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126 CEREAL CHEMISTRY
Fermented Wheat Bran as a Functional Ingredient in Baking
Kati Katina,1,2 Riikka Juvonen,1 Arja Laitila,1 Laura Flander,1 Emilia Nordlund,1 Susanna Kariluoto,3
Vieno Piironen,3 and Kaisa Poutanen1
ABSTRACT Cereal Chem. 89(2):126–134
The aim of the current study was to identify factors influencing the
technological functionality of fermented bran. The influences of fermen-
tation type and type of wheat bran on the microbial community, bioactiv-
ity, arabinoxylans (AX), and activity of xylanases were studied in the
bran ferments. Furthermore, technological quality of ferments was estab-
lished by using them to replace wheat in baking with a 20% substitution
level. Solubilization of AX and endogenous xylanase activity of bran
were influenced by the type of bran, fermentation type, and conditions.
Peeled bran had a clearly reduced microbial load and different microbial
community in comparison to native bran. Bran from peeled kernels con-
tained 10-fold lower activities of endogenous xylanases in comparison to
native bran. Yeast fermentation of bran from peeled kernels increased the
level of folates (+40%), free phenolic acids (+500%), and soluble AX
(+60%). Bread containing yeast-fermented peeled bran had improved vol-
ume (+10–15%) and crumb softness (25–35% softer) in comparison to un-
fermented counterparts. Solubilization of AX during the 20 hr fermenta-
tion and decreased endogenous xylanase activity are proposed as the main
reasons for the improved technological functionality of fermented bran.
Consumption of foods rich in whole grains and cereal fiber has
been shown to reduce the risk of obesity, diabetes, inflammation,
cardiovascular disease, and some types of cancer in epidemiologi-
cal studies (Slavin 2003; De Munter et al 2007; Anderson et al
2009). However, there is a wide gap between the dietary recom-
mendations and current intake of whole grains and dietary fiber
(Lang and Jebb 2003). The real challenge is to develop technolo-
gies that lead to lower consumer barriers to frequent use of high-
fiber and whole-grain foods.
One of the most common raw materials for increasing the level
of dietary fiber in baking is wheat bran. Dietary fiber and bioac-
tive compounds such as alkylresorcinols, lignans, phenolic acids,
phytosterols, tocopherols, tocotrienols, and folates are concen-
trated in the bran fraction of cereals (Liukkonen et al 2003;
Kamal-Eldin et al 2009). Thus, bran can be considered a superior
raw material for development of nutritionally optimized cereal
foods and new ingredients. The main part of dietary fiber in bran
is insoluble, which influences the digestibility and bioavailability
of nutrients and phytochemicals. The outer layers of grain contain
cellulose and lignin, which have negative influence on the taste
and mouthfeel of the bran. The properties of bran thus restrict its
full exploitation in different consumer foods. Use of native bran
in wheat baking is a technological challenge because of the detri-
mental effect of bran on the gluten network and subsequent tex-
tural attributes of bread (Noort et al 2010).
Fermentation with well-characterized starter cultures, yeast or
lactic acid bacteria (LAB), is a potential means to improve the
palatability and processability of brans and whole-meal flours
(Salmenkallio-Marttila et al 2001). Furthermore, bran fermenta-
tion could assist in the management of indigenous microbes and
improvement of the microbiological safety of bran. Brans con-
tain more microbes and their metabolites than do endosperm
flours, and they could also be a source of spoilage bacteria and
fungi (Rosenkvist and Hansen 1995). Cereal fermentations are
always combined actions of added starter cultures and the indige-
nous microbes naturally present in grains. Therefore, the knowl-
edge of microbial dynamics during bran fermentations is highly
important.
The endogenous and microbial enzymes are concentrated in the
outer grain layers (Gys et al 2004; Dornez et al 2006b) and have
thus the possibility for action during the bran fermentation. Both
acidification of the matrix and especially activity of xylan-degrad-
ing enzymes were shown to contribute to the solubilization of
arabinoxylans (AX) during the baking process of whole-meal rye
(Boskov Hansen et al 2002). Solubilization of pentosans and es-
pecially transformation of water-unextractable AX to water-extract-
able AX have been reported to improve bread volume and texture
in wheat baking (Courtin and Delcour 2002), and it is assumed
also to take place in high-fiber wheat baking (Katina et al 2006).
Our previous study showed that the solubilization of pentosans in
rye bran fermentations was dependent on the type of bran as well
as on fermentation conditions (Katina et al 2007a). The fate of
AX, however, has not yet been studied in wheat bran fermenta-
tions. Furthermore, the role of different indigenous microbes in
bran modifications has not yet been clarified.
Many biochemical changes that influence nutritional quality as
well as the texture and flavor of wheat flour occur during sour-
dough baking, as reviewed by Katina et al (2005) and Poutanen et
al (2009). Levels of folate and easily extractable phenolic com-
pounds increase (Liukkonen et al 2003; Kariluoto et al 2004),
whereas levels of phytate (Lioger et al 2007), alkylresorcinols,
and tocopherols decrease in sourdough baking processes. Fermen-
tation of rye bran has significantly increased the level of folates
and free ferulic acid (Katina et al 2007a). These biochemical
changes may have significant impact on nutritional properties of
bran; for example, fermented bran was recently shown to have
improved bioavailability of ferulic acid both in vitro and in vivo
(Mateo Anson et al 2009, 2010).
The goal of the current study was to identify factors influencing
wheat bran fermentation, with the ultimate aim of using fer-
mented bran as a functional ingredient in wheat baking. In par-
ticular, our aim was to establish the link between the state of AX
and endogenous xylanases of wheat bran and the technological
behavior of bran in baking. Furthermore, we wanted to assess the
role of the microbial community in bran modifications.
