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Review
REVIEW New Biotechnology �Volume 32, Number 6 �December 2015
The protein fraction from wheat-baseddried distiller’s grain with solubles (DDGS):extraction and valorizationM.F. Villegas-Torres, J.M. Ward and G.J. Lye
The Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, Gordon Street, WC1H 0AH London, UK
Nowadays there is worldwide interest in developing a sustainable economy where biobased chemicals
are the lead actors. Various potential feedstocks are available including glycerol, rapeseed meal and
municipal solid waste (MSW). For biorefinery applications the byproduct streams from distilleries and
bioethanol plants, such as wheat-based dried distiller’s grain with solubles (DDGS), are particularly
attractive, as they do not compete for land use. Wheat DDGS is rich in polymeric sugars, proteins and
oils, making it ideal as a current animal feed, but also a future substrate for the synthesis of fine and
commodity chemicals. This review focuses on the extraction and valorization of the protein fraction of
wheat DDGS as this has received comparatively little attention to date. Since wheat DDGS production is
expected to increase greatly in the near future, as a consequence of expansion of the bioethanol industry
in the UK, strategies to valorize the component fractions of DDGS are urgently needed.
Contents
Wheat proteins and varieties used in UK distilleries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 607
Wheat DDGS protein and its extraction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 608
Wheat DDGS protein and hydrolysate valorization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 609
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 610
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 610
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 610
Agenda 21 was established in 1992 and described an action plan
towards the consolidation of a green economy [1]. One of the
program goals was to increase the availability of renewable raw
materials and the development of biotechnological processes for
the production of sustainable biomass-based chemicals. These
could replace petroleum-based resources and/or expand the range
of accessible compounds at industrial scale [2]. To achieve this, it is
necessary to: (1) understand the various biomass feedstocks avail-
able for such purposes; (2) improve and develop biorefinery-basic
technologies for the fractionation of raw material; and (3) improve
and develop conversion methods of each fraction towards chemi-
cal production [2,3].
Corresponding author: Lye, G.J. ([email protected])
www.elsevier.com/locate/nbt
606 1871-6784/� 2015 The Authors. Published by Elsevier B.
In 2007 the UK Government established the Industrial Biotech-
nology Innovation and Growth Team (IB-IGT) to identify oppor-
tunities (and challenges) for future competitiveness in industrial
Biotechnology (IB). In 2009, their report ‘IB 2025: Maximising UK
Opportunities from Industrial Biotechnology in a Low Carbon Economy’
identified five crucial recommendations towards the consolida-
tion of IB in the UK. These included: to speedup IB knowledge and
innovation transfer; to attract and retain IB experts in science,
engineering and management; and to create a supportive envi-
ronment at both public and private level. The team also estimated
that up to £12 billion per year could be added to the UK economy
from IB innovation [4,5].
To date, several bio-based chemicals have been successfully pro-
duced at industrial scale from various raw materials, for example,
http://dx.doi.org/10.1016/j.nbt.2015.01.007
V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
New Biotechnology �Volume 32, Number 6 �December 2015 REVIEW
Review
biodiesel from plant oil and bioethanol from sucrose and starch.
However, the use of byproduct streams is of special interest as they
do not interfere with the debate on alternative land uses. Their
composition can be highly variable depending on the source, but
generally they are composed of oils, polysaccharides, lignin and/or
proteins. Due to the high content of polysaccharides and lignin
across different sources most of the recent research has focused on
valorization of these fractions [6]. In contrast, little attention has
been paid to the protein fraction, except for its nutritional value as
animal feed. Various byproduct streams that contain high quanti-
ties of protein, that is, >10% (w/w), come from the following
industries: (1) distilleries and first generation biofuel production,
that is, DDGS; (2) agriculture, such as stover, straw, leaves and hay;
and (3) oil and biodiesel production, that is, meals and seedcakes.
Their protein content can vary from as little as 3% (w/w) found in
maize stover, to 65% (w/w) present in jatropha seed meal [7].
