U.S. patent application number 14/002849 was filed with the patent office on 2014-07-31 for ice crystallization inhibitor derived from plant seed.
The applicant listed for this patent is Naoki Arai, Hidehisa Kawahara, Hideaki Kegasa. Invention is credited to Naoki Arai, Hidehisa Kawahara, Hideaki Kegasa.
Application Number | 20140213663 14/002849 |
Document ID | / |
Family ID | 45873168 |
Filed Date | 2014-07-31 |
United States Patent
Application |
20140213663 |
Kind Code |
A1 |
Kawahara; Hidehisa ; et
al. |
July 31, 2014 |
ICE CRYSTALLIZATION INHIBITOR DERIVED FROM PLANT SEED
Abstract
The objective to be solved by the present invention is to
provide a novel ice crystallization inhibitor which is industrially
useful, which can be efficiently and stably produced in a safe
process suitable for a food production without difficulty and which
has excellent functions and properties. Also, the objective of the
present invention is to provide an antibody which specifically
reacts with the ice crystallization inhibitor, and a composition, a
food, a biological sample protectant and a cosmetic which contain
the ice crystallization inhibitor. Furthermore, the objective of
the present invention is to provide a peptide which gives an
indication of a protein having an ice crystallization inhibitory
activity. The ice crystallization inhibitor according to the
present invention is characterized in comprising a seed protein
derived from a plant belonging to genus Vigna in Leguminosae, an
allied species thereof or an improved species thereof. The ice
crystallization inhibitor according to the present invention is
characterized in comprising a seed protein derived from a plant
belonging to genus Vigna in Leguminosae, an allied species thereof
or an improved species thereof.
Inventors: |
Kawahara; Hidehisa;
(Suita-shi, JP) ; Kegasa; Hideaki; (Takasago-shi,
JP) ; Arai; Naoki; (Takasago-shi, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Kawahara; Hidehisa
Kegasa; Hideaki
Arai; Naoki |
Suita-shi
Takasago-shi
Takasago-shi |
|
JP
JP
JP |
|
|
Family ID: |
45873168 |
Appl. No.: |
14/002849 |
Filed: |
March 2, 2012 |
PCT Filed: |
March 2, 2012 |
PCT NO: |
PCT/JP2012/055460 |
371 Date: |
September 3, 2013 |
Current U.S.
Class: |
514/773 ; 252/70;
404/17; 405/263; 426/321; 426/654; 435/1.1; 435/2; 530/324;
530/327; 530/389.1 |
Current CPC
Class: |
A23L 3/37 20130101; H04N
5/2254 20130101; E01C 21/00 20130101; G02B 13/0095 20130101; G02B
13/14 20130101; G02B 26/0875 20130101; C09K 5/20 20130101; H04N
5/349 20130101; H01L 27/14621 20130101; H01L 27/14625 20130101;
G02B 3/0031 20130101 |
Class at
Publication: |
514/773 ; 252/70;
530/324; 530/327; 530/389.1; 435/1.1; 435/2; 426/321; 426/654;
404/17; 405/263 |
International
Class: |
C09K 5/20 20060101
C09K005/20; E01C 21/00 20060101 E01C021/00; A23L 3/37 20060101
A23L003/37 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 4, 2011 |
FR |
1151790 |
Claims
1-13. (canceled)
14. A method for inhibiting ice crystallization, comprising the
step of adding a seed protein derived from a plant belonging to
genus Vigna in Leguminosae, an allied species thereof or an
improved species thereof.
15. The method according to claim 14, wherein the seed protein is
added to a food.
16. The method according to claim 14, wherein the seed protein is
added as a cryoprotective agent to movable part of machinery.
17. The method according to claim 14, wherein the seed protein is
added as a cryoprotective agent to road.
18. The method according to claim 14, wherein the seed protein is
added as a cryoprotective agent to ground.
19. The method according to claim 14, wherein the seed protein is
added to a cosmetic.
20. The method according to claim 14, wherein the seed protein is
added to a biological sample.
21. An ice crystallization inhibitor, comprising a seed protein,
wherein an amino-acid sequence of the seed protein is a sequence of
SEQ ID NO: 1, or an amino-acid sequence corresponding to the
sequence of SEQ ID NO: 1 with one or more amino-acid deletions,
substitutions or additions and containing an indicative sequence of
a protein having an ice crystallization inhibitory activity.
22. An ice crystallization inhibitor, comprising a seed protein,
wherein an amino-acid sequence of the seed protein is a sequence of
SEQ ID NO: 2, or an amino-acid sequence corresponding to the
sequence of SEQ ID NO: 2 with one or more amino-acid deletions,
substitutions or additions and containing an indicative sequence of
a protein having an ice crystallization inhibitory activity.
23. An antibody, specifically responding with the ice
crystallization inhibitor containing a seed protein derived from a
plant belonging to genus Vigna in Leguminosae, an allied species
thereof or an improved species thereof for inhibiting ice
crystallization.
24. A food, comprising the ice crystallization inhibitor containing
a seed protein derived from a plant belonging to genus Vigna in
Leguminosae, an allied species thereof or an improved species
thereof for inhibiting ice crystallization.
25. A biological sample protectant, comprising the ice
crystallization inhibitor containing a seed protein derived from a
plant belonging to genus Vigna in Leguminosae, an allied species
thereof or an improved species thereof for inhibiting ice
crystallization.
26. A cosmetic, comprising the ice crystallization inhibitor
containing a seed protein derived from a plant belonging to genus
Vigna in Leguminosae, an allied species thereof or an improved
species thereof for inhibiting ice crystallization.
27. A peptide, having a sequence of SEQ ID NO: 2, or an amino-acid
sequence corresponding to the sequence of SEQ ID NO: 2 with one or
more amino-acid deletions, substitutions or additions and
containing an indicative sequence of a protein having an ice
crystallization inhibitory activity.
Description
TECHNICAL FIELD
[0001] The present invention relates to an ice crystallization
inhibitor which is derived from a plant seed; an antibody which
specifically responds with the ice crystallization inhibitor; a
composition, a food, a biological sample protectant and a cosmetic
which contain the ice crystallization inhibitor; and a peptide
which provides an indication of a protein having an ice
crystallization inhibitory activity.
BACKGROUND ART
[0002] It is known that an organism which lives under the condition
of low temperature produces an ice crystallization inhibitor such
as an antifreeze protein and uses such an ice crystallization
inhibitor to protect itself from freezing of the cells.
Hereinafter, an antifreeze protein is referred to as "AFP". AFP is
a protein which has functions such as thermal hysteresis, and can
inhibit an aqueous solution from freezing and control the figure of
ice crystal. AFP has been found from an organism such as a fish, an
insect, a plant, a fungi and a microorganism.
[0003] In the past, AFP has been found in a fish such as a
Cottidae, an insect such as a mealworm, a microorganism such as
Flavobacterium and the like, and the AFPs have a high ice
crystallization inhibitory activity (Patent Documents 1 to 3). In
addition, AFP also has been found in a plant such as a winter rye
and a carrot (Non-patent Documents 1 and 2).
