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\begin{document}

\title{Select functional properties of protein isolates obtained from canola
meals modified by solid-state fermentation}



\author[1]{Chenghao Li}%
\author[1]{Dai Shi}%
\author[2]{Andrea Stone}%
\author[3]{Janitha Wanasundara}%
\author[1]{Takuji Tanaka}%
\author[1]{Michael Nickerson}%
\affil[1]{University of Saskatchewan College of Agriculture and Bioresources}%
\affil[2]{University of Saskatchewan}%
\affil[3]{Agriculture and Agri-Food Canada}%


\vspace{-1em}



  
  \date{June 17, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
The present study investigated the effect of solid-state fermentation
(SSF) of cold-pressed (CP) and hexane-extracted (HE) canola meals with
Aspergillus niger NRRL 334 and A. oryzae NRRL 5590 on the
functionalities of protein products extracted from them. After SSF,
proteins were recovered using alkaline extraction-isoelectric
precipitation (AE-IP) or salt extraction-dialysis (SE). SSF of the two
meal types reduced the protein content of the extracts produced by
AE-IP. There were varied effects to solubility, foaming, and emulsifying
properties as a result of SSF under the combined influence of
functionality pH, strain, meal type, and protein extraction method. The
protein isolate produced from CP meal using SE had increased solubility
at pH 7 (from 51.8 to 90.7\%) when the meal was fermented with A.
oryzae. Both strains resulted in an over 2-fold increase in the
emulsifying activity index (at pH 7) of AE-IP products from CP meal. For
both protein extraction methods, the protein products from A. niger
fermented HE meal had better foaming capacity (FC) at pH 7 than the
controls (non-fermented), but reduced FC at pH 3. Overall, regardless of
meal fermentation, the SE products were richer in protein and had higher
oil holding capacity (OHC), whereas the water holding capacity (WHC) was
higher for AE-IP isolates. SSF of the meals generally improved the O/WHC
of the extracted proteins. The findings suggest that canola protein
functionality could be effectively modulated by SSF with different
microbial strains under various processing conditions to enhance their
applicability in the food industry.%
\end{abstract}%



\sloppy


\textbf{Select functional properties of protein isolates obtained from
canola meals modified by solid-state fermentation}

Chenghao Li\textsuperscript{1}, Dai Shi\textsuperscript{1}, Andrea K.
Stone\textsuperscript{1}, Janitha P. D. Wanasundara\textsuperscript{2},
Takuji Tanaka\textsuperscript{1}, and Michael T.
Nickerson\textsuperscript{1}*

\textsuperscript{1}Department of Food and Bioproduct Sciences,
University of Saskatchewan, 51 Campus Drive, Saskatoon, SK, S7N 5A8,
Canada

\textsuperscript{2}Agriculture and Agri-food Canada -- Saskatoon
Research Station, 107 Science Pl, Saskatoon, SK, S7N 0X2, Canada

\textbf{*Correspondence}

Michael Nickerson, Department of Food and Bioproduct Sciences,
University of Saskatchewan, 51 Campus Drive, Saskatoon, SK, S7N 5A8,
Canada

Email: Michael.Nickerson@usask.ca

Short running title: Canola protein from fermented meals

\textbf{Abstract}

The present study investigated the effect of solid-state fermentation
(SSF) of cold-pressed (CP) and hexane-extracted (HE) canola meals
with\emph{Aspergillus niger} NRRL 334 and \emph{A. oryzae} NRRL 5590 on
the functionalities of protein products extracted from them. After SSF,
proteins were recovered using alkaline extraction-isoelectric
precipitation (AE-IP) or salt extraction-dialysis (SE). SSF of the two
meal types reduced the protein content of the extracts produced by
AE-IP. There were varied effects to solubility, foaming, and emulsifying
properties as a result of SSF under the combined influence of
functionality pH, strain, meal type, and protein extraction method. The
protein isolate produced from CP meal using SE had increased solubility
at pH 7 (from 51.8 to 90.7\%) when the meal was fermented with \emph{A.
oryzae} . Both strains resulted in an over 2-fold increase in the
emulsifying activity index (at pH 7) of AE-IP products from CP meal. For
both protein extraction methods, the protein products from \emph{A.
niger} fermented HE meal had better foaming capacity (FC) at pH 7 than
the controls (non-fermented), but reduced FC at pH 3. Overall,
regardless of meal fermentation, the SE products were richer in protein
and had higher oil holding capacity (OHC), whereas the water holding
capacity (WHC) was higher for AE-IP isolates. SSF of the meals generally
improved the O/WHC of the extracted proteins. The findings suggest that
canola protein functionality could be effectively modulated by SSF with
different microbial strains under various processing conditions to
enhance their applicability in the food industry.

\textbf{KEYWORDS}

\emph{Aspergillus} \emph{niger, Aspergillus} \emph{oryzae} , canola
protein, functionality, protein extraction, solid-state fermentation

\textbf{1 INTRODUCTION}

Canola meal, the by-product of the canola oil extraction process, is
gaining tremendous interest in the food industry due to its high protein
content (35\%-45\%) and low cost. The use of canola meals as an
alternative protein source can provide more protein options to the
plant-based food industry. Canola proteins are dominated by two major
proteins, a salt-soluble globulin protein (cruciferin, 12\emph{S} , MW
\textasciitilde{}300 kDa) and a smaller water-soluble albumin (napin,
1.7-2\emph{S} , MW \textasciitilde{}14--17 kDa). The amino acid
composition of canola meal is well balanced with a relatively high
protein efficiency ratio (PER) of 2.64 and therefore can be used for
human nutrition (Aider \& Barbana, 2011). In recent research, canola
proteins have presented high solubility, foaming capacity and stability,
and comparable or moderate emulsifying properties compared to other
plant proteins such as soybean and pea protein (Chang \emph{et al.} ,
2015; Cheung \emph{et al.} , 2014; Khattab \& Arntfield, 2009; Tan
\emph{et al.} , 2011a; Wu \&Muir, 2008). These characteristics make
canola protein a potential ingredient for the food industry.

Canola oil can be extracted by hexane extraction (HE) or cold-pressing
(CP). The conventional way, HE, is a chemical process involving the use
of non-polar solvents, e.g., hexane. During HE, the seeds are ground up
and washed with hexane under controlled conditions to release oil
molecules stored within. A desolventizing step is followed to remove the
hexane by distilling at approximately 100-110\textsuperscript{o}C, after
which the solvent is recycled and reused (Cheng et al., 2019). The HE
process efficiently produces a high yield of oil, however, the use of
chemicals and the application of heat lead to considerate damage to the
proteins within the meal, resulting in reduced functionality (e.g.,
emulsifying properties) (\selectlanguage{ngerman}Östbring et al., 2019). The CP technique, on
the other hand, solely utilizes mechanical forces (e.g., crushing)
without the application of organic solvents or high heat for their
subsequent removal. The seeds are pressed at low temperatures
(\textless{}40\textsuperscript{o}C), leaving nutrients in the remaining
meal in a less disturbed form compared to HE, however, the oil yield was
lower in HE with a high level of residual oil (6-20\%) (Hickling, 2007;
Östbring et al., 2019).

Fermentation is the process accomplished by the metabolism of
microorganisms that catalyze nutrients, synthesize secondary
metabolites, and complete other physiological activities under anaerobic
or aerobic conditions. Solid-state fermentation (SSF) is a popular
process to modify the functionality and nutritional composition of
protein ingredients. As opposed to submerged fermentation (SmF), there's
no free water in SSF, making it suitable for microorganisms that don't
require high moisture or high water activity to grow, for example,
fungi. Recently, SSF has been applied to canola meals to improve their
quality. Pal Vig and Walia (2001) used \emph{Rhizopus oligosporus} as
the fermenting culture to produce a high-protein product from HE canola
meal. The results showed a significant decline in contents of
glucosinolates (GLS) (\textasciitilde{}43\%), thiooxazolidones
(\textasciitilde{}31 \%), phytic acid (\textasciitilde{}42\%) and fibre
(\textasciitilde{}26\%) along with a \textasciitilde{}65\% increase in
crude protein after 10 days. Croat \emph{et al.} (2016) studied the use
of SSF on HE and CP canola meals to modify their nutritional
composition. The strains \emph{Trichoderma reesei} , \emph{Aspergillus
pullulans} , and \emph{A. pullulans} improved protein content by 22.9,
16.9 and 15.4\%, while reducing the total GLS content from 60.6 to 1.0,
3.2 and 10.7 \selectlanguage{greek}μ\selectlanguage{english}mol/g, respectively. Significantly higher content of dry
matter yield was reported for the HE meal compared to the CP meal, which
is mainly due to the high oil residues left by CP. Much work has been
done on enhancing the nutritional quality of canola meals, however, the
use of SSF to alter the functionality of proteins in HE or CP canola
meals has been limited.

Several methods, including alkaline extraction-isoelectric precipitation
(AE-IP), salt extraction-dialysis (SE), protein micellation method
(PMM), and low pH extraction combined with membrane separation and
ultrafiltration (UF), have been widely used for the production of canola
protein isolates (Tan \emph{et al.} , 2011a, 2011b; Wanasundara, 2011).
The structural composition and functionality of the canola proteins may
vary significantly depending on the extraction method used (Can
Karaca\emph{et al.} , 2011; Hoglund \emph{et al.} , 1992; Wu \& Muir,
2008). In addition, the extraction of canola proteins is especially
difficult due to the differences in protein fractions (widely differing
isoelectric points of pH 4-11 and molecular weights of 13-320 kDa) and
the presence of antinutrients (GLS, phytic acid, and polyphenols),
pigments, and fibre in the canola meal (Wu \& Muir, 2008). It is
expected that pre-treatment, such as fermentation, and specific
purification processes may improve protein solubility in the meals and
consequently protein extractability.

