Journal of Cereal Science 95 (2020) 103043

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Journal of Cereal Science

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Production of syrup rich in arabinoxylan oligomers and antioxidants from

wheat bran by alkaline pretreatment and enzymatic hydrolysis, and
applicability in baking
Ville Pihlajaniemi *, Outi Mattila , Taru Koitto 1, Markus Nikinmaa , Raija-Liisa Heinio
€,
Lotta Sorsam€ aki , Matti Siika-aho , Emilia Nordlund
VTT Technical Research Centre of Finland Ltd., Finland, P.O. Box 1000, FI-02044, VTT, Finland

A R T I C L E I N F O A B S T R A C T

Handling editor: Prof. John R.N. Taylor The target of this work was to develop a novel, industrially applicable process for simultaneously releasing
different valuable components from wheat bran, including carbohydrates, oligomeric arabinoxylan and anti­
Keywords: oxidants. The process was based on alkaline pretreatment with potassium hydroxide (KOH) and subsequent
Lignocellulose hydrolysis enzymatic hydrolysis. Increasing KOH-dosage and thermal severity in pretreatment promoted carbohydrate
Soluble dietary fibre
solubilisation in hydrolysis, reaching glucose and arabinoxylan yields up to 86% and 76%, respectively. Release
Sensory evaluation
of antioxidants was particularly promoted by increasing KOH-dosage, while both the pretreatment severity and
Techno-economic analysis
KOH-dosage promoted the release of oligomeric arabinoxylan in enzymatic hydrolysis. Two bran syrups, with or
without KOH-treatment, were tested in bread making by substituting added sugar in the dough with bran syrup.
The KOH-derived KCl also substituted 30% of NaCl in the bread formulation. The addition of bran syrup did not
affect the baking properties of wheat bread dough. However, a decrease in bread flavour balance was observed
with addition of syrup from KOH-pretreated bran. Conceptual level techno-economic assessment indicated that
production of bran syrup would be economically feasible at a minimum selling price of 770 €/t and 1030 €/t with
KOH-pretreatment and without KOH, respectively.

1. Introduction carbohydrate in bran, and it is associated with varying levels of

phenolic acids, particularly ferulic acid, which is a mediator of hemi­
Wheat bran is an abundant side stream generated in the wheat cellulose interchain and lignin crosslinking. The hemicellulose of the
milling process, comprising the outer layers of wheat grain. Of the wheat aleurone cell walls is arabinoxylan with a low level of substitution,
grain weight, 14–27% ends up as bran, leading to a yearly estimated whereas the highest level of substitution is found in the outer pericarp
global output 90–50 million metric tons (Reisinger et al., 2013). (Antoine et al., 2003; Mandalari et al., 2005).
Although applications for bran in food industry exist, the majority of Wheat bran has been associated with several health benefits in
bran is still used as low value animal feed, signifying it as a potential human consumption (Arte et al., 2015; Coda et al., 2014) specially due
biomass feedstock for further valorisation. to its high dietary fibre content that originates from the lignocellulosic
Wheat bran is structured in distinct layers including the outer peri­ and hemicellulose components, as well as due to associated bioactive
carp, composed mainly of hemicellulose and cellulose with a low lignin components, such as phenolic compounds. In particular, wheat bran
content, the lignocellulosic inner pericarp and the seed coat with the arabinoxylan has been shown to provide prebiotic effects (Broekaert
highest lignin content, and the aleurone layer rich in protein and et al., 2011), whereas the phenolic compounds exert antioxidant activity
hemicellulose, and practically free of lignin (Antoine et al., 2003). and other health benefits (Mateo Anson et al., 2008). The benefits of
Additionally, bran contains 10–25% starch resulting from incomplete bran are challenged by low applicability of bran in food applications,
separation of the endosperm. Hemicellulose is the major non-starch thus limiting the use of bran for human consumption. Different physical,

* Corresponding author.
E-mail address: [email protected] (V. Pihlajaniemi).
1
Current address: aAalto University, School of Chemical Technology, Department of Bioproducts and Biosystems, Espoo, Finland, P.O. Box 16300, FI-00076 Espoo,
Finland.

https://doi.org/10.1016/j.jcs.2020.103043
Received 27 March 2020; Received in revised form 26 June 2020; Accepted 29 June 2020
Available online 6 July 2020
0733-5210/© 2020 Elsevier Ltd. All rights reserved.
V. Pihlajaniemi et al. Journal of Cereal Science 95 (2020) 103043

