The present invention relates to natuxally-derived emulsifiers
with enhanced emulsifying power, and specifically it relates to a soybean-derived
protein and polysaccharides conjugate and to emulsifiers and emulsions containing
Emulsifiers used in food products include surfactants such
as soybean lecithin, glycerin fatty acid esters, sucrose fatty acid esters and the
like. These low molecular emulsifiers are widely used for preparation of oil-in-water
emulsions which have oil stably dispersed in an aqueous phase, forming stably dispersed
emulsions with the hydrophobic regions in the emulsifiers adsorbed onto the oil-phase
interface and the hydrophilic regions directed toward the aqueous phase. These emulsifiers
exhibit very powerful emulsifying power, allowing preparation of satisfactory emulsions
with small emulsified particle sizes.
Such emulsions maintain stable emulsified states when used
under non-diluted conditions such as in creams. However, in the case of food products
consisting of highly diluted emulsions, such as emulsified flavorings in beverages,
the emulsified state readily disintegrates and the heating stability and acid stability
which are a requirement for emulsifiers are insufficient, and for these reasons
they have not been used very much in practice.
Currently, gum arabic is commonly used as a natural macromolecular
emulsifier for emulsified flavorings (Non-patent documents 1-2).
Gum arabic is reported to be a glycoprotein comprising a polypeptide chain composed
of hydroxyproline and serine as the major constituent amino acids bonded to an arabinogalactan
chain consisting of the acidic sugar glucuronic acid and its 4-0-methylated form
as constituent sugars (Non-patent document 3).
The sugar chains of gum arabic function as hydrophilic
groups while the polypeptides bonded to the sugar chains function as hydrophobic
groups, thereby stabilizing O/W emulsions (Non-patent document 4).
The sugar chains of emulsions prepared using gum arabic form thick hydrophilic layers
at oil droplet interfaces, and since there is little separation of the gum arabic
at the oil droplet interfaces even with high degrees of dilution, it is an excellent
emulsifier for emulsified flavorings. However, because the Acacia sap gum arabic
is only produced in certain regions and is susceptible to weather conditions, it
has not been possible to sustain stable volumes and prices.
On the other hand, soybean-derived water-soluble polysaccharides
function as natural macromolecular emulsifiers (Patent document 1), and are used
in the field of food products as a substitute for the emulsifier function of gum
arabic. However, the "emulsifying power" for obtaining emulsions with small particle
sizes is currently insufficient compared to synthetic low molecular emulsifiers
and the like, and therefore a need exists for natural macromolecular emulsifiers
that exhibit not only high emulsifying stability but also strong emulsifying power.
Conjugation of proteins and sugars by aminocarbonyl reaction has been proposed as
a method of improving functions such as emulsification (Patent document 2), but
this is still impractical in terms of both production and function.
Disclosure of the Invention
Problems to be Solved by the Invention
- Patent document 1:
Japanese Unexamined patent Publication No. 6-121922
- Patent document 2:
Japanese Unexamined Patent Publication No. 9-107886
- Non-patent document 1:
Ray et al., Food Hydrocolloids, 9(2), 123-131, 1995
- Non-patent document 2:
Trubiano, Flavor Technology: ACS Symposium Series 610. Washington D.C.: American
Chemical Society. P198-209, 1995
- Non-patent document 3:
Williams, P.A. et al., Gum arabic. In Handbook of hydrocolloids
- Non-patent document 4:
Glicksman, Food Hydrocolloids. Boca Raton, Fla.: CRC Press. P7-30, 1983
Natural macromolecular emulsifiers have excellent emulsifying
stability allowing emulsions to be rendered stable for long periods, but much room
for improvement still remains in regard to "emulsifiability" for reducing emulsified
particle sizes. It is an object of the present invention to provide natural macromolecular
emulsifiers with improved "emulsifiability" in order to obtain emulsions with small
emulsified particle sizes. Means for Solving the Problems
As a result of much diligent research on water-soluble
soybean polysaccharides as natural macromolecular emulsifies to improve emulsifiability,
with the goal of solving the problems described above, the present inventors obtained
a fraction from soybean in which polysaccharides form conjugates with specific proteins
and discovered that these conjugates of polysaccharides with specific proteins having
high proline contents contribute to the emulsifying function of water-soluble soybean
polysaccharides, on which discovery the present invention has been completed.