In our previous study, both CP and HE canola meals had a
\textasciitilde{}45\% degree of protein hydrolysis upon 72 hours of
fermentation with \emph{Aspergillus niger} NRRL 334 and\emph{Aspergillus
oryzae} NRRL 5590. In this study, those fermented canola meals were used
in the wet fractionation process to produce protein products. Two wet
extraction processes, AE-IP and SE, were employed. The resulting protein
products were characterized for select functional properties and
compared with protein products extracted from meals that were not
pre-treated by fermentation. We hypothesize that pre-treatment with SSF
has the potential to improve the protein extractability from canola meal
because of partial protein hydrolysis and the loosening of
protein-carbohydrate interactions while also modifying protein
functionality. Moreover, the CP meals are expected to yield protein
products with higher functional properties than those processed by HE
due to the lack of heat and chemical damage to the proteins. The
findings of this work will provide a relatively comprehensive view of
the effect of SSF of canola meals on the functionality of extracted
proteins with direct comparisons between the test fungal strains, meal
type, and protein fractionation techniques.

\textbf{2 MATERIALS AND METHODS}

\textbf{2.1 Materials}

Canola meals (CP from Pleasant Valley Oil Mills (Clive, AB, Canada) or
HE from Bunge Canada (Harobe, MB, Canada)) of 150 g were fermented
separately with \emph{Aspergillus niger} NRRL 334 and \emph{Aspergillus
oryzae} NRRL 5590 (each at 10\textsuperscript{7} spores/g meal) for
three days (72 h) with moisture maintained at 50\% and temperature at
30. The strains were obtained from the Agricultural Research Service
(ARS) Culture Collection (Northern Regional Research Laboratory (NRRL),
Peoria, IL, USA). The fermented meals were immediately processed for
protein extraction using the AE-IP and SE methods (as described in the
following sections) to obtain canola protein products. All the reagents
and chemicals used were of analytical grade and obtained from
Sigma-Aldrich Canada Ltd. (Oakville, ON, Canada).

\textbf{2.2 Preparation of canola protein products}

\textbf{2.2.1 Alkaline extraction-isoelectric precipitation}

The fermented (72 h) CP and HE meals (100 g) were extracted at a 1:10
(w/v, meal:water) ratio at pH 11 (adjusted using 2 N NaOH) for a 1-h
period at room temperature (21-23) according to Aider and Barbana
(2011). The suspension was then centrifuged (Sorvall RC Plus Superspeed
Centrifuge, Thermo Fisher Scientific, Asheville NC, USA) (4,500 \selectlanguage{ngerman}×
\emph{g} at 4\selectlanguage{english} for 15 min), and the supernatant was vacuum filtered with
No. 1 Whatman filter paper (Whatman International Ltd., Maidstone, UK)
to remove any floating particles. The supernatant was then adjusted to
pH 5 using 2 N HCl to precipitate the proteins. The protein precipitate
was collected as a pellet after centrifugation at 4500 x \emph{g} at 4\selectlanguage{english}
for 15 min and freeze dried. The crude protein content of meals and
protein products was determined using the micro-Kjeldahl method (AOAC
960.52) with a conversion factor of 6.25 (AOAC, 2005).

\textbf{2.2.2 Salt extraction-dialysis (SE)}

SE described by Klassen \emph{et al.} (2011) and Chang \emph{et al.}
(2015) was employed with minor modification to obtain protein products
from fermented CP and HE canola meals. In brief, a 0.05 M Tris--HCl
buffer solution (pH 7.0) containing 0.1 M NaCl was mixed with fermented
canola meal at a 1:10 (w/v) ratio and stirred at 500 rpm for 2 h at room
temperature (21-23\selectlanguage{english}). The supernatant was then collected by centrifuging
(Sorvall RC Plus Superspeed Centrifuge, Thermo Fisher Scientific,
Asheville NC, USA) at 6,000 x \emph{g} for 30 min, followed by vacuum
filtration with No.1 Whatman filter paper (Whatman International Ltd.,
Maidstone, UK). The filtrate was dialyzed to remove NaCl and other small
molecules using a Spectra/Por molecular porous membrane tubing (6--8 kDa
cut off, Spectrum Medical Industries, Inc., USA) at 4\selectlanguage{english} for 72 h against
distilled water (distilled water was refreshed twice a day). The
dialyzed protein extract was then centrifuged at 3,000 x \emph{g} for 30
min at 4\selectlanguage{english}. The supernatant was discarded, and the protein pellet was
collected and freeze dried. The protein content was determined using the
same protocol for AE-IP samples.

\textbf{2.2.3 Protein product controls}

The canola protein products were extracted directly from unfermented CP
and HE meals (without defatting) using both the AE-IP and SE methods as
described above and used as the control protein products for the study.
In addition, a defatted CP meal was also prepared as a control to study
the effect of residual oil on protein extractability. In brief, the
unfermented CP meal was defatted with a meal:hexane ratio of 1:3 (w:v)
over a 1.5 h period of stirring at room temperature, followed by vacuum
filtration with No. 1 Whatman filter paper (Whatman International Ltd.,
Maidstone, UK) to recover the defatted solids. The above defatting
process was repeated twice to reach an oil content of \textless{}2\%,
and the resulting meal was used to extract the unfermented defatted CP
control (DCP) protein products using both the AE-IP and SE methods. In
total, three canola protein product controls were prepared (from HE, CP,
and defatted CP meals) for each extraction method (AE-IP and SE)
resulting in six total control (unfermented) protein products.

\textbf{2.3 Functional analysis}

\textbf{2.3.1 Solubility}

Protein solubility was studied as a function of pH according to Can
Karaca \emph{et al.} (2011) and Chang \emph{et al.} (2015) with minor
modifications. In brief, 20 mL of 0.25\% (w/w) protein (corrected by
protein content) solution at the required pH (3, 5, and 7) was prepared
and allowed to stir for 1 h at room temperature (21-23\selectlanguage{english}). The protein
solution was then transferred to a 15-mL centrifuge tube and centrifuged
(VWR clinical centrifuge 200, VWR International, Mississauga, ON,
Canada) at 4,180 x \emph{g} for 10 min at room temperature. The
supernatant was carefully collected, followed by the Bradford method to
determine the protein content. In detail, a 50 \selectlanguage{greek}µ\selectlanguage{english}L of supernatant was
added to a 1.5-mL centrifuge tube and 1.5 mL~of~Coomassie~dye (Thermo
Fisher Scientific, Madison, WI, USA) was added. The mixture was vortexed
for 10 s vigorously and then left to stand for 5 min. The absorbance of
the sample~was measured at 595 nm using a UV-visible spectrophotometer
(Genesys 20, Thermo Scientific, Madison, WI, USA), and Milli-Q water was
used as a blank while a mixture of 50~\selectlanguage{greek}µ\selectlanguage{english}L~of~0.5 N NaOH~and 1.5 mL
of~Coomassie~dye was used as the sample blank.~Different concentrations
of bovine serum albumin (BSA) were used to create a standard curve. The
total protein content was determined by dissolving 20 mg of dry protein
extract in 20 mL of 0.5 N NaOH solution and allowing the solution to
stir for 1 h at room temperature, followed by centrifugation at 12,100
\selectlanguage{ngerman}×\emph{g} for 30 min, and the total protein content of the supernatant
was determined using the Bradford method. The protein solubility (\%)
was determined by dividing the protein content in the solutions at
different pH levels by the total protein in the sample (N\% × 6.25),
multiplied by 100\%. All measurements were reported as the mean ± one
standard deviation (n=3).

\textbf{2.3.2 Water and oil holding capacity}

The water and oil holding capacity (WHC and OHC) were determined
according to Stone \emph{et al.} (2015). In brief, 0.5 g of protein
(corrected by protein content) was mixed with 5.0 g of water or oil in a
50 mL screw cap centrifuge tube. The mixture was vortexed for 10 s every
5 min for a total period of 30 min, followed by centrifugation (VWR
clinical centrifuge 200, VWR International, Mississauga, ON, Canada) at
1,000 × \emph{g} for 15 min. The remaining pellet was weighed after
carefully decanting the supernatant. The WHC and OHC were calculated by
dividing the weight gain by the original sample weight (g/g). All
measurements were reported as the mean ± one standard deviation (n=3).

\textbf{2.3.3 Emulsifying properties}

The emulsifying activity index (EAI, m\textsuperscript{2}/g) and
emulsion stability index (ESI, min) were determined according to Pearce
and Kinsella (1978) and Cheung\emph{et al.} (2014). In brief, a 0.25\%
(w/v) protein solution (corrected by protein content) was prepared at pH
3, 5, and 7 and stirred at room temperature (21-23\selectlanguage{english}) overnight. To
prepare the emulsion, 5.0 g of the protein solution was added to 5.0 g
of canola oil, followed by homogenization using an Omni Macro
Homogenizer (Omni International, Marietta, GA, USA) with a 20 mm saw
tooth generating probe positioned at the oil/water interface at speed 4
(\textasciitilde{}7,200 rpm) for 5 min. Immediately after
homogenization, 50 \selectlanguage{greek}μ\selectlanguage{english}L of the emulsion from the bottom of the tube was
transferred to 7.5 mL of 0.1 \% (w/v) sodium dodecyl sulphate (SDS),
followed by vortexing for 10 s. The absorbance of the diluted emulsion
was determined using a Genesys 20 UV-visible spectrophotometer (Thermo
Scientific, Madison, WI, USA) at 500 nm using plastic cuvettes. A second
absorbance reading was taken from the dilution after 10 min. The EAI and
ESI were calculated using the following equations:

\begin{equation}
EAI\ (\frac{m^{2}}{g}\mathrm{)}=\frac{2\times 2.203\times A_{0}\times N\ \times l}{c\times\varphi\times 100}\ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ (1)\nonumber \\
 \end{equation}\begin{equation}
\text{ESI\ }\left(\min\right)=\frac{A_{0}}{A}\times t\ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ (2)\nonumber \\
 \end{equation}

where \emph{A} \textsubscript{0} is the absorbance of the diluted
emulsion immediately after homogenization, \emph{N} is the dilution
factor, \emph{l} is the 0.01 m path length of the plastic
cuvette,\emph{c} is the weight of protein per volume (g/mL), \selectlanguage{greek}\emph{φ} \selectlanguage{english}is
the oil volume fraction of the emulsion, \selectlanguage{greek}Δ\selectlanguage{english}\emph{A} is the difference in
absorbance between 0 and 10 min (\emph{A} \textsubscript{0} -\emph{A}
\textsubscript{10}), and t is the time interval (10 min). All
measurements were reported as the mean \selectlanguage{ngerman}± one standard deviation (n=3).

