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\begin{document}

\title{Effect of solid-state fermentation on select antinutrients and protein
digestibility of cold-pressed and hexane-extracted canola meals}



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


\vspace{-1em}



  
  \date{May 21, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
In this study, the effects of solid-state fermentation (SSF), including
strain (\emph{Aspergillus niger} NRRL 334 and \emph{A. oryzae} NRRL
5590) and fermentation time (24, 48, and 72 h) on the nutritional value
of cold-pressed (CP) and hexane-extracted (HE) canola meals were
examined. SSF increased the protein content of both types of meals (from
\textasciitilde{}36 to \textasciitilde{}40\%) while reducing the oil
content of CP meals (from \textasciitilde{}12 to 9\%). There was a
significant reduction (\textasciitilde{}80\%) in the phytic acid content
of both types of meals after fermentation using either fungi. Overall,
fermented samples showed a decrease in the total phenolic content from
2.7-3.1 to \textasciitilde{}1.0 mg gallic acid equivalents (GAE)/g DM (a
\textasciitilde{}65\% reduction), of which specifically the HE meal
fermented with \emph{A. niger} sample had the greatest decrease from 3.1
to 0.6 mg GAE/g DM (\textasciitilde{}81\% reduction). Seventy-two hours
of fermentation decreased the \emph{in vitro} protein digestibility
(IVPD) of the meals. In contrast, a shorter fermentation time (24 h)
increased the IVPD as compared to the controls (from
\textasciitilde{}73\% to 77-81\%), with the exception of the CP meal
fermented with \emph{A. niger} which had decreased IVPD at all
fermentation times. Overall, the changes indicate that SSF using
\emph{A. niger} or \emph{A. oryzae} can be useful to positively modify
the composition of different canola meals and improve their nutritional
value by significantly increasing the protein content, decreasing the
levels of antinutrients, while only slightly reducing IVPD.%
\end{abstract}%



\sloppy


\textbf{Effect of solid-state fermentation on select antinutrients and
protein digestibility of cold-pressed and hexane-extracted canola meals}

Chenghao Li\textsuperscript{1}, Dai Shi\textsuperscript{1}, Andrea K.
Stone\textsuperscript{1}, YiXiao Wang\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: Nutrition of fermented canola meals

\textbf{Abstract}

In this study, the effects of solid-state fermentation (SSF), including
strain (\emph{Aspergillus niger} NRRL 334 and \emph{A. oryzae} NRRL
5590) and fermentation time (24, 48, and 72 h) on the nutritional value
of cold-pressed (CP) and hexane-extracted (HE) canola meals were
examined. SSF increased the protein content of both types of meals (from
\textasciitilde{}36 to \textasciitilde{}40\%) while reducing the oil
content of CP meals (from \textasciitilde{}12 to 9\%). There was a
significant reduction (\textasciitilde{}80\%) in the phytic acid content
of both types of meals after fermentation using either fungi. Overall,
fermented samples showed a decrease in the total phenolic content from
2.7-3.1 to \textasciitilde{}1.0 mg gallic acid equivalents (GAE)/g DM (a
\textasciitilde{}65\% reduction), of which specifically the HE meal
fermented with \emph{A. niger} sample had the greatest decrease from 3.1
to 0.6 mg GAE/g DM (\textasciitilde{}81\% reduction). Seventy-two hours
of fermentation decreased the \emph{in vitro} protein digestibility
(IVPD) of the meals. In contrast, a shorter fermentation time (24 h)
increased the IVPD as compared to the controls (from
\textasciitilde{}73\% to 77-81\%), with the exception of the CP meal
fermented with \emph{A. niger} which had decreased IVPD at all
fermentation times. Overall, the changes indicate that SSF using\emph{A.
niger} or \emph{A. oryzae} can be useful to positively modify the
composition of different canola meals and improve their nutritional
value by significantly increasing the protein content, decreasing the
levels of antinutrients, while only slightly reducing IVPD.

