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J.
Agric.
Food
Chem.
1981,
29,
11-15
11
Topps,
J.
H.;
Kay,
R.
N.
B.
Proc.
Nutr.
SOC.
1969,
28,
23A.
Received for review
October
20,1978.
Revised
September
8,1980.
Accepted October
8,
1980.
Arizona
Agricultural Experiment
Station Journal Article No.
2917.
Pazur, J.
H.;
Ando, T.
J.
Biol.
Chem.
1959,
234,
1966.
Steel,
R.
G.
D.;
Torrie,
J.
H.
"Principles
and
Procedures
of
Statistics"; McGraw-Hill: New York,
1960;
p
72.
Thivend, P.; Mercier,
C.;
Guilbot,
A.
In
"Methods
in
Carbohydrate
Chemistry"; Whistler,
R.
L., Ed.; Academic Press:
New
York,
1972;
Vol.
VI,
pp
100-105.
Effects of Ultra-High-Temperature Pasteurization
on
Milk Proteins
Frederic W. Douglas, Jr.,* Rae Greenberg, Harold M. Farrell, Jr., and Locke
F.
Edmondson'
Ultra-high-temperature (UHT) pasteurization of
skim
milk
(148
"C for
3
s)
has been found
to
inactivate
effectively foot and mouth disease virus. For determination of the effect of UHT pasteurization on milk
proteins, the composition and properties of proteins from milk after this treatment were compared with
those from conventional
high-temperature-short-time
pasteurized (HTST
=
71.7
"C for
15
s)
and raw
skim milks.
Vacuum-dried-acid-precipitated
caseins and freeze-dried-dialyzed whey proteins were
prepared from each product. Functional properties of casein such
as
solubility, viscosity, emulsifying
capacity, and electrophoretic mobility were compared. For both casein and whey proteins, compositional
comparisons were made among molar ratios of amino acids,
total
protein,
and
chemically available lysine.
The solubility of milk caseins was reduced by UHT pasteurization. Whey protein nitrogen analyses
show significant protein denaturation.
No
significant losses in nutritive value are indicated, and dif-
ferences in viscosity and emulsification capacity are small.
Earlier studies (Burrows and Dawson,
1968;
Hedger and
Dawson,
1970;
Sellers,
1969;
Terbriiggen,
1932)
indicated
that
the
virus
from foot and mouth disease
(FMD)
infeded
cows can survive in milk and derived milk products such
as
cheese (Blackwell,
1975)
and casein (Cunliffe and
Blackwell,
1977).
FMD
virus
in whole milk,
skim
milk, and
cream from experimentally infected dairy cows (Blackwell
and Hyde,
1976)
can survive conventional high-tempera-
ture-short-time (HTST
=
71.7
"C for
15
s)
pasteurization.
Cunliffe et
al.
(1978, 1979)
showed the persistence of in-
fectious FMD virus up to
42
days in heat-dried caseins
produced by acid precipitation of HTST-pasteurized skim
milk from infected cows. Milk-borne FMD
virus
may pose
a serious threat to FMD-free countries, and reliable pro-
cedures are needed
to
inactivate FMD
virus
in milk. Heat
treatments considerably above pasteurization have been
referred to as ultra-high-temperature (UHT) processes.
According to the International Dairy Federation
(1972),
UHT processes refer to pasteurization techniques with
temperatures of at least
130
"C in a continuous flow and
holding times of
-1
s
or
more. Cunliffe et al.
(1979)
reported that UHT pasteurization effectively inactivates
FMD virus in milk when carried out at
148
"C for
3
s
or
longer. It was of interest to determine the effect of this
regimen on the properties of caseins and whey proteins
prepared from UHT-pasteurized
skim
milk and
to
compare
them with those prepared from HTST-pasteurized and raw
skim
milk. The results indicated that UHT pasteurization
Eastern Regional Research Center, Agricultural Re-
search, Science and Education Administration,
US.
De-
partment of Agriculture, Philadelphia, Pennsylvania
19118.