chemical and biochemical strategies have been studied to improve the content were subtracted from gravimetric lignin. Cellulose content was
applicability of bran, including treatments with enzymes and microbes determined as the difference of total polymeric glucose and starch. The
(Arte et al., 2015; Coda et al., 2014; Zhang et al., 2019). crude protein content was calculated as 6.25 x nitrogen content,
An alternative approach to bran modification is isolation of partic­ analyzed by a FLASH 2000 series elemental analyzer (Thermo Scienti­
ular high value components from bran, such as arabinoxylans, pheno­ fic). Ash content was determined gravimetrically after combustion at
lics, protein and platform chemical precursors (Apprich et al., 2014). 550 � C. Compositional analysis procedures were carried out in dupli­
Among the technologies for fractionation of bran, thermochemical and cate. The enzymes used in this study included commercial cellulase
enzymatic treatments have been applied for extracting arabinoxylan. (Flashzyme Plus, Roal Oy; with a specific activity of 0.56 Filter Paper
Extraction by strong alkali has been applied as an analytical procedure Units/mg protein, α-amylase (Grindamyl A14000, Dupont; 129 CU/mg,
for sequential isolation of cell wall arabinoxylan and phenolics of bran Ceralpha-unit by Megazyme standard method (K-CERA 08/16), amylo­
(Mandalari et al., 2005; Zhang et al., 2011), and has been associated glucosidase (Grindamyl Plusweet, Dupont; 542 nkat/mg and xylanase
with decreasing arabinose substitution level of arabinoxylan in fractions (Depol 740L, Biocatalysts Ltd; 611 nkat/mg. For the baking experi­
with increasing alkali concentration. For process scale extraction of bran ments, wheat flour (Helsingin Mylly, Finland), compressed yeast (Suo­
arabinoxylan, an alkaline peroxide process has been reported (Maes and men Hiiva, Finland), rapeseed baking fat (Raisio, Finland) and caster
Delcour, 2001) and combined with extensive purification steps for sugar (Dansukker, Finland) were used.
production of high purity arabinoxylan (Hollmann and Lindhauer,
2005), while leaving cellulose untouched. Hydrothermal treatment is 2.2. Laboratory scale treatments
frequently applied as a pretreatment improving enzyme efficacy within
lignocellulosic biorefinery concepts, and this approach is also effective Bran pretreatment reactions were carried out with 0–10% KOH per
for solubilising bran hemicellulose (Reisinger et al., 2013; van den Borne bran DM at 60–90 � C for 1–24 h, with a bran dry matter (DM) concen­
et al., 2012). However, the high temperatures and acidic conditions of tration of 15% a total reaction volume of 100 ml in sealed 200 ml metal
hydrothermal pretreatments lead to formation of sugar degradation containers, in a rotating water bath incubator (Linitest Plus, Atlas,
products, which are undesirable in food applications. Enzymatic hy­ Germany) at 45 rpm. Pretreatments at 120 � C were performed in glass
drolysis has also been applied directly to bran, but with lower efficiency flasks heated in an autoclave. After pretreatment the pH was adjusted to
(Arte et al., 2016; Petersson et al., 2013). Alkaline pretreatments are 5.0 with 37% HCl. The thermal severity of the pretreatment was
effective for improving enzymatic hydrolyzability of biomass at lower described with the severity factor Log(R0), which was calculated as the
thermal severities compared to hydrothermal treatments, while as a combined effect of reaction time t (min) and temperature T (oC) ac­
trade-off, costly recycling of alkaline chemicals is usually required (Chen cording to Eq. (1) (Overend and Chornet, 1987).
et al., 2013). However, alkaline pretreatment has so far not been sys­ � �
tematically studied for improving enzymatic hydrolysis of bran. In LogðR0 Þ ¼ Log10 t * e 14:75 (1)
T 100

addition to arabinoxylan solubilisation, alkaline treatment contributes

to increasing antioxidant potential of the hydrolysate by cleavage of Twenty g of pretreated bran suspension was weighed into 50 ml
ferulic acid ester linkages (Antoine et al., 2003). Nevertheless, conical tubes. Na-azide was added to a final concentration of 0.2 mg/mL
alkaline-aided extraction studies of bran have mainly focused on single to prevent microbial growth. Enzymes were added to a final reaction
components, and a so far no reports exist on a scalable process targeting volume of in 22 ml, leading to a solids concentration of 13.6% in hy­
extraction of all carbohydrates while maximizing oligomeric arabinox­ drolysis. An enzyme dosage (as enzyme protein/DM) of 10 mg/g cellu­
ylan and antioxidant content. lase, 0.05 mg/g α-amylase and 2.5 mg/g amyloglucosidase was applied
The target of this work was to develop a novel process for high yield unless stated otherwise. This α-amylase and amyloglucosidase dosage
extraction of multifunctional syrup from wheat bran, combining was found to be sufficient for hydrolysing the starch present in bran
maximal total carbohydrate yield with a high content of oligomeric (Fig. S1, Supplementary material). The tubes were incubated in a tilted
arabinoxylan and antioxidants in the syrup. Alkaline pretreatment with position on a shaker (150 rpm) for 24 h or 96 h at 50 � C. Hydrolysate
potassium hydroxide (KOH) and subsequent enzymatic hydrolysis was samples were immediately placed in boiling water for 10 min, cooled
hypothesized to be a favourable combination for facilitating starch and and centrifuged, and the supernatants were analyzed for mono- and
cellulose hydrolysis in addition to the release of arabinoxylan and an­ oligomeric sugars and antioxidants. Laboratory scale reactions were
tioxidants. The effect of KOH-loading, pretreatment severity and enzy­ performed in duplicate, unless stated otherwise. Eight repetitions were
matic hydrolysis conditions on the yields of these components were performed of the KOH-treatment at 90 � C, 24 h at 4% KOH per DM
determined. Applicability of the syrups in wheat bread making was serving as the reference reaction in different experiments.
evaluated in terms of baking quality and sensory quality. Furthermore,
the techno-economic feasibility of an industrial scale plant for the pro­ 2.3. Treatment scale-up for bread making trials
duction of functional bran syrup was assessed. In our understanding, this
is the first report evaluating economic feasibility of a bran-syruping To produce hydrolysates for bread making, wheat bran was pro­
process and applicability of bran syrup in bread. cessed in a 10 L temperature controlled mixing reactor with a helical
stirrer (Tankki Oy, Aht
€ € ari, Finland). Batches of 2 kg of bran (DM basis)
2. Materials and methods were treated at an initial solids concentration of 20%, with 4% KOH/DM
(“KOH-syrup”) for 24 h or without KOH (“H2O-syrup”) for 1 h at 90 � C
2.1. Materials and compositional analysis (mixing 130 rpm). After pretreatment, the material was cooled and the
pH was adjusted to 5.0 using 37% HCl. Cellulase (10 mg/g DM),
Wheat bran from Lantma €nnen Cerealia AB (Sweden) was used in this α-amylase (0.05 mg/g DM) and amyloglucosidase (2.5 mg/g DM) was
study. The total carbohydrate content of bran (as polysaccharides) was added and hydrolysis was carried out under mixing (130 rpm) for 24 h at
49.8%, comprising 13.7% starch, 10.6% cellulose, 13.7% xylose, 8.1% 50 � C. After hydrolysis, enzymes were inactivated by heating the reactor
arabinose, 1.0% galactose, 0.5% mannose and 2.2% fructose. Other to 90 � C for 10 min. After cooling, the hydrolysates were centrifuged at
constituents included 15.9% protein, 10.0% lignin, 5.3% ash and 4.5% 3963 RCF (Sorvall RC12BP, rotor H-12000) for 20 min and the super­
lipophilic extractives. The bran starch content was analyzed with the natant was collected.
Total starch analysis kit (Megazyme). The total carbohydrate content,
extractives and lignin were analyzed according to the NREL biomass
compositional analysis standard (Sluiter et al., 2011). Ash and protein