\textbf{KEYWORDS}

Antinutritional factors, \emph{Aspergillus} \emph{niger,
Aspergillusoryzae} , canola meal, protein digestibility, solid-state
fermentation

\textbf{1 INTRODUCTION}

Canola meal, leftover from oil extraction process is rich in protein
(\textasciitilde{}36-39\%) and total dietary fibre
(\textasciitilde{}33\%) and is typically used as animal feed. The meal
byproduct is prepared using a conventional process involving solvent
extraction (e.g., hexane extraction (HE), 60\selectlanguage{ngerman}°C) and a desolventization
step (110°C) (Kalaydzhiev et al., 2019), or a cold pressing (CP) process
involving mechanical crushing of seeds at low temperatures (50-60°C)
(Febrianto et al., 2011). The former achieves higher levels of oil
extraction, however, leaves the remaining meal protein severely heat
treated, whereas the latter achieves sub-optimal oil extraction but
leaves proteins of higher quality in the meal. In addition to having
different compositions and physicochemical properties, the two meal
types have been reported to vary in their protein digestibility
properties (Kasprzak et al., 2016).

In general, proteins of canola meal have a favorable pattern of
essential amino acids and are considered a suitable feedstock for
further processing for human consumption. However, food uses of canola
meal are limited due to naturally present compounds such as phytic acid,
sinapine, phenolic acids and polyphenols (Croat et al., 2017). These
compounds can lead to poor protein digestibility, interfere with mineral
absorption, and give undesirable colour and taste attributes (Wu \&
Muir, 2008). Phenolic compounds are the major antinutritional compounds
found in canola meals with levels of \textasciitilde{}5 mg gallic acid
equivalents (GAE)/g dry meal in common canola meals, which is almost
30-times higher than in soybean meals (Shahidi \& Naczk, 1992). Thus, it
is necessary to further process canola meals to minimize the levels of
or eliminate these phytochemicals to make the meals more suitable for
human consumption.

Solid-state fermentation (SSF) is a clean label process where there is
nearly no free water in the solid substrate, making it acceptable for
fungi growth. Recently, researchers have employed SSF of canola meals
with various fungal strains to achieve significant reductions in
glucosinolates (Shi et al., 2015), thiooxazolidones, phytic acid
(El-Batal \& Abdel, 2001), total phenolic compounds, and neutral
detergent fibre (NDF) (Pal Vig \& Walia, 2001) levels, with an
enhancement in the protein content. Furthermore, Shi et al. (2015) found
improved amino acid in-vitro digestibility after A. niger SSF on a
composite substrate containing rapeseed cake and wheat bran. Hyphae can
penetrate into structural matrices to loosen them and to allow higher
utilization of nutrients entrapped in the matrices by fungi. Canola
meals contain ligno-cellulosic materials and other hard-to-digest
matrices that are hardened by the heating process during oil extraction
of canola.

The direct comparison of the effect of SSF on the nutritional attributes
of differently processed meals (CP and HE) has not yet been
investigated. In this study, SSF with two types of fungal strains
(Aspergillus niger NRRL 334 and Aspergillus oryzae NRRL 5590, both have
generally recognized as safe (GRAS) status) was used to ferment CP and
HE extracted meals to different times to improve their nutritional
value. It was hypothesized that SSF would induce protein hydrolysis and
produce canola meals with increased protein content, decreased levels of
antinutritional compounds, and improved protein digestibility with
variations as a function of fungus and meal type.

\textbf{2 MATERIALS AND METHODS}

\textbf{2.1 Materials}

CP meal was obtained from Pleasant Valley Oil Mills (Clive, AB, Canada).
Commercial HE meal was gifted by Bunge Canada (Harobe, MB, Canada).
Fungal strains of \emph{Aspergillus niger} NRRL 334 and\emph{Aspergillus
oryzae} NRRL 5590 were of GRAS status and obtained from Agricultural
Research Service (ARS) Culture Collection (Northern Regional Research
Laboratory (NRRL), Peoria, IL, USA). All media, reagents, and chemicals
used were of analytical grade and obtained from Sigma-Aldrich Canada
Ltd. (Oakville, ON, Canada).

\textbf{2.2 Sample preparation}

\textbf{2.2.1 Preparation of fungi spore suspensions}

Both the \emph{A. niger} NRRL 334 and \emph{A. oryzae} NRRL 5590 were
incubated on potato dextrose agar (PDA) plates at 30\selectlanguage{english} for 7 days. Spore
suspensions were prepared using deionized water, followed by spore
counting using direct microscopy (Leica, Model 97 S6E, Wetzlar, Germany)
with a hemocytometer (Bright-Line, Horsham, PA, USA). Spore suspensions
were prepared before the inoculation.

