J. Nutr.
CARBON
DIOXIDE-PROTEIN A GAS-SOLID
Sci. Vitaminol.,
21, 151-162,
INTERACTION PHASE
1975
IN
Hisateru MITSUDA,1 Fumio KAWAI,2Aijiro YAMAMOTO,1 and Kenji NAKAJIMA1 1Departmentof Food Scienceand Technology, Facultyof Agriculture, KyotoUniversity,Kyoto 2Laboratoryof FoodScienceand Technology, ResearchInstitutefor ProductionDevelopment, Kyoto (ReceivedMay 17, 1975) Summary
In
under
the
were by
found the
gas
to
was
in
this
to
casein
and
the
while between gas
less in
tyramine failed
gas-solid a
mode phase
gas-liquid
mode
It is generally 1 満 田久 輝
on
the
adsorption
of
interaction
was phase. of
of
the
and
discussed Large
chemical carbon
recognized
between
contribution
The
(free
ex base),
dioxide
dioxide acids
and
(free
protein
results
in
obtained
adsorption
chemisorption
gas
observed
carbon
amino
dioxide the
showed were
were
the
and
physical
dioxide
were
and assumed
interaction.
is one of the nutrients
, 2 河 合文 雄, 1 山 本 愛 二 郎, 1 中 島 謙 二 151
almost
adsorbed
also
carbon
of
with of
or
dioxide-protein
that protein
of
carbon
be
them.
L-arginine
amines
better others
amines
however,
comparing
reaction
gelatin
and
amount
by
the and
gas of
and
carbon
of carbon
of
reversibility
those
be
to
content
differences,
hair of
found
base),
dried
rabbit
to
amount
(free
dioxide
proteins, the
gluten
acids
state
obtained
carbon
dioxide
moisture
a large
and
results
revealed
carbon
foods
solid
pressure
hydrolyzates
Some
proteins
as
was of
L-lysine
so.
purified
partial
amino
dependence by
contribution the
do
of
were
a
of
hemoglobin,
the
adsorbed
to
The
such
adsorption
partial
these,
temperature
The
silk
Amount
greater and
Among
adsorption
bases).
raw
This
the
high
albumin,
depended
is,
the
Oligo-peptides,
and others
and egg
gas.
Peptones
too.
histamine
in
gelatin
gram
protein
in
100-1,000ƒÊl
by
in
of
proteins
materials
placed
with
packaging
gradually.
that
24hr
gelatin
adsorbability.
amined
a
were
dioxide
moisture
increase.
the
for
experiment.
carbon
gas
proteinous
comparing
specific
lower
25•Ž
Casein,
the various
indicated
other
they
gas.
tested
gas
at and
developing
dioxide
manometry
when
adsorbents
of
atmosphere,
carbon
adsorbed
dioxide
by
adsorb
foods silk
course
dioxide
Warburg
protein raw
the
carbon
that
is in critical
152
H.
MITSUDA,
F. KAWAI,
A. YAMAMOTO,
and
K. NAKAJIMA
supply as the demands for meetingthe needs of an ever-increasingpopulation inten sity. The authors have carried out detailed researches in utilizing microbial cells as a source of protein for improving the world's protein food supply in the future (1). Microbial isolated proteins (MIPRON) will be used in future as a raw ingre dient such as dried egg white and defatted soybean meals. They should have the good properties such as high solubility, mild odor, low bacterial count, good qualities for the processing and so on. The major problems faced in the pre servation of these protein foods have been the development of mold growth, dis coloration, loss in texture and spring of unpleasant odor. Mold growth and oxidative deterioration are known to take place primarily in the presence of oxygen. In addition to this, many investigators have reported that dehydrated protein foods were very susceptibleto the attack by the molecular oxygen(2,3). The obvious.solution to these problems would appear to be the packaging and preserving them in the absence of oxygen. In other words, inert gas preservation is effectiveto retain the qualities. Carbon dioxide has been found to have pre servativeproperties at higher pressures than normally encountered in the atmos phere. The authors discovered the carbon dioxide gas adsorption phenomenon of cereal grains and developed a novel technique for skin-packaging,the Carbon Dioxide ExchangeMethod (CEM) (4,5). The principle of this techniquebases on the physical adsorption phenomenon of carbon dioxide gas by cereal grains. Examinations of this adsorption phenomenon indicated the possibilityof the con tribution of capillary condensation at the porous parts of cereal grains (5). In the course of developingthe CEM skin-packagingof protein foods, an additional fact was found that dried protein foods and other proteinous materials also adsorb a fair amount of carbon dioxide gas. In this paper, the results obtained by the CEM skin-packagingtest of protein and the Warburg manometry on the carbon dioxide gas adsorption by proteins are reported and the mode of interaction between carbon dioxide and protein is discussed. EXPERIMENTAL Materials. All materials used in this study were in a solid state. Whole milk was obtained from Meiji Milk Products Co., Ltd. Hydrocarbon-assymilating yeast was presented by Kyowa Hakko Kogyo Co., Ltd. Raw silk was obtained from the Yokohama Raw Silk Inspection Office. Chinese white rabbit hair was obtained from FPEC Ltd. Rice bran was purchased from local rice dealer's shop. Amino acid mixture which was specially presented by Tanabe Seiyaku Co., Ltd. was prepared by mixing the individual amino acid powder in proportion to the amino acid composition of casein (17). Partial hydrolyzates of gelatin were pre pared at the Institute from gelatin powder which was purchased from Yasu
CO2 ADSORPTION Kagaku
Co.,
then
the
Ltd.
lyophilized. 1,000,
fraction
B to
and
prepared
cylinders
is
flask thermal
from an
the
and
barometer
which
pressure.
The
the
pressure, value
of
for
air
content
NaCl of
been
them
and
was
of per
days
in
and
KNO3
for
carbon unit
determined
each
dioxide
a mean
desiccators
air-oven
Sigma
dioxide .