\textbf{2.3.4 Foaming properties}

The foaming properties were determined according to Stone \emph{et
al.}(2015). In brief, a 1.0\% (w/w) protein solution (corrected by
protein content) was prepared at pH 3, 5, and 7 and stirred overnight at
4\selectlanguage{english}. For each protein solution, 15 mL (\emph{V} \textsubscript{li}) was
transferred into a 400 mL tall glass beaker (inner diameter = 69 mm;
height = 127 mm; as measured with a digital caliper) and foamed using an
Omni Macro homogenizer (Omni International, Marietta, GA, USA) with a 20
mm saw tooth generating probe for a total of 5 min at speed 4
(\textasciitilde{}7,200 rpm). The foam was immediately transferred to a
50- or 100-mL graduated cylinder (inner diameter = 26 mm) after
homogenization. The foam volume was recorded at time zero (\emph{V}
\textsubscript{fi}) and after 30 min (\emph{V} \textsubscript{ft}) at
room temperature. The foaming capacity (FC) and foaming stability (FS)
were determined using following the equations:

\(\%FC=\frac{V_{\text{fi}}}{V_{\text{li}}}\times 100\%\ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ (3)\)

\(\%FS=\frac{V_{\text{ft}}}{V_{\text{fi}}}\times 100\%\ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ (4)\)

where V\textsubscript{fi} is the volume of foam immediately after
homogenization, and V\textsubscript{ft} is the volume of foam remaining
after 30 min.

\textbf{2.3.5} \textbf{Statistical analysis}

All protein products were prepared in triplicate on separate fermented
meals (n=3). The results were reported as mean +- one standard deviation.
A two-way analysis of variance (ANOVA) was used to study the statistical
differences in protein functionality using a Tukey's test with a
significance level of \emph{p} \textless{}0.05. A simple Pearson
correlation (\emph{r} ) was used to describe the relationship between
the functional properties. The statistical analysis was performed using
the IBM SPSS Version 28.0 software (IBM Corp. NY, IL, USA).

\textbf{3 RESULTS AND DISCUSSION}

\textbf{3.1 Protein content of canola protein products}

The protein products obtained using AE-IP from unfermented HE and CP
(non-defatted) meals had protein levels of 86.5\% and 68.9\%,
respectively (Table 1). After the defatting process, an 81.6\% protein
content was achieved for the CP meal, indicating that the high oil
content negatively influenced protein extraction by AE-IP. The canola
protein products obtained from the CP meal fermented with \emph{A.
niger} NRRL 334 and \emph{A. oryzae} NRRL 5590 had protein levels of
62.7\% and 57.9\%, respectively. The protein products from the HE meals
fermented with \emph{A. niger} and \emph{A. oryzae} had protein levels
of 56.3\% and 58.4\%, respectively. Both the CP and HE meals, when
fermented with either of the fungal strains, gave products with lower
protein content upon enrichment using AE-IP.

In contrast, the SE method resulted in higher protein levels
(\textgreater{}90\%) for all unfermented and fermented canola protein
products than those produced by AE-IP (56.3\%-86.5\%). The lower protein
content found in the AE-IP samples may be due to the heterogeneous
nature of canola proteins (cruciferin and napin), which were proven to
have different isoelectric points that complicate their precipitation
(Wu \& Muir, 2008). Fermentation did not increase the protein content of
SE products for either type of meal (CP and HE). Of the protein products
extracted from fermented meals, the SE process using the CP meal
fermented with \emph{A. oryzae} resulted in the highest protein content
(99.0\%).

As these findings refute the hypothesis that SSF could improve the
protein extraction process, a few theories are proposed. Fermentation
can lead to partial hydrolysis of the protein, which is hypothesized to
increase the protein surface hydrophobicity (decrease in protein
solubility) and in turn create stronger protein-lipid interactions.
Also, the large quantity of hyphae produced by fungi may act like a
`glue' to connect the solid substrates tightly and affect the protein
extraction by limiting the soluble protein dissolving into the
surrounding aquatic system. After the SSF, the degradation of fibre
might also release insoluble or soluble carbohydrates (polysaccharides
or starch) and make it difficult to separate the proteins from the
complex (protein-carbohydrate interaction). In addition, the oil could
largely affect the protein extraction process, which may be the case for
the CP meals (Krause \& Schwenke, 2001), although adding a defatting
step to the unfermented meal resulted in slightly lower protein content
(99.6 \emph{vs.} 96.5\%) of the SE products.

\textbf{3.2 Protein solubility}

The protein solubility is one of the most important physicochemical
properties, as it is generally related to other functional properties,
which may affect the rheological, hydrodynamic, and surface activity
characteristics (Zayas, 1997). As shown in Table 2, the AE-IP CP control
had a significantly higher (\emph{p} \textless{}0.05) solubility at pH 3
where it was 88.9\% soluble protein compared to the defatted CP and HE
controls (76.2\% and 81.4\%, respectively). Similar to the results at pH
3, at pH 5 the AE-IP CP control showed higher solubility (53.5\%) than
the defatted CP (36.9\%) and HE (2.0\%) controls, whereas at pH 7 the HE
control (70.0\%) had a higher solubility than both the CP control
(27.7\%) and the defatted CP control (37.8\%). As for the SE products,
all of the controls had a higher solubility (\selectlanguage{english}[?]94\%) at pH 3 than the
controls produced by AE-IP. At pH 5, a higher solubility was found for
the SE HE control (29.4\%) compared to AE-IP (2.0\%), whereas it was
lower than the SE CP control (37.7\%) and defatted CP control (37.3\%),
which had similar solubility values. At pH 7, both the SE CP (51.8\%)
and defatted CP control (49.3\%) had a higher solubility than the
respective AE-IP product however, the SE HE control (43.2\%) was less
soluble than the AE-IP HE control. Overall, the SE products showed
higher solubility at pH 3 and 7. Stone \emph{et al.} (2015) also found a
lower solubility of canola protein products obtained by AE-IP compared
to the SE process. Similar solubility values were found for both the CP
and defatted CP controls at every pH and tended to be higher than the HE
control.

The canola protein products prepared from fermented meals showed a lower
solubility at each pH compared to the control products. This is
hypothesized to be due to partial hydrolysis of protein during
fermentation which led to the exposure of previously buried hydrophobic
groups. At pH 3, all AE-IP and SE controls showed higher solubility than
products from fermented meals. However, the solubility of products
from\emph{A. oryzae} fermented meals was significantly higher than
products from \emph{A. niger} fermented meals. This indicated that
fermentation with \emph{A. oryzae} was able to maintain the solubility
(pH 3) at a relatively higher level than \emph{A. niger} after SSF. As
such, using\emph{A. oryzae} may be favorable to obtain a more soluble
protein product at pH 3. After fermentation, most extracted samples
showed significant decreases (\emph{p} \textless{}0.05) in solubility
compared to the controls at pH 5 and had overall low solubility values
ranging from 4.5\% to 11.8\%. However, there was an increase in
solubility at pH 5 from 2.0\% for the AE-IP HE control to 11.8\% and
5.8\% when fermented using \emph{A. niger} and \emph{A. oryzae} ,
respectively. According to a previous study, the isoelectric point at pH
6.2 and low solubility at both pH 5 (\textasciitilde{}37\%) and pH 7
(\textasciitilde{}23\%) were reported for a SE canola protein isolate
produced from unfermented HE meal (Chang \emph{et al.} , 2015). Our
results indicated that SSF processing decreased the protein solubility
at pH 5 which may be due to a change in the isoelectric point (close to
pH 5) by partial hydrolysis of the proteins, causing the shift in
solubility with pH value.

At pH 7, the solubility of AE-IP canola protein products showed
significantly lower (\emph{p} \textless{}0.05) solubility than SE
products obtained from CP control meals. The inverse was found for the
products extracted from the HE control meal. Unlike at pH 3 and 5, the
SSF showed the ability to improve the protein solubility of the
extracted products at pH 7. For instance, the AE-IP product from
the\emph{A. niger} fermented CP meal showed a significant
(p\textless{}0.05) increase in solubility (to 47.5\%) compared to both
the AE-IP CP controls. In addition, the same level of solubility
(70.0\%) was found for the AE-IP HE control and the HE product from
the\emph{A. niger} fermented meal. However, there was a decrease in
solubility (\emph{p} \textless{}0.05) for protein products extracted
(AE-IP) from \emph{A. oryzae} fermented CP and HE meals compared to the
controls, which indicated that \emph{A. niger} was preferred to maintain
or increase the solubility of AE-IP protein products at pH 7. Some
opposite results were found for SE protein products, as products
from\emph{A. oryzae} fermented meals resulted in higher solubility
values (90.7\% for \emph{A. oryzae} CP and 55.6\% for \emph{A. oryzae}
HE) compared to the controls (p\textless{}0.05). An increase in the
solubility of the SE product from \emph{A. niger} fermented HE meal
(48.5\%) was also observed compared to the HE control
(p\textless{}0.05). However, for the CP meals a decrease in solubility
was reported when using SE on the \emph{A. niger} fermented meal
(37.2\%) compared to the CP controls. When extracting proteins via SE,
both \emph{A. niger} and \emph{A. oryzae} can be acceptable inoculums
for meals to maintain or increase the protein solubility at pH 7,
whereas \emph{A. niger} might be a better culture choice for AE-IP
products at pH 7.

The results above indicate the differences between AE-IP and SE canola
protein (protein fraction, the percentage of napin and cruciferin) and
the hydrolysis mechanism of \emph{A. niger} and \emph{A.
oryzae}including possible different proteinase, length of peptides,
structure of hydrolyzed protein, and synthesized metabolism. The protein
products extracted using SE showed higher solubility than the AE-IP
products mainly due to the low content of non-protein compounds,
possible differences in protein fraction, and possible interactions of
protein and carbohydrate in the AE-IP products. In addition, the protein
products extracted from CP meals showed higher solubility than products
from the HE meal. Protein denaturation during the hexane-extraction
(heat treatment) could explain the solubility reduction. Heat treatments
could result in the exposure of hydrophobic groups, which contribute to
the reduction in protein solubility (Khattab \& Arntfield, 2009). The
partial denaturation of proteins can alternate the balance of protein
hydrophobicity/hydrophilicity and further affect solubility
(Moure\emph{et al.} , 2006).