\textbf{2.2.2 Solid-state fermentation}

The HE and CP canola meals (200 g of each) were fermented with \emph{A.
niger} NRRL 334 and \emph{A. oryzae} NRRL 5590 spore suspensions. Both
spore suspensions were standardized to a spore concentration of
10\textsuperscript{7} colony forming units (CFU) to apply on per gram of
meal and used as the starter culture for fermentation as described by
Croat et al\emph{.} (2016; 2017) and Shi et al. (2015). The canola meal,
spore suspension, and deionized water were mixed at speed 5 for 3 min
using a commercial stand mixer (Pro 600, KitchenAid, Benton Harbor, MI,
USA) before being spread out thinly (\textless{}1.5 cm) and evenly onto
a stainless-steel sheet pan. The fermentation was performed at a 50\%
(w/w) moisture content at 30\selectlanguage{english} over a 72-h period in an Isotemp incubator
(Fisher Scientific, Model 650D, Waltham, MA, USA). Mill-Q water was
added based on the weight loss each day to maintain the 50\% (w/v)
moisture content. Samples (\textasciitilde{}50 g) were collected on
random spots from each batch at the time of initial inoculation (0), 24,
48, and 72 h, freeze-dried (Benchtop Pro 8L ZL-105, SP Scientific,
Warminster, PA, USA) and saved for later analyses. All the meals were
frozen at --20degC to terminate the fermentation until further
freeze-drying. The dried powders were stored at 4\selectlanguage{english} until used in
testing.

\textbf{2.3 Physicochemical analysis}

\textbf{2.3.1 pH determination}

The pH value was determined for the fermented and unfermented meal
slurries using a pH meter. The slurries were prepared by mixing 1 g of
the dried meal and Milli-Q water at a 1:10 (w/v) ratio and allowed to
stir for 30 min at room temperature (21-23\selectlanguage{english}). Measurements were made in
triplicate on each of the replicate batches (n=3) and reported as mean +-
one standard deviation.

\textbf{2.3.2 Degree of protein hydrolysis}

The degree of protein hydrolysis was measured as the released free amino
groups using the 2,4,6-trinitrobenzene sulfonic acid (TNBS) method
according to Adler-Nissen (1979) and Jung et al. (2005). In brief, a
freeze-dried meal containing 1 g of protein (corrected by protein
content) was added to distilled water, followed by stirring for 30 min
at room temperature (21-23\selectlanguage{english}). The mixture was centrifuged (Sorvall RC-6
Plus centrifuge, Thermo Scientific, Asheville, NC, USA) at 6,000
x\emph{g} for 15 min at room temperature, and the supernatant was
collected. A 250-\selectlanguage{greek}μ\selectlanguage{english}L aliquot of the supernatant was added to 2 mL 20 mM
sodium phosphate buffer (pH 7.8), followed by the addition of 2 mL
0.01\% TNBS solution. After mixing using a vortex for 15 s, the mixture
was covered by aluminum foil and placed in a water bath at 50 for 1 h.
The reaction was stopped by adding 4 mL 0.1 N HCl, and the sample was
allowed to cool for 10 min. The absorbance of the reaction mixture was
measured at 340 nm using a spectrophotometer (Genesys 10S UV-VIS, Thermo
Scientific, Madison, WI, USA). The total protein hydrolysis achieved by
hydrolyzing proteins with acid was also measured as part of the degree
of hydrolysis calculations. The total protein hydrolysis was determined
by adding 24 mg of sample (corrected by protein content) to a screw cap
Pyrex tube containing 15 mL of 6.0 N HCl. The tubes were incubated at
room temperature for 20 h. The dispersion was adjusted to pH 7.0 with 2
M NaOH, followed by filtration through the Whatman Grade 3 filter paper
(Whatman International Ltd., Maidstone, UK). A 250 \selectlanguage{greek}µ\selectlanguage{english}L aliquot of the
above sample was then added to 2.0 mL 1\% SDS solution in buffer. This
was followed by the addition of 250 \selectlanguage{greek}µ\selectlanguage{english}L of the mixed solution to 2.0 mL
10 mM sodium phosphate buffer (pH 7.8). This mixed solution was
performed in triplicate and analyzed according to the procedures above.
The sample blank was prepared by adjusting a solution of 6.0 N NaOH and
6.0 N HCl to pH 7.0. A 1.5 mM glycine solution was used to create the
standard curve to calculate the
\(\alpha\)-NH\textsubscript{2}-glycine equivalents of each
sample for use in the following equations to calculate the degree of
protein hydrolysis:

