CA 02872863 2014-12-01
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OAT PROTEIN GELS
Field of the Invention
[0001] The present invention relates oat protein gels and methods of making
and using the
same.
Background
[0002] Gelation is an important functional property of proteins as it provides
texture and
support in foods. Generally, thermal gelation of globular proteins involves
unfolding of the
protein molecules by heating, which leads to exposure of hydrophobic amino
acid residues.
Later, unfolded molecules re-arrange and aggregate irreversibly via disulfide
bridges,
hydrogen bonds, hydrophobic and/or van der Waals interactions. Finally,
aggregation carries
on with association of protein particles and if the protein concentration is
sufficiently high, a
three-dimensional network is created (Lefevre & Subirade, 2000). This process
only takes
place in the presence of suitable environmental conditions, such as pH,
temperature and ionic
strength (Totosaus, Montejano, Salazar, & Guerrero, 2002). (Twomey, Keogh,
Mehra, &
O'Kennedy, 1997, Ziegler, & Foegeding, 1990).
[0003] Plant proteins are normally considered inferior to animal proteins in
terms of gelling
properties. Gelatin, egg white and whey proteins are widely used as gelling
agents in the food
industry, particularly in meat and dairy based systems. In recent years,
proteins derived from
plant sources are becoming an important ingredient segments owing to health
(no Bovine
Spongiforme Encephalopathy concern), religious and cost reasons. For a long
time, soy
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protein has been the major plant protein gelling ingredient in the market. Yet
there is an
opportunity for other novel gelling ingredients of plant origin to meet the
increasing market
requirement for different functionalities and sensory attributes.
[0004] Oat is commonly used as an animal feed and only a small percentage of
the grain is
currently used for human consumption. Recently the human food market for oat
has been
gaining momentum mainly due to the growing public awareness of the health
benefits of fl-
glucan. This soluble dietary fiber component of oat is known to reduce blood
cholesterol
(Braaten, Wood, & Scott, 1994), and regulate blood glucose levels (Wood,
Scott, Riedel,
Wolynetz, & Collins, 1994). Several techniques have been developed to isolate
3-glucan from
oat grain as a health ingredient in food products. The remaining components
such as protein
and starch are awaiting research to develop their full value (Inglett, Lee, &
Stevenson, 2008).
[0005] Oat has the highest protein level (12-20%) (Mohamed, Biresaw, Xu,
Hojilla-
Evangelista, & Rayas-Duarte, 2009) among cereals, with a superior amino acid
profile due to
higher amounts of limiting amino acids lysine and threonine (Mose & Arendt,
2012). This is
related to the fact that in most cereals the major storage proteins are
alcohol-soluble
prolamines whereas in oat, globulins represent 70-80% of the total protein
fraction (Robert,
Nozzolillo, Cudjoe, & Altosaar, 1933). The major fraction in oat protein is
the 12S globulin,
which consists of two major subunits with molecular weight of about 32 and 22
kDa called
the A- and B-subunits, where the A-subunit is an acidic polypeptide and the B-
subunit is a
basic polypeptide. The A- and B-subunits are disulfide bonded in the native
globulin, forming
a dimer with a molecular weight of 54 kDa, which further associates into a
hexamer through
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noncovalent forces (Burgess, Shewry, Matlashewski, Altosaar, & Miflin, 1983).
The 7S and
3S are the minor fractions. 7S globulins are polypeptides with molecular
weight of 55 kDa,
and some minor components with a molecular weight of 65 kDa are also present.
The 3S
fraction entails at least two major components with molecular weight of about
15 and 21 kDa
(Klose & Arendt, 2012).
[0006] Two previous publications demonstrated that oat protein could form gels
(Ma &
Harwalkar, 1987; Ma, Khanzada, & Harwalkar, 1988). But at acidic and neutral
pH, very
weak gels with poor water holding capacity were obtained. The gel properties
improved after
pH 8, but strong gels could only be prepared at p1-Is 9-10. The gel hardness
was greatly
increased by both acetylation and succinylation (Ma & Wood, 1986, 1987). The
authors
suggested that the changes in the functional properties of oat protein after
modification
resulted from altered conformation and increase in net charge (Ma, 1984, 1985;
Ma & Wood,
1986, 1987). This was later confirmed with the study of the thermal
aggregation of oat
globulin by Raman spectroscopy (Ma, Rout, & Phillips, 2003). In this work,
changes in
protein interactions and conformation were induced by the addition of protein
structure
modifying agents such as ehaotropic salts, sodiumdodecyl sulfate or
dithiothreitol, which can
either enhance or inhibit thermal gelation of oat globulin.
[0007] Enzymatic hydrolysis is a preferable tool to alter functional
properties of proteins
because of milder processing conditions required, easier control of reaction
and minimal
formation of by-products (Mannheim & Cheryan, 1992). Recent research has
reported the
effect of enzymatic hydrolysis over the gelling properties of proteins
including soy protein
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(Hou & Zhao, 2011), rice bran protein (Yeom, Lee, Ha, Ha, & Bae, 2010),
sunflower protein
(Sanchez & Burgos, 1997), and canola protein (Pinterits & Amtfield, 2007).
Results from
these studies indicate that improvement of the gelling capacity is highly
enzyme specific. The
gelling properties of oat protein treated with trypsin were studied in
previous work (Ma &
Wood, 1986, 1987), however, weak gel structure was obtained due to the short
size of the
protein molecules, which may no longer be able to associate to form a strong
gel matrix. Since
the final composition and thus the use of the hydrolysates will depend on the
type of enzyme
used and the hydrolysis conditions (Benitez, lbarz, & Pagan, 2008), a
systematic investigation
of the effect of various proteases over the gelling capacity of oat protein is
required. Such
information has not been available, however important for the development of
new
modification strategy to improve oat protein gelling properties.
1,00081 Modification of protein conformation can also be achieved through
limited hydrolysis,
as changes in the secondary and tertiary structure can be produced. This can
alter the surface
exposure of reactive amino acids, leading to an increase in interactions
favoring aggregation
(Foegeding & Davis, 2011) and three-dimensional network formation.
[0009] Cold-set gelation as alternative gelling method opens an interesting
opportunity for
proteins in development of functional food ingredient, such as protecting heat
sensitive
bioactive compounds. This process consists of two consecutive steps. The first
step is
preheating protein above denaturation temperature to induce protein unfolding,
exposure of
reactive groups, and subsequent aggregation at solution pH far from protein
isoelectric point
(IEP) and at a concentration below a critical value. In this step, protein
remains as soluble
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aggregates due to the high electrostatic repulsive forces. For the second
step, addition of salt
(Ca2') or altering solution pH induce the formation of three-dimensional gel
network (Bryant
and McClements, 1998, Alting, de Jongh, Visa:hers, & Simons, 2002; Ailing,
Hammer, de
Kruif, & Visschers, 2003a; Campbell, Gu, Dewar, & Euston, 2009). Generally,
two kinds of
cold-set gels, particulate and filamentous gels, can be achieved depending on
processing
conditions (Lefevre, and Subirade, 2000; Maltais, Remondetto, Gonzalez,
Subirade, 2005;
Maltais, Remondetto, Subirade, 2008). Filamentous gel is formed by linearly
linked protein
aggregates maintained by hydrophobic interactions at low ionic strength or pH
far from
protein IEP, which exhibits regular network structure with more or less linear
strands. In
coniTast, particulate gel is created by random aggregation of protein units
mainly through van
der Waals interaction at high ionic strength or pH near protein IEP, which
composes of large
and almost spherical aggregates. These different predominated interactions and
gel network
structures lead to various gel mechanical properties and applications
(Remondetto, Neyssac, &
Subirade, 2004).
[0010] Extensive works have focused on salt-induced whey protein and soy
protein gels in
terms of gel properties, formation mechanism and applications (Maltais,
Remondetto,
Subirade, 2010; Barbut, & Foegeding, 1993, Foff, and Roegeding, 1996; Zhang,
Liang, Chen,
Subirade, 2012). These cold-set gels were used to improve the texture and
stability of food
products (Hongsprabhas, & Barbut, 1999), or play as carrier of bioactive
compounds or
divalent cations (Maltais, Remondetto, & Subirade, 2010; Remondetto, Beyssac,
& Subirade,
2004; Vazquez da Silva, et al, 2010).
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[0011] Another commonly used method to form cold-set gel is altering solution
pH towards
- protein IEP. It can be achieved by adding organic acids or acidulants, or
lactic acid
fermentation, which lead to the reduction of electrostatic repulsion forces
between protein
aggregates (Venugopal, Doke, & Nair, 2002; Riebroya, Benjakula, Visessanguanb,
Eriksonc,
& Rustad, 2009; Xu, Xia, Yang, Kim & Nie, 2010). Among them, glucono-d-lactone
(GDL)
as an acidulant has been widely used in food products (Tseng & Xiong, 2009;
Chawla,
Venugopal, & Mair, 1996). GDL can be slowly hydrolyzed to glueonic acid in
water, which
resulted in a gradual decrease of pH to neutralize negatively charged protein
aggregates and
create gel with homogeneous porous structure (Malaki, Nik, Alexander, -Poysa,
Woodrow, &
Corredig, 2011). However, the gelation mechanism and protein conformational
changes at
different GDL concentrations were not completely elucidated.
[0012] The gelling properties of proteins can be affected by interaction with
other
components, such as polysaccharides. Protein and polysaccharide are often
mixed to develop
food products with novel textural properties. The interactions developed among
protein and
polysaccharide will define the microstructure of food product and thus the
resulting texture or
mechanical properties. Interactions between protein and polysaccharides can be
either
associative or segregative depending on the molecular characteristics of the
contributing
polymers and the medium conditions such as pH, or ionic strength. As
electrostatic
interactions are produced under associative conditions between a protein and
an ionic
polysaccharide of opposite charge, a complex coacervate structure is obtained.
When no
strong interactions exist between protein and polysaccharide, interpenetrating
networks arc
formed, where each polymer is in its own continuous network. Phase-separated
networks are
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formed when interactions between polymers are repulsive or when there are no
electrostatic
forces to drive the association, This results in a bi-continuous phase or a
continuous
supporting phase containing inclusions of the other phase.
Summary Of The Invention
[0013] We have demonstrated that partial enzymatic hydrolysis can improve oat
protein
gelling properties under specific conditions. Thus, it is believed that oat
protein and its
hydrolysatcs could form gels of plant origin with similar properties as those
from animal
proteins such as egg white. Therefore, this work aims to complete a systematic
study of the
thermal gelation of oat protein and its hydrolysates under different
environmental conditions
=
with an emphasis on the gel mechanical strength and water-holding capacity
which are the
most important gel characteristics for food applications. If the defined gel
physical properties
would be in the range of similar properties of animal protein derived gels,
value-added
opportunities would exist for oat protein to be used as a new gelling
ingredient in food
formulations such as meat binder and fat replacer to create food with improved
quality and
nutritive value, or used in meat analogues for vegetarian foods. In this way,
additional revenue
return could be generated to oat producers and processors to enhance their
sustainability.
[0014] The effects of partial hydrolysis and the environmental conditions (pH
and
temperature) on the gelling properties of oat protein isolate (OP1) were
investigated. OPI was
treated with flavourzyme, alcalase, pepsin and trypsin.
[0015] The changes in protein structure were observed by SDS-PAGE, size
exclusion high
performance liquid chromatography (SE-HPLC) and amino acid analysis. Gel
mechanical
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properties were evaluated by textural profile analysis (TPA). The results
revealed that the
acidic polypeptides (12S-A) of oat globulin exerted great influence over the
gelling ability of
oat protein. Partial hydrolysis by flavourzyme and trypsin could significantly
improve oat
protein gel strength, especially at pHs 8-9 by modulating the balance between
the
electrostatically repulsive force and the hydrophobic attractive force among
polypeptide
chains during the gelling process. The gels prepared with flavourzyme and
trypsin treated oat
proteins have comparable or higher mechanical strength than soy protein gels
at neutral pH.
At pH 9 the gel made of trypsin treated oat protein even showed comparable
mechanical
strength to egg white protein gels under the same pH. Both oat protein and its
hydrolysate gel
exhibited excellent water-holding capacity at neutral or mildly alkaline
conditions. The results
of this study indicate that oat protein may be has a gelling ingredient of
plant origin to provide
texture and structure in food products.
[0016] Therefore, in one aspect, the invention may comprise a gel formed from
oat protein
hydrolysate, having a degree of hydrolysation less than about 20, or less than
about 15, or less
than about 10. In one embodiment, the oat protein hydrolysate is formed by
partially
hydrolyzing oat protein with a protease, which may comprise flavourzymeõ
trypsin or alcalase.
[0017] In another aspect, the invention may comprise a method of forming an
oat protein gel,
comprising the step of partially hydrolyzing oat protein at a pH of about 7 or
higher,
preferably about pH 8 or about pH 9, and forming a gel from the hydrolysate.
In one
embodiment, the degree of hydrolysation is less than about 10, and preferably
less than about
8.
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[0018] In another aspect, the invention may comprise a gel formed from oat
protein and an
organic acid or acidulant. In one embodiment, the organic acid or acidulant
comprises GDL.
[0019] In another aspect, the invention comprises a method of forming an oat
protein gel,
comprising the step of mixing oat protein with an organic acid or acidulant,
and forming the
gel. In one embodiment, oat protein is added at about 5 or about 7% w/v, and
organic acid or
acidulant at less than about 20% w/w of protein, or less than about 15%, or
less than about
10%, or less than about 5%, at a pH less than about 9, or less than about 8,
at about 20 C. In
one specific embodiment, the method of claim 9 wherein the oat protein is
added about 7%
w/v, and GDL at about 10% w/w of protein, at a pH of about 8.
[0020] In another aspect, the invention may comprise a gel formed from oat
protein and
inulin.
[0021] In another aspect, the invention may comprise a method of forming an
oat protein gel,
comprising the step of mixing oat protein with inulin. In one specific
embodiment, the oat
protein is mixed at about 15% w/v with inulin less than about 1.0% w/v,
heating at 100 C at a
pH of less than 8.
[0022] In another aspect, the invention may comprise a method of forming an
oat protein gel
comprising the steps of heating the oat protein at less than about 100 C and
microwaving the
oat protein. The microwaved mixture may include inulin and/or a fatty acid.
[0023] Brief Description Of The Drawings
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[0024] Section 1
[0025] Fig. 1. SDS polyacrylamide gel electrophoresis of oat protein and its
hydrolysates.
Lanes: Standard protein markers, 2. OPI, 3. OPT-F, 4. OPT-A, 5. OPI-P, and 6.
OPT-T.
[0026] Fig. 2. Size exclusion chromatograms of oat protein and its
hydrolysates.
[0027] Fig. 3. Mechanical properties of oat protein derived gels prepared at
120 C. a.
Hardness (N), b. cohesiveness and c. springiness (mm).
[00281 Fig. 4. SEM images of the cross section of oat protein derived gels
prepared at 120 C.
Scale bar represents 5 Rm. a. ON (pH 7), b. OPI-F (pH 7), c. OPT-F (pH 9), d.
OPI-T (pH 7),
and e,.OPI-T (pH 9).
[0029] Section 2 Drawings have brief descriptions appended thereto.
[0030] Section 3 Drawings have brief descriptions appended thereto.
[0031] Section 4 Drawings have brief descriptions appended thereto.
Detailed Description
[0032] As will be apparent to those skilled in the art, various modifications,
adaptations and
variations of the following specific disclosure can be made without departing
from the scope
of the invention claimed herein. The various features and elements of the
described invention
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may be combined in a manner different from the combinations described or
claimed herein,
without departing from the scope of the invention.
[00331 As used herein, the recited terms have the following meanings. All
other terms and
phrases used in this specification have their ordinary meanings as one of
skill in the art would
understand. Such ordinary meanings may be obtained by reference to technical
dictionaries,
such as Hawley 's Condensed Chemical Dictionary 14th Edition, by R.J. Lewis,
John Wiley &
Sons, New York, N.Y., 2001.
[0034] References in the specification to one embodiment", "an embodiment",
etc., indicate
that the embodiment described may include a particular aspect, feature,
structure, or
characteristic, but not every embodiment necessarily includes that aspect,
feature, structure, or
characteristic. Moreover, such phrases may, but do not necessarily, refer to
the same
embodiment referred to in other portions of the specification. Further, when a
particular
aspect, feature, structure, or characteristic is described in connection with
an embodiment, it is
within the knowledge of one skilled in the art to affect or connect such
aspect, feature,
structure, or characteristic with other embodiments, whether or not explicitly
described.
[0035] The singular forms "a," "an," and "the" include plural reference unless
the context
clearly dictates otherwise. Thus, for example, a reference to "a plant"
includes a plurality of
such plants. It is further noted that the claims may be drafted to exclude any
optional element.
As such, this statement is intended to serve as antecedent basis for the use
of exclusive
terminology, such as "solely," "only," and the like, in connection with the
recitation of claim
elements or use of a "negative" limitation.
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[0036] The term "and/or" means any one of the items, any combination of the
items, or all of
the items with which this term is associated. The phrase "one or more' is
readily understood
by one of skill in the art, particularly when read in context of its usage.
[0037] The term "about" can refer to a variation of + 5%, 10%, 20%, or
25% of the
value specified. For example, "about 50" percent can in some embodiments carry
a variation
from 45 to 55 percent. For integer ranges, the term "about" can include one or
two integers
greater than and/or less than a recited integer at each end of the range.
Unless indicated
otherwise herein, the term "about" is intended to include values and ranges
proximate to the
recited range that are equivalent in terms of the functionality of the
composition, or the
embodiment. As will be understood by the skilled artisan, all numbers,
including those
expressing quantities of reagents or ingredients, properties such as molecular
weight, reaction
conditions, and so forth, are approximations and are understood as being
optionally modified
in all instances by the term "about." These values can vary depending upon the
desired
properties sought to be obtained by those skilled in the art utilizing the
teachings of the
descriptions herein. It is also understood that such values inherently contain
variability
necessarily resulting from the standard deviations found in their respective
testing
measurements.