[0004] Furthermore, as AFP derived from a fungus, AFPs derived from
a basidiomycete such as Typhula ishikariensis and Flammulina
velutipes KUAF-1 are known (Patent Documents 4 and 5).
[0005] Recently, there was an attempt at using AFP for maintaining
the quality of a frozen confection product and a frozen food, such
as an ice cream, by industrially utilizing the above-described
properties (Patent Documents 6 and 7).
PRIOR ART DOCUMENT
Patent Document
[0006] Patent Document 1: JP 2004-83546 A [0007] Patent Document 2:
JP 2002-507889 T [0008] Patent Document 3: JP 2004-161761 A [0009]
Patent Document 4: JP 2004-24237 A [0010] Patent Document 5: JP
2004-275008 A [0011] Patent Document 6: WO 92/22581 [0012] Patent
Document 7: WO 94/03617
Non-Patent Document
[0012] [0013] Non-Patent Document 1: Plant Physiology, vol. 119,
pp. 1361-1369 (1999) [0014] Non-Patent Document 2: Biochem. J.,
vol. 340, pp. 385-391 (1999)
SUMMARY OF THE INVENTION
Problems to be Solved by the Invention
[0015] Regardless of the above-described properties, it is
difficult to completely remove an odor from AFP derived from a
fish. In addition, AFP derived from an insect and a microorganism
is not suitable for a food, since it is difficult to use an insect
and a microorganism as a raw material for a food.
[0016] On the other hand, AFP derived from a plant is expected to
be used for a food, since a plant is used as a raw material of a
food. However, AFP derived from a plant has been rarely used
industrially, since the binding ability thereof to an ice is weaker
than those of other AFPs and the APF is not sufficiently stable
against heat.
[0017] Under the above-described situation, the objective to be
solved by the present invention is to provide a novel ice
crystallization inhibitor which is industrially useful, which can
be efficiently and stably produced in a safe process suitable for a
food production without difficulty and which has excellent
functions and properties. Also, the objective of the present
invention is to provide an antibody which specifically reacts with
the ice crystallization inhibitor, and a composition, a food, a
biological sample protectant and a cosmetic which contain the ice
crystallization inhibitor. Furthermore, the objective of the
present invention is to provide a peptide which gives an indication
of a protein having an ice crystallization inhibitory activity.
Solutions to the Problems
[0018] The present inventors intensively studied so as to solve the
above problems. As a result, the inventors completed the present
invention by finding that an ice crystallization inhibitor which
has excellent properties can be obtained from a seed of a plant
belonging to genus Vigna in Leguminosae.
[0019] The ice crystallization inhibitor of the present invention
is characterized in comprising a seed protein derived from a plant
belonging to genus Vigna in Leguminosae, an allied species thereof
or an improved species thereof.
[0020] As the plant belonging to genus Vigna in Leguminosae, an
adzuki bean or a mung bean is preferred. The present inventors
experimentally found that a seed of the above plants may contain a
seed protein which has very excellent ice crystallization
inhibitory activity.
[0021] As the seed protein, a grain filling protein, a LEA protein:
Late embryogenesis abundant protein, and a protein having an
amino-acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or an
amino-acid sequence corresponding to the sequence of SEQ ID NO: 1
or SEQ ID NO: 2 with one or more amino-acid deletions,
substitutions or additions and containing an indicative sequence of
a protein having an ice crystallization inhibitory activity are
preferred. The excellent ice crystallization inhibitory activity of
the above seed proteins is experimentally demonstrated by the
present inventors.
[0022] The seed protein which is contained in the ice
crystallization inhibitor according to the present invention may
form a complex with other protein. In the seed protein which was
found by the present inventors and which exhibits an ice
crystallization inhibitory activity, there is a protein which has a
structural feature of a complex with other denatured protein.
[0023] The antibody according to the present invention is
characterized in specifically responding with the above-described
ice crystallization inhibitor.
[0024] The composition, food, biological sample protectant and
cosmetic according to the present invention are characterized in
comprising the above-described ice crystallization inhibitor of the
present invention.
[0025] The peptide according to the present invention is
characterized in having a sequence of SEQ ID NO: 2, or an
amino-acid sequence corresponding to the sequence of SEQ ID NO: 2
with one or more amino-acid deletions, substitutions or additions
and containing an indicative sequence of a protein having an ice
crystallization inhibitory activity.
Effect of the Invention
[0026] The ice crystallization inhibitor according to the present
invention can be stably supplied, since the ice crystallization
inhibitor can be efficiently produced from a seed of a plant
belonging to genus Vigna in Leguminosae, an allied species thereof
or an improved species thereof with no difficulty. In addition, the
ice crystallization inhibitor can be used for a food at ease, since
the ice crystallization inhibitor has no problem of odor and is
obtained from a plant. Therefore, for example, the ice
crystallization inhibitor according to the present invention can be
used for maintaining a quality of a frozen food or the like. In
addition, the ice crystallization inhibitor can be effectively used
as a cosmetic, a protectant when an organ, a cell, blood or
platelet is preserved in a frozen state, or the like.
BRIEF DESCRIPTION OF THE DRAWING
[0027] FIG. 1 is a magnified photograph of an ice crystal of which
figure was controlled by the ice crystallization inhibitor
according to the present invention.
[0028] FIG. 2 is a magnified photograph of an ice crystal of which
figure was controlled by the ice crystallization inhibitor
according to the present invention.
MODES FOR CARRYING OUT THE INVENTION
[0029] Hereinafter, the present invention is described in more
detail.
[0030] In the present invention, the term "ice crystallization
inhibitor" means a substrate which adsorbs on a crystal face of an
ice crystal and inhibits a growth of the ice crystal. In addition,
as the result of the adsorption on a crystal face, free water is
inhibited from further adsorbing to the ice crystal and ice
recrystallization is prohibited. In other words, the ice
crystallization inhibitor according to the present invention means
a substance having an ice crystallization inhibitory activity which
is confirmed by any one of public methods such as measurement of
ice recrystallization inhibitory activity, measurement of a thermal
hysteresis and observation of the structure of ice crystal.
[0031] For example, an ice crystallization inhibitory activity can
be measured by the following condition. First, an aqueous solution
of an ice crystallization inhibitor containing 30 w/v % sucrose is
cooled down to -40.degree. C., and then the temperature is raised
up to -6.degree. C. After 30 minutes, an average area of observed
ice crystals is measured. As a control, 30 w/v % sucrose aqueous
solution is similarly treated and an average area of ice crystals
is measured. For example, an average area of ice crystals can be
measured by analyzing a microscopic image using a commercially
available image analysis process soft, such as Image Factory
manufactured by Imsoft, ADOBE PHOTOSHOP ELEMENT 8 manufactured by
Adobe and Scion Image manufactured by Scion, and dividing a total
area of the ice crystals in the image by the number of the ice
crystals in the image. Next, an ice crystallization inhibitory
activity can be quantitatively evaluated by using a value obtained
by dividing the measured average area of the ice crystals by the
average area of the control as the indicator. Since an average area
of ice crystals is smaller as an ice crystallization inhibitory
activity is stronger, when the formulation of ice crystal is
inhibited if only a little by adding the ice crystallization
inhibitor in comparison with the control, the above-described value
is smaller than 1. In such a case, it is judged that the ice
crystallization inhibitor has an ice crystallization inhibitory
activity.
