Downloaded from orbit.dtu.

dk on: thg 9 28, 2021

Robust processing of airborne laser scans to plant area density profiles

Arnqvist, Johan; Freier, Julia; Dellwik, Ebba

Published in:
Biogeosciences

Link to article, DOI:

10.5194/bg-17-5939-2020

Publication date:
2020

Document Version
Publisher's PDF, also known as Version of record

Link back to DTU Orbit

Citation (APA):
Arnqvist, J., Freier, J., & Dellwik, E. (2020). Robust processing of airborne laser scans to plant area density
profiles. Biogeosciences, 17(23), 5939-5952. https://doi.org/10.5194/bg-17-5939-2020

General rights
Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright
owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights.

 Users may download and print one copy of any publication from the public portal for the purpose of private study or research.
 You may not further distribute the material or use it for any profit-making activity or commercial gain
 You may freely distribute the URL identifying the publication in the public portal

If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately
and investigate your claim.
 Available online at www.sciencedirect.com
ScienceDirect
Enzymes in grain processing
Marie Sofie Møller and Birte Svensson
Enzymes are central in producing compounds from grains that
are desirable in modern food and beverages. Some of these
enzymes occur naturally in grains, while others are exogenous,
often of microbial origin, and either supplied as agents for
pretreatment and manufacture or introduced into the grains
using genetic techniques. Enzymes can add value by utilising
raw materials more efficiently, enhance the impact of food and
beverages on human health and nutrition, serve to eliminate
antinutrients, facilitate techno-functional performance and be
inspiration in pioneering handling of industrial waste from crop
grains. The review focuses on recent findings on grains, grain
fractions, flour and extracted grain components with emphasis
on starch, proteins and cell wall polysaccharides subjected to
industrially relevant enzyme-catalyzed processing.
Address
Enzyme and Protein Chemistry, Department of Biotechnology and
Bioengineering, Technical University of Denmark, Søltofts Plads Bldg.
224, DK 2800 Kgs. Lyngby, Denmark
Corresponding author: Svensson, Birte (bis@bio.dtu.dk)
Current Opinion in Food Science 2021, 37:153–159
This review comes from a themed issue on Food bioprocessing
Edited by Adriano Brandelli
https://doi.org/10.1016/j.cofs.2020.10.016
2214-7993/ã 2020 Elsevier Ltd. All rights reserved.
Introduction
The field of enzymes in grain processing is extremely
broad with regard to raw materials and concerned enzyme
catalyzed processes occurring either in the grain in situ or
on pretreated grains, grain fractions or extracted molecu-
lar constituents. The areas where the reaction products
are applied are also diverse spanning from food, health,
nutrition, beverages and animal feed to bioethanol,
energy and biomaterials. Additionally, a wide range of
relevant methods include analytical and techno-function-
ality assays, genetic modification and gene editing,
molecular structure/function investigations and protein
engineering. Mostly key hydrolytic, biosynthetic and
redox-active enzymes are targeted for brewing, baking,
and production of healthy compounds, for example, pre-
biotic oligosaccharides and phenolics. The present review
focuses on trends, recent progress and development in
www.sciencedirect.com
application of enzymes in grain processing related to food
science. Other currently important areas involving
enzyme treatment of grains are energy feedstock,
bioethanol [1,2], biomaterials and edible films [3], and
sustainable utilization of biomass [4]. Generally, the
targeted enzymes act on polysaccharides, proteins or
lipids, in manipulating and exploiting starch structure,
or are oxidoreductases useful in food and beverage
industries.
Enzymes active in grain processing can be exogenous,
commonly of microbial origin, or endogenous catalyzing
natural reactions during germination and sprouting
including enzymes from microbes thriving on plant hosts,
for example, fungal xylanases [5]. Overall, enzymes used
to advance areas of food, feed, beverages or nutrition can
i) mobilize or protect valuable compounds, for example,
dietary fibers (DF), ii) facilitate industrial processes, for
example, baking and brewing, or iii) detoxify gluten and
antinutrients, for example, phytate. The most common
substrate molecules include starch, cell wall polysacchar-
ides, lignin, and storage proteins. We shall describe
enzymes added for processing of grain components,
enzymes present in germinated and/or mature grains,
and enzymes introduced into grains using gene modifica-
tion technologies (see Figure 1 for an overview of areas
covered). Notably, grain crops have been transformed for
more than two decades, and recently gene edited to
encode key enzymes in biosynthetic or degradative pro-
cesses (for a review see Ref. [6]). Their actual use for food
and beverages still depends on legislation.
Exogenous enzymes for processing grains for
food
Grains or products derived thereof constitute a very
important part of human diet and form the basis of a
wide variety of foods providing caloric energy, DF,
molecular building blocks and phytonutrients. To
improve the nutritional value an array of exogenous
enzymes are added to grains, grain fractions, flour and
the industrial residues, brewers spent grains (BSG) [7]
and dried distillers’ grains with solubles (DDGS) from
production of potable spirits and bioethanol [8]. Spoiled
grains can be feedstock in bioethanol production and the
DDGS still have potential for use in food and feed [9].
Enzyme processing can modify and enhance starch
digestibility by facilitating interaction between starch
and non-starch components, generally adversely influenc-
ing access of digestive enzymes and hence starch diges-
tion rates (for a review see Ref. [10]). Moreover, removal
of proteins by protease treatment increased in vitro starch
Current Opinion in Food Science 2021, 37:153–159
154 Food bioprocessing