\textbf{3.3 Water holding capacity}

The ability of a protein to absorb and retain water is critical in
stabilizing the structure, improving flavour retention and mouth feel,
and reducing moisture loss of food products (Khattab \& Arntfield,
2009). As shown in Table 3(a), the AE-IP CP control protein product
showed higher WHC (2.1 g/g) than the products of the HE control (1.8
g/g), which may be due to the thermal and chemical changes of HE
proteins that occurred during the solvent extraction processing, leading
to an increase in the protein-protein aggregations and limiting the
protein-water or protein-oil interactions. However, the value of the
defatted CP control (2.7 g/g) was higher than that of non-defatted ones
(2.1 g/g), which was mainly due to the presence of residual oil that
impacted the hydrophilic ability. Similar results were found in the SE
controls of which the defatted CP control (2.1 g/g) had a higher WHC
value than the HE (1.3 g/g) and non-defatted CP controls (1.1 g/g) as
shown in Table 3(b).

After a period of 72-h fermentation of the source meal, a significant
increase (\emph{p} \textless{}0.05) in the WHC of AE-IP products was
found. The WHC increased from 2.1 g/g (CP control) and 2.7 g/g
(non-defatted CP control) to 3.1 g/g (\emph{A. niger} fermented) and 3.0
g/g (\emph{A. oryzae} fermented). No significant difference (\emph{p}
\textgreater{}0.05) was found between the strains for AE-IP CP products.
In addition, \emph{A. oryzae} showed a higher increase in WHC of AE-IP
HE products (3.2 g/g) than \emph{A. niger.} (2.9 g/g). Similar results
were found for SE HE products as SSF increased the WHC values from 1.3
g/g to 2.3 g/g (\emph{A. niger} ) and 2.7 g/g (\emph{A. oryzae} ). The
partial protein hydrolysis during the fermentation process may change
the compact protein structure and expose more hydrophilic and
hydrophobic sites (additional binding sites available for water), which
made it easier for the protein to adsorb and hold water (Kinsella,
1982). In contrast, the WHC values of the SE CP products remained stable
(\emph{p} \textgreater{}0.05) after SSF using \emph{A. niger} (1.3 g/g)
and \emph{A. oryzae} (1.4 g/g) compared to the non-defatted CP control
(1.1 g/g) and decreased when compared to the defatted CP control (2.1
g/g). For the type of strains, \emph{A. oryzae} was preferred to improve
the WHC of both AE-IP and SE HE products. In addition, all HE products
showed higher increases in WHC than the CP products.

Overall, the defatted CP controls showed higher WHC values than the
non-defatted CP controls and the HE controls. As for the difference
between the AE-IP and SE products, the AE-IP protein products showed a
slightly higher WHC value than the SE ones. This was possibly due to the
lower protein level in AE-IP products, which led to a larger sample
amount of AE-IP products used for testing than the SE products
(corrected by protein content). The non-protein compounds in AE-IP
products such as the soluble fibre and polysaccharides may contribute to
the ability of water to absorb and bind. Thus, it is important to take
the possibly contained fibre and polysaccharides into consideration. The
presence of these compounds can enhance the overall water holding
capacity of canola products (Aider \& Barbana, 2011).

\textbf{3.4 Oil holding capacity}

Fats act as a flavour carrier and mouthfeel enhancer, so the ability of
the protein to absorb and retain oil is important in food formulations
(Kinsella, 1982). Fat adsorption of proteins mainly depends on the
physical entrapment of oil through a capillary-attraction process
(Kinsella, 1982). As shown in Table 3(a), the AE-IP CP control showed a
higher OHC value (2.9 g/g) than the HE control (2.3 g/g), possibly due
to the thermal and chemical changes of proteins that occurred during
solvent extraction, leading to an increase in protein-protein
aggregations and limited protein-water or protein-oil interactions.
Similar results were observed for the products obtained using SE in
Table 3(b). The CP control had a higher OHC value (3.2 g/g) than the
products of the HE control (2.4 g/g). The OHC value of the AE-IP
defatted CP control (2.4 g/g) was lower than that of the non-defatted CP
control (2.9 g/g), whereas the SE defatted CP control (3.9 g/g) had a
higher OHC value than the CP non-defatted control (3.2 g/g).

After the 72-h SSF, the OHC of AE-IP CP products increased to 2.8 g/g
(both \emph{A. niger} and \emph{A. oryzae} ) compared to the defatted CP
control (2.4 g/g, p\emph{\textless{}} 0.05), whereas it was similar to
that of the non-defatting CP control (2.9 g/g, p\textgreater{}0.05). The
AE-IP HE products also showed an increase (p\textless{}0.05) from 2.3
g/g to 3.1 g/g (\emph{A. niger} ) and 3.3 g/g (\emph{A. oryzae} ). As
for the SE products, an increase in OHC was found for both the AE-IP and
the SE protein products compared to the controls (p\textless{}0.05). For
the SE CP products, the OHC values increased from 3.2 g/g (CP control)
and 3.9 g/g (defatted CP control) to 4.6 g/g (\emph{A. niger} ) and 4.7
g/g (\emph{A. oryzae} ), while the value increased from 2.4 g/g (HE
control) to 4.5 /g (\emph{A. niger} ) and 3.9 g/g (\emph{A. oryzae} )
for HE products. The partial hydrolysis of proteins during fermentation
may change the compact protein structure and expose more hydrophilic and
hydrophobic sites, making it easier for proteins to adsorb and hold oil.
The above results were agreed by Kinsella (1982) that the increase in
fat absorption could be associated with the hydrolysis and denaturation
of proteins due to the exposure of non-polar residues buried inside the
protein molecules. Comparing the type of strains, there is no
significant difference (p\textgreater{}0.05) between \emph{A. niger}
and\emph{A. oryzae} except for the SE HE products, where \emph{A.
niger}was preferred to improve the OHC of products\emph{.} Similar to
the WHC, a higher increase was found in the HE meal compared with the CP
meal after SSF.

Whereas the WHC results showed higher capacities with the AE-IP
isolates, canola protein products obtained using SE showed higher OHC
compared to those prepared by AE-IP. It may indicate that a higher level
of protein (\textasciitilde{}95\% for SE products and
\textasciitilde{}60-80\% for AE-IP products) is preferred to obtain
protein products with enhanced OHC. It could also be concluded that the
proteins of SE might be more hydrophilic than those from AE-IP, hence a
higher protein solubility for the SE products. The lower solubility and
greater OHC suggest that the proteins recovered from AE-IP could obtain
a more hydrophobic nature. The partial hydrolysis of proteins during the
fermentation process may change the compact protein structure and expose
more hydrophilic and hydrophobic sites (additional binding sites
available) buried inside the protein molecule, which may decrease the
solubility of canola proteins while increasing the ability of the
proteins to adsorb and hold water/oil (Kinsella, 1982).

\textbf{3.5 Emulsifying properties}

The emulsifying properties are critical functional attributes of food
proteins. Several factors affect the emulsifying properties, including
the type and concentration of protein, pH, ionic strength, and viscosity
of the solvating medium. Many chemical and physical factors are involved
in the formation, stability, and textural properties of oil-water
emulsions stabilized by protein (Chang \emph{et al.} , 2015). The EAI is
a measurement of the interfacial area coated by protein during the
formation of an emulsion and acts as a good predictor for the surface
activity of the protein, whereas the ESI is a measurement of the
stability of the diluted emulsion over a fixed period of time (Can
Karaca \emph{et al.} , 2011).

As shown in Table 4(a), the AE-IP HE control had a higher EAI value
(13.2 m\textsuperscript{2}/g) than the CP control (11.4
m\textsuperscript{2}/g) and the defatted CP control (8.3
m\textsuperscript{2}/g) at pH 3, which may indicate a higher
hydrophobicity value of the HE products compared to the CP controls at
pH 3. Similarly for pH 7, a higher EAI value was found for the HE
control (18.3 m\textsuperscript{2}/g) than the CP control (11.7
m\textsuperscript{2}/g) and the defatted CP control (11.6
m\textsuperscript{2}/g) at pH 3. As for pH 5, the defatted CP control
(5.0 m\textsuperscript{2}/g) had a higher EAI value than the CP control
(4.1 m\textsuperscript{2}/g, \emph{p} \textgreater{}0.05) and the HE
control (3.0 m\textsuperscript{2}/g, \emph{p} \textless{}0.05). As for
the SE products, the defatted CP control (11.0, 9.4, and 14.5
m\textsuperscript{2}/g at pH 3, 5, and 7, respectively) showed the
highest EAI values compared to the CP (5.6, 5.8, and 12.9
m\textsuperscript{2}/g at pH 3, 5, and 7, respectively) and the HE
control (5.9, 6.8, and 13.9 m\textsuperscript{2}/g at pH 3, 5, and 7,
respectively). The difference between the AE-IP and SE products may be
due to the different protein fractions and the nature of products
resulting from the two methods. As for pH, the EAI values tended to be
low at pH 5, ranging from 1.3-5.5 m\textsuperscript{2}/g due to the low
protein solubility. Higher EAI values were found at pH 7 compared to pH
3 for all protein products obtained from unfermented control meals,
which was possibly related to the higher solubility at pH 7 than at pH
3.

