Industrial Crops and Products 61 (2014) 202–210

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Industrial Crops and Products

journal homepage: www.elsevier.com/locate/indcrop

Sequential proteolysis and cellulolytic hydrolysis of soybean hulls for

oligopeptides and ethanol production
Mayerlenis J. Rojas a , Paula F. Siqueira a,b , Liceres C. Miranda b , Paulo W. Tardioli a,c ,
Raquel L.C. Giordano a,c
a
Postgraduate Program of Chemical Engineering of the Federal University of São Carlos (PPGEQ/UFSCar), Rod. Washington Luiz, km 235, PO Box 676,
13565-905 São Carlos, SP, Brazil
b
IMCOPA—Imp. Exp. Ind. de Óleos S.A., Av. das Araucárias, 5899, 83707-000 Araucária, PR, Brazil
c
Department of Chemical Engineering, Federal University of São Carlos, Rod. Washington Luiz, km 235, PO Box 676, 13565-905 São Carlos, SP, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: The present study concerns the recovery of proteins from soybean hulls, mainly as oligopeptides, and the
Received 4 January 2014 production of ethanol from the remaining lignocellulosic fraction. These goals were achieved using the
Received in revised form 2 June 2014 following steps: hydrolysis of the soybean hulls by endoprotease Novo-ProD® , acid hydrolysis of the lig-
Accepted 2 July 2014
nocellulosic residue to remove hemicellulose, hydrolysis of cellulose by cellulolytic enzymes (Accellerase
Available online 22 July 2014
1500® ), and alcoholic fermentation of the saccharified liquor. The proteolysis of the soybean hulls resulted
in recovery of around 60% of the proteins as small peptides (86% with molar mass less than 6.5 kDa). The
Keywords:
acid pretreatment removed more than 60% of the hemicelluloses, allowing conversion of around 55% of
Soybean hulls
Proteolysis
the cellulose to glucose in the enzymatic hydrolysis. In the fermentation with Saccharomyces cerevisiae, a
Lignocellulosic biomass yield of around 90% was achieved, relative to the theoretical yield, representing an ethanol productivity
Cellulolytic hydrolysis of around 0.20 g/L/h for the integrated process. The results of this work indicate that soybean hulls might
Ethanol be a promising feedstock for the production of cellulosic ethanol and a high-value protein hydrolysate
composed mainly of low molecular weight oligopeptides.
© 2014 Elsevier B.V. All rights reserved.

1. Introduction
Global ethanol demand is growing continuously due to expan-
sion in the human population and industrialization (Saxena et al.,
2009). Currently, ethanol is mainly produced from corn and sug-
arcane (in the USA and Brazil, respectively), accounting for 66% of
worldwide production (Taherzadeh and Karimi, 2008). However, in
recent years, there has been increasing interest in cellulosic ethanol
production because lignocellulosic biomass is an abundant feed-
stock (the fourth largest primary energy resource in the world, after
coal, crude oil, and gas) that is inexpensive and has a high cellulosic
content (Mielenz, 2001; Saxena et al., 2009).
Lignocellulosic biomass mainly consists of cellulose, hemi-
celluloses, lignin, and small amounts of extractives (fatty acids,
waxes, terpenes, essential oils, aromatic compounds, and resid-
ual sucrose). The structure of cellulose allows the formation of
intramolecular and intermolecular hydrogen bonds, which gen-
erates organized rigid crystalline regions known as “elementary
microfibrils” (Delmer and Amor, 1995; Sjöström, 1993).
∗ Corresponding author. Tel.: +55 16 3351 8264; fax: +55 16 3351 8266.
E-mail addresses: pwtardioli@ufscar.br, pwtardioli@hotmail.com (P.W. Tardioli).
The main sources of this biomass are perennial energy crops
such as switch grass, crop residues (including sugarcane bagasse
and corn stover), and agricultural co-products (including soybean
hulls). Soybean hulls, which represent about 8% of the whole seed
and are the main byproduct of the soybean processing industry,
are obtained in the initial processing steps (Gnanasambandam and
Proctor, 1999). Brazil is the second largest soybean producer in the
world, with annual production of around 81.5 million tons (data
for the 2012/2013 harvest, CONAB, 2013), resulting in an estimated
generation of approximately 7.0 million tons of soybean hulls.
The chemical composition of soybean hull depends on the effi-
ciency of the dehulling process. If soybean meal with high protein
content is required, the dehulling process is more intense in order to
avoid contamination of the meal with pieces of hull. Consequently,
the soybean hulls may contain variable amounts of cellulose
(38–51%), hemicelluloses (20–25%), lignin (4–8%), pectins (4–8%),
proteins (11–15%), and minor extractives (Mielenz et al., 2009;
Yoo et al., 2011). Due to its composition, this biomass is widely
used as animal feed (Chee et al., 2005; Cole et al., 1999). However,
the low lignin content also makes it an attractive source of fer-
mentable sugars for cellulosic ethanol production (Karuppuchamy
and Muthukumarappan, 2009; Mielenz et al., 2009). In addi-
tion, the enzymatic hydrolysis of soybean hulls by endoproteases

