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. (g.lye@ucl.ac.uk)

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].

www.elsevier.com/locate/nbt 607

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

www.elsevier.com/locate/nbt 609

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).

References

[1] Gaor UN. Agenda 21: programme of action for sustainable development; 1992.[2] Kamm B, Kamm M. Principles of biorefineries. Appl Microbiol Biotechnol

2004;64:137–45. http://dx.doi.org/10.1007/s00253-003-1537-7.[3] Sanders J, Scott E, Weusthuis R, Mooibroek H. Bio-refinery as the bio-inspired

process to bulk chemicals. Macromol Biosci 2007;7:105–17. http://dx.doi.org/10.1002/mabi.200600223.

[4] Industrial Biotechnology Innovation and Growth Team. Maximising UK oppor-tunities from industrial biotechnology in a low carbon economy. Departmentfor Business Enterprise & Regulatory Reform; 2009.

[5] Paul Bond BSU. The industrial biotechnology innovation and growth team;2010, http://webarchive.nationalarchives.gov.uk/+/http:/www.berr.gov.uk/whatwedo/sectors/chemicals/ibigt/page44395.html (accessed 10.09.14).

[6] Sheldon RA. Green and sustainable manufacture of chemicals from biomass: stateof the art. Green Chem 2014;16:950–63. http://dx.doi.org/10.1039/C3GC41935E.

[7] Lammens TM, Franssen MCR, Scott EL, Sanders JPM. Availability of protein-derived amino acids as feedstock for the production of bio-based chemicals.Biomass Bioenergy 2012;44:168–81. http://dx.doi.org/10.1016/j.biombioe.2012.04.021.

New Biotechnology �Volume 32, Number 6 �December 2015 REVIEW

Review

[8] Alberici S, Toop G. Overview of UK biofuel producers. ECOFYS; 2014.[9] Department for Environment Food and Rural Affairs. Environmental and nutri-

tional benefits of bioethanol co-products; 2010.[10] Olukosi OA, Adebiyi AO. Chemical composition and prediction of amino acid

content of maize- and wheat-distillers’ dried grains with soluble. Anim Feed SciTechnol 2013;185:182–9. http://dx.doi.org/10.1016/j.anifeedsci.2013.08.003.

[11] Veraverbeke WS, Delcour JA. Wheat protein composition and properties ofwheat glutenin in relation to breadmaking functionality. Crit Rev Food Sci Nutr2002;42:179–208. http://dx.doi.org/10.1080/10408690290825510.

[12] Luo C, Griffin WB, Branlard G, McNeil DL. Comparison of low- and highmolecular-weight wheat glutenin allele effects on flour quality. Theor ApplGenet 2001;102:1088–98. http://dx.doi.org/10.1007/s001220000433.

[13] Swanston JS, Smith PL, Agu RC, Brosnan JM, Bringhurst TA, Jack FR. Variation,across environments within the UK, in grain protein and grain hardness, inwheat varieties of differing distilling quality. Field Crops Res 2012;127:146–52.http://dx.doi.org/10.1016/j.fcr.2011.11.016.

[14] Kindred DR, Smith TC, Sylvester-Bradley R, Ginsberg D, Dyer CJ. Optimisingnitrogen applications for wheat grown for the biofuels market. HGCA; 2007.

[15] Swanston JS, Newton AC. Mixtures of UK wheat as an efficient and environ-mentally friendly source for bioethanol. J Ind Ecol 2005;9:109–26. http://dx.doi.org/10.1162/1088198054821708.

[16] Dong FM, Rasco BA, Gazzaz SS. A protein quality assessment of wheat and corndistillers’ dried grains with solubles. Cereal Chem 1987;64:327–32.

[17] Han J, Liu K. Changes in composition and amino acid profile during dry grindethanol processing from corn and estimation of yeast contribution towardDDGS proteins. J Agric Food Chem 2010;58:3430–7. http://dx.doi.org/10.1021/jf9034833.