Carbon
gram
the the
of
and
air
cylinder
.
vessel
was
measured
at
in
dioxide
addition
to
carbon an
empty
temperature
carbon were
saturated between at
One
volume
the
was
was of
. in
measuring
used
adsorbed
humidities an
used
. and
were
content with
at relative
were
carbon
for
5min,
in
24hr value
which
system
in
gas
moisture
in
the
changes
per
bacto
materials Ltd
used
from
after
sample the
. Co.,
Ltd.),
purification
protein
18ml
manometers
weight
various the
in
any
derived
usually
measure
from
with
was
air
by
about
gas
pressure
higher than the method
those
liquid
was
gas
of
dioxide
two
to
manometer
14
the
in
used
volume
flask
than
moisture.
method
achieved,
experiment,
was
samples 3 to
Mg(NO3)2,
one
STP)
Protein them
this
0 .05%
dioxide
carbon
has
In
atmosphere
and
by
atmosphere
hour.
carbon
manometric
out
equilibrium
than
lower
.
and
without
.,
Other
Chemical
ethylene
manometric of
the
less
were
and and
Kagaku
Co
furthermore
used
be
Eiyo
Kogyo
media.
purifing
and
to
Daigo
Nakarai
oxygen,
Warburg
in
purged
without
and
of
Laboratories)
from
obtained
desorption
placed
was
used
purity
Difco
trypsin Diafilter
and fraction C to be in our laboratory by
culture
purchased
hydrogen,
The and
protein
all
were
99.95%
Methods.
of
and the
assumed
Seiyaku
bacteriological
commercially
were
adsorption
in
were
nitrogen, were
dioxide
use
They
Helium,
Kyokuto
(products
for
was
(product
of
pepsin
through
A
10 ,000, prepared
Polypeptone
bacto-tryptone
Ltd.
and
153
with
filtration
fraction
were
(product
experiment
Chemical
1,000
(1).
especially
this
of
Chlorella
Kyokuto-peptone
peptone
of
of
PROTEIN
hydrolyzed by
weight
between
cells
previously
Ltd.),
off
be
partially
fractionated
molecular
Bleached
described
was
were
The
10,000.
in
Gelatin
hydrolyzates
BY
(at
and
standard
calculated
gas
prepared
by
11 and
the
After closed
intervals gas
thermo barometric
temperature by
dioxide
solutions
dried in
of 88%
substracting manometers
.
equilibrating LiCl .
, K2CO3, Moisture
105•Ž.
RESULTS
CEM skin-packaging of protein foods and others Packaging and preservation tests have been carried out in order to find the usefulness of the CEM technique which was developed during the packaging and preservation of cereal grains. Various protein such as herring roe , shrimp, "Fu" (light cake of wheat gluten), gelatin powder, casein powder, silk, woolen cloth and so on were packaged with plastic laminated film under the carbon dioxide gas atmosphere. The packages thus obtained became skin-tight when these materials were packaged with the carbon dioxide gas. In Fig. 1, skin-tightness of CEM packages was compared with it of the conventional ones . Most typical
154
H.
difference not
could
only
the
products led
us
The
to
for
be
F. KAWAI,
seen
protein
were
packaged the
product
the
packages
of
but
also
protein
of
preservation
shown
in
"Fu."
As
powder, CEM
temperature
2.
fibers
gas
or that
Boiled
and
4•Ž
in
protein.
a refrigera in
larval
were
their
findings
and
resulted dried
technique
fact, and
These
dioxide
skin-packages
CEM
interesting
technique.
room
the
the
animal
carbon
Fig.
by
dried
the
CEM
packaged
and K. NAKAJIMA
between at
typical
were roe
by
preserved
Some
herring
pure
interaction
were
weeks.
and
A. YAMAMOTO,
skin-tightly mode
skin-packages several
shrimp
in
foods
examine
CEM
tor
MITSUDA,
improved anchovy,
revealed
to
retain
their original colors and appearances, while those packaged by the conventional method
lost
vation
their
was
Adsorption
of
In As
Fig.
shown
while
air under
in
in
various
3,
gas
this
ethylene
proximate
original
retarded
gases volume
figure,
and
also the
same
examined conditions
Color
case
of
CEM
by
protein
adsorbed carbon
oxygen
equilibrium
were
qualities. the
was
by
less
obtained
but in
casein
dioxide
were
none
of
the
case
changes
of
raw
silk
during
the
preser
skin-package.
gas
was was
than
200
after
24hr.
them of
and
was carbon
plotted
adsorbed
against more
time than
50ƒÊl/g/24hr Helium, adsorbed dioxide
in
respectively. nitrogen, considerably
hours.
500ƒÊl/g/24hr Ap
hydrogen by
and casein
gas.
Adsorption of carbon dioxide gas by protein Adsorption of carbon dioxide gas by protein was investigated by the Warburg manometry. The amount of carbon dioxide gas adsorbed by protein was shown in Table 1. Various proteins were found to adsorb carbon dioxide gas when they were placed in the high partial pressure of the gas concerned. Simple protein such as egg albumin, zein, gelatin and conjugated protein such as casein (phos phoprotein), hemoglobin (chromoprotein), protein foods and proteinous materials showed the similar behavior to the carbon dioxide gas. Casein, gelatin and raw silk were revealed to be the better adsorbents comparing with others. Adsorption of carbon dioxide gas by protein hydrolyzates As shown in Table 2, various protein hydrolyzates which have been prepared by hydrolysis of protein with proteinases were revealed to adsorb the considerable amount of carbon dioxide gas. Amounts of carbon dioxide gas adsorbed by peptone and tryptone which were obtained from various companies were almost the same except one, bacto-peptone (Difco). In the series of gelatin partial hydrolyzates, the adsorption amount seemed to be in inverse proportion to the molecular weight of them. Adsorption of carbon dioxide gas by amino acids, peptides and amines Naturally occuring amino acids have been tested for their ability of the carbon dioxide gas adsorption. The results were shown in Table 3. Amino acid mixture which was composed of the casein-like pattern of amino acids adsorbed about
CO2 ADSORPTION
BY PROTEIN
155
Fig. 1. Comparison of the skin-tightness of the packages. Left and middle, conventional packages; right, CEM skin-packages, Materials, (A) Fu, dried (light cake of wheat gluten), (B) woolen cloth, (C) gelatin powder, and (D) casein powder.
156
H. MITSUDA,
F. KAWAI,
A. YAMAMOTO,
and
K. NAKAJIMA
Fig. 2. Some CEM skin-packages that have resulted in improved product preservation. Left, conventional packages; right, CEM skin-packages. Materials, (A) herring roe, (B) shrimp, (C) larval anchovy, boiled and dried, and (D) silk.