[00381 As will be understood by one skilled in the art, for any and all
purposes, particularly in
terms of providing a written description, all ranges recited herein also
encompass any and all
possible sub-ranges and combinations of sub-ranges thereof, as well as the
individual values
making up the range, particularly integer values. A recited range (e.g.,
weight percents or
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carbon groups) includes each specific value, integer, decimal, or identity
within the range.
Any listed range can be easily recoglized as sufficiently describing and
enabling the same
range being broken down into at least equal halves, thirds, quarters, fifths,
or tenths. As a
non-limiting example, each range discussed herein can be readily broken down
into a lower .
third, middle third and upper third, etc.
[0039] As will also be understood by one skilled in the art, all language such
as "up to", "at
least", "greater than", ''less than", "more than", ''or more", and the like,
include the number
recited and such terms refer to ranges that can be subsequently broken down
into sub-ranges
as discussed above. In the same manner, all ratios recited herein also include
all sub-ratios
falling within the broader ratio. Accordingly, specific values recited for
radicals, substituents,
and ranges, are for illustration only; they do not exclude other defined
values or other values
within defined ranges for radicals and substituents.
[0040] One skilled in the art will also readily recognize that where members
are grouped
together in a common manner, suCh as in a Markush group, the invention
encompasses not
only the entire group listed as a whole, but each member of the group
individually and all
possible subgroups of the main group. Additionally, for all purposes, the
invention
encompasses not only the main group, but also the main group absent one or
more of the
group members. The invention therefore envisages the explicit exclusion of any
one on more
of members of a recited group. Accordingly, provisos may apply to any of the
disclosed
categories or embodiments whereby any one or more of the recited elements,
species, or
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embodiments, may be excluded from such categories or embodiments, for example,
as used in
an explicit negative limitation.
SECTION 1
[0041] Methods and Materials
[0042] Naked oat grains (Avena nuda) were purchased from Wedge Farms Ltd.,
Manitoba,
Canada. The protein content was 17.2%. Flavourzyme (>500 U/g), alcalase (2.4
U/g), pepsin
(>250 U/mg), trypsin (1462 U/mg), sodium dodecyl sulfate (SDS) and Tri-nitro
benzene
sulfonic acid (TNBS) were obtained from Sigma-Aldrich Canada (Oakville, ON,
Canada). E-
Z run pre-stained protein ladder/marker was purchased from Fisher Scientific
(Whitby, ON,
Canada).
[0043] Oat grains were ground to flour using a mill (Ultra Centrifugal ZM 200
Retsch, PA)
equipped with a 0.5 mm screen. The flour was then defatted with hexane at room
temperature.
Globular protein was extracted from the defatted oat flour according to the
method reported
by Wu, Sexson, Cluskey, and lnglett (1977) with some modifications. Briefly,
defatted oat
flour was dispersed in an alkali solution adjusted to pfl 9.2 using sodium
hydroxide at a flour-
to-solvent ratio of 1:6 and mixed for 1 h at room temperature. The slurry then
passed through
a 300 pm wire mesh and the permeated mixture was centrifuged at 7000 xg for 15
min. Then,
the supernatant was collected and pH was adjusted to 5 with 1 M HC1, followed
by
centrifugation at 7000 xg for 15 min. The pellet corresponding to the
precipitated protein was
washed with distilled water and freeze-dried for later use. Protein content of
the extracted oat
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protein was determined using the Leco nitrogen analyzer (FP-428, Leco
Corporation, St
Joseph,MI) and a nitrogen to crude protein conversion factor of 6.25 was used.
[0044] A 2% (w/v) protein suspension was prepared with distilled water. The pH
and
temperature of the suspension were adjusted to the optimum condition for each
enzyme.
Hydrolysis with flavourzyme was carried out at pH 7 and 50 C, alcalase at pH
8 and 50 C,
pepsin at pH 2 and 37 C and trypsin at pH 8 and 37 'C. The enzyme/substrate
ratio was set at
10/100 for all treatments. Over the hydrolysis period (30 min) the pH was kept
constant with 1
M 1-IC1 or 1 M NaOH. At the end of the hydrolysis, the solution was heated at
90 C for 10
mm to inactivate the enzyme. Hydrolysate samples were collected, freeze-dried
and stored for
further experiments. The protein content of the dried hydrolysates was also
determined using
the Leco nitrogen analyzer (FP-428, Leco Corporation, St. Joseph, MI).
[0045] Degree of hydrolysis (DH) was determined by the TNBS assay (Adler-
Nissen, 1979).
Total number of amino groups was determined in a sample completely hydrolyzed
with 6 N
HC1 at 110 C for 24 h. The DH was calculated with the following equation.
= Tr¨
'14 ftat
where h (hydrolysis equivalents) is the amount of peptide bonds cleaved during
hydrolysis,
which is expressed as millimole equivalents per gram of protein (mmol/g of
protein) and hto, is
the total amount of peptide bonds in the protein substrate. L-Leucine (0-1.5
mM) was used to
generate a standard curve (R2 --- 0.99).
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[0046] SDS-polyacrylamide gel electrophoresis (SDS-PAGE)was perfomaed to study
the
molecular weight of the oat protein subunits. Protein samples were mixed with
sample buffer
(0.125 M Tris¨HC1 pH 6.8,4% w/v SDS, 20% v/v glycerol, 0.5% 2-mercaptoethanol
and 1%
bromophenol blue w/v) and heated at 100 C for 5 min, then cooled to room
temperature.
After cooling, 30 gt sample (1 mg/mL)was loaded on 4% stacking gel and 12%
separating gel
and subjected to electrophoresis at a constant voltage of 160 V. After
electrophoresis the gels
were stained with 0.1 A (w/v) Coomassie Brilliant Blue R-250 in
water¨methanol¨acetic acid
(4:5:1, v:v:v).The molecular weight distribution of the samples was determined
using a size
exclusion high performance liquid chromatography (SE-HPLC) system (Agilent
1200 series)
equipped with a BiosuiteTM 125/5 tun HR-SEC column (7.8 A.¨ 300 nun, Water
Corp. MA,
USA). The eluent used was 0.2 M phosphate buffer with 0.2 M NaCl (pH 7) at a
flow rate of
0.5 mL/min and room temperature. Samples (50 RL) were injected into the system
and elution
was monitored at 220 nm. Standard molecular markers were used to calculate Mw
of the oat
protein samples. A calibration curve was made from the log Mw of the markers
and their
respective elution times (R2 = 0.97).
[0047] Amino acid composition analysis of the samples was performed using the
Waters
AccQ-TagTm precolumn method. Dried samples were hydrolyzed under vacuum and
after
derivatization were loaded on a reversed phased column. The AccQ reagent, 6-
aminoquinolyi-
Nhydrozysuccinimidyl carbamate, is an N-hydroxysuccinimide-activated
heterocyclic
carbamate, which converts both primary and secondary amino acids to stable
fluorescent
derivatives.
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[0048] The denaturation temperature of OPT and its hydrolysates was determined
using a
differential scanning calorimeter Q1000 (TA Instruments, New Castle, DE, USA).
Approximately 10 iL of a 15% protein (w/v) suspension was weighed on a pre-
weighed
aluminumpan and hermetically sealed. An empty hermetic pan was used as
reference. The
sample was heated at a 10 C/min, over a temperature range of 30-160 C. The
protein
suspensions (15% protein) were prepared at pHs 5, 7 and 9 to study the effect
of pH on the
protein denaturation temperature. The denaturation temperature (Td) was
computed from the
endothermic peaks observed in the thermograms using computer software.
[0049] 2.5. Gel preparation Gels were prepared by heating the protein sample
suspension
(15%, w/v) at pHs 5, 7 and 9. The pH of the suspension was adjusted using 1 N
NaOH or 1 N
HCI. Vacuum was applied to remove air bubbles. Test tubes containing the
suspension were
tightly closed and placed in an oil bath at 110 C and 120 C for 15 min. Once
heat treatment
was completed, the tubes were cooled in an ice bath and stored in the
refrigerator overnight.
[0050] 2.6. Textural profile analysis (TPA) The mechanical properties of the
gels Prepared
above were evaluated using an Instron 5967 universal testing machine (Instron
Corp.,
Norwood, MA, USA). Gels were released from test tubes and cut into cylindrical
pieces (-10
mm height, ¨14 mm diameter). A two cycle compression test using a 50 N load
cell was
performed at room temperature at a rate of 1 min/min to evaluate their
mechanical properties.
Each sample was compressed to 50%, since deformation levels between 20 and 50%
have
been commonly applied in several works on gel food systems. At tins level the
sample does
not break, but it is still possible to obtain valuable information on
important parameters (Pons
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& Fiszman, 1996). The textural profile parameters including, hardness,
springiness and
cohesiveness were calculated. These parameters were determined form the
typical Instron
force¨time curve inwhich hardness is calculated as the peak compression force
in the 1st bite
cycle, and cohesiveness is the ratio of the area under the first and second
compression peaks.
Springiness is the distance calculated from the area under the second
compression peak.
[0051] 2.7. Scanning electron microscopy (SEM) The morphology observation of
the gels
was carried out with a Phillips XL-30 scanning electron microscope (FEI
Company, Oregon,
USA). The samples were frozen in liquid nitrogen and freeze-dried before
observation. Dry
samples were coated with gold and platinum and a scanning electron microscope
was used to
observe the microstructure of the gels.
[0052] 2.8. Water holding capacity (WI-IC) A gel sample (0.9-1.2 g) was placed
into a
Vivaspin 20 centrifugal filter unit (GE Healthcare Bio-Sciences AB, Uppsala,
Sweden) and
centrifuged at 290 A¨g for 5 min at 15 C. The weight of the gel was recorded
before (Wi)
and after (Wf) centrifugation to the nearest 0.0001 mg and the percentage of
water loss after
centrifugation was expressed as:
(1.¨Wr
ZW.HC = 10.0 = ') x100
[0053] 2.9. Statistical analysis All data were analyzed for significant
differences, with
minimum significance test set at the 5% level (p b 0.05) with Tukey's test to
compare all
=
means, using CiraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA). All
18
CA 02872863 2014-12-01
experimentswere performed at least in three independent trials and the results
were reported as
mean standard deviation.
[0054] 3. Results and discussion
[0055] The protein content of the final oat protein isolate (OPI) was 91.2%
2.4. Oat protein
was partially hydrolyzed using flavourzyme, alcalase, pepsin and trypsin,
which are common
in industrial applications (Benitez et al., 2008). After 30 mm of enzymatic tr
eatment, limited
hydrolysis was achieved with the final DT-i% value reaching 7.1% 0.54, 5.8%
0.44, 5.5%
0.41 and 5.3% 0.40 for flavourzyme, alcalase, pepsin and trypsin hydrolysates
and the
samples were labeled as OPT-F, OPI-A, OPT-P and OPI-T, respectively.
[0056] 3.1. Characterization of oat protein and its hydrolysates
[0057] 3.1.1. SDS-PAGE
[0058] The SDS-PAGE pattern in Fig. 1 demonstrates that the predominating
protein fraction
in the extracted oat protein is 12S globulin (Lane 2). The acidic (12S-A) and
basic (12S-B)
polypeptides are easily identified. The bands between 43 and 72 kDa and below
17 kDa
correspond to the 7S and 3S fractions, respectively.
[0059] Hydrolysis with flavourzyme partially affected the acidic polypeptide
as the 12S-A
fraction bands shifted to a lower molecular weight region (26-34 kDa) (Lane
3). In contrast,
alcalase had a much stronger effect upon the 12S-A fraction as it disappeared
in OPT-A
sample (Lane 4). This 12S-A was less susceptible to trypsin as most of the
original bands
remained, although the low end of the band shifted to a lower molecular weight
range (Lane
19
CA 02872863 2014-12-01
6). Flavourzyme is a protease composed of a mixture of endoprotease and
exopeptidase,
which has been used to prepare short chain peptides and free amino acids
(Marambe, Shand,
& Wanasundara, 2008). Alcalase is an endoprotease composed of a mixture of
different
proteases, each with different specificities (Sukan & Andrews, 1982), thus it
has a broad
specificity toward peptide bonds. The extensive degradation of the 12S-A
subunit can be
explained by this broad specificity. In addition, Glu is an abundant amino
acid in oat protein
and especially in the acidic subunit of oat globulin (Brinegar & Peterson,
1982; Burgess et al.,
1983). A Glu-specific endopeptidase has been isolated from a commercial
preparation of
alcalase (Svendsen & Breddam, 1992), therefore the presence of Glu in thel2S-A
fraction
favors its alcalase degradation.
[0060] From the enzymes selected in this study, trypsin is probably the most
specific toward
its substrate. Furthermore, it cleaves peptides on the C terminal side of Lys
and Arg (Chen et
al., 2012), thus the acidic polypeptides 12S-A was less susceptible to trypsin
digestion. On the
other hand, the 12S-B was fairly resistant to all enzymes tested and only a
minor shift in the
molecular weight was noticed. The resistance of the basic polypeptide to
enzymatic hydrolysis
could be explained by the fact that this subunit is buried at the interior of
the structure, thus is
not as readily accessible as acidic subunit (Plietz, Zirwer, Schlesier, Gast,
& Damaschun,
1984; Yin et al., 2008). A similar result has been reported for peanut protein
isolate in which
the acidic subunit of arachin was more susceptible to hydrolysis, whereas the
basic subunit
was maintained (Zhao, Liu, Zhao, Ren, & Yang, 2011).
CA 02872863 2014-12-01
[0061] The case of pepsin is an exception as only faint bands were observed
after hydrolysis
(Lane 5), indicating that both 12S-A and 12S-B were digested by pepsin. This
might be
related to the low pH required for pepsin treatment, under which oat protein
could be partially
unfolded and thus both acidic and basic polypeptides are accessible and
susceptible to
proteolysis (Brinegar & Peterson, 1982; Burgess et al., 1983). Therefore both
units are rapidly
digested during 30 min of pepsin treatment.
[0062] 3.1.2. Size exclusion high performance liquid chromatography (SE-HPLC)
[0063] SE-HPLC chromatograms of OPT and its hydrolysate samples are shown in
Fig. 2,
divided into three regions, comprising region I (656-22.4 kDa), region 11(21.4-
0.4 kDa) and
region 111 (b0.4 kDa).
[0064] Oat protein isolate shows a dominant peak in region I with molecular
weight (Mw) of
approximately 190 kDa. This peak almost disappeared in hydrolysates by
alcalase and pepsin,
meanwhile hydrolysates by -flavourzyme and trypsin show a peak dramatically
reduced in
height, indicating that the oat protein was hydrolyzed by pepsin and alcalase,
but only partially
by flavourzyme and trypsin. In region II, OPI shows a group of small peaks
with molecular
weights ranging from 2.4 to 0.7 kDa, whereas this group of peaks dominated in
all the oat
protein hydrolysate samples, confirming hydrolysis of the oat protein.
[0065] Hydrolysates by trypsin, pepsin and alcalase showed a major peak at
¨0.7 kDa and a
shoulder with larger molecular weight. In contrast, the flavourzyme treated
sample showed a
different contour including a sharp peak with molecular weight of 0.9 kDa and
a dramatically
=
reduced shoulder peak, indicating that flavourzyme was more effective at
producing low
21
CA 02872863 2014-12-01
molecular weight polypeptides. Oat protein isolates did not show any peaks in
region III. On
the other hand, all hydrolysates contained small peptides, particularly those
treated with
flavourzyme as a sharp peak of approximately 70 Da was observed at the end of
the
chromatogram. This confirms the ability of flavourzyme to produce small
peptides and even
free amino acids. Peptides from this region are not likely to participate in
the gel formation
process due to their small molecular weight, thus the fractions of main
interest are contained
in regions I and II. Both SDS-PAGE and SE-HPLC observations confirmed that
flavourzyme .
,
:
and trypsin hydrolysates maintained greater integrity of the original
structure of oat protein,
!
when compared to alcalase and pepsin hydrolysates.
Tall*Ill
AlLiiiin zed co Waal ti i L of :::41: pititseiThistiklAeriliislityiLtalyzAs,
:
Ez..411. !i:: It in I
CI, N OPIT G014 oprp, clepi,,,T
,
Pe Yr 5,5 Ei MD 7:1 6,4
4,4 42 4.2
224 164, 142 155 2U.
C Iy::IIIA 77 8,4 8,7 24, 22
Ilk Odin wil, 32. Iii4.. RA, At
Ai6.ill ill.::! 62 a2, a6 &5, 7.9
TI.F.,n 0.i.ile 35: 37 15 .41,3 3.5
e",U4 it.ii:,9, C7 605 611 7.2 62
,
V idilli, 7.5 15 53:
541 5.7
24; 1.6 13 15
:
Ty I or:: ine 22 Mg 3.2 17
ILI iiro 77 13 17 74 6.$1
?FM! i AIM 21 14:
iLyi ille 33 43 3.7 4.21
,
,
Ix) le,103fte 4,6 5.6 6-,'.1. 52 53
Lewd io. Sii 1, ....i as al
;tut ,Il :. 1 µ, i mt. =A ,:'
.,,Ik-1. ,
,
b tik
,
,
22
CA 02872863 2014-12-01
[0066] 3.1.3. Amino acid analysis
[0067] As shown in Table 1, oat protein and its hydrolysates contain high
levels of Glx (Gin +
Gin), since glutamic acid is the most abundant amino acid in oat protein
(Brinegar & Peterson,
1982; Burgess et al., 1983; Liu et al., 2009). Other amino acids at high
levels are Gly, Len and
Val. The amount of Gin Glnwas noticeably reduced in the hydrolysate samples.