Figure 1

5
Resistant starch
Biopolished grains Extracted molecular components Dietary fibers
Phenolics
Starch
1 2 8
Germinated grains Grains Grain fractions/flour Syrups
3
4 6
Gluten removal

Brewing and distilling Baking

Health and nutrition

7
Spent grains (BSG and DDGS)

Current Opinion in Food Science

Overview of the different areas of grain processing involving enzymes covered in the present review. Enzymes used in the numbered processes: 1)
CE, EG, and XYN; 2) a-AMY, b-AMY, EG, CE, CEL, pectinases, proteases, and XYN; 3) ABF, a-AMY, a-GLU, b-AMY, CEL, EG, lipase, protease,
LD, PUL, and XYN; 4) EP, PEP, proteases, and TG; 5) CE, EG, and XYN; 6) MAase, NTS, and XYN; 7) a-AMY, NTR, PEP, proteases, and XYN; 8)
b-AMY and GA.
Abbreviations: ABF, arabinofuranosidase; a-AMY, a-amylase; a-GLU, a-glucosidase; b-AMY, b-amylase; CE, carbohydrate esterase; CEL,
cellulase; EG, endo-b-glucanase; EP, endoproteinase; GA, glucoamylase; LD, limit dextrinase; MAase, maltogenic amylase; NTR, NADPH-
dependent thioredoxin reductase; NTS, NTR and thioredoxin system; PEP, prolyl endopeptidase; PUL, pullulanase; TG, transglutaminase; XYN,
xylanase.

digestibility of rice flour [11]. Various initiatives modelled
starch degradation kinetics yielding model data allowing
prediction of digestibility [12,13]. In this case, while
purified starches and flours are digested in a single-phase
process, starch amylolysis in macroparticles followed a
two-phase reaction reflecting differences in substrate
accessibility. Grain starch is also the raw material of
glucose-containing and maltose-containing syrups and
in this context EFSA has approved Aspergillus niger glu-
comylase and wheat b-amylase for food production
[14,15].
Bread making covers a broad range of manufacturing
methods, where endogenous and exogenous enzymes
play prominent roles to deliver the best combination of
healthy food components, good technological perfor-
mance and long shelf-life. Use of xylanases for extraction
of arabinoxylans from rye bran provides an ingredient to
improve dough rheology and gel formation [16]. Com-
mercial endo-b-(1,4)-xylanases from glycoside hydrolase
families (GHs) 10 and 11 (http://www.cazy.org/; [17]) are
commonly applied in baking: GH11 xylanases displaying
high activity and selectivity for unsubstituted xylan
regions as opposed to GH10 enzymes able to act on
xylose linkages closer to side chain decorations. Wheat
and other cereals, however, contain proteinaceous inhi-
bitors of microbial xylanases. This problem has been
overcome by structure-guided engineering of a five-
amino acid residue loop into GH10 xylanases from Peni-
cillium canescens and Trichoderma reesei relieving their
sensitivity to the inhibitory proteins [18]. Antistaling
of bread is a well-established trait that can be conferred by
adding microbial maltogenic amylases to the flour result-
ing in suppression of starch retrogradation by shortening
polysaccharide chains in the starch polymers [19].
Various agents can manipulate the gluten network struc-
ture developed in bread dough by disulphide bond for-
mation. A recent procedure used recombinant barley
NADPH-dependent thioredoxin reductase (NTR) and
thioredoxin (Trx), composing a so-called NTS system
that is naturally implicated in grain filling and germina-
tion [20]. Notably, functional wheat Trx in the flour is
also recruited by the added barley NTR. Baking tests
confirmed the potential of NTS and NTR as tools for
weakening strong doughs, or in flat product baking [20].
Mechanical polishing of cereal grains is a conventional
process typically applied on rice, which improves cooking
performance, but removes the bran rich in fibers and other
bioactive molecules. This was recently upgraded to bio-
polishing of cereal grains by treatment with cellulases,
xylanases, b-glucanases, esterases, amylases, proteases or
pectinases in a way that opens up and mobilizes soluble