[0032] A thermal hysteresis is expressed as a difference between a
melting temperature of ice and a freezing temperature of a solution
in the presence of the ice crystallization inhibitor. The higher
the value is, the higher the activity of the ice crystallization
inhibitor is. For example, a thermal hysteresis is measured by the
following condition.
[0033] A glass petri dish is maintained in a temperature of
-20.degree. C. using a phase contrast microscope which can control
a temperature in a low temperature range, 1 mL of a sample is
placed on the glass petri dish, and a temperature is decreased to
-40.degree. C. at a speed of 100.degree. C./min to form ice
crystal. The ice crystal is warmed up to -5.degree. C. at a speed
of 100.degree. C./min, and then further warmed up at a speed of
5.degree. C./min to be a single crystal. Next, the temperature is
decreased at a speed of 1.degree. C./min, and the time at which the
single crystal starts to grow is measured. A thermal hysteresis can
be calculated in accordance with the following formula.
Thermal hysteresis (.degree. C.)=[60-1](.degree.
C./sec).times.measured time (sec)
[0034] When the calculated value is 0.02.degree. C. or more, it is
judged that a measured sample has an ice crystallization inhibitory
activity. A thermal hysteresis is preferably not less than
0.025.degree. C. and more preferably not less than 0.03.degree.
C.
[0035] Also, it can be judged by observing a structure of ice
crystal in water or a solution which contains a test sample whether
the test sample is an ice crystallization inhibitor or not.
Specifically, a figure of ice crystal which is obtained by cooling
usual water is like a flat disc. On the other hand, an ice crystal
which is obtained by adding an ice crystallization inhibitor takes
various forms such as hexagonal-shaped form, i.e. flat hexagonal
column form, and bipyramid form depending on a crystal face of an
ice crystal on which the inhibitor adsorbs. Therefore, when a form
of an ice crystal is other than flat disc form due to an addition
of a substance, it is judged that the substance is an ice
crystallization inhibitor.
[0036] The ice crystallization inhibitor according to the present
invention contains a seed protein derived from a plant belonging to
genus Vigna in Leguminosae, an allied species thereof or an
improved species thereof.
[0037] A seed of a plant belonging to genus Vigna in Leguminosae is
exemplified by, in the Japanese name, a large-size adzuki bean
(Dainagon adzuki bean) such as Hokuto Dainagon, Toyomi Dainagon,
Akane Dinagon, Kamui Dinagon, Beni Dainagon and Sahoroshouzu; a
normal-size adzuki bean such as Erimoshouzu, Shumarishouzu,
Kitanootome and Sahoroshouzu; a white adzuki bean such as
Kitahotaru and Hokkaishiroshouzu; a mung bean; a black-eyed pea. An
adzuki bean is preferred and a large-size adzuki bean is more
preferred among the above examples, since the beans are widely
eaten for a long time and readily available. In addition, an ice
crystallization inhibitory activity and a thermal hysteresis
activity per unit weight of an extract obtained from the beans are
excellent.
[0038] For example, the term "allied species of a plant belonging
to genus Vigna in Leguminosae" in the present invention means a
breed of a plant which similarly belongs to family Leguminosae but
does not belong to genus Vigna and which is close to genus Vigna in
an academical classification. A specific allied species of a plant
belonging to genus Vigna in Leguminosae means a breed of a plant
which is close to genus Vigna in Leguminosae in an academical
classification. The term "improved species of a plant belonging to
genus Vigna in Leguminosae" means a specific plant belonging to
genus Vigna in Leguminosae improved by artificial selection,
hybridization, mutation, gene recombination and the like.
[0039] The above seed protein is not particularly limited as long
as the protein is a seed protein extracted from the above plant,
and may be a fraction which contains the seed protein.
Specifically, the above seed protein may be various grain filling
proteins. Such a grain filling protein is generally defined as a
protein which is synthesized and accumulated during a process of
grain filling. The term "grain filling" means that a plant seed
matures and enlarges. Such a grain filling protein is exemplified
by various seed storage proteins such as albumin, globulin,
glutelin and prolamin; a stress protein such as a heat shock
protein; and a LEA protein (Late embryogenesis abundant protein).
In particular, a LEA protein is preferred in the above grain
filling proteins.
[0040] The ice crystallization inhibitor according to the present
invention may contain only one of the above seed proteins or a
plurality of the above seed proteins. The above seed protein may
form a complex with other protein in the ice crystallization
inhibitor of the present invention. It is particularly known that a
LEA protein interacts with other denatured protein. A denatured
protein which forms a complex with the seed protein according to
the present invention may be derived from a seed containing the
seed protein or derived from other than the seed. In addition, the
number of the denatured protein may be only one or not less than
two.
[0041] The above LEA protein is a group of highly hydrophilic
proteins of which expression is specifically induced when a water
stress is produced in a cell, and is synthesized and accumulated
during a process of grain filling, which means maturing and
enlargement of a plant seed. It is sometimes observed that a LEA
protein is accumulated in a leaf, a root, pollen or the like other
than a seed. A protein which is contained in other than a seed is
included in the range of the present invention as long as the
protein has a homology with a seed protein of a plant belonging to
genus Vigna in Leguminosae and is an ice crystallization
inhibitor.
[0042] A molecular weight of the ice crystallization inhibitor
according to the present invention is not particularly limited, and
for example, an average molecular weight measured by gel filtration
chromatography is preferably not less than 1 kDa and not more than
200 kDa. When the molecular weight is 1 kDa or more, an ice
crystallization inhibitory activity may be sufficiently exhibited.
On the other hand, when the molecular weight is too large, a
viscosity of the solution may become too high in some cases.
Therefore, the molecular weight is preferably 200 kDa or less. The
molecular weight is more preferably not more than 180 kDa and even
more preferably not more than 160 kDa. The ice crystallization
inhibitor may be possibly a complex, and the above average
molecular weight may be an average molecular weight of a monomer of
the complex. In addition, various decomposed substances of the ice
crystallization inhibitor which has the above molecular weight are
also included in the range of the present invention as long as such
decomposed substances exhibit an ice crystallization inhibitory
activity and the like.
[0043] It is preferred that an amino-acid sequence of the ice
crystallization inhibitor of the present invention is each of
sequences of SEQ ID NO: 1 to SEQ ID NO: 3, or an amino-acid
sequence corresponding to each of sequences of SEQ ID NO: 1 to SEQ
ID NO: 3 with one or more amino-acid deletions, substitutions or
additions and containing an indicative sequence of a protein having
an ice crystallization inhibitory activity.