\(h=\left(h_{t}-\ h_{c}\right)\ \times\ DF\) (1)

\(DH=\left(\frac{h}{h_{t}}\right)\ \times\ 100\%\) (2)

Where \(h\) is the net mM concentration
of\(\alpha\)-NH\textsubscript{2}-glycine equivalents,
\(h_{t}\) is the mM concentration of
\(\alpha\)-NH\textsubscript{2}-glycine equivalents at the time
of testing, \(h_{c}\) is the mM concentration
of\(\alpha\)-NH\textsubscript{2}-glycine equivalent at the time
of initial inoculation before the microorganism was added,
h\textsubscript{tot} is the mM concentration of
\(\alpha\)-NH\textsubscript{2}-glycine equivalents after total
acid hydrolysis, and DF is the dilution factor.
Measurements were made in triplicate on each of the replicate batches
(n=3) and reported as the mean \selectlanguage{ngerman}± one standard deviation.

\textbf{2.3.3 Proximate composition}

The crude protein content was determined according to AACC method
46-30.01 using a combustion nitrogen analyzer (FP-628 CAN, LECO Corp.,
Saint Joseph, MI, USA) with a nitrogen conversion factor of 6.25 (AACC,
2010). The content of ash was determined using 2 g of sample in a muffle
furnace at 600\selectlanguage{english} for a 2-h period according to AOAC method 943.05 (AOAC,
2005). The crude lipid was measured using the Goldfisch apparatus
according to AOAC method 920.39 (AOAC, 2005). Measurements were made in
triplicate on each of the replicate batches (n=3) and reported as the
mean +- one standard deviation.

\textbf{2.4. Antinutritional components: Phytic acid and total phenolic
content}

The phytic acid content (PAC) was determined using the phytic acid assay
kit (Megazyme Inc., Sydney, Australia). All measurements were done in
triplicate (n=3) and reported as the percentage of phytic acid on a dry
weight basis (d.b.).

The total phenolic content (TPC) was determined using the
Folin-Ciocalteu method according to Olukomaiya et al. (2020) with some
modifications. In brief, 0.5 g of sample was extracted using 5 mL 80\%
(v/v) methanol for 15 min (stirring at 500 rpm and room temperature).
The supernatant was collected after centrifugation at 5000 x \emph{g}
for 10 min and filtered with the Whatman No. 1 filter paper (Whatman
International Ltd., Maidstone, UK). The remaining pellet was extracted
again using 80\% (v/v) methanol, followed by centrifugation and
filtration under the same conditions to obtain an aqueous phenolic
extract for analysis. A 0.1 mL aliquot of the extract was mixed with 2.5
mL 10\% (v/v) Folin-Ciocalteu reagent. After a 2-min reaction time, 2.5
mL 7.5\% (w/w) sodium carbonate was immediately added to the sample
mixture and allowed to incubate in the dark for 30 min at room
temperature. The absorbance was measured at 750 nm using a
spectrophotometer (Genesys 10S UV-VIS, Thermo Scientific, Madison, WI,
USA). Gallic acid (reagent grade) was used as the standard. All
measurements were done with triplicate independent assays (n=3) and
reported as milligrams of gallic acid equivalents (GAE) per gram of dry
meal (mean +- standard deviation).

\textbf{2.5 \emph{In vitro} protein digestibility (IVPD)}

The IVPD of control and fermented meals was determined using the pH-drop
method according to Tinus et al. (2012). A three-enzyme assay containing
31 mg chymotrypsin (bovine pancreas \selectlanguage{english}[?] 40 units/mg protein), 16 mg
trypsin (porcine pancreas 13,000--20,000 BAEE units/mg protein), and 13
mg protease (\emph{Streptomyces griseus} \selectlanguage{english}[?] 15 units/mg solid) in 10 mL
of water was prepared (fresh daily), kept in a 37\selectlanguage{english} water bath, and
adjusted to pH 8.0 +- 0.05 with 0.1 M NaOH and HCl. The sample
suspensions were prepared by dispersing 62.5 +- 0.5 mg protein in 10 mL
water. The samples were stirred at 37\selectlanguage{english} for 1 h before pH adjustment to
pH 8.0 +- 0.05 with 0.1 M NaOH and HCl. The protein digestibility
measurements were done by adding 1 mL of the enzyme cocktail into the
sample suspensions and recording changes in pH every 30 s for a total of
10 min. The equation below was followed to calculate the IVPD of each
sample:

\(\text{IVPD\ }\left(\%\right)=\ 65.66+18.10\ \times\ \text{pH}_{10\ min}\)(3)

Where \(\text{pH}_{10\ min}\) is the changes in pH since the addition of the
enzyme solution to the end of the 10 min measuring period.

\textbf{2.6} \textbf{Statistical analysis}

The fermentation was made in triplicate using separate plates and spore
suspensions (n=3). A three-way analysis of variance (ANOVA) was used to
study the statistical differences between samples as a function of meal
type, fungal strains, and fermentation time with a significance level
of\emph{p} =0.05. A post-hoc Tukey's test was used to detect statistical
differences in fermentation time. All statistics were performed using
the SPSS Version 28.0 software (IBM Corp. NY, IL, USA).

\textbf{3 RESULTS AND DISCUSSION}

\textbf{3.1 pH, degree of protein hydrolysis, and proximate composition}

The HE and CP canola meals were fermented with\emph{A. niger} NRRL 334
and\emph{A. oryzae} NRRL 5590 over a 72-h period. After 48 h of
fermentation, significant growth of hyphae was observed, and spores
started to form on the surface after 72 h of fermentation as shown in
Figure 1. For pH upon fermentation (Table 1a), significant differences
(\emph{p} \textless{}0.001) between meal types (CP vs. HE) and among
fermentation time groups were observed. The pH for all fermented samples
increased with fermentation time, with the CP meals showing changes
after 48 h while changes occurred earlier (24 h) for HE meals. The HE
meals had an overall higher pH (\textasciitilde{}8.2) than CP meals
(\textasciitilde{}7.6) at the end of the fermentation period (72 h),
regardless of the strains used. In contrast to the result of the present
study, Olukomaiya et al. (2020) found a reduction in the pH value from
6.26 to 6.13 after SSF for 7 days using \emph{A. ficuum} , \emph{A.
sojae} , and co-culture \emph{Lactobacillus salivarius} on autoclaved,
solvent-extracted canola meal. The authors described this to be caused
by an increase in organic acid content during SSF, especially when
using\emph{L. salivarius} . Aljubori et al. (2017) also reported
relatively stable or decreased pH values when using \emph{L. salivarius}
. The differences in results may be explained by the variations in
canola meals, fungi strains, and fermentation conditions used in these
studies in comparison to the present study. In addition, the organic
acids might have been used as nutrients by \emph{A. niger} and \emph{A.
oryzae}during SSF, leading to increases in pH.

The protein hydrolysis, measured as the degree of hydrolysis (DH),
increased throughout the fermentation period (Table 1b). Meal type,
strain used, fermentation time, and their interactions were all
significant factors (\emph{p} \textless{}0.001) affecting DH. The DH of
the meals increased from zero (0 h) to 15-30\% (24 h), then to
\textasciitilde{}40\% (48 h), and finally to 45-50\% after 72 h. For the
first two days (0-48 h), the DH increased quite rapidly due to the large
surface area that was exposed to oxygen, which helped with hyphae
growth. The proteins were most likely hydrolyzed partially by the
proteases synthesized by fungal cells, resulting in increased DH with
the release of free amino acids. The relatively compact nature of the
lower layers of the meal significantly lowered the speed of growth as
fungi could not penetrate easily to the bottom of the solid substrate,
hence the lower rate of DH increase at 72 h. The increase in DH was
slower for HE meals (15.8-23.4\%) as compared to CP meals (28.3-30.1\%)
on the first day (24 h) of fermentation, with the strain \emph{A.
oryzae} being less efficient at protein hydrolysis, particularly in the
HE meal. At the end of the fermentation cycle, the DH was similar for CP
meals and the HE meal fermented by \emph{A. oryzae} (45.1-46.4\%), while
that of the \emph{A. niger} HE meal was higher (52.3\%).