'Retired.
of skim milk resulted in interaction of casein and whey
proteins, a reduction in solubility of casein at neutral pH
or below, a decrease in chemically available lysine in whey
protein, and a
56%
denaturation of whey protein in the
skim milk.
EXPERIMENTAL SECTION
Sample Preparation.
A
schematic for the preparation
of UHT-pasteurized, HTST-pasteurized, and raw caseins
is shown in Figure
1.
Fresh raw whole milk was obtained
from a local dairy and separated cold. The raw skim milk,
which contained
0.06%
fat, was divided into three lots.
Casein was prepared from
9.1
kg of lot no.
1
at
40
"C by
precipitation at pH
4.6,
with the addition of
1025
mL of
0.5
N
HC1 added slowly from a buret over a period of
15
min with stirring. After an additional
5
min of stirring,
the casein settled and the whey was decanted and filtered.
The casein was washed
4
times with water at pH
5.0.
Water temperatures were
35, 45
(twice), and
-23
OC.
Approximately
38.6
kg of lot no.
2
was
pasteurized at
76.7
"C for
15
s
in a triple tube heater, and the HTST-pas-
teurized casein was prepared
as
above from
9.1
kg
of milk.
Approximately
38.6
kg of lot no.
3
was sterilized in a tu-
bular heat exchanger at
148.5
"C for
2.5
s,
and
9.1
kg was
used
to
prepare UHT casein
as
above except the
skim
milk
was heated
to
47.5
"C and
982
mL
of
0.5
N
HC1
was
added
over a period of
7
min with stirring. Stirring was continued
for an additional
10
min. The caseins from the raw,
HTST-pasteurized, and UHT-pasteurized milks were dried
in a shelf dryer under high vacuum.
Figure
2
shows the procedure for preparation of raw,
HTST, and UHT freeze-dried whey proteins. Samples of
all three wheys were dialyzed for
36
h in running chilled
tap water and then for
12
h in distilled water. The dialyzed
wheys were concentrated to approximately half
of
the
This article not subject
to
US.
Copyright. Published
1981
by the American Chemical Society
12
J.
Agric.
Food
Chem.,
Vol.
29,
No.
1,
1981
Douglas
et
al.
raw whole milk
1
i
cold separate
raw skim milk
pasteurize at
148
OC
for
2.5
s,
cool and store
1.
"-I
store raw skim milk
pasteurize at
77
OC
for
15
s,
cool and store
heat to
47.5
OC
1
1
1
.1
heat to
40
OC
add
0.5
N
HC1
and
1
stir
about
20
min
/
decant wheys and store
!
heat to
40
OC
wash caseins four
times
dry in shelf dryer
1
Figure
1.
Schematic
for
the
preparation
of
raw,
HTST-pasteurized,
and
UHT-pasteurized
caseins.
whey (raw, HTST-pasteurized, and IJHT-pasteurized)
1
1
1
1
dialyze
36
h in running chilled tap water
dialyze
12
h
in
distilled water
condense to one-half volume
freeze dry overnight
Figure
2.
Schematic
for
the
preparation
of
raw,
HTST-pas-
teurized,
and
UHT-pasteurized whey proteins.
original volume and then freeze-dried overnight.
Reagents and Buffers. Potassium phosphate (mono-
basic)-sodium hydroxide buffers at pH 6.0, 6.8, and
8.0
were prepared by the procedure in Association of Official
Analytical Chemists (1965a), sections 13.023 and 13.024.
These buffers were made 0.15
M
with respect to sodium
chloride. Boric acid-potassium chloride buffer at pH 9.0
was prepared by the procedure listed in Association of
Official Analytical Chemists (1965b), sections 13.023 (c,
d), 13.024, and 33.047 (a). Corn oil was purchased from
a local swermarket. All other reagents used were ana-
lytical grade.