http://dx.doi.org/10.1016/j.indcrop.2014.07.002
0926-6690/© 2014 Elsevier B.V. All rights reserved.
 M.J. Rojas et al. / Industrial Crops and Products 61 (2014) 202–210
can release oligopeptides that have beneficial nutritional proper-
ties for human and animal consumption (Nielsen, 1997). Protein
hydrolysates composed of oligopeptides can be used as nitrogen
fortification agents in specialist beverages, as pre-digested ingre-
dients for enteral/parenteral nutrition of nursing infants and sick
adults, and as ingredients in cell growth media, cosmetics, and
healthcare products (Gonzales-Tello et al., 1994; FitzGerald and
O’Cuinn, 2006; Sun, 2011). Soy protein hydrolysates are reported to
have several activities beneficial to human health, such as antioxi-
dant capacity (Darmawan et al., 2010; Park et al., 2010), anticancer
properties (Mora-Escobedo et al., 2009; Rayaprolu et al., 2013),
hypocholesterolemic effects (Yoshikawa et al., 2000), and anti-
inflammatory properties (Vernaza et al., 2012).
The production of cellulosic ethanol involves four steps: alka-
line and/or acid pretreatment, enzymatic hydrolysis, fermentation,
and distillation. The main purpose of the pretreatment is to disrupt
the natural lignocellulosic complex, reducing the degree of crys-
tallinity and releasing the amorphous fraction of the cellulose for
the enzymatic attack (Mosier et al., 2005; Taherzadeh and Karimi,
2008; Alvira et al., 2010).
Physicochemical pretreatments of soybean hulls for hemicellu-
lose removal have been reported previously. Cassales et al. (2011)
evaluated different acid concentrations in order to achieve high
sugar release and low generation of toxic compounds. Yoo et al.
(2011) investigated the pretreatment of soybean hulls by thermo-
mechanical extrusion. Karuppuchamy and Muthukumarappan
(2009) studied the effect of microwave irradiation during the
pretreatment of soybean hulls in order to improve enzymatic sac-
charification. Mielenz et al. (2009) obtained high yields of ethanol
by simultaneous saccharification and fermentation (SSF) of soy-
bean hulls, without pretreatment (due to the low lignin content).
However, the fermentation time was very long (around 9 days). It
was found that the removal of carbohydrates from soybean hulls
resulted in a 2.5-fold increase in the concentration of protein in
the biomass. Earlier work has already demonstrated the feasibility
of ethanol production from soybean hulls. However, to our knowl-
edge, no studies have been published concerning the recovery of
proteins from soybean hulls and subsequent ethanol production
from the deproteinized biomass. The present work describes a pro-
cess for the recovery of protein and the production of ethanol from
soybean hulls. The process consists firstly of submitting the soybean
hulls to the action of an endoprotease to solubilize the proteins
as oligopeptides, after which the hemicellulose is removed using
acid hydrolysis. The remaining lignocellulosic biomass is sacchari-
fied with commercial cellulases, and finally the sugars released are
fermented using Saccharomyces cerevisiae yeast.
2. Materials and methods
Materials
Soybean hulls were supplied by IMCOPA (Araucária, Brazil).
The endoprotease Novo-ProD® (subtilisin from B. licheniformis),
with a declared activity of 16 KNUPU-S/g, was supplied by
Novozymes® Latin America Ltda (Araucária, Brazil). Accellerase®
1500 from Trichoderma reesei was supplied by Genencor®
(Palo Alto, CA). Chymotrypsin, protein standards (albumin,
carbonic anhydrase, aprotinin, gastrin, and Ser-Gly-Gln-Ser-Trp-
Arg-Pro-Gln-Gly-Arg-Phe-NH2), arabinose, 5-methylfurfural (MF),
5-hydroxymethylfurfural (HMF), glucuronic acid, and galacturonic
acid were acquired from Sigma-Aldrich Co. (St. Louis, MO). Glucose
and xylose were acquired from Synth (São Paulo, Brazil). Saccha-
romyces cerevisiae (baker’s yeast, Fleischmann) was purchased in a