MATERIALS AND METHODS
Raw Materials
Experiments were carried out with Finnish commercial wheat
bran fractionated from native kernel (Melia Ltd., Raisio, Finland)
and bran from peeled wheat kernels (Buhler, Uzwil, Switzerland).
* The e-Xtra logo stands for “electronic extra” and indicates that Fi
g
ures 1 and 2
appear in color online.
1 VTT Biotechnology and Food Research, P.O. Box 1500, FIN-02044 VTT, Finland.
2 Corresponding author. Phone: 358-405763426. Fax: 358-207227111. E-mail:
kati.katina@vtt.fi
3 Department of Food and Environmental Sciences, University of Helsinki, Finland.
http://dx.doi.org/10.1094/ CCHEM-08-11-0106
© 2012 AACC International, Inc.
e-Xt
r
a*
Vol. 89, No. 2, 2012 127
Approximately 3.5% of the wheat kernel was peeled off before
the bran separation. Commercial white wheat flour (0.6% ash
content, 13.2% protein content) for baking was obtained from
Helsingin Mylly (Helsinki, Finland), and the fresh yeast used in
bran fermentations was obtained from Suomen Hiiva (Lahti,
Finland). Instant active dry yeast (Fermipan Red, Gist-Brocades,
Delft, The Netherlands), table salt, sugar (Suomen Sokeri,
Finland), and emulsifier Panodan M2020 (Danisco Ingredients,
Copenhagen) were used in baking.
Bran Fermentations
Eight different types of bran fermentations were studied: 1) na-
tive bran fermented with yeast at 20°C for 20 hr; 2) native bran
fermented with yeast at 32°C for 20 hr; 3) bran from peeled ker-
nels fermented with yeast at 20°C for 20 hr; 4) bran from peeled
kernels fermented with yeast at 32°C for 20 hr; 5) native bran
fermented without yeast starter (spontaneous fermentation) at
20°C for 20 hr; 6) native bran fermented without yeast starter
(spontaneous fermentation) at 32°C for 20 hr; 7) bran from peeled
kernels fermented without yeast starter (spontaneous fermenta-
tion) at 20°C for 20 hr; and 8) bran from peeled kernels fermented
without yeast starter (spontaneous fermentation) at 32°C for 20
hr. Conditions were chosen based on modeling of rye bran fer-
mentation to present two extreme conditions for yeast-started bran
fermentations (Katina et al 2007b).
Dough yield of 450 g was obtained by mixing 600 g of wheat
bran (native or peeled), 2,100 g of tap water, and 7.5 g of fresh
baker’s yeast (107 colony-forming units [CFU] per gram) (Suomen
Hiiva, Lahti, Finland) in a large beaker, which was covered with
aluminum foil and fermented for 20 hr at 20 or 32°C. Spontane-
ous fermentations were carried out similarly but without yeast
addition. Fresh samples were taken from unfermented and fer-
mented bran for microbiological analyses. In addition, samples
were frozen for later measurements of pH and total titratable acid-
ity (TTA). Fermented bran samples were also freeze-dried for
analysis of bioactive compounds. Fermentations were done in
duplicate and performed twice (so four times altogether).
Microbiological Analyses
Samples for microbiological analyses were taken from the
commercial baker’s yeast and from unfermented and fermented
brans. The following microbial groups were analyzed: aerobic
heterotrophic bacteria, LAB, yeasts, and molds. The bran samples
of 10 g were homogenized for 10 min with 90 mL of sterile saline
in a Stomacher 400 lab blender (Seward Medical, London) before
the cultivation. Aerobic heterotrophic bacteria were determined
on plate count agar (PCA, Difco Laboratories, Detroit, MI), which
was incubated under aerobic conditions at 30°C for two-to-three
days. LAB were cultivated on de Man–Rogosa–Sharpe (MRS) agar
(Oxoid, Basingstoke, U.K.) under anaerobic conditions at 30°C for
five days. PCA and MRS media were supplemented with 0.001%
cycloheximide (Sigma Chemical, St. Louis, MO) to prevent fun-
gal overgrowth of bacterial colonies. Yeast and molds were deter-
mined on YM agar (Difco Laboratories). Samples were incubated
under aerobic conditions at 25°C for three-to-five days. Chlortetra-
cycline and chloramphenicol (both at 0.01%) were added to the
medium to prevent bacterial growth, and 0.02% Triton-X 100
(BDH, Poole, U.K.) was used to limit the spreading of fungal colo-
nies. To determine viable counts of bacterial spores, 5 mL of ho-
mogenized sample was heated in a water bath of 80°C for 10 min
for the inactivation of vegetative cells. Aerobic spore-forming bac-
teria were enumerated on tryptone soy agar (Oxoid), and plates
were incubated at 30°C for three days. The bacteria and yeast re-
sults are expressed as colony-forming units per gram (CFU/g).
Isolation and Identification of Microbes
LAB and yeasts were isolated from unfermented and fermented
bran samples for species identification. LAB were isolated from
MRS plates. Ten colonies were randomly picked from each sam-
ple whenever possible. The bacterial isolates were characterized
by Gram-staining and the catalase test and were grouped by DNA
fingerprinting. Isolates representing different fingerprint types
were purified by successive subculturing on an antibiotic-free
MRS medium. DNA extraction, random amplification of poly-
morphic DNA (RAPD) polymerase chain reaction (PCR) with
OPA-2 primer (5′-TGCCGAGCTG-3′), and partial 16S rRNA
gene sequencing of pure cultures were performed as described by
Katina et al (2007a). Yeasts were selected based on colony mor-
phology (diameter, shape, color, and surface). The yeast isolates
were grouped by PCR fingerprinting with M13 microsatellite
primer (5′-GAGGGTGGCGGTTCT-3′), and representative strains
were identified by sequence analysis of the D1/D2 domain of the
26S rRNA gene as described by Laitila et al (2006). Similarity of
>99% to rRNA gene sequences was used as a criterion for species
identification.
pH and TTA
Frozen bran samples were thawed overnight in a refrigerator.