CO2 ADSORPTION
Fig.
3.
Adsorption
at
rate
various
gases
by
casein
determined
Determined
1.
by
Adsorption
the
warburg
of carbon
manometry
dioxide
120ƒÊl/g/24hr casein
were
gas
free ƒÃ-amino to
show
listed
in
more the
gas
were
used
they
carbon
of
gas.
at
L-lysine
dioxide
This
amino
25•Ž
the
warburg
manometry
f
could
not they
the and gas
salts
do were of
24hr.
is obviously
tested,
of so.
and
carbon
HCl
as or
.
than
gas
L-lysine
free
that
L-arginine dioxide
Although
used
bases
, they
adsorbed (free
bases)
while
other
and
L-arginine
didn't
adsorb
L-glutamate
guanidino-groups adsorption
lower
L-lysine
200ƒÊl/g/24hr
when as
value
acids
than
Table
dioxide
groups the
the
adsorb
carbon when
dioxide
Among
to
acids
adsorbed the
of carbon itself.
found
amino
by
gas by dried proteins .
or
by
157
25•Ž.
Table
a
of
BY PROTEIN
of Other
. This implied that L-arginine were essential
amino
acids
such
as
DL-
158
H.
MITSUDA,
Table
2.
F. KAWAI,
Adsorption
a Gelatin hydrolyzates
A. YAMAMOTO,
of carbon
were prepared
dioxide
and K . NAKAJIMA
gas by dried protein
hydrolyzates.
by partial hydrolysis with proteinases and they
were fractionated by filtration through Diafilter and lyophilized. Molecular weight assumed are: (A) lower than 1,000, (B) between 1,000 and 10,000, (C) higher than 10,000. Table 3.
Adsorption of carbon dioxide gas by dried amino acid, peptide and amine powders.
a Amino acid mixture was patterned after the amino acid composition of casein (17) . b Negative value means that the value obtained from CO2 gas manometer is higher than that from air one under the condition described in EXPERIMENTAL. methionine, adsorb
L-leucine, carbon
As carbon
for
the
and
gas. - asparagine
gas
glycine
Among
the
Tyramine
base),
L-tryptophan
glycylglycylglycylglycine adsorption
didn't
amines, also
(free
and
L-tyrosine
didn't
gas.
peptides,
dioxide
glycine
L-histidine
dioxide
and
showed
glycylglycylglycine
the
largest
followed
value it
while
of
the
glycyl
adsorb. histamine
adsorb
and ƒÁ-amino-n-butyric
adsorbed more
than acid
didn't
an
extensive
500ƒÊl/g/24hr do
so.
amount while
of
carbon L-glutamine,
dioxide L
CO2 ADSORPTION
BY PROTEIN
159
Dependence on moisture content of the carbon dioxide gas adsorption by protein In Fig. 4, the amount of carbon dioxide gas adsorbed by casein and gelatin have been plotted against the moisture content of them. Gas volume in this figure was shown on dry weight of sample while it was shown on wet weight in other figures and tables. The lower the moisture is, the greater the adsorption amount of carbon dioxide increases.
Fig. 4. Effect of moisture and gelatin.
content
on the amount
of carbon
dioxide
gas adsorbed
by casein
Reversibility of the carbon dioxide gas adsorption Reversibility of the carbon dioxide gas adsorption by casein, gelatin, L-lysine (free base), L-arginine (free base) and histamine was examined. More than 90% of carbon dioxide gas which had been adsorbed by casein and gelatin desorbed when they were put into the atmosphere of low partial pressure of carbon dioxide but only a few per cent desorbed in the case of amino acid (free base) and histamine . DISCUSSION The studies on the carbon dioxide-protein interaction have been done mainly in a gas-liquid phase and a few report was presented about the studies in a gas solid phase. In some cases, the results obtained in a gas-liquid phase might be applied to those in a gas-solid phase. In a gas-liquid phase, carbon dioxide is thought to combine with free amino groups of proteins. This has been demonstrated with hemoglobin (7), and it is possible that the inhibition of plant succinic oxidase by high partial pressure of carbon dioxide (8) occurs by an analogous mechanism. Catalatic activity was also reported to be sensitive to carbon dioxide. The probability that the inhibition was due to the action of unhydrated and unionized carbon dioxide ,
160
H. MITSUDA,
was
presented
reaction
by
of
groups
of
F. KAWAI,
using
carbon
carbonic
dioxide
aliphatic
A. YAMAMOTO, anhydrase
with
amines,
(9).
ammonia,
amino
acids
According
and and
and K. NAKAJIMA to
primary
ROUGHTON
and
(10),
secondary
amino
proteins,
RR•LNH+CO2_??_RR•LNCOO-+H+ is
rapid,
more
requires
no
Reaction acid,
species
by
a
of
tion
the
is:
the
by
of
carbon proteins. The
be,
gas
dioxide with
there
directly
to
no
carbon
of
to
carbonic
be
examined
dioxide
gas
dioxide
gas
carbon
that
thought
Growe
by
and
groups.
groups.
been
moisture.
acid,
amino
evidence
amino
has
carbonic
uncharged
was
with
gas
hydration
of
value
and
partial
proteins
and
remarkably acid
absorbed
a
"snugging
packaging, (11).
and
The
reac
different
from gas
gas
(free
base),
that
of
adsorbed
temperature to was
pressure
of gelatin
amino
this,
protein
not
desorbed
carbon was acids
carbon
dioxide
in
gas
which
more
(free
seemed
the
be
amines
and
90%
"chemical
of
and
the
reaction"
gas
protein,
guanidino amounts (free
was
of
increased and amino the
gas or
carbon as
the
gelatin. acids low
adsorbed
dioxide
base)
somewhat
amount
in
be are
carbamation
large
placed
carbon
to
groups by
the
bases) casein
were
designed
adsorption
of
tested,
peptones
L-lysine
The
in
carbon
acids
Although
(free
case by
between to
lysine
gelatin.
materials than
of
tyramine,
acids
adsorbed
these
Interaction
and amino
adsorption.
adsorption by
the
which simple
amino
directly
acids.