The changes to the proximate composition were similar for CP and HE
meals fermented with both strains. For protein, lipid and ash contents,
significant impacts (\emph{p} \textless{}0.001) from meal type and
fermentation time were observed. As shown in Table 2a, the crude protein
level increased from \textasciitilde{}34 to \textasciitilde{}37\% (a
9-11\% increase) after 72 h of fermentation in the CP meals, regardless
of the strain used. The protein levels were higher in the HE meals,
ranging from 39.7-41.5\%, and remained relatively constant upon
fermentation (though still increased by \emph{A. niger} ). In contrast
to protein, the crude lipid levels decreased from \textasciitilde{}12.5
to \textasciitilde{}9\% for CP meals, whereas the contents in the HE
meals decreased from \textasciitilde{}2.5 to \textasciitilde{}1\% (Table
2b). There was a small increase in the ash content for CP meals from
\textasciitilde{}6 to \textasciitilde{}7\% after 72 h of fermentation,
while that of HE meals increased from \textasciitilde{}8 to
\textasciitilde{}10\% (Table 2c). Compared to HE meals, CP meals had a
lower initial crude protein content due to the higher proportion of oil;
this trend remained even after a 72-h period of SSF. The high levels of
residual oil in the CP meals can be inhibitory to fungal growth and lead
to similar results found by Simon et al. (2017). They introduced and
compared several strains (\emph{Aureobasidium pullulans} NRRL-58522
3.0,\emph{Aureobasidium pullulans} NRRL-Y-2311-1, \emph{Trichoderma
reesei}NRRL 3653, \emph{Fusarium venenatum} NRRL 26139,
\emph{Paecilomyces variotti} NRRL 1115, \emph{Rhizopus microspores} var.
\emph{oligosporus}NRRL 2710, \emph{Neurospora crassa} NRRL 2332,
\emph{Mucor circinelloides} , and \emph{Pichia kudriavzeii} ) as SSF
cultures on HE and CP meals and concluded that fungal strains preferred
HE meals due to the lower level of residual oil.

The increase in the levels of crude protein and ash is attributed to the
effect of concentration. The carbohydrates, including sucrose and fibre,
acted as carbon sources for the fungi to grow, and carbon dioxide
(CO\textsubscript{2}) was produced as a result, concentrating the
remaining compounds (protein and ash). Wang et al\emph{.} (2012)
reported a 27\% increase in the crude protein content using composite
strains with \emph{Candida tropicalis} CICIM Y0079(T). The results
agreed with those of Plaipetch and Yakupitiyage (2014), who found a 9\%
increase in the crude protein content in \emph{Saccharomyces
cerevisiae}yeast-fermented canola meal. Simon et al. (2017) reported an
increase in crude protein from 42 to 44-50\% (d.b.) in fermented HE meal
and a \textasciitilde{}14\% increase in fermented CP meal, depending on
the strains. The increases in the nitrogen content of 11.6, 23.1, 34.2,
47.6, and 65.4\% after 1, 2, 5, 8 and 10 days of incubation,
respectively, were reported by Pal Vig and Walia (2001).

\textbf{3.2 Contents of phytic acid and total phenolic compounds}

The levels of PAC and TPC in the canola meals are presented in Table 3.
All fermented samples showed decreases in PAC and TPC. Significant
influences were observed only from fermentation time for PAC (\emph{p}
\textless{}0.001), whereas fermentation time, strain used, meal type x
strain, meal type x time, strain x time, and the three-way interaction
of meal type x strain x time were all statistical factors affecting TPC
(\emph{p} \textless{}0.05). The reduction of PAC from
\textasciitilde{}5.9 to \textasciitilde{}1.3\% (a 74-85\% reduction) was
achieved by phytase synthesized by \emph{A. niger} and \emph{A.
oryzae}during SSF. In contrast, decreases in TPC upon fermentation as
well as the corresponding increases have both been reported in the
literature as the enzymatic breakdown of polyphenol complexes (e.g.,
lignins, tannins, flavonoids) leads to the accumulation of smaller
units, and sometimes the formation of new bioactive compounds (Adebo \&
Medina-Meza, 2020). Depending on seed composition and structure (e.g.,
ease of cell wall breakdown), microbial strains employed, and the
choices of the assay (and standard) for TPC determination, fermentation
can bring different outcomes to the contents of ANF. All fermented
samples showed a decrease in TPC from 2.7-3.1 to 1.0 mg GAE/g DM (a
\textasciitilde{}65\% reduction) except for the HE meal fermented by
\emph{A. niger} . This sample had an even greater decrease in TPC from
3.1 to 0.6 mg GAE/g DM (a \textasciitilde{}81\% reduction). Overall, the
reduction in PAC and TPC was correlated with fungal growth, indicating
that the high residual oil in the CP meal was still suitable for
\emph{A. niger} NRRL 334 and\emph{A. oryzae} NRRL 5590 to grow without
affecting the reduction of ANF levels.