[0044] In the above ice crystallization inhibitor, the number of an
amino-acid which is deleted, substituted or added is preferably not
less than 1 and not more than 5, more preferably not less than 1
and not more than 3, and even more preferably 1 or 2.
[0045] In the above ice crystallization inhibitor, the sentence of
"containing an indicative sequence of a protein having an ice
crystallization inhibitory activity" means that the protein having
a sequence with deletion, substitution or addition has an ice
crystallization inhibitory activity which is measured by any one of
publicly known method such as a measurement of an ice
recrystallization inhibitory activity, a measurement of a thermal
hysteresis and an observation of an ice crystal structure.
[0046] As a method for obtaining the ice crystallization inhibitor
of the present invention, a method of extracting from a seed of the
above-described plant belonging to genus Vigna in Leguminosae is
exemplified. Hereinafter, a specific production method is
described.
[0047] The ice crystallization inhibitor may be further induced by
preliminarily acclimatizing a seed of a plant belonging to genus
Vigna in Leguminosae which is subjected to extraction to low
temperature. For example, a seed may be stored at 20.degree. C. or
lower for 3 days or more. However, the ice crystallization
inhibitor can be sufficiently obtained from a seed of a plant
belonging to genus Vigna in Leguminosae even without acclimatizing
to low temperature.
[0048] The form of a seed of a plant belonging to genus Vigna in
Leguminosae which is subjected to extraction may be a fractured
form, a crushed form, a grinded form or the like. The fractured
form or the like may be obtained by fracturing a raw seed or a
dried seed, or the like.
[0049] An extraction efficiency can be enhanced by fracturing a
seed or the like. As a means for fracturing a plant seed, various
general means can be applied. For example, a physical fracturing
means such as pressurized fracture, mechanical grinding, sonication
and homogenizer can be used. A specific fracturing means is
exemplified by a homogenizer such as Potter-Elehjem homogenizer, a
blender such as Waring blender, a mil such as dyno mil, French
press, a mortar and a muddler, a pulverizing machine, freezing and
pulverization by using liquid nitrogen, and sonication
treatment.
[0050] After a seed is roughly fractured, the fractured seed may be
further grinded or finely crushed using a mortar and a muddler, a
ball mil, a hammer mil or the like.
[0051] A component is extracted by dispersing a fractured seed or
the like in an extracting solvent. When a dried seed is used, it is
preferred that a dried seed is preliminarily immersed in an
extracting solvent in order to readily extract a component.
[0052] It is preferred that the target substance is hardly
denatured due to an extracting solvent and toxicity of an
extracting solvent is low. A preferable extracting solvent is
exemplified by water; brine; a buffer solution such as a sodium
acetate buffer solution, a phosphate buffer solution and a
tris-hydrochloric acid buffer solution; a hydrophilic organic
solvent such as methanol, ethanol and acetone; other organic
solvent such as ethyl acetate; and a mixed solvent of the above
solvents. When it is taken into account that the ice
crystallization inhibitor of the present invention is applied to a
food, a preferred solvent is water, brine, a sodium acetate aqueous
solution or an arbitrarily-mixed solution of the solvents. A
composition, pH or the like of an extracting solvent may be
arbitrarily selected. In general, extracting solvent is preferably
adjusted to be neutral, for example, the pH is adjusted to about
not less than 5 and not more than 9.
[0053] An amount of an extracting solvent relative to a seed may be
arbitrarily selected, and is generally adjusted so that a seed is
totally immersed in the extracting solvent. For example, an amount
of an extracting solvent relative to a used seed may be
approximately adjusted to not less than 1 mL/g and not more than 5
mL/g.
[0054] For extraction, a raw seed or a dried seed may be mixed with
an extracting solvent, and the seed may be fractured in the
extracting solvent using a mixer or the like. In addition, a seed
may be immersed only, extraction may be carried out in a reduced
pressure, or a mixture may be stirred during extraction.
[0055] A temperature for extraction may be arbitrarily adjusted.
For example, a temperature for extraction may be not less than
0.degree. C. and not more than 100.degree. C., and more preferably
not less than 2.degree. C. and not more than 10.degree. C. In
particular, when the temperature is 10.degree. C. or lower,
degradation of a protein may be inhibited more surely by decreasing
an activity of a protease which is contained in a seed. With
respect to regulation of temperature, a temperature of a mixture of
a seed and an extracting solvent may be adjusted to a predetermined
temperature for extraction, or an extracting solvent of which
temperature is adjusted to a predetermined temperature may be added
and an extraction may be carried out with maintaining the
temperature. For example, after a seed is immersed in water or the
like and the mixture is maintained at a temperature of not less
than 2.degree. C. and not more than 10.degree. C. for a sufficient
time, water is substituted by an extracting solvent and extraction
carried out with fracturing the seed.
[0056] A time for extraction may be also arbitrarily adjusted, and
is generally not less than 5 minutes and not more than 100
hours.
[0057] After extraction, obtained extract may be separated by
filtration, centrifugation or the like, and the separated extract
may be used as the ice crystallization inhibitor. Further, an
extraction residue is repeatedly subjected to a similar extraction
treatment, the obtained extracts are brought together, and the
extract may be used as the ice crystallization inhibitor.
[0058] An alkaline treatment step in which a base is added to the
obtained extract may be carried out. By such an alkaline treatment
step, an ice crystallization inhibitory activity of a protein may
be increased in some cases.
[0059] A condition of an alkaline treatment is not particularly
limited and may be arbitrarily set. For example, as a base, an
alkali metal hydroxide such as sodium hydroxide and potassium
hydroxide may be used. The value of pH is preferably not less than
10.0 and more preferably not less than 10.5. The upper limit of pH
is not particularly limited, and pH is preferably not more than
13.0 and more preferably not more than 12.0.
[0060] After the pH is adjusted to higher as the above, the mixture
may be neutralized using hydrochloric acid or the like so that the
pH may be adjusted not less than about 6.0 and not more than about
8.0.
[0061] The ice crystallization inhibitor obtained as the above may
be further purified as necessary. For example, a solid substance
may be removed by a preferable combination of decantation,
filtration, centrifugation and the like. In addition, for example,
precipitation by salting-out or using an organic solvent, affinity
chromatography, ion-exchange column chromatography, gel filtration,
purification by binding to ice using a low-speed refrigerator,
concentration by dialysis or ultrafiltration, and the like may be
properly combined.
[0062] A form of the ice crystallization inhibitor of the present
invention may be variously selected depending on the use
application, and may be a solid, a solution, a concentrated liquid,
a dispersion or the like. As necessary, the ice crystallization
inhibitor may be further formed into an arbitrary form such as a
powder, a granule or a tablet. A method of formulation is not
particularly limited, and is exemplified by a method for
powderizing the above extract with a conventional method such as
spray drying and freeze drying, a method for powderizing or
granulating the extract by adsorbing and supporting the extract on
an excipient, and the like. Such methods are well-known for the
person skilled in the art, and the skilled person can select a
proper method to be used depending on the intended use
application.