Among the above feedstocks, wheat DDGS is of particular
importance to the UK after the recent establishment of two
wheat-bioethanol refineries; one by Ensus, established in 2010
with a maximum capacity of 400,000 m3 of bioethanol and
350,000 ton of DDGS per year, and one by Vivergo, established
in 2013 with a maximum production of 420,000 m3 of bioetha-
nol and 500,000 ton per year. Before 2010, there was an annual
production of 250 kt/annum of wheat DDGS from the UK
distillery industry. This will increase at least fourfold by the time
these two bioethanol plants are fully operational, and is expected
to rise even further if more companies build plants in the UK,
FIGURE 1
Wheat DDGS composition and options for valorization of the different fractions.
such as Vireol: planned for 2016 with a capacity of 200 million
liters per year [8,9]. This increased supply is expected to saturate
the animal feed market resulting in a lower value for this feed-
stock. The UK Government thus has an interest to identify
alternative uses and opportunities for valorization of wheat
DDGS.
Wheat DDGS is composed of polymeric sugars (cellulose and
hemicellulose), oils, protein and other materials for example, ash.
Their proportion varies according to the production process and the
wheat variety initially used, but on average is 46%, 5%, 38% and
11% (w/w), respectively [10]. Each fraction can be further valorized
(Fig. 1), yet despite the wide range of compounds that can be
obtained from it, the protein fraction has received limited attention
to date. This review therefore focuses on protein extraction and
valorization opportunities from wheat-based DDGS as a UK priority.
Wheat proteins and varieties used in UK distilleriesWheat protein is mainly composed of gluten (80–85% (w/w) of
total protein), while the remaining fraction comprises a heteroge-
neous group of globulins and albumins. The latter are soluble in
aqueous salt solutions, and in their monomeric forms are <25 kDa
except for a very small portion that can reach up to 70 kDa. In
contrast, gluten proteins are soluble in ethanol due to the high
content of non-polar and/or uncharged side chain amino acids.
When solubilized, two main fractions are recognized: (1) mono-
meric gliadins of around 30–80 kDa; and (2) glutenin subunits (GS)
that can vary between 80 and several million kDa [11].
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REVIEW New Biotechnology �Volume 32, Number 6 �December 2015
FIGURE 2
The amino acid sequence of a glutenin from wheat showing a biased amino acid composition towards glutamine (35% of the total amino acid sequence
highlighted in red), glycine (20% highlighted in green) and proline (13% highlighted in blue) The illustrated protein is from Triticum aestivum with the GenBank
accession number: CAA27052.1. This example is 838 amino acids in length.
Review
GS are more difficult to solubilize than gliadins as the various
subunits are bound through disulfide-bonds. For their solubiliza-
tion, a reducing pre-treatment is thus required. In solution, low
molecular weight (LMW-GS) and high molecular weight (HMW-
GS) subunits can be differentiated [11]. The weight ratio HMW-GS
to LMW-GS varies between 0.18 and 0.74, indicating that LMW-GS
are in a molar excess to HMW-GS, possibly due to the size differ-
ence and the number of coding genes within the genome [12].
There are more than 20 different HMW-GS varying among
wheat varieties. Within each variety, there are normally three to
five different HMW-GS expressed simultaneously. They are all
biased in their amino acid composition towards glutamine, pro-
line and glycine, which are present in repetitive sequences within
the central domain of the protein (Fig. 2) forming overlapping b-
reverse-turns. At the ends of this sequence, cysteine residues are
present to form disulfide bonds introducing some flexibility to the
central rigid structure. An example of a wheat glutenin is shown in
Fig. 2. This particular protein has 292 glutamine residues (35% of
the total amino acids in the protein), 164 glycine (20%), 107
proline (13%) and 46 tyrosine (5%) residues. In the case of
LMW-GS, 40 different variants have been identified and in one
single variant 7-16 different LMW-GS can be expressed simulta-
neously. LMW-GS is rich in glutamine and proline located in
repetitive sequences at the N-terminal, forming b-reverse turns;
and the non-repetitive sequences at the C-terminal forms an
608 www.elsevier.com/locate/nbt
a-helical secondary structure. Within this domain at least six
cysteine residues are found, which are responsible for the globular
structure of the C-terminal in comparison to the N-terminal [11].