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V. Pihlajaniemi et al. Journal of Cereal Science 95 (2020) 103043

2.4. Antioxidant analysis was substituted by the calculated level of KCl present in the KOH syrup.
The amount of flours in the recipes were also adjusted to compensate for
Antioxidant analysis was based on a combination of the 2,2- the DM content of the syrups and the reduced level of salt in the KOH
diphenyl-1-picrylhydrazyl (DPPH) assay (Brand-Williams et al., 1995) syrup bread.
and spectrophotometry. DPPH-assay was performed on selected samples Baking trials were carried out in duplicate, and for both baking trials,
covering the experimental range of severity and KOH dosage. Four di­ seven bread loaves were produced for each bread type. The dough was
lutions of each sample and a Trolox standard were incubated in meth­ mixed for 7 min by Diosna spiral mixer to a final dough temperature of
anol for 30 min in the presence of 0.0121 μM DPPH, after which the 27 � C � 0.5 � C. After resting (15 min, 35 � C, 75 RH%), the dough was
absorbance of the unreacted DPPH was measured at 515 nm. The exact divided into 350 g pieces, moulded mechanically and put into pans for
sample dilution leading to 50% depletion of DPPH was linearly inter­ proofing (45 min, 35 � C, 75 RH%). Breads were baked for 20 min at 200
polated from adjacent dilutions. This sample dilution was converted into �
C with 15 s steam in the beginning, and left to cool down at room
Trolox-equivalent (Trolox/bran DM, μmol/g) by dividing the Trolox temperature for approximately 1.5 h before the analysis of weight loss (5
concentration with the volume fraction of hydrolysate in the dilution. To replicates), specific volume (4 replicates, by BreadVolScan, Backaldrin,
allow efficient analysis of a large number of laboratory scale hydroly­ Austria), lightness of colour (10 replicate measurements of crumb and
sates, a direct spectrophotometric method was developed (Supplemen­ crust, by recording L* values using Minolta chroma meter cr-200, Japan)
tary material, chapter 2). Absorbance at 290 nm was selected to and crumb hardness (10 replicates, by TA-XT2 Texture Analyzer using
represent antioxidant activity based on linear correlation with Texture Profile Analysis test; cylindrical crumb samples of 2.4 mm
Trolox-equivalent (R2 ¼ 0.94) and signal strength, and used for deter­ diameter and 2.5 cm thickness were analyzed with a 25 mm diameter
mination of Trolox-equivalent of all laboratory scale hydrolysates. aluminium probe using a pre-test, test and post-test speed of 1.7 mm/s,
trigger force of 5 g and 40% sample deformation). Two bread loaves
2.5. Determination of soluble carbohydrates, protein and molecular from each baking trial were frozen in plastic bags for subsequent sensory
weight distribution evaluation.

Monomeric sugars of syrups and compositional analysis samples 2.7. Sensory evaluation of bread
were determined by HPAEC with pulse amperometric detection (Dionex
ICS 3000 equipped with CarboPac PA1 column). Total sugars in syrups Bread loaves were defrosted at 6 � C overnight. Sensory evaluations of
were determined after hydrolysis with 4% H2SO4 for 1 h at 120 � C and the three bread samples (1) control bread, 2) H2O-syrup bread and 3)
oligomeric sugars were determined as the difference of total and KOH syrup bread were carried out by a trained panel with proven skills
monomeric sugars. Protein content of the syrups from the scale-up re­ at the sensory laboratory of VTT, which fulfils the requirements of the
actions were calculated from the total nitrogen content (N ¼ 6.25) ISO standards (ISO 2007 and 2017). The protocol for performing the
measured using a Kjeldahl autoanalyzer (Foss Tecator Ab, Ho €gana€s, sensory evaluation has been accepted by the Ethical Committee of VTT
Sweden). The sugar and protein yields of the high consistency scale-up (see Supplementary material). Panelists were VTT employees belonging
reactions were calculated from the total liquid phase volume Vl of the to in-house food and beverage sensory panel, and all assessors gave their
hydrolysate, which was calculated from the initial water mass mw by prior informed consent to be involved in the trial, and that they had been
correcting with hydrolysate supernatant density ρ, kg/L and DM content made aware that the enzyme preparation (0.0026 ml/g bread) used in
(dmÞ, kg/kg according to Eq. (2). baking did not have approval for food use, and that the enzyme itself was
mw inactivated in the process. Furthermore, the panelists were instructed
Vl ¼ (2) not to swallow the samples. In accordance with EU General Data Pro­
ρ�ð1 dmÞ
tection Regulation GDPR (2016/679), necessary individual information
Hydrolysis response to pretreatment conditions were illustrated as of the members of the panel is collected in the Data protection registry,
2nd degree polynomial response surfaces, fitted with Matlab R2015a and the panelists have also given their consent for this. The sensory
(Mathworks). panel consisted of 10 trained assessors. The sensory method was
Molecular weight distribution of the soluble compounds in the descriptive analysis (Lawless and Heymann, 2010), the evaluated at­
syrups was analyzed by size exclusion chromatography (SEC) using PSS tributes being sweetness, saltiness, flavour balance, intensity of possible
MCX 1000 & 100000 Å columns with a pre-column, with 0.1 M NaOH as off-flavour, and aftertaste intensity. The attribute intensities (0–10)
the eluent (0.5 ml/min, 25 � C). The samples were diluted with 0.1 M were rated on continuous graphical intensity scales, verbally anchored
NaOH and filtered (0.45 μm) before the measurement. Two different from both ends, where 0 ¼ attribute not existing, 10 ¼ attribute very
detectors were used, including Waters 2414 Refractive index detector clear. The sample slices were served to the assessors coded with
for total solubles and Waters 2998 Photodiode Array detector at 280 nm three-digit numbers in random order in two replicate sessions. Water
for detecting phenolic compounds. The molar mass distributions (MMD) was provided to the assessors for cleansing the palate between the
were calculated against polystyrene sulphonate (10 x PSS, samples. The scores were recorded and collected using a computerized
1600–267200 g/mol) standards by UV and against 8 x pullulan Compusense Five data system, Ver. 5.6 (Compusense, Guelph, Canada).
(6100–708000 g/mol) standards by RI, using Waters Empower 3
software. 2.8. Statistical analysis