of
in
was
group
amino
those
with
free
of
So the
adsorb
comparing
gas
histamine,
casein and
when
desorbed. bases)
as
decreased
dioxide even
all by
amines it
dioxide
dependence
such
these while
the
of
adsorbed
amino
explained
as ƒÃ-amino
temperature
by
increased,
addition
such
to
the
Generally
case
hydration
is observed
tend
gas
that
adsorption
their
gelatin
composition
next.
carbon be
of
which
Among
dioxide
which on
the
group
were
of do.
the
3 are
content.
tendency
to
the gas
1, 2 and
characteristics
carbon of
that
dioxide
Tables
adsorption
opposite in
imply
moisture
acids
cannot
protein
carbon
the
gas is
obtained
the
hydrolyzates amino
to
study
and ƒ¿-amino
dioxide
clear
situated
greatly this
results This
in
consider
dioxide
tendency
(5).
arbitrary
to
mixture base)
of
shown
adsorbs
(free
the
the
of
present
admitted
than
groups
cause
gas
Peptones
in
rice
in the
doesn't
quite
to
paddy
main
each
contribute
L-arginine
the
tested
arginine
carbon
not
carbon
but
similar by
dioxide
obtained
amino
dioxide
is
that
samples
quite
carbon
base)
to
results free
gas
L-arginine
expected
is
adsorption
Amino
and
clearly of
materials
proteins.
casein
indicates content
This
of
more
(free
4
gas
however,
values.
L-lysine
and
and
produced
the
the
by of
may
dioxide
and
CO2,
dioxide
was
Fig.
amount
comparison
bases)
only
conteins
by
moisture
dioxide
obtained
and
carbon
reacted
which caused
dioxide
by
group
be
carbon that
Growe.
carbon
but
to
carbonate
film
study,
assumed
and
reported
of occurs
CO2+H2O_??_H2CO3
on
these
hydration reaction
phase,
it is
this
depends
are
the
Direct
product
that
In
the
or
any
estimated
of
were
gas-solid
almost
effect"
of
than
bicarbonate, In
It
rapid
catalyst.
by and
"chemisorption"
In (free partial casein amines
CO2 ADSORPTION
BY PROTEIN
161
rather than "physical adsorption." This may also indicates that reactivity of car bon dioxide with free amino groups in amino acids, peptides, amines and proteins are differentwith each others in a gas-solidphase. It is reported that amino acid carbamates obtained in a gas-liquid phase are not always stable. On exposure to air at room or at elevated temperatures, they liquefy owing to their hygroscopic nature, and decomposewith visible evolution of carbon dioxide gas (12). A little reversibilityfound in the case of amino acid (free base) and histamine may be caused by this decomposition. Quantitative analysis of amines and free amino groups in the protein samples tested in this study might present another in formation about the interaction between carbon dioxide gas and proteins. Dependenceon moisture content of the carbon dioxide adsorption by protein calls us attention to the similar interaction between molecular oxygen and dehy drated foods. In the latter case, monomolecularlayer of moisture is assumed to prevent the attack of molecular oxygen to the various active groups and free radicals in the dehydrated foods (13). On the other hand, RIEPEand WANG(14, 15) examined the infrared spectra of carbonic anhydrase solutions equilibrated with CO2+N2O mixtures and infered that the CO2 (or N2O) is bound in a hydrophobic cavity of the protein. SCHULTZ and RANDALL (16) reported the interestingpaper in which they examined the distributioncoefficientsfor alcohols and estersbetween the liquidcarbon dioxide and water. They stated that as the molecular weight (namely,number of carbon atoms) increase within a homologous series, a greater part of the solute tend to concentrate in the liquid carbon dioxide phase. This higher affinity of liquid carbon dioxide to the carbon chain of alcohols and esters suggeststhe possibility of hydrophobic interaction between carbon dioxide gas and peptide chain of protein in the carbon dioxide gas adsorption by proteins. A conclusioncannot be drawn due to an insufficientnumber of experimentalsamples,results obtained with the carbon dioxide gas adsorption by glycine,glycylglycine,glycylglycylglycine and glycylglycylglycylglycine showed the possibilityof this interaction. REFERENCES 1) 2) 3) 4) 5) 6) 7) 8) 9) 10)
MITSUDA, H., J. Nutr. Sci. Vitaminol.,19, 1 (1973). SIDWELL, C. G., SALWIN, H., and KOCH,R. B., J. Food Sci., 27, 255 (1962). HALLER, H. S. and HOLM,G. E., J. Dairy Sci., 30, 197 (1947). MITSUDA,H., KAWAI,F., KUGA,M., and YAMAMOTO, A., J. Japanese Soc. Food Nutr., 25, 627 (1972). MITSUDA, H., KAWAI,F., KUGA,M., and YAMAMOTO, A., J. Nutr. Sci. Vitaminol., 19, 71 (1973). MITSUDA, H., KAWAI,F., and YAMAMOTO, A., Mem. Coll. Agr. Kyoto Univ.,100, 49 (1971). WYMAN, Jr. J., Advances in Protein Chem., 4, 485 (1948). RAMSON, S. L., WALKER, D..A., and CLARKE, I. D., Biochem.J., 66, 57 (1971). MITSUDA, H., KAWAI,F., YASUMOTO, K., and HIROTANI, K., Bull. Inst. Chem.Research, Kyoto Univ., 36, 145 (1958). ROUGHTON F. J. W., Harvey Lect., 39, 96 (1943).
162
11) 12) 13) 14) 15) 16) 17)
H. MITSUDA,
F. KAWAI,
A. YAMAMOTO,
and K.
NAKAJIMA
GROWE,T. H., Modem Packaging, Oct., 183 (1969). BAILEY, J. L., J. Chem. Soc., 1950, 3461. SALWIN,H., Food Technol.,13, 594 (1959). RIEPE,M. E. and WANG,J. H., J. Amer. Chem.Soc., 89, 4229 (1967). RIEPE,M. E. and WANG,J. H., J. Biol. Chem., 243, 2779 (1968). SCHULTZ, W. G. and RANDALL, J. M., Food Technol.,24, 1282 (1970). ITOH,H., KISHI,T., EMA,M., and CHIBATA, I., J. Nutr. Sci. Vitaminol.,20, 431 (1974).