local market. All other chemicals (analytical grade) were purchased
from Synth (São Paulo, Brazil) and Quemis (São Paulo, Brazil).
203
Analytical methods
The protein content of the soybean hulls (pretreated and in
natura) was determined by the Kjeldahl method, using a Buchi
323 nitrogen analyzer. The total protein content was calculated
by multiplying the nitrogen content of the sample by 6.25. The
molar mass distribution of the hydrolyzed proteins was deter-
mined by SE-HPLC (size exclusion liquid chromatography), using
a Shimadzu LC-6A chromatograph equipped with a UV-visible
detector (Shimadzu SPD-10AV) set at 214 nm. The hydrolyzed
proteins were separated on an Amersham Biosciences Superdex
75 column (3000–70,000 Da), at room temperature, using 50 mM
sodium phosphate buffer with 0.15 M of NaCl (pH 7) as eluent, at a
flow rate of 0.5 mL/min. The injected sample volume was 25 ␮L.
Albumin (66,000 Da), carbonic anhydrase (29,000 Da), aprotinin
(6512.14 Da), gastrin (2859 Da), and Ser-Gly-Gln-Ser-Trp-Arg-Pro-
Gln-Gly-Arg-Phe-NH2 (1304 Da) were used as molecular mass
standards.
Ethanol and hydrolysis products (glucose, xylose, arabinose,
organic acids, etc.) were quantified by HPLC using a Shimadzu
SCL-10A chromatograph equipped with a refractive index detector
(RID 10-A). The compounds were separated on an HPX-87H col-
umn (300 × 7.8 mm, Bio-Rad, Hercules, CA), at 45 ◦ C, using 5 mM
sulfuric acid as the mobile phase, at a flow rate of 0.6 mL/min.
The hydrolyzed samples were pre-filtered through extraction car-
tridges (Sep-Pak C-18, Waters Corp., Milford, MA) to remove the
phenolic compounds. Degradation sugars (MF and HMF) were
also analyzed by HPLC, using a ␮Bondapack C-18 column (10 ␮m,
3.9 × 300 mm, Waters Corp., Milford, MA), at 25 ◦ C, and acetoni-
trile/water (1:8, v/v) with 1% acetic acid as the mobile phase, at a
flow rate of 0.8 mL/min.
Chemical characterization of soybean hulls
The chemical composition of in natura and pretreated soybean
hull (after proteolysis and chemical pretreatments) was evaluated
using the methodology described by Gouveia et al. (2009). Briefly,
the biomass was placed in a cellulose cartridge and extracted with
boiling 95% ethanol (using a biomass/solvent ratio of 1:100) for 8 h
in a Soxhlet system. One gram of soybean hull (dried to less than
10% moisture content) containing no extractives was hydrolyzed
for 7 min using 10 mL of 72% (v/v) sulfuric acid at 45 ◦ C. The concen-
tration of sulfuric acid was adjusted to 3% (v/v), and the hydrolysis
was performed at 121 ◦ C for 30 min. The carbohydrates, organic
acids, methylfurfural (MF), and hydroxymethylfurfural (HMF) were
determined by HPLC in order to estimate the contents of cellulose,
hemicellulose, and pectin. The acid-soluble lignin was determined
by UV spectrophotometry at 280 nm, and insoluble lignin was
determined from the difference in weight before and after drying
the biomass samples to a constant weight at 105 ◦ C. The ash frac-
tion was determined from the difference in weight before and after
incineration of the biomass samples in a muffle furnace at 400 ◦ C
for 1 h, followed by 800 ◦ C for 2 h.
The biomass samples were dried to less than 10% moisture con-
tent because excessive moisture would affect the selected acid
concentrations (NREL/TP-510-42618, 2014).
Proteolysis of in natura soybean hulls
The biomass was hydrolyzed with the Novo-ProD® endopro-
tease (1%, mass of enzyme per mass of protein in the soybean
hulls). The hydrolyses were performed for 5 h using a Titrino pH-
stat (Metrohm, Switzerland) equipped with a mechanical stirrer,
at controlled pH (9.0) and temperature (60 ◦ C). A solid/liquid ratio
of 1:10 was employed. After the hydrolysis, the remaining solid
fraction was filtered, dried at 60 ◦ C for 12 h, and used to evaluate
204 M.J. Rojas et al. / Industrial Crops and Products 61 (2014) 202–210