Effect of the Invention
Specifically, the invention provides a soybean-derived conjugate comprising polysaccharides
and a protein having an amino acid composition of ≥10% proline, which are
found in soybean, wherein the molecular weight of the protein forming the conjugate
is composed mainly of the fraction from 50,000 Da to 80,000 Da, the conjugate contains
the protein at 3% or greater, the polysaccharides forming the conjugate is acidic
polysaccharides containing at least 20 wt% galacturonic acid as a constituent sugar,
and the molecular weight of the conjugate is 100,000 Da or greater. The invention
further provides emulsifiers comprising the conjugate as an active ingredient and
emulsions containing the conjugate.
According to the invention, it is possible to obtain emulsifiers
with improved emulsifying power, allowing formation of emulsions with small emulsified
particle sizes using a natural macromolecular compound composition.
Best Mode for Carrying Out the Invention
The protein and polysaccharides conjugate according to
the invention is composed of two components, polysaccharides and protein. The polysaccharides
is preferably any of various polysaccharides obtained from soybean as the source,
but the source is not necessarily limited to soybean. These polysaccharides are
also preferably acidic polysaccharides containing at least 20 wt% galacturonic acid
as the main constituent sugar, and the molecular weight of the glycoprotein conjugate
is preferably 100,000 Da or greater. A smaller molecular weight will tend to reduce
the function of the macromolecular emulsifier, while lower acidic sugars content
among the constituent sugars will tend to reduce electrostatic repulsion between
the emulsion particles, thus leading to inferior emulsifiability.
The protein as one of the components of the conjugate is
the protein of a specific fraction in soybean, and it has a proline content of 10
wt% or greater and more preferably 15% or greater among its constituent amino acids.
Soybean protein usually contains 5-6% proline overall, and even well-known soybean
protein components contain proline at about 7% as 11S globulin or basic 7S globulin,
but no soybean protein is known that contains proline at 10% or greater. The molecular
weight of the protein is also preferably 50,000 Da-80,000 Da. Proline is an amino
acid that significantly alters the tertiary structure of a protein, and it is conjectured
that the high proline content is responsible for a different tertiary structure
than ordinary protein and results in strong emulsifiability.
Thus, a lower proline content will tend to provide insufficient improvement in emulsifiability,
while a small protein molecular weight will lead to inferior function as a macromolecular
The molecular weight of the conjugate or polysaccharides
referred to here may be measured by gel filtration HPLC using standard pullulan
(Showa Denko K.K.) as the standard substance. Measurement of galacturonic acid was
performed using a Shodex SH-1821 column (Showa Denko K.K.), and measurement of neutral
sugars was performed by GLC after alditol acetate conversion. The protein molecular
weight was analyzed by SDS polyacrylamide electrophoresis, and the amino acid composition
was analyzed using an amino acid analyzer (L-8500A, Hitachi, Ltd.).
The method of preparing the polysaccharides and protein
conjugate of the invention is not particularly restricted, but water-soluble soybean
polysaccharides may be obtained by high-temperature extraction from a soybean starting
material, and especially a soybean protein-extracted residue ("okara"), in the acidic
range (Patent document 1), and subsequently removing the low molecular components
or impurity proteins which do not form conjugates with the polysaccharides. The
resulting polysaccharides and protein bond together strongly, forming stable glycoprotein
conjugates. Generally speaking, the bonds between sugars and proteins range from
weak hydrogen bonds to strong glycoside bonds between serine hydroxyl groups and
the reduced ends of saccharides. The bonding form in the invention product is believed
to consist of fairly strong bonds since the conjugation is maintained even in the
presence of denaturing agents as described below. It is conjectured that these conjugates
provide the powerful emulsifiability and emulsion stability not found in the prior
In regard to the polysaccharides and protein, soybean protein
does not increase emulsifiability when simply mixed with polysaccharides, and instead
the protein constitutes an impurity that lowers the quality of the emulsifier. It
is essential that a specific proline-rich protein be conjugated with the polysaccharide.
The protein contains at least 10% proline as the amino acid composition of the protein,
as mentioned above, but in terms of the weight ratio of the polysaccharides and
protein, the effect of improved emulsifiability will be reduced if the protein weight
is too low, and therefore the protein content of the conjugate is preferably at
least 3%. There is no particular upper limit on the protein content of the conjugate,
but if it is too high the effect will not increase, and a content exceeding 50%
will instead be counterproductive.
As regards the molecular weight of the conjugate, emulsifying
power will be exhibited with any size cf macromolecule but lower molecular weights
will not provide strong emulsifying power, and the emulsifying stability will also
be impaired. The molecular weight may generally be from several tens of thousand
to about one million, but molecular weights of greater than 100,000 are preferred
for uses requiring high emulsifying power.
An emulsifier comprising the conjugate as an active ingredient
may be suitably used in emulsified flavorings instead of gum arabic, but it may
also be used as an emulsifier for creams such as whipped cream or coffee whitener,
or for mayonnaise or dressings.