Current Opinion in Food Science 2021, 37:153–159 www.sciencedirect.com


fibers and phenolics of the bran yielding healthier, more
palatable and consumer acceptable products (for a review
see Ref. [21]).
Endogenous enzyme activities in grains for
food processing
Seed germination serves as a bioprocessing practice to
mobilize and enhance selected enzyme activities for
hydrolysis and release of storage macromolecules, starch
and proteins, as well as cell wall polysaccharides and
phenolics. In cereals amylolytic activity represented by
de novo synthesized a-amylase, limit dextrinase (LD), and
a-glucosidase together with mobilized b-amylase,
increases during germination as do activities of cell wall
deconstructing enzymes, b-glucanase and xylanase, pro-
teases and lipases (for a review see Ref. [22]). Germinated
seeds and malts have wide application in foods to gain
nutritional value and palatability [23]. Barley and wheat
malts are used to standardize enzyme activities in baker’s
flour because certain levels of amylolytic activities are
required for optimal baking performance. Malt also adds
flavor and aroma that influence on sensory properties.
Other flour replacements include malted or sprouted rye,
oat, sorghum, millet, and legumes, for example, cowpea
and chickpea [23,24].
A major problem in particular in temperate climates is
presented by late maturity a-amylase (LMA) and prehar-
vest sprouting (PHS), which are genetic defects in wheat
causing a decrease in falling number (FN) reflecting
adverse biopolymer degradation in the grain. LMA and
PHS effects cannot be discriminated by FN analysis, but
notably overexpression of a wheat a-amylase in the grain
to mimick LMA, resulted in good baking performance
with improved loaf volume and increased Maillard-
related browning [25]. Generally, however, PHS leads
to reduced loaf volume (for a review see Ref. [26]).
In barley, hydrolases catalyzing cell wall degradation are
required for normal starch mobilization as demonstrated
through chemical genetics using an iminosugar inhibitor
for arabinoxylan arabinofuranohydrolase possibly allow-
ing rapid diffusion of amylolytic enzymes [27]. The same
approach has enabled distinguishing individual roles of
different barley a-glucosidases in starch degradation of
which some appeared specific for hydrolyzing maltose to
glucose while others were assumed to participate in
endosperm starch degradation [28].
Germination of the drought tolerant crop sorghum is also
accompanied by degradation of starch via elevated
a-amylase activity and overall serves to improve the
nutrient digestibility [29]. Markedly increased digestibil-
ity of sorghum starch was observed to stem from a low
abundant allele encoding a variant of the starch debranch-
ing enzyme LD. An N-terminal putative starch binding
domain of this LD carried a few substituted amino acid
www.sciencedirect.com
Enzymes in grain processing Møller and Svensson 155
residues compared to LD encoded by the prominent
allele [30,31]. Mutational analysis of the equivalent
domain in barley LD confirmed the role of these positions
in activity towards starch, but did not mimick the higher
activity observed for the rare allele in sorghum [32].
Enzyme processing of grains for health and
nutrition
The major cereal crops wheat, rye and barley, contain
gluten proteins causing celiac disease (CD) with a preva-
lence of about 1% of the world population (for a review
see Ref. [33]). While life-long gluten-free diets provide
one solution, another is to minimize the content of gluten
in foods and beverages. This can be done by aid of
proteases capable of degrading proline and glutamine
rich immunoreactive sequences to avoid ingestion of
peptides that trigger CD autoimmune reactions.
Recently, a glutamine-specific endoproteinase from bar-
ley (EP-B2) and a prolyl endopeptidase from Flavobacter-
ium meningosepticum (Fm-PEP) when expressed in com-
bination in wheat endosperm were capable of detoxifying
immunogenic gluten peptides under simulated gastroin-
testinal conditions. EP-B2 cleavage sites predicted in
barley C hordeins highlight the undesirable sequences
[34]. Wheat genotypes have been developed by transfor-
mation to contain EP-B2 and Fm-PEP [35], but these
‘glutenases’ are limited by poor thermostability and were
therefore subjected to sequence-guided site-saturation
mutagenesis resulting in enzyme variants thermostable
at 60 C and 90 C, respectively, and further improvment
to about 100 C will allow to package the proteases in
grains for application in the industrial processes [36].
Other strategies to obtain gluten-free grain products
apply barley cysteine proteases, including enzymes of
the papain-like C1A family and the C13 family also called
legumains, to destroy epitopes eliciting CD [37], or a
combination of prolyl endoproteases from Aspergillus niger
(AN-PEP) and Sphingomonas capsulate (SC-PEP) with EP-
B2 [38]. In addition, gluten-free beer has been produced
from cereals and pseudocereals by applying EP-B2 for
hordein detoxification. Proteases are simple to extract
from malt and no genetic engineering is needed, but
the lack of thermostaility might be a problem. Moreover,
AN-PEP [34] and microbial transglutaminase [39] were
used to produce gluten-free beer.
Wheat bran is low-cost and valuable for different products
in biotechnology including enzyme-extracted phenol
acids used to scavenge free radicals (for a review see
Ref. [40]). Ferulic acid is released by esterases while
xylanase treatment can produce feruloyl oligosaccharides
from insoluble DF [41–43]. Interest in DF as healthy
ingredients in processed foods motivated isolation by
extraction using b-glucanase in a cocktail with other cell
wall degrading enzymes to release different sized soluble
b-glucan from oat and barley fractions suitable for inclu-
sion in beverages [44].