the mass yield (Eq. (1)) and the chemical composition (in terms Table 1
Chemical composition of soybean hulls.
of lignin, cellulose, hemicellulose, and protein). The liquid fraction
was used to determine the content of soluble protein and the size Component Composition (% m/m, dry basis)
distribution of the peptides.
This worka Mielenz et al. Cassales et al.
mfinal (2009) (2011)
= × 100 (1)
minitial Cellulose 35.8 ± 0.6 29–51 31
Hemicellulose 23.1 ± 0.4 10–20 26
where  is the mass yield, mfinal is the biomass mass (dry basis) Acid-soluble lignin 4.3 ± 0.3 2–8 5.7
after hydrolysis, and minitial is the biomass mass (dry basis) before Acid-insoluble lignin 4.8 ±1.0 Nd 3.4
hydrolysis. Extractives 5.0 ± 1.0 Nd 3.2
Ash 4.0 ± 0.3 1–4 0.6
Proteins 15.4 ± 0.6 9–14 13.1
Acid pretreatment for hemicellulose removal Pectins 4.2 ± 0.2 6–15
Total 96.6 ± 4.4

The soybean hulls were pretreated with dilute sulfuric acid (3%,
v/v) for removal of hemicellulose, using the experimental condi-
tions described by Fogel et al. (2005) for sugarcane bagasse. The
assays were performed in an autoclave at 120 ◦ C and 1 atm, for
40 min, using a solid/liquid ratio of 1:4. The solid and liquid frac-
tions were separated by vacuum filtration. The liquid fraction was
analyzed for soluble protein, hemicellulose (xylose, arabinose, and
glucose), and organic acids. The solid fraction was washed with
distilled water until neutral pH was reached, prior to chemical char-
acterization in terms of protein, lignin, hemicellulose, and cellulose.
The mass yield was calculated using Eq. (1).
Enzymatic saccharification
One gram of pretreated biomass (dry basis) was suspended in
10 mL of 50 mM sodium citrate buffer at pH 4.8 (a solid/liquid ratio
of 1:10 was used in all assays). Accellerase® 1500 was added (7 or
20 FPU/g of cellulose) and the suspension was stirred at 200 rpm
and 50 ◦ C in a shaker (MA-832, Marconi) for 72 h. Samples were
collected at 0, 3, 6, 24, 48, and 72 h for analysis of glucose in the
supernatant. At the end of the period, the suspension was cen-
trifuged at 2500 rpm and 4 ◦ C for 10 min, and the supernatant
(saccharified liquor) was stored for further use in the fermentation
process.
The enzymatic conversion of cellulose to glucose (xg ) was cal-
culated using the following equation:
Mg × 0.9
xg = × 100 (2)
Mi yi
where Mg is the mass of glucose in the supernatant, 0.9 is the
hydrolysis factor for conversion of glucose to cellulose, Mi is the ini-
tial mass of soybean hulls (dry basis), and yi is the cellulose content
(% mass fraction, dry basis) of the lignocellulosic biomass.
Fermentation
The saccharified liquor was employed in the alcoholic fermen-
tation with Saccharomyces cerevisiae yeast. Two grams (dry basis)
of yeast cells were reactivated into 200 mL of culture medium con-
taining the following nutrients: yeast extract (2 g/L), Na2 SO4 (1 g/L),
MgSO4 ·7H2 O (0.25 g/L), urea (2 g/L), CaCl2 ·2H2 O (0.5 g/L), K2 HPO4
(0.5 g/L), and glucose (20 g/L). The pH was adjusted to 4.8. The
medium was transferred to a 500 mL Erlenmeyer flask and stirred at
250 rpm and 30 ◦ C in a shaker (MA-830, Marconi) for 2 h. The yeast
cells were then recovered by vacuum filtration, and an appropriate
mass of cells was transferred to 15 mL of saccharified liquor, using a
ratio of 0.25 g (dry basis) of cells/g of glucose. The nutrient concen-
trations and fermentation conditions were the same as described
above. The assays were carried out in 50 mL conical flasks in order
to ensure anaerobic fermentation conditions, and the concentra-
tions of glucose and ethanol were monitored using HPLC analyses.
Samples of the supernatant were diluted and filtered using syringe
a
The values correspond to the average of three replicates ± standard deviation.
filters containing 0.2 ␮m pore size cellulose acetate membranes
(Sartorius, Germany), before injection into the chromatograph.
3. Results and discussion
3.1 Chemical composition of soybean hulls
The chemical composition of soybean hulls usually depends
on the type of grain processing, cultivation characteristics, and
genetic factors. Differences may therefore be observed for soybean
hulls obtained from different soybean processing industries. Table 1
shows the chemical composition of soybean hulls obtained from
IMCOPA (Araucária, Brazil).
The cellulose and hemicellulose contents (35.8 and 23.1%,
respectively) were within the ranges reported previously (Cassales
et al., 2011; Mielenz et al., 2009). The hemicellulose fraction con-
tained 18.3 ± 1.2% xylose and 5.5 ± 0.4% arabinose. These data show
that soybean hull is a promising biomass for ethanol production,
using either hexose or pentose fermentation. Furthermore, soybean
hulls have a lower lignin content (9.1%) than sugarcane bagasse
(21.7%), which is the main biomass studied in Brazil for ethanol pro-
duction (Carvalho et al., 2013). The low lignin content of soybean
hulls can enable enzymatic cellulose hydrolysis to proceed with-
out the need for alkaline pretreatment to remove lignin, avoiding
the presence of degradation products (aromatic compounds) that
could inhibit the yeast during the fermentation step (Larsson et al.,
1999; Ximenes et al., 2010).
The fraction of pectin (4.2%), the third most important car-
bohydrate found in plant cell walls, was close to the value
reported by Mielenz et al. (2009). This carbohydrate has many
industrial applications. Its isolation from soybean hulls and its
characterization have been the subject of several previous stud-
ies (Gnanasambandam and Proctor, 1999; Monsoor and Proctor,
2001).
Soybean hulls also have a high protein content (15.4%), which
differentiates this biomass from other materials such as wood and
sugarcane bagasse. The protein recovered could be used in applica-
tions including animal feed formulations and as a nitrogen source
in culture media (Cassales et al., 2011). Moreover, the recovery of
proteins as oligopeptides by using endoproteases can improve their
solubility and functional properties, and consequently extend the
range of their applications (Adler-Nissen, 1986; Nielsen, 1997).
Other components present in minor quantities, grouped as
extractives and ash (5 and 4%, respectively), showed compositions
comparable to those reported by Cassales et al. (2011).
3.2. Enzymatic solubilization of proteins from soybean hulls
Solubilization assays were performed under different condi-
tions to evaluate the removal of proteins from the soybean hulls.
 M.J. Rojas et al. / Industrial Crops and Products 61 (2014) 202–210 205

Table 2
Solubilization of soybean hull proteins in water at 60 ◦ C, expressed as the mass of protein in the liquid phase as a percentage of the maximum theoretical mass. Novo-ProD®
was added to the reaction medium at a concentration of 1% (m/m).

Solubilized protein (%, mass basis)

Assay 1: pH 9.0 during all assay with Assay 2: stabilization of pH, adjustment to 9.0 Assay 3: equal to Assay 2 but without
Novo-ProD® (1%, m/m) and addition of enzyme enzyme addition (control)