During SSF, the partial hydrolysis of proteins loosens the compact
protein structure and exposes more hydrophobic sites buried inside the
protein molecule. A higher hydrophobicity value can be achieved.
Townsend and Nakai (1983) suggested a positive correlation between
hydrophobicity and the emulsifying properties of proteins. Results of
the present study showed that canola products extracted from fermented
meals have better or at least unchanged emulsifying properties compared
to the controls. Among the AE-IP products, the EAI values increased to
13.8 m\textsuperscript{2}/g (\emph{A. niger} ) and 21.1
m\textsuperscript{2}/g (\emph{A. oryzae} ) at pH 3 for the CP products
(p\textless{}0.05). In addition, only \emph{A. oryzae} improved the EAI
of the HE product (17.6 m\textsuperscript{2}/g), while a decrease (8.5
m\textsuperscript{2}/g) was found using \emph{A. niger} at pH 3. At pH
7, the AE-IP products from \emph{A. niger} and \emph{A. oryzae}fermented
meals had either improved or unchanged EAI values. In detail, the EAI
values of AE-IP CP products extracted from \emph{A. niger} and\emph{A.
oryzae} fermented CP meals significantly increased (\emph{p}
\textless{}0.05) to 25.8 and 27.6 m\textsuperscript{2}/g, respectively.
Similar to the AE-IP HE products, the EAI values were significantly
increased to 18.0 and 25.5 m\textsuperscript{2}/g when fermented using
\emph{A. niger} and \emph{A. oryzae} . The EAI values of the AE-IP
products at pH 5 remained relatively low, ranging from 1.3-5.5
m\textsuperscript{2}/g for both the AE-IP CP and the HE products,
regardless of whether fermented or not, may be due to the low protein
solubility (pH 5). In addition, a low ESI was reported for all protein
products and ranged from 1.1-4.5 min. For the SE products, \emph{A.
niger} and \emph{A. oryzae} improved the EAI values of the CP products
at pH 3 to 8.5 m\textsuperscript{2}/g (\emph{A. niger} ) and 9.7
m\textsuperscript{2}/g (\emph{A. oryzae} ) compared to the CP control
(5.6 m\textsuperscript{2}/g), however, they were lower than the defatted
CP control (11.0 m\textsuperscript{2}/g). The increases in the SE HE
product at pH 3 after SSF were also reported as 8.5
m\textsuperscript{2}/g (\emph{A. niger} ) and 11.3
m\textsuperscript{2}/g (\emph{A. oryzae} ) compared to the HE control
(5.9 m\textsuperscript{2}/g). However, SSF decreased the EAI of the CP
and HE products to 1.8 m\textsuperscript{2}/g (CP, \emph{A. niger} ),
4.6 m\textsuperscript{2}/g (CP, \emph{A. oryzae} ), 4.1
m\textsuperscript{2}/g (HE, \emph{A. niger} ), and 3.0
m\textsuperscript{2}/g (HE, \emph{A. oryzae} ) at pH 5. At pH 7, a
decrease in the EAI of SE CP and HE products was also reported to 12.2
m\textsuperscript{2}/g (CP, \emph{A. niger} ), 14.9
m\textsuperscript{2}/g (CP, \emph{A. oryzae} ), 11.4
m\textsuperscript{2}/g (HE, \emph{A. niger} ), and 7.6
m\textsuperscript{2}/g (HE, \emph{A. oryzae} ). The strain \emph{A.
oryzae} was able to more positively modify the EAI values of canola
protein products than \emph{A. niger} while the enhanced properties were
more frequently observed in AE-IP isolates than SE.

The above EAI and ESI results reported were lower than those from
previous studies by Cheung \emph{et al.} (2014) and Chang \emph{et
al.}(2015). Cheung \emph{et al.} (2014) suggested that high solubility
was positively correlated with the ESI values due to the additional
protein precipitation and adherence to the viscoelastic film surrounding
the droplets. A lower solubility was reported compared to previous
studies on canola protein, which may result in a reduction in ESI. In
contrast, Kinsella and Melachouris (1976) explained that high protein
solubility and high fat-adsorption capacity were positively correlated
with the ability of emulsifying (form and stabilize emulsions). Wu and
Muir (2008) and Cheung \emph{et al.} (2014) examined the emulsifying
properties of two major canola proteins, cruciferin and napin. It has
been reported that the emulsion prepared with cruciferin showed a
significantly higher specific surface area and a lower particle size
than that with napin. The study by Wu and Muir (2008) indicated that the
presence of napin could detrimentally affect the emulsion stability of
canola protein isolates. The differences may be a result of the canola
cultivar, the preparation of canola meals (oil extraction method) and
protein products (extraction methods and conditions), and the analytical
methods employed. These differences might result in variations in
protein fractions and the levels of protein that further affect the
emulsifying properties. In addition, the released short peptides from
fermentation may also have an impact on the protein functionality such
as a reduction in emulsion activity and stability (Kristinsson \& Rasco,
2000).

\textbf{3.6 Foaming properties}

The foaming properties of proteins are important in the production of a
variety of foods (Hettiarachchy \emph{et al.} , 2012). Foaming
properties can be defined as two parts, the ability to form a two-phase
system consisting of air cells surrounded by a thin continuous liquid
layer (the lamellar phase) and the ability to stabilize the foams. All
canola protein products in this study regardless of the source of meal
or fermentation treatment showed FC of 131.1-480.0 \% and moderate FS
ranging from 68.0-89.0\% at pH 3, 5, and 7.

Among the controls, the defatted AE-IP CP control (306.7\% at pH 3 and
243.8\% at pH 7) had higher FC than the CP (224.4\% pH 3 and 165\% at pH
7) and the HE control (244.4\% at pH 3 and 154.4\% at pH 7) possibly due
to the residual oil in the CP control and a low protein content
(\textasciitilde{}60\%) of both controls. As for the SE products, the CP
control had a higher foaming capacity (322.2\%) than the defatted CP
(246.7\%) and the HE (266.7\%) control at pH 3. At pH 7, the SE defatted
CP control showed the highest FC in all products (480.0\%), higher than
the SE CP (464.4\%) and the HE (145.6\%) control. At pH 5, only the CP
(241.1\% for AE-IP and 211.1\% for SE) and the defatted CP control
(131.1\% for AE-IP and 222.2\% for SE) showed the ability to form stable
foams for both AE-IP and SE products. However, the poor solubility at pH
5 still had an adverse impact on the foaming properties compared to the
values at pH 3 and pH 7. Besides, the high temperature and the exposure
to hexane during oil extraction may be the factors that contribute to
the loss of foaming ability of the HE control at pH 5. In addition, the
SE products showed significantly higher FC values than AE-IP. This might
be due to the higher purity of protein in the SE products
(\textgreater{}93\%) than AE-IP (\textless{}80\%) and possible
differences in protein composition (napin and cruciferin).

Among the AE-IP products, only the AE-IP CP protein products by \emph{A.
oryzae} fermented meals showed the ability to form foams (228.9\%) with
a low FS of 33.0\% at pH 3 after SSF. At pH 7, all fermented AE-IP
products showed foaming properties of 230.7\% (CP, \emph{A. niger} ),
180.0\% (CP, \emph{A. oryzae} ) 244.2\% (HE, \emph{A. niger} ), and
136.7\% (HE, \emph{A. oryzae} ) with comparable FC compared to the
controls ranging 72.0-84.6\%. The AE-IP HE samples pre-treated
with\emph{A. niger} even showed a significantly higher FC value
(244.2\%) compared to the HE control (154.4\%). As for the SE products,
all samples were able to form foams at pH 3 and 7. In detail, the FC
values of CP products from fermented meals decreased to 196.5\%
(\emph{A. niger} at pH 3), 175.6\% (\emph{A. oryzae} at pH 3), 191.1\%
(\emph{A. niger} at pH 7), and 153.3\% (\emph{A. oryzae} at pH 7)
compared to the CP control at pH 3 and 7. For the HE products, decreases
were also found for products at pH 3, at 195.6\% (\emph{A. niger} ) and
135.6\% (\emph{A. oryzae} ), while significant increases were reported
at pH 7 as 306.7\% (\emph{A. niger} ) and 155.6\% (\emph{A. oryzae} )
compared to the HE control (266.7\% at pH 3 and 155.6\% at pH 7). In
addition, the FS decreased to \textasciitilde{}52\% for all products
from fermented meals at pH 3, while they remained relatively unchanged
(77.6-88.4\%) at pH 7. At pH 5, none of the products extracted from
fermented meals showed the ability to form stable foams. All proteins
had poor foaming properties at pH 3 and 5 and could only form weak foams
with large bubbles that disappeared within 5 min. This was possibly due
to the extremely low protein solubility (\textless{}10\%) of proteins
extracted from fermented meals at pH 5. The SSF was found to decrease
the solubility of canola protein products to \textless{}10\% at pH 5,
which could have further adverse impacts on the foaming properties.
Overall, protein products prepared from the fermented HE meals using
both extraction methods showed better FC and FS than the HE controls,
while opposite results were found for CP canola meal products.

The above results indicate the potential of SSF for improving the
foaming properties at pH 7 of protein products extracted from HE meal,
especially when using \emph{A. niger} . In other words, \emph{A.
niger}was preferred to provide protein products with better foaming
properties compared to \emph{A. oryzae} . The CP products had better
foaming properties demonstrated by some foam when non was observed for
HE isolates under certain conditions. Although the FC and FS values
decreased after SSF, proteins extracted from the fermented CP meal
displayed acceptable foaming properties compared to other plant proteins
like soybean, pea, and lentil (\textasciitilde{}200\%) (Chabanon\emph{et
al.} , 2007; Stone \emph{et al.} , 2015). However, they still could not
produce comparable protein products similar to the ones from unfermented
CP meals.