[18] Liu B, Thacker P, McKinnon J, Yu P. In-depth study of the protein molecularstructures of different types of dried distillers grains with solubles and theirrelationship to digestive characteristics. J Sci Food Agric 2013;93:1438–48.http://dx.doi.org/10.1002/jsfa.5912.

[19] Yu P, Nuez-Ortın WG. Relationship of protein molecular structure to metabolisableproteins in different types of dried distillers grains with solubles: a novel approach.Br J Nutr 2010;104:1429–37. http://dx.doi.org/10.1017/S0007114510002539.

[20] Lookhart G, Bean S. Separation and characterization of wheat protein fractionsby high-performance capillary. Electrophoresis 1995;72:527–32.

[21] Qian Y, Preston K, Krokhin O, Mellish J, Ens W. Characterization ofwheat gluten proteins by HPLC and MALDI TOF mass spectrometry. J Am

Soc Mass Spectrom 2008;19:1542–50. http://dx.doi.org/10.1016/j.jasms.2008.06.008.

[22] Zilic S, Barac M, Pesic M, Dodig D, Ignjatovic-Micic D. Characterization ofproteins from grain of different bread and durum wheat genotypes. Int J Mol Sci2011;12:5878–94. http://dx.doi.org/10.3390/ijms12095878.

[23] Cookman DJ, Glatz CE. Extraction of protein from distiller’s grain. BioresourTechnol 2009;100:2012–7. http://dx.doi.org/10.1016/j.biortech.2008.09.059.

[24] Shukla R, Cheryan M. Zein: the industrial protein from corn. Ind Crops Prod2001;13:171–92. http://dx.doi.org/10.1016/S0926-6690(00)00064-9.

[25] Dong Y, Li Q, Gao N, Chen X, Cheng Y, Lu J, et al. Optimization of proteinextraction from DDGS using response surface methodology and the propertiesof the protein. In: 2012 Fifth Int. Conf. Intell. Comput. Technol. Autom.,ICICTA. 2012. p. 242–5. http://dx.doi.org/10.1109/ICICTA.2012.67.

[26] Dong Y, Shi H, Chen X, Liu C, Liu L, Zhao N, et al. Artificial intelligence basedoptimization of the extracting process of protein from DDGS using alkalimethod. In: Zhu E, Sambath S, editors. Inf. Technol. Agric. Eng. Springer BerlinHeidelberg; 2012. p. 135–41.

[27] Sari YW, Alting AC, Floris R, Sanders JPM, Bruins ME. Glutamic acid productionfrom wheat by-products using enzymatic and acid hydrolysis. Biomass Bioe-nergy 2014;67:451–9. http://dx.doi.org/10.1016/j.biombioe.2014.05.018.

[28] Werpy T, Petersen G. Top value added chemicals from biomass. Results ofscreening for potential candidates from sugars and synthesis gas, vol. I. Golden,CO, USA: National Renewable Energy Lab; 2004.

[29] Tuck CO, Perez E, Horvath IT, Sheldon RA, Poliakoff M. Valorization of biomass:deriving more value from waste. Science 2012;337:695–9. http://dx.doi.org/10.1126/science.1218930.

[30] Scott E, Peter F, Sanders J. Biomass in the manufacture of industrial products –the use of proteins and amino acids. Appl Microbiol Biotechnol 2007;75:751–62.http://dx.doi.org/10.1007/s00253-007-0932-x.

[31] Gupta P, Nayak KK. Characteristics of protein-based biopolymer and its appli-cation. Polym Eng Sci 2014;1:14. http://dx.doi.org/10.1002/pen.23928.

[32] Pallos FM, Robertson GH, Pavlath AE, Orts WJ. Thermoformed wheat glutenbiopolymers. J Agric Food Chem 2006;54:349–52. http://dx.doi.org/10.1021/jf051035r.

[33] Zuo M, Song Y, Zheng Q. Preparation and properties of wheat gluten/methyl-cellulose binary blend film casting from aqueous ammonia: a comparison withcompression molded composites. J Food Eng 2009;91:415–22. http://dx.doi.org/10.1016/j.jfoodeng.2008.09.019.

www.elsevier.com/locate/nbt 611