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Digestion of barley malt porridges in a gastrointestinal model: Iron
dialysability, iron uptake by Caco-2 cells and degradation of b-glucan
Ann-Katrin Haraldssona,*, Lena Rimstenb, Marie Almingera,
Roger Anderssonb, Per Amanb, Ann-Sofie Sandberga
aDepartment of Chemical and Biological Engineering/Food Science, Chalmers University of Technology, P.O. Box 5401, SE-402 29 Goteborg, SwedenbDepartment of Food Science, Swedish University of Agricultural Sciences, P.O. Box 7051, SE-750 07 Uppsala, Sweden
Received 22 December 2004; revised 7 April 2005; accepted 13 April 2005
Abstract
Iron availability and degradation of (1/3,1/4)-b-D-glucan (b-glucan) in three whole grain porridges made from two optimised barley
malts and unprocessed barley were studied in a dynamic gastrointestinal model. The malting processes, with steeping at 15 or 48 8C with
lactic acid (LA), enabled a complete reduction of phytate by subsequent soaking of ground malt, still with well preserved b-glucan. Iron
dialysability and iron uptake by Caco-2 cells were higher in phytate reduced porridges, compared to the reference porridge. During simulated
digestion, the extractability of b-glucan increased and the Calcofluor average molecular weight decreased for all porridges, indicating a
gradual degradation during passage through the model. The degradation rate, however, appeared lower in porridge prepared from malted
barley steeped at 48 8C with LA. The gastrointestinal model ranked iron availability according to human absorption data and showed high
repeatability when evaluating changes in b-glucan. The results indicate the potential for using high temperature steeping with LA to yield
improved iron availability combined with reduced degradation of b-glucan in the small intestine, maintaining the beneficial properties
of barley.
q 2005 Elsevier Ltd. All rights reserved.
Keywords: Iron availability; Phytate; (1/3,1/4)-b-Glucan; In vitro digestion; Barley
1. Introduction
The role of whole grain in the maintenance of human
health has attracted considerable scientific interest. Several
epidemiological studies have shown an inverse association
between whole grain consumption and risk of coronary heart
diseases (Jacobs et al., 1998; Liu et al., 1999) and type-2
diabetes (Meyer et al., 2000; Pereira et al., 2002). While
0733-5210/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jcs.2005.04.002
Abbreviations Cfu, colony forming units; DMEM, Dulbecco’s modified
eagle medium; DF, dialysis fluid sample; HPIC, high performance ion
chromatography; HPSEC-FD, high performance size exclusion-fluor-
escence detection; ID, ileal delivery sample; IP6KIP3, hexa- to tri-inositol
phosphate; LSC, liquid scintillation counting; Mcf , calcofluor average
molecular weight of b-glucan; NEAA, non-essential amino acids; PEST,
pencillin/streptomycin; phytate-P, phytate-phosphorous derived from
SIP6KIP3; TGA, tryptone glucose agar.* Corresponding author. Tel.: C46 31 3355600; fax: C46 31 833782.
E-mail address: mlw@fsc.chalmers.se (A.-K. Haraldsson).
the protective mechanisms of whole grain are not yet clear
(Slavin et al., 1999) much attention is focused on cereal fibre
and the cholesterol lowering (Newman et al., 1989; Davids-
son et al., 1991) and hypoglycaemic effects (Cavallero et al.,
2002; Jenkins et al., 2002) of soluble dietary fibre. It is
primarily (1/3,1/4)-b-D-glucan (b-glucan), an endo-
sperm cell wall polysaccharide consisting of long, linear
chains of b-glucose residues joined through both (1/3)- and
(1/4)-linkages, that is associated with these effects, but
arabinoxylan may also be involved (Newman et al., 1992).
The effects are believed to originate in the upper gastroin-
testinal tract and to be related to the viscosity that b-glucan
and arabinoxylan bring to a solution. Two important factors
that influence the viscosity of a solution are the molecular
size and concentration of b-glucan.