\textbf{4 CONCLUSIONS}

The present study investigated the effect of treating canola meals (CP
or HE) with SSF by \emph{A. niger} and \emph{A. oryzae} on the
functionality of extracted protein products (AE-IP or SE). In summary of
the findings, protein purity in the resulting isolates was negatively
influenced by fermentation, and the magnitude of reduction was greater
for AE-IP products than SE. The protein content was also lowered in the
presence of residual fat in the CP samples compared to HE when the
protein products were produced by AE-IP. There were significant
differences in canola protein functionalities extracted from fermented
meals compared to the controls. For AE-IP proteins, meal fermentation
had an overall negative effect on solubility however some improvements
were seen with \emph{A. niger} fermentation. Pretreatment with \emph{A.
oryzae} increased the protein solubility at pH 7 when extracting
proteins via SE. Grouped together, the SE protein products were more
soluble than those by AE-IP, while those extracted from CP meals showed
higher solubility than those from HE. Fermentation of the meals improved
the WHC and OHC of most protein products with strain difference most
evident in SE proteins from HE meals. For emulsifying properties, the
strain \emph{A. oryzae} was shown to enhance or maintain the EAI of the
isolates more effectively than \emph{A. niger} , whereas ESI was largely
unchanged upon fermentation, regardless of the strains; the AE-IP
prepared isolates responded more positively to SSF than those by SE.
Foaming properties at pH 3 and 5 were generally diminished by SSF while
some increases at pH 7 were observed. \emph{A. niger} was slightly
preferred over \emph{A. oryzae} to improve or minimize the decreases,
while the CP products, control or fermented, tended to foam better than
those processed by HE. In short, no overarching trend could be reported
for the effect of meal fermentation on the resulting functionalities of
extracted canola proteins, as most functional attributes were under the
combined influence of pH, test strains, and oil and protein extraction
techniques. However, the findings indicate that by modifying processing
conditions, functionalities of canola meal protein products could be
tailored accordingly to suit potential applications, and SSF by \emph{A.
niger} and \emph{A. oryzae} remains a promising practice to expand the
utilization of canola proteins.

\textbf{ACKNOWLEDGEMENTS}

The financial support for the present study was provided by the
Saskatchewan Agriculture Development Fund.

\textbf{AUTHOR CONTRIBUTIONS}

Chenghao Li carried out the experiments, analyzed data, and wrote the
first draft of the manuscript. Dai Shi and Andrea K. Stone performed
additional data analysis and interpretation, and critical revision of
the manuscript. Janitha P. D. Wanasundara and Michael T. Nickerson were
responsible for the design of the study, supervision of the research,
and revision of the manuscript drafts. Takuji Tanaka provided scientific
consultation on the results and manuscript-writing.

\textbf{CONFLICT OF INTEREST}

The authors declare that they have no conflict of interest.

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Zayas JF. Solubility of Proteins. In: Functionality of Proteins in
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doi:10.1007/978-3-642-59116-7\_2

\textbf{Table 1 Protein content (\%, d.b.) of protein products obtained
by alkaline extraction-isoelectric precipitation (AE-IP) and salt
extraction-dialysis (SE) prepared from fermented and unfermented
(control) cold-pressed (CP) and hexane-extracted (HE) canola meals.}\selectlanguage{english}
\begin{longtable}[]{@{}llllllll@{}}
\toprule
& \textbf{CP} & \textbf{CP} & \textbf{CP} & \textbf{CP} & \textbf{HE} &
\textbf{HE} & \textbf{HE}\tabularnewline
\midrule
\endhead
& Unfermented & Unfermented & Fermented & Fermented & Unfermented &
Fermented & Fermented\tabularnewline
& No defatting & With defatting* & \emph{A. niger} & \emph{A. oryzae} &
Control & \emph{A. niger} & \emph{A. oryzae}\tabularnewline
\textbf{AE-IP} & 68.9 \selectlanguage{ngerman}± 0.3\textsuperscript{c} & 81.6 \selectlanguage{ngerman}±
1.1\textsuperscript{b} & 62.7 \selectlanguage{ngerman}± 0.2\textsuperscript{d} & 57.9 \selectlanguage{ngerman}±
0.2\textsuperscript{f} & 86.5 \selectlanguage{ngerman}± 0.2\textsuperscript{a} & 56.5 \selectlanguage{ngerman}±
0.2\textsuperscript{g} & 58.5 \selectlanguage{ngerman}± 0.2\textsuperscript{e}\tabularnewline
\textbf{SE} & 99.6 \selectlanguage{ngerman}± 1.0\textsuperscript{a} & 96.5 \selectlanguage{ngerman}±
1.3\textsuperscript{d} & 93.3 \selectlanguage{ngerman}± 2.2\textsuperscript{f} & 99.0 \selectlanguage{ngerman}±
1.2\textsuperscript{b} & 97.7 \selectlanguage{ngerman}± 3.3\textsuperscript{c} & 95.9 \selectlanguage{ngerman}±
2.3\textsuperscript{e} & 95.8 \selectlanguage{ngerman}± 4.2\textsuperscript{e}\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{ngerman}

Notes:

*Defatted CP control - canola protein products extracted from defatted
CP meal using AE-IP or SE. Data was reported as mean ± one standard
deviation across each extraction process (n=3). Significant difference
exists between data with different letters in a row (p\textless{}0.05).

\textbf{Table 2} \textbf{Protein (nitrogen-based) solubility (\%) of
protein products obtained by alkaline extraction-isoelectric
precipitation (AE-IP) and salt extraction-dialysis (SE) from fermented
and unfermented (control) canola meals.}\selectlanguage{english}
\begin{longtable}[]{@{}lllllll@{}}
\toprule
& \textbf{AE-IP} & \textbf{AE-IP} & \textbf{AE-IP} & \textbf{SE} &
\textbf{SE} & \textbf{SE}\tabularnewline
\midrule
\endhead
& \textbf{pH 3} & \textbf{pH 5} & \textbf{pH 7} & \textbf{pH 3} &
\textbf{pH 5} & \textbf{pH 7}\tabularnewline
CP control & 88.9 \selectlanguage{ngerman}± 3.7\textsuperscript{a} & 53.5 \selectlanguage{ngerman}±
2.6\textsuperscript{a} & 27.7 \selectlanguage{ngerman}± 2.2\textsuperscript{d} & 94.0 \selectlanguage{ngerman}±
0.4\textsuperscript{a} & 37.7 \selectlanguage{ngerman}± 0.2\textsuperscript{a} & 51.8 \selectlanguage{ngerman}±
1.1\textsuperscript{bc}\tabularnewline
DCP control* & 76.2 \selectlanguage{ngerman}± 3.6\textsuperscript{cd} & 36.9 \selectlanguage{ngerman}±
2.1\textsuperscript{b} & 37.8 \selectlanguage{ngerman}± 2.4\textsuperscript{c} & 97.1 \selectlanguage{ngerman}±
0.5\textsuperscript{a} & 37.3 \selectlanguage{ngerman}± 0.8\textsuperscript{a} & 49.3 \selectlanguage{ngerman}±
0.8\textsuperscript{c}\tabularnewline
CP \emph{A. niger} & 25.9 \selectlanguage{ngerman}± 1.9\textsuperscript{f} & 5.9 \selectlanguage{ngerman}±
0.9\textsuperscript{d} & 47.5 \selectlanguage{ngerman}± 2.9\textsuperscript{b} & 21.3 \selectlanguage{ngerman}±
1.6\textsuperscript{e} & 4.5 \selectlanguage{ngerman}± 0.2\textsuperscript{d} & 37.2 \selectlanguage{ngerman}±
0.1\textsuperscript{e}\tabularnewline
CP \emph{A. oryzae} & 72.7 \selectlanguage{ngerman}± 1.2\textsuperscript{d} & 7.5 \selectlanguage{ngerman}±
0.5\textsuperscript{cd} & 25.6 \selectlanguage{ngerman}± 0.9\textsuperscript{d} & 83.4 \selectlanguage{ngerman}±
0.2\textsuperscript{b} & 6.6 \selectlanguage{ngerman}± 0.4\textsuperscript{c} & 90.7 \selectlanguage{ngerman}±
3.8\textsuperscript{a}\tabularnewline
HE control & 81.4 \selectlanguage{ngerman}± 0.9\textsuperscript{b} & 2.0 \selectlanguage{ngerman}±
0.9\textsuperscript{e} & 70.0 \selectlanguage{ngerman}± 1.4\textsuperscript{a} & 94.6 \selectlanguage{ngerman}±
1.7\textsuperscript{a} & 29.4 \selectlanguage{ngerman}± 2.3\textsuperscript{b} & 43.2 \selectlanguage{ngerman}±
1.1\textsuperscript{d}\tabularnewline
HE \emph{A. niger} & 10.1 \selectlanguage{ngerman}± 1.0\textsuperscript{g} & 11.8 \selectlanguage{ngerman}±
1.0\textsuperscript{c} & 70.0 \selectlanguage{ngerman}± 1.1\textsuperscript{a} & 43.8 \selectlanguage{ngerman}±
0.9\textsuperscript{d} & 5.9 \selectlanguage{ngerman}± 0.4\textsuperscript{c} & 48.5 \selectlanguage{ngerman}±
1.6\textsuperscript{c}\tabularnewline
HE \emph{A. oryzae} & 53.0 \selectlanguage{ngerman}± 1.1\textsuperscript{e} & 5.8 \selectlanguage{ngerman}±
0.4\textsuperscript{d} & 38.5 \selectlanguage{ngerman}± 3.2\textsuperscript{c} & 49.8 \selectlanguage{ngerman}±
0.7\textsuperscript{c} & 6.5 \selectlanguage{ngerman}± 0.3\textsuperscript{c} & 55.6 \selectlanguage{ngerman}±
2.7\textsuperscript{b}\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{ngerman}

Notes:

*DCP control: canola protein control prepared from defatted unfermented
CP meal using AE-IP or SE. Data was reported as mean ± one standard
deviation (n=3). Significant difference exists between data with
different letters across each pH (p\textless{}0.05).