The pH value was measured from an aliquot of 10 g of fermented
bran blended with 100 mL of distilled water in a TitroLine autoti-
trator (Alpha 471217, Schott, Mainz, Germany). For the determi-
nation of TTA, this suspension was titrated with 0.1M NaOH to a
final pH of 8.5 with the TitroLine Alpha autotitrator. TTA was
expressed as the amount of NaOH used (mL). All samples were
analyzed in duplicate.
Chemical Analysis of Raw Materials
The dry matter contents of the wheat flour and bran were deter-
mined following AACC International Approved Method 44-15.02
(AACCI 2010). Dietary fiber content of brans was analyzed by
AOAC method 991.43 (Prosky et al 1988). AX content was
analyzed by the gas chromatographic method, as described by
Blakeney et al (1983), with corresponding standard compounds
and internal standard (myo-inositol) (method detailed later). Pro-
tein content was analyzed following the Kjeldahl method (AOAC
method 14.068 [1980]) and starch by AOAC method 996.11
(McCleary et al 1994); ash was analyzed gravimetrically as an
inorganic residue after burning samples at 550°C to remove water
and organic material. The chemical analyses were performed in
duplicate.
Analysis of Folates and Phenolic Compounds
Folates were analyzed by a microbiological assay method includ-
ing extraction and trienzyme treatment, as described by Kariluoto et
al (2004). Total phenolic acids were analyzed as alkaline and free
phenolic acids (ferulic acid, sinapic acid, and coumaric acid) af-
ter ethyl acetate extraction by HPLC (Bartolomé and Gómez-
Cordovés 1999). All analyses were done in duplicate, and the
obtained average values of bioactive compounds were expressed
on a dry weight basis.
Content of Total and Soluble AX
The water-extractable AX fraction was obtained by extracting
1 g of the cereal sample with 4 mL of cold water (4°C). After that,
the obtained water-soluble fraction was hydrolyzed with 1.2 mL
of 7.5N H2SO4 in a boiling water bath for 2 hr. To measure the
total amount of AX, 50 mg of cereal sample was prehydrolyzed
with 1.56 mL of 26N H2SO4 at room temperature (25°C) for 30
min. After that, samples were diluted with 15.6 mL of Milli-Q
water (Millipore, Billerica, MA) and hydrolyzed in a boiling wa-
ter bath for 2 hr. After cooling, the solutions were made neutral by
adding appropriate volume of 4M NaOH.
The sugars obtained from the hydrolysis steps and the mono-
saccharide standards (50 mg/mL; glucose, arabinose, xylose,
galactose, and mannose) were analyzed as their alditol acetates,
as described by Blakeney et al (1983). The dilutions for the stan-
128 CEREAL CHEMISTRY
dard curves were made from these monosaccharide solutions.
Myo-inositol was used as the internal standard (0.5 mg/mL sam-
ple). The acetylated monosaccharides were analyzed with gas
chromatography (GC) using an Agilent 6890 GC (Palo Alto,
CA) equipped with a flame ionization detector (FID). The col-
umn was DB-225 (30 m × 0.32 mm, film thickness 0.15 µm,
Agilent). Helium was used as the carrier gas at 1.2 mL/min.
Split injection (1:3) was performed at 250°C, and the FID was
operated at 250°C. The analytes were separated at 220°C for 15
min. The monosaccharides were identified according to their
retention times and quantified with a standard curve. Free hex-
ose sugars were corrected by a factor of 0.9 to anhydro sugars,
and pentose sugars by factor of 0.88. All analyses were made in
duplicate.
Xylanase Activity
Xylanase activities were determined by the Xylazyme-AX
method (Megazyme, Bray, Ireland), as described by Gys et al
(2004), with cross-linked azo-dyed wheat AX as the substrate.
Bran material (1.0 g) was suspended in 10.0 mL of sodium ace-
tate buffer (100 mM, pH 5.0), extracted by constant mixing for 60
min at room temperature, and centrifuged (10,000 × g, 30 min,
6°C). The extracts (0.5 mL) were equilibrated for 10 min at 50°C
before adding an azurine cross-linked AX (AZCL-AX) tablet and
were incubated with the tablet for 60–240 min at 50°C. The reac-
tion was terminated by addition of 2.0% (w/v) tris(hydroxy-
methyl)aminomethane (TRIS) solution (5.0 mL) and mixing on a
vortex mixer. After 5 min at room temperature, the samples were
shaken vigorously and filtered through Whatman no. 1 filter pa-
per. The A590 values of the sample solutions were measured
against a control, that is, the nonenzymatic color release from the
AZCL-AX tablets, prepared by incubating the extracts without
the substrate tablet and by adding the substrate tablet after adding
1.0% TRIS solution to the extract. The soluble azo-wheat AX
substrate (Megazyme) was also tested to determine the enzyme
activities on the soluble AX. With this soluble substrate, incuba-
tion of 120 min was used to measure the xylanase activity of the
bran materials. Xylanase activities were expressed as absorbance
units per hour, that is, increase in A590 of a 1.0 g sample in 1 hr
under the assay conditions.
Baking
The following six bread types were studied: 1) reference bran
bread made with native bran; 2) reference bran bread made with
bran from peeled kernels; 3) bran bread with yeast-fermented
native bran, fermented at 20°C; 4) bran bread with yeast-fer-
mented native bran, fermented at 32°C; 5) bran bread with yeast-
fermented bran from peeled kernels, fermented at 20°C; and 6)
bran bread with yeast-fermented bran from peeled kernels, fer-
mented at 32°C.