CARBON
DIOXIDE-PROTEIN A GAS-SOLID
Sci. Vitaminol.,
21, 151-162,
INTERACTION PHASE
1975
IN
Hisateru MITSUDA,1 Fumio KAWAI,2Aijiro YAMAMOTO,1 and Kenji NAKAJIMA1 1Departmentof Food Scienceand Technology, Facultyof Agriculture, KyotoUniversity,Kyoto 2Laboratoryof FoodScienceand Technology, ResearchInstitutefor ProductionDevelopment, Kyoto (ReceivedMay 17, 1975) Summary
In
under
the
were by
found the
gas
to
was
in
this
to
casein
and
the
while between gas
less in
tyramine failed
gas-solid a
mode phase
gas-liquid
mode
It is generally 1 満 田久 輝
on
the
adsorption
of
interaction
was phase. of
of
the
and
discussed Large
chemical carbon
recognized
between
contribution
The
(free
ex base),
dioxide
dioxide acids
and
(free
protein
results
in
obtained
adsorption
chemisorption
gas
observed
carbon
amino
dioxide the
showed were
were
the
and
physical
dioxide
were
and assumed
interaction.
is one of the nutrients
, 2 河 合文 雄, 1 山 本 愛 二 郎, 1 中 島 謙 二 151
almost
adsorbed
also
carbon
of
with of
or
dioxide-protein
that protein
of
carbon
be
them.
L-arginine
amines
better others
amines
however,
comparing
reaction
gelatin
and
amount
by
the and
gas of
and
carbon
of carbon
of
reversibility
those
be
to
content
differences,
hair of
found
base),
dried
rabbit
to
amount
(free
dioxide
proteins, the
gluten
acids
state
obtained
carbon
dioxide
moisture
a large
and
results
revealed
carbon
foods
solid
pressure
hydrolyzates
Some
proteins
as
was of
L-lysine
so.
purified
partial
amino
dependence by
contribution the
do
of
were
a
of
hemoglobin,
the
adsorbed
to
The
such
adsorption
partial
these,
temperature
The
silk
Amount
greater and
Among
adsorption
bases).
raw
This
the
high
albumin,
depended
is,
the
Oligo-peptides,
and others
and egg
gas.
Peptones
too.
histamine
in
gelatin
gram
protein
in
100-1,000ƒÊl
by
in
of
proteins
materials
placed
with
packaging
gradually.
that
24hr
gelatin
adsorbability.
amined
a
were
dioxide
moisture
increase.
the
for
experiment.
carbon
gas
proteinous
comparing
specific
lower
25•Ž
Casein,
the various
indicated
other
they
gas.
tested
gas
at and
developing
dioxide
manometry
when
adsorbents
of
atmosphere,
carbon
adsorbed
dioxide
by
adsorb
foods silk
course
dioxide
Warburg
protein raw
the
carbon
that
is in critical
152
H.
MITSUDA,
F. KAWAI,
A. YAMAMOTO,
and
K. NAKAJIMA
supply as the demands for meetingthe needs of an ever-increasingpopulation inten sity. The authors have carried out detailed researches in utilizing microbial cells as a source of protein for improving the world's protein food supply in the future (1). Microbial isolated proteins (MIPRON) will be used in future as a raw ingre dient such as dried egg white and defatted soybean meals. They should have the good properties such as high solubility, mild odor, low bacterial count, good qualities for the processing and so on. The major problems faced in the pre servation of these protein foods have been the development of mold growth, dis coloration, loss in texture and spring of unpleasant odor. Mold growth and oxidative deterioration are known to take place primarily in the presence of oxygen. In addition to this, many investigators have reported that dehydrated protein foods were very susceptibleto the attack by the molecular oxygen(2,3). The obvious.solution to these problems would appear to be the packaging and preserving them in the absence of oxygen. In other words, inert gas preservation is effectiveto retain the qualities. Carbon dioxide has been found to have pre servativeproperties at higher pressures than normally encountered in the atmos phere. The authors discovered the carbon dioxide gas adsorption phenomenon of cereal grains and developed a novel technique for skin-packaging,the Carbon Dioxide ExchangeMethod (CEM) (4,5). The principle of this techniquebases on the physical adsorption phenomenon of carbon dioxide gas by cereal grains. Examinations of this adsorption phenomenon indicated the possibilityof the con tribution of capillary condensation at the porous parts of cereal grains (5). In the course of developingthe CEM skin-packagingof protein foods, an additional fact was found that dried protein foods and other proteinous materials also adsorb a fair amount of carbon dioxide gas. In this paper, the results obtained by the CEM skin-packagingtest of protein and the Warburg manometry on the carbon dioxide gas adsorption by proteins are reported and the mode of interaction between carbon dioxide and protein is discussed. EXPERIMENTAL Materials. All materials used in this study were in a solid state. Whole milk was obtained from Meiji Milk Products Co., Ltd. Hydrocarbon-assymilating yeast was presented by Kyowa Hakko Kogyo Co., Ltd. Raw silk was obtained from the Yokohama Raw Silk Inspection Office. Chinese white rabbit hair was obtained from FPEC Ltd. Rice bran was purchased from local rice dealer's shop. Amino acid mixture which was specially presented by Tanabe Seiyaku Co., Ltd. was prepared by mixing the individual amino acid powder in proportion to the amino acid composition of casein (17). Partial hydrolyzates of gelatin were pre pared at the Institute from gelatin powder which was purchased from Yasu
CO2 ADSORPTION Kagaku
Co.,
then
the
Ltd.
lyophilized. 1,000,
fraction
B to
and
prepared
cylinders
is
flask thermal
from an
the
and
barometer
which
pressure.
The
the
pressure, value
of
for
air
content
NaCl of
been
them
and
was
of per
days
in
and
KNO3
for
carbon unit
determined
each
dioxide
a mean
desiccators
air-oven
Sigma
dioxide .