In the UK different soft milling wheat varieties are used for
distilling: Beluga, Viscount, Zebedee, Invicta, Glasgow, Cassius,
Rodigus, Istabraq, Alchemy, Claire, Scout, and Warrior [13]. Their
main characteristics are their softness for milling and their low
protein content because of the inverse relationship between protein
and starch content in wheat seed [14]. On average the total protein
percentage in each variety is between 10 and 12% (w/w) (Source:
HGCA – Cereals and Oilseeds Division of the Agriculture and
Horticulture Development Board (AHDB)), but the percentage of
a particular type/group of proteins in each variety has not been
reported to date. In addition, wheat varieties are normally mixed for
bioethanol production [15], thus it is not possible to reliably predict
the distribution of different proteins in the resultant wheat DDGS.
Wheat DDGS protein and its extractionThe wheat DDGS protein fraction is not only composed of wheat
protein, namely gluten, globulins and albumins, but also of yeast
protein. This is because following the fermentation step the re-
covered DDGS is normally mixed, and dried, with the light con-
centrated slurry to improve its animal feed value. The contribution
of yeast protein to wheat DDGS is not well documented, but
previous studies have shown that wheat fermentation for the
New Biotechnology �Volume 32, Number 6 �December 2015 REVIEW
Review
production of bioethanol or beer does not affect the amino acid
composition of DDGS [16]. In the case of corn-based DDGS,
however, up to 20% (w/w) yeast protein has been reported in
the total protein content [17]. In the same study, an exhaustive
analysis of the amino acid distribution was performed throughout
corn processing including yeast, ground corn and corn DDGS. The
results indicated that some amino acids increased, others de-
creased and others remained unchanged, but that their individual
contribution is not higher than 2%. It thus appears that despite the
contribution of yeast protein, the overall amino acid distribution
is unaltered [17].
Wheat processing for beer/bioethanol and DDGS production
will influence protein degradability. This is due to enzymatic
modification during fermentation and/or the DDGS heat-drying
step during beer/bioethanol processing [18,19]. Since there is no
change in amino acid composition it can be expected that the
solubility characteristics of the wheat proteins are unaltered.
Consequently, alcohol-based solvents could be implemented to
solubilize the gluten, as well as a reduction step before extraction
to disrupt the disulfide bonds.
While there have been numerous reports on the processing of
milled seed for the (selective) extraction of protein, to the best
of our knowledge there have been none on the use of wheat DDGS
for such purposes [20–22]. In the case of the milled wheat seed
there is generally an initial step for albumin and globulin extrac-
tion carried out using a 0.5 M NaCl solution [22]. If albumin and
globulin are to be separated, then a pre-treatment step using
deionized water must be implemented to first extract the albumin
[20]. The gliadin fraction is then obtained using 70% (v/v) aqueous
ethanol, while the glutenin is extracted using 50% (v/v) 1-propa-
nol plus 1% (v/v) dithiothreitol (DTT) [20,22]. In a separate study a
single step extraction using 50% (v/v) 1-propanol mixed with 1%
(v/v) DTT at 608C was used to obtain a mixture of gliadins and
glutenins of different molecular weights [21].
A type of DDGS that has received some attention for protein
extraction, particularly in the United States [23], is maize-based
DDGS. In contrast to wheat, maize protein is composed of: zein,
glutelin, globulin and albumin. Among these four protein classes,
zein is the equivalent to wheat gliadin however it is deficient in
essential amino acids, with a bias towards glutamic acid, leucine,
proline and alanine. It is soluble in aqueous alcohol, urea, high pH
or anionic detergents. Zein, like gliadin, arranges in globular
structures linked together by disulfide bonds between different
zein peptides of various length, solubility and charge.
Zein has been extracted direct from maize since 1939, mainly
employing aqueous ethanol solutions between 50 and 90% (v/v).