Current Opinion in Food Science 2021, 37:153–159
156 Food bioprocessing
Cell walls form a physical barrier to nutrients in grains and
xylanase treatment of different wheat milling fractions
has been demonstrated to result in increased starch
digestibility [45]. Cell walls thus slow down the action
of pancreatic amylase and create a type of resistant starch
(RS). Maintaining cell walls intact is a way to retard in
vitro starch digestion, whereas xylanase degradation of
cell walls in wheat raised the starch digestion rate [45].
Proteins associated with starch also act as barrier and
protease treatment can increase the digestibility [10].
Starch is a major nutrient and enzyme management offers
opportunities both to increase digestibility and to produce
RS as a DF metabolized by human gut microbiota to host-
beneficial short chain fatty acids. Notably, chewing influ-
ences availability of starch for oral and gastrointestinal
digestion and salivary a-amylase hydrolysis of starch led
to elevated postprandial blood glucose levels. A chewing
step should therefore be considered in in vitro studies
evaluating starch digestibility [46].
Numerous studies describe transgenic cereal lines with
modified starch structures obtained by chain transfer,
branching, phosphorylation and hydrolysis [47] resulting
in altered in vitro starch digestibility as for high-amylose
rice starch granules, which are more slowly degraded by
porcine pancreatic a-amylase than normal starch granules
[48]. Genetically modified crops are widely explored in
production of RS by manipulating steps in the biosyn-
thetic pathway [6].
Enzyme processing in grains for alcoholic
beverages
Malted barley is the main source of fermentable sugars in
beer production and malt can also serve as the source of
enzymes in brewing using raw barley or other grains,
referred to as adjunct for production of the wort (see
for a review in Ref. [49]). Using adjunct is cost saving and
can benefit from local grains like barley and corn in
Europe, sorghum in Africa, rice in Asia and corn in
America. It is possible to make wort from 100% raw grains
using commercial brewing enzyme cocktails of b-gluca-
nase, xylanase, endoprotease, a-amylase, pullulanase,
lipase, arabinoxylanases or cocktails for high adjunct
brewing of amylase, b-glucanase, cellulase and protease
[49,50]. Sensory attributes of beers brewed from 100%
barley are typically light and with little body and mouth-
feel, albeit their foam stability is good due to low proteo-
lytic activity [51].
Enzyme catalyzed processes occurring during barley ger-
mination have been studied in great detail. However,
certain reactions and conversions are not fully understood
or even unrecognized. Recently, individual steps in beer
production were monitored as viewed from the side of the
polysaccharide substrate using a semi-quantitative carbo-
hydrate microarray technology based on monoclonal
Current Opinion in Food Science 2021, 37:153–159
antibodies recognizing cell wall polysaccharide epitopes
[52]. This resulted in an unprecedented level of insight
into the underlying enzyme catalyzed molecular conver-
sions throughout the brewing processes involving
(1,3;1,4)-b-glucans, arabinoxylans, mannans, galactoman-
nans, xyloglucans and pectin components. This study also
ranked 60 different beers in a way that may prove valu-
able for making choices on specific enzyme additions
during mashing for cost-effective brewing.
Barley grain morphology and distribution of enzyme
activities related to cell wall hydrolysis were tracked
during simulated commercial malting comparing differ-
ent cultivars by using fluorescent immunohistochemical
imaging along with biochemical, enzyme activities and
transcript data [53]. Betts et al. [53] included only tran-
script data on cell wall degrading enzymes since barley
genome sequencing revealed important enzyme families
involved in starch and protein hydrolysis to be larger and
more complex than previously anticipated. While com-
paring cultivars indicated similar expression patterns for
most genes encoding (1,3;1,4)-b-endoglucanase, b-glucan
glucohydrolase, xylanase and arabinoxylan arabinofura-
nohydrolase, the findings indicated a previously unrecog-
nized (1,3;1,4)-b-endoglucanase isozyme to differ
between cultivars and possibly contributing to superior
malting quality [53].
A review on enzymes active in starch biosynthesis and
degradation in barley focuses on the activity of the
debranching enzymes, isoamylase (ISA) and LD and their
impact on amylopectin structure [54]. ISA and LD play
different roles in acting on a-1,6-glucosidic bonds result-
ing in restructuring amylopectin branching pattern during
biosynthesis and in hydrolysis of branch points in limit
dextrins, respectively. It is discussed that no data are
available that link structure of the substrate to the profile
of fermentable sugar in wort [54]. LD seems a limiting
factor in brewing as about 75% of the branch points in
amylopectin are detected in oligosaccharides in beer.
Importantly, the LD activity is regulated by an endoge-
nous limit dextrinase inhibitor (LDI) having picomolar
affinity for LD. The crystal structure of the LD-LDI
complex suggested protein-protein intermolecular con-
tact points for protein engineering of LD to suppress its
high affinity for LDI [55], thus increasing LD activity
during germination and mashing.
For production of potable alcohol by distilling cereal
grains, rye and barley together with malts thereof are
major raw materials and sources of amylolytic enzymes to
release fermentable sugars from starch, mostly maltose
followed by glucose and maltotriose. These agricultural
distillates were characterized by low contents of the
undesirable products, acetaldehyde and methanol [56].
The use of grain residues from distilling will be discussed
below.
www.sciencedirect.com