This is in
agreement with the enzymatic susceptibility of the acidic polypeptide of oat
globulin,
considering the acidic polypeptide is rich in Gin. The amount of Asx (Asp
Asn) in the
alcalase hydrolysate is significantly higher compared to the other
hydrolysates. This confirms
that the remaining unit is the basic polypeptide, considering that this
polypeptide is rich in
Asp (Burgess et al., 1983). If most of the Glx is considered as Glu, such
amino acid
composition modification could alter the charge of the polypeptide chains, and
thus their
functionalities in different pH environments. Reduced Pro residue was also
observed in oat
protein hydrolysates which could potentially impact the protein properties as
this amino acid
is believed to play an important role in the stabilization of protein
structure due to hydrogen
bonding with hydroxyl groups (Gamez-Gaillen et al., 2002).
[0068] 3.1.4. Differential scanning calorimetry (DSC)
[0069] As shown in Table 2 the extracted oat protein isolate had a
denaturation temperature
(Td) value of 112.4 C, which is in agreement with previous reports (Ma &
Harwalkar, 1987;
Ma et al., 1988). The highest Td valueswere observed at pH 7. Its Td and the
enthalpy of
denaturation (AH) value decreased slightly at pHs 5 and 9, possibly due to
partial denaturation
of oat protein under acidic or alkali conditions (Sun & Amtfield, 2011). It is
interesting to
23
CA 02872863 2014-12-01
notice that the oat protein hydrolysates showed significantly increased AI-1
values although
their Td values remained almost unchanged. It is possible to speculate that
some oat protein
fractions, such as 12S have a configuration composed of loosely arranged
segments and
tightly packed segments. Normally, these tightly packed segments are highly
hydrophobic and
are located at the interior of the structure, whereas the loose segments are
at the exterior, being
1.0 more accessible to hydrolysis. After enzymatic cleavage of the exterior
loose part, most of the
hydrophobic core structure remained in the hydrolysate samples, which could be
much more
stable against heat treatment. Thus higher energy is required to disrupt
intramolecular bonds
to achieve complete denaturation. This type of reaction is called a zipper
reaction (Adler-
Nissen, 1986) and it is not unusual to observe the formation of resistant
polypeptides even
after prolonged hydrolysis due to their compact structure.
lEflx.t:dirataraltedimmturstkfit itkinktrattut andeilidimet [le dIVI:
iiAtypintly.
:111
rJi LI1. Cri iA!)
-.I I 2 CI 7CA !1L5Ril
07.1 101.9.13. 11,2.52. 3,1173 110.14 1,111a;
011,,TLk8 .34L IOU!
24
CA 02872863 2014-12-01
Divie 3;
Cap.rotirt cleaved piiis_trawd a.1111. diffesent i LoflidFfiru
TIE 5-
1.0,tii c 12 a nz: n 117 1.23
CERMT
0pp
0171-A, X
X X
011,-T xty.o:
araffina.
[0070] 3.2. Thermal gelation of OPT and derived hydrolysates
[0071] In this work, the initial thermal gelation test was conducted at two
temperatures near
or above oat denaturation temperature (Td) (110 and 120 C) at three different
pHs (5, 7, 9).
The purpose was to screen samples and conditions that allow gel network
formation, which is
defined for this work as the establishment of a self-supporting structure
showing no flow upon
inversion after thermal treatment and cooling.
[0072] As shown in Table 3, the oat protein formed gels under all tested
conditions. Oat
protein hydrolysed by flavourzyme and trypsin formed gels under almost all
conditions,
except at pHs 5 and 7 at 110 C. Possibly, this temperature was not sufficient
to unfold the
compact structure of these hydrolysates extensively enough to expose reactive
groups that
could participate in crosslinking and form a self-supporting structure;
conversely at a higher
temperature the gelation took place. At pH 9, the protein structure could be
more readily
opened to expose hydrophobic patches, due to disruption of hydrogen bonds and
dissociation
of hydrogen from carbonyl and sulfate groups at alkaline conditions, thus a
lower energy input
was required to favor protein interactions, allowing gel formation at 110 C.
Oat protein
CA 02872863 2014-12-01
hydrolysates by alcalase were able to form gels only at pH 9 while those by
pepsin did not
form gels under any conditions. It is possible that the 12S-A fraction of oat
globulin exerts
great influence over the gelling ability of oat protein, especially under
acidic and neutral pH,
as sampleswith well-preserved 12S-A subunits demonstrated good gelling
properties in such
pH ranges. This might be partially related to the larger molecular weight of
the acidic fraction
compared to the basic polypeptide, which allows exposure of more reactive
sites on a single
polypeptide chain for intermolecular interaction development.
[0073] Whereas smaller fractions expose little reactive sites limiting the
aggregation step,
essential for the gel network formation (Handa, Hayashi, Shidara, & Kuroda,
2001). The
formation of gel for alcalase hydrolysate at pH 9 might be attributed to the
low surface charge
of the basic polypeptide under alkaline condition which has an isoelectric
point of 8-9. Thus
the limited net charge could favor network formation via hydrophobic
interactions due to
,=
reduced repulsive forces compared to those at pHs 5 and 7 (Totosaus et al.,
2002). The
hydrolysate prepared with pepsin could not form gels due to loss of both
acidic and basic
subunits.
[0074] Gels prepared from oat protein and its hydrolysates by flavourzyme and
trypsin were
selected for the following experiments, as these samples were able to form
gels under a broad
range of conditions.
[0075] 3.3. Textural profile analysis (TPA)
[0076] The mechanical properties of the gels prepared with oat protein isolate
and its
hydrolysates at 120 C were then studied, including hardness (force required
to attain a given
26
CA 02872863 2014-12-01
deformation), cohesiveness (work required to overcome the internal bonding of
thematerial)
and springiness (rate at which a deformed material recovers to its original
condition after
removal of deforming force) (Yuan & Chang, 2007). Results of TPA are shown in
Fig. 3.a.
Oat protein isolate formed strong gels at pHs 5 (1.90 N) and 7 (1.92 N) at 120
C. In contrast,
softer gels were observed at pH 8 (0.80 N) and very weak gels were obtained at
pH 9 (0,07 N).
The network structure of a heat-denatured globular protein gel depends greatly
on the balance
of attractive (hydrogen and hydrophobic interactions) and repulsive
(electrostatic) forces
among the protein molecules, as determined by pH and ionic strength (Bryant &
McClements,
2000; Ma et al., 1988). Thus the right balance between the electrostatically
repulsive force and
the hydrophobic attractive force should explain the strong gels obtained at pH
5 and pH 7.
However, beyond the optimal pH, disproportionate repulsive forcesmay have led
to fewer
protein interactions, since very weak gels were formed at pH 9 and
intermediate hardness
values were observed at pH 8. Similar behavior was observed in f3-
lactoglobulin gels prepared
at pH 8. In this case, excessive repulsive forces created a high energy
barrier preventing
denatured protein molecules from associating and forming a strong self-
supporting structure
(Mulvihill, Rector, & Kinsella, 1991). It is interesting to note that
flavourzyme hydrolysates
formed stronger gels than oat protein isolate under comparable conditions.
This improvement
was especially significant at pHs 7-9 as the gel hardness increased from 2.80
N to 4,80 N.
100771 Significant increases in gel hardness were not detected at pHs 5-7 for
trypsin
hydrolysates but hardness values increased to 3.03 N at pH 8, then
dramatically improved to
8.80 N at pH 9. The fact that oat protein hydrolysates produced very strong
gels at pHs 8-9
indicates that the balance between the electrostatically repulsive force and
the hydrophobic
27
CA 02872863 2014-12-01
attractive force changed as a result of enzymatic hydrolysis. Since enzymatic
hydrolysis
reduces the amount of Glu (acidic amino acid), the net charge of the
hydrolysates at pHs 8-9
could be lower than that of the oat protein isolate, leading to decreased
repulsive forces among
polypeptide chains. In addition, the augmented hydrophobicity of the peptide
chains after
partial hydrolysis could contribute to the increased gel strength. A similar
observation was
reported by Ma(1985), in which both surface and exposed hydrophobicity of oat
protein
increased after trypsin hydrolysis. As mentioned earlier, the acidic
polypeptide with
hydrophilic character covers the basic polypeptide which has a more
hydrophobic character,
thus as hydrolysis progresses the acidic polypeptide is broken down and the
overall
hydrophobicity of the remaining fraction is increased (Kuipers & Gruppen,
2008). Thus, the
reduced electrostatically repulsive forces and the increased hydrophobicity
attractive force are
equilibrated at this producing the right balance to develop a gel with
enhanced hardness. It
has been reported that soy protein gels had hardness values of around 2.1 N-
2.6 N at neutral
pH (Lamsal, Jung, & Johnson, 2007; Molina, Defaye, & Ledward, 2002). Gels
prepared with
oat protein isolate, showed a slightly lower value, but those gels prepared
with flavourzyme
and trypsin hydrolysates were comparable or higher than soy protein gels at
the same pH. Gels
prepared with flavourzyme and trypsin hydrolysates at pH 9 showed enhanced
hardness, and
the results corresponding to trypsin hydrolysate gels are even comparable to
egg white protein
gels (8.70 N) under the same pH (Hammershoj & Larsen, 2001). It is understood
that gels
prepared at pH 9 could have a limited application as most food products have a
pH value
between 3 and 8. Nevertheless, egg white has a pH of 7.6-9.7 depending on the
storage time
and temperature (Banerjee, Keener, & Lukito, 2011), and yet it is commonly
used in different
28
CA 02872863 2014-12-01
applications. In addition, strong oat protein gels were also obtained at pHs 7
and 8 after
limited flavourzyme hydrolysis, and at pH 8 after limited trypsin hydrolysis.
[0078] These gels can be more widely used in different food products. The
effect of trypsin
hydrolysis upon the gelling capacity of oat protein was previously studied by
Ma and Wood
(1986, 1987). The result indicated that trypsin treatment leads to a weak gel
structure,
probably due to reduction in the size of the protein molecules, which may no
longer be able to
associate to form a strong gel matrix. Whereas in this work, gels with
significantly improved
hardness were obtained at pHs 8 and 9 after limited trypsin hydrolysis due to
maintenance of
appropriate level of peptide size, allowing formation of good three-
dimensional networks.
[0079] The detrimental effect of trypsin hydrolysis was also observed as part
of the
preliminary experiments for this work (data not shown), in which those
hydrolysates produced
after long periods of enzymatic treatment would not form a gel at all. Gelling
conditions also
significantly affect the properties of the resulting gels. The protein
concentration and
temperature selected for this study were higher than those applied in the work
reported by Ma
and Wood (1986, 1987).
[0080] Oat protein isolate gels displayed good cohesiveness (Fig. 3.b.) with
values of 0.6-0.8
at pHs 5-9. Similar values have been reported for soy protein isolate gels
(Molina et al.,
2002). Gels prepared with flavourzyme and trypsin hydrolysates also presented
good
cohesiveness, although slightly lower values were observed ranging from 0.7 to
0.4. The
cohesiveness values reported in this study indicate that the gels maintained
the integrity of
their internal bonds when compressive forces were applied. A low cohesiveness
value
29
CA 02872863 2014-12-01
indicates damage to the internal bonds and thus a tendency to fracture under
stress. Most of
gels prepared in this study showed good resistance to disintegration due to
compression.
[0081] Both oat protein isolate and its hydrolysate gels showed good
springiness (Fig. 3.c.)
under the conditions tested. The impact of pH on gel springiness did not show
an obvious
trend.
1.0 [0082] 3.4. Gel morphology
[0083] The gel morphology observed using SEM shows the effect of pH on the gel
microstructure (Fig. 4). It was expected that two types of structures would be
observed, either
fine-stranded or particulate gels, however the SEM micrographs showed a
polymer gel
structure for oat protein at pH 7 and for its hydrolysates at pH 9. Typical
particulate gels were
formed for both oat protein and its hydrolysate gels at pH 5 (SEM micrograph
not shown) and
for hydrolysate gels at pH 7. Only some specific protein gels, such as
gelatin, can be
considered polymer gels. Oat protein may have relatively flexible molecular
chains, which
allow formation of bridges between the interaction points when the balance
between
electrostatic repulsive forces and hydrophobic forces among polypeptide chains
is achieved.
This explains the strong mechanical property of oat protein gel at neutral pH
and the
significantly enhanced hardness of gels made from hydrolysates in mild
alkaline pH. The gel
morphology also clearly shows the effect o'f partial enzymatic hydrolysis on
the gel
microstructure. Since enzymatic hydrolysis reduces the amount of Glu (acidic
amino acid), the
net charge of the hydrolysates at pH 7 could be lower than that of the oat
protein isolate,
leading to decreased repulsive forces among polypeptide chains. Therefore
these polypeptide
CA 02872863 2014-12-01
chains could aggregate rapidly via hydrophobic interactions during heating
treatment. Later,
these aggregates associated to form particulate networks. Whereas at 9, the
increased
charge on the polypeptide chains led to strong repulsive force to prevent
rapid protein
aggregation, thus allowed formation of bridges between the interaction points
on the
polypeptide chains to create polymer gel.
[0084] 3.5. Water holding capacity (WI-IC)
[0085] Water holding capacity is another important property of food gels and
the separation of
liquid from the gel network can produce physical modifications such as
shrinking or
alterations in the palatability of the product due to reduced moisture (Mao,
Tang, & Swanson,
2001).
[0086] These changes can reduce the quality and acceptability of the product
and for this
reason a high WI-IC is required in gels destined for food applications. All
gels demonstrated
excellent WI-IC (82.8-95.5%) at pHs 7-9 as shown in Fig. 5. Significantly
reduced WI-IC
values (61.5-65.2%) were observed at pH 5. According to previous literature,
particulate gels
formed at pH near the isoelectric point are characterized by an increased pore
size that leads to
a decrease in capillary forces and therefore a higher water loss
(Chantrapornehai &
McClements, 2002).
[0087] The WI-IC of gels prepared with oat protein isolate at pH 9 could not
be determined as
they were very weak. The WI-IC values reported in this work are comparable to
soy protein
(82.2%) (Wu, Hua, Lin & Xiao, 2011) and whey protein (-88%) (Yamul & Lupano,
2003).
31
CA 02872863 2014-12-01
[0088] 4. Conclusion
[0089] This study has demonstrated that partially hydrolyzed oat protein could
form gels with
similar mechanical strength and water-holding capacity as those from animal
proteins such as
egg white. Thus we can conclude that oat protein gels have potential of
replacing those
derived from animal proteins to provide texture and structure in food
products. This may
provide value-added opportunities for oat producers and processors, and at the
same time,
manufacturers could have access to a new and cost-effective gelling ingredient
of plant origin
in diversified food formulations.
SECTION 2 Cold Gelation
[0090] The above description demonstrates gelling properties of oat protein
isolate (OPT)
upon heat treatment. The mechanical properties of resultant gels were
comparable to those of
egg white protein gels (Nieto-Nieto, T. V., Wang, Y. X., Ozimek, L., and Chen,
L. 2014).
Unlike typical particulate or filamentous gel network structure of globular
protein, oat protein
gels exhibited a polymer-like network structure at pH 7 and 9, which was
similar to gelatin
gels and explained their strong mechanical properties. Strong plant protein
gels are interesting
for applications in providing texture and structure for food products, or
acting as carrier of
bioactive compounds to resist deformation during processing, so as to
potentially replace
animal protein gels. It will be even more interesting if such strong gels
could be formed by
cold-gelation. However, cold-set gelation of oat protein has never been
reported, and the
formation mechanism at molecular and/or supramblecular levels of its unique
polymer-like
network structure is unclear.
02
CA 02872863 2014-12-01
[0091] In this work, the structure, properties and formation mechanism of cold-
set oat protein
gels prepared with glucono-6-lactone (GDL) were systematically investigated.
Molecular basis
of oat protein aggregation was examined using Fourier transform infrared
(FTIR), dynamic
light scattering (DLS), and atomic force microscopy (AFM). Oat protein gel
supramolecular
characterization was then conducted using rheological measurement and a
scaling model,
which linked gel macroscopic elastic properties to microscopic structural
parameters to
determine the gel fractal dimension (1-l4wara, Kumagai, & Matsunaga, 1997,
Renkema, &
van Vliet, 2004).
[0092] 2.1 Materials Naked oat grains Ovena nude) (crude protein 17.2%) were
purchased
from Wedge Farms Ltd., Manitoba, Canada, Glucono-6-lactone (GDL) was obtained
from
Sigma-Aldrich Canada (Oakville, ON, Canada). Other chemicals used in the
experiment were
all analytical grade and from Fisher Scientific (Whitby, ON, Canada). Milli-Q
water was used
in all experiments. Oat protein isolate (OPI) was extracted from deflated oat
flour using
alkaline and isoelectric point precipitation method according to our previous
work (Nieto-
Nieto, et al., 2014). The protein content of OPI was 91.2% 2.4 determined by
Leco nitrogen
analyzer (FP-428, Leco Corporation, St Joseph, MI) and a nitrogen to crude
protein
conversion factor of 6.25 was used.
[0093] 2.2 Rheological properties and pH value of OPI solutions with GDL
[0094] Dynamic rheology experiment was carried out on a DPIR-3rheometer (TA
Instruments,
DE, USA) to study the gelation process of OPI solutions with the addition of
GDL. Parallel
plate geometry with a gap of liniu was used to measure dynamic viscoelastic
parameters
33
CA 02872863 2014-12-01
(shear storage modulus G' and loss modulus G"). The value of the strain
amplitude for all
samples was set as 1%, which was within a linear viscoelastic regime. The
preheated OPT (5
and 7%, w/v) solutions with different amount GDL (3, 5, 10, and 15%, w/w,
based on the dry
weight of protein) were placed on the plate immediately after the addition of
GDL and the
dynamic time sweep measurements were performed at an angular frequency of 1 Hz
at 25oC
over a period'of 20 h. A frequency sweep was subsequently conducted as a
function of angular
frequency (co) from 0.1 to 100 rad s-1 at 25 C to study gel shear strength. A
thin layer of low-
viscosity silicone oil was applied to prevent dehydration during the test. The
change of pH
value during OPT gelation was monitored simultaneously after GDL addition
using pH meter
(Thermo Scientific Orion 3 Star pH Meter, MA, USA) and the pH value did not
change after
aging for 20 h.
[0095] 2.3 Gel preparation
[0096] Gels were prepared by mixing preheated OPT solutions with different
amount of GDL.