[0063] The ice crystallization inhibitor according to the present
invention can be utilized for the purpose of removing impediment
caused by crystallization of water in various fields where such an
impediment is present. For example, the inhibitor can be utilized
in the fields of a food, machinery, civil engineering, cosmetics,
and medicine in which a biological sample is used.
[0064] The ice crystallization inhibitor of the present invention
may have various forms depending on the use application thereof.
The ice crystallization inhibitor may be used as it is, or may be
in the form of a solution, a concentrated solution, a suspension, a
freeze-dried product, a powder, a granule, a tablet and the like.
In addition, the ice crystallization inhibitor may be mixed with an
excipient to be used as a composition.
[0065] The antibody according to the present invention reacts
specifically with the above-described ice crystallization
inhibitor. The antibody therefore can be used for confirming the
presence or absence of the ice crystallization inhibitor in a
basidiomycete or the culture medium therefor, and specifying a
polysaccharide having an ice crystallization inhibitory activity
from a culture medium for a basidiomycete.
[0066] The antibody according to the present invention may be
produced according to a conventional method. For example, a mouse,
rat or the like is immunized with the above-described ice
crystallization inhibitor, and the antibody-producing cell or the
splenocyte is fused with a myeloma cell to obtain a hybridoma. The
hybridoma is cloned, and the clone producing an antibody which is
reactive specifically with the above-described ice crystallization
inhibitor is screened. The clone is cultured, and a secreted
monoclonal antibody may be purified.
[0067] In the field of a food, it is possible to prevent the
deterioration of taste and others by suppressing ice
crystallization of water contained in a food. For example, when
water in a food is crystallized to be an ice, protein, fat
component, oil component and the like are physically compressed,
and the structure of the components is changed. As a result, taste,
quality and the like of a food is deteriorated. When the ice
crystallization inhibitor is added to a food, such deterioration
can be inhibited and the quality of a frozen food and the like can
be improved.
[0068] In the fields of machinery and civil engineering, the ice
crystallization inhibitor according to the present invention can be
utilized as a cryoprotective agent for movable part of machinery,
road, ground and the like.
[0069] In the field of cosmetics, the ice crystallization inhibitor
according to the present invention can be utilized as an additive
agent for preventing the reduction in quality of cosmetics. For
example, when cosmetics containing an oil component or a fat
component are frozen, water contained in the cosmetics is
crystallized to be an ice. As a result, the oil component and fat
component may be physically compressed and the structure thereof
may be destroyed, whereby the quality and impression from use of
the cosmetics become deteriorated. When the ice crystallization
inhibitor according to the present invention is used, the reduction
in quality and the like can be avoided since ice crystallization of
water is prevented and the structure of an oil component and a fat
component is maintained.
[0070] In the field of medicine, the ice crystallization inhibitor
according to the present invention can be utilized as a protectant
when a biological sample is cryopreserved. For example, when a
biological sample such as a cell, blood, an organ, a bioactive
protein, a bioactive peptide and a low-molecular compound derived
from a living body is cryopreserved in a conventionally and
publicly known preservation solution, water in the preservation
solution freezes to generate ice crystals. The ice crystals may
damage the biological sample. On the other hand, when the ice
crystallization inhibitor according to the present invention is
added thereto, the biological sample can be protected from the
damage caused by ice crystals since generation and growth of ice
crystal can be suppressed.
[0071] The present application claims the benefit of the priority
date of Japanese patent application No. 2011-048318 filed on Mar.
4, 2011, and all of the contents of the Japanese patent application
No. 2011-048318 filed on Mar. 4, 2011, are incorporated by
reference.
EXAMPLES
[0072] Hereinafter, the present invention is described in more
detail with Examples. However, the present invention is not limited
to the following Examples in any way.
[0073] In the following Examples, a concentration of a protein was
measured by a bicinchoninic acid (BCA) method using a commercially
available measurement kit (BCA Protein Assay, manufactured by
Thermo SCIENTIFIC K. K.). A measurement condition was set in
accordance with an ordinary method, and bovine serum albumin (BSA)
was used as a standard protein.
Example 1
[0074] Into a 1,000 mL beaker, commercially available Dainagon
adzuki bean (100 g) was added. Distilled water was added until the
bean was immersed and the mixture was maintained at 4.degree. C.
for 48 hours. The distilled water was removed, and then 5 mM
Tris-HCl buffer solution (pH: 8.0, 500 mL) was added thereto. The
bean was fractured at 4.degree. C. using a mixer, and then the
mixture was filtered using a gauze and centrifuged at 4.degree. C.
and at 8,000.times.g for 30 minutes. A supernatant (460 mL) after
centrifugation was obtained as a crude extract. A concentration of
protein in the obtained crude extract was measured by BCA method;
as a result, the concentration was 42 mg/mL.
Example 2
[0075] To the crude extract (460 mL) obtained in Example 1, acetone
which was cooled down to -30.degree. C. was added dropwise little
by little until a concentration of acetone became 25 v/v %. Then,
an acetone solution was separated from an insoluble component
(insoluble fraction 1). An ice crystallization inhibitory activity
of the insoluble fraction 1 was not detected. Next, acetone was
added to the above acetone solution until an acetone concentration
became 75 v/v %, and an insoluble component (insoluble fraction 2)
was separated from 75 v/v % acetone solution. The obtained
insoluble fraction 2 was dissolved in 5 mM Tris-HCl buffer solution
(pH: 8.0, 100 mL). The obtained solution was subjected to dialysis
using a dialysis membrane (molecular weight cut off: 14,000), and
then the solvent was replaced with the same buffer solution to
obtain a solution (230 mL) as an acetone fraction. A concentration
of protein in the acetone fraction was measured by BCA method; as a
result, the concentration was 8.3 mg/mL.
Example 3
[0076] The solution (230 mL) obtained in Example 2 was charged in a
DEAE column (product name: DEAE TOYOPEARL 650M, manufactured by
TOSOH Corporation) which was equilibrated using the above buffer
solution, and eluted at a flow rate of 2.0 mL/min with NaCl
gradient of 0 to 0.5 M to obtain non-adsorbed fraction as an active
fraction. The obtained fraction was subjected to dialysis using a
dialysis membrane (molecular weight cut off: 14,000), and then the
solvent was replaced with 20 mM sodium acetate buffer solution (pH:
5.0) to obtain a solution (485 mL). A concentration of protein in
the obtained DEAE column chromatography active fraction was
measured by BCA method; as a result, the concentration was 1.8
mg/mL.