Y
Solubility Characteristics. The procedure of Lawhon
and Cater (1971) was modified and used to determine
solubility characteristics of the caseins. Essentially the
method consisted of dispersing
1
g of each casein in 100.0
mL of each buffer and stirring for 5 min. The slurry
was
readjusted to the original buffer pH, stirred an additional
5
min, with the pH readjusted when necessary; stirring
continued for an additional 5 min. The pH adjustments
of the casein slurry were made with 0.01
N
HC1 and 0.015
N
NaOH. Samples were centrifuged for 20 min at
2000
rpm and then filtered. Aliquots of 10.0 mL of supernatant
were analyzed for nitrogen by the micro-Kjeldahl method
(Association of Official Analytical Chemists, 1965~).
Viscosity Measurements. Casein solutions (2.5%) in
buffers at pH 6.8,
8.0,
and 9.0 were prepared by the
modified technique of Lawhon and Cater (1971). The
viscosity measurements of the casein solutions were de-
termined
in
Ostwald-Cannon-Fenske viscometers. Samples
were equilibrated 10 min in the viscometers at 30.4 OC
prior to being measured.
Emulsifying Capacity.
A
modification of the proce-
dure of Pearson et
al.
(1965) was used
to
determine the
emulsifying capacity of the caseins. The vacuum-dried
caseins were dispersed in buffer solutions at pH 6.8, 8.0,
and 9.0. Samples were stirred for 15 min, and the pH was
readjusted with 0.5
N
NaOH. After dissolving,
all
samples
were readjusted
to
pH 6.8. The emulsifying capacity was
determined with corn oil with 25.0-mL aliquots
of
casein
solutions containing from 120 to 250 mg of protein. The
oil was added slowly from a buret to the sample in a
beaker, with stirring at 1600 rpm. Just enough oil was
added to break the emulsion as shown by a sudden drop
in viscosity.
Gel Electrophoresis. Polyacrylamide gel electropho-
resis of the casein and whey protein samples was carried
out according to the method of Thompson et al. (1964)
with 7% polyacrylamide in 4.5% M urea at pH 9.2, after
the samples were reduced with
10%
2-mercaptoethanol.
An
eight-slot gel was
run
and samples were applied
so
that
each slot received 83 pg of the sample on a dry weight
basis.
Whey Protein Nitrogen. Whey protein nitrogen de-
terminations of raw, HTST-pasteurized, and UHT-pas-
teurized skim milks were by the Methods of Laboratory
Analysis for Dry Whole Milk, Nonfat Dry Milk, Dry
Buttermilk and Whey (1961), with a modification of the
Harland-Ashworth test used for whey protein nitrogen.
Available Lysine. Chemically available lysine was
determined by the method of Kakade and Liener (1969)
as modified by Greenberg et al.
(1977).
Effects of
UHT
Pasteurization on Milk Proteins
J.
Agric.
Food
Chem.,
Vol.
29,
No.
1,
1981
13
Table
I.
Percentage
of
Soluble
Nitrogen
of
Caseins
%
of
soluble
nitrogen"
PH
raw
HTST UHT
6.0 78.78
f
0.11 74.76
f
0.29 50.98
f
0.16
6.8 88.15
f
0.41 84.11
f
0.46 71.72
f
0.32
8.0
94.38
f
0.16 86.85
f
0.21 78.05
f
0.14
a
f
f
u
for
two
determinations.
Table
11.
Relative
Viscosity
of
2.5%
Casein
Solutions
viscosity measurementsa
PHb
raw
HTST UHT
6.8 1.53 1.45 1.29
8.0
1.66 1.57
1.38
9.0 1.60 1.52 1.55
a
Viscosity relative to
water.
K
for
three
determina-
tions.
Standard
errors
were
+
0.01
or
less.
Buffers
at
pH
6.8
and
8.0
were
potassium
phosphate-sodium
hy-
droxide.
At
pH
9,
a
boric
acid-potassium
chloride
buffer
was
used.
Amino Acid Composition.