Besides this method, pH variation and hydrolytic enzymes have
also been implemented in protein extraction from maize kernel.
pH values lower than 1, using HCl, or higher than 12, using NaOH,
are normally applied, but such pH variations can degrade the
protein. Proteases have proven to be an alternative method
[24], but enzyme costs limit the industrial application of this
approach. Analogous methods have been described when using
maize DDGS as feedstock for zein extraction. Some used NaOH
addition and elevated temperatures [25,26], while others
employed ethanol-based extraction with or without the addition
of NaOH after a reductive pre-treatment step. In addition, direct
enzyme extraction using a mixture of proteases has also been
performed [23]. In each case more than 70% (w/w) of the protein
has been solubilized.
Considering the above comparison, it is clear that gluten
extraction from wheat, and zein extraction from maize kernel,
or maize DDGS, are performed using comparable methodologies.
It is therefore reasonable to expect that protein fractionation from
wheat DDGS can be performed using equivalent methods to those
already published using maize and similar yields can be expected.
Thus, an average of 265 kton/year of extracted protein would be
initially available to produce high-value products (based on a
1000 kton/year of DDGS production from Ensus and Vivergo,
and a 70% protein extraction efficiency).
For protein extraction at large scale, as opposed to analytical
and laboratory scales, it will also be necessary to consider the
environmental impact of the process and its sustainability. This
will restrict the choice of chemicals used and the energy require-
ments of individual unit operations. The predicted increases in
wheat DDGS production in the UK suggest it is now imperative to
optimize these methods and also to consider options for valori-
zation of the protein fraction in this important biorefinery feed-
stock.
Wheat DDGS protein and hydrolysate valorizationUp to now, only one initial attempt to produce glutamic acid from
wheat protein, as a representation of wheat DDGS valorization,
has been reported in the literature [27]. Glutamic acid is consid-
ered one of the top twelve building block [28] chemicals as it can
be converted into diols, diacids and aminodiols, which are mono-
mers of polyesters and polyamides widely employed as polymers
[28]. The current production process of glutamic acid requires a
neutralization step with further purification and conversion from
the sodium salt to the free amino acid with high environmental
and financial costs [28]; thus, alternative production processes are
required. In the above-mentioned report, Sari et al. [27] tested
acid hydrolysis, enzymatic hydrolysis and their combination to
obtain glutamic acid. According to their results, the most sustain-
able method included enzymatic hydrolysis, using a mixture of
Validase FP Concentrate and Peptidase R, followed by a diluted
acid hydrolysis giving an 80% (w/w) yield. Consequently, this
work supports the idea of using wheat DDGS protein as feedstock
for the production of commodity and fine chemicals of industrial
relevance.
Besides glutamic acid, various amino acids have proven to be
useful in biocatalytic syntheses. For example: L-phenylalanine has
been converted to cinnamic acid, by a deamination step, and can
be further decarboxylated to synthesize styrene; likewise L-lysine
has been employed in the conversion of 1,5-diaminopentane,
caprolactam, and 5-amino valeric acid [29]. These studies suggest
that other amino acids, and not just glutamic acid and glutamine,
are of interest when designing wheat DDGS protein valorization
strategies.
Wheat DDGS protein presents a biased amino acid composition
(Fig. 2) where glutamine (residues in highlighted in red) is in the
highest proportion, followed by proline (residues in highlighted in
blue), leucine, aspartic acid, phenylalanine, valine, serine, argi-
nine, and glycine (residues in highlighted in green) [10] as previ-
ously mentioned. Among them, leucine, phenylalanine, and
valine are essential amino acids while the others are considered
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REVIEW New Biotechnology �Volume 32, Number 6 �December 2015
Review
non-essential. To maintain the feed value of wheat DDGS, the
non-essential amino acids can be further valorized, while the
essential ones should remain in the residual material [6,29].
Proline can be chemically transformed to pyrrolidone, a
precursor of N-vinylpyrrolidone, a 15,000 s/ton polymer used
in the cosmetic and medical sector. Aspartic acid can give rise to
acrylamide used in wastewater treatment [30], and to maleic and
fumaric acid employed in the food and paper industries in salt
form as chelating agents and sweeteners [28,30]. Serine, after an
a-decarboxylation step, produces ethanolamine used in several
industries with an annual demand of several hundred kilotonnes.