In addition to its content of dietary fibre, whole grain is
also a dietary source of minerals. Because of the
considerable amount of mineral-complexing phytate
(myo-inositol hexaphoshate, IP6) in cereal products, they
are often considered to have low mineral availability.
Mainly concentrated in the nutritious outer layers of
Journal of Cereal Science 42 (2005) 243–254
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A.-K. Haraldsson et al. / Journal of Cereal Science 42 (2005) 243–254244
the kernel, phytate is associated with the major fibre fraction
of the whole grain. The molecule is highly negatively
charged at neutral pH values and chelates divalent minerals,
preventing their absorption in the small intestine (Reddy et
al., 1982). Iron and zinc absorption from cereal meals is
improved when the phytate content is reduced by food
processing (Brune et al., 1992; Fredlund et al., 2003; Hurrell
et al., 2003; Larsson et al., 1996).
Processes that are used to remove phytate in cereals often
involve soaking, germination or fermentation, and these
processes are very likely to result in extensive degradation
of b-glucan. However, the positive nutritional aspects of
b-glucan is a reason to attempt to preserve them. In two
previous publications, high temperature steeping and the use
of lactic acid during steeping were found useful for obtaining
substantial phytate hydrolysis while preventing an extensive
degradation of b-glucan (Haraldsson et al., 2004; Rimsten
et al., 2002). In the present study, these conditions were
adopted and used in pilot scale malting processes.
The properties and behaviour of food components during
passage through the gastrointestinal tract and the effect of
food processing can be studied in vitro. A computerised
gastrointestinal model has been developed to simulate upper
gastrointestinal transit, pH, composition and rate of
secretions and absorption of digested products (Minekus
et al., 1995). This model was used here to investigate iron
dialysability, the change in the molecular weight and
extraction yield of b-glucan, and the change in the
molecular size of extractable arabinoxylan during digestion
of a processed barley malt porridge. Iron availability was
further assessed by combining the in vitro model with
estimation of iron uptake by the human intestinal cell line,
Caco-2 (Hidalgo et al., 1989).
The aim was to elucidate whether manipulation of
processing conditions is a practical feasible way to improve
the nutritional value of whole meal barley with respect
to increased mineral availability while preserving high
molecular weight b-glucan.
2. Material and methods
2.1. Chemicals
Pepsin A from porcine stomach mucosa (2260 units/mg,
P-7012), trypsin from bovine pancreas (7500 N-a-benzoyl-
L-arginine ethyl ester (BAEE) units/mg, T-4665), bile
extract from porcine (B-8631) and pancreatin from porcine
pancreas (4!U.S.P., P-1750) were from Sigma–Aldrich
(Stockholm, Sweden), and Rhizopus lipase
(150,000 units/mg F-AP 15) was from Amano Enzyme,
Inc. (Nagoya, Japan). Dulbecco’s modified Eagle medium
(DMEM) with 4.5 g/l and L-glutamine, non-essential amino
acids (NEAA), pencillin/streptomycin (PEST) and trypsin-
EDTA and foetal calf serum were from PAA Laboratories
GmbH (Linz, Austria), radioactive isotopes (55FeCl3) from
NEN Life Science Products (PerkerElmer Life Science,
Inc., Zaventem, Belgium) and ULTIMA-FLO AP liquid
scintillation counting (LSC) cocktail from Packard
Bioscience B.V. (Groningen, The Netherlands). All other
chemicals were from Sigma–Aldrich (Stockholm, Sweden)
or Scharlau Chemie S.A. (Barcelona, Spain).
2.2. Pilot scale malting of barley kernels
Hull-less barley (cultivar SW1290, Svalof Weibull AB,
Svalov, Sweden), was malted on a 50-kg scale (OY Lahden
Polttimo, Lahti, Finland) under conditions based on
Rimsten et al. (2002) and Haraldsson et al. (2004). Two
different malt types were produced: malt A was made by
steeping whole kernels at 15 8C with 0.8% lactic acid until a
water content of 39.2% was obtained, followed by
germination at 15 8C for 72 h and kilning at increasing
temperatures of 50–60 8C, for 21 h. Malt B was steeped at
48 8C with 0.8% lactic acid until a water content of 42.7%
was obtained, germinated at 15 8C for 72 h and kilned at
increasing temperatures of 50–82 8C, for 21 h. The water
content of both malt types was kept at approximately 42%
during germination. For determination of total bacterial
count in the final product, homogenised samples were
spread on tryptone glucose agar (TGA) plates and colony
forming units (cfu) were counted after 3 d at 30 8C.