2.6. Baking trials Data from pretreatments, baking trials and sensory evaluation were
subjected to analysis of variance using IBM SPSS Statistics, Ver. 25 (IBM
Three different breads were prepared: 1) control bread (with sugar), Corporation, New York, USA), and statistically significant differences (p
2) H2O-syrup bread and 3) KOH-syrup bread. Optimal dough water < 0.05 unless stated otherwise) between individual means were iden­
contents for the flour and for the flour-syrup-mixtures were determined tified by Tukey’s test. For pairwise comparison to a reference, Student’s
by Farinograph analyses (the water content required to reach FU-value T-test was applied.
of 500) and by test bakings. The recipe for the control bread was (as % of
flour weight) flour 100, water 59.6, salt 1.5, sugar 2.0, yeast 5.0 and fat 2.9. Techno-economic analysis (TEA)
6.0. In the syrup breads, sugar and part of water were replaced by the
syrups. The syrup was dosed based on its content of glucose and fructose, Conceptual-level TEA of the bran syruping process was conducted,
as these are readily fermentable by baker’s yeast. In addition, part of salt assuming a production plant capacity of 30 t (metric ton) wheat bran/

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V. Pihlajaniemi et al. Journal of Cereal Science 95 (2020) 103043

day. The plant was assumed to be located at an existing syrup 3. Results and discussion
manufacturing facility, exploiting partially existing equipment and re­
sources. Mass and energy balances were simulated using a steady-state 3.1. Effect of KOH-pretreatment on bran hydrolyzability
computer simulation software Balas®. The simulated process consisted
of pretreatment (with or without KOH) and hydrolysis in a single A syruping process was developed for wheat bran and optimized as a
reactor, followed by separation of syrup with two decanter centrifuges in two-step reaction, starting with alkaline pretreatment with KOH, fol­
series with washing between (1 kg water/1 kg DM) and concentration to lowed by enzymatic hydrolysis of carbohydrates. The alkaline pre­
70% DM by evaporation. The process parameters and yields were based treatment (0–10% KOH/DM, 60–120 � C) and subsequent enzymatic
on the scale-up experiments, with the cellulase dosage decreased to 5 hydrolysis (10 mg/g cellulase, 0.05 mg/g amylase and 2.5 mg/g amy­
mg/g DM and pretreatment time with 4% KOH reduced to 12 h. loglucosidase) led to total sugar yields of 58.2–82.1% of bran carbohy­
Investment costs are presented in table Table S3 (Supplementary drates, and the maximum yield was obtained with the 10% KOH dosage
material), including combined pretreatment and enzymatic hydrolysis at maximum treatment severity (Table 1). Sugar yields of 72.5–77.4%
reactor, buffer tank for the bran hydrolysate and decanter centrifuges. were reached at a KOH dosage of 4% at 90 � C or above. Pretreatment
Tanks and reactors were assumed to be purchased as used equipment. considerably increased the total sugar yield compared to the 40.2% yield
Syrup concentration step was expected to utilize existing evaporator from hydrolysis of untreated bran, where the low glucose release
capacity at the syrup manufacturer facility. showed incomplete starch hydrolysis (<66.7%), accompanied by low
Production costs are presented in Table S4 (Supplementary mate­ release of hemicellulosic oligomers. On the other hand, relatively high
rial). Electricity consumption of S/L-separation was 3 kWh/t. Steam and total sugar yields up to 60.0% and 65.6% were obtained after aqueous
electricity consumptions during syrup concentration were 2.4 t and 0.23 heat treatment without KOH at 60 � C and 90 � C, respectively, and the
kWh/t of evaporated water, respectively. Heating and cooling duties difference of the total sugar yield after pretreatment at 90 � C with or
during pretreatment or enzymatic hydrolysis and heat recovery were not without 4% KOH was not statistically significant. Monomeric sugar
included. yields ranged from 44.4% to 55.3% with pretreated bran, showing only
The total capital investment of the functional bran syrup process small and mostly statistically insignificant differences between pre­
consisted of the fixed capital investment (FCI), including the purchased treatment conditions. In contrast, the yield of oligomeric arabinoxylan
equipment cost and the construction cost of the plant, and a working was significantly increased in correlation with severity and KOH-
capital of 6.5% of the FCI. The FCI was estimated using a factor of 1.6 as loading, so that up to 38% of the total sugar yield was composed of
the ratio of FCI to the sum of purchased equipment cost (Lang-factor), as oligomeric sugars. Analysis of selected samples directly after 90 � C
suggested for food industry (Marouli and Maroulis, 2005). The fixed pretreatment suggested that the majority of oligomers were released
capital investment was annualized with a 10-year life time, 94% plant during hydrolysis, whereas only 15.3% total and 2.7% monomeric sugar
utilization degree and 5% interest rate. The total annual production cost dissolution occurred in average during the pretreatments (Fig. S3,
(TAPC) comprise the raw material, utility and labour cost (five opera­ Supplementary material).
tors), maintenance, insurance and capital charges (Table S4, Supple­ The glucose yields reached after pretreatment with 10% KOH-dosage
mentary material). The solid residue was assumed to be sold as animal were similar to previous reports on alkaline peroxide extraction (Maes
feed. The minimum selling price of syrup was defined as the price at and Delcour, 2001) and on hydrothermal treatment followed by enzy­
which the annual revenue equals TAPC. matic hydrolysis (Reisinger et al., 2013), whereas somewhat higher
arabinoxylan yield (76%) was reached in the current study compared to
the previous reports (~70% yields). A clearly smaller arabinoxylan yield
(50%) was reported by Hollmann and Lindhauer (2005) in a scale-up
study of alkali peroxide extraction.
The arabinose to xylan ratio (A/X) of the hydrolysate after the lowest