Briefly, OPT (5 and 7%, w/v) was dissolved in distilled water and stirred
overnight. The
solution pH was adjusted to 8 using 1 M NaOH before heating. Then the OPT
solution was
tightly sealed in glass vial and heated at 115 C (above denaturation
temperature) in oil bath
for 15 mm, followed by cooling them down to room temperature and addition of
different
amount of GDL (3, 5, 10, 15% w/w, based on the dry weight of protein). Then,
the
suspensions were stored at 4 C for 20 h to form OPI gels. The obtained gels
were coded as
0G5-3, 005-5, 0G5-10, 005-15, 007-3, 0G7-5, 0G7-10, and 0G7-15, corresponding
to
the different OPT and GDL concentrations, respectively.
34
CA 02872863 2014-12-01
[0097] 2.4 Gel properties
[0098] Mechanical properties of the obtained gels with about 10 mm in length
and 12 mm in
diameter were determined using an Instron 5967 Universal testing instrument
(Instron Corp.,
Norwood, MA, USA) equipped with a 50N load cell. All gel samples were
compressed twice
to 50% of their original height at room temperature and the constant crosshead
speed of 1
mm/min. Two texture parameters including compressive stress and springiness
were
computed by software (Buie Hill 2) These parameters were determined from the
typical
Instron force-time curve. Compressive stress indicates the gel firmness
calculated as the
compressive force (hardness, N) over the cross-sectional area of the gel.
Springiness indicates
how well a gel physically springs back after the first compression, which is
measured by the
distance of down stroke of the second compression (Bourne, 2002).
[0099] Water holding capacity (WHC) of OPT gels was measured according to the
method of
Kocher and Foegeding (1993) with modifications. Gel samples (0.8 -1.0 g) were
placed into
Vivaspin 20 centrifugal filter unit (GE Healthcare Bio-Sciences AB, Uppsala,
Sweden) with
5m filter membrane and then centrifuged at 2000 rpm for 5 min at room
temperature. The
weight of the gels was recorded before (Wt) and after (We) centrifugation. The
centrifuged gel
was dried in oven at 60oC overnight and weighted (Wd). WI-IC was calculated
using equation
(1),
We- TaTd.
wHC % =x 100%
Tart-11M
35
CA 02872863 2014-12-01
[00100] 2.5 Gel morphology
[001011 The morphology observation of OPI gels was carried out with a Philips
XL-30
scanning electron microscope (SEM) at an acceleration voltage of 6 kV. The
samples were
frozen in liquid nitrogen and then freeze-dried. The surface of the gels was
then sputter-coated
with gold, observed and photographed.
[00102] 2.6 Controlled release
[00103] Riboflavin was selected as a bioactive molecule model to investigate
the in vitro
release properties of GDL-induced OPI gels. Drag-loaded 0G7-10 gels were
prepared by
dispersing riboflavin in pre-heated OPI solutions before adding GDL, and the
riboflavin
content was 7.1% (w/w) based on the dry weight of protein. The drag-loaded
gels were cut
into small pieces (2 x 2 x 2 mm) and dried at room temperature for 48 h. The
release kinetics
was then assessed with a 2100C dissolution system (Distek Inc., NJ, USA) in
four dissolution
mediums: HCI-saline solution (pH 1.2); phosphate-buffered saline (PBS, pH
7.4); simulated
gastric fluid (SGF, pH 1.2) with 0.1% pepsin (\v/v); and simulated intestinal
fluid (SIF, pH
7.4) with 1.0% pancreatin (w/v). One piece of drug-loaded gel was placed in 50
iriL HC1-
saline solution or SGF at 37 C and stirred at 100 nnp.
[00104] After 2 h, the gel was washed and transferred into 50 mL PBS or SIF
for another 15 h
at the same conditions. The riboflavin content in the release mediums was
monitored with S-
3100 UV-vis spectrophotometer (Seine Co. Ltd., Japan) at a wavelength of 445
urn.
[00105] 2.7 Protein structures in OPI gels
36
=
CA 02872863 2014-12-01
[001061 The conformational changes of unheated, preheated OPI and acid-induced
OPI gels
were characterized by FTIR. OPI (1%, w/v) were dispersed in H20 and adjusted
to pH 8 using
1% Na0H, The OPI solution was heated at 115 C for 15 min, followed by cooling
it down.
Then, different amount of GDL (3, 5, 10, 15%, w/w) was added into OPI
solutions,
respectively, and stored for 20 h. The samples were placed between two CaF2
windows
separated by 25 im polyethylene terephthalate film spacer for FTIR
measurement. 1120 with
or without GDL was used as background. The spectra of samples were recorded
using a
Nicolet 6700 spectrophotometer (Thermal Fisher Scientific Inc., Pittsburgh,
PA, USA) in the
range of wavenumber from 400 to 4000 cm-1 during 128 scans with 2cm-1
resolution. The
spectrophotometer was continuously purged with dry air from a lab gas
generator (Parker
Hannifin Corp., USA). For amide I band region (1700-1600 cm-1), Fourier self-
deconvolution
was performed using Omnic 8.1 software at a bandwidth of 24 cm-1 and an
enhancement
-factor of 2.5. The established wavenumber ranges reported by Byler, and Susi
(1986) were
used as reference to assign the amide I band components to secondary structure
motifs.
[001071 Dynamic light scattering measurement was performed using a Zetasizer
Nano-ZS
(Malvern Instruments Ltd., UK) equipped with a 633 mu He-Ne laser to determine
the size
change of ON. A total of three averaged sub-runs were analyzed at a fixed 90o
scattering
angle. Unheated, preheated, and GDL added OPI suspensions (1%, w/v) were
diluted to 0.25
mg/mL in Milli-0 water before analysis. The apparent particle size was
obtained by CONTIN
mode analysis.
37
CA 02872863 2014-12-01
[00108] The morphology of unheated, preheated, and GDL added OPT suspensions
were
=
=
determined by atomic force microscopy (AFM). OPT suspensions were diluted to
0.025
nag/mL. Then, 20 III, sample solution was deposited onto freshly cleaved mica
and dried at
room temperature. Tapping mode AFM images were collected by AFM MRF-3D (Asylum
research, Oxford Instrument Company, Santa Barbara, CA, USA) and Inverted
Optical
Microscope Olympus 70/71 (Olympus Co., USA) under ambient conditions. The
system was
installed in an acoustic hood to minimize vibrational noise. A silicon-etched
cantilever with a
tip radius of 20-30 nm was driven at oscillation frequencies in the range of
580-600 kHz. The
collected images were -flattened using AS software for further analysis.
[00109] 2.8 Determination of fractal dimension
[00110] Strain sweep measurements for 5-8% (w/v) cold-set OPT gels formed at
various GDL
concentrations (3, 5, 10, 15%, w/w) were carried out on a DlR-3 rheometer (TA
Instruments,
DE, USA). Each gel was cut into 10 mm height before test. The gel was
compressed to 80%
of original height (8 mm) using parallel plate geometry. The G' value of each
gel was
evaluated as a function of strain from 0.1 to 100% with an angular frequency
of 1 Hz. Initial
G' value, GO, was calculated as the average value of G' at the strain range
from 0.1 to 1%.
Over a certain strain, G' decreased corresponding to the breakdown of the
gels. The critical
strain (70) was calculated as the criticaLpoint of strain at 95% of GO.The
calculated GO and 70
were then used for -fractal analysis of OPT gels.
[00111] The scaling model modified by Wu and Morbidelli (2001) was selected to
determine
the elastic contributions of both inter- and intra-floc links using a
microscopic elastic constant
38
CA 02872863 2014-12-01
(a, 0 < a < 1). It allows the identification of gelation regime prevailing in
the system and
indicates the importance of inter- and intra-fioc links. The expressions of
scaling model
dependence of G' and 70 are as follows:
0,01(d¨D.0 ()
et.--P-131/41¨Dti (3)
f3 = ¨ :2) + (2 -1-3c)(1. ¨ ict) (4)
[00112] Where d is the Euclidean dimension (d=3 in three-dimensional systems);
Dir is the
-fractal dimension of the system; fl is an auxiliary parameter; x is the
fractal dimension of the
floc backbone or tortuosity of the network range of [1, 1.3]. Based on the
equations (2) and
(3), Df and j3 can be directly calculated through the slopes (power-law
exponents) of the log-
log plots (G' vs oandy0 vs 0).
[00113] The values of a will be determined through subsequent substitution of
the fi value to
equation (4) with the assumed backbone -fractal dimension x= 1 and x= 1.3,
respectively, to
identif the prevalent gelation regime in the system.
[00114] 2.9 Statistical analysis
[00115] All experiments were performed at least in triplicate. Results were
expressed as mean
standard deviation. Statistical analysis was conducted using the Statistical
Analysis System
(SAS for windows, Release 9.0, SAS Institute Inc., Cary, NC). Analysis of
variance
(ANOVA) was chosen to analyze the effects of GDL concentration on gel
mechanical
properties, Tukey test was used to compare multiple means. A probability
ofp<0.05 was
considered to be statistically significant.
39
CA 02872863 2014-12-01
[00116] 3. Result and discussion
[00117] 3.1 Cold-set OPI gel characterization
[00118] 3.1.1 Gelation of OPI suspensions with GM,
[00119] Relatively low concentrations of OPI (5 and 7%, w/v) and high
electronic repulsion
condition (pH 8) were Chosen to achieve the cold-set gelation rather than the
thermally
induced gelation. The addition of GDL to the pre-heated OPI solutions resulted
in the decrease
of pH and neutralization of the negatively charged OPI molecules. The
gradually weakened
repulsive force allowed the establishment of connections among OPI molecules
to form a
continuous three-dimensional network.
[00120] The pH changes during acidification of preheated OPI solutions at
various GDL
concentrations as a function of time are shown in Figs. 1a and lb. For OPI
solutions with low
GDL concentrations (3 and 5% GDL), pH decreased rapidly in first 100 min;
while for the
samples with relatively high GDL concentrations (10 and 15% GDL), fast pH
reduction was
found during first 210 min, followed by slow pH decrease. After 1200 min,
steady-state pH
values were achieved, indicating that the GDL hydrolysis reached equilibrium
situation. Thus,
an aging time of 1200 min (20 h) was selected for the formation of GDL-induced
OPI gels.
Increasing GDL content led to faster pH reducing rate and lower final pH
values due to the
high level of &conic acid production. This result agreed with the trend of
acid-induced whey
protein isolate gels (Cavallieri, & Cunha, 2008). Both 5 and 7% OPI solutions
had similar pH
alteration trends and final pH values, suggesting that the GDL content was an
important key to
control the final pH of the system, as a consequence impacted the gel
structure and properties.
CA 02872863 2014-12-01
According to our preliminary works, the final pH values of OPT solutions were
set as 6.75,
6.25 (higher than IEP), 5.15 (around EP) and 4.35 (lower than IEP) by adding
different
concentrations of GDL (3, 5, 10, and 15%, w/w), respectively.
1001211 The OPT gelation process triggered by GDL hydrolysis was monitored by
advanced
rheometer. Figs. lc and ld illustrate the evolution of storage modulus (G') of
OPT solutions
with different GDL concentration as a function of time. G' and G" values
indicate the
evolution of the solid elements and viscous elements in the system,
respectively. The initial G'
was always higher than G" for oat protein, suggesting that an elastic modulus
predominated
the system (Tunick, 2010). In this case, only G' was chosen as the indicator
to display the OPT
gelation process at different GDL concentrations. For all samples, G'
significantly increased
after the addition of GDL within a certain time, then presented a plateau-like
behavior which
indicated the transition from a viscoelastic fluid to a viscoelastic solid
(Barbut, & Foegeding,
1993). Comparing Figs. la with lc or lb with ld, the development of G' was
correlated to the
gradual decrease of pH value, and the points where G' researched plateau were
found at pH
6.80 for all the samples. The suspensions reached these points faster at
higher amount of
GDL. However, the highest final G' values (1200 min) were observed when GDL
content was
10% and the final pH value was around IEP. It could be supposed that the final
pH of the
system as modulated by GDL amount played an important role in determining the
structure
and properties of OPT gels.
[00122] 3.1.2 Gel mechanical properties and water holding capacity
41
CA 02872863 2014-12-01
[00123] Figs. 2a and 2b show the frequency dependence of G' and G" curves of
OPT gels
prepared with different GDL concentrations. For all the samples, G' was higher
than G". The
lowest G' values for both 5% and 7% OPI gels were found at 3% GDL. The G' and
G" curves
of OG5-3 were frequency dependent and even had crossover at high frequency,
which
indicated the weak gel shear strength (Nunes, Raymund , Sousa, 2006.
Savadkoohi,
Faraimaky, 2012). G' values of other gels formed at 5, 10, and 15% GDL were
frequency
independent, indicating strong gel shear strength (Zhang, Jiang, Wang, 2007).
The highest gel
shearing strength was observed at the GDL concentration of 10%. In addition,
gels formed
with higher protein concentration (7%, w/v) exhibited stronger shear strength.
=
[00124] Compressive stress of OPT gels is shown in Fig. 2c, which indicates
the gel firmness.
None of the gels broke when com-pressed to 50% of their original height. GDL
contents
significantly impacted gel compressive stress. As shown in Fig. 2c, the
weakest gel was
observed at 3% GDL concentration, where the final gel pH was 6.75. With
increasing GDL
concentration to 10%; the compressive stress of OPT gels dramatically raised
from 2.5 kPa to
12.5 kPa where the final pH value decreased to around 5.15. Further increase
of GDL
concentration to 15% led to the decrease of compressive stress to 9.0 kPa and
a lower pH
value of 4.35. Gels prepared with 7% protein exhibited significantly higher
compress stress
than those prepared with 5% protein when the GDL content was same. This trend
was in
accordance to the result of frequency sweep test. Similar phenomenon was also
reported -for
fish protein gels (Hamaguchi, & Tanaka, 2003, Fretheim, et al. 1985) and diary
protein gels
(Jacob, M, Nobel, S., Jams, D., & Rohm, 1-1. 2011). The gel springiness, which
indicates how
well a gel physically springs back after the first compression, is shown in
Fig. 2d. All the gels
42
CA 02872863 2014-12-01
exhibited good springiness, since they could spring back to 3.4 - 4.3 mm after
first
compression of 5 mm. GDL amounts did not significantly impact the gel
springiness when
gels were prepared with 5% OPT. However, when prepared with 7% OPT, the gel
with 10%
GDL showed lowest springiness 3.5 mm).
[00125] Water holding capacity ('WHC) is another important property of gel to
evaluate its
acceptability. Losing water caused by intrinsic instability, external forces,
or temperature
fluctuation may result in the shrinkage of gels, changing texture and reducing
quality (Mao,
Tang, & Swanson, 2001). Thus, the gels with high WHC are usually required for
food and
non-food applications. As shown in Fig. 3, all the gels prepared with 7% OPII
demonstrated
excellent WHC of around 89-92%, which were generally higher than those
prepared with 5%
OPI (68-80%). Moreover, the gel with 3% GDL exhibited higher WHC compared to
the gel
with 10% GDL,
[00126] It was worth noting that 0G7-10 gel showed the highest compressive
stress of 30
kPa, which is comparable to that of egg white protein gels (22-32 kPa)
(Hammershoj &
Larsen, 2001). In addition, these cold-set OPT gels were prepared at
relatively low protein
concentration, but most of them possessed superior gel strength comparing with
many other
plant protein gels. For example, thermal-induced oat protein gels (15% w/v)
had hardness
values of around 13 kPa at pH 7 and 5 (Nieto-Nieto, et al. 2014); legume
protein isolate gels
(20 wt%) showed the compressive stress of approximate 5.0-8,0 kPa when
compressed to
70% deformation (Makri, Papalamprou, & Doxastakis 2006); the stress at rupture
for cold-set
whey protein (8%)/flaxseed gum (0.3%) bi-polymeric gel and soy protein (3%)
gella.n gum
43
CA 02872863 2014-12-01
(0.7%) bi-polymeric gel was around 25 and 20 kPa, respectively, when
compressed to 80% of
iteir original height (Kuhn, Cavallieri, & Cunha, 2011; Vilela, Cavallieri, &
Cunha, 2011); the
GDL-induced 7% whey protein isolate gel had stress of around 22 kPa at rupture
when
compressed to 80% of its original height (Cavallieri, & Cunha,2008,). GDL-
induced OPT gels
also exhibited excellent WHC, which were comparable not only to thermal-
induced oat
protein gels (-- 90%) and soy protein gels (¨ 82%) (Nieto-Nieto, et al. 2014,
Wu, Hua, Lin, &
Xiao, 2011), but also to the cold-set whey protein gels (-92%) (Vilela, et
al., 2011). The gels
maintain their good mechanical properties and water-holding capacity after
microwave
treatment.
[00127] 3.1.3 Gel morphology
[00128l In general, globular protein forms gels with particulate or
filamentous
microstructures depending on processing conditions, such as pH, ionic
strength, etc. As shown
in Fig. 4, polymer-like network structure was observed for all cold-induced
OPT gels, which
could be the reason of their strong mechanical properties. However, various
pore size and wall
thickness were observed depending on the final pH value modulated by different
GDL
contents. Network structure with large pores was observed at pH 6.25 and 4.35
when 5% and
15% GDL were added into the system, whereas the pore size was much smaller at
pH 6.75
and 5.15 with addition of 3% and 10% GDL. Especially, the walls were obviously
thicker
when prepared at 10% GDL compared to those at 3% GDL (insets of Figs. 4a and
4c). The
alteration of gel network structure well explained the change of their
mechanical properties.
The most compact network structure with small pore size and thick walls formed
at 10% GDL
44
CA 02872863 2014-12-01
resulted in the strongest gel, while the moderate mechanical properties were
observed for
those with 5 and 15% GDL due to the relatively loose network structure.