The procedure of Moore
and Stein (1963) was used for automated amino acid
analysis. Samples were hydrolyzed at 110 "C for 24 h with
5.7
N
HCl containing phenol (10 pL/mL), in sealed
evacuated tubes.
Radial Immunodiffusion.
The distribution of 0-lac-
toglobulin in the various fractions was determined by ra-
dial immunodiffusion. For this work it was necessary to
obtain antibodies specific for 0-lactoglobulin. The protein
was purified by the method of Aschaffenberg and Drewry
(1957). Antibodies to 0-lactoglobulin were produced in
rabbits by Cappel Laboratories, Cochraneville,
PA.
The
antiserum produced was highly specific; it gave no cross-
reactions
to
purified caseins or
to
a-lactalbumin by either
Ouchterlony diffusion or immunoelectrophoresis (Crowle,
1973). Standard proteins and samples of each fraction
were dissolved in
50
mM
Tris,
4
mM citrate,
5
M urea, and
10 mM 2-mercaptoethanol.
A
3-pL aliquot was withdrawn
to assay for 0-lactoglobulin content by the method of
Mancini et at. (1965) with 0-lactoglubulin in urea-2-
mercaptoethanol used
as
the standard.
A
100-pL
aliquot
was taken for estimating of the protein content of each
fraction by the Coomassie dye binding method (Bradford,
1976), with either whole casein or 0-lactoglobulin in the
urea buffer used as the standard.
RESULTS AND DISCUSSION
Solubility Characteristics.
The solubility of the ca-
seins
as
shown in Table
I
provides evidence that there is
a marked reduction in the solubility of casein from UHT-
pasteurized milk compared with caseins from raw and
HTST-pasteurized milks when determinations were made
at neutral pH or below. The differences were less when
the caseins were first dispersed in buffer at pH 8.0 and
then titrated back to the appropriate pH. Low solubility
of the UHT-treated sample is no doubt related to the
heat-induced
~-lactoglobulin-K-casein
interaction (Zittle
et
al.,
1962). This complex formed by the sulfhydryl-di-
sulfide interchange tends to dissociate at high pH, as was
observed in this case. For some food uses, solubility may
not
be
an important factor, but for applications where high
solubility is needed, redispersion of UHT-pasteurized ca-
sein at an elevated pH followed by adjustment to the de-
sired pH level is recommended.
Viscosity.
Viscosity is the principal means for char-
acterizing the flow of a fluid. The three caseins were
dissolved in buffers at pH 6.8,8.0, and
9.0
to make
2.5%
solutions. Viscosity of the casein solutions was determined
Table
111.
Emulsifying
Capacity
of
Caseinates
mL
of
oil/mg
caseinate
of
protein
pH
concn,mg/25mL
raw
HTST UHTa
6.8 120 1.75 1.67
160 1.31 1.25
200 1.05 1.00
250 1.36
0.88
8.0
-*
6.8 120 1.75 1.75 1.67
160 1.31 1.22 1.19
200 1.05 0.95 1.05
250 0.96 1.30 1.08
9.0
-*
6.8 120 1.67 1.75 1.71
250 1.28 1.24 1.28
The
UHT
casein
sample
did
not completely
dissolve
at
pH
6.8;
therefore,
no
emulsion
was
run.
Table
IV.
Total Protein
and
Chemically
Available
Lysine
in Caseins and
Whey
Proteins
protein,"
g/lOO
available
lysine,
sample
e
of
sample
a/100
g
of
protein*
casein
raw
93.75 6.73
f
0.16
HTST
95.24 6.95
f
0.17
UHT
92.06 6.94
f
0.15
raw
90.40 8.99
f
0.20
HTST
84.86 8.75
f
0.18
UHT
69.96
8.18
f
0.17
whey
protein
a
Moisture-free
basis.
x
f
u
for
duplicate
samples
of
available
lysine.
at 30.4 "C. Although there
is
a trend toward lower viscosity
with increasing heat treatment (Table
11),
this is most
likely contributed by protein interaction (Zittle et at., 1962)
in the pasteurized samples causing low solubility at pH 6.8.