Table 1
Total, oligomeric and monomeric yields of total carbohydrates, glucose (G) and arabinoxylan (AX), as % of each component in bran. Arabinose to xylose ratio (A/X)
and released antioxidants as Trolox-equivalent, μmol/g bran DM. Letters under total yields indicate statistically different groups (p < 0.05), i.e. total yields sharing a
letter do not differ significantly.
Temp No 60 � C 90 � C 120 � C

Time Treat- 1h 24 h 1h 24 h 1h
KOH ment 0% 2% 10% 4% 0% 2% 4% 0% 2% 4% 10% 4%
Log(R0) 0a 0.60 0.60 0.60 1.98 1.48 1.48 1.48 2.86 2.86 2.86 2.86 2.37
Total sugars 40.21 60.13 58.19 69.24 63.19 65.64 64.08 72.50 55.79 65.06 74.30 82.06 77.43
a bcd bc cdefg bcde bcdef bcde defg b bcdef efg g fg
G 37.25 74.81 72.83 82.42 69.43 79.25 78.08 86.13 65.14 79.21 86.72 85.34 87.55
AX 35.94 42.28 40.70 53.30 53.85 49.00 47.30 54.37 43.92 47.84 59.66 75.68 65.68
A/X 0.41 0.32 0.35 0.50 0.48 0.35 0.37 0.46 0.37 0.43 0.51 0.68 0.58
Total oligomers 5.65 11.89 14.70 22.04 21.30 14.79 21.22 22.63 12.84 22.72 25.79 31.05 27.03
a ab abc bcd bcd abc bcd bcd abc bcd bcd d cd
G 2.88 7.64 5.72 7.02 5.94 3.13 12.29 8.38 2.22 13.68 10.06 1.40 7.37
AX 12.82 20.35 23.82 33.29 33.01 22.93 26.43 30.71 20.88 28.01 36.87 56.01 43.40
A/X 0.38 0.53 0.52 0.79 0.79 0.68 0.65 0.71 0.75 0.69 0.79 0.93 0.87
Total monomers 34.56 48.24 44.44 51.54 46.44 54.36 46.13 54.36 46.97 46.20 51.98 55.28 54.48
a ab ab b ab b ab b ab ab b b b
G 34.37 67.17 67.11 75.40 63.49 76.12 65.79 77.75 62.91 65.53 76.66 83.93 80.18
AX 23.12 21.93 16.88 20.02 20.84 26.06 20.87 23.67 23.04 19.83 22.78 19.67 22.28
A/X 0.43 0.17 0.16 0.19 0.17 0.15 0.13 0.23 0.14 0.17 0.20 0.22 0.22
Antioxidants 7.1 6.3 6.6 14.8 12.7 7.4 8.4 11.6 9.0 9.5 11.2 27.7 13.1
a a a e cde a ab bcde ab abc bcd f de
Number of repetitions 2 4 4 2 2 2 3 2 2 2 8 2 2
a
Log(R0) is not defined for the untreated sample and was assigned the value of 0.

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V. Pihlajaniemi et al. Journal of Cereal Science 95 (2020) 103043