Although 3% GDL
gels also had small pore size, the thickness of wall was much thinner. Thus,
ON gels formed
at 3% GDL exhibited the weakest gel strength. The same trend was observed for
both systems
with protein concentration of 5% and 7%. But in general, more solid and
compact structure
was observed at 7% protein due to stronger protein-protein interactions, thus
the gels with 7%
OPI contents were stronger than 5% OPI gels.
[00129] Environmental pH value impacts the balance between attractive force
(hydrogen
bonding and hydrophobic interaction) and repulsive force (electrostatic) in
the gel system. As
a consequence, it influences the gel network structure and gel strength
(Bryant & McClement,
2000; Ma, nanzada., Harwalkar, 1988). When the final pH of gel was far from
IEP (pH 5) of
oat protein, such as OG5-3 and 007-3 (pH 6.75), the high repulsive forces
between protein
molecules which might resist the development of protein molecular attractive
interactions
resulted in gel network structure with small pores and thinnest walls. Thus,
the gels prepared
with 3% GDL exhibited the lowest gel strength. As the pH decreased to 6.25 at
5% GDL, the
reduced repulsive forces between protein molecules caused the increase of pore
size and wall
thickness, which accounted for higher gel shear strength and compressive
stress compare to
the one prepared with 3% GDL. When the pH was near oat protein IEP, the
electrostatic
repulsive forces reached a minimum value which facilitated development of
protein attractive
= interactions to reinforce the gel networks (Denis Renard, Fred
vandeVelde, & Visscher, 2006,
Totosaus, Montejano, Salazar, & Guerrero, 2002). Therefore, OH gels induced by
10% GDL,
where the final pH was around 5.15, showed dense OPI gel network structure
with rough wall
CA 02872863 2014-12-01
consisted of high level of protein aggregates (internal figure of Figs. 4a and
4c) and small
pores, resulting in the highest shear strength and compressive stress. In
addition, as a.
consequence of reduced electrostatic repulsions at this condition, the protein-
protein
interactions increased and protein-water interactions decreased (Puppo,
Lupano, & Afion,
1995, Chantrapornchai, & MeClements, 2002), so that the weaken of capillary
forces between
protein and water molecules was responsible for the relatively low WI-IC value
of OPT gels
with 10% GDL. Likewise, the stronger interactions or cross-links within 0G7-10
gel
restricted the flexibility of the protein aggregates, and caused the less
springiness and more
rigid structure (Ngapo, et al, 1996). While at pH lower than IEP (gel formed
with 15% GDL,
pH 4.35), the pore size of gel network became large again due to the regained
electrostatic.
repulsive forces. Thus, the gel shear strength and compressive stress
decreased again. In
addition to GDL content, protein concentration also significantly influenced
the gel
mechanical properties and water holding capacities. The stronger gels with
better water
holding capacities were obtained in system with higher protein concentration,
where more
protein molecules were involved to build up the gel network structure and had
capacity to
interact with water. Unlike soy protein and whey protein gels, cold-set OPI
gels formed the
expected polymer-like structure, which largely contributed to the enhanced gel
properties.
[00130] 3.1.4 In vitro release behavior
[00131] Gels have been extensively studied in food and nutraceutical
applications, due to
their ability to protect and deliver bioactive compounds (Chen, et al, 2006;
.Buwalda, Noere,
Dijkstra, Feijen, Vermonden, and Hennink, 2014). The porous gel network
structure along
46
CA 02872863 2014-12-01
with retaining high water content allows gels to encapsulate water-soluble
molecules with
high drug loading efficiency (Kashyap, Kumar, Ravi Kumar, 2005; Gangyly,
Chaturvedi,
More, Nadagouda, Aminabhavi, 2014).
[001321 To investigate the controlled release property of GDL-induced OPI
gels, the strongest
gel 0G7-10 with small pores and dense wall was chosen as the matrix and
riboflavin was
selected as the model bioactive molecule. As shown in Fig. 5, 0G7-10 gel
exhibited a slow
release rate in the mediums without digestion enzymes, where only 11.4% and
37.2%
riboflavin was detected in HC1-saline after 120 ruin and in PBS after 1020
min, respectively.
Understanding of the release mechanisms is important to design nutraceutical
delivery system
efficiently. In general, the mechanisms of drug release from a polymer matrix
can be
categorized in three ways (Arifin, Lee, & Wang, 2006): (1) diffusion from the
non-degraded
polymer (diffusion-controlled system); (2) enhanced drug diffusion due to
polymer swelling
(swelling-controlled system); and (3) release by polymer degradation and
erosion (erosion-
controlled system). The Korsmeye Peppas semi-empirical equation was applied to
identify
the mechanism of riboflavin release from 0G7-10 gel (Chen, Remondetto,
Rouabhia, &
Subirade, 2008; Wang, & Chen, 2012; Wang, & Chen, 2014):
(5)
[00133] Where Mt /M is the fraction of the model molecule released after time
t relative to
the amount of model molecule released at infinite time, k is a constant and n
is the diffusional
exponent. Inferences about the release mechanism are based on the fit of this
equation to the
model molecule release data through 60% dissolution and comparison of the
value of n to the
47
CA 02872863 2014-12-01
semi-empirical values for slab geometry reported by Peppas, where n = 0.43
indicates Fickian
diffusion, 0.45 <ii < 0.89 indicates non-Fickian transport, and n = 0.89 or
higher indicates
case II transport. The 0G7-10 gel in HC1-saline buffer (before 120 min) had a
12 value of 0.23
(R2 = 0.93), while in PBS (after 120 min) the n value was 0.68 (R2 0.98). This
result
revealed that riboflavin release followed a diffusion-controlled mechanism in
the acid
condition at first, while the enhanced riboflavin diffusion happened due to
the swelling of the
gel in PBS.
[00134] The release behavior of riboflavin from 007-10 gel in the simulated
gastro-intestinal
tract with the presence of digestive enzymes was also evaluated. Drag-loaded
gels were
immersed in SGF for 120 min and then in SIF for another 900 min. As shown in
Fig. 5, the
release of riboflavin was only 22.6% after the incubation in SGF,
demonstrating a good
barrier property of OPT gel. When transfer to SIP', the remaining riboflavin
was released
slowly and completed after 960 mita due to the gel matrix degradation. The
controlled release
of riboflavin in PBS or simulated gastro-intestinal fluids could be due to the
well-established
polymer-like network structure with dense wall and small pores, which
inhibited the
penetration of PBS and/or digestive enzymes and slowed down the leaching out
of bioactive
molecules. Thus, it could be concluded that the GDL-induced OPT gels had the
ability to
protect and deliver bioactive molecules to the small intestine.
[00135] 3.2 Cold-set OPI gel formation mechanism
[00136] Unlike many other globular proteins, oat protein formed polymer-like
structure. Such
unique structure endowed the OPT gels strong mechanical properties, high water
holding
48
CA 02872863 2014-12-01
capacity and controlled release behavior, which suggested wide food and non-
food
applications. Especially, they were formed at mild conditions. This triggered
our interest to
study the OPI gel formation mechanism, because a better understanding of the
correlation of
gel structure and functional properties will allow designing gel with
desirable applications at
molecular and/or supramolecular level.
[00137] 3.2.1 A molecular study - Protein conformational changes
[00138] Amide I band, which corresponds mainly to the C=0 stretching vibration
of the
peptide backbone, is sensitive to the alterations in protein secondary
structures (Byler & Susi,
1986 Surewicz &mantsch, 1988, Renugopalakrislman, Chandrakasan, Moore, Hutson,
Berney, & Bhatnagar, 1989). Four main components relate to particular
secondary structure
including a-helix, fl-sheet, 13-turn, and random coil (Surewica, Mantsch, &
Chapman, 1993,
An-ondo, Muga, Castresana, & Coni, 1993). These individual component peaks can
be
achieved through Fourier self-deconvolution within a range of 1600-1700 cm-1.
[001391 The deconvoluted spectrum in the amide I band region of I% (w/v) OPI
solutions
without heating treatment is shown in Fig 6a. Unheated OPI exhibited several
bands which
had been previously assigned to protein secondary structures: 1691 cm-1 (fl-
sheets/turns),
1670 cm-1 (fl-sheets/turns), 1646 cm-1 (a-helix and random coil), 1638 and
1628cm-1 (13-
Sheets), and 1608 cm-1 (vibration of amino acid residues) (Byler et al., I
b86; Boye, Ma,
Ismail, & Alli, 1996; Liu, Li, Shi, Wang, Chen, Liu, et al., 2009). The
deconvoluted spectrum
of OPI solution heated to 115 oC (Fig. 6a) significantly differed from the
native one. It should
be noticed that two new peaks appeared at 1682 and 1618 cm-I. The band of 1618
cm-1
49
CA 02872863 2014-12-01
corresponded to intermolecular 3-sheets caused by aggregation via hydrogen
bonding (Clark,
Saunderson, & Suggett, 1981), while the one at 1682 cm-1 indicated the
antiparallel 13-sheets
(Bandekar, 1992). Moreover, the high intensity at the wavenumbers ranged from
1625 to 1675
cm-1 could be observed. Within this range, the band at 1631 cm-1 was
corresponded to
disordered 13-strands, the high intensity at the wavenumbers of 1642 cm-1 was
attributed to
polypeptide segments in random coil configuration, and the weak peak at 1659
cm-I could be
associated with the segments of a-helix. After cooling the preheated solutions
(Fig. 6a), the
peak at 1670 cm-1 disappeared and the intensity of peaks at 1659, 1642, and
1618 cm-1_
increased.
[001401 The increase of intermolecular and antiparallel 13-sheets structures
and the changes of
a-helix, 13-strands, and random coils contents suggested the conformation
rearrangement and
realignment of molecular segments within the network during heating and
cooling process.
Fig. 6b presents the deconvoluted spectra of OPI solutions with various GDL
concentrations.
Interestingly, the spectra of OPT solution with 3% GDL was very similar to
that of preheated
OPT solution without GDL (Fig. 6b), suggesting that the molecular stnicture of
OPT did not
significantly change after the addition of 3% GDL. With the further increase
of GDL
concentration, the significant decrease of intensity between wavenumbers of
1625 to 1675 cm-
1 could be found, and the aggregation peak at 1618 cm-1 became larger and
broader which
even shifted to 1610 cm-1.
[00141] Based on these results, it was understood that oat protein got
denatured and then
aggregated during heating. Through cooling process, more aggregation was
observed since the
CA 02872863 2014-12-01
peak at 1618 cm-1 became larger. Similar to the conformational changes of whey
protein,
intermolecular and antiparallel I3-sheets increased during heating and cooling
(Painter, and
Koenig, 1976; Ma, Rout, Philips, 2003; Remondetto, G. E., Subirade, M. 2003).
Nevertheless,
in spite of aggregation structures, the considerable amount of secondary
structures still
existed. It was different from BSA, soy and whey proteins, which exhibited
most aggregates
after heating and cooling (Murayama, & Tomida, 2004; Lefevre, & Subirade,
2000;
Remondetto, G. E., Subirade, M. 2003). This difference might relate to the
amino acid
composition of oat protein, which contained high level of Glx (Glu + Gin).
Such amino acid
composition could alter the charge of the polypeptide chains, so that prevent
protein severe
aggregation at pH far from its IEP. After adding GDL, Obvious aggregation
between protein
molecules was monitored by FTIR. However, because of the existence of
secondary
components, the association might also occur between the polypeptide segments
at
supramolecular level in addition to small aggregates. Unfortunately, this
change could not be
=
reflected by FTIR test.
[00142] 3.2.2 A supramolecular study -- Size and microstructure analysis
[00143] Fig. 7a illustrates the size distribution of native, preheated, and
GDL added OPT
suspensions using dynamic lighter scattering technique. The native (Fig. 7a,
BEI) OPT
displayed two peaks, which indicated the approximate hydrodynamic radius of
59.59 and
620.40 mu, respectively. After heating at 115 C for 15 min, only one peak was
observed and
the hydrodynamic radius of OPT significantly decreased to around 15.12 n-m.
Addition of GDL
caused OPT hydrodynamic radius increased again to 43.85 nm. The changes of
particle size of
51
CA 02872863 2014-12-01
native, preheated, and GDL added OPI suspensions were further determined by
atomic force
microscopy. As shown in Fig. 7b, the native OPI displayed the heterogeneous
aggregates with
disordered structures on the mica slide. Notable differences were found in
preheated oat
proteins (Fig. 7c) that those large aggregates dissociated into small ones
with size range from
approximately 20 to 300 urn. It should be noticed that the size of these
particles observed by
AFM was larger than that determined by DLS. It was due to the air drying
process during
sample preparation, which aroused aggregation of the protein molecules or
association of the
aggregates. The addition of GDL into preheated OPI solution significantly
increased the
particle size (Fig. 7d). Although no continuous network structure was observed
due to the
dilute protein concentration, these large aggregates could be considered as
precursors of gel
network. It was interesting that the size of OPI decreased after heating at
115 oC for 15 min,
which was contrary to soy protein and whey protein that their hydrodynamic
radius increased
after heating due to the thermal-induced molecular aggregation (Jones,
Adamcik, Handschin,
Bolisetty, & Mezzenga, 2010; Maltais, Remondetto, & Subirade, 2008). The
significantly
decreased hydrodynamic radius of preheated OPI was attributed to the
dissociation of OPI
hexamers (Zhao, Mine, & Ma, 2004; Runyon, Nilsson, Alftran, & Bergenstahl,
2013). When
heating at high temperature (100 or 110 oC), the monomers even existed as
extended
conformation, which has similar size with trimmers of around 5.9 urn. In this
work, the size of
preheated OPI was in the range of 7 to 20 urn, so it might consist of extended
monomers,
oligomers, and small aggregates. This could explain the result of FTIR that
considerable
amount of secondary structures mainly with random coil polypeptide segments
still existed in
addition to aggregates after heating and cooling process. After adding GDL,
these polypcptide
52
=
CA 02872863 2014-12-01
segments could be observed as revealed by FTIR, but the particle size
significantly increased
as shown in DLS and AFM results. Thus these monomers and oligomers should
directly
associate to form gel structure at supramolecular level, which confirmed the
assumption
proposed in FTIR test.
[00144] According to the above results, it could be deduced that both
association of OPI
oligomers and aggregates occurred at the same time after adding GDL. Normally,
globular
protein forms soluble aggregates first, followed by association into insoluble
aggregates and
thus a three-dimensional network. However, the three-dimensional network of
oat protein was
formed through the link of monomers, oligomers and small aggregates. It was
worth noting
that the association was initiated at pH 8, where relatively strong
electrostatic repulsive forces
existed between OPT. Thus, the monomers and oligomers could have high
potential to grow in
an order way into polymer-like microstructures following the nucleation-
dependent
polymerization process, similar to protein fibril formation. The first step
was the initial slow
nucleation phase, in which the nuclei (oligomers and/or small aggregates) were
formed from
extend monomers. Then the elongation stage began with addition or condensation
in an
orderly fashion of monomers and oligomers to form protofibrils, and finally,
the association of
protofribils led to polymer-like network microstructure (Harper, & Lansbury,
1997; Lee,
Culyba, Powers, 8z, Kelly, 2011; Benseny-Cases, Klementieva, & Cladera, 2012).
[00145] 3.2.3 A supramoleeular study-- Sealing behavior and fractal analysis
[001461 Rheological test was then applied for fractal analysis, since it
provides another
insight into the microstructure of gel based on the macro-mechanical
properties. The scaling
53
CA 02872863 2014-12-01
model developed by Wu et al. (2001) was chosen in this study which relates the
structure of
gels to the theological properties. Using this model, the fractal, dimensions
(Di) and gelation
regime prevailing in the system could be estimated and identified. Calculation
of the Df and
determination of the gelation regime using this model closely relies on the
double logarithmic
plot of elastic modulus (G') and critical strain (70) versus protein
concentration (go).
[00147] Fig. 8a shows the modulus-strain profile of 3% GDL-induced OPT gels.
At all protein
concentrations, G' values remained almost constant as strain increased and
then suddenly
decreased beyond a certain strain value, which indicated a breakdown of bonds
within the gel
network and a transition from linear to non-linear behavior (Ould Eleya, &
Gunasekaran,
2004). The strain amplitude at which G' began to decrease by 5% from its
maximum value, as
shown in the inset of Fig. 8a, was taken as a measurement of the limit of
linearity or critical
strain 70 of the gel (Rueb & Zukoski, 1997; Shill, Shill, Kim, Liu, & Aksay,
1990). The gels
formed with 5 and 10% GDL had similar transition trends where the critical
strain decreased
with the increase of protein concentration, while gel produced by 15% GDL had
an opposite
transition trend. According to the obtained critical strain, scaling behaviors
of y0 versus cp for
gels prepared with different amount of GDL were plotted in Fig. 8b.The average
0' values in
the linear region of strain sweep measurements were calculated and plotted as
a function of
protein concentration in Fig. 8c. As shown in Figs. 8b and 8c, both 70 vs (f)
and G' vs cp for all
the gels exhibited power-law relationship and the slopes which indicated as
the exponents
(Table I, A and B) were applied to obtain Df and a values through equations
(2), (3) and (4).