As the pH increases,
so
does the viscosity for
all
of the
samples. The viscosity for all three samples at
all
three
pH values
falls
well within the range of skim milk (1.33
at 30 OC and 1.54 at 25 "C) (Whitaker et al., 1927). The
differences in viscosity of the
three
samples probably would
not affect most food uses of the caseins.
Emulsifying Capacity.
In many food applications,
emulsifying properties of ingredient proteins are important
and are commonly discussed in terms of emulsifying ca-
pacity (EC). The EC denotes the maximum amount of oil
that is emulsified under specified conditions by a standard
amount of protein (Pearce and Kinsella, 1978). EC of the
three vacuum-dried caseins was measured as a ratio of
milliliters of oil per milligram of protein (Table
111).
At
pH 6.8, the UHT-pasteurized casein sample did not dis-
solve completely; therefore, no emulsion test was run on
this sample. Although the UHT casein is more difficult
to dissolve, once solution is effected at pH 8.0 or 9.0 and
then readjusted to pH 6.8, the emulsifying properties
compare favorably with those of raw and HTST-pasteu-
rized caseins at all concentrations.
Chemically Available Lysine and Total Protein.
The results of determinations for total protein and chem-
ically available lysine are listed in Table IV. The lyo-
philized whey protein samples have low total protein
values, reflecting inefficient lactose removal by dialysis of
the whey fraction. The available lysine levels when cal-
culated on a gram per 100 g of protein basis are not sig-
nificantly different for the raw, HTST-pasteurized, and
UHT-pasteurized caseins. This indicates
that
as
measured
by this parameter there is no loss of nutritional value for
the casein fractions. The UHT-pasteurized whey protein
does show a small decrease
(<lo%)
in available lysine as
14
J.
Agric.
FwdChe”,
VoI.
29.
No. 1,
1981
CASEIN
WHEY
--
WHEY
1
-
-
r\,
PROTEINS
CASEINS
-
-
-
-
Douglas
et
al.
E-LACTOGLOBULIN
a
L~CTALBUMIN
X-
12315678
Figure
3.
Gel electrophoresis
of
caseins and wheys. Slots
1
and
4
are raw casein, slot
2
is
HTST-pasteurized casein, and slot
3
is UHT-pasteurized
castein.
Slots
5
and
8
are
raw
whey proteins,
slot
6
is
HTST-pasteurized whey
protein,
and slot
7
is
UHT-
pasteurized
whey
protein.
Table
V.
Radial Immunodiffusion
Quantitation
of
the
Percent
@-Lactoglobulin
in
Casein
and
Whey
Protein
Fractions
mg
of
0-lactoglobulin
x
100“
sample
mg
of
total
protein
casein
.-.~...
raw
HTST
UHT
0.817
f
0.072
1.56
f
0.085
11.9
f
1.4
whey
protein
raw
85.9
f
7.1
HTST
67.6
f
4.5
UHT
23.3
t
0.9
Ox
f
o
for
four
determinations.
compared
to
the raw whey sample, hut this decrease is not
enough to diminish significantly its nutritional value.
A
PER
test
on the whey and casein
as
well would provide
confirmation of this hut
at
this point there is no reason
to
expect otherwise.
Electrophoretic Mobility. Gel electrophoresis pat-
tems
of
the
raw, HTST-pasteurized, and UHT-pasteurized
caseins and whey proteins are shown in Figure
3.
The
major casein fractions,
as-
and @-caseins are unaffected by
the heat treatment in samples 1,2,3, and
4.
Close exam-
ination of wasein bands in sample
3
shows
a
diminution
in their intensity when compared with those of samples
2 and
4.