Carbon
gram
the the
of
and
air
cylinder
.
vessel
was
measured
at
in
dioxide
addition
to
carbon an
empty
temperature
carbon were
saturated between at
One
volume
the
was
was of
. in
measuring
used
adsorbed
humidities an
used
. and
were
content with
at relative
were
carbon
for
5min,
in
24hr value
which
system
in
gas
moisture
in
the
changes
per
bacto
materials Ltd
used
from
after
sample the
. Co.,
Ltd.),
purification
protein
18ml
manometers
weight
various the
in
any
derived
usually
measure
from
with
was
air
by
about
gas
pressure
higher than the method
those
liquid
was
gas
of
dioxide
two
to
manometer
14
the
in
used
volume
flask
than
moisture.
method
achieved,
experiment,
was
samples 3 to
Mg(NO3)2,
one
STP)
Protein them
this
0 .05%
dioxide
carbon
has
In
atmosphere
and
by
atmosphere
hour.
carbon
manometric
out
equilibrium
than
lower
.
and
without
.,
Other
Chemical
ethylene
manometric of
the
less
were
and and
Kagaku
Co
furthermore
used
be
Eiyo
Kogyo
media.
purifing
and
to
Daigo
Nakarai
oxygen,
Warburg
in
purged
without
and
of
Laboratories)
from
obtained
desorption
placed
was
used
purity
Difco
trypsin Diafilter
and fraction C to be in our laboratory by
culture
purchased
hydrogen,
The and
protein
all
were
99.95%
Methods.
of
and the
assumed
Seiyaku
bacteriological
commercially
were
adsorption
in
were
nitrogen, were
dioxide
use
They
Helium,
Kyokuto
(products
for
was
(product
of
pepsin
through
A
10 ,000, prepared
Polypeptone
bacto-tryptone
Ltd.
and
153
with
filtration
fraction
were
(product
experiment
Chemical
1,000
(1).
especially
this
of
Chlorella
Kyokuto-peptone
peptone
of
of
PROTEIN
hydrolyzed by
weight
between
cells
previously
Ltd.),
off
be
partially
fractionated
molecular
Bleached
described
was
were
The
10,000.
in
Gelatin
hydrolyzates
BY
(at
and
standard
calculated
gas
prepared
by
11 and
the
After closed
intervals gas
thermo barometric
temperature by
dioxide
solutions
dried in
of 88%
substracting manometers
.
equilibrating LiCl .
, K2CO3, Moisture
105•Ž.
RESULTS
CEM skin-packaging of protein foods and others Packaging and preservation tests have been carried out in order to find the usefulness of the CEM technique which was developed during the packaging and preservation of cereal grains. Various protein such as herring roe , shrimp, "Fu" (light cake of wheat gluten), gelatin powder, casein powder, silk, woolen cloth and so on were packaged with plastic laminated film under the carbon dioxide gas atmosphere. The packages thus obtained became skin-tight when these materials were packaged with the carbon dioxide gas. In Fig. 1, skin-tightness of CEM packages was compared with it of the conventional ones . Most typical
154
H.
difference not
could
only
the
products led
us
The
to
for
be
F. KAWAI,
seen
protein
were
packaged the
product
the
packages
of
but
also
protein
of
preservation
shown
in
"Fu."
As
powder, CEM
temperature
2.
fibers
gas
or that
Boiled
and
4•Ž
in
protein.
a refrigera in
larval
were
their
findings
and
resulted dried
technique
fact, and
These
dioxide
skin-packages
CEM
interesting
technique.
room
the
the
animal
carbon
Fig.
by
dried
the
CEM
packaged
and K. NAKAJIMA
between at
typical
were roe
by
preserved
Some
herring
pure
interaction
were
weeks.
and
A. YAMAMOTO,
skin-tightly mode
skin-packages several
shrimp
in
foods
examine
CEM
tor
MITSUDA,
improved anchovy,
revealed
to
retain
their original colors and appearances, while those packaged by the conventional method
lost
vation
their
was
Adsorption
of
In As
Fig.
shown
while
air under
in
in
various
3,
gas
this
ethylene
proximate
original
retarded
gases volume
figure,
and
also the
same
examined conditions
Color
case
of
CEM
by
protein
adsorbed carbon
oxygen
equilibrium
were
qualities. the
was
by
less
obtained
but in
casein
dioxide
were
none
of
the
case
changes
of
raw
silk
during
the
preser
skin-package.
gas
was was
than
200
after
24hr.
them of
and
was carbon
plotted
adsorbed
against more
time than
50ƒÊl/g/24hr Helium, adsorbed dioxide
in
respectively. nitrogen, considerably
hours.
500ƒÊl/g/24hr Ap
hydrogen by
and casein
gas.
Adsorption of carbon dioxide gas by protein Adsorption of carbon dioxide gas by protein was investigated by the Warburg manometry. The amount of carbon dioxide gas adsorbed by protein was shown in Table 1. Various proteins were found to adsorb carbon dioxide gas when they were placed in the high partial pressure of the gas concerned. Simple protein such as egg albumin, zein, gelatin and conjugated protein such as casein (phos phoprotein), hemoglobin (chromoprotein), protein foods and proteinous materials showed the similar behavior to the carbon dioxide gas. Casein, gelatin and raw silk were revealed to be the better adsorbents comparing with others. Adsorption of carbon dioxide gas by protein hydrolyzates As shown in Table 2, various protein hydrolyzates which have been prepared by hydrolysis of protein with proteinases were revealed to adsorb the considerable amount of carbon dioxide gas. Amounts of carbon dioxide gas adsorbed by peptone and tryptone which were obtained from various companies were almost the same except one, bacto-peptone (Difco). In the series of gelatin partial hydrolyzates, the adsorption amount seemed to be in inverse proportion to the molecular weight of them. Adsorption of carbon dioxide gas by amino acids, peptides and amines Naturally occuring amino acids have been tested for their ability of the carbon dioxide gas adsorption. The results were shown in Table 3. Amino acid mixture which was composed of the casein-like pattern of amino acids adsorbed about
CO2 ADSORPTION
BY PROTEIN
155
Fig. 1. Comparison of the skin-tightness of the packages. Left and middle, conventional packages; right, CEM skin-packages, Materials, (A) Fu, dried (light cake of wheat gluten), (B) woolen cloth, (C) gelatin powder, and (D) casein powder.
156
H. MITSUDA,
F. KAWAI,
A. YAMAMOTO,
and
K. NAKAJIMA
Fig. 2. Some CEM skin-packages that have resulted in improved product preservation. Left, conventional packages; right, CEM skin-packages. Materials, (A) herring roe, (B) shrimp, (C) larval anchovy, boiled and dried, and (D) silk.