As fats and oils to be used in the emulsion there may be
mentioned animal and vegetable fats and oils such as soybean oil, corn oil, cottonseed
oil, sunflower oil, carthamus oil, coconut oil, palm oil, palm kernel oil, rapeseed
oil, peanut oil, rice bran oil, cacao butter, milk fat, beef tallow, lard, butter
and the like, as well as processed fats and oils obtained by hardening, transesterification
or separation thereof, and even fat-soluble aromas such as orange oil, lemon oil,
milk flavor and the like, or waxes. The effect may be further enhanced by combination
with known emulsifiers. As emulsifiers to be combined, there may be mentioned combinations
with one or more from among lecithin, enzyme-treated lecithin, fatty acid glycerin
esters and their derivatives, sorbitan fatty acid esters, propylene glycol fatty
acid esters and sucrose fatty acid esters (sugar esters). The amounts of such additional
components may be 0.01-2 fold and preferably 0.02-1 fold with respect to the fat
or oil. A sufficient emulsifying effect may not be achieved if they are added in
very small amounts, while the flavor and texture may be impaired by increased viscosity
if they are added in very large amounts.
The present invention will now be explained in greater
detail by examples, with the understanding that the invention is not restricted
by these examples. The "parts" and "%" values in the examples are all based on weight.
<Preparation of water-soluble soybean polysaccharides (control)>
A two-fold amount of water was added to 100 parts of okara
(80% moisture content) obtained from production of isolated soybean protein, and
the pH was adjusted to 3.0 with hydrochloric acid prior to hot extraction at 120°C
for 1.5 hours. After cooling, the pH was adjusted to 5.0, centrifugation was performed
(10,000 G × 30 min) and the supernatant and precipitate were separated. An
equivalent amount of water was added to the obtained precipitate for rinsing, and
the rinsed precipitate was centrifuged again. This supernatant was pooled with the
previous supernatant to obtain an extract which was then subjected to desalting
by electrodialysis and purification with an active carbon column, after which it
was spray dried to obtain water-soluble soybean polysaccharides A.
<Fractionation of water-soluble soybean polysaccharides (Preparation
of invention conjugate 1)>
A 20 mL portion of a 10% aqueous solution of water-soluble
soybean polysaccharides A was applied to a gel filtration column (Sephacryl S-400HR;
5.0 cm × 65 cm) using 50 mM acetate buffer (pH 4.5) as the mobile phase for
molecular weight fractionation, and the peak in the vicinity of 300,000 Da molecular
weight was recovered, dialyzed and freeze-dried to obtain water-soluble soybean
polysaccharides fraction B. The fraction was subjected to sugar detection by RI
and protein detection by UV (280 nm), by which the fraction was shown to be a protein-polysaccharides
conjugate. The yield of the fraction was 25% with respect to the water-soluble soybean
polysaccharides A. The gel filtration chart is shown in Fig. 1.
<Fractionation of water-soluble soybean polysaccharides fraction
in the presence of denaturing agent (Preparation of invention conjugate 2)>
A 15 mL portion of a 2% aqueous solution of water-soluble
soybean polysaccharides fraction B was applied to a gel filtration column (Sepharose
CL-6B; 2.5 cm × 80 cm) using 1% SDS as the mobile phase, to obtain a fractionated
product. Upon detecting the sugars by RI and the protein by UV (280 nm), protein
was found only in the macromolecular fraction, and the macromolecular fraction was
recovered and dialyzed against water to remove the denaturing agent to obtain water-soluble
soybean polysaccharides fraction C. The gel filtration chart for the molecular weight
fractionation in the presence of the denaturing agent is shown in Fig. 2.
<Amino acid composition of protein in soybean polysaccharides
<Emulsifiability of polysaccharides or polysaccharides conjugate>
(Table 1) Amino acid composition of water-soluble soybean polysaccharides
composition (fraction B)
soybean polysaccharides A
Water-soluble soybean polysaccharides A and water-soluble
soybean polysaccharides fractions B and C were used to prepare emulsions by the
following method. To 25 parts of soybean oil (Refined Soybean Oil, product of Fuji
oil Co., Ltd.) there were added 75 parts of different sample solutions dissolved
in 20 mM sodium citrate buffer (pH 4) to be 5%, and emulsification was carried out
at 0°C for 1 minute with an ultrasonic disruptor (Ultrasonic Disruptor 5281
by Kaijo Co.). After one hour, the emulsified particle size was measured with a
laser particle size distribution meter (SALD-2000 by Shimadzu Corp.).