The recipe for both the reference bran breads, in parts by
weight, was wheat flour (80), wheat bran (20), yeast (1.5), sugar
(1.5), salt (1.5), fat (6), emulsifier Panodan M2020 (0.48), and
water (76). The optimal water addition was determined by farino-
graph measurement. If bran ferment was used, 88 g of water/
100 g of total dough water was prefermented with bran for 20 hr
(bran ferment) at either 20 or 32°C, and the remaining ingredients
were added to the bran ferment and mixed as described later.
Breads without bran ferment were prepared by mixing flour,
water, sugar, salt, yeast, and emulsifier for 3 min at low speed
with a Diosna spiral mixer (SP 12 F, Dierks & Söhne, Osnabrück,
Germany). Bran was then incorporated into the dough, and further
mixing was carried out for 5 min at high speed. If bran ferment
was utilized, the remaining ingredients were mixed with bran
ferment for 8 min (3 + 5). After a floor time of 10 min at 28°C
and 76% rh, the dough was divided into 400 g loaves and molded
mechanically. The loaves were proofed in pans (55 min at 35°C,
76% rh) and baked at 200°C for 25 min. After 2 hr of cooling,
bread volume was determined by a BreadVolScan device (Back-
aldrin, Asten, Austria). For shelf-life measurements, breads were
stored for six days at room temperature (20°C, 50% rh) in plastic
bags.
Crumb Firmness Measurements
Crumb firmness was measured on days 0, 1, 3, and 6 to assess
the shelf life of the breads. Bread crumb firmness during storage
was determined as maximum compression force (40% compres-
sion, modified AACCI Approved Method 74-09.01) with the tex-
ture profile analysis test. Eight bread slices (originating from
three loaves) were measured, and results were expressed as mean
values. The thickness of each bread slice was 2.5 cm, and edges
of the slice were cut off before measurement.
Statistical Analysis
In analysis of the results, the significance of each instrumental
or chemical measurement in discriminating between the samples
was analyzed with analysis of variance (ANOVA) and Tukey’s
honestly significant difference test (significance of differences at
P < 0.05). A one-way ANOVA was applied as the general linear
model procedure for the bread and dough samples with SPSS
software (version 10.0, SPSS, Chicago, IL). ANOVA was used to
test the statistical differences in instrumental measurements and
chemical measurements between the samples. When the differ-
ence among the samples in ANOVA was statistically significant,
pairwise comparisons of these samples were analyzed with
Tukey’s test.
RESULTS
Chemical Properties of Bran from Native and Peeled Kernels
Bran from peeled kernels contained higher amounts of starch,
protein, and ferulic acid and lower amounts of dietary fiber and
folates compared with bran from native kernels (Table I). The
amount of soluble AX was also slightly lower in the bran from
peeled kernels.
Impacts of Bran Fermentations on Microbial Growth
The viable cell counts of different microbial groups were enu-
merated from the raw materials and the bran fermentations (Ta-
bles I and II). Peeling clearly reduced the load of aerobic bacteria
and fungi in the brans. The levels of LAB and aerobic spore-form-
ing bacteria were low in both types of brans. Fresh baker’s yeast
TABLE I
Chemical and Microbiological Composition of Commercial Wheat Bran
and the Wheat Bran from Peeled Kernelsa
Components Commercial
Native From Peeled
Kernels
Macrocomponents (g/100 g dw)
Ash ND 6.9
Protein 13.0 ± 0.22 19.8 ± 0.34
Starch 12.0 ± 0.22 17.5 ± 0.19
Dietary fiber 48 ± 0.14 41.5 ± 0.20
Arabinoxylans 32.5 ± 0.1 22.5 ± 0.2
of which soluble 0.52 ± 0.02 0.48 ± 0.01
Bioactive compounds
Ferulic acid (mg/100 g dw)
Total 32.5 ± 0.35 39.7 ± 0.42
Free (water extractable) 2.0 ± 0.01 4.0 ± 0.05
Folates (µg/100 g dw) 121 ± 13 114 ± 8
Microbial groups (CFU/g)
Total aerobic heterotrophic bacteria 8 × 105 <5 × 103
Aerobic spore-forming bacteria 2 × 102 1 × 102
Lactic acid bacteria 2 × 102 2 × 102
Yeasts 1 × 103 2 × 102
Molds 5 × 101 <5 × 101
a ND = not determined; dw = dry weight; and CFU = colony-forming unit.
Vol. 89, No. 2, 2012 129
contained high amounts of LAB (7 × 108 CFU/g). Furthermore,
rather high numbers of aerobic heterotrophic bacteria were found
(1 × 105 CFU/g). Some of the aerobic bacteria were spore form-
ing (2 × 102 CFU/g).
The growth of bacteria and yeast was dependent on the process
conditions. High numbers of aerobic bacteria (>108 CFU/g) and
LAB were detected in spontaneous fermentations at 32°C, whereas
limited bacterial and yeast growth was observed in spontaneous
fermentations carried out at 20°C. Higher fermentation tempera-
ture also promoted indigenous yeasts. Approximately 10-fold
higher yeast counts were detected after a 20 hr fermentation at
32°C compared with the fermentations performed at 20°C.
In the yeast-initiated fermentations, the commercial baker’s
yeast, added at a level of 107 CFU/g, dominated the bran fermen-
tations. The high initial number of LAB (1 × 106 CFU/g) reached
109 CFU/g after a 20 hr fermentation at 32°C. Addition of baker’s
yeast restricted the growth of aerobic bacteria and indigenous
yeasts.
Intensive LAB growth led to the acidification of ferments (Ta-
ble II). Acid formation (measured as decreased pH values and
increased TTA) was more pronounced at the fermentation tem-
perature of 32°C. In both spontaneous and yeast-initiated fermen-
tations, high TTA values (13.6 and 14) were measured after 20 hr
fermentations. In spontaneous and yeast-initiated fermentations of
both bran types, pH decreased from 6.6 to 4.9–5.2 after a 20 hr
fermentation at 32°C (Table II), whereas pH remained at 6.4–6.5
after a 20 hr fermentation at 20°C. More restricted production of
acids occurred in fermentation of bran from peeled grains, as com-
pared with fermentation of the native brans.
