:1185.

Morr, C. V. and Josephson, R. V. 1968. Effect of Calcium,
N—Ethylmaleimide Upon Heat-Induced Whey Protein and
Calcium Aggregation. J. Dairy Sci. ‘66:1349.

Morr, C. V. 1967. Effect of Oxalate and Urea Upon Ultra-
centrifugation Properties of Raw and Heated Skimmilk
Casein Micelles. J. Dairy Sci. 66:1744.

Morr, C. V. 1967. Effect of Urea upon the Physical
Properties of B-Lactoglobulin A and B. J. Dairy
Sci. 50:1752.

Oser, B. L. 1959. An Integrated Essential Amino Acid
Index for Predicting the Biological Value of Proteins.
66_Albanese, A. A. (ed.) Protein and Amino Acid
Nutrition. Academic Press, New York. p. 281.

 

Oser, B. L. 1951. Method for Integrating the Essential
Amino Acid Content in the Nutritional Evaluation of
Protein. J. Am. Dietet. Assoc. 66:396.

Pataki, G. 1968. Techniques 66 Thin-Layer Chromatography
12 Amino Acid and Peptide Chemistry. Ann Arbor
Science Publishers, Ann Arbor. 218 pp.

 

 

 

Patton, A. R. 1950. Present Status of Heat-Processing
Damage to Protein Foods. Nutr. Reviews. 6:193.

Patton, A. R.; Hill, E. G.; and Foreman, E. M.‘ 1948.
Amino Acid Impairment in Casein Heated with Glucose.
Science 107:623.

Peterson, R. F. 1963.‘ High Resolution of Milk Proteins
Obtained by Gel Electrophoresis. J. Dairy Sci. 46:
1136.

131

Porter, J. W. G. 1964. Nutritive Value of Milk Proteins.
J. Dairy Res. 66:201.

Rice, E. E., Beuk, J. F. 1953. The Effects of Heat upon
the Nutritive Value of Protein. In Mrak, E. M. and
Stewart, G. F. (ed.) Advances iH_Food Research.
666: 66. Academic Press, New YOFEL p. 233.

 

 

Rick, W. 1963. Pepsin, Pepsinogen, Uropepsinogen. 66
Bergmeyer, H. U. (ed.) Methods of Enzymatic
Analysis. Academic Press, New YOFEL p. 819.

 

 

Rippon, W. P. 1959. A Comparison of Several Methods for H
Estimating the Nutritional Value of Proteins. Brit. ?
J. Nutr. 66:243.

Roach, A. G.; Sanderson, P. and Williams, D. R. 1967.
Comparison of Methods for the Determination of Avail—
able Lysine Value in Animal and Vegetable Protein
Sources. J. Sci. Food Agric. 66:274. A

 

Rose, D. 1962. Factors Affecting the Heat Stability of
Milk. J. Dairy Sci. 66:105.

Rupp, J. R.; Neimann, C.; and Hein, G. E. 1966. The
Primary Specificity of Chymotrypsin. Further Evi—
dence for "Wrong—way" Binding. Biochemistry 6:4100.

Shahani, K. M. 1966. Milk Enzymes, Their Role and Signifi—
cance. J. Dairy Sci. 46:907.

Sheffner, A. L.; Eckfeldt, G. A. and Spector, H. 1956. The
Pepsin-Digest-Residue (PDR) Amino Acid Index of Net
Protein Utilization. J. Nutr. 66:105.

Smith, E. L. and Hill, R. L. 1960. Leucine Aminopeptidase.
66 Boyer, P. D.; Lardy, H.; and Myrbach, K. (ed.)
The Enzymes, Vol. 4. Academic Press, New York, p. 37.

 

Squibb, R. L. 1963a. An Improved Technique for the Pre-
paration and Scanning of Thin-Layer Chromatograms.
Nature 198:317.

Squibb, R. L. 1963b. Thin-Layer Chromatographic Separation
and Quantitative Determination of Several Free Amino
Acids of Avian Liver. Nature 199:1216.

Summers, J. D. and Fisher, H. 1961. Net Protein Values
for the Growing Chicken as Determined by Carcass
Analysis: Exploration of the Method. J. Nutr.

16:435.

132

Swaisgood, H. E. and Brunner, J. R. 1962. Characteriza-
tion of k-Casein Obtained by Fractionation with
Trichloroacetic Acid in a Concentrated Urea Solution.
J. Dairy Sci. 46:1.

Swaisgood, H. E.; Brunner, J. R.; and Lillevik, H. A. 1964.
Physical Parameters of k-Casein from Cow's Milk.
Biochemistry 6:1616.

Swaisgood, H. E. and Timasheff, S. N. 1968. Association
of d -Casein C in the Alkaline pH Range. Arch.
Biocfiem. Biophys. 125:134. wQET*‘

Tanford, C.; Buckley, C. E.; De, P. K.; and Lively, E. D.
1962. Effect of Ethylene Glycol on Conformation of
y—Globulin and B-Lactoglobulin. J. Biol. Chem. 237:
1168.

W?“ '

Tanford, C.; Kawahara, K.; and Lapanje, S. 1967. Proteins
as Random Coils. I. Intrinsic Viscosoties and I
Sedimentation Coefficients in Concentrated Guanidien “
Hydrochloride. J. Am. Chem. Soc. 66:729.

 

Thompson, M. P.; Tarrasuk, N. P.; Jenness, R.; Lillevik,
H. A.; Ashworth, U.S.; Rose, D. 1965. Nomenclature
of the Proteins of Cow's Milk.r-Second Revision.

J. Dairy Sci. 466159.

Trautman, J. C. and Swanson, A. M. 1959. Protein Complex
Formation in Evaporated Milk. J. Dairy Sci. 46:
8950

Warner, R. C. and Polis, E. 1945. On the Presence of a
Proteolytic Enzyme in Casein. J. Am. Chem. Soc.

61:529.

Waugh, D. F. 1961. Casein Interactions and Micelle Forma-
tion. J. Phys. Chem. 6651793.

Wollenweber, P. 1962. Dfinnschicht-chromatographische
Trennungen von Aminoséuren an Cellulose-Schichten.
J. Chromatog. 6:369.

World Health Organization. 1965. Protein Requirements.
Report of a Joint FAO/WHO Expert Group. WHO
Technical Report Series No. 301.

Yamauchi, K. and Tsugo, T. 1961. ElectrOphoretic Changes
of Casein by Heating and Influence of Sugars and
Whey Protein. (in Japanese, English summary.)
Japanese J. Zootechnical Sci. 66:311.

133

Zebrowska, T. 1968. The Course of Digestion of Different
Food Proteins in the Rat. Fractionation of the
Nitrogen in Intestinal Contents. Brit. J. Nutr°
22:483.

Zittle, C. A. 1961. Stabilization of Calcium-Sensitive
(a ) Casein by Kappa-Casein: Effect of Chymotrypsin
an Heat on Kappa-Casein. J. Dairy Sci. 44:2101

Zittle, C. A. 1965. Purification of Protease in Cow's
Milk. J. Dairy Sci. 46:771.

Zittle, C. A. and Custer, J. H. 1963. Purification and
some of the Properties of ds-Casein and k-Casein.
J. Dairy Sci. 46:1183. 1

 

 

 

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