Example 4
[0077] The solution (485 mL) obtained in Example 3 was charged in a
cation-exchange column (product name: CM TOYOPEARL 650M,
manufactured by TOSOH Corporation) which was equilibrated using the
above buffer solution, and eluted at a flow rate of 2.0 mL/min with
NaCl gradient of 0 to 0.5 M to obtain an adsorbed fraction as an
active fraction. The obtained fraction was subjected to dialysis
and desalination using a dialysis membrane (molecular weight cut
off: 14,000), and then the solvent was replaced with 20 mM
phosphate buffer solution (pH: 6.0) to obtain a solution (132 mL).
A concentration of protein in the obtained active fraction (CM1
fraction) was measured by BCA method; as a result, the
concentration was 0.73 mg/mL.
Example 5
[0078] The solution (132 mL) obtained in Example 4 was charged in a
cation-exchange column (product name: CM TOYOPEARL 650M,
manufactured by TOSOH Corporation) which was equilibrated using the
above buffer solution, and eluted at a flow rate of 2.0 mL/min with
NaCl gradient of 0 to 0.5 M to obtain a non-adsorbed column
fraction (CM2 non-adsorbed fraction) and an adsorbed column
fraction (CM2 adsorbed fraction) respectively. The adsorbed column
fraction was subjected to dialysis and desalination using a
dialysis membrane (molecular weight cut off: 14,000), and then the
solvent was replaced with 20 mM phosphate buffer solution (pH:
6.0). Concentrations of protein in the obtained CM2 non-adsorbed
fraction (126 mL) and CM2 adsorbed fraction (64 mL) were measured
by BCA method; as a result, the concentrations were respectively
0.22 mg/mL and 0.29 mg/mL.
Example 6
[0079] The CM2 non-adsorbed fraction and CM2 adsorbed fraction
obtained in Example 5 were respectively dissolved in 20 M phosphate
buffer solution. Concentrations of protein of each obtained aqueous
solution (1000 .mu.L) were respectively 5000 .mu.g/mL and 5000
.mu.g/mL. Each of aqueous solutions was charged in a gel filtration
column (product name: Sephacrl 5300 HR, manufactured by GE Health
Care) as a sample, and eluted using 5 mM Tris-HCl buffer solution
(pH: 8.0, 0.15 M NaCl was contained) at a flow rate of 0.5 mL/min
in a temperature condition of 4.degree. C. A non-adsorbed fraction
was eluted and detected by an absorption wavelength of 215 nm. In
addition, standard proteins of which molecular weight were
different each other were eluted in the same conditions.
[0080] In the above-described column chromatography, single peaks
were measured at molecular weight of about 160,000 and 130,000
respectively. Each fraction (CM2 non-adsorbed 5300 fraction and CM2
adsorbed 5300 fraction) was respectively obtained, and subjected to
dialysis using water. Then, the fractions were concentrated using a
filter for removing particle (product name: Centricut, manufactured
by KURABO INDUSTRIES Co., Ltd., molecular weight cut off: 10,000),
and 3.9 mg and 2.9 mg of purified samples were respectively
obtained. The samples were subjected to Native-PAGE; as a result,
single bands were respectively detected. In addition, the samples
were subjected to SDS-PAGE; as a result, multiple bands mainly at
46 kDa and 52 kDa were respectively detected. From the above
result, it was demonstrated that at least a part of the purified
substances formed a complex.
Test Example 1
Measurement of Ice Crystallization Inhibitory Activity
[0081] An ice crystallization inhibitory activity of each solution
obtained in Examples 1 to 6 was measured. Specifically, each
solution obtained in Examples 1 to 6 was diluted to adjust a
protein concentration as described in Table 1, and then the diluted
solutions were mixed with 60 w/v % sucrose aqueous solution in
equal amount to obtain 30 w/v % sucrose solution of the sample. In
order to evaluate an ice crystallization inhibitory activity, under
a microscope having a stage with a function to control a low
temperature, 30 w/v % sucrose solution of the sample was cooled
down to -40.degree. C. and then warmed to -6.degree. C. to melt the
ice crystals. The ice crystals were observed for 30 minutes with
maintaining the temperature at -6.degree. C., and an average area
was measured. The average area of ice crystals was measured by
analyzing a microscopic image using a commercially available image
analysis process soft (Image Factory manufactured by Imsoft) and
dividing a total area of the ice crystals in the image by the
number of the ice crystals. In addition, with respect to 30 w/v %
sucrose aqueous solution as a control, a similar measurement was
carried out and an average area of ice crystals was calculated.
[0082] An average area of ice crystals is smaller as an ice
crystallization inhibitory activity is stronger. Therefore, the
average area was divided by the average area of ice crystals of 30
w/v % sucrose aqueous solution as a control, and an ice
crystallization inhibitory activity was quantitatively evaluated
using the calculated value (RI value) as an indicator. In addition,
a concentration of protein in the sample was measured by BCA
method, a specific activity was obtained by dividing the reciprocal
of RI value by the protein concentration, and a value relative to a
crude extract of Example 1 was calculated. The result is
demonstrated in Table 1.
TABLE-US-00001 TABLE 1 Ice Protein crystallization Specific concen-
inhibitory activity tration activity (Relative Sample (mg/mL) (RI
value) value) Example 1 crude excract 3.0 0.33 1 Example 2 acetone
fraction 0.5 0.40 5.0 Example 3 DEAE fraction 0.4 0.29 8.5 Example
4 CM1 fraction 0.2 0.25 19.8 Example 5 CM2 non-adsorbed 0.12 0.33
25.0 fraction Example 5 CM2 adsorbed 0.14 0.37 19.1 fraction
Example 6 CM2 non-adsorbed 0.13 0.27 28.2 S300 fraction Example 6
CM2 adsorbed 0.13 0.22 34.6 S300 fraction
[0083] From the result described in Table 1, it was clearly
demonstrated that an extract which has an excellent ice
crystallization inhibitory activity can be obtained from
commercially available Dainagon adzuki bean and an ice
crystallization inhibitory activity per protein, i.e. specific
activity, is remarkably improved by fractionation and purification
of Examples 2 to 6.
Test Example 2
Measurement of Thermal Hysteresis
[0084] The CM2 non-adsorbed 5300 fraction and CM2 adsorbed 5300
fraction obtained in Example 6 were respectively dissolved in water
so that a concentration of protein became 0.25 mg/mL, and a thermal
hysteresis was measured as follows. Specifically, a glass petri
dish was maintained in a temperature of -20.degree. C. using a
phase contrast microscope (product name: L600A, manufactured by
Olympus Corporation) which could control a temperature in a low
temperature range, 1 mL of the sample was placed on the glass petri
dish, and a temperature was decreased to -40.degree. C. at a speed
of 100.degree. C./min to form ice crystals. The ice crystals were
warmed up to -5.degree. C. at a speed of 100.degree. C./min, and
then further warmed up at a speed of 5.degree. C./min to be single
crystals. Next, the time at which the single crystals started to
grow was measured by decreasing a temperature at a speed of
1.degree. C./min, and a thermal hysteresis was calculated in
accordance with the following formula.