CO2 ADSORPTION
Fig.
3.
Adsorption
at
rate
various
gases
by
casein
determined
Determined
1.
by
Adsorption
the
warburg
of carbon
manometry
dioxide
120ƒÊl/g/24hr casein
were
gas
free ƒÃ-amino to
show
listed
in
more the
gas
were
used
they
carbon
of
gas.
at
L-lysine
dioxide
This
amino
25•Ž
the
warburg
manometry
f
could
not they
the and gas
salts
do were of
24hr.
is obviously
tested,
of so.
and
carbon
HCl
as or
.
than
gas
L-lysine
free
that
L-arginine dioxide
Although
used
bases
, they
adsorbed (free
bases)
while
other
and
L-arginine
didn't
adsorb
L-glutamate
guanidino-groups adsorption
lower
L-lysine
200ƒÊl/g/24hr
when as
value
acids
than
Table
dioxide
groups the
the
adsorb
carbon when
dioxide
Among
to
acids
adsorbed the
of carbon itself.
found
amino
by
gas by dried proteins .
or
by
157
25•Ž.
Table
a
of
BY PROTEIN
of Other
. This implied that L-arginine were essential
amino
acids
such
as
DL-
158
H.
MITSUDA,
Table
2.
F. KAWAI,
Adsorption
a Gelatin hydrolyzates
A. YAMAMOTO,
of carbon
were prepared
dioxide
and K . NAKAJIMA
gas by dried protein
hydrolyzates.
by partial hydrolysis with proteinases and they
were fractionated by filtration through Diafilter and lyophilized. Molecular weight assumed are: (A) lower than 1,000, (B) between 1,000 and 10,000, (C) higher than 10,000. Table 3.
Adsorption of carbon dioxide gas by dried amino acid, peptide and amine powders.
a Amino acid mixture was patterned after the amino acid composition of casein (17) . b Negative value means that the value obtained from CO2 gas manometer is higher than that from air one under the condition described in EXPERIMENTAL. methionine, adsorb
L-leucine, carbon
As carbon
for
the
and
gas. - asparagine
gas
glycine
Among
the
Tyramine
base),
L-tryptophan
glycylglycylglycylglycine adsorption
didn't
amines, also
(free
and
L-tyrosine
didn't
gas.
peptides,
dioxide
glycine
L-histidine
dioxide
and
showed
glycylglycylglycine
the
largest
followed
value it
while
of
the
glycyl
adsorb. histamine
adsorb
and ƒÁ-amino-n-butyric
adsorbed more
than acid
didn't
an
extensive
500ƒÊl/g/24hr do
so.
amount while
of
carbon L-glutamine,
dioxide L
CO2 ADSORPTION
BY PROTEIN
159
Dependence on moisture content of the carbon dioxide gas adsorption by protein In Fig. 4, the amount of carbon dioxide gas adsorbed by casein and gelatin have been plotted against the moisture content of them. Gas volume in this figure was shown on dry weight of sample while it was shown on wet weight in other figures and tables. The lower the moisture is, the greater the adsorption amount of carbon dioxide increases.
Fig. 4. Effect of moisture and gelatin.
content
on the amount
of carbon
dioxide
gas adsorbed
by casein
Reversibility of the carbon dioxide gas adsorption Reversibility of the carbon dioxide gas adsorption by casein, gelatin, L-lysine (free base), L-arginine (free base) and histamine was examined. More than 90% of carbon dioxide gas which had been adsorbed by casein and gelatin desorbed when they were put into the atmosphere of low partial pressure of carbon dioxide but only a few per cent desorbed in the case of amino acid (free base) and histamine . DISCUSSION The studies on the carbon dioxide-protein interaction have been done mainly in a gas-liquid phase and a few report was presented about the studies in a gas solid phase. In some cases, the results obtained in a gas-liquid phase might be applied to those in a gas-solid phase. In a gas-liquid phase, carbon dioxide is thought to combine with free amino groups of proteins. This has been demonstrated with hemoglobin (7), and it is possible that the inhibition of plant succinic oxidase by high partial pressure of carbon dioxide (8) occurs by an analogous mechanism. Catalatic activity was also reported to be sensitive to carbon dioxide. The probability that the inhibition was due to the action of unhydrated and unionized carbon dioxide ,
160
H. MITSUDA,
was
presented
reaction
by
of
groups
of
F. KAWAI,
using
carbon
carbonic
dioxide
aliphatic
A. YAMAMOTO, anhydrase
with
amines,
(9).
ammonia,
amino
acids
According
and and
and K. NAKAJIMA to
primary
ROUGHTON
and
(10),
secondary
amino
proteins,
RR•LNH+CO2_??_RR•LNCOO-+H+ is
rapid,
more
requires
no
Reaction acid,
species
by
a
of
tion
the
is:
the
by
of
carbon proteins. The
be,
gas
dioxide with
there
directly
to
no
carbon
of
to
carbonic
be
examined
dioxide
gas
dioxide
gas
carbon
that
thought
Growe
by
and
groups.
groups.
been
moisture.
acid,
amino
evidence
amino
has
carbonic
uncharged
was
with
gas
hydration
of
value
and
partial
proteins
and
remarkably acid
absorbed
a
"snugging
packaging, (11).
and
The
reac
different
from gas
gas
(free
base),
that
of
adsorbed
temperature to was
pressure
of gelatin
amino
this,
protein
not
desorbed
carbon was acids
carbon
dioxide
in
gas
which
more
(free
seemed
the
be
amines
and
90%
"chemical
of
and
the
reaction"
gas
protein,
guanidino amounts (free
was
of
increased and amino the
gas or
carbon as
the
gelatin. acids low
adsorbed
dioxide
base)
somewhat
amount
in
be are
carbamation
large
placed
carbon
to
groups by
the
bases) casein
were
designed
adsorption
of
tested,
peptones
L-lysine
The
in
carbon
acids
Although
(free
case by
between to
lysine
gelatin.
materials than
of
tyramine,
acids
adsorbed
these
Interaction
and amino
adsorption.
adsorption by
the
which simple
amino
directly
acids.