L-Arginine has been hydrolyzed and decarboxylated to 1,4-
diaminobutane, a 4,6-nylon precursor [29] while glycine can be
converted to oxalic acid used as bleaching agent in the textile and
pulp industries and in wastewater treatment [30]. Hence, there
would appear to be significant potential for the valorization of
non-essential amino acids present in wheat DDGS.
One of the main bioprocess limitations to achieving valoriza-
tion of all the non-essential amino acids is their separation after
hydrolysis. They can be separated by size, charge and hydrophobic
characteristics; differential reactivity with other chemicals produc-
ing compounds that are easier to separate [29]; or a process of
electrodialysis coupled to chemical or enzymatic conversion
which is currently under development. Nevertheless, further work
is required, as the above methods would not be cost-effective at
industrial scale at present [7].
An alternative strategy to valorize wheat DDGS protein without
prior hydrolysis and purification steps is to employ the protein
and/or extracted peptides in the manufacture of protein-based
biomaterials. Different proteins have been employed in the man-
ufacture of biopolymers such as wheat, zein, casein and soy. They
are biodegradable, can increase soil fertility, and are easily dispos-
able. As a result, they are important as packing materials, and can
also be employed in coating industries and agriculture. To date
they have been successfully employed in the synthesis of edible
food packing, but further work is required to improve film prop-
erties aimed at mimicking those of synthetic ones. This has proven
to be a major challenge due to limitations over the control of bond
formation and the quality of protein crosslinking [31].
Wheat biopolymers are characterized by their elastic behavior,
and are efficient barriers for oxygen, carbon dioxide and aromatic
compounds; however, their mechanical properties are not ideal.
Thermoformation [32], plasticizers [33], and UV crosslinking [31]
have been successfully applied in their manufacture. Material
elasticity is inversely correlated to the level of crosslinking trans-
lated into the number of S–S bonds present as demonstrated by
Pallos et al. [32]. In this study gluten was mixed with non-cereal
proteins that contained higher amounts of cysteine residues,
610 www.elsevier.com/locate/nbt
resulting in a material with reduced elasticity and increased
disulfide bonds. Mixtures including acids, saturated fatty acids,
or formaldehyde affect the biomaterial properties in terms of
elasticity and water vapor permeability [32]. An interesting bio-
polymer blend is the wheat gluten/methylcellulose film. This
particular mixture has shown improved properties at different
ranges depending on the proportion of each component: higher
moisture barrier, tensile strength and better water vapor perme-
ability [33].
To date, the use of wheat DDGS extracted protein in poly-
merization processes has not been reported. Nevertheless, its
potential use is clear, as it would be a cheap gluten source.
During protein extraction, the solubilization of other materials
such as lignin, cellulose, and hemicellulose would affect the
biomaterial properties, as it was previously reported in the case
of methylcellulose [33]. Additionally, the DDGS production
process and the extraction methods used can be expected to
have an impact on protein structure [11] and its subsequent
polymerization. Hence it may be worth investigating film
formation from wheat DDGS extracted protein, and how the
extraction conditions affect the mechanical properties of the
resulting biopolymer.
ConclusionsPrevious studies employing wheat DDGS protein have mainly
focused on analyzing its nutritional value as animal feed. Howev-
er, there is now growing interest in valorization of the protein
fraction into a range of potential products. To progress this re-
search, and to establish industrial processes for the manufacture of
protein-based chemicals and biomaterials, efficient protein extrac-
tion methods must be established. This review suggests that rapid
initial progress could be made by considering previous research on
protein extraction from maize DDGS. Once the protein is extracted
there appear to be numerous opportunities for its valorization
including the extraction of individual amino acids with the po-
tential to be transformed into building blocks for polymers or,
pharmaceuticals, or the solubilization of the whole or partially
hydrolyzed protein for use in the formation of wheat-based films.
In either case it will require the development of new chemical and
biological processing techniques that are the focus of our current
research.
AcknowledgementsThe authors would like to thank the UK Biotechnology and
Biological Sciences Research Council (BBSRC) and the Engineering
and Physical Sciences Research Council (EPSRC) for financial
support of this work as part of the Integrated Biorefining Research
and Technology Club grant (BB/J019445/1).
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