Enzyme catalysed processing of spent grains
The left-overs from distilling and brewing; distillers dried
grains with solubles (DDGS) and brewers spent grains
(BSG) can be used in animal feed, but occasionally also in
food. Spent grains contain valuable carbohydrates, pro-
teins, lipids and micronutrients to which enzymes can
open up and provide access [57]. Valorization of BSG by
xylanase hydrolysis producing prebiotic oligosaccharides
has been verified by monitoring effects on probiotic
bacteria and a human fecal fermentation model [58].
In particular protein from BSG is useful in both food and
feed [7]. Barley malt rootlets form a sidestream in brewing
rich in phenolics and enzyme pretreatment was one
technique supporting their extraction [59].
Enzymes generally recognized as safe (GRAS) are avail-
able for applications in fermentation to potable or fuel
alcohol with co-products destined for animal feed [60].
Because of the high costs of energy in pig diets, use of
DDGS has been increased and here enzymes can reduce
the contents of fibers causing negative impact on growth
performance of pigs [61].
A different approach monitored microbiota in buffaloes for a
core group of more than fifty rumen bacteria present in
animals fed various levels of total digestible nutrients
(TDN) in corn grains/wheat straw mixtures showing
decreasing abundance from Prevotella, Bacteroides, Clos-
tridium, Ruminococcus, Eubacterium, Parabacteroides,
Fibrobacter, Butyrivibrio and so on. [62]. The diversity of
enzymes from GH families 23, 28, 39, 97, 106 and 127, which
are active in starch and fiber hydrolysis, were significantly
higher on a 100% TDN diet containing larger amounts of
grains, whereas hydrolysis of ester bonds between carbohy-
drate and lignin by carbohydrate esterase family 14 increased
for lower TDN (70%). Thus ester bond cleavage was better
in animals fed a diet higher in wheat straw [62].
Barley spoiled by fungal (e.g. Fusarium) infection can be
treated with commercial xylanase and amylolytic
enzymes to improve bioethanol production and the
DDGS is added lactic acid bacteria with antimicrobial
effects to make the residue suitable for feed by freeing it
from toxins [9]. RS which is beneficial in DDGS used for
feed, can be retained in grains after supply of amylolytic
enzymes for production of fermentable sugars for bioetha-
nol production [8].
So-called first wave enzymes present in mature grains can
be utilized to increase nutrient availability in liquid feed
for livestock, which by addition of NADPH-dependent
thioredoxin reductase showed higher susceptibility to
proteolysis for monogastric animals [63].
Conclusions and prospects
The current trend to replace meat with plant-based food
draws attention to upgrading grains as attractive palatable
www.sciencedirect.com
Enzymes in grain processing Møller and Svensson 157
and health-promoting raw materials. The nutritional
value of grains, however, typically depends on pretreat-
ments for which various biotechnological solutions have
been developed. Indeed a large number of studies report
on enzyme-catalyzed processing of grains applied in or
with potential to become applied in food and beverage
industries on larger scale. While most studies described in
this review are quite straightforward and adopt essentially
well-established strategies, one can expect transgenic
crops to gain attention in the light of increasing awareness
of sustainability and the high cost of resources such as
externally administred commercial enzymes compared to
endogenous enzymes present in genetically manipulated
grains. This will obviously depend not just on technolog-
ical progress but also on regulations and consumer atti-
tudes. Another area may include healthy food forming
synbiotics with probiotic bacteria capable of producing
metabolites stimulating human gut bacteria associated
with good nutritional and metabolic performance for
healthy normal subjects as well as for individuals with
challenging gut conditions.
Conflict of interest statement
Nothing declared.
Acknowledgement
This work was supported by the Independent Research Fund Denmark |
Natural Sciences (FNU) (Grant No. 6108-00476B).
References and recommended reading
Papers of particular interest, published within the period of review,
have been highlighted as:
 of special interest
 of outstanding interest
1. Iram A, Cekmecelioglu D, Demirci A: Distillers’ dried grains with
solubles (DDGS) and its potential as fermentation feedstock.