LAB and Yeast Diversity During Bran Fermentations
Ninety-nine LAB colonies and 37 yeast colonies were isolated
from spontaneous fermentations, and 120 LAB colonies and three
yeast colonies from yeast-initiated fermentations. Moreover, 13
LAB and 14 yeast isolates were recovered from the native bran
and bran from peeled kernels. LAB were subjected to RAPD fin-
gerprinting, and the isolates representing different fingerprint
types were further identified by partial 16S rRNA gene sequenc-
ing. Yeast isolates were first discriminated with PCR fingerprint-
ing with M13 microsatellite primer. Yeasts representing different
fingerprint types were identified by sequence analysis of the D1/D2
domain of the 26S rRNA gene.
The small initial LAB community in native unfermented brans
was mainly composed of Pediococcus pentosaceus and Weissella
viridescens. In brans from peeled kernels, an Enterococcus sp.
and Lactobacillus curvatus/graminis were detected. L. plantarum/
pentosus, L. curvatus/graminis, and P. pentosaceus were identi-
fied as the dominant LAB species in the commercial baker’s
yeast. Among 219 LAB isolates from bran fermentations, 13 dif-
ferent species altogether were identified (Tables III and IV). The
fermented bran contained 4–8 LAB, of which 1–4 species made
up 15% or more of the total LAB community. The species diver-
sity generally increased with the use of commercial baker’s yeast
and with fermentation temperature (Table IV). Different types of
LAB communities developed during spontaneous and yeast-initi-
ated fermentations. Obligately heterofermentative LAB, mainly
Weissella spp., with facultatively heterofermentative cocci domi-
nated in spontaneous fermentations (Table III). Facultatively het-
erofermentative lactobacilli were the major species in yeast-initi-
ated fermentations. Low temperature favored the growth of L.
curvatus/graminis, whereas facultatively heterofermentative cocci
dominated at 32°C. The use of brans from peeled grains selected
for the growth of facultatively over obligately heterofermentative
species.
Among 54 yeast isolates from the bran raw materials and fer-
mented brans, 13 species were found. Native brans carried
higher species diversity compared with the bran from peeled
grains. The main species identified were basidiomycetous yeasts
Aureobasidium pullulans and a Rhodotorula sp. from the R.
glutinis species group, whereas the yeast population in brans
from peeled kernels primarily consisted of Cryptococcus al-
bidus. The baker’s yeast dominated yeast-initiated fermenta-
tions, with minor amounts of Issatchenkia orientalis, which was
also found from the baker’s yeast sample. In spontaneous fer-
mentations, a minor yeast population consisting of basidiomyce-
tous species developed. The yeast communities in spontaneous
fermentations at 20°C were mainly composed of C. albidus and
Saccharomyces cerevisiae. The fermented brans from peeled
kernels also contained C. diffluens and R. mucilaginosa. By
contrast, the Rhodotorula sp. in the R. glutinis species group
was found from fermented native brans.
Impacts of Bran Fermentations on Bioactive Compounds
The amount of folates increased by 20–40% depending on the
fermentation temperature after yeast fermentation of both bran
types (Table V) in comparison to the level of folates in unfer-
mented bran. In spontaneous fermentations, folate levels de-
creased by 8–50% with both bran types. Amounts of total pheno-
lic acids did not change in any of the bran ferments. However, the
amount of free phenolic acids increased threefold to fivefold in
both yeast and spontaneous fermentations at 20°C, depending on
the type of bran (Table V).
Impacts of Bran Fermentations on AX and Xylanase Activity
Native bran contained a clearly higher amount of total AX in
comparison to the bran from peeled kernels. The content of total
TABLE II
Microbial Counts, pH, and Total Titratable Acidity (TTA) Before and After Fermentation of Brans from Native and Peeled Wheat Kernela
Aerobic Heterotrophic Bacteria
Sample and
Fermentation Type Fermentation
Tem p. (° C) Fermentation
Time (hr) Total Spores
Ye as t s
Molds
LAB
pH
TTA
Native bran
Spontaneous … 0 2 × 105 <5 × 101 8 × 102 <5 × 101 7 × 102 6.6 ± 0.02 2.8 ± 0.09
Spontaneous 20 20 3 × 107 <5 × 101 1 × 103 5 × 101 2 × 106 6.5 ± 0.02 4.6 ± 0.2
Spontaneous 32 20 5 × 108 <5 × 101 9 × 104 <5 × 101 2 × 109 5.0 ± 0.05 13.6 ± 0.4
Yeast … 0 2 × 105 <5 × 101 2 × 107 <5 × 101 1 × 106 6.6 ± 0.02 2.8 ± 0.09
Yeast 20 20 2 × 106 <5 × 101 6 × 107 <5 × 101 1 × 108 6.4 ± 0.02 5.9 ± 0.2
Yeast 32 20 7 × 105 <5 × 101 5 × 107 <5 × 101 2 × 109 4.9 ± 0.04 14.0 ± 0.4
Bran from peeled kernels
Spontaneous … 0 <5 × 104 <5 × 101 8 × 101 <5 × 101 2 × 102 6.6 ± 0.02 2.8 ± 0.09
Spontaneous 20 20 2 × 106 <5 × 101 2 × 103 <5 × 101 2 × 106 6.6 ± 0.13 3.8 ± 0.2
Spontaneous 32 20 2 × 108 <5 × 101 1 × 104 <5 × 101 1 × 109 5.1 ± 0.2 12.8 ± 1.2
Yeast … 0 2 × 104 <5 × 101 1 × 107 <5 × 101 1 × 106 6.6 ± 0.02 2.8 ± 0.09
Yeast 20 20 <5 × 104 <5 × 101 6 × 107 <5 × 101 1 × 108 6.4 ± 0.01 5.9 ± 0.2
Yeast 32 20 <5 × 104 <5 × 101 4 × 107 <5 × 101 2 × 109 5.2 ± 0.04 11.7 ± 0.4
a LAB = lactic acid bacteria. Microbial counts are measured in colony-forming units per gram (n = 2). TTA is measured in mL of 0.1M NaOH per 10 g.