2.3. Preparation of meals for digestion experiments
Malted barley kernels were ground and portions of 20 g
malt flour and 100 ml preheated water (MilliQ) were
incubated at 48 8C in a shaking water bath for 4 h. Five
portions of flour and incubation water were pooled and
immediately frozen at K40 8C. Porridge A was prepared by
thawing incubated flour of malt A in a microwave oven at low
power (450 W) followed by heating at high power (800 W)
with occasional stirring until gelatinisation (4–5 min). The
water used during incubation was also used when the
porridges were prepared (to prevent loss of nutrients by
leakage). Porridge B was prepared from incubated flour of
malt B using the same procedure as for porridge A. As a
reference, a porridge was prepared from ground unprocessed
barley by mixing 100 g of flour with 500 ml water,
immediately followed by heating in the microwave oven
using the same procedure as for malt porridges.
A meal containing 210 g of freshly made porridge and
90 g of water was prepared prior to the start of each
digestion experiment. All porridge meals were sup-
plemented with 3.5 mg iron (625 ml FeSO4 0.1 M in 0.1 M
HCl) to allow detectable concentrations of iron in the large
volumes of dialysates collected.
2.4. The gastrointestinal model
The gastrointestinal model is described in detail else-
where (Minekus et al., 1995) and has previously been used
Fig. 1. A schematic diagram of the gastrointestinal model: (a) stomach, (b) duodenum, (c) jejunum, (d) ileum, (e) ileo-ceacal valve and (f) hollow-fibre
membrane devices. All samples of dialysis fluid (DF) and ileal deliveries (ID) are summarised to view time of sampling (min), sampling positions in the model
(Roman numerals) and relevant analyte(s) for each sample.
A.-K. Haraldsson et al. / Journal of Cereal Science 42 (2005) 243–254 245
for assessment of iron availability (Larsson et al., 1997;
Salovaara et al., 2003). Briefly, the model comprises four
compartments that represent the stomach, duodenum,
jejunum and ileum (Fig. 1a–d). Each compartment consists
of a glass exterior with a flexible, inner silicon tubing,
connected by peristaltic valves that determine the transport
rate of the food between the different compartments. To
simulate peristalsis, the tubes are squeezed periodically by a
pumping action on the surrounding water kept at physio-
logical temperature (37G1 8C). Secretion of digestive
juices and pH adjustment in each section are simulated
according to physiological data (Minekus et al., 1995). All
parameters are computer controlled and a protocol for
medium transport time of the food, i.e. half of the meal was
delivered from the stomach to the duodenum after 70 min,
was chosen in the present study to simulate a semi-solid
meal. For simulation of absorption of potentially available
iron, the jejunal and ileal compartments are connected with
semi-permeable hollow fibre membrane units (Hospal
hemodialyzer HG-400 (cut-off of 3–5 kD), Gambro, Renal
Products, Lund, Sweden) (Fig. 1f). Each experiment was
terminated after 360 min and at this time approximately
80% of the food had passed the ileo-ceacal valve of the
model (Fig. 1e). Each type of porridge was run in the model
in at least three separate digestion experiments. All utensils
were washed with nitric acid (3%) prior to each experiment.
The composition of the different digestive juices used in the
model was according to Salovaara et al. (2003).
2.4.1. Samples collected during digestion
The samples collected during each digestion experiment
are listed in Fig. 1. Dialysis fluids (DF) were continuously
collected every 2 h in aliquots from the jejunal and ileal
compartments (Fig. 1, I and II), sampled after 120 min
(DF0–120), 240 min (DF120–240) and 360 min (DF240–360).