severity treatment without KOH was 0.31, corresponding to the A/X of bran antioxidants were dissolved by the 10% KOH-treatment at
ratio of arabinoxylan in the aleurone layer (Antoine et al., 2003). The maximum severity. Hydrolysis can also increase antioxidant content of
A/X was increased up to 0.67 with increasing treatment severity and the solid residue of bran (Zhang et al., 2019), suggesting overall increase
KOH-loading, together with increased release of antioxidants, as shown in antioxidant capacity by degradation.
in 3.2. This indicates that the KOH-treatment particularly improved the
hydrolysis of the outer pericarp arabinoxylan (A/X of 1.14 (Antoine
3.3. Characterization of enzymatic hydrolysis
et al., 2003)) through cleavage of the esterified substituents, including
ferulic ester crosslinks.
In order to screen for improvement potential in the enzymatic hy­
The sugar yields showed consistent responses to the thermal severity
drolysis step, the effect of different hydrolysis parameters was studied
factor Log(R0) (Fig. 1), indicating that the reaction temperature and
after a constant pretreatment with a KOH-dosage of 4% of DM at 90 � C
time were somewhat interchangeable. This could provide flexibility for
for 24 h. First, the effect of varying the cellulase dosage and hydrolysis
technical implementation and asepticity control of the process.
time was determined. Hydrolysis without cellulases showed glucose
Increasing the severity and alkali dosage promoted the release of olig­
release of 24% in 24 h, corresponding to nearly complete starch hy­
omeric arabinoxylan which was not monomerized during hydrolysis. A
drolysis (Fig. 2A). Hemicellulosic oligomers were additionally released,
similar extent of unhydrolyzable soluble hemicellulosic oligomers have
reaching to a total sugar yield of as high as 50% and 65% after 24 h and
also been observed after low-severity hydrothermal treatments (160 � C
96 h of hydrolysis, respectively. Sugar yield showed a positive response
or below) of wheat bran (Reisinger et al., 2013), and accordingly, van
to cellulase dosage in the range of 2.5–10 mg/g, whereas 20 mg/g
den Borne et al. (2012) reported only a minor effect of hydrothermal
showed only a small further improvement. Maximum carbohydrate
severity on enzymatic monomerization of wheat bran xylan. The present
solubilisation of 76% was reached in 24 h with cellulase dosages of 10
data indicated relatively high A/X ratios (up to 0.96) in the unhydro­
and 20 mg/g, whereas similar yields could also be reached with 2.5 mg/
lyzed oligomers, and maximum 19% monomerization of bran arabinose.
g and 5 mg/g when hydrolysis time was increased to 96 h. The maximum
This proposes that the A-X linkages at AX branching sites were not hy­
monomeric sugar yield (57%) was reached in 96 h with the enzyme
drolyzed, increasing the oligomer yield. In accordance, a high level of
dosage of 10 mg/g (Fig. 2B). These results suggest that the cellulase
arabinose substitution in known to limit the efficacy of enzymatic hy­
dosage could be set in the range of 2.5–5 mg/g in order to minimize
drolysis of arabinoxylan (McCleary et al., 2015). This also indicates that
enzyme costs without compromising total sugar yield. This corresponds
no extensive cleavage of arabinose substituents took place during
to a dosage of 23–47 mg/g cellulose, which is comparable with the
alkaline treatment, in line with previous alkaline peroxide extraction of
dosage of ~20 mg/g cellulose considered adequate for materials with a
arabinoxylan by (Hollmann and Lindhauer, 2005), although loss of
higher lignocellulose content (Klein-Marcuschamer et al., 2012).
substitution has been associated with analytical strong alkali extractions
Doubling the dosage of amylolytic enzymes did not improve hydro­
(Mandalari et al., 2005).
lysis of KOH-pretreated bran (Fig. 2C). An additional 5 mg/g xylanase
(Depol 740) led to a small but significant increase in arabinoxylan
3.2. Antioxidant activity of bran syrups dissolution and monomeric sugar yield, compared to the reference. The
release of antioxidants was also increased by xylanase addition, sug­
Antioxidant activity was determined from the bran hydrolysates as gesting further ferulic acid release parallel with arabinoxylan hydroly­
Trolox-equivalent, μmol/g bran DM. Trolox-equivalent correlated posi­ sis. Arabinoxylan dissolution ranged from 55% to 62% with or without
tively with pretreatment severity, and particularly with KOH-dosage additional xylanases or amylolytic enzymes, whereas previously less
(Fig. 1C), reaching the maximum of 27.7 μmol/g with 10% KOH, than half of arabinoxylan could be dissolved by xylanases alone
while up to 13.1 μmol/g and 9.0 μmol/g were reached with 4% KOH and (Petersson et al., 2013), indicating that the presence of cellulases im­
0% KOH, respectively (Table 1). Bound ferulic acid is the major anti­ proves arabinoxylan release from bran. Generally, it can be stated that
oxidant compound in bran, and it can be efficiently liberated by alkaline sugar yields were not significantly improved by further addition of en­
hydrolysis (Antoine et al., 2003), suggesting that ferulic acid was a zymes, whereas further optimization could be targeted at reducing
major factor in increasing syrup’s antioxidant activity in the present enzyme consumption and improving the release of antioxidants.
study. Besides ferulic acid and other phenolic acids, dissolved Performing starch hydrolysis as a separate process step was studied,
lignin-derived phenolics can contribute to increased antioxidant activ­ since it would allow application of thermostable amylolytic enzymes.
ity, as alkali-degraded lignin also carries antioxidant properties (Kaur However, starch pre-hydrolysis of 5 h prior to the addition of cellulases
and Uppal, 2015). Previously reported Trolox-equivalents from different had an adverse effect total sugar yield (Fig. 2C). This suggests that cel­
native wheat bran samples have been obtained by extraction with polar lulases and amylolytic enzymes work synergistically during bran hy­
solvents, showing maxima of 33.1 μmol/g (Brewer et al., 2014) and 15.3 drolysis, while pre-hydrolysis of starch may lead to higher product
μmol/g or 60.0 μmol/g depending on the radical system used for anal­ inhibition of lignocellulolytic enzymes. Starch may also restrict enzyme
ysis (Zhou and Yu, 2004). These numbers suggest that a large proportion access to the lignin rich intermediate layers of bran (Antoine et al.,

Fig. 1. Total sugar yield (A), oligomeric sugar yield (B) and antioxidant release (C) after enzymatic hydrolysis of pretreated wheat bran as a function of pretreatment
severity and KOH-loading. The response surfaces present a 2nd degree polynomial fit to the experimental averages shown as dots, and the black bars represent
distance from surface (residuals).