Thermal hysteresis (.degree. C.)=[60-1](.degree.
C./sec).times.measured time (sec)
[0085] The higher calculated value is, the higher an ice
crystallization inhibitory activity is. The result is demonstrated
in Table 2. In addition, a magnified photograph of an ice crystal
of which figure was controlled by CM2 non-adsorbed S300 fraction is
demonstrated as FIG. 1, and a magnified photograph of an ice
crystal of which figure was controlled by CM2 adsorbed S300
fraction is demonstrated as FIG. 2.
TABLE-US-00002 TABLE 2 Sample Thermal hysteresis (.degree. C.) CM2
non-adsorbed S300 fraction 0.036 CM2 adsorbed S300 fraction
0.055
[0086] From the result described in Table 2, it was clearly
demonstrated that the purified protein obtained in Example 6 had an
excellent thermal hysteresis as an ice crystallization
inhibitor.
[0087] In addition, as FIGS. 1 and 2, both of ice crystals obtained
by adding the purified protein of Example 6 had forms of
non-cutting hexagonal, and the forms were different from a flat
disc form of an ice crystal obtained from ordinary water or aqueous
solution. It was demonstrated by such a change in form that the
obtained purified protein bound to a crystal face of an ice
crystal. Therefore, the experimental result provides evidence that
the purified proteins are ice crystallization inhibitors. The
photograph of FIG. 1 represents a side direction of a hexagonal
crystal.
Test Example 3
Determination of Amino-Acid Sequence of Purified Protein
[0088] The CM2 adsorbed 5300 fraction (4.0 .mu.g) obtained in
Example 6 was dissolved in distilled water (5 .mu.L), and the
solution was subjected to a protease treatment in accordance with
an ordinary method. Then, the solution was mixed with a sample
buffer (product name: EzApply, manufactured by ATTO corporation) (5
.mu.L) in a volume ratio of 1:1, and the mixture was heated at
99.degree. C. for 3 minutes. The sample after the above heat
treatment was applied on 10 to 20% polyacrylamide gel (product
name: e-Page I, manufactured by ATTO corporation), and SDS-PAGE was
carried out at 20 mA for 85 minutes. The gel after SDS-PAGE was
transferred on a PVDF membrane (product name: Immobilon PSQ,
manufactured by Millipore Corporation) by semi-dry method, and CBB
staining was carried out. Multiple bands which mainly contained 52
kDa digested by protease were cut out, and amino-acid sequence was
determined by Edman method using a protein sequencer (product name:
PPSQ-33A, manufactured by Shimadzu Corporation). The determined
sequences are demonstrated as SEQ ID NO: 1 to 3.
[0089] The sequences had a high homology with a part of an
amino-acid sequence of a LEA protein (Late embryogenesis abundant
protein) which was an already-known seed protein. It was clearly
demonstrated from the above result that the ice crystallization
inhibitor obtained from an adzuki bean was a seed protein.
Example 7
[0090] Crude extracts were produced from commercially available
mung bean, white adzuki bean and adzuki bean produced in Tokachi
which belonged to genus Vigna in Leguminosae as similarly to
Example 1. A concentration of protein was adjusted to 2.0 mg/mL,
and an ice crystallization inhibitory activity was measured
similarly to the method of Test Example 1. The result is
demonstrated in Table 3.
TABLE-US-00003 TABLE 3 Ice crystallization inhibitory activity Raw
material (RI value) mung bean 0.24 white adzuki bean 0.18 adzuki
bean produced in Tokachi 0.33
[0091] It was demonstrated by the result described in Table 3 that
a seed protein obtained from a seed of the above-described plant
belonging to genus Vigna in Leguminosae has excellent ice
crystallization inhibitory activity.
Example 8
[0092] A crude extract obtained from a mung bean in Example 7 was
frozen at -20.degree. C., the frozen extract was melted and
subjected to centrifugation, and the supernatant was recovered to
remove deposition. A concentration of protein in the obtained crude
extract was measured by BCA method; as a result, the concentration
was 15.5 mg/mL. The pH of the crude extract (200 mL) was adjusted
to 11.0 by adding sodium hydroxide. Then, the crude extract was
neutralized and the pH was adjusted to 7.0 by adding hydrochloric
acid. The mixture was subjected to dialysis using a dialysis
membrane (molecular weight cut off: 14,000), and then the solvent
was replaced with water to obtain a solution (230 mL). A
concentration of protein in the obtained solution was measured by
BCA method; as a result, the concentration was 10.0 mg/mL.
Example 9
[0093] The crude extract (100 mL) after an alkaline treatment
obtained in Example 8 was subjected to ice crystallization
inhibitor concentration process (a cold finger (CF) method)
described below. Specifically, a cooling bar having a cylinder
shape was inserted into the crude extract of Example 8 containing
an ice crystallization inhibitor, and the solution around the
cooling bar was cooled at a low cooling speed (from 0.5.degree. C.
to -1.5.degree. C. for 16 hours). After 16 hours, ice which
generated around the cooling bar and on which ice crystallization
inhibitor was adsorbed was recovered. The above process was
repeated two times, and a solution (50 mL) after CF treatment was
obtained. A concentration of protein in the obtained solution was
measured by BCA method; as a result, the concentration was 4.4
.mu.g/mL. The solution was subjected to SDS-PAGE; as a result, a
single band was detected at the position of about 48 kDa.
Example 10
[0094] To the crude extract (230 mL) after an alkaline treatment
obtained in Example 8, acetone which was cooled down to -30.degree.
C. was added dropwise little by little until an acetone
concentration became 25 v/v %. Then, an acetone solution was
separated from an insoluble component (insoluble fraction 3). Next,
after acetone was added to the acetone solution until an acetone
concentration became 50 v/v %, and an acetone solution was
separated from an insoluble component (insoluble fraction 4).
Further, after acetone was added to the acetone solution until an
acetone concentration became 75 v/v %, an acetone solution was
separated from an insoluble component (insoluble fraction 5). The
above insoluble fractions 3 to 5 were dissolved in 5 mM Tris-HCl
buffer solution (pH 8.0). The solutions were subjected to dialysis
using a dialysis membrane (molecular weight cut off: 14,000), and
then the solvent was replaced with the same buffer solution. With
respect to insoluble fraction 3 and insoluble fraction 5, an ice
crystallization inhibitory activity could not be confirmed. On the
other hand, it was confirmed that insoluble fraction 4 (30 mL) had
a strong ice crystallization activity. Therefore, insoluble
fraction 4 was recovered as an acetone fraction. A concentration of
protein in insoluble fraction 4 was measured by BCA method; as a
result, the concentration was 3.0 mg/mL.