130 CEREAL CHEMISTRY
AX remained unaltered after yeast fermentation and decreased
slightly after spontaneous fermentation of native bran (Table VI).
The content of soluble AX increased almost threefold when
peeled bran was fermented with S. cerevisiae at either 20 or 32°C
(Table VI) and increased slightly after spontaneous fermentation
of peeled bran. With native bran, the content of soluble AX in-
creased after yeast fermentation by 25–35% but remained unal-
tered after spontaneous fermentation.
The bran from native wheat kernels had approximately 10-fold
higher xylanase activity when compared with the bran made from
peeled wheat kernels (Table VI). The unfermented bran materials
had higher activity than the fermented bran when determined
using the Xylazyme-AX tablets. The differences between the
treatments were, however, low.
Impacts of Bran Ferments on Bread Volume and Shelf Life
The reference bran breads made with unfermented bran had the
lowest specific volumes (3.8–3.9 mL/g). Type of bran (native
versus peeled) did not affect the volume of the reference breads.
Yeast fermentation of native bran either at 20 or 32°C did not
improve loaf volume significantly. Fermentation of peeled bran
both at 20 and 32°C improved volume from 3.9 to 4.3 mL/g, pro-
viding the best volume of bran breads (Fig. 1).
Fermentation of peeled bran was the most effective treatment in
improving crumb texture, measured as hardness of crumb (Fig. 2).
Of the fresh breads (day 0), the reference bran breads (made both
with native bran and with bran from peeled kernels) and the bread
containing native bran fermented at 32°C had the hardest crumb.
Yeast fermentation of both types of bran effectively improved
crumb softness (Fig. 2). On the day of baking, the softest bread
was obtained by using fermented bran from peeled kernels or the
native bran fermented at 20°C. After storage for four and six days,
the crumbs of the breads baked with fermented bran from peeled
kernels or with native bran fermented at 20°C were significantly
softer in comparison to the control bread with unfermented bran.
The softest crumb texture after six days, however, was obtained
by using fermented bran from peeled kernels (Fig. 2).
DISCUSSION
Development of the indigenous LAB and yeast communities
during bran fermentation was strongly dependent on the tem-
perature, type of bran, and added baker’s yeast. Only low num-
bers of LAB were detected in the bran fractions, but their num-
ber increased greatly during fermentation at 32°C, especially in
bran fermentation of native brans, which resulted in intensive
acidification. Baker’s yeast was a significant source of LAB,
and thus yeast could be considered as a multifunctional starter
consisting of yeast and LAB. Lower acid production associated
with bran from peeled grains compared with native brans was
correlated with a higher fraction of facultatively heterofermenta-
tive cocci and with lower endogenous and microbial xylanase
TABLE IV
Distribution (%) of Isolated Lactic Acid Bacteria Species in Yeast-Initiated Fermentationsa
Native Bran Bran from Peeled Kernels
Metabolism and Identification NF, 0 hr 20°C, 20 hr 32°C, 20 hr NF, 0 hr 20°C, 20 hr 32°C, 20 hr
Facultatively heterofermentative
Lactococcus lactis … 5 … … 5 25
Pediococcus pentosaceus … … 25 25 10 30
Lactobacillus casei/paracaseib 15 … 10 15 … 15
Lactobacillus coryniformis 10 … … … 5 …
Lactobacillus curvatus/graminisb 15 55 30 25 65 30
Lactobacillus plantarum/pentosusb 30 20 5 25 5 …
Lactobacillus paraplantarum … … 5 … 5 …
Lactobacillus sakei … 5 … … … …
Obligately heterofermentative
Lactobacillus fermentum 30 10 5 10 … …
Lactobacillus brevis … … 10 … 10 …
Leuconostoc pseudomesenteroides … 5 … … 5 …
Weissella confusa … … 5 … … …
120 total isolates 20 20 20 20 20 20
12 total species 5 6 8 5 8 4
a NF = not fermented.
b The identification was not conclusive at the species level.
TABLE III
Distribution (%) of Isolated Lactic Acid Bacteria Species in Spontaneous Fermentationsa
Native Bran Bran from Peeled Kernels
Metabolism and Identification NF, 0 hr 20°C, 20 hr 32°C, 20 hr NF, 0 hr 20°C, 20 hr 32°C, 20 hr
Facultatively heterofermentative
Lactococcus lactis 11 30 5 50 75 35
Pediococcus pentosaceus 11 … 20 … … 5
Lactobacillus curvatus/graminisb 68 5 15 50 5 …
Lactobacillus plantarum/pentosusb 10 … … … … 5
Lactobacillus sakei … … 5 … … 5
Obligately heterofermentative
Lactobacillus brevis … … … … 10 …
Weissella confusa … 40 30 … 5 20
W. cibaria … 25 25 … 5 25
99 total isolates 13 20 20 6 20 20
8 total species 4 4 6 2 5 6
a NF = not fermented.
b The identification was not conclusive at the species level.
Vol. 89, No. 2, 2012 131
activity, thereby supporting our earlier results obtained with rye
brans (Katina et al 2007a). Mild acidity provides a technological
advantage for bran from peeled grain, because strong acidic or
pungent cereal food flavor does not appeal to most consumers,
which thus limits the use of fermented wheat bran as a food in-
gredient.