\textbf{Table 3 Water (WHC) and oil holding capacity (OHC) (g/g) of
protein products obtained by alkaline extraction-isoelectric
precipitation (AE-IP) (a) and salt extraction-dialysis (SE) (b) from
fermented and unfermented (control) canola meals.}

\textbf{a) AE-IP}\selectlanguage{english}
\begin{longtable}[]{@{}llllllll@{}}
\toprule
& \textbf{CP} & \textbf{CP} & \textbf{CP} & \textbf{CP} & \textbf{HE} &
\textbf{HE} & \textbf{HE}\tabularnewline
\midrule
\endhead
& Unfermented & Unfermented & Fermented & Fermented & Unfermented &
Fermented & Fermented\tabularnewline
& No defatting & With defatting* & \emph{A. niger} & \emph{A. oryzae} &
Control & \emph{A. niger} & \emph{A. oryzae}\tabularnewline
\textbf{WHC} & 2.1 \selectlanguage{ngerman}± 0.0\textsuperscript{d} & 2.7 \selectlanguage{ngerman}±
0.1\textsuperscript{c} & 3.1 \selectlanguage{ngerman}± 0.1\textsuperscript{ab} & 3.0 \selectlanguage{ngerman}±
0.0\textsuperscript{b} & 1.8 \selectlanguage{ngerman}± 0.0\textsuperscript{e} & 2.9 \selectlanguage{ngerman}±
0.1\textsuperscript{b} & 3.2 \selectlanguage{ngerman}± 0.0\textsuperscript{a}\tabularnewline
\textbf{OHC} & 2.9 \selectlanguage{ngerman}± 0.0\textsuperscript{b} & 2.4 \selectlanguage{ngerman}±
0.1\textsuperscript{c} & 2.8 \selectlanguage{ngerman}± 0.1\textsuperscript{b} & 2.8 \selectlanguage{ngerman}±
0.1\textsuperscript{b} & 2.3 \selectlanguage{ngerman}± 0.1\textsuperscript{c} & 3.1 \selectlanguage{ngerman}±
0.1\textsuperscript{ab} & 3.3 \selectlanguage{ngerman}± 0.1\textsuperscript{a}\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{ngerman}

\textbf{b) SE}\selectlanguage{english}
\begin{longtable}[]{@{}llllllll@{}}
\toprule
& \textbf{CP} & \textbf{CP} & \textbf{CP} & \textbf{CP} & \textbf{HE} &
\textbf{HE} & \textbf{HE}\tabularnewline
\midrule
\endhead
& Unfermented & Unfermented & Fermented & Fermented & Unfermented &
Fermented & Fermented\tabularnewline
& No defatting & With defatting* & \emph{A. niger} & \emph{A. oryzae} &
Control & \emph{A. niger} & \emph{A. oryzae}\tabularnewline
\textbf{WHC} & 1.1 \selectlanguage{ngerman}± 0.1\textsuperscript{c} & 2.1 \selectlanguage{ngerman}±
0.1\textsuperscript{b} & 1.3 \selectlanguage{ngerman}± 0.1\textsuperscript{c} & 1.4 \selectlanguage{ngerman}±
0.1\textsuperscript{c} & 1.3 \selectlanguage{ngerman}± 0.1\textsuperscript{c} & 2.3 \selectlanguage{ngerman}±
0.1\textsuperscript{b} & 2.7 \selectlanguage{ngerman}± 0.0\textsuperscript{a}\tabularnewline
\textbf{OHC} & 3.2 \selectlanguage{ngerman}± 0.1\textsuperscript{d} & 3.9 \selectlanguage{ngerman}±
0.1\textsuperscript{c} & 4.6 \selectlanguage{ngerman}± 0.1\textsuperscript{a} & 4.7 \selectlanguage{ngerman}±
0.0\textsuperscript{a} & 2.4 \selectlanguage{ngerman}± 0.1\textsuperscript{e} & 4.5 \selectlanguage{ngerman}±
0.0\textsuperscript{b} & 3.9 \selectlanguage{ngerman}± 0.1\textsuperscript{c}\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{ngerman}

Notes:

*Defatted CP control: canola protein product extracted from defatted
cold-press meal using AE-IP or SE. Data was reported as mean ± one
standard deviation (n=3). Significant difference exists between data
with different letters in a row (p\textless{}0.05).

\textbf{Table 4 Emulsifying activity index (EAI, m\textsuperscript{2}/g)
and emulsion stability index (ESI, min) of protein products obtained by
alkaline extraction-isoelectric precipitation (AE-IP) (a) and salt
extraction-dialysis (SE) (b) of fermented and unfermented (control)
canola meals.}

\textbf{a) AE-IP}\selectlanguage{english}
\begin{longtable}[]{@{}lllllll@{}}
\toprule
& \textbf{EAI (m\textsuperscript{2}/g)} & \textbf{EAI
(m\textsuperscript{2}/g)} & \textbf{EAI (m\textsuperscript{2}/g)} &
\textbf{ESI (min)} & \textbf{ESI (min)} & \textbf{ESI
(min)}\tabularnewline
\midrule
\endhead
& \textbf{pH 3} & \textbf{pH 5} & \textbf{pH 7} & \textbf{pH 3} &
\textbf{pH 5} & \textbf{pH 7}\tabularnewline
CP control & 11.4 \selectlanguage{ngerman}± 0.9\textsuperscript{cd} & 4.1 \selectlanguage{ngerman}±
0.3\textsuperscript{a} & 11.7 \selectlanguage{ngerman}± 1.5\textsuperscript{c} & 4.5 \selectlanguage{ngerman}±
1.4\textsuperscript{ab} & 1.4 \selectlanguage{ngerman}± 0.1\textsuperscript{ab} & 1.2 \selectlanguage{ngerman}±
0.1\textsuperscript{b}\tabularnewline
DCP control* & 8.3 \selectlanguage{ngerman}± 0.6\textsuperscript{d} & 5.0 \selectlanguage{ngerman}±
0.4\textsuperscript{a} & 11.6 \selectlanguage{ngerman}± 1.7\textsuperscript{c} & 1.5 \selectlanguage{ngerman}±
0.1\textsuperscript{ab} & 1.6 \selectlanguage{ngerman}± 0.1\textsuperscript{ab} & 1.4 \selectlanguage{ngerman}±
0.2\textsuperscript{b}\tabularnewline
CP \emph{A. niger} & 13.8 \selectlanguage{ngerman}± 0.7\textsuperscript{c} & 2.1 \selectlanguage{ngerman}±
0.3\textsuperscript{bc} & 25.8 \selectlanguage{ngerman}± 1.9\textsuperscript{a} & 4.8 \selectlanguage{ngerman}±
2.9\textsuperscript{a} & 1.1 \selectlanguage{ngerman}± 0.1\textsuperscript{b} & 1.4 \selectlanguage{ngerman}±
0.2\textsuperscript{b}\tabularnewline
CP \emph{A. oryzae} & 21.1 \selectlanguage{ngerman}± 1.4\textsuperscript{a} & 5.5 \selectlanguage{ngerman}±
0.1\textsuperscript{a} & 27.6 \selectlanguage{ngerman}± 1.8\textsuperscript{a} & 1.8 \selectlanguage{ngerman}±
0.3\textsuperscript{ab} & 1.5 \selectlanguage{ngerman}± 0.1\textsuperscript{ab} & 1.6 \selectlanguage{ngerman}±
0.1\textsuperscript{ab}\tabularnewline
HE control & 13.2 \selectlanguage{ngerman}± 1.6\textsuperscript{c} & 3.0 \selectlanguage{ngerman}±
0.5\textsuperscript{b} & 18.3 \selectlanguage{ngerman}± 1.6\textsuperscript{b} & 3.1 \selectlanguage{ngerman}±
0.6\textsuperscript{ab} & 2.2 \selectlanguage{ngerman}± 0.7\textsuperscript{a} & 2.0 \selectlanguage{ngerman}±
0.3\textsuperscript{a}\tabularnewline
HE \emph{A. niger} & 8.5 \selectlanguage{ngerman}± 0.8\textsuperscript{d} & 1.3 \selectlanguage{ngerman}±
0.1\textsuperscript{c} & 18.0 \selectlanguage{ngerman}± 1.9\textsuperscript{b} & 1.6 \selectlanguage{ngerman}±
0.4\textsuperscript{ab} & 1.3 \selectlanguage{ngerman}± 0.2\textsuperscript{b} & 1.3 \selectlanguage{ngerman}±
0.2\textsuperscript{b}\tabularnewline
HE \emph{A. oryzae} & 17.6 \selectlanguage{ngerman}± 1.9\textsuperscript{b} & 4.8 \selectlanguage{ngerman}±
0.6\textsuperscript{a} & 25.5 \selectlanguage{ngerman}± 2.5\textsuperscript{a} & 1.2 \selectlanguage{ngerman}±
0.1\textsuperscript{b} & 1.3 \selectlanguage{ngerman}± 0.1\textsuperscript{b} & 1.2 \selectlanguage{ngerman}±
0.2\textsuperscript{b}\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{ngerman}