Aggregation of protein particles behaves as stochastic mass-fractal on a
length scale larger
than the size of primary particles (Hagiwara, kumagai, & Nakamura, 1998;
Marangoni,
54
CA 02872863 2014-12-01
Barbut, McGauley, Marcone, & Narine, 2000). Then these highly disordered
fractal
aggregates grow to form three-dimensional continuous network or gel when
protein
concentration is large enough. The Df value is used to quantify the disordered
structure of
aggregated particles, which indicates the relation between the number of
particles in the
aggregates and their typical size (Jullien and Botet, 1987; Vreeker, Hoekstra,
den Boer, and
Agterof, 1992). The estimated Df values of OPT gels are listed in Table 1,
ranging from 1.99
to 2.31, which agree well within the range of Df (¨ 1.5-2.8) of other protein
gels (Bremer, et
al. 1990; Hagiwara, et al. 1998, Marangoni, et al, 2000; Bi, Li, Wang, &
Adhikari, 2013; Ould
Eleyaõ et al., 2004). Normally, the higher Df value means more compact and
dense aggregate
structure (Vreeker, Hoekstraõ den Boer, and Agterof, 1992; Kontogiorgos,
Vaikousi,
Lazaridou, and Biliaderis, 2006). It was necessary to note that the gel
induced by 3% GDL had
higher Df value of 2.23 comparing to 5% GDL (Df = 1.99), which meant the
structure of 3%
GDL induced gel was more compact and dense. However, the high-energy barrier
caused by
strong electrostatic repulsion forces due to the pH far from protein 1EP at 3%
GDL condition
could result in the high Df values (Lin, Lindsay, Weitz, Ball, Klein, &
meakin, 1990). Thus,
the high Df value of 3% GDL induced gel overestimated relationships between
OPT
molecules. With raising GDL concentration from 5 to 15%, Df values increased
from 1.99 to
2.31, which implied that the protein aggregates became more compact and
denser. As GDL
induced pH reduction from pH 8 and Df value growth, micro- and macro-syneresis
within the
system might occur. For the gel, induced by 5% GDL (pH 6.25), small amount of
repulsive
charges among OPT molecules were existed. Thus the rearrangement of protein
aggregates was
believed to happen, also known as micro-syneresis, which was initiated at
molecular level by
CA 02872863 2014-12-01
binding of the flexible branches of the clusters (Mellema, Walstra, van
Opheusden, & van
Vliet, 2002). This process resulted in bigger cluster and larger pores,
leading to a gradual
coarsening of the structure and a change in the firmness of gels, which well
explained the
network structure of 5% GDL induced ON gel and gel strength revealed in SEM
image and
mechanical test. At 10% GDL, the pH was around oat protein IEP where the
repulsive forces
were almost diminished and higher Df value was observed. Accordingly, macro-
syneresis
occurred, which behaved as compacting and shrinking gel, inducing denser
aggregates, and
forcing out liquid (Mahais, et al. 2008, Mellema, et al, 2002). As a
consequence, the walls of
0G5-10 and 0G7-10 gel network structure became denser and rougher, and the
pore size
became smaller than 5% GDL induced gel (internal figures of Figs. 4a and 4c),
which
eventually resulted in the improved gel strength as revealed by the mechanical
test. For 15%
GDL gel, the highest Df value 2.31 was estimated, indicating the most compact
and densest
system structure. This was revealed by the internal images of SEM result
(Figs. 4c and 4d)
that 15% GDL had thicker and rougher walls.
1001481 However, at 15% GDL condition, the final pH of system was around 4.35,
the pore
size of gel network structure became larger due to the existence of repulsive
forces, as shown
in SEM result, so that the gel strength of 15% GDL induced gel was weaker than
10% GDL
gel. The micro-elastic parameter, a, of the model is also presented in Table
1, which
distinguishes the type of the gel and implies the relative contribution of
inter- and intra-flock
links in the gel network. When a = 0, it indicates strong inter-floc link gel;
when a = 1, it
indicates a weak inter-floc link gel (stronger intra-flock link); when 0 <a<
1, it suggests a
transition regime with comparable contributions from inter- and intra-floc
links to the gel. The
56
CA 02872863 2014-12-01
value of a is estimated using two x values, 1 and 1.3, which are commonly used
to provide
approximation of fractal dimension of the backbone of colloidal aggregates
(Ould Eleya, &
Gunasekaran, 2004, Wu, et al. 2001). In this work, gelation of OPT solution
induced by GDL
was initiated at pH 8, followed by decrease of pH to form gel network
structure. The pH of the
initial network formation was around 6.80 as indicated by the result of time
sweep (Fig. 1)
where the point that G' almost reached plateaus. Thus, gel formed with 3% GDL,
where the
pH was around 6.75, could be considered as gel with initial network structure.
The a values
of 3% GDL induced gel, 0.34 (x = 1) and 0.40 (x = 1.3), were in the transition
regime,
suggesting a comparable contributions of inter- and intra-floc bonding. These
comparable
links allowed floes approach to each other and linked in a linear way due to
the gradual
neutralization of charges between protein aggregates (Maltais, et al. 2008).
Eventually, this
linear link of flocs contributed to the formation of polymer-like network
structures. With
increase GDL concentration to 10%, a value increased slightly to 0.42 (x = 1)
or 0.47 (x = 1.3)
due to the reduced pH and repulsive forces, which still in the transition
regime. It should be
noted that a value was raised to 0.81/0.83 at the GDL concentration of 15%.
The change of a
values indicated that the aggregation of protein molecules at supramolecular
level was toward
weak inter-floc link regime as GDL concentration increasing. A dramatic
decrease in
repulsion forces on the surface of structural units of 15% GDL one, as
indicated by the fast pH
decreasing in Figs. la and lb, allowed the energy barrier to be lowered enough
to increase the
probability of interaction between two units (Lin et al, 1990), which
increased the possibility
for strong intra-floc link. Consequently, the protein aggregates were prone to
associate in a
random way in all directions, resulting thicker and rough walls as shown in
the SEM images
57
CA 02872863 2014-12-01
1
1
(internal figure of Fig. 4d). Undoubtedly, decreasing rate of pH value was an
important factor
to influence intra- and inter-floe links and thus impacted gel network
structure. Nevertheless,
in this work, OPT gelation was initiated at pH S, and .Formed initial network
structure at pH
6.80 at any GDL concentration. Although the rapid decreasing rate of pH at
higher GDL
concentration might enhance the potential of OPI molecules associate randomly,
oat protein
molecules approached one another in an ordered way to form a structure at
first during the pH
decreasing process. This association approach contributed to the polymer-like
structure
formation instead of particulate structure.
[001491 Therefore, all the OPT gels exhibited polymer-like network structures,
but the one
prepared with higher GDL concentration had thicker and denser walls. Fig. 9
summarizes the
steps involved in the formation mechanism of cold-set OPT gels. In the first
step, heating
treatment caused oat protein dissociated from hexamers to trimers and
monomers. Only
partial trimers and extended monomers associated into oligomers and small
aggregate and the
others retained the structure during heating and cooling. In the second step,
the addition of
GDL caused pH decrease gradually, so that the reduction of repulsive forces
between protein
molecules promoted the non-covalent interactions, mainly based on hydrophobic
interaction
and hydrogen bonding, which resulted in the association of monomers, oligomers
and small
aggregates at both molecular and supramolecular levels with an ordered way
because of
comparable inter- and intra-floc interactions. Consequently, a three-
dimensional polymer-like
network could be fabricated rather than particulate and filamentous structure.
Specifically, at
the 3% GDL concentration where the final pH was 6.75, the repulsive forces
between OPT
molecules were relatively high, promoting the association of monomers,
oligomoers
58
CA 02872863 2014-12-01
(polypeptide segments) and small aggregates to form both the nucleated
conformation phase
and the "protofibrils" backbone at elongation phase. Then protein molecular
aggregation
occurred preferentially and the comparable inter and intra-floc interactions
allowed the
association mainly along an ordered approach. Eventually, a polymer-like
network structure
with thin walls and small pores was fabricated. Such kind of structure
resulted in a relatively
weak gel strength, but high water holding capacity. This structure was
considered as the initial
OPI gel network structure. Since the gel induced by 5, 10, and 15% GDL
concentration started
at pH 8 and went through pH 6.75, the development of network structure of 5,
10, and 15%
GDL induced gel based on this initial structure. When increasing GDL
concentration, non-
covalent interactions developed between OPI molecules after the initial
structure formation as
the further reduction of repulsive forces due to the -final pH reaching/near
oat protein IEP. At
the same time, micro- and/or macro-syneresis of OPI molecules occurred due to
various GDL
concentration.
[00150] Micro-syneresis of OPI aggregates at 5% Gin concentration caused the
pore size of
gel network structures larger than 3% GDL induced gel, so that the gel
strength was improved
and has excellent water holding capacity as well. While at 10% GDL
concentration, the
micro- and macro-syneresis occurred at the same time, resulting in a polymer-
like structure =
with thicker and rougher walls and small pore size. This kind of structure
largely improved the
gel strength, and maintained the good water holding capacity. For the gel
induced by 15%
GDL, the final pH was apart from OPI IEP.
59
CA 02872863 2014-12-01
! [001511 Therefore, even the thick and rough walls was formed, the pore size
of gel network
structure was larger than 10% GDL induced gel, which resulted in decreased gel
strength.
According to the results discussed above, a schematic description of the
formation mechanism
of cold-set OPI gels is proposed. The addition of GDL in OPI solution allowed
it to form a
polymer-like gel network sit-act-La-0 with differ mechanical properties and
water holding
capacities.
Table- L Ex-pm-him/nally measured Theological data and derived microscopic
structural parameters of
OPI gels prepared at 'different GD-Lconcentrations.
[GM.] c:-/0 Power-law
(wlw) moments Model of Wu and meibidelli (2001)
ac at x =
.Dic
d !at x. 1.0 1.3 Regime
Transition
3.86 -124 2.23 2.97 034 0.40
=
Transition
5 2.92 -0.93 1.99 2.94 0.35 04l gel
Transition
10 2.87 -0.77 2.04 2.73 0.42 0.47 gel
Transition
9.23 0.65 9..31 1.56 0.81 0-.83 gel
a Slope from log-log plot of G' vs: Concentration.
' Slope from log-log plot of strain vs Conconfration.
Values of DAP and n based on the model of Wu and Modaidelli (2001).
[00152] 4. Conclusion
[00153] Cold-set OPI gels at 5 and 7% (w/v) protein concentrations were
prepared with the
15 addition of GDL. These gels were formed at mild conditions (room
temperature and pH 8) and
low protein concentration, but exhibited strong mechanical. properties and
great water holding
capacity. Moreover, all the gels exhibited polymer-like microstructures.
Heating and cooling
process, gel final pH, and pH decreasing rate were responsible for such
network structures.
CA 02872863 2014-12-01
Firstly, heating and cooling process caused OPT hexamers dissociation,
followed by the
association of OPI monomers and oligomers to form "protofibrils" backbone at
elongation
phase. Then the addition of GDL resulted in the further association of small
aggregates and
oligomers orderly at the same time due to comparable intra- and inter-floc
interactions.
Depending on the different GDL concentrations and the final pH values, gels
with polymer-
like structure exhibited various pore size and wall thickness. At 3% GDL
concentration,
where the final pH was higher than OP1 IEP and the pH decreased slowly, oat
protein
fabricated a polymer-like structure with small pore size and thin wall and
relatively weak gel
strength.
[001541 With increasing GDL concentration, micro- and macro-syneresis occurred
which
resulted in compact network structures with large pore size and thick wall and
stronger gel
strength. The strongest gel, 0G7-10, was even comparable to egg white protein
gel. This gel
with good strength, elasticity and water holding capacity demonstrated the
capacity to
controlled release riboflavin in buffers and simulated gastro-intestinal
fluids. Thus, such cold-
set gels Using plant resource could be developed for food and non-food
applications,
especially in terms of delivery vehicle for heat sensitive compounds,
development of food
texture, design as facial mask, scaffolds in tissue engineering, and dressings
for wound
healing, etc.
61
CA 02872863 2014-12-01
SECTION 3- OPT and INULIN
[00155] As described above, oat protein is suitable as a gelling agent. Oat
protein forms
strong gels at alkaline conditions, for example, trypsin-treated oat protein
could form gels
with comparable mechanical strength to egg white protein at pH 9. However, oat
protein gels
are relatively weak when formed under acidic and neutral pH. This could be a
limiting factor
in the application of oat protein in food systems that normally have pH values
in the range of
2.5 to 7. Therefore alternative approaches to enable formation of stronger oat
gels within a -
more appropriate for
food processing are desirable to promote the utilization of oat protein
as a gelling agent.
[00156] inulin is a non-digestible polysaccharide naturally occurring in
several edible fruits
and vegetables. It is formed by fructose molecules linked by [3-(2-1)
glycosidic bonds,
generally with a terminal glucose unit connected to the last fructose by an a-
(1-2) bond. Due
to the -unique nature of inulin bonds, digestive enzymes in the human gut
cannot hydrolyze
this polysaccharide. Inulin reaches the colon undigested and produces a
prebiotic effect since
it is fermented by lactic acid bacteria. Additionally, inulin has other
interesting biological
properties such as enhancing mineral absorption, and reducing both lipid
levels and the risk of
colon cancer. The utilization of inulin in the food industry is not limited to
its biological
properties; it is also incorporated in food formulations as a fat replacer Or
hulking agent, such
as in table spreads, baked goods, sauces and yogurt. Such a wide range of
applications are
related to its capacity to form microcrystals that interact with each other
forming small
aggregates, which immobilize a great amount of water, creating a fine creamy
texture that
62
CA 02872863 2014-12-01
provides a mouth sensation similar to that of fat. Previous reports have
investigated the
influence of inulin, soy protein gels, yogurt and cheese, finding that the
protein-inulin system
had improved gelling properties. Nonetheless the effect of inulin addition on
the gelation
properties of oat protein has never been reported. Thus it is hypothesized
that inulin addition
can produce a synergistic effect which will enable the formation of
strengthened oat protein
gels.
1001571 The aim of this work is to investigate the effect of oat protein and
inulin interactions
on the gelation properties of oat protein isolate. Mechanical and theological
properties of oat
protein gels were determined as were their microstructures. Improvement of the
gelling
properties of oat protein at acidic and/or neutral p1-I may create broad
applications of this
plant-sourced gelling ingredient in foods. These value-added opportunities may
represent very
significant sources of revenue to oat producers and processors to enhance
their sustainability.
[001581 2. Experimental Materials
1001591 Naked oat grains (Avena nuda) were purchased from Wedge Farms Ltd.,
Manitoba,
Canada. The protein content was 16.6% 0.64 as determined by Leco nitrogen
analyzer (FP-
428, Leco Corporation, St Joseph, MI) using a protein calculation factor of
6.25. Oat protein
isolate (OP I) was extracted according to our previous work and the protein
content was
determined to be 90.40 % 0.59 using the same Leco nitrogen analyzer. Inulin
was extracted
from chicory root with an average degree of polymerization of according to
product
specifications; 2-mercaptoethanol, urea, sodium dodecyl sulfate, fluorescein
isothiocyanate
63
CA 02872863 2014-12-01
(FITC), Rhodamine B and dimethyl sulfoxide (DIVISO) were obtained from Sigma-
Aldrich
Caiyada (Oakville, ON, Canada).
[00160] Gel preparation
[00161] To study the effect of inulin addition upon gel properties, gels were
prepared by
- heating the protein-inulin suspensions at pH 2.5, 5 and 7 adjusted with 0.1
N NaOH or HC1.
The concentration of OPT in the mixtures was kept constant at 15% (w/v), which
was revealed
to be the optimized oat protein concentration for gel formation (Nieto-Nieto
et al. 2014). The
concentration of inulin varied from 0 to 0.5% (w/v) in the mixture, these
values were selected
based in preliminary trials. Samples were labeled as OPI, OPT-I 0.1%, OPT-I
0.25% and OPI-I
0.5%, representing inulin content of 0%, 0.1%, 0.25% and 0.5% respectively.
Test tubes
containing the suspension were tightly closed and placed in an oil bath at 100
C for 30 min.
Once the heat treatment was completed, the tubes were cooled in an ice bath
and stored in the
refrigerator overnight.
[00162] Textural profile analysis (TPA)
[00163] The mechanical properties of the gels were evaluated using an instron
5967 universal
testing machine (Instron Corp., Norwood, MA, USA). Gels were dismounted from
test tubes
and cut into cylindrical pieces (-10 mm height, ¨14 mm diameter). A two cycle
compression
test using a 50 N load cell was performed at room temperature at a rate of 1
mm/min and 50%
compression to evaluate their mechanical properties. The textural profile
parameters were
determined from the typical Instron force-time curve in which compressive
stress was
calculated as the peak compression force in the 1st bite cycle, divided by the
initial cross-
64
CA 02872863 2014-12-01
section area of the gel sample, and cohesiveness is the ratio of the area
under the first and
second compression peaks. Springiness is the distance calculated from the area
under the
second compression peak and gumminess is the product of peak compression force
in the 1st
bite cycle multiplied by cohesiveness.
[00164] Water Holding Capacity (WHC)
[00165] A gel sample (0.9 ¨ 1.2 g) was placed into a Vivaspin 20 centrifugal
filter unit (GE
Healthcare Bio-Sciences AB, Uppsala, Sweden) and centrifuged at 290 x g for 5
min at 15 C.
The weight of the gel was recorded before (W1) and after (W) centrifugation to
the nearest
0.0001 mg and the percentage of water loss after centrifugation was expressed
as:
( ¨ Wf)
Z=VHCi.00 - ___________________ x100
= -1
[00166] Scanning electron microscopy (SEN1)
[00167] The morphology observation of the gels was carried out with a Phillips
XL-30
scanning electron microscope (FEI Company, Oregon, USA) at an acceleration
voltage of 6
kV. The samples were frozen in liquid nitrogen and freeze-dried before
observations. The
cross-section and surfaces of the dry gels were sputtered with gold and
platinum, observed and
photographed.
[00168] Rheological measurements
CA 02872863 2014-12-01
[00169] The theological measurements were done with a TA Discovery HR-3
rheometer (TA
instruments, New Castle, DE, USA). Approximately 1 mL sample was loaded in the
bottom
plate of the parallel plate geometry; the upper plate was lower to the
appropriate geometry
gap. To avoid evaporation during heating a solvent trap was used and a thin
layer of silicone
oil was applied. The temperature of the bottom plate was controlled with a
Peltier system. To
study the changes in viscoelastic properties as a function of temperature, OPT
and OPI-inulin
suspensions were subject to a temperature rally from 25 to 95 C, then cooled
down to 25 C at
a rate of 1.5 C/min. Sample conditioning took place before and after each
temperature ramp
for a period of 3 min. The temperature ramp was not run up to the gelling
temperature used in
other experiments described in this paper (100 C) as preliminary experiments
reaching 100 C
produced readings with intense disparities due to water boiling, thus the
maximum
temperature used was 95 C. All theological measurements were done within a
predetermined
linear visco elastic region, which was determined in preliminary experiments,
setting the strain
value at 0.05%.