At the time of sampling, the dialysis fluids from the two
compartments were pooled and an aliquot was taken and
kept frozen (K18 8C) until analysis. Ileal delivery (ID)
samples were collected on ice every 2 h in aliquots at the
ileo-ceacal valve (Fig. 1, III). The total volume of
intestinal contents delivered during the respective time
periods was sampled after 120 min (ID0–120), 240 min
(ID120–240) and 360 min (ID240–360). Additionally, a small
sample (1 ml) was removed from the ID0–120 pool at
60 min (ID0–60). Three small samples (1 ml) were also
collected from the duodenal, jejunal and ileal compart-
ments (Fig. 1, IV–VI). Duo1⁄2was taken at tZ70 min,
when approximately 50% of the meal was delivered from
the stomach, Jej1⁄2at tZ145 min, when approx. 50% of
the meal had passed through the jejunum and Ile1⁄2at tZ
220 min, when approx. 50% of the meal had passed
through the ileum. All ileal delivery and half-time samples
were immediately frozen (K18 8C), freeze-dried and kept
frozen until analysed.
2.5. The Caco-2 cell model
Caco-2 cells were purchased from American Type
Culture Collection (HTB-37, Manassas, VA, USA) and
stock cultures were maintained and uptake experiments
performed as described by Salovaara et al. (2003), with
minor modifications. For uptake studies, cells were seeded
(250,000 cells/well) in collagen-treated 12-well plates
(3.66 cm2/well) (TPP, Trasadingen, Switzerland) and exper-
iments were conducted using differentiated cultures at 13–15
days post seeding. 55FeCl3 was added to dialysates collected
between 0 and 120 min (DF0–120) to give 37 kBq/ml, the
radioactively labelled samples (1 ml) were applied to
A.-K. Haraldsson et al. / Journal of Cereal Science 42 (2005) 243–254246
the cells in duplicate or triplicate wells and the cells
incubated for 1 h at 37 8C. After incubation, non-absorbed
iron was removed from the cell surface according to Glahn et
al. (1995, 1998). Cells were lysed by the addition of NaOH
(0.5 M), transferred to a scintillation vial with LSC cocktail
and absorbed radioactive iron analysed in a Tri-Carb
1900CA liquid scintillation analyzer (Packard Instrument,
Meriden, CT, USA).
The amount of absorbed iron was calculated by
multiplying the fraction of absorbed radioactive iron with
the amount of non-radioactive iron applied to the cells and
the exact volume of the collected dialysate. The values are
reported as mg hK1 and are means of 12 measurements, i.e.
dialysates from two separate digestion experiments with
each porridge type were studied in triplicate in two
separate cell batches (passages 29–31 and 34–36,
respectively).
2.6. Determination of phytate and lower inositol phosphates
Inositol phosphates were extracted from freeze-dried,
ground samples with HCl (0.5 M) and quantified by high
performance ion chromatography (HPIC) (Carlsson et al.,
2001). To allow analyses of the IP3KIP5 inositol phos-
phates, samples were concentrated by evaporating aliquots
to dryness and re-dissolving them in water. Dialysis fluids
were concentrated by using ion exchange resin (AG I-X8,
200–400 mesh chloride form, BioRad Laboratories,
Hercules, CA, USA). Inositol phosphates were determined
as mmol IPi gK1 dm.
2.7. Measurement of enzyme activities
Phytase was extracted and the activity measured in
crude extracts as described by Bergman et al. (2000). The
enzyme extract was incubated for 15 min at 47 8C with a
sodium phytate (phytic acid dodecasodium salt hydrate,
Aldrich Chemical Company inc. Milwaukee, WI, USA)
solution (15 mmol/ml, pH 5.2) and citrate buffer (0.13 M,
pH 5.2). Phytase activity was calculated as mmol degraded
IP6 gK1 minK1 and related to raw material as percentage
activity.