Example 11
[0095] The acetone fraction (3.0 mg) obtained in Example 10 was
charged in a DEAE column (product name: DEAE TOYOPEARL 650M,
manufactured by TOSOH Corporation) which was equilibrated using 5
mM Tris-HCl buffer solution (pH 8.0), and eluted at a flow rate of
2.0 mL/min with NaCl gradient of 0 to 0.5 M. A peak was detected
using absorption wavelength of 215 nm. An adsorbed fraction which
was obtained when a peak was detected was recovered as an active
fraction. The obtained fraction was subjected to dialysis and
desalination using a dialysis membrane (molecular weight cut off:
14,000), and the solvent was replaced with deionized water. A
concentration of protein in the obtained fraction was measured by
BCA method; as a result, the concentration was 12 .mu.g/mL (yield:
30 mL).
Test Example 4
Measurement of Ice Crystallization Inhibitory Activity
[0096] With respect to each solution which was obtained by
extracting from a mung bean in Examples 7 to 11, an ice
crystallization inhibitory activity was measured by a similar
method as Test Example 1. From protein concentrations of each
solution measured by BCA method and RI value which is the indicator
of an ice crystallization inhibitory activity, a specific activity
per protein was calculated in accordance with the formula
represented in Test Example 1 and a value relative to the crude
extract of Example 7 was calculated. In addition, a total mass of
protein extracted from 100 g of a mung bean was calculated from a
solution amount and a protein concentration. The result is
demonstrated in Table 4 and Table 5.
TABLE-US-00004 TABLE 4 Specific activity Total protein Sample
(Relative value) mass (mg) Example 7 crude extract 1 3100 from mung
bean Example 8 crude extract 1.3 2300 from mung bean after alkaline
treatment Example 9 crude extract 1.5 1 from mung bean after CF
treatment
TABLE-US-00005 TABLE 5 Specific activity Total protein Sample
(Relative value) mass (mg) Example 7 crude extract 1 3100 from mung
bean Example 8 crude extract 1.3 2300 from mung bean after alkaline
treatment Example 10 acetone fraction 2 89 Example 11 DEAE fraction
5.9 11
[0097] As the result demonstrated in Table 4, the ice
crystallization inhibitor derived from a mung bean was purified by
purification processes of Examples 7 to 9 so that a specific
activity became 1.3 times and a protein concentration became
1/3100. In addition, as the result demonstrated in Table 5, the ice
crystallization inhibitor derived from a mung bean was purified by
purification processes of Examples 10 and 11 so that a specific
activity became 5.9 times and a protein concentration became
11/3100.
[0098] Further, as the result demonstrated in Tables 4 and 5, an
ice crystallization inhibitory activity was increased as much as
1.3 times by an alkaline treatment of Example 8. It was
demonstrated by the result that an ice crystallization inhibitory
activity can be increased in the condition that protein is
denatured, such as an alkaline treatment. The above result is
related to the feature of a LEA protein, which is known to prohibit
a denatured protein from being aggregated. Accordingly, the
following Test Example 5 and Test Example 6 were carried out.
Test Example 5
Inhibiting Effect on Aggregation of Protein Using Purified
Fraction
[0099] Commercially available LDH: lactate dehydrogenase (15 .mu.g)
was dissolved in 1.0 mL of water. In the solution, 9 .mu.g of
purified mung bean protein after CF treatment obtained in Example 9
was dissolved. An aqueous solution of LDH in which a purified
protein after CF treatment was not added was used as control. Each
solution was frozen using liquid nitrogen, and the frozen solution
was melted at room temperature. An absorbance of the melted
solution at 340 nm was measured. The result is demonstrated in
Table 6.
TABLE-US-00006 TABLE 6 Sample Absorbance (340 nm) LDH aqueous
solution (control) 0.21 LDH aqueous solution + CF purified protein
0.06
[0100] A turbidity of LDH aqueous solution as control was
increased, since LDH protein was denatured and aggregated due to
freezing and thawing. On the other hand, in case of the solution
containing the purified protein after CF treatment derived from a
mung bean, an increase of turbidity was remarkably inhibited. It
was demonstrated from the result that the purified mung bean
protein obtained in Example 9 has an inhibitory effect on
aggregation of protein, which inhibitory effect is characteristic
of a LEA protein, and the purified mung bean protein can protect a
protein and inhibit denaturation of protein due to freezing and
thawing.
Test Example 6
Increase of Ice Crystallization Inhibitory Activity of Crude Mung
Bean Extract, Associated with Inhibitory Effect on Aggregation
[0101] A protein concentration of a crude mung bean extract
obtained in Example 7 was adjusted to 60 mg/mL. To 240 .mu.L of the
solution, 30 .mu.g of the purified mung bean protein after CF
treatment obtained in Example 9 was dissolved. The crude mung bean
extract to which the purified protein after CF treatment was not
added was used as control. Each solution was frozen using liquid
nitrogen, and the frozen solution was melted at room temperature.
The melted solution was subjected to centrifugation, and then a
protein concentration was adjusted to 0.95 mg/mL. An ice
crystallization inhibitory activity of the solution was measured by
a similar method to Test Example 1. The result is demonstrated in
Table 7.
TABLE-US-00007 TABLE 7 Ice crystallization inhibitory activity Raw
material (RI value) crude extract from mung bean 0.53 (control)
crude extract from mung bean + 0.32 CF purified protein
[0102] An ice crystallization inhibitory activity of the crude mung
bean extract was remarkably increased by adding the purified mung
bean protein after CF treatment obtained in Example 9 in a slight
amount, and freezing and thawing the mixture. Such an increase of
an ice crystallization inhibitory activity was not observed when
the purified mung bean protein after CF treatment was merely added.
Therefore, it was thought that an ice crystallization inhibitory
activity was increased by interacting the purified mung bean
protein with a protein and the like in the crude extract when
stress of freezing and thawing was applied.
Example 12
[0103] The DEAE fraction obtained in Example 11 (4.5 mg) was
charged in a gel filtration column (product name: Sephacryl 5300
HR, manufactured by GE Health Care) as a sample, and eluted using 5
mM Tris-HCl buffer solution (pH: 8.0, 0.15 M NaCl was contained) at
a flow rate of 0.5 mL/min in a temperature condition of 4.degree.
C. A non-adsorbed fraction was eluted and detected by an absorption
wavelength of 215 nm. In addition, standard proteins of which
molecular weight were different each other were eluted in the same
conditions. By the above column chromatography, an absorption peak
was detected at the same molecular weight (160,000 and 130,000) as
the case of Dainagon adzuki bean which was subjected to
purification in Example 6.
Sequence CWU 1
1
3125PRTVigna angularis 1Met Ala Val Gln Glu Ala Val Ala Gly Lys Lys
Glu Ser Val Pro1 5 10 15 Ala Lys Ala His Thr Thr Glu Val Ile His 20
25 211PRTVigna angularis 2Thr Leu Asn Lys Met Gly Glu Tyr Lys Asp
Tyr1 5 10 322PRTVigna angularis 3Met Lys Glu Gly Lys Asp Ala Thr
Leu Asn Lys Met Gly Glu Tyr1 5 10 15 Lys Asp Tyr Thr Ala Glu Lys
20
* * * * *