A lower reduction in PAC and TPC was reported by Olukomaiya et al.
(2020) where a \textasciitilde{}20\% reduction in PAC and a
\textasciitilde{}17\% reduction in TPC were achieved using \emph{A.
sojae} ATCC 9362 and \emph{A. ficuum} ATCC 66876 on solvent-extracted
(45\% moisture, 30\selectlanguage{english}) canola meal, even though their fermentation period
(7 days) was longer than in the current study. The initial values of PAC
and TPC in their meals were 27.06 mg/g DM and 9.31 mg GAE/g DM,
respectively, which were significantly lower and higher than those in
samples from the present study (\textasciitilde{}59 mg/g DM and
\textasciitilde{}2.9 mg GAE/g DM), respectively. Pal Vig and Walia
(2001) also reported a similar result that a maximum decrease in PAC (a
42.4\% reduction from the initial content of 29.3 mg/g DM) occurred
after a 10-day fermentation using \emph{Rhizopus oligosporus} with a
water:rapeseed meal ratio of 1:3 at 25\selectlanguage{english}. The degradation of ANF in
canola meals might require multiple enzymes. The lower reduction in the
ANF levels in those studies compared to the present study could be
explained by the lack of certain enzymes specific for degrading the
particular ANF or the insufficient level of enzymes produced when using
other strains. Cultivar differences in the starting materials (meals)
should also be considered as cultivar and growing environment can
greatly influence the composition and distribution of these compounds in
the seed and therefore meals (Bueckert et al., 2011).

\textbf{3.3 \emph{In vitro} protein digestibility}

The IVPD of control and fermented meals is presented in Table 4. Meal
type, strain used, fermentation time and all their interactions were
significant (\emph{p} \textless{}0.05) factors affecting IVPD. At time 0
h, HE meals and the CP meal fermented by \emph{A. oryzae} had similar
IVPD of \textasciitilde{}72.7\%, while that for CP by \emph{A. niger}was
much higher (80.2\%). The \emph{A. niger} fermented CP meal also
followed a different trend than the other three meals after 24 h of
fermentation as it had a decrease in IVPD (a \textasciitilde{}5.7\%
decrease) whereas the IVPD of all other meals increased (increases of
\textasciitilde{}4.8, 10.6 and 11.9\% for CP by \emph{A. oryzae} , and
HE by \emph{A. oryzae} and \emph{A. niger} , respectively). The three
aforementioned meals reached their maximum IVPD value after 24 h, after
which the values started decreasing as fermentation continued (towards
48 and 72 h). At the end of the fermentation period (72 h), all
fermented samples had similar IVPD values of \textasciitilde{}69.7\%,
statistically lower compared to the respective controls for CP meals and
the HE meal fermented by \emph{A. oryzae} , whereas that for HE
by\emph{A. niger} remained relatively unchanged. The most substantial
decrease was for \emph{A. niger} fermented CP meal (by
\textasciitilde{}12.8\%), while those for CP meal fermented with\emph{A.
oryzae} (by \textasciitilde{}3.6\%) and HE meal with \emph{A. niger} (by
\textasciitilde{}5.2\%) were less consequential.