\textbf{b) SE}\selectlanguage{english}
\begin{longtable}[]{@{}lllllll@{}}
\toprule
& \textbf{EAI (m\textsuperscript{2}/g)} & \textbf{EAI
(m\textsuperscript{2}/g)} & \textbf{EAI (m\textsuperscript{2}/g)} &
\textbf{ESI (min)} & \textbf{ESI (min)} & \textbf{ESI
(min)}\tabularnewline
\midrule
\endhead
& \textbf{pH 3} & \textbf{pH 5} & \textbf{pH 7} & \textbf{pH 3} &
\textbf{pH 5} & \textbf{pH 7}\tabularnewline
CP control & 5.6 \selectlanguage{ngerman}± 1.1\textsuperscript{c} & 5.8 \selectlanguage{ngerman}±
0.5\textsuperscript{bc} & 12.9 \selectlanguage{ngerman}± 0.5\textsuperscript{b} & 1.7 \selectlanguage{ngerman}±
0.2\textsuperscript{b} & 1.2 \selectlanguage{ngerman}± 0.1\textsuperscript{d} & 3.4 \selectlanguage{ngerman}±
0.3\textsuperscript{a}\tabularnewline
DCP control* & 11.0 \selectlanguage{ngerman}± 0.4\textsuperscript{a} & 9.4 \selectlanguage{ngerman}±
0.2\textsuperscript{a} & 14.5 \selectlanguage{ngerman}± 0.4\textsuperscript{a} & 1.2 \selectlanguage{ngerman}±
0.0\textsuperscript{b} & 1.2 \selectlanguage{ngerman}± 0.0\textsuperscript{d} & 1.6 \selectlanguage{ngerman}±
0.1\textsuperscript{b}\tabularnewline
CP \emph{A. niger} & 8.5 \selectlanguage{ngerman}±0.5\textsuperscript{b} & 1.8
\selectlanguage{ngerman}±0.2\textsuperscript{d} & 12.2 \selectlanguage{ngerman}± 0.7\textsuperscript{b} & 4.2 \selectlanguage{ngerman}±
1.0\textsuperscript{a} & 2.8 \selectlanguage{ngerman}± 0.4\textsuperscript{a} & 1.8 \selectlanguage{ngerman}±
0.2\textsuperscript{b}\tabularnewline
CP \emph{A. oryzae} & 9.7 \selectlanguage{ngerman}± 0.4\textsuperscript{ab} & 4.6 \selectlanguage{ngerman}±
0.5\textsuperscript{c} & 14.9 \selectlanguage{ngerman}± 0.3\textsuperscript{a} & 1.2 \selectlanguage{ngerman}±
0.0\textsuperscript{b} & 1.2 \selectlanguage{ngerman}± 0.1\textsuperscript{d} & 1.7 \selectlanguage{ngerman}±
0.1\textsuperscript{b}\tabularnewline
HE control & 5.9 \selectlanguage{ngerman}± 0.5\textsuperscript{c} & 6.8 \selectlanguage{ngerman}± 0.4\textsuperscript{b}
& 13.9 \selectlanguage{ngerman}± 0.7\textsuperscript{ab} & 1.6 \selectlanguage{ngerman}± 0.2\textsuperscript{b} & 2.4 \selectlanguage{ngerman}±
0.3\textsuperscript{ab} & 4.3 \selectlanguage{ngerman}± 1.3\textsuperscript{a}\tabularnewline
HE \emph{A. niger} & 8.5 \selectlanguage{ngerman}± 0.5\textsuperscript{b} & 4.1 \selectlanguage{ngerman}±
0.6\textsuperscript{cd} & 11.4 \selectlanguage{ngerman}± 0.4\textsuperscript{b} & 1.3 \selectlanguage{ngerman}±
0.1\textsuperscript{b} & 1.9 \selectlanguage{ngerman}± 0.1\textsuperscript{bc} & 1.5 \selectlanguage{ngerman}±
0.1\textsuperscript{b}\tabularnewline
HE \emph{A. oryzae} & 11.3 \selectlanguage{ngerman}± 0.9\textsuperscript{a} & 3.0 \selectlanguage{ngerman}±
0.3\textsuperscript{d} & 7.6 \selectlanguage{ngerman}± 0.8\textsuperscript{c} & 1.2 \selectlanguage{ngerman}±
0.1\textsuperscript{b} & 1.4 \selectlanguage{ngerman}± 0.2\textsuperscript{cd} & 1.2 \selectlanguage{ngerman}±
0.0\textsuperscript{b}\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{ngerman}

Notes:

*DCP control: canola protein product extracted from defatted cold-press
meal using AE-IP or SE. Data was reported as mean ± one standard
deviation (n=3). Significant difference exists between data with
different letters within each pH (p\textless{}0.05).

\textbf{Table 5 Foaming capacity (\%) and foam stability (\%) of
alkaline extraction-isoelectric precipitation (AE-IP) (a) and
salt-extraction dialysis (SE) (b) canola concentrates and isolates from
different fermented and control canola meals.}

\textbf{a) AE-IP}\selectlanguage{english}
\begin{longtable}[]{@{}lllllll@{}}
\toprule
& \textbf{Foaming capacity (\%)} & \textbf{Foaming capacity (\%)} &
\textbf{Foaming capacity (\%)} & \textbf{Foam stability (\%)} &
\textbf{Foam stability (\%)} & \textbf{Foam stability
(\%)}\tabularnewline
\midrule
\endhead
& \textbf{pH 3} & \textbf{pH 5} & \textbf{pH 7} & \textbf{pH 3} &
\textbf{pH 5} & \textbf{pH 7}\tabularnewline
CP control & 224.4 \selectlanguage{ngerman}± 20.4\textsuperscript{c} & 241.1 \selectlanguage{ngerman}±
16.4\textsuperscript{a} & 165.0 \selectlanguage{ngerman}± 5.2\textsuperscript{bc} & 93.0 \selectlanguage{ngerman}±
2.3\textsuperscript{a} & 83.2 \selectlanguage{ngerman}± 5.5\textsuperscript{a} & 68.0 \selectlanguage{ngerman}±
1.4\textsuperscript{d}\tabularnewline
DCP control* & 306.7 \selectlanguage{ngerman}± 20.0\textsuperscript{a} & 131.1 \selectlanguage{ngerman}±
7.7\textsuperscript{b} & 243.8 \selectlanguage{ngerman}± 8.1\textsuperscript{a} & 87.0 \selectlanguage{ngerman}±
1.9\textsuperscript{b} & 32.3 \selectlanguage{ngerman}± 4.2\textsuperscript{b} & 78.9 \selectlanguage{ngerman}±
0.6\textsuperscript{abc}\tabularnewline
CP \emph{A. niger} & NF & NF & 230.7 \selectlanguage{ngerman}± 6.8\textsuperscript{a} & NF & NF
& 82.3 \selectlanguage{ngerman}± 3.1\textsuperscript{ab}\tabularnewline
CP \emph{A. oryzae} & 228.9 \selectlanguage{ngerman}± 3.8\textsuperscript{c} & NF & 180.0 \selectlanguage{ngerman}±
3.3\textsuperscript{b} & 33.0 \selectlanguage{ngerman}± 1.1\textsuperscript{c} & NF & 84.6 \selectlanguage{ngerman}±
4.0\textsuperscript{a}\tabularnewline
HE control & 244.4 \selectlanguage{ngerman}± 10.2\textsuperscript{bc} & NF & 154.4 \selectlanguage{ngerman}±
6.9\textsuperscript{cd} & 17.3 \selectlanguage{ngerman}± 1.6\textsuperscript{d} & NF & 75.2 \selectlanguage{ngerman}±
3.8\textsuperscript{bcd}\tabularnewline
HE \emph{A. niger} & NF & NF & 244.2 \selectlanguage{ngerman}± 12.4\textsuperscript{a} & NF & NF
& 72.0 \selectlanguage{ngerman}± 3.0\textsuperscript{cd}\tabularnewline
HE \emph{A. oryzae} & NF & NF & 136.7 \selectlanguage{ngerman}± 8.8\textsuperscript{d} & NF & NF
& 79.6 \selectlanguage{ngerman}± 3.0\textsuperscript{ab}\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{ngerman}

\textbf{b) SE}\selectlanguage{english}
\begin{longtable}[]{@{}lllllll@{}}
\toprule
& \textbf{Foaming capacity (\%)} & \textbf{Foaming capacity (\%)} &
\textbf{Foaming capacity (\%)} & \textbf{Foam stability (\%)} &
\textbf{Foam stability (\%)} & \textbf{Foam stability
(\%)}\tabularnewline
\midrule
\endhead
& \textbf{pH 3} & \textbf{pH 5} & \textbf{pH 7} & \textbf{pH 3} &
\textbf{pH 5} & \textbf{pH 7}\tabularnewline
CP control & 322.2 \selectlanguage{ngerman}± 7.7\textsuperscript{a} & 211.1 \selectlanguage{ngerman}±
20.4\textsuperscript{b} & 480.0 \selectlanguage{ngerman}± 11.5\textsuperscript{a} & 87.2 \selectlanguage{ngerman}±
1.5\textsuperscript{a} & 87.2 \selectlanguage{ngerman}± 4.0\textsuperscript{a} & 83.8 \selectlanguage{ngerman}±
0.7\textsuperscript{ab}\tabularnewline
DCP control* & 246.7 \selectlanguage{ngerman}± 17.6\textsuperscript{b} & 222.2 \selectlanguage{ngerman}±
7.7\textsuperscript{a} & 464.4 \selectlanguage{ngerman}± 20.4\textsuperscript{b} & 84.2 \selectlanguage{ngerman}±
0.4\textsuperscript{a} & 87.4 \selectlanguage{ngerman}± 4.2\textsuperscript{a} & 89.0 \selectlanguage{ngerman}±
2.0\textsuperscript{a}\tabularnewline
CP \emph{A. niger} & 196.5 \selectlanguage{ngerman}± 6.6\textsuperscript{c} & NF & 191.1 \selectlanguage{ngerman}±
13.9\textsuperscript{d} & 54.6 \selectlanguage{ngerman}± 0.1\textsuperscript{c} & NF & 88.4 \selectlanguage{ngerman}±
1.2\textsuperscript{a}\tabularnewline
CP \emph{A. oryzae} & 175.6 \selectlanguage{ngerman}± 10.2\textsuperscript{c} & NF & 153.3 \selectlanguage{ngerman}±
8.8\textsuperscript{e} & 51.0 \selectlanguage{ngerman}± 1.1\textsuperscript{c} & NF & 89.0 \selectlanguage{ngerman}±
4.3\textsuperscript{a}\tabularnewline
HE control & 266.7 \selectlanguage{ngerman}± 6.7\textsuperscript{b} & NF & 145.6 \selectlanguage{ngerman}±
10.2\textsuperscript{e} & 53.4 \selectlanguage{ngerman}± 4.9\textsuperscript{c} & NF & 77.2 \selectlanguage{ngerman}±
5.4\textsuperscript{b}\tabularnewline
HE \emph{A. niger} & 195.6 \selectlanguage{ngerman}± 13.9\textsuperscript{c} & NF & 306.7 \selectlanguage{ngerman}±
17.6\textsuperscript{c} & 51.8 \selectlanguage{ngerman}± 2.0\textsuperscript{c} & NF & 86.4 \selectlanguage{ngerman}±
4.6\textsuperscript{ab}\tabularnewline
HE \emph{A. oryzae} & 135.6 \selectlanguage{ngerman}± 10.2\textsuperscript{d} & NF & 155.6 \selectlanguage{ngerman}±
3.8\textsuperscript{de} & 51.3 \selectlanguage{ngerman}± 1.4\textsuperscript{d} & NF & 77.6 \selectlanguage{ngerman}±
2.9\textsuperscript{ab}\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{ngerman}

Notes:

*DCP control: canola protein product extracted from defatted cold-press
meal using AE-IP or SE. NF: no foam, or foam disappeared within 5 min.
Data was reported as mean ± one standard deviation (n=3). Significant
difference exists between data with different letters across each pH
(p\textless{}0.05).

\selectlanguage{english}
\FloatBarrier
\end{document}