In addition, sample
3
shows
a
hand
(X)
helow
8-casein. Compared with samples
5-8,
this hand
(X)
represents @-lactoglobulin complexed with the casein as
a
result of the heat treatment (Zittle et al., 1962). Quan-
titation by single radial immunodiffusion carried out with
antiserum specific for @-lactoglobulin gives further evidence
that @-lactoglobulin is complexed with the casein in the
UHT-pasteurized sample. Table
V,
comparing the levels
of
@-lactoglobulin in raw and UHT casein, shows that the
raw casein contains only 0.817% @-lactoglobulin in contrast
to
the high level (11.9%) in the UHT-pasteurized sample.
Also, raw whey protein contains
a
high level of &lacto-
globulin
(85.9%)
as
compared to only 23.3% in the
UHT-pasteurized sample. The complex precipitates with
the casein, but the conditions under which the gel is run
allows for the separation of the complex. Comparison of
samples
7
and
8
of Figure
3
indicates that much of the
8-lactoglobulin
is
carried out of the whey. The ratio of
Blactoglobulin
to
a-lactalbumin is diminished in sample
7,
and on
a
dry weight basis (Table
V)
the whey protein
was subsequently reduced from 85.9% @-lactoglobulin for
raw dialyzed whey to 23.3% for UHT whey protein. The
bovine serum albumin has been affected by the heat as
well.
Table
VI.
Molar
Ratios
of
Amino Acids
in
Raw,
HTST-Pasteurized,
and
UHT-Pasteurized
Caseins
and
Whey
Proteins‘
molar
ratios
caseins
wheys
amino
acids
raw
HTST
UHT
raw
HTST
UHT
1.72 1.74 1.88 3.13 3.03 3.62
1.09 1.18 1.17 1.67 1.65 1.81
Etb
1.60 1.60 1.63 1.70 1.73 2.03
Glu
5.04 4.73 4.83 4.49 4.62 4.98
PI0
3.34 3.05 3.00 1.66 1.75 2.06
Gly
0.80 0.81 0.83 1.03 1.04 1.01
Ala
1.07 1.10 1.23 1.99 1.99 1.48
‘/,-Cys
0.02 0.15
0.77
0.72 0.69
Val
1.81 1.97 1.83 1.79 1.80 1.57
Met
0.59 0.59 0.45 0.62 0.39 0.35
Ile
1.30 1.39 1.40 1.66 1.87 2.00
Leu
2.33 2.47
2.57
3.45 3.61 3.29
Tyr
1.01 0.90 0.99 0.78
0.75
0.73
Phe
1.02 1.01 1.00 0.91 0.84 0.99
LYS
1.79 1.76 1.90 2.58 2.72 3.04
His
0.63 0.62
0.60
0.53 0.55 0.72
Arg
0.69 0.69 0.68
0.66
0.69 0.62
Ratios
were
determined
by
averaging
data
using
Lys,
Arg,
and
Phe
as
divisors.
Whey Protein Nitrogen. The principal interest in
protein denaturation centers around the effects of heat
treatment, which considerably affects the whey proteins.
In
the natural
state,
the milk whey proteins have a definite
configuration which, when exposed
to
heat above
a
certain
critical level,
is
disrupted and the characteristic properties
of the protein are altered (Jenness and Patton, 1959).
Analysis for whey protein nitrogen in the skim milks used
to
produce the three casein and whey protein samples
showed
a
56%
denaturation in the UHT-pasteurized milk,
a
0.4%
denaturation in the HTST-pasteurized milk,
and
no denaturation in the raw milk.
Molar Ratios
of
Amino Acids.
The
amino acid
com-
position
as
shown in Table
VI
provides further evidence
of the interaction of whey proteins and casein during UHT
pasteurization. The molar ratios of amino acids in UHT-
pasteurized casein, compared with those in the raw and
HTST-pasteurized samples, indicate elevated levels of
aspartic acid, alanine, and cystine. Each of these
is
present
in higher ratios in raw whey.