Appl Microbiol Biotechnol 2020, 104:6115-6128.
2. Razdan N, Kocher GS: Utilization of damaged and spoiled
wheat grains for bioethanol production. Biosci Biotechnol Res
Commun 2018, 11:658-673.
3. Baranzelli J, Kringel DH, Mallmann JF, Bock E, Mello El Halal SL,
Prietto L, da Rosa Zavareze E, Zavariz de Miranda M, Renato
Guerra Dias A: Impact of wheat (Triticum aestivum L.)
germination process on starch properties for application in
films. Starch 2019, 71:1800262.
4. Herrmann KR, Ruff AJ, Schwaneberg U: Phytase-based
phosphorus recovery process for 20 distinct press cakes. ACS
Sustain Chem Eng 2020, 8:3913-3921.
5. Sultan A, Frisvad JC, Andersen B, Svensson B, Finnie C:
Investigation of the indigenous fungal community populating
barley grains: Secretomes and xylanolytic potential. J
Proteomics 2017, 169:153-164.
6. Ansari WA, Chandanshive SU, Bhatt V, Nadaf AB, Vats S,
Katara JL, Sonah H, Deshmukh R: Genome editing in cereals:
approaches, applications and challenges. Int J Mol Sci 2020,
21:4040.
7. Wen C, Zhang J, Duan Y, Zhang H, Ma H: A mini-review on
brewer’s spent grain protein: Isolation, physicochemical
properties, application of protein, and functional properties of
hydrolysates. J Food Sci 2019, 84:3330-3340.
8. Li J, Vasanthan T, Gao J, Naguleswaran S, Zijlstra RT, Bressler DC:
Resistant starch escaped from ethanol production: evidence
Current Opinion in Food Science 2021, 37:153–159
158 Food bioprocessing
from confocal laser scanning microscopy of distiller’s dried
grains with solubles (DDGS). Cereal Chem 2014, 91:130-138.
9. Juodeikiene G, Cernauskas D, Vidmantiene D, Basinskiene L,
Bartkiene E, Bakutis B, Baliukoniene V: Combined fermentation
for increasing efficiency of bioethanol production from
Fusarium sp. contaminated barley biomass. Catal Today 2014,
223:108-114.
10. Khatun A, Waters DLE, Liu L: A review of rice starch digestibility:
effect of composition and heat-moisture processing. Starch
2019, 71 1900090.
11. Ye J, Hu X, Luo S, McClements DJ, Liang L, Liu C: Effect of
endogenous proteins and lipids on starch digestibility in rice
flour. Food Res Int 2018, 106:404-409.
12. Edwards CH, Warren FJ, Milligan PJ, Butterworth PJ, Ellis PR: A
novel method for classifying starch digestion by modelling the
amylolysis of plant foods using first-order enzyme kinetic
principles. Food Funct 2014, 5:2751-2758.
13. Warren FJ, Zhang B, Waltzer G, Gidley MJ, Dhital S: The interplay
of a-amylase and amyloglucosidase activities on the digestion
of starch in in vitro enzymic systems. Carbohydr Polym 2015,
117:185-191.
14. EFSA Panel on Food Contact Materials Enzymes FAPAC, Silano V,
Bolognesi C, Castle L, Cravedi J, Fowler P, Franz R, Grob K,
Gürtler R, Husøy T et al.: Safety evaluation of a b-amylase food
enzyme obtained from wheat (Triticum spp.). EFSA J 2017, 15
e04754.
15. EFSA Panel on Food Contact Materials EPAC, Silano V, Barat
Baviera JM, Bolognesi C, Brüschweiler BJ, Cocconcelli PS,
Crebelli R, Gott DM, Grob K, Lampi E et al.: Safety of the food
enzyme glucoamylase from a genetically modified Aspergillus
niger (strain NZYM-BF). FSA J 2018, 16:5450.
16. Bender D, Nemeth R, Wimmer M, Götschhofer S, Biolchi M,
Török K, Tömösközi S, D’Amico S, Schoenlechner R:
Optimization of arabinoxylan isolation from rye bran by
adapting extraction solvent and use of enzymes. J Food Sci
2017, 82:2562-2568.
17. Lombard V, Ramulu HG, Drula E, Coutinho PM, Henrissat B: The
carbohydrate-active enzymes database (CAZy) in 2013.
Nucleic Acids Res 2014, 42:490-495.
18. Denisenko YA, Gusakov AV, Rozhkova AM, Zorov IN,
 Bashirova AV, Matys VY, Nemashkalov VA, Sinitsyn AP: Protein
engineering of GH10 family xylanases for gaining a resistance
to cereal proteinaceous inhibitors. Biocatal Agric Biotechnol
2019, 17:690-695.
Protein engineering of xylanase to loose sensitivity for cereal proteinac-
eous xylanase inhibitors.
19. Woo SH, Shin YJ, Jeong HM, Kim JS, Ko DS, Hong JS, Choi HD,
Shim JH: Effects of maltogenic amylase from Lactobacillus
plantarum on retrogradation of bread. J Cereal Sci 2020, 93
102976.
20. Navrot N, Buhl Holstborg R, Hägglund P, Povlsen I, Svensson B:
 New insights into the potential of endogenous redox systems
in wheat bread dough. Antioxidants 2018, 7:190.
Improving wheat dough characteristics by treatment with barley thior-
edoxin reductase/thioredoxin system.
21. Singh A, Sharma V, Banerjee R, Sharma S, Kuila A: Perspectives
 of cell-wall degrading enzymes in cereal polishing. Food Biosci
2016, 15:81-86.