<Emulsifiability evaluation of other macromolecular emulsifiers>
Using the same method as the aforementioned preparation
of emulsions with the polysaccharides or polysaccharides conjugates, emulsions were
obtained using sodium caseinate (EM-LV by DMV Co.), whey protein (Purified WPI by
NZMP Co.) and gum arabic (HP grade, product of T. Hasegawa Co., Ltd.) instead of
polysaccharides. One hour after preparation, the emulsified particle sizes of the
emulsions were measured in the same manner with a laser particle size distribution
meter (SALD-2000 by Shimadzu Corp.). The results are shown in Table 2.
(Table 2) Emulsion evaluation results
Emulsion particle size (µm)
Water-soluble soybean polysaccharides A
Water-soluble soybean polysaccharides fraction B
Water-soluble soybean polysaccharides fraction C
Whey protein Gum arabic
The proline-rich water-soluble soybean polysaccharides
fractions B and C were confirmed to be satisfactory emulsions with much higher emulsifying
power than the other macromolecular emulsifiers, and smaller particle sizes than
the conventionally known soybean water-soluble polysaccharides A. Emulsified particle
size differs even for the same emulsifier and emulsion composition, depending on
the emulsifying conditions. Thus, comparison of the emulsified particle sizes under
the same emulsifying conditions allows comparative judgment of the emulsifiability
of each emulsifier.
<Composition of water-soluble soybean polysaccharides fraction
The composition of the water-soluble soybean polysaccharides
fraction B is shown in Table 3 below.
(Table 3) Composition of water-soluble soybean polysaccharides fraction B
Dry content (%)
Content in sugar (%)
The following reference experiment was carried out to demonstrate
the properties of the polysaccharides and protein conjugate.
<Protease decomposition and emulsifiability of water-soluble
soybean polysaccharides fraction B>
To 80 g of a 5% aqueous solution of water-soluble soybean
polysaccharides B there was added 0.5 ml (13.6 kunits) of a trypsin aqueous solution
(from bovine pancreas, Sigma Co.), and reaction was conducted at pH 7, 30°C.
After 1, 3, 6 and 24 hours, the enzyme was inactivated by heating at 90°C for
20 minutes, 20 g of soybean oil was added for 5 minutes of pre-emulsification with
a Power Gen125, and then main emulsification was carried out with a homogenizer
(EmulsiFlex-C5, Avestin Co.) at 40 Mpa. The particle size of the emulsion was measured
with a Mastersizer X (Malvern Instruments Ltd), yielding the results shown in Table
4. The trypsin-added water-soluble soybean polysaccharides A increased in particle
size with time, losing its emulsifiability. This result suggests that protein plays
an important role in the emulsifiability of water-soluble soybean polysaccharides.
<SDS-Polyacrylamide electrophoresis of water-soluble soybean
polysaccharides fraction C>
Enzyme treatment time (h)
Emulsion particle size (µm)
The sugar chains of water-soluble soybean polysaccharides
fraction C were decomposed using an enzyme (Hemicellulase M, product of Tanabe Seiyaku
Co., Ltd.) to obtain water-soluble soybean polysaccharides decomposition product
D. When fraction C and decomposition product D were applied to SDS-polyacrylamide
electrophoresis, fraction C was found to be a macromolecular component that completely
failed to enter the gel, while the decomposition product D showed three protein
bands at 52,200 Da, 60,400 Da and 78,500 Da. This is illustrated in Fig. 3.
The water-soluble soybean polysaccharides fraction C had
very powerful emulsifiability, and even with gel filtration in the presence of SDS
the polysaccharides and protein composing the conjugate failed to dissociate and
behaved as a macromolecular fraction which also failed to enter the gel in SDS electrophoresis,
while the decomposition product D obtained by enzyme decomposition of the polysaccharides
showed three clear protein bands. Consequently, it was concluded that fraction C
and its constituent fraction B are the sources of emulsifiability, and that the
conjugate consisted of the polysaccharides and protein in strong linkage. In addition,
with the high galacturonic acid content in the sugar composition of fraction B,
it is believed that the highly polar sugar chains increased the electrostatic repulsion
and thereby further stabilized the emulsion.
Furthermore, the constituent amino acids of the protein
include abundant proline, and this is significantly different from the constituent
protein of gum arabic that is rich in hydroxyproline and serine.
Brief Description of the Drawings
- Fig. 1 is a graph showing the gel filtration chart for water-soluble soybean
- Fig. 2 is a graph showing the gel filtration chart for water-soluble soybean
polysaccharides fraction B with molecular weight fractionation in the presence of
a denaturing agent.
- Fig. 3 is a photograph from electrophoresis of water-soluble soybean polysaccharides