[00170] .To evaluate the molecular interactions involved in the formation of
OPI and OPT-
inulin gels, a frequency sweep analysis was conducted. Gels were prepared as
previously
described in the gel preparation section at pH 2.5, 5 and 7 and cut into
approximately 1 cm
(height) sections. The resulting gel disk were submerged for 48 h in solutions
of 2-
mercapthoethanol (2-ME) (0.2 M), urea (6 M) and 109 sodium dodecyl sulfate
(SDS) (1%
w/v), which could disrupt disulfide bonds, hydrogen bonds and hydrophobic
interactions
respectively. A frequency sweep test was done to evaluate the dependence of G'
to frequency
(0.1 ¨ 100 rad/s) on gels compressed to 80% of its original height.
66
CA 02872863 2014-12-01
[00171] Confocal laser scanning microscopy (CSLAI)
[00172] CSLM was used to observe the distaibution of inulin within the protein
network. A
laser scanning confocal microscope Zeiss LSM710 (Carl Zeiss Microscopy, Jena,
Germany)
was used with a 63x oil immersion objective. Inulin was labeled covalently
with FITC. For
this, 0.5 g of inulin and 10mL of DMSO were stirred overnight. Later, 7mg of
FITC were
added to the inulin-DMSO mixture. The reaction mixture was protected from the
light, heated
at 90 C for 2 hours and dialyzed extensively against distilled water in the
dark and freeze-
dried. Rhodamine B was used for non-covalent labeling of oat protein. A 15%
protein
suspension was prepared, and 40111.., of Rhodamine B (5mg/mL) were added to 1
ml of protein
suspension. The mixture was stirred for 2 h at room temperature, dialyzed
against distilled
water in the dark and freeze-dried. Once protein and inulM were labeled, 0.PI-
inulin
suspensions were prepared as described previously in the gel preparation
section. Samples
were place into a concave microscope glass slide, covered with a lamella,
which was sealed
with nail polish and heated for 15 min at 40, 60, 80, and 100 C. Once the heat
treatment was
completed samples were cooled in an ice bath and store in the refrigerator
overnight.
Measurements at 25 C referred to unheated sample. The Iluorescent images were
analyzed
simultaneously at wavelengths of 488 urn and 516 mu. Images were processed
with ZEN 2009
LE software (Carl Zeiss AG, Oberkoehen, Germany).
[00173] Particle size measurements
[00174] A Zetasizer Nano ZS ZEN1600 system (Malvern Instruments, U.K.) was
used to
study the evolution of particle size distribution as a function of increasing
temperature. For
67
CA 02872863 2014-12-01
particle size measurements the OPI and OPI-inulin suspensions were heated at
40, 60, 80 and
100 C for 30 min. Measurements at 25 C referred to unheated samples. Samples
were
immediately cooled in an ice bath to room temperature, after completing the
heating period.
Samples were then diluted to a total concentration of 0.1% (w/v) 134 and
passed through a
0.45 lam pore size filter prior to measurement. Number-based particle size
distribution was
measured to identify the total number of particles of a given size.
[00175] Fourier transform infrared (FTIR) spectroscopy
[00176] In order to observed changes in protein conformation during heating,
the infrared
spectra of OPI and OPI-inulin suspensions was recorded using a Nicolet 6700
spectrometer
(Thermo Fisher Scientific Inc., MA, USA). OPI and OPI-inulin suspensions (5%,
v/w) were
dissolved in D20. To ensure complete H/D exchange, samples were prepared 48 h
before =
infrared measurements. Suspensions were placed in between two CaF2 windows
separated by
a 25 uM polyethylene terephthalate spacer in a temperature controlled infrared
transmission
cell. Temperature was regulated by a Peltier controller (Thermo Fisher
Scientific Inc., MA,
USA). Samples were heated from 20 to 80 C, and every 10 C, the sample was 144
equilibrated and the spectra were automatically recorded. As the Peltier
controller was not
able to reach 100 C, samples heated at 100 C were prepared by the KBr-disk
method. For this,
the gels were prepared as previously described and freeze-dried. The dried gel
was crush into
powder, vacuum¨dried at 40 C overnight and mixed with K.Br powder (1:100 w/w),
the
mixtures was compressed to 13 mm discs and used for spectroscopy measurements.
To study
the amide 1 region of the protein (1700-1600 cm Fourier self-deconvolutions
were performed
68
CA 02872863 2014-12-01
using the software provided with the spectrometer (Omnic 8.1.210 software).
Each spectrum
was the result of 128 scans; band narrowing was achieved with a full width at
half maximum
of 20-25 cm and with resolution enhancement factor of 2.0-2.5 cm . During
measurements
Nitrogen was continuously run through the spectrometer. Band assignment in the
amide I
region was made based on previous literature reports.
[00177] Statistical analysis
[00178] All data were analyzed for significant differences, with minimum
significance test set
at the 5% level (p <0.05). Tukey's multiple comparison test was used to
establish statistical
significance using GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA).
All
experiments were performed at least in three independent batches and the
results were
reported as the mean standard deviation.
[001.79] 3. Results and Discussion
[00180] Gel formation and textural profile analysis
[00181] The mechanical properties of the gels including compressive stress,
cohesiveness,
springiness and gumminess are summarized in Table 1. Compressive stress
indicates the
capacity of the material to withstand a given deformation. ha the case of OPT,
the compressive
stress values of the gels were the highest at pH 5 and 7 (10.19 and 11.29
kPa), these values
dropped at pH 2.5 (1.53 kP a). At pH 5 and 7, oat protein gels show a good
balance between
electrostatic repulsive forces mid hydrophobic attractive forces allows
formation of strong gel
networks, which could withstand higher compression force applied. Hydrogen and
disulfide
69
CA 02872863 2014-12-01
bonds can also participate in addition to hydrophobic forces in the
stabilization of the protein
network by balancing the electrostatic repulsive forces. Under acidic
conditions, cysteine
,
shows low reactivity, and thus, disulfide bonds are unlikely to take form,
explaining why gels .
,
,
,
,
,
,
prepared at pH 2.5 showed lower compressive stress values. Moreover, it is
possible that at .
,
,
.
.
,
,
pH 2.5 fewer interactions were developed, as attractive forces could not
counterbalance the ,
,
,
,
strong electrostatic repulsive forces produced by positively charge amino acid
residues. ,
,
,
,
Table 1 Mechanical properties of OPI and OPI-inulin get; prepareast 1.00C
.
pH .2.5 pH 5. pH 7
,
=
. .
.
Compressive stress (IzPa)
CPI 1.53 - 0.27 10.19 +1.32 11.29 +3_49
=
DPI-I 1.47 - 0:11 9.0 1.03 1.3.93 *1.95
DPI-I 2.07 0.31 10.92 2.12 14:41 1.39
DPI-I . 239 +038 , 14.16 . +2.85 µ 22.98 ,
+1.12
Cohesiveness
OK 0.41 +0.03 037 0.01 0.55 0.04
.
DPI-I 0.47 - 0.06 0.53 0.07 0.56 0.05
DPI-I 0.39 0.04 0.70 0.02 0.55 0.06
OPI-I. , 0.26 . 4{03 . 037 . +0.05 . 0.55 , +0.08
Springiness fjrnm)
DPI 130.20 5.37. 211.13 T74 163..05 15_4
-OPI-I 159.83 21 A 193.13 6.14 228..21 204
OPLI 143.73 21.1 208.16 4.76 220.73 9.85
0.PI-I 197_82 +17.6 .208.88 +.12.7 217.81 17.4
Gumminess,: (N)
DPI 0.08 4-0.01 0.90 +0.09 0.73 0.12
.
OPI-1. 0.09 Ø02 0.80 0.10 0.90 034
DPI-I 0.10 0.02 0.94 0.15 1.03 0.10
DPI-I 0.08 +0.01 1.16 0.13 1.68 -0.18
Values are means stanplard deviation
[001821 At pH 7, addition of a small amount of inulin (0.67% -3.33 %, based on
dry weight
of protein) greatly increased the compressive stress. This effect is
especially strong at the
CA 02872863 2014-12-01
highest level of inulin addition as compressive stress values increased form
13.93 kPa to
14.41 kPa and 22.98 kPa OPI-I 0.1%, OPI-I 0.25% and OPT-I 0.5%, respectively.
Since inulin
is incapable of forming a gel on its own under any of the concentration
utilized in this
experiment, any improvement on the mechanical properties was the result of a
synergistic
effect of inulin and OPT. Inulin addition may have produced a more densely
cross-linked
network, leading to higher compressive stress. At pH 5 the addition of inulin
produced a slight
increment of the compressive stress values, however this change was not a
significant
improvement (p <0.05). Gels prepared at p1-I 5 with and without inulin were
prone to syneresis
(once the heating step was completed, a water layer on the top of the gel was
observed). The
compressive stress values reported for gels at p1-I 5 could be overestimated,
since the
exudation of water resulted in a higher solid content in the actual gel
network. Moreover,
water release was observed after the compression cycle was completed for these
samples. At
pH 2.5, the addition of inulin did not produce a significant improvement
(p<0.05) of the
compressive stress value either, which ranged from 1.53 to 2.19 kPa. Earlier
research reported
the effect of pH and temperature over the chemical stability of inulin,
showing that heating of
inulin under acidic conditions caused intensive hydrolysis, whereas, heating
of inulin under
neutral or alkaline conditions produced very little change in the content of
reducing sugars.
Thus the stability of inulin could be lost at acidic explaining why at
2.5 the
improvement of the compressive stress did not take place. Gels prepared with
OPT and OPT-
inulin mixtures at pH 7 had higher or comparable compressive stress values to
gels obtained
with soy protein/gellan gum (-12.5 kPa) and soy protein/loctus bean gum (-20
kPa). The
cohesiveness value indicates the integrity of the internal bonds after a
compressive force was
CA 02872863 2014-12-01
applied. Cohesiveness values close to 1 indicate little damage to the internal
bonds of the
structure and thus high resistance to deformation. The highest value (0.67)
was obtained at pH
5, followed by that at pH 7 (0.55). The lowest value of cohesiveness (0.41)
was recorded at
pH 2.5. The addition of inulin did not significantly influence the
cohesiveness of the oat
protein gels. Springiness relates to how fast the structure can recover from
the deforming
force. A higher springiness value indicates that the sample can quickly
recover from the
deformation. Gumminess represents the energy required to disintegrate
semisolid food to a
ready for swallowing state. The gels prepares at pH 2.5 had the lowest
springiness values
among all samples, indicating these gels were more affected by the compressive
force and
took longer to recover. Gels prepared at pH 5 and 7 showed similar springiness
values (193-
228 mm). Gels prepared at pH 7 had the highest gumminess values, suggesting
that these gels
require more energy to be disintegrated. Gels prepared at pH 2.5 had the
lowest gumminess
values and gels prepared at pH 5 were at an intermediate level. The addition
of inulin led to
significant increase in springiness and gumminess for gels formed at pH 7
(p<0.05) and the
gumminess value in the presence of 0.5% inulin was 2.3 fold of the value
observed for OPI
gel alone. Thus, the addition of a small amount of inulin also provides the
opportunity to tailor
other properties of oat protein gels such as springiness and gumminess to meet
different
sensory requirements.
[00183] Water holding capacity (WHC)
[00184] WHC is a key property of gels and low values often result in dry
products and thus
low texture stability. As shown in Figure 1, WI-IC values ranging from 85.09
to 93.29% were
72
CA 02872863 2014-12-01
recorded for all gels except for those prepared at pH 5 with values around
60%. This is in
agreement with the syneresis and observation of water released after the
compressive tests at
pH 5. With increase of inulin, a slight increasing trend was observed for WI-
IC value at pH 5.
At pH 2.5 and 7 the addition of inulin did not significantly impact (p<0,05)
the gel WI-IC. The
OPI and OPI-inulin gels exhibited higher or comparable WFIC values to those of
soy
protein/loctus bean gtilia (>60%), whey protein/cassava starch (>85%), and egg
white
protein/konjac glucomamian (90.2%), previously reported. The gels maintain
their strength
and water-holding capacity after freeze-thaw cycle treatment and microwave
heating.
[00185] Scanning electron microscopy (SEM) A clear distinction has been
established in
the morphology of particulate or -fine-stranded gels, as protein gels are
expected to form one
. of these structures depending on the pH and ionic strength of the medium. At
pH 5 (Figure 2)
bundles of large spheroid aggregates are randomly distributed along the
network forming a
characteristic particulate gel. The characteristic morphology of this type of
gels is related to
the restricted intermolecular repulsion that protein molecules exhibit when
the pH is near the
isoeleetric point. Thus, the low net charge of the protein produced minimal
repulsion and
protein molecules unsystematically aggregate to favor the development of
protein-protein
interactions, whereas protein-water interactions are limited which results
into a gel network
with low WI-IC due to increased pore size and decreased capillary forces, and
consequently a
higher water loss. In contrast, fine-stranded gels are generally stronger and
have higher water
holding capacity, as these gels are formed at pHs far from the isoelectric
point of the protein.
However, SEM micrographs of oat protein gels (Figure 2) show a unique
structure at pH 2.5
and 7, similar to that formed by gelatin. At pH 2.5, protein aggregates array
in such fashion
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CA 02872863 2014-12-01
that hollow cells are formed between thin vertical walls. These cells are
almost tubular in
shape. The cell walls at this pH (Figure 3) seem to be thin and flaky and the
presence of inulin
does not apparently alter the structure. At pH 7, a similar structure to that
formed at pH 2.5
was produced, except that in this case cell walls seem thicker, smoother and
highly
interconnected. Such thicker walls were probably developed by stronger
interactions that
resulted in a reinforced structure. The addition of inulin at pH 7 led to
thicker walls and
increased junction zones, as the gel formed with 0.5% inulin showed a highly
cross-linked
network (Figure 3.). This could explain the greatly improved mechanical
strength and
gumminess for the gel OPI-I 0.5%. The polymer-like network structure
maintained after
freeze-thaw cycle treatment and microwave heating.
[00186] We realized this unique gel structure may have specific gelling
applications in a
variety of foods. This triggered our interest to further investigate the
molecular mechanism by
which oat protein forms such structures. Also, we wanted to know better how
small amounts
of inulin could greatly improve the gel properties especially at neutral pH,
an environmental
condition highly convenient for food applications. So we further investigated
the gels with
experiments using rheological measurements, Fourier transformed infrared
spectroscopy and
laser light scattering to study the molecular events taking place during the
gel formation
process itself.
[001871 Rheological measurements in order to further investigate the
development of the gel
network as a function of heating, a temperature ramp was run for OPT (15%
protein) and OP1-
inulin suspensions with addition of 0.1 and 0.5 % inulin. Both G' and G" were
examined upon
74
CA 02872863 2014-12-01
heating and cooling, however only G' values are shown as G" always showed
lower values
than G', even as a solution. This means that the elastic behavior dominated at
all stages of the
network development. A similar phenomenon has been observed on egg white,
which showed
gel-like properties (G'>G") over the entire temperature range probed and even
at low
temperature the native protein already forms a weak network that can propagate
stress. At pH
7 (Figure 4A) the G' value for OPT was initially stable then decreased at
around 50 C and a
plateau was formed until the temperature reached ¨75 C. This decrement could
be attributed
to weakened hydrogen bonding, since heat disrupts hydrogen bonds and
electrostatic
interactions but enhances hydrophobic interaction and accelerates molecular
motion. From
¨75 C onward, G' increased with increasing temperature and then further
increased during the
cooling stage, reaching a maximum of 304.57 Pa. In the case of OPT-I 0.1% and
OPT-I 0.5%
the G' value increased abruptly at ¨70 C until the maximum temperature was
reached. During
the cooling stage G' further increased to reach a maximum value of 6,823.96 Pa
and
24,758.65 Pa for OPT-I 0.1% and OPT-I 0.5%, respectively. The sharp increase
of G' from ¨70
to 95 C indicates that the formation of a rigid gel network occurred, as heat
prompts protein
unfolding, exposing reactive groups of the molecule that enable the molecular
interactions
such as hydrophobic forces and disulfide bonds to reinforce the gel network. .
It is Clear that
part of the development of the gel network also took place during the cooling
stage. The
addition of inulin also produced two phases in the development of the gel
network. According
to previous reports, the first stage (-50 C) could be related to the
development of an inulin
network by entanglement of molecules through hydrogen bonds and van der Waals
forces.
The second stage (-70 C) could be related to the development of the protein
network.
CA 02872863 2014-12-01
Apparently the development of the gel network started at a lower temperature
for samples
containing inulin (-70 C) compared to OPT gels (-75 C). A sharper increase of
G' can also be
observed in the case of OPI-inulin mixtures and the more inulin the higher the
G' value,
confirming that inulin had a synergistic effect on the development of the gel
network.
[00188] At pH 2.5 OPT showed (Figure 5B) a low G' value (0.15 Pa) at 25 C, but
increased to
8.55 Pa and 3.80 Pa after addition of 0.1% and 0.5% inulin, respectively. A
decrease in G'
value was also observed for OPT at 45-60 C, but was less evident and prolonged
as at pH 7.