Stabilized pH 23.1 23.1

5.6
Initial pH 9.0 43.4 43.5
Final (5 h) pH 56.9 56.9 45.6
9.0

According to the manufacturer’s specifications, Novo-ProD® is an Table 3

Molar mass distribution of soybean hull proteins solubilized in water at 60 ◦ C, in the
alkaline endoprotease that under alkaline conditions can hydrolyze
presence or absence of Novo-ProD® (1%, m/m).
a fraction of the protein. A series of assays were performed with the
aim of separating the chemical and enzymatic effects. In Assay 1, Molar mass range Molar mass distribution (%)
soybean hulls were added to water at 60 ◦ C, the pH was immedi- Sample 1 Sample 2 Sample 3 Sample 4
ately adjusted to 9.0 with 5 M NaOH, and the enzyme was added.
MM > 67 kDa 0 7.0 10.7 1.9
In Assay 2, soybean hulls were added to water at 60 ◦ C and the sus- 67 KDa <MM < 29 kDa 2.3 3.4 3.8 1.5
pension was stirred until it reached a constant pH (5.6). The pH was 29 kDa < MM <6.5 kDa 4.5 7.8 10.4 10.4
then adjusted to 9.0 with 5 M NaOH and the enzyme was added. In 6.5 kDa < MM < 2.9 kDa 17.4 36.8 46.9 60.7
Assay 3, the conditions were the same as described for Assay 2, but 2.9 kDa < MM < 1.3 kDa 5.4 9.9 9.4 9.0
MM< 1.3 kDa 70.2 35.0 18.9 16.5
without enzyme addition (control). A reaction time of 5 h was used
for all assays. Sample 1: proteins solubilized in water until pH 5.6; sample 2: proteins solubilized at
pH 9.0; sample 3: proteins solubilized after 5 h without enzyme; sample 4: proteins
Table 2 shows the results of protein solubilization, expressed
solubilized after 5 h at pH 9.0 using the Novo-ProD® endoprotease.
as the mass of protein in the liquid phase as a percentage of the
maximum theoretical mass. Approximately 23% of the total pro-
tein content was solubilized in water at 60 ◦ C, which decreased the other hand, the enzymatic hydrolysis performed using the Novo-
pH to 5.6. It is likely that this protein fraction was already soluble, ProD® endoprotease was able to solubilize almost 60% of the total
because hydrolysis of proteins is not expected to occur under these protein, and a large fraction of the hydrolysate produced consisted
mild conditions. When the pH was increased to 9.0, the protein of small peptides (86% with molecular mass <6.5 kDa), with only
content in the liquid fraction increased to 43.4%. After 5 h (with- a very small fraction of large proteins (1.9% with molecular mass
out the endoprotease), the solubilized protein content reached >67 kDa). These findings suggest that the enzymatic hydrolysate
45.6%, indicating that there was hydrolysis of approximately 23% produced from soybean hull proteins may possess improved func-
of the protein, which became soluble. This behavior was expected, tional characteristics (such as high digestibility and hypoallergenic
because alkaline extraction is frequently used to solubilize proteins, properties). However, this possibility would need to be confirmed
although under more drastic conditions, such as incubation at pH by tests conducted in vivo.
12 using 3 M NaOH for 1 h (Connor et al., 1976; Saunders et al., Table 4 shows the mass yield () and the chemical composition
1975). The use of proteolytic hydrolysis led to an increase of 11.3% of the soybean hulls after each solubilization assay. The best con-
in the total amount of solubilized proteins, yielding 56.9% protein ditions for protein solubilization were provided by the enzymatic
recovery from the soybean hulls. Although the increase in the frac- hydrolysis with immediate adjustment of pH to 9.0, which resulted
tion extracted by the enzymatic route was only 11.3%, it is expected in no significant losses of the cellulose, hemicellulose, and lignin
that enzymatic hydrolysis not only increases solubilization of the contents. When the adjustment of the pH to 9.0 was performed only
proteins remaining in the soybean hull, but also greatly reduces after stabilization of the initial pH to 5.6 (30 min after immersion
the molecular mass of the proteins already present in the liquid, of the soybean hulls in water at 60 ◦ C), there were significant losses
hence improving the properties of the final product. The molar (∼10%) of cellulose and hemicellulose. According to Taherzadeh
mass distributions of the proteins from soybean hulls solubilized and Karimi (2008), lignocellulosic materials contain a crystalline
under different conditions are shown in Table 3. The protein frac- cellulose fraction that is more resistant to hydrolysis, together with
tion solubilized in pure water was composed of 70% small peptides an amorphous fraction that is easily hydrolyzed at acidic pH. Hence,
(<1.3 kDa), confirming the supposition that this fraction consisted if it were desired to obtain a protein hydrolysate with minimal
only of soluble proteins. However, this fraction corresponded to contaminants, the best experimental conditions would be those
only 23% of the total protein contained in the soybean hulls. On the described for Assay 1.

Table 4
Mass yield (, expressed in relation to initial mass) and chemical composition (%, mass basis) of the solid residue after solubilization of the soybean hull proteins.