Despite the rather substantial drop in ANF contents (TPC and PAC with
\textasciitilde{}65-85\% reductions, Table 3), the IVPD decreased
overall upon fermentation in the present study. Literature concerning
changes in protein digestibility of fermented canola meals is scarce.
Shi et al. (2015) reported a significantly enhanced amino acid \emph{in
vitro} digestibility of rapeseed meal (from China) after SSF
with\emph{A. niger} ; however, wheat bran was included with the meal as
a carbon substrate for fungal growth. Fermentation, in general, has been
reported to improve protein digestibility in various legume, cereal, and
oilseed samples. Employing a similar fermentation approach (SSF
by\emph{A. oryzae} and \emph{A. niger} ), Kumitch et al. (2020) reported
an increased IVPD of pea-protein enriched flour (from
\textasciitilde{}74.8 to \textasciitilde{}80.2\%) over the fermentation
period (6 h). Natural fermentation (16-20 h) using a yogurt culture
containing\emph{Lactobacillus delbrueckii} subsp. \emph{bulgaricus}
and\emph{Streptococcus thermophilus} improved the IVPD (a
\textasciitilde{}9.5\% increase) of desi chickpea flour; however, the
fermentation process did not alter the values for kabuli chickpea or
faba bean flours, according to Chandra-Hioe et al. (2016). An increased
IVPD of sorghum flours coupled with reduced phytic acid, trypsin
inhibitors, and tannins after traditional (natural) fermentation (24 h)
was observed by Osman (2004).

The improved protein digestibility upon fermentation in the
aforementioned studies is likely due to the breakdown of protein
molecular structures that enhanced the accessibility of digestive
enzymes, while the reduction/elimination of ANF also resulted in
weakened crosslinking activities, allowing more intensive proteolytic
attacks. However, depending on the nature of processing (e.g., types of
fermentation and strains utilized) and the starting material (e.g.,
structural differences among species and cultivars), such microbial
modification of matrix structures may also cause blockage in certain
passages and limit the function of digestive enzymes (Chandra-Hioe et
al., 2016; Emkani et al., 2022; Skalickova et al., 2022) which is
hypothesized to have occurred in the present study. It is interesting to
note that during the period where the IVPD of most samples initially
increased (0-24 h), the DH was also increasing at its fastest rate
(Table 1). We therefore hypothesize that within that time frame,
effective hydrolysis of large protein molecules into smaller subunits
might have overcome the influence of passage blockage of the digestive
enzymes and as such an overall positive effect on the protein
digestibility of the fermented meals was seen. The primary factor
influencing IVPD in the canola meals was not the presence of ANF in our
case but the specific actions of the selected fungal strains and the
sensitivity of sample materials to fungal fermentation using SSF.

\textbf{4 CONCLUSIONS}

SSF using \emph{A. niger} NRRL 334 and \emph{A. oryzae} NRRL 5590 was
applied on CP and HE canola meals to modify their nutritional value with
\textasciitilde{}50\% DH achieved after 72 h of fermentation. While HE
meal naturally contains more protein than CP meal, 72 h SSF with
\emph{A. niger} increased the protein content of CP meal to levels
comparable to unfermented HE meal. Similar fermentation conditions did
not result in the same increase in protein in HE meals. The SSF lowered
the lipid content of both meals. The levels of phytic acid and total
phenolics decreased significantly after fermentation with \% reduction
(after 72 h) being 74-85\% for PAC and 65-81\% for TPC in both CP and HE
meals. Enzymes from fungal cells, including lipase, proteinase and
phytase, played critical roles in changing meal composition. In terms of
fungal strain, \emph{A. niger}performed better to reduce PAC in CP meals
and TPC in HE meals. The effective reduction of PAC and TPC, as well as
similar DH values between meals, indicates that the high residual oil
content in CP meals did not interfere with the growth of the fungi. SSF
for 72 h brought decreases in IVPD to all meals except for HE by
\emph{A. niger} (unchanged) whereas 24 h fermentation improved IVPD for
both HE meals and the CP meal fermented with \emph{A. oryzae} . The
recommended fungal strain and fermentation time for canola meal will
depend on the desired outcome as increased protein content, decreased
ANFs, and increased IVPD could not all be achieved at the same time
under the conditions tested.

\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, Takuji Tanaka, and Michael T.
Nickerson were responsible for the design of the study, supervision of
the research, and revision of the manuscript drafts.

\textbf{CONFLICT OF INTEREST}

The authors declare that they have no conflict of interest.

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\textbf{LIST OF FIGURES}

\textbf{Fig. 1} Cold-pressed (CP) and hexane-extracted (HE) canola meals
fermented with \emph{Aspergillus niger} NRRL 334 and \emph{Aspergillus
oryzae} NRRL 5590

\textbf{Hosted file}

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