Comprehensive review on contents of enzymes in a wide range of
germinating cereals.
22. Guzmán-Ortiz FA, Castro-Rosas J, Gómez-Aldapa CA, Mora-
Escobedo R, Rojas-León A, Rodrı́guez-Marı́n ML, Falfán-
Cortés RN, Román-Gutiérrez AD: Enzyme activity during
germination of different cereals: a review. Food Rev Int 2019,
35:177-200.
23. Mäkinen OE, Arendt EK: Nonbrewing applications of malted
cereals, pseudocereals, and legumes: a review. J Am Soc Brew
Chem 2015, 73:223-227.
24. Guardado-Félix D, Lazo-Vélez MA, Pérez-Carrillo E, Panata-
Saquicili DE, Serna-Saldı́var SO: Effect of partial replacement of
Current Opinion in Food Science 2021, 37:153–159
wheat flour with sprouted chickpea flours with or without
selenium on physicochemical, sensory, antioxidant and
protein quality of yeast-leavened breads. LWT - Food Sci
Technol 2020, 129 109517.
25. Ral JP, Whan A, Larroque O, Leyne E, Pritchard J, Dielen AS,
Howitt CA, Morell MK, Newberry M: Engineering high a-amylase
levels in wheat grain lowers Falling Number but improves
baking properties. Plant Biotechnol J 2016, 14:364-376.
26. Olaerts H, Courtin CM: Impact of preharvest sprouting on
endogenous hydrolases and technological quality of wheat
and bread: a review. Compr Rev Food Sci Food Saf 2018, 17:698-
713.
27. Andriotis VME, Rejzek M, Barclay E, Rugen MD, Field RA,
Smith AM: Cell wall degradation is required for normal starch
mobilisation in barley endosperm. Sci Rep 2016, 6:33215.
28. Andriotis VME, Rejzek M, Rugen MD, Svensson B, Smith AM,
Field RA: Iminosugar inhibitors of carbohydrate-active
enzymes that underpin cereal grain germination and
endosperm metabolism. Biochem Soc Trans 2016, 44:159-165.
29. Singh A, Sharma S, Singh B, Kaur G: In vitro nutrient digestibility
and antioxidative properties of flour prepared from sorghum
germinated at different conditions. J Food Sci Technol 2019,
56:3077-3089.
30. Gilding EK, Frère CH, Cruickshank A, Rada AK, Prentis PJ,
Mudge AM, Mace ES, Jordan DR, Godwin ID: Allelic variation at a
single gene increases food value in a drought-tolerant staple
cereal. Nat Commun 2013, 4:1483.
31. Møller MS, Windahl MS, Sim L, Bøjstrup M, Abou Hachem M,
Hindsgaul O, Palcic M, Svensson B, Henriksen A:
Oligosaccharide and substrate binding in the starch
debranching enzyme barley limit dextrinase. J Mol Biol 2015,
427:1263-1277.
32. Andersen S, Svensson B, Møller MS: Roles of the N-terminal
domain and remote substrate binding subsites in activity of
the debranching barley limit dextrinase. Biochim Biophys Acta -
Proteins Proteomics 2020, 1868.
33. Cela N, Condelli N, Caruso MC, Perretti G, Di Cairano M, Tolve R,
 Galgano F: Gluten-free brewing: issues and perspectives.
Fermentation 2020, 6:53.
Comprehensive review on brewing for gluten-free beer compatible with
celiac disease.
34. Kerpes R, Fischer S, Becker T: The production of gluten-free
beer: degradation of hordeins during malting and brewing and
the application of modern process technology focusing on
endogenous malt peptidases. Trends Food Sci Technol 2017,
67:129-138.
35. Osorio CE, Wen N, Mejias JH, Liu B, Reinbothe S, von Wettstein D,
Rustgi S: Development of wheat genotypes expressing a
glutamine-specific endoprotease from barley and a prolyl
endopeptidase from Flavobacterium meningosepticum or
Pyrococcus furiosus as a potential remedy to celiac disease.
Funct Integr Genomics 2019, 19:123-136.
36. Osorio CE, Wen N, Mejı́as JH, Mitchell S, von Wettstein D,
 Rustgi S: Directed-mutagenesis of Flavobacterium
meningosepticum prolyl-oligopeptidase and a glutamine-
specific endopeptidase from barley. Front Nutr 2020, 7:11.
Protein engineering of thermostability of barley glutamine-specific endo-
proteinase EP-B2 and prolyl endopeptidase from Flavobacterium menin-
gosepticum (Fm-PEP) for detoxification of gluten.
37. Martinez M, Gómez-Cabellos S, Giménez MJ, Barro F, Diaz I, Diaz-
 Mendoza M: Plant proteases: from key enzymes in germination
to allies for fighting human gluten-related disorders. Front
Plant Sci 2019, 10:721.
Application of plant cysteine proteases and expression in endosperm to
reduce the amount of indigestible gluten peptides to generate gluten-free
wheat to manufacture gluten free products.
38. Cavaletti L, Taravella A, Carrano L, Carenzi G, Sigurtà A, Solinas N,
De Caro S, Di Stasio L, Picascia S, Laezza M et al.: E40, a novel
microbial protease efficiently detoxifying gluten proteins, for
the dietary management of gluten intolerance. Sci Rep 2019,
9:1-11.
www.sciencedirect.com