a Protein Cellulose Hemicellulose Lignin

In natura soybean hulls 1.00 15.4 ± 0.6 35.8 ± 0.6 23.1 ± 0.6 9.1 ± 1.2
Solid residue after stabilization of pH to 5.6 (30 min) 0.79 9.9 ± 0.5 26.1 ± 1.8 13.6 ± 1.9 12.3 ± 1.5
Solid residue from Assay 2b 0.75 5.9 ± 0.8 22.5 ± 0.3 13.6 ± 0.5 5.4 ± 1.7
Solid residue from Assay 3c 0.68 6.3 ± 0.6 24.6 ± 0.7 11.3 ± 0.6 11.1 ± 1.2
Solid residue from Assay 1d 0.75 5.9 ± 0.8 32.4 ± 0.3 17.3 ± 0.3 7.2 ± 1.2
a
 was evaluated by Eq. (1).
b
Stabilization of pH to 5.6, adjustment to 9.0 and addition of the Novo-ProD® endoprotease (1%, m/m).
c
Equal to Assay 2 but without enzyme (control).
d
pH 9.0 during all assay with the Novo-ProD® endoprotease (1%, m/m).
206 M.J. Rojas et al. / Industrial Crops and Products 61 (2014) 202–210
Fig. 1. Chemical composition of in natura soybean hulls—biomass 1; solid residue
after hydrolysis using Novo-ProD® (2% m/m, 60 ◦ C, pH 9.0, 5 h)—biomass 2; and
after sequential hydrolysis using chymotrypsin (1% m/m, 55 ◦ C, pH 8.0, 5 h) and
Novo-ProD® (1% m/m, 60 ◦ C, pH 9.0, 5 h)—biomass 3. The calculated mass fractions
correspond to the in natura soybean hulls.
3.3. Enzymatic solubilization of proteins from soybean hull using
specific and non-specific proteases
It was reported previously (McDougall et al., 1996) that aromatic
residues (tyrosine and tryptophan) from soybean hull proteins can
form cross-links with phenolic compounds derived from cell walls.
If these linkages are broken, a larger fraction of proteins could be
solubilized.
Chymotrypsin is a highly specific endoprotease that preferen-
tially hydrolyzes peptide bonds involving hydrophobic residues
at the carboxyl side in the polypeptide chain, such as those of
tryptophan, tyrosine, and phenylalanine (Tardioli et al., 2003). Sol-
ubilization of the soybean hull proteins was performed using this
endoprotease at a concentration of 1% (m/m), employing the condi-
tions described by Tardioli et al. (2003) for the hydrolysis of proteins
from cheese whey (55 ◦ C and pH 8.0). Despite its specificity, the
enzyme was able to solubilize 44% of the soybean hull proteins
after 5 h. Subsequent hydrolysis using Novo-ProD® (1% m/m, 60 ◦ C,
pH 9.0, 5 h) increased protein solubilization to 77%. A similar result
(74%) was achieved using Novo-ProD® alone, at a higher concentra-
tion (2% m/m, 60 ◦ C, pH 9.0, 5 h). However, both routes led to losses
of approximately 10% in the cellulose and hemicellulose contents
(Fig. 1). It seems that the action of chymotrypsin not only released
the proteins, but also caused solubilization of the carbohydrates
adjacent to them in the cell wall. Therefore, these routes were not
suitable for the recovery of proteins free from contaminants.
3.4. Acid pretreatment for hemicellulose removal
The acid-catalyzed hydrolysis of glycoside and peptide bonds
has been widely reported (Dagnino et al., 2013; Saha et al., 2005;
Tsugita and Scheffler, 1982). Hemicellulose is a branched het-
eropolymer that is more susceptible to acid hydrolysis, compared
to cellulose (Taherzadeh and Karimi, 2008). Therefore, hydrolysis
of lignocellulosic biomass with dilute acid can be used to release
only hemicellulose, so that the remaining cellulose becomes more
accessible to attack by cellulolytic enzymes (Chen et al., 2012; Fogel
et al., 2005; Torget et al., 1991).
Acid pretreatment was applied to in natura and deproteinized
soybean hulls (in natura soybean hulls hydrolyzed by 1% and 2%
Fig. 2. Profiles of the conversion of cellulose to glucose at 50 ◦ C, pH 4.8, and 200 rpm,
using 7 FPU/g of cellulose. Soybean hull substrates: () in natura, (䊉) deproteinized,
() in natura pretreated with dilute acid, () deproteinized and pretreated with
dilute acid.
(m/m) Novo-ProD® ), using 3% (v/v) H2 SO4 at 120 ◦ C for 20 min. The
acid hydrolysis of the deproteinized biomass released more than
60% of the hemicellulose (Table 5), mainly as xylose and arabi-
nose, with very low amounts of degradation compounds such as
furfural and hydroxymethylfurfural (Table 6). Cassales et al. (2011)
reported a 67% release of xylose from soybean hulls, using 2.7%
(w/w) H2 SO4 at 153 ◦ C for 20 min, with amounts of furfural and
soluble lignin that were below toxic levels. In the present work,
even using a higher concentration of sulfuric acid, low amounts of
furfural, hydroxymethylfurfural, and soluble lignin were produced,
although compounds inhibitory to cellulolytic enzymes were prob-
ably released (Ximenes et al., 2010).
The acid hydrolysis of in natura soybean hulls released more
than 80% of the hemicellulose, but there were also significant losses
of cellulose. Acid hydrolysis released around 90% of the proteins
from the soybean hulls (Table 5). However, different to the proteo-
lysis, the hydrolysate contained high concentrations of free amino
acids (aspartic and glutamic acids, aspargine, glycine, and proline).
In addition, the hydrolysate was contaminated with hemicellu-
lose monomers (mainly xylose and arabinose). If it were desired to
obtain a protein hydrolysate composed of oligopeptides, in order
to improve osmotic properties (Moreno and Cuadrado, 1993), it
would be preferable to use enzymatic hydrolysis (rather than acid
hydrolysis).
3.5. Enzymatic hydrolysis of cellulose and fermentation of sugars
Fermentable sugars for ethanol production were produced by
cellulolytic hydrolysis of in natura and pretreated soybean hulls.
Fig. 2 shows that acid pretreatment was essential for the hydrolysis
of cellulose. Conversions of around 40% were achieved after 72 h for
in natura and deproteinized soybean hulls treated with dilute acid.
However, when in natura soybean hulls were used without acid
pretreatment, the maximum conversion was only 4% at 24 h, after
which the conversion decreased to around 1% at 72 h. It is likely
that the high protein content in the biomass promoted a Maillard
reaction that decreased the glucose concentration in the reaction
medium. Low conversion (∼15%) was also observed for soybean
hulls that were treated with endoprotease alone. However, in this
case, the lower conversion was due to the high content of hemi-
cellulose, which hindered access of the cellulolytic enzymes to the
 M.J. Rojas et al. / Industrial Crops and Products 61 (2014) 202–210 207

Table 5
Mass yield () and chemical composition (% mass) of the acid-pretreated soybean hulls (using 3% (v/v) sulfuric acid at 120 ◦ C for 40 min).

Acid-pretreated soybean hulls a Proteins Cellulose Hemicellulose Lignin Pectins

In natura (before pretreatment) 1.00 15.4 ± 0.6 35.8 ± 0.6 23.1 ± 0.4 9.1 ± 1.0 4.2 ± 0.2
In natura (after pretreatment) 0.40 1.6 ± 0.3 24.8 ± 2.8 3.7 ± 0.6 8.2 ± 1.1 0.1 ± 0.1
Deproteinized (1% Novo-ProD® ) 0.74 1.3 ± 0.1 35.8 ± 0.5 6.9 ± 0.2 8.2 ± 1.1 1.5 ± 0.1
Deproteinized (2% Novo-ProD® ) 0.68 2.1 ± 0.5 33.4 ± 0.6 8.8 ± 0.2 15.6 ± 0.3 0.3 ± 0.1
a
 was evaluated by Eq. (1).