Possibly less hydrogen bonds were present in oat protein at pH 2.5 due to
partial protein
unfolding and/or dissociation caused by the acidic environment. A sharp
increase of G' was
observed at around 60 C until the maximum temperature was reached. The two-
phase network
development was also observed at pH 2.5 in OPI-inulin mixtures, but not, as
well defined as
at pH 7. In the case of OPI-I 0.1%, G' increased sharply from 60 C to ¨85 C
then G'
increased once again until the end of the heating stage. For OPT-I 0.5%, the
first enlargement
of G' occurred at a slightly lower temperature (-55 C), then a sharper
increment was observed
at ¨8 5 C up to the maximum temperature. Additional enlargement of G' values
was observed
during the cooling stage for all samples. As discussed earlier, at this acidic
pH the stability of
inulin is low, in addition heat treatment could further break down inulin into
shorter inulin
chains at such pH. The strengthening effect of inulin was limited compared to
the effect
produced at pH 7. Nonetheless addition of inulin produced gels with higher G'
values. The
final G' value for OPT was 7,113.13 Pa, whereas the values for OPT-I 0.1% and
OPI-I 0.5%
were 16,422.62 Pa and 25,350.60 Pa, respectively. At pH 5 (Figure 5C) the
initial G' value =
was 3.79 Pa for OPT but increased to 18.12 Pa for OPI-I 0.1% and 10.95 Pa for
OPI-I 0.5%. A
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CA 02872863 2014-12-01
slight increase in G' was observed during the heating and cooling stages. The
inclusion of
inulin did not produce a siglificant improvement in the final G' values which
were 200.57 Pa,
113.13 Pa, 294 and 156.85 Pa for OPI, OPT-I 0.1% and OPT-I 0.5%, respectively.
These values
were much lower than the corresponding values of gels prepared at pH 2.5 and
7. Considering
the low G' value observed for gels formed at pH 5 it was confirmed that the
high compressive
stress values reported for gels at pH 5 were overestimated since the exudation
of water
produced a higher solid content in the actual gel network.
11001891 in order to determine the type of interactions involved in the
development the gel
network, a frequency sweep test was conducted on gels treated with different
dissociating
reagents including urea to interrupt hydrogen bonding, 2-ME to dissociate
disulfide bonds and
SDS to destroy hydrophobic interactions. The frequency sweep test on gels
prepared at pH 2.5
was not performed as these gels swelled severely, then ruptured in the
presence of dissociating
reagents, which did not permit an appropriate measurement. This observation
also confirms
that the strength of the interactions formed at pH 2.5 were inferior in
comparison to those
present in gels formed at pH 5 and 7. -Figure 5 shows the response of G' to
the variations in
frequency of OPT and OPI-I 0.5% gels. At pH 7, OPI gels were strongly affected
by 2-ME as
the integrity of the gels was lost, which did not allow the appropriate
measurement of the G'
response to frequency. This indicates that disulfide bonds performed an
essential role for
development of the gel network. Gels in contact with urea and SDS, showed very
similar
response and in both cases G' values were reduced significantly, suggesting
that hydrogen
bonds and hydrophobic interactions also contribute to the development of the
three-
dimensional network structure. In the case of OPT-I 0.5%, gels were affected
by 2-ME in the
77
CA 02872863 2014-12-01
same way as the gels prepared with OPI. Thus, disulfide bonds also play an
important role in
the gel network formation. In presence of urea and SDS the gel structure was
affected to a
greater extent as a significant drop of the G' value was observed. This
indicates that addition
of inulin may strengthen hydrogen bonding and hydrophobic interactions to
further improve
the gel network structure and mechanical properties. In the case of OPI at pH
5 there was not a
significant contribution of disulfide bonds since the frequency response of
the gel submerged
in 2-ME showed very similar trend compared to the control sample. On the other
hand,
samples submerged in urea and SDS had lower G' values. This suggests that
hydrogen bonds
and hydrophobic interactions played important roles in the maintenance of the
gel structure at
phi 5. OPI-I 0.5% at pH 5 showed a similar outcome. Disulfide bonds had very
little
contribution to the development of the protein network. The gel submerged in
SDS had a
comparable response to the gel prepared with OPI alone. But the gel submerged
in urea was
apparently less affected as the G' value reduced to a lower extent. Therefore,
both hydrogen
bonds and hydrophobic interactions are the ruling forces in the establishment
of the OPI-
inulin gel structure at pH 5, whereas disulfide bonds are not developed
probably due to the
compact structure of the protein near its isoelectrie point, where the sulfur
hydroxyl groups are
hidden inside the protein structure.
[00190] Confocal laser scanning microscopy (CSLM) The microstructure of OPI
and OPI-
inulin gels was also observed with CSLM. Red color in the micrographs
correspond to
rhodamine-B labeled protein, whereas, bright green regions indicate FITC
labeled inulin. Thus
the distribution of oat protein and inulin in the gel system could be
observed. Under the
conditions tested in this work, only protein could form a gel, as the amount
of inulin included
78
CA 02872863 2014-12-01
;
in the system was under its minimum concentration required for gel formation,
which has
been reported to be 10% homogeneous, conceivably due to the low concentration
of inulin
compared to the protein concentration, thus at this protein/polysacchatide
ratio the rate of de
mixing could be very low. As temperature increased the protein aggregates grew
gradually,
and around 60 C phase separation began. This suggests increased thermodynamic
incompatibility of the components probably due to the excluded volume effect
when the
protein started to unfold or change conformation at an elevated temperature
temperature
onward it was possible to see two phases, in which the protein network formed
a continuous
phase comprised of solid inulin particles ranging in size from several
hundreds nanometers to
¨I pm distributed within the network. In the case of OPI-I 0.5%, early signs
of phase
separation were noted at 40 C since a small green dot was observed among the
expanded
protein aggregates. The development of the protein network in OPI suspensions
was similar to
that of OPI-inulin mixtures, indicating that inulin did not interfere with the
development of
the protein network; nonetheless it did reinforce the structure. At pH 2.5
(Figure 7), less inulin
particles were observed through the heating stage as well as at the final gel.
Inulin has poor
stability under acidic conditions and high temperature, which causes it to
break down into
shorter inulin chains or reducing sugars. Only a few reinforcement sections or
junction zones
were formed, explaining the limited improvement observed in the reported
compressive stress
values. Perhaps the inulin rich sections observed were formed by some inulin
chains that were
more resistant to the acidic conditions. In the ease of the gels prepared at
pH 5, larger
aggregates with larger void spaces were formed (Figure 8). At this pH, no
phase separation
was seen, not even with increasing temperature. In this case the net charge of
the protein was
79
CA 02872863 2014-12-01
close to zero, therefore both protein and polysaccharide might form a
compatible system and
no phase-separation was produced. Apparently, a main protein network was
formed and
inulin was covered inside the protein structure. Gug,gisberg et al. (2009)
evaluated the effect
of inulin addition as a fat replacer in yogurt and their CLSM images suggested
that an inulin
system could be built in the protein network, since inulin was not visible by
CSLM. The larger
void spaces indicates large pores, which led to the release of water from the
network, a
phenomenon consistent with the low water holding capacity of the gels prepared
at this pH.
[001911 Gels prepared at pH 7 showed promising application potential due to
their excellent
mechanical strength and very unique structure. Samples prepared at this pH
were selected to
further investigate the effect of inulin addition in the gel formation
mechanism.
[001921 Size distribution measurement
[00193] The changes in the size distribution of protein molecules were studied
as a function
of heating temperature. As shown in Figure 9, progressive reduction of the
mean particle size
occurred during heating with the same trend for all samples, regardless of the
inulin level
added. The main peak of the native protein gradually shifted towards a lower
particle size with
increasing temperature. At 100 C the peak value (diameter) detected for ON
gels was 10.1
urn, and 8.72 mu and 11.7 rim for OPI-I 0.1% and 369 OPI1 0.5% respectively.
According to
previous literature, a diameter value of 11.8 nm was estimated for oat
globulin monomers
with an extended conformation. Thus the recorded peak values in the current
study suggest
dissociation of oat protein hexamers down to monomers. The thermal aggregation
of oat
globulin has been previously studied and the changes produced by heat have
been described
CA 02872863 2014-12-01
globulin hexamers dissociating first into trimmers and then into monomers that
are highly
reactive. These then rapidly associate to larger and more stable molecules. It
is hypothesized
that the monomers formed after the heating treatment serve as building blocks
in the
establishment of the three-dimensional network.
[00194] Fourier transform infrared (FTIR) spectroscopy
[00195] A FTIR study was performed to study how changes in the proteini
conformation are
influenced by the heating process and the presence of inulin. Amide 1(1700-
1600 cm-1) band
components were assigned to protein secondary structure according to previous
reports in
literature. Figure 10 shows the de-convoluted spectra of OPI and OPI-inulin
mixtures (0.1
and 0.5%) at different temperatures when pD was set at 7, including the gel
sample prepared
at 100 C. In the case of OPI, the amide I band at 25 C showed five clear
components with
strong absorption, including f3-turn (1670 and 1658 cm'), a-helix (1649 cm -
1), random coil
(1640 0m-1),(3-sheet (1630 cm-1) and vibration of amino acid residues(1609 cm -
1). This is in
agreement with a previous report that indicated the a-helix and random coils
are the major
secondary structures in oat globulin, followed by 13-sheet and 13-turn. A
small peak was also
observed at 1618 cm-1 which was assigned to intermolecular 13-sheet and is
believed to be
related to protein aggregation via the exposed reactive groups. As temperature
increased from
to 80 C, no major changes in the secondary structure were detected; this could
be related to
the high heat stability of oat protein with a denaturation temperature of
112.4 C as revealed by
differential scanning calorimetry in our previous work. The absorption at 1690
((3-sheet),
25 1660 (turns) and 1619 cm-1 (intermolecular 3-sheet) increased gradually
with temperature,
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CA 02872863 2014-12-01
suggesting more aggregates were formed probably due to some exposure of the
reactive sites.
The amide I band was significantly altered when the OPI was heated to 100 C.
In order to test
whether the aggregation caused by the dry process impacted the protein
secondary structure,
the dried gel powder formed at temperatures below 100 C were tested. The
results indicated
that the dried gels formed at these conditions had similar peaks with the
heated suspensions.
Thus the protein secondary changes can be attributed to heating at 100 C. The
absorption at
1619 cm-lvanished, whereas a peak appeared at 1627 cm-1. A similar transition
(from 1623 to
1630 cm-1) has been related to the dissociation of the dimeric form of
p¨lactoglobulin into
monomers. This transition agrees with previous particle size results in which
a progressive
reduction of the mean particle size was observed as function of temperature,
implying the
dissociation of oligomers down to the monomeric form. Increased absorption
intensity was
observed at 1694, 1683, 1671 and 1659 cm-1 and two peaks appeared at-1649 and
1638 cm-1
Such changes suggest partial protein unfolding during heating at 100 C
followed by re-
organization of protein secondary structure during gel formation process.
Heating of OPI-I
0.1% and 0.5% from 80 to 100 C showed similar elements in the final spectrum
at 100 C as
well as the transition related to the oligomer-monomer dissociation. In all
cases, shifts in
wavenumber compared to the sample at 25 C were observed, implying
reorganization of the
polypeptide chain within the protein. This indicates that addition of inulin
did not significantly
interfere or alter the protein network construction. The Kra images show very
similar heat
associated transition at the secondary structural level of the protein.
[001961 Proposed formation mechanism of OPI-inulin gels at neutral pH
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CA 02872863 2014-12-01
1.4(1971 Based on fractal theory, protein particles form a fractal structure,
ultimately leading
to a gel network built of fractal clusters. Three factors have been found
relevant to the type of
structure formed: 1) the effective size of the building block of the fractal
structure, 2) the
amount of protein incorporated in the fractal clusters at the moment of the
gel is formed, and
3) the way in which the fractal clusters are linked together. For globular
proteins in general
such as whey and soy protein the formation of heat-induced globular protein
gels, involves
unfolding of the protein molecules by heating, leading to the exposure of
active amino acid
residues. This is then followed by protein aggregation and dissociation of
these aggregates to
form either filament or particulate gels depending on and ionic strength.
The unique
structure formed by oat protein gels at neutral pH may be associated with
monomers as the
predominant basic building blocks of the gel network. These smaller units are
highly reactive,
which would allow the development of the protein gel network at a near
molecular level,
resulting in development of strong interaction in oat protein gels with high
mechanical
strength. The formation of fibril network structures of oat protein gels can
be described in two
stages; in the first stage or nucleation phase, monomers aggregate into larger
particles Or
oligomers, which increase their size as the nucleation stage comes to an end.
Then the
elongation stage begins with addition or condensation in an orderly fashion of
monomers and
oligomers to form protofibrils, and .finally, the association of protofribils
leads to fibril
formation. At pH 7, heat caused the dissociation of oat globulin hexamers by
breaking
disulfide bonds linking the acidic and basic subunits. This allowed the re-
formation of
disulfide bonds possibly during the heating process. Hence disulfide bonds
contributed to the
83
CA 02872863 2014-12-01
stabilization of the gel network as a major supporting force, which could
further strengthen
the gel network and mechanical properties.
[00198] Addition of inulin led to formation of phase separated gels during
heating processing.
This was expected, as it is highly unlikely for the OP1-inulin dispersions to
establish
electrostatic interactions; the protein has a negative charge at pH 7, whereas
inulin has no
charge. In this case protein-protein or inulin-inulin interactions require
less energy than
protein-inulin interactions; thus the system separated into a protein-rich and
an inulin-rich
phase. In fact, each polymer is excluded from a volume occupied by the other
polymer; hence
the effective concentration of both polymers is increased sub-micron
crystalline structures that
are able to immobilized large amounts of water. The addition of a small amount
of inulin can
force the protein into a smaller volume through the excluded volume effect. In
this way an
effective higher protein concentration is produced, which causes more
intermolecular
interactions. In a different study, incorporation of a low concentration of
guar gum (neutral
polysaccharide) improved the theological properties of whey protein gels
formed by thermal
gelation. This was attributed to segregative interactions due to thermodynamic
incompatibility
of guar gum and whey protein on the gels strength. The addition of glueomannan
to whey
proteins increased the rate of gelation and G' values of heat-set gels. It was
speculated that
this improvement was attributed to localized changes in osmotic pressure which
caused an
apparent concentration of the protein phase greatly improved gel properties
attributed to the
addition of inulin in small amounts, can be explained by the strengthening
effect of inulin
nanoparticies homogeneously distributed inside the protein network. According
to the
rhcological test results in section 3.4, addition of inulin may increase
hydrogen bonding and
84
CA 02872863 2014-12-01
hydrophobic interactions to further improve the oat protein gel network
structures and
mechanical properties. Therefore, it is possible that in the OPI-inulin
system, hydrogen bonds
and hydrophobic interaction could occur in the border between the continuous
network and
the discontinuous phase. Hydrophobic interactions between inulin and other
proteins such as
casein and 13¨lactoglobulin have been previously suggested due to the fact
that inulin is able to
form a a-helix in solution and may contain a hydrophobic center that binds to
the hydrophobic
regions of the oat protein, Another important consideration is that inulin is
rich in hydroxyl
groups that are able to take part in supra-molecular interactions, in
particular hydrogen
bonding. Hence hydroxyl groups have considerable capacity for hydrogen bond
formation
with polar residues of the protein. Thus, additional hydrogen bonds and
hydrophobic
interactions may develop in the border between the continuous network. and
discontinuous
phase that work as a junction zones and provide extra support to the
structure. This would
explain the reinforcing effect obtained with a small amount of inulin. In
summary, three forms
of gel reinforcement by inulin at pti 7 are proposed here (Figure 12): 1)
Increased interactions
among the protein network by phase separation due to increased protein
concentration,
causing More intermolecular interactions; 2) Laulin performs a filling effect
by occupying the
void spaces of the protein network once the phase separation has been promoted
by
temperature and builds junctions zones; 3) Localized interactions such as
hydrogen and
hydrophobic bonds take place between protein and inulin at the phase borders.
1001991 4. Conclusion The strength of oat protein gels was significantly
improved by
addition of small amounts of inulin. The enhancement on the mechanical
properties of the gels
is likely due to the increased concentration of protein produced by the
excluded volume effect.
CA 02872863 2014-12-01
Particle size distribution observations indicated that heating at 100 C causes
the dissociation
of the oat globulin hexamers into monomer, which become the main reactive unit
in the gel
network development. Additionally, inulin contributed to the formation
ofjunction zones in
which hydrogen bonds and hydrophobic forces may participate in leading to a
highly
crossl inked gel network with a reinforced structure.
[002001 Protein and polysaccharides are often used in combination in the
development of
food products. This research is critically important for future progress in
the development of
food products including OPI-irnilin mixtures. Novel products are now designed
based on the
understanding of a growing consumer preference for natural and healthy foods,
and oat
protein-derived ingredients show excellent potential to be well adopted by
consumers. The
acceptance and utilization of oat and oat-inulin mixtures as value-added
ingredients to
produce food products of different textures, could potentially contribute to
the growth of the
food and agricultural industry. Also this research demonstrates the potential
of oat protein as
an appropriate food gelling agent since the strongest gels were formed at
neutral conditions; in
previous reports this was only achieved at alkaline conditions.
SECTION 4¨ MICROWAVE TREATMENT
[002011 The work above shows that oat protein formed strong gels when heated
above 100 C
for 15-20 minutes. We have found that gelling temperature may be reduced by
combining
heating and microwave technology, for example, by heating at less than about
100 C for
about 3 min and 15 see microwave treatment . In one embodiment, microwave
heating is
done in an ordinary commercial microwave oven, at a power level of about 1000
W to about
86
CA 02872863 2014-12-01
1500W. Microwave radiation may cause a higher degree of protein unfolding and
may also
contribute to some hydrolysis of the protein. The microwave treatment may
occur
simultaneously or consecutively with the heating step.
[00202] In one embodiment, the OPT gels were prepared with 25% (w/v) protein
by 3min
heating at 80 C followed by 15 sec microwave treatment exhibited compressive
stress of
5kPa. Adding 1-2% (w/v) fatty acids such as sodium dodecanoate may increase
the
compressive stress to 6-12kPa. Addition of 1% inulin (w/w based on dry weight
of protein) to
oat protein in the presence of fatty acids may further enhance the compressive
stress to 14kPa.
The gels also showed excellent water holding capacity (>90%) and good
springiness (4-
5mm).The gels maintained their strength and water-holding capacity after
freeze-thaw cycle
treatment and microwave heating.
[00203] The gels with combined heat and microwave treatment also demonstrated
polymer
like network structures, unlike many other globulin protein gels (e.g. whey
and soy), as seen in
the photographs for Section 4. The polymer like network structure is seen to
remain after
freeze-thaw cycle treatment and microwave heating.
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