Also,
UHT-pasteurized
casein
contains depressed ratios of proline and histidine, with raw
whey
again
reflecting lower levels of these residues. These
comparisons suggest
an
admixture of whey proteins,
thereby diluting the values for UHT-pasteurized casein
compared with those of the unpasteurized sample. In the
same fashion, UHT-pasteurized whey exhibits higher
values of glutamic acid, proline, phenylalanine, and his-
tidine, compared with those of raw whey and concomitant
lower values for alanine and cystine. These results are
consistent with the polyacrylamide gel patterns showing
whey proteins in casein fractions hut also indicate the
presence of casein in the UHT-pasteurized whey fraction.
ACKNOWLEDGMENT
We thank Frederick Adair for determining the emulsi-
fying capacity of the caseins, Brien Sullivan for assisting
in Kjeldahl determinations of nitrogen, and Harold Dower
for assisting in amino acid analysis.
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Reference
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US.
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of
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similar nature not mentioned.
Effects of Salts and Denaturants
on
Thermocoagulation of Proteins
Kazuko Shimada* and Setsuro Matsushita
Thermocoagulation of proteins containing different amounts of hydrophobic amino acids was investigated
with regard
to
the effects of
salts
and denaturants. The formation of thermocoagulum of egg albumin
was enhanced by the addition of salts, and the effect of salts followed the lyotropic series.
For
bovine
serum albumin, salts of the higher order on the lyotropic series enhanced fromation of coagulum but
those of the lower order inhibited it when the salt concentrations were increased. For soybean protein,
an increase in turbidity at alkaline pH was observed when
salts
were added, while formation of ther-
mocoagulum was inhibited. Guanidine hydrochloride enhanced the coagulum formation in a manner
similar
to
that of salts. Sodium dodecyl sulfate and urea suppressed thermocoagulation. From the effects
of
salts
and denaturants on thermocoagulation of these proteins, the mechanism of coagulum formation
can be surmised from the standpoint of the hydrophobic amino acid content of proteins.
Thermocoagulation of proteins is widely utilized in food
processing. However, there has been very little past re-
search on this subject. Heat-treated solutions of serum
albumin have been found
to
form an opaque coagulum, a
clear gel, or an intermediate clot depending on the pH of
the medium (Jensen et
al.,
1950). Seideman et al. (1963)
reported that some factors (pH, addition of sucrose, etc.)
affected the coagulation temperature of egg white. Dif-
ferences in coagulation of egg white resulting from ap-
plication of conventional heat and electronic exposure were
compared by Baldwin et al. (1967). It has been observed
that the microstructures of soybean protein curds and
yeast protein curds which were examined by an optical
microscope and a scanning electron microscope varied
according
to
pH (Lee
and
Rha,
1978;
Tsintsadze et
al.,
1978).
The three-dimensional network of the protein coagulum
is believed to be formed by hydrophobic interactions,
hydrogen bonds, and ionic attractions, but the mechanism
Research Institute for Food Science, Kyoto University,
Kyoto, Japan.
of formation is still not well understood. Our previous
paper showed
that
the amount of hydrophobic amino acids
in proteins easily forming thermocoagulum differed from
those in proteins forming thermoreversible gel (Shimada
and Matsushita, 1980b). In this paper, we report the ef-
fects of
salts
and denaturants on thermocoagulation of
proteins containing various amounts of hydrophobic amino
acids. Egg albumin which coagulates on heating and bo-
vine serum albumin and soybean protein which gel on
heating were chosen as models in this study.
MATERIALS
AND
METHODS
Materials.
Egg albumin was purchased from Nakarai
Chemicals
Ltd.,
Kyoto. Bovine serum albumin (BSA)
(demineralized) was obtained from Povite Producten N.V.
(Amsterdam, Holland). These proteins were defatted by
acetone before use. Soybean protein solution, prepared
from an aqueous extract of defatted soybean meal, was
powdered by acetone treatment. Other chemicals were
reagent grade.
Heat Treatment and Turbidimetry.
Heat treatment
was carried out with
5
mL of protein solution in a glass
tube (105
X
15
mm). Protein solutions were heated in a
0021-8561/81/1429-0015$01.00/0
0
1981 American Chemical Society