39. Taylor JP, Jacob F, Arendt EK: Fundamental study on the impact
of transglutaminase on hordein levels in beer. J Am Soc Brew
Chem 2015, 73:253-260.
40. Katileviciute A, Plakys G, Budreviciute A, Onder K, Damiati S,
Kodzius R: A sight to wheat bran: High value-added products.
Biomolecules 2019, 9:14-25.
41. Long L, Wu L, Lin Q, Ding S: Highly efficient extraction of ferulic
acid from cereal brans by a new type A feruloyl esterase from
Eupenicillium parvum in combination with dilute phosphoric
acid pretreatment. Appl Biochem Biotechnol 2020, 190:1561-
1578.
42. Wu H, Li H, Xue Y, Luo G, Gan L, Liu J, Mao L, Long M: High
efficiency co-production of ferulic acid and
xylooligosaccharides from wheat bran by recombinant
xylanase and feruloyl esterase. Biochem Eng J 2017, 120:41-48.
43. Dupoiron S, Lameloise ML, Bedu M, Lewandowski R, Fargues C,
Allais F, Teixeira ARS, Rakotoarivonina H, Rémond C: Recovering
ferulic acid from wheat bran enzymatic hydrolysate by a novel
and non-thermal process associating weak anion-exchange
and electrodialysis. Sep Purif Technol 2018, 200:75-83.
44. Aktas-Akyildiz E, Sibakov J, Nappa M, Hytönen E, Koksel H,
 Poutanen K: Extraction of soluble b-glucan from oat and barley
fractions: process efficiency and dispersion stability. J Cereal
Sci 2018, 81:60-68.
Enzyme assisted extraction of soluble b-glucan fibers and techno-eco-
nomic analysis with a feed-stock design capacity of 100 kg/h.
45. Korompokis K, De Brier N, Delcour JA: Differences in endosperm
cell wall integrity in wheat (Triticum aestivum L.) milling
fractions impact on the way starch responds to gelatinization
and pasting treatments and its subsequent enzymatic in vitro
digestibility. Food Funct 2019, 10:4674-4684.
46. Tamura M, Okazaki Y, Kumagai C, Ogawa Y: The importance of
an oral digestion step in evaluating simulated in vitro
digestibility of starch from cooked rice grain. Food Res Int
2017, 94:6-12.
47. Hebelstrup KH, Sagnelli D, Blennow A: The future of starch
bioengineering: GM microorganisms or GM plants? Front Plant
Sci 2015, 6:247.
48. Man J, Yang Y, Zhang C, Zhang F, Wang Y, Gu M, Liu Q, Wei C:
Morphology and structural characterization of high-amylose
rice starch residues hydrolyzed by porcine pancreatic
a-amylase. Food Hydrocoll 2013, 31:195-203.
49. Kok YJ, Ye L, Muller J, Ow DSW, Bi X: Brewing with malted
 barley or raw barley: what makes the difference in the
processes? Appl Microbiol Biotechnol 2019, 103:1059-1067.
Use of commercial brewing enzyme cocktails in high raw grain and all raw
grain adjunct brewing.
50. Zhuang S, Shetty R, Hansen M, Fromberg A, Hansen PB,
Hobley TJ: Brewing with 100 % unmalted grains: barley, wheat,
oat and rye. Eur Food Res Technol 2017, 243:447-454.
51. Steiner E, Auer A, Becker T, Gastl M: Comparison of beer quality
attributes between beers brewed with 100% barley malt and
100% barley raw material. J Sci Food Agric 2012, 92:803-813.
52. Fangel JU, Eiken J, Sierksma A, Schols HA, Willats WGT, Harholt J:
 Tracking polysaccharides through the brewing process.
Carbohydr Polym 2018, 196:465-473.
www.sciencedirect.com
Enzymes in grain processing Møller and Svensson 159
Carbohydrate semi-quantitative microarray monitoring of cell wall poly-
saccharides at all steps in beer manufacture and assessment for 60 dif-
ferent beers as a tool to decide on enzyme addition.
53. Betts NS, Wilkinson LG, Khor SF, Shirley NJ, Lok F, Skadhauge B,
Burton RA, Fincher GB, Collins HM: Morphology, carbohydrate
distribution, gene expression, and enzymatic activities related
to cell wall hydrolysis in four barley varieties during simulated
malting. Front Plant Sci 2017, 8:1-15.
54. Gous PW, Fox GP: Review: amylopectin synthesis and
hydrolysis – understanding isoamylase and limit dextrinase
and their impact on starch structure on barley (Hordeum
vulgare) quality. Trends Food Sci Technol 2017, 62:23-32.
55. Møller MS, Vester-Christensen MB, Jensen JM, Hachem MA,
Henriksen A, Svensson B: Crystal structure of barley limit
dextrinase-limit dextrinase inhibitor (LD-LDI) complex reveals
insights into mechanism and diversity of cereal type inhibitors.
J Biol Chem 2015, 290:12614-12629.
ska-Kubczak U,
56. Balcerek M, Pielech-Przybylska K, Dziekon
Patelski P, Stra?k E: Fermentation results and chemical
composition of agricultural distillates obtained from rye and
barley grains and the corresponding malts as a source of
amylolytic enzymes and starch. Molecules 2016, 21:1320.
57. Connolly A, Cermeño M, Crowley D, O’Callaghan Y, O’Brien NM,
FitzGerald RJ: Characterisation of the in vitro bioactive
properties of alkaline and enzyme extracted brewers’ spent
grain protein hydrolysates. Food Res Int 2019, 121:524-532.
58. Sajib M, Falck P, Sardari RRR, Mathew S, Grey C, Karlsson EN,
 Adlercreutz P: Valorization of Brewer’s spent grain to prebiotic
oligosaccharide: production, xylanase catalyzed hydrolysis,
in-vitro evaluation with probiotic strains and in a batch human
fecal fermentation model. J Biotechnol 2018, 268:61-70.
Brewer’s spent grain oligosaccharide hydrolysates are fermented by
human gut microbiota at the same rate as fructooligosaccharides and
are valorized as a promising functional food ingredient.
59. Budaraju S, Mallikarjunan K, Annor G, Schoenfuss T, Raun R:
Effect of pre-treatments on the antioxidant potential of
phenolic extracts from barley malt rootlets. Food Chem 2018,
266:31-37.
60. Sewalt VJ, Reyes TF, Bui Q: Safety evaluation of two a-amylase
enzyme preparations derived from Bacillus licheniformis
expressing an a-amylase gene from Cytophaga species. Regul
Toxicol Pharmacol 2018, 98:140-150.
61. Rojas OJ, Stein HH: Processing of ingredients and diets and
effects on nutritional value for pigs. J Anim Sci Biotechnol 2017,
8:48.
62. Kala A, Kamra DN, Kumar A, Agarwal N, Chaudhary LC, Joshi CG:
Impact of levels of total digestible nutrients on microbiome,
enzyme profile and degradation of feeds in buffalo rumen.
PLoS One 2017, 12 e0172051.
63. Sultan A, Andersen B, Christensen JB, Poulsen HD, Svensson B,
Finnie C: Quantitative proteomics analysis of barley-based
liquid feed and the effect of protease inhibitors and NADPH-
dependent thioredoxin reductase/thioredoxin (NTR/Trx)
system. J Agric Food Chem 2019, 67:6432-6444.
Current Opinion in Food Science 2021, 37:153–159