Addition of baker’s yeast had the ability to dominate and mod-
ify the natural microbial community of brans. Accordingly, in
spontaneously fermented brans, different microbial populations
were dominant. In spontaneous fermentations, obligately hetero-
fermentative species such as W. confusa and W. cibaria dominated
the microbial community, especially in ferments made with native
bran. Weissella species, especially W. c i baria and W. confusa, are
encountered in the cereal kernels and flour and have been isolated
from a variety of traditional and spontaneous sourdoughs, particu-
larly from wheat, and may even dominate some processes (De
Vuyst et al 2002; Catzeddu et al 2006; Corsetti and Settanni 2007;
Corsetti et al 2007; Robert et al 2009). In yeast-initiated fermenta-
tions, Weissella species were outnumbered by mainly facultatively
heterofermentative LAB species.
In agreement with our previous investigations of whole-grain
rye and rye bran (Liukkonen et al 2003; Katina et al 2007b),
wheat bran was also a rich source of folates, containing at least
double the folates of wheat flour. The amount could even be fur-
ther increased by yeast fermentation, because of folate production
by yeast (Liukkonen et al 2003; Kariluoto et al 2004; Jägerstad et
al 2005). Hjortmo et al (2005) reported that folate content in
yeast-fermented foods could be further increased by choosing a
proper yeast strain. They showed that folate production in S. cere-
visiae varied extensively between different strains. In the present
study, yeast addition could not totally explain the increase in
folates in bran fermentations. The highest folate level was obtained
when the growth of indigenous LAB was pronounced (fermen-
tation for 20 hr at 32°C), which may indicate a supportive role of
indigenous LAB and other indigenous bacteria in folate synthesis,
as was also suggested by Herranen et al (2010). Regular wheat
flour contains 27–66 μg/100 g of folates (Arcot et al 2002; Piironen
et al 2008). Supplementation of wheat flour with 20% optimally
fermented wheat bran would increase the folate content of the
flour-bran mixture to 72–97.8 μg/100 g. Thus, the wheat bread
supplemented with yeast-fermented bran would have 32–62%
higher folate content as compared with regular white bread.
Phenolic compounds, especially ferulic acids, are partly respon-
sible for the insolubility of cell wall structures of cereal kernels,
because ferulic acid can form cross-links between AX polysaccha-
rides and lignin (Faulds and Williamson 1999). In the present study,
the highest level of free ferulic acid was obtained in bran fermenta-
tion carried out at 20°C, which conditions also led to the modest
LAB growth and acidification (pH value of 6–6.5). This level is
very near to the reported optimum pH (7) of cinnamoyl esterases of
whole-grain flour (Boskov Hansen et al 2002). Accordingly, the
lowest level of free ferulic acid was in fermentation conditions pro-
viding strong acidity (3.9–4.1), which inhibits the cinnamoyl es-
terase. Because similar increased amounts of free ferulic acid were
obtained in all types of fermentations that led to higher pH values,
TABLE VI
Amount of Arabinoxylans (AX) and Endogenous Xylanase Activity in Different Bran Ferments After 20 hr Fermentation (n = 2)a
Fermentation Type Fermentation Temperature (°C) Total AX (% dw) Soluble AX (% dw) Xylanase Activity (A590/hr)
Native bran
Not fermented … 32.3 ± 0.06a 0.52 ± 0.0a 0.98 ± 0.13a
Yeast 20 33.30 ± 0.6a 0.80 ± 0.13c 0.76 ± 0.04a
Yeast 32 33.20 ± 1.43a 0.70 ± 0.03bc 0.81 ± 0.05a
Spontaneous 20 26.8 ± 0.50b 0.50 ± 0.01a 0.81 ± 0.12a
Spontaneous 32 29.0 ± 0.90c 0.60 ± 0.01ab 0.75 ± 0.14a
Bran from peeled kernels
Not fermented … 21.5 ± 2.11a 0.40 ± 0.04a 0.10 ± 0.01a
Yeast 20 22.7 ± 1.18a 1.1 ± 0.02c 0.08 ± 0.00a
Yeast 32 23.8 ± 1.27a 1.0 ± 0.14c 0.08 ± 0.01a
Spontaneous 20 20.9 ± 1.0a 0.60 ± 0.02b 0.08 ± 0.01a
Spontaneous 32 20.9 ± 1.0a 0.70 ± 0.03b 0.07 ± 0.00a
a Not fermented indicates that water, bran, and yeast were mixed, and the sample was taken at the time point 0 hr. Values in the same column with the same letter
are not significantly different (P < 0.05) within groups (native bran and bran from peeled kernels). dw = dry weight.
Fig. 1. Influence of bran type and fermentation conditions on the specific
volume of wheat breads supplemented with 20% bran addition. Columns
with the same letter are not statistically different from each other at P <
0.05. Fermented refers to yeast-initiated fermentation.
TABLE V
Amounts of Folates and Phenolic Acids in Different Bran Ferments
After 20 hr Fermentation (n = 2)a
Fermentation Type
Tem p .
(°C)
Folates
(μg/100 g)
Tot al
Phenolic
Acids
(mg/100 g)
Free Ferulic
Acid
(mg/100 g)
Native bran
Not fermented … 139 ± 8a 315 ± 70a 2.0 ± 0a
Yeast 20 172 ± 0c 344 ± 50a 11.0 ± 4b
Yeast 32 225 ± 11d 283 ± 40a 3.0 ± 1a
Spontaneous 20 129 ± 5a 324 ± 50a 6.0 ± 1ab
Spontaneous 32 100 ± 9b 339 ± 60a 2.0 ± 1a
Bran from peeled kernels
Not fermented … 131 ± 4a 361 ± 70a 4.0 ± 1a
Yeast 20 170 ± 13c 397 ± 40a 19.0 ± 1b
Yeast 32 219 ± 13d 391 ± 50a 3.0 ± 1a
Spontaneous 20 70 ± 4b 398 ± 50a 17.0 ± 8b
Spontaneous 32 72 ± 1b 315 ± 60a 2.0 ± 1a
a Not fermented indicates that water, bran, and yeast were mixed, and the
sample was taken at the time point 0 hr. Values in the same column with the
same letter are not significantly different (P < 0.05). Comparison made in
group 1 (native) and in group 2 (bran from peeled kernels). Temp. =
fermentation temperature.