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V. Pihlajaniemi et al. Journal of Cereal Science 95 (2020) 103043

3.4. Scale-up of the bran syruping process

In order to produce bran syrup for baking trials, the syruping process
was scaled up to 10 L scale for the two distinct pretreatment conditions
with good yields in the laboratory scale experiments, including treat­
ment with 4% KOH for 24 h at 90 � C (“KOH-syrup”) and water for 1 h at
90 � C (“H2O-syrup”). Detailed syrup compositions are presented in
Table S1 (Supplementary material). The total and oligomeric sugar
yields of the scale-up reactions were similar to those of the laboratory
scale reactions (Fig. 3), and thus, the higher consistency (20%) in the
scale-up compared to the laboratory scale reactions (15%) did not have
an apparent effect on the sugar yields. In the KOH-syrup, total and
oligomeric sugar yields of 77% and 28% were reached, whereas the
corresponding yields in the H2O-syrup were 67% and 14%, respectively.
The scale-up reactions showed lower antioxidant yields compared to the
laboratory scale reactions, for which no apparent explanation is
currently available. Still the KOH treatment showed a higher antioxidant
release compared to the water-only reaction in both cases.
For further characterization of the oligomers in the KOH- and H2O-
syrup, their molecular weight (Mw) distribution was studied by size
exclusion chromatography. According to refractive index, both syrups
showed a major peak at the monomeric sugar region (Fig. S4, Supple­
mentary material), and a broader signal ranging molecular weights
corresponding to arabinoxylan oligomers with a degree of polymeriza­
tion (dp) of approximately 2–16 units. In both syrups the oligomeric
range showed a peak corresponding to approximately five pentose units.
This peak, however, coincided with the single peak in the UV-signal,
suggesting that phenolics such as ferulic acid may be incorporated in
the oligomers. The 4% KOH-syrup showed a broad shift towards larger
molecular weight oligomers compared to the H2O-syrup.
Besides the carbohydrates, the solubilisation of proteins from the
scale-up samples was analyzed. The alkaline conditions with 4% KOH
led dissolution of 63% of bran protein (excluding enzyme protein
addition), while the water-only process dissolved 38% of protein, in
accordance with the previously reported increase in the solubility of
bran protein in alkaline conditions (Idris et al., 2003). Protein could be a
desirable side-product of the process and several studies have assessed
wheat bran protein utilization using protease enzymes and thermo­
chemical treatments (Arte et al., 2016; Idris et al., 2003; van den Borne
et al., 2012).

Fig. 2. Effect of cellulase dosage on the total (A) and monomeric (B) sugar yield
from KOH-pretreated bran as sugar yields from total bran carbohydrates. The
effect of additional amylases and xylanases, a separate destarching step and the
combination of each on 24 h hydrolysis yields of KOH-pretreated wheat bran
(C). Sugar yields are presented as % from original bran carbohydrates and
antioxidant capacity as Trolox-equivalent (mg/g bran DM). Error bars repre­
sent � standard error.

Fig. 3. Scalability of bran syruping process, showing sugar yields and antiox­
2003), initially reducing harmful cellulase interactions with lignin. idant yield (Trolox-equivalent) in scale-up (single reactions) and laboratory
Combining additional amylase and xylanase with separate destarching scale reactions. Error bars represent � standard error of laboratory scale ex­
did not show further improvement. periments; n ¼ 8 for 4% KOH and n ¼ 2 for reactions without KOH. Laboratory
scale experiments showed statistically significant (p < 0.05) differences be­
tween the two process conditions in the yields of oligomeric sugars and anti­
oxidants, but not for total sugars.

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V. Pihlajaniemi et al. Journal of Cereal Science 95 (2020) 103043

3.5. Effect of bran syrups on the baking quality of wheat bread breads, for example, wholegrain and sourdough breads. Furthermore,
the use of the syrups as a fibre and antioxidant enriched ingredient is not
The aim of the baking experiments was to examine the applicability limited to bread and could be considered particularly for food applica­
of the produced syrups in wheat bread baking by substituting sugar with tions where smoky and tar-like nuances of the KOH-syrup could be
the KOH-syrup or the H2O-syrup. Based on the Farinograph analyses, the desirable.
optimal water contents of the control dough, H2O-syrup dough and
KOH-syrup dough were 59.6%, 61.5% and 66.0% (of flour weight of the 3.7. Utilizing KOH-syrup to reduce NaCl in bread
control dough), respectively. However, for the final baking trials the
same dough water content (59.6%) was used for all bread types, which A notable viewpoint on the composition of the KOH-syrup is the
was found better in terms of dough processability (lower stickiness) and content of KCl that can be utilized as salt source for partially replacing
product quality compared to the 66% water content in test baking of the NaCl in bread. The cost of chemical recycling normally constrains the
KOH-syrup bread. The high Farinograph result for KOH syrup dough use of alkaline pretreatments in lignocellulosic biorefineries (Chen et al.,
could be due to the high level of oligosaccharides, which may have 2013). Therefore, the ability of completely utilizing the neutralized
increased viscosity and torque during the Farinograph analysis. pretreatment chemical in the product is an important synergistic
There was no statistically significant (p < 0.05) difference in weight advantage of the bran syruping process. KCl is used in conjunction with
loss between the control, H2O-yrup and KOH-syrup breads (12.2 � 1.0 NaCl in commercial low-salt applications, and it has been reported that
(standard deviation), 11.9 � 0.4 and 12.5 � 0.8, respectively). KOH- replacing 30% of NaCl by K-as in white bread did not alter sensory
syrup bread had a significantly higher specific volume (4.9 � 0.2 ml/ acceptability of white bread compared to 1.8% NaCl of flour weight
g) compared to the H2O-syrup and control bread (4.6 � 0.2 and 4.5 � (Braschi et al., 2009). This suggests that about 0.5% of KCl of flour
0.2, respectively). The hardness of bread crumb was 34.8 � 4.0, 32.2 � weight can be added without introducing a bitter taste of KCl to wheat
3.6 and 26.9 � 3.4 for the control, H2O-syrup and KOH syrup breads, bread. This limit was not exceeded in the present study, as the KCl
respectively, showing that the KOH syrup bread was significantly (p < addition was 0.45% of flour weight in the KOH-syrup bread and
0.01) softer than the other breads. Both syrups, particularly the KOH- substituted 29.6% of NaCl in the recipe. However, this does not include
syrup, introduced a darker colour to the bread, as the observed light­ potential dissolution of ash components of bran, which may include
ness values for the control, H2O-syrup and KOH-syrup breads were 72 � potassium and magnesium (Reisinger et al., 2013). Nevertheless, no
2, 64 � 3 and 61 � 2 for the bread crust and 81 � 1, 79 � 1 and 71 � 1 changes in saltiness were observed, while the other observed flavour
for the bread crumb, respectively. Generally, the data indicated that it deviations likely resulted from treatment-derived hydrolysis products e.
was possible to produce technically acceptable wheat bread with both g. phenolic compounds and amino acids, rather than KCl.
bran syrups and the quality differences between the breads were rather
small. 3.8. Techno-economic analysis of the syruping process