The activity of (1/3,1/4)-b-D-glucan hydrolase
(b-glucanase) was determined in the porridges prior to
digestion and in the accompanying ID240–360 samples by
extracting enzymes with 6 ml of buffer (40 mM sodium
acetate and 40 mM sodium phosphate at pH 4.6) for 15 min
as in the method of McCleary and Sheeran (1987). After
centrifugation (1200g for 10 min), 0.5 ml of the supernatant
was mixed with 0.5 ml of a solution of purified barley
b-glucan (MW 250,000, Megazyme, Wicklow, Ireland).
The samples were directly and repeatedly injected onto a
high performance size exclusion chromatography system
with fluorescence detection (HPSEC-FD) every 90 min for
22.5 h. The inverse molecular weight for each injection was
plotted against time after mixing. Enzyme solutions used in
the gastrointestinal model were also tested with this
procedure for traces of b-glucanase activity.
2.8. Analysis of iron content
The iron content in samples was analysed by HPIC
coupled with UV–visible detection (Fredrikson et al., 2002).
Homogenised, freeze-dried samples were completely dis-
solved by microwave digestion (Milestone microwave
laboratory system, EthosPlus, Sorisole, Italy). All DF
samples were spiked with 0.1 mg Fe/ml sample to improve
the accuracy of the chromatography.
2.9. b-Glucan molecular weight determination
b-Glucan was extracted from samples (10 mg) with 2 ml
of deionised water containing CaCl2 (0.28 mg/ml) and 20 ml
of thermostable alpha-amylase at 100 8C for 2 h and
analysed using the HPSEC-FD system according to Rimsten
et al. (2003). Molecular weights of b-glucan were
determined as average molecular weight of b-glucans over
10,000 g molK1 ð �Mcf Þ. To describe the distribution of the
molecular weight, percentiles were calculated describing
the molecular weight at which 10, 50 and 90% of the
distribution was below the calculated value. Samples from
two separate digestion experiments were analysed in
duplicate, and values presented are means of two digestion
experiments, each analyzed in duplicate.
2.10. Arabinoxylan extraction
Samples (150 mg) were mixed with 6 ml of deionised
water with CaCl2 (0.28 mg/ml) and 20 ml of thermostable
alpha-amylase (EC 3.2.1.1, 3000 U/mL, Megazyme, Wick-
low, Ireland) and incubated at 100 8C with occasional
mixing for 2 h. After cooling, samples were mixed on a
vortex mixer and centrifuged (1200g, 10 min). Duplicate
samples were taken (2 ml) from each supernatant. To one of
these duplicates, 3 ml of 99.5% EtOH (fraction 60%) was
added, and 19 ml of 99.5% EtOH (fraction 90%) was added
to the other. All samples were kept on ice for 4 h, and
precipitates were collected by centrifugation (1200g,
10 min) and freeze-dried. The content of arabinose, xylose
and galactose in the ethanol precipitates was determined
using the method of Andersson et al. (1999), which is a
modified method of Theander et al. (1995).
2.11. Statistical evaluation
Data were analysed by ANOVA, and significant
differences between groups were determined by Tukey
HSD multiple rank test. A p-value of !0.05 was considered
significant. Standard error (SE) was calculated by dividing
the standard deviation of replicates withffiffiffi
np
.
A.-K. Haraldsson et al. / Journal of Cereal Science 42 (2005) 243–254 247
3. Results and discussion
Two different barley porridge meals were designed to
enable removal of phytate to be combined with a
preservation of the molecular weight of b-glucan. Addition-
ally, iron availability and degradation of the b-glucan were
studied simultaneously during passage through a simulated
dynamic gastrointestinal tract.