of
in
was
group
amino
those
with
free
of
So the
adsorb
comparing
gas
histamine,
casein and
when
desorbed. bases)
as
decreased
dioxide even
all by
amines it
dioxide
dependence
such
these while
the
of
adsorbed
amino
explained
as ƒÃ-amino
temperature
by
increased,
addition
such
to
the
Generally
case
hydration
is observed
tend
gas
that
adsorption
their
gelatin
composition
next.
carbon be
of
which
Among
dioxide
which on
the
group
were
of do.
the
3 are
content.
tendency
to
the gas
1, 2 and
characteristics
carbon of
that
dioxide
Tables
adsorption
opposite in
imply
moisture
acids
cannot
protein
carbon
the
gas is
obtained
the
hydrolyzates amino
to
study
and ƒ¿-amino
dioxide
clear
situated
greatly this
results This
in
consider
dioxide
tendency
(5).
arbitrary
to
mixture base)
of
shown
adsorbs
(free
the
the
of
present
admitted
than
groups
cause
gas
Peptones
in
rice
in the
doesn't
quite
to
paddy
main
each
contribute
L-arginine
the
tested
arginine
carbon
not
carbon
but
similar by
dioxide
obtained
amino
dioxide
is
that
samples
quite
carbon
base)
to
results free
gas
L-arginine
expected
is
adsorption
Amino
and
clearly of
materials
proteins.
casein
indicates content
This
of
more
(free
4
gas
however,
values.
L-lysine
and
and
produced
the
the
by of
may
dioxide
and
CO2,
dioxide
was
Fig.
amount
comparison
bases)
only
conteins
by
moisture
dioxide
obtained
and
carbon
reacted
which caused
dioxide
by
group
be
carbon that
Growe.
carbon
but
to
carbonate
film
study,
assumed
and
reported
of occurs
CO2+H2O_??_H2CO3
on
these
hydration reaction
phase,
it is
this
depends
are
the
Direct
product
that
In
the
or
any
estimated
of
were
gas-solid
almost
effect"
of
than
bicarbonate, In
It
rapid
catalyst.
by and
"chemisorption"
In (free partial casein amines
CO2 ADSORPTION
BY PROTEIN
161
rather than "physical adsorption." This may also indicates that reactivity of car bon dioxide with free amino groups in amino acids, peptides, amines and proteins are differentwith each others in a gas-solidphase. It is reported that amino acid carbamates obtained in a gas-liquid phase are not always stable. On exposure to air at room or at elevated temperatures, they liquefy owing to their hygroscopic nature, and decomposewith visible evolution of carbon dioxide gas (12). A little reversibilityfound in the case of amino acid (free base) and histamine may be caused by this decomposition. Quantitative analysis of amines and free amino groups in the protein samples tested in this study might present another in formation about the interaction between carbon dioxide gas and proteins. Dependenceon moisture content of the carbon dioxide adsorption by protein calls us attention to the similar interaction between molecular oxygen and dehy drated foods. In the latter case, monomolecularlayer of moisture is assumed to prevent the attack of molecular oxygen to the various active groups and free radicals in the dehydrated foods (13). On the other hand, RIEPEand WANG(14, 15) examined the infrared spectra of carbonic anhydrase solutions equilibrated with CO2+N2O mixtures and infered that the CO2 (or N2O) is bound in a hydrophobic cavity of the protein. SCHULTZ and RANDALL (16) reported the interestingpaper in which they examined the distributioncoefficientsfor alcohols and estersbetween the liquidcarbon dioxide and water. They stated that as the molecular weight (namely,number of carbon atoms) increase within a homologous series, a greater part of the solute tend to concentrate in the liquid carbon dioxide phase. This higher affinity of liquid carbon dioxide to the carbon chain of alcohols and esters suggeststhe possibility of hydrophobic interaction between carbon dioxide gas and peptide chain of protein in the carbon dioxide gas adsorption by proteins. A conclusioncannot be drawn due to an insufficientnumber of experimentalsamples,results obtained with the carbon dioxide gas adsorption by glycine,glycylglycine,glycylglycylglycine and glycylglycylglycylglycine showed the possibilityof this interaction. REFERENCES 1) 2) 3) 4) 5) 6) 7) 8) 9) 10)
MITSUDA, H., J. Nutr. Sci. Vitaminol.,19, 1 (1973). SIDWELL, C. G., SALWIN, H., and KOCH,R. B., J. Food Sci., 27, 255 (1962). HALLER, H. S. and HOLM,G. E., J. Dairy Sci., 30, 197 (1947). MITSUDA,H., KAWAI,F., KUGA,M., and YAMAMOTO, A., J. Japanese Soc. Food Nutr., 25, 627 (1972). MITSUDA, H., KAWAI,F., KUGA,M., and YAMAMOTO, A., J. Nutr. Sci. Vitaminol., 19, 71 (1973). MITSUDA, H., KAWAI,F., and YAMAMOTO, A., Mem. Coll. Agr. Kyoto Univ.,100, 49 (1971). WYMAN, Jr. J., Advances in Protein Chem., 4, 485 (1948). RAMSON, S. L., WALKER, D..A., and CLARKE, I. D., Biochem.J., 66, 57 (1971). MITSUDA, H., KAWAI,F., YASUMOTO, K., and HIROTANI, K., Bull. Inst. Chem.Research, Kyoto Univ., 36, 145 (1958). ROUGHTON F. J. W., Harvey Lect., 39, 96 (1943).
162
11) 12) 13) 14) 15) 16) 17)
H. MITSUDA,
F. KAWAI,
A. YAMAMOTO,
and K.
NAKAJIMA
GROWE,T. H., Modem Packaging, Oct., 183 (1969). BAILEY, J. L., J. Chem. Soc., 1950, 3461. SALWIN,H., Food Technol.,13, 594 (1959). RIEPE,M. E. and WANG,J. H., J. Amer. Chem.Soc., 89, 4229 (1967). RIEPE,M. E. and WANG,J. H., J. Biol. Chem., 243, 2779 (1968). SCHULTZ, W. G. and RANDALL, J. M., Food Technol.,24, 1282 (1970). ITOH,H., KISHI,T., EMA,M., and CHIBATA, I., J. Nutr. Sci. Vitaminol.,20, 431 (1974).