Table 6
Chemical composition of the liquid phase of acid hydrolysates from in natura and deproteinized soybean hulls. Acid hydrolysis was performed for 40 min using 3% (v/v)
sulfuric acid at 120 ◦ C.

Acid-pretreated soybean hulls Xylose (g/L) Arabinose (g/L) HMF (g/L) MF (g/L) Protein (g/L)

Deproteinized with 1% (m/m) Novo-ProD® 2.62 1.13 0.01 0.06 0.88

Deproteinized with 2% (m/m) Novo-ProD® 1.65 1.25 0.02 0.07 0.96
In natura 2.82 1.00 0.01 0.12 2.47

cellulose. Pretreatment is necessary to break down the crystalline
structure of the lignocellulosic biomass and allow the cellulases to
attack the cellulose. The present findings are in agreement with pre-
vious work concerning the pretreatment of soybean hulls (Corredor
et al., 2008; Karuppuchamy and Muthukumarappan, 2009).
When the enzyme loading was increased from 7 to 20 FPU per
gram of cellulose, the conversion reached approximately 55% after
48–72 h (Fig. 3). The total hydrolysis of cellulose requires the syn-
ergistic action of three enzymes: endoglucanases that randomly
cleave internal ␤-glycosidic bonds, exoglucanases that release cel-
lobiose units from the ends of the chains, and ␤-glucosidases that
cleave cellobiose, releasing two glucose molecules (limiting step)
(Sun and Cheng, 2002; Teeri, 1997).
The hydrolysis medium was supplemented with ␤-glucosidase
(120 IU/g of cellulose) and pectinase (1%, m/m) in an attempt
to increase the cellulose conversion. Nevertheless, similar cellu-
lose conversion (49–55%) was achieved for all conditions (Fig. 4).
The commercial Accellerase 1500® enzyme already contained ␤-
glucosidase activity (450–775 pNPG U/g, according to DuPont,
2014) to ensure almost complete conversion of cellobiose to glu-
cose. Hence, the hydrolysis step should not be limited by the action
of this enzyme.
Conversions of cellulose to glucose ranging from 50 to 80% have
been reported previously (Puri et al., 2013; Modenbach and Nokes,
2013; Wang et al., 2012). The conversion depends on a variety
of factors, such as the source of the biomass, type of pretreat-
ment, conditions used for cellulolytic hydrolysis, and loadings of
solids and enzymes in the reactor, amongst others. The partial con-
version of cellulose to glucose obtained for soybean hulls in this
work (around 55%) could have been due to several effects. These
include the non-productive binding of enzymes (endoglucanases
and/or exoglucanases) with lignin (Ximenes et al., 2010; Bragatto
et al., 2013), which must be removed in order to enhance the
cellulolytic hydrolysis. In addition, cellulolytic enzymes (endoglu-
canases, exoglucanases, and beta-glucosidase) can be inhibited
by phenolic compounds such as vanillin, syringaldehyde, trans-
cinnamic acid, and hydroxybenzoic acid, which are present in the
plants and are released during the pretreatment and hydrolysis of
cellulosic biomass (Ximenes et al., 2010). These inhibitory phenols
must be removed from the pretreated cellulose in order to maxi-
mize enzyme activity and consequently increase the conversion of
cellulose to glucose. Besides these effects, inhibition of cellulolytic
enzymes by substrate and products, particle size effects, and trans-
fer resistance should be considered. Ximenes et al. (2010) reported

Fig. 3. Profiles of the conversion of cellulose to glucose at 50 ◦ C, pH 4.8, and 200 rpm,
using enzyme loads of (䊉) 7 FPU/g of cellulose and () 20 FPU/g of cellulose. The
substrate was soybean hull sequentially hydrolyzed with 1% (m/m) Novo-ProD®
and dilute acid.
Fig. 4. Profiles of the conversion of cellulose to glucose at 50 ◦ C, pH 4.8, and 200 rpm,
using () 20 FPU/g of cellulose. The medium was also supplemented with (䊉) ␤-
glucosidase at 120 IU/g of cellulose and 20 FPU/g of cellulose, and () ␤-glucosidase
(120 IU/g of cellulose) plus pectinase (1%, m/m). The substrate was soybean hull
sequentially hydrolyzed with 1% (m/m) Novo-ProD® and dilute acid.
208 M.J. Rojas et al. / Industrial Crops and Products 61 (2014) 202–210