An industrial scale bran syrup production process was designed and

3.6. Effect of bran syrup on the sensory quality of wheat bread simulated for the KOH-syrup and H2O-syrup. The total capital invest­
ment was estimated to be 980,000 € (95 €/annual ton of bran) and
The sensory characteristics of the control bread and the H2O-syrup 830,000 € (80 €/annual ton of bran) for KOH-syrup and H2O-syrup,
bread were not deviating significantly from each other in any evaluated respectively. The total annual production cost (TAPC) for KOH-syrup
attribute, suggesting that the H2O-syrup would be directly applicable in and H2O-syrup were 5,015,000 €/a and 4,463,000 €/a (Table S3, Sup­
wheat baking (Fig. 4). The KOH syrup breads showed to have statisti­ plementary material). The annual production rates for KOH-syrup and
cally significantly less balanced flavor (p < 0.01) and have more intense H2O-syrup were 7490 t and 4930 t, resulting in a unit production cost of
off-flavor (p < 0.001) and aftertaste (p < 0.01) than the other breads. 670 €/t and 905 €/t, respectively. The cost distribution of TAPC is
The off-flavor was described as being musty, pungent, bitter, yeast-like, presented in Fig. 5A and B, showing that the largest part of the TAPC
tar-like, smoky and unbalanced. Any of the breads did not deviate sta­ were enzyme cost (37–42%), bran cost (24–27%) and energy (21%). It
tistically significantly from each other in sweetness or saltiness. Since can be concluded that optimizing the enzyme dosage is an important
the KOH-syrup led to observable flavor deviations in the rather mild- parameter whereas the capital charge only has a minor effect on eco­
flavored wheat bread, it could be more suitable for more flavor-rich nomic feasibility of the process.
The minimum selling price for the investment to break even was 766
€/t for KOH-syrup and 1029 €/t for H2O-syrup (Table S5, Supplementary
material). These are considerably higher than the market selling price
estimates of 400–650 €/t (Da �vila et al., 2014) for normal glucose syrup.
While a direct price reference for a functional syrup could not be found,
additional value is expected to result from the content of
arabinoxylo-oligomers, antioxidants, and in the case of KOH-syrup, KCl.
For example, value of arabinoxylan is ~7000 €/t (Misailidis et al.,
2009). The additional value resulting from the arabinoxylan content
(13% and 8%) would be 910 €/t for KOH-syrup and 560 €/t for
H2O-syrup. In this light, there is potential for economic feasibility of the
concept.
Sensitivity analysis was carried out on the major variables affecting
the profitability of the process (Fig. 5C and D). Variation in enzyme and
raw material costs has a considerable impact on the unit production cost,
indicating that further optimization of the hydrolysis step could be
beneficial by reducing enzyme consumption and improving the effi­
ciency of raw material utilization. The variation in cost of steam also has
a notable effect on the unit production cost, whereas changes in labour
Fig. 4. Sensory evaluation of the bran syrup wheat breads, n ¼ 2 � 10. Sig­ cost and capital charges did not affect the unit production cost of syrup
nificance level of difference (p) or no difference (ns). remarkably.

7
V. Pihlajaniemi et al. Journal of Cereal Science 95 (2020) 103043

Fig. 5. Cost distribution of total annual production cost of a bran syruping plant producing KOH-syrup (A) and water-syrup (B). Sensitivity analysis of the unit
production cost of KOH-syrup (C) and water-syrup (D) on key economic parameters.

4. Conclusions Writing - original draft, Writing - review & editing. Outi Mattila:
Investigation, Writing - original draft. Taru Koitto: Investigation.
A process consisting of alkaline pretreatment and subsequent enzy­ Markus Nikinmaa: Investigation, Writing - original draft. Raija-Liisa
matic hydrolysis was found effective for producing wheat bran syrup Heinio€ : Investigation, Writing - original draft. Lotta Sorsama
€ki: Formal
high in antioxidants and arabinoxylo-oligomers. KOH-pretreatment analysis, Writing - original draft. Matti Siika-aho: Supervision,
improved total carbohydrate yield as well as antioxidant release from Conceptualization, Funding acquisition, Writing - review & editing.
bran, and it was also found to increase the proportion of arabinoxylo- Emilia Nordlund: Supervision, Conceptualization, Funding acquisition,
oligomers in the syrup, compared to pretreatment with water. The Writing - review & editing.
KOH used in the syruping process could be completely utilized as a
source of salt in the breadmaking product, allowing partial replacement Acknowledgements
of Na with K in the bread recipe. However, syrup from the KOH-process
generated flavour deviations in the bread in baking trials of wheat Business Finland is acknowledged for funding of this work (Waste­
bread, and thus, the syrup from the KOH-process is postulated to suit bake-project). We wish to thank Atte Mikkelson, Marita Ikonen, Ulla
better for products having a higher taste intensity. The economic po­ Vornamo, Eero Mattila, Leila Kostamo and Riitta Pasanen for excellent
tential of the process relies on the valuation of arabinoxylan and anti­ technical assistance.
oxidants, encouraging further application studies on functional bran
syrup. Appendix A. Supplementary data

Declaration of competing interest Supplementary data to this article can be found online at https://doi.
org/10.1016/j.jcs.2020.103043.
The authors declare that no competing interests exist regarding this
work. References

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