132 CEREAL CHEMISTRY
pH-mediated activation of endogenous enzymes of wheat is the
most probable explanation for the observed phenomena.
The observed solubilization of AX in wheat bran fermentation
is in accordance with our previous results obtained with fermenta-
tion of rye bran from peeled kernels (Katina et al 2007a). Solubi-
lization of AX seemed to be limited in spontaneous fermentations,
although acidity levels and endogenous xylanase activities were
not different from those in the yeast-fermented counterparts. The
lower level of soluble AX in spontaneously fermented brans com-
pared with yeast-fermented brans may partly be explained by
differences in their microbiota. Heterofermentative LAB were
among the major LAB only in spontaneous fermentations. From
previous studies, it is known that obligate heterofermenters, such
as L. fermentum and L. brevis, are able to cometabolize arabinose
and xylose with maltose (Gobbetti et al 1999). W. cib aria , the
species found in all spontaneous fermentations, can also utilize
both arabinose and xylose (Bounaix et al 2010). Hence, indige-
nous enzymes of bran may have first solubilized AX (high content
of soluble AX) in spontaneous fermentation and then degraded
them to monosaccharides that were used by the heterofermenta-
tive LAB, thereby reducing the apparent AX content. In yeast-
started fermentations, potential AX-utilizing LAB were a minor
group, especially with fermented brans from peeled kernels (with
low xylanase activity), leading to a higher content of soluble AX.
Thus, microbial activity greatly appeared to determine the overall
fate of solubilized AX during bran fermentations. Therefore, the
means to control bran microbiota are of the utmost importance for
the technological functionality of bran. However, determination
of molecular weight (MW) of AX after fermentation was not part
of this study, and MW most likely has an additional important
role in determining functionality of fermented wheat brans.
Endogenous xylanase activity was clearly higher in the native
bran in comparison to the peeled bran. Microbial xylanases are only
or almost entirely located in the outer layers of grain (Dornez et al
2008, 2009). Thus, debranning greatly reduces endogenous xy-
lanase activity (Gys et al 2004).
The lower amount of solubilized AX in native bran in compari-
son to peeled bran could also be because the bran from peeled
kernels contained relatively more AX originating from aleurone,
and removal of the pericarp probably made this AX more
accessible for enzymatic degradation. Structure of AX is differ-
ent in pericarp and aleurone fractions, as the AX in pericarp
(outer bran) are highly substituted (arabinose to xylose [A/X]
ratio ≈ 1.0), whereas AX in the aleurone layer (A/X = 0.4–0.5)
and the nucellar epidermis (A/X ≈ 0.1) have a lower A/X ratio
and are less substituted, which facilitates enzymatic hydrolysis
of AX (Antoine et al 2003). Furthermore, xylanase inhibitors
from bran can inhibit microbial xylanases, which are probably
partly responsible for the higher endogenous activity of native
bran (Dornez et al 2009).
Both types of fermented bran, native and peeled, provided sig-
nificant endogenous xylanase activities to the wheat dough, but
with different activity levels and also most probably with different
types of xylanases. Native fermented bran provided a high level
of (microbial) xylanases, which may have had a negative impact
on the final bread quality owing to breakdown of soluble AX to
smaller MW (Dornez et al 2006a). Peeled fermented bran pro-
vided a much more modest endogenous xylanase activity to the
dough, which has been suggested to have a positive effect on
bread quality (Dornez et al 2007).
A positive influence of solubilization of AX has been reported
in wheat-flour baking (Maat et al 1992; Courtin and Delcour
2002), which could explain the 10–14% increase in the volume of
breads made with fermented bran containing an increased amount
of soluble AX. Wheat dough with fermented bran from peeled
kernels (20% substitution level of bran) contained 0.62% soluble
AX, whereas the control dough with untreated bran had a soluble
AX content of 0.48%. A 15–22% increase in the amount of solu-
ble AX has been linked to improved volume of wheat breads (5–
14%) by several researchers (Rouau et al 1994; Trogh et al 2004).
Increased softness of the bread crumb with both types of fer-
mented bran was not directly linked to the bread volume because
breads with fermented native brans also provided significantly
softer crumb without improved volume. The improved shelf life is
probably because bread containing fermented bran had altered
water distribution between starch and gluten and retarded starch
retrogradation, as reported by Katina et al (2006).
CONCLUSIONS
Yeast-started fermentation improved the bioactivity and baking
properties of wheat bran prepared from peeled kernels. Use of
Fig. 2. Influence of bran type and fermentation conditions on the hardness of wheat breads supplemented with 20% bran addition during six days o
f
storage. Columns marked by the same letter are not statistically different at P< 0.05 (for comparisons made at the same measuring points, e.g., after fou
r
days of storage). Fermented refers to yeast-initiated fermentation.
Vol. 89, No. 2, 2012 133
bran from peeled kernels and added yeast starter enabled better
control of the fermentation process in terms of microbial activity.
This tailored fermentation resulted in solubilization of AX during
a 20 hr fermentation and diminished final endogenous xylanase
activity. These effects are proposed as the main reason for the
improved technological functionality of fermented bran.
ACKNOWLEDGMENTS
Arja Viljamaa is thanked for skillful technical assistance. Bühler AG,
Switzerland, is gratefully thanked for providing raw material for this study.
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[Received August 27, 2011. Accepted February 28, 2012.]