that glucan hydrolysis is also inhibited by hemicellulose, pectin, and

soluble products from hydrolyzed xylan, including xylooligosac-
charides and xylose.
From the above discussion, it can be seen that for a cellulosic
ethanol process to be cost-effective, optimization of the integrated
process is crucial. The cellulolytic hydrolysis reactor should be
operated at a high solids concentration (≥20%, w/w) in order to
produce saccharified liquor with a glucose concentration of at least
8% (w/w). This can then be used to produce a fermented wine
with a titer (≥4%, w/w) that is sufficient for the ethanol distilla-
tion process to be energy efficient (Modenbach and Nokes, 2013).
The pretreatment should be able to provide effective removal of
lignin and hemicellulose from the biomass, and inhibition of the
cellulolytic enzymes must be avoided or minimized. Powerful enzy-
matic cocktails and higher enzyme dosages could compensate the
loss of activity caused by phenolic compounds, hydrolysis products
of hemicellulose, and starch inhibition, and lead to higher cellulose-
to-glucose conversions (Ximenes et al., 2010).
The liquor produced from soybean hulls pretreated with Novo-
ProD® (1%, m/m) and dilute sulfuric acid (3%, v/v), and saccharified Fig. 5. Fermentation profile (glucose consumption and ethanol production) for
with Accellarase 1500® (20 FPU/g of cellulose), was used in the saccharified liquor resulting from (a) soybean hulls pretreated by acid hydroly-
sis and cellulolytic hydrolysis (7 FPU/g cellulose, pH 4.8, 50 ◦ C), and (b) soybean
fermentation process with S. cerevisiae yeast. A maximum ethanol
hulls pretreated by proteolysis, acid hydrolysis, and cellulolytic hydrolysis (20 FPU/g
concentration of 12.8 g/L (Fig. 5) was achieved after 2 h, with a yield cellulose, pH 4.8, 50 ◦ C). Fermentation conditions: 30 ◦ C, pH 4.8, stirring at 250 rpm.
of 89.2% (compared to the theoretical yield). A similar ethanol con-
centration (13.4 g/L) and yield (∼93.7%) were achieved using the
saccharified liquor produced in the cellulolytic hydrolysis of the in Fig. 6 shows a mass balance for the integrated process used
natura soybean hulls pretreated with dilute acid alone. The results in this work, consisting of the following sequential steps: proteo-
showed that the removal of protein did not influence the pro- lysis (5 h, 60 ◦ C, pH 9.0, 1% (m/m) Novo-ProD® , solid/liquid ratio
duction of ethanol from the soybean hulls. Although the ethanol of 1:10), weak acid hydrolysis (40 min, 120 ◦ C, 3% (v/v) H2 SO4 ,
titer was too low to enable the distillation to be energy efficient solid/liquid ratio of 1:4), cellulolytic hydrolysis (48–72 h, 50 ◦ C, pH
(Ximenes et al., 2010; Modenbach and Nokes, 2013), strategies that 4.8, Accellerase® 1500 at 20 FPU/g of cellulose, solid/liquid ratio of
could be used to make the ethanol production economically feasible 1:10), and alcoholic fermentation (2 h, 30 ◦ C, pH 4.8, 0.25 g of yeast
include increasing the loading of cellulosic substrate and mixing the cells per gram of glucose). In the main streams of this process, every
cellulolytic hydrolysate with a sugar-rich stream from a first gener- 100 g of in natura soybean hulls produced 8.8 g of proteins, 8.5 g
ation ethanol plant (Wang et al., 2012; Puri et al., 2013; Pirota et al., of xylose, and 16.7 g of glucose. The glucose concentration of the
2014). In the case of the soybean hulls, the saccharified liquor could last stream was around 30 g/L, resulting in ethanol productivity for
be mixed with the soy molasses (which are rich in soluble carbo- the integrated process of around 0.20 g/L/h. Although the process
hydrates) generated during the concentration of the protein meal. adopted in this work enabled the production of two added-value
In Brazil, the company IMCOPA (Araucária, Paraná) produces com- products (oligopeptides and ethanol) from a feedstock currently
mercial soy molasses (Imcosoy) (IMCOPA, 2014), used in animal used as animal feed, the economically viable production of cellu-
nutrition and as source of carbohydrates for fermentation processes losic ethanol would require application of the biorefinery concept.
and biofuel. This is a set of processes whereby lignocellulosic biomass fractions

Fig. 6. Mass balance for an integrated process consisting of the following sequential steps: proteolysis (5 h, 60 ◦ C, pH 9.0, 1% (m/m) Novo-ProD® , solid/liquid ratio of 1:10),
weak acid hydrolysis (40 min, 120 ◦ C, 3% (v/v) H2 SO4 , solid/liquid ratio of 1:4), cellulolytic hydrolysis (48–72 h, 50 ◦ C, pH 4.8, Accellerase® 1500 at 20 FPU/g of cellulose,
solid/liquid ratio of 1:10), and alcoholic fermentation (2 h, 30 ◦ C, pH 4.8, 0.25 g of yeast cells per gram of glucose).
 M.J. Rojas et al. / Industrial Crops and Products 61 (2014) 202–210
can be used to produce energy, chemicals, and byproducts with
industrial applications (Bragatto et al., 2013). In order to achieve
this, it would be crucial to add value to the hemicellulose stream.
Xylan-rich hemicellulose heteropolymers could be used to produce
xylose, xylitol, ethanol, and xylooligosaccharides (XOS), which are
potential probiotic ingredients for functional foods offering ben-
efits to human health including reduction of cholesterol levels,
control of precancerous lesions, and improved biological calcium
availability (Bragatto et al., 2013).
4. Conclusions
Soybean hull is a rich source of proteins and cellulose (contents
of 15.4 and 35.8%, respectively, on a dry mass basis). Despite
having current commercial value as animal feed, this biomass
could be used for the production of high-value products, includ-
ing an important commodity (ethanol). The findings demonstrated
that enzymatic processes could be used to produce a protein
hydrolysate composed mainly of small peptides, which should have
improved nutritional properties, compared to products obtained in
the absence of enzymatic extraction. In addition, the deproteinized
biomass resulting from acid and enzymatic pretreatments could be
used for efficient production of ethanol.
Acknowledgements
The authors thank the Brazilian agencies ANP (PRH-44), CAPES,
and CNPq for financial support. We are also grateful to IMCOPA
(Imp. Exp. Ind. de Óleos S.A., Araucária, Brazil) for financial sup-
port and donation of soybean hulls, and the Postgraduate Program
in Chemical Engineering of the Federal University of São Carlos
(PPGEQ/UFSCar), where this work was undertaken.
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(accessed May 2014).