3.1. Characteristics of malted barley grain and barley
porridge meals
3.1.1. Phytate content and phytase activity
The phytate content in unprocessed barley was
13.0 mmol gK1 (2.5 mg phytate-P derived from IP6KIP3
per gram), which was reduced to 8.5 mmol gK1 (1.7 mg
phytate-P gK1) in malt A and 4.0 mmol gK1 (1.1 mg phytate-
P gK1) in malt B during the pilot scale malting. This
reduction was in good agreement with results obtained in
laboratory scale malting (Haraldsson et al., 2004). However,
since concentrations above 0.5 mmol IP6 gK1 (Sandberg and
Svanberg, 1991) or 10 mg phytate-P per meal (Brune et al.,
1992; Hallberg et al., 1989) are considered to impair iron
Untreated
Ref
eren
ce
Incuba
Mal
t A
104
105
106
Mal
t B
104
105
Molecular we
Fig. 2. Molecular weight distributions of b-glucan in untreated raw material and m
porridges made from the respective materials. The replicates are individually sho
absorption in humans a more extensive phytate reduction is
needed. Absorption of zinc is on the other hand significantly
improved at levels below 50 mg phytate-P per meal
(Sandstrom and Sandberg, 1992) and the reduced phytate
content in malt B (70%) is likely to result in improved zinc
availability. Malt A can also be expected to have improved
zinc availability since the zinc absorption in human
subjects was significantly higher from a malted barley
meal with a phytase activity of 138% of that of raw material
and a relatively high phytate content (approx. 6.3 mmol
IP6 gK1 dm) as compared to unprocessed barley meal with
deactivated phytase (Fredlund et al., 2003).
One objective of malting the barley was to obtain malt
with high phytase activity, that would successfully hydro-
lyse phytate in a subsequent process. The phytase activities
in malts A and B were 131 and 62%, respectively, of that of
the raw material. After the incubation at 48 8C for 4 h, the
phytase activity in both malt types effectively reduced the
IP6 content to !0.1 mmol gK1 in both the A and B porridges
(Table 1). By comparison, incubation of unprocessed barley
flour under the same conditions reduced the IP6 content to
1.4 mmol gK1. This is unlikely to result in a meaningful
improvement of iron absorption.
ted Porridge
106
ight (g/mol)
104
105
106
alted barley (malt A and malt B) and replicates of incubated material and
wn as full or dotted lines.
Tab
le1
To
tal
con
ten
to
fir
on
and
ino
sito
lp
ho
sph
ates
(IP
i)in
mea
ls,to
tal
amo
un
to
fd
ialy
zab
leir
on
,ir
on
up
tak
ep
erh
ou
rb
yC
aco
-2ce
lls
fro
md
ialy
sis
flu
idco
llec
ted
bet
wee
n0
and
12
0m
ino
fd
iges
tio
n(D
F0–120)
and
esti
mat
edto
tal
iro
nab
sorp
tio
np
erh
ou
rfr
om
the
wh
ole
mea
l
Mea
lty
pe
Mea
lp
rop
erti
esa
Iro
nd
ialy
sabil
ity
aIr
on
up
tak
efr
om
DF
0–120
bE
stim
ated
abso
rp-
tio
nfr
om
the
wh
ole
mea
lc
(mg
/h)
(%o
fto
tal)
Fe
con
ten
t
(mg
/po
rtio
n)
IP6
con
ten
t
(mm
ol/
po
rtio
n)
SIP
3K
IP6
in
mea
l(m
mol/
po
rtio
n)
Dia
lysa
ble
Fe
in
the
wh
ole
mea
l
(mg
)(%
of
tota
l)
Fe
con
ten
tin
DF
0–120b
SIP
3K
IP6
in
DF
0–120
(mm
ol
into
tal
vo
l.)
Fe
abso
rpti
on
per
ho
ur
from
DF
0–120
d
(mg
)(m
g/m
l)(%
abs
55F
e)(m
g/h
)
Po
rrid
ge
A(n
Z4
)4
77
8G
10
0!
31
3.1
G1
.83
75
G3
1(7
.9%
)2
13
G2
10
.091
06
.01
.95
G0
.17
4.4
0G
0.3
67
.0(0
.15
%)
Po
rrid
ge
B(n
Z3
)4
78
4G
11
5!
31
47
G1
92
71
G1
1(5
.7%
)1
63
G0
.48
0.0
68
25
71
.61
G0
.10
2.6
3G
0.1
74
.3(0
.09
%)
Ref
eren
ce(n
Z3
)4
77
8G
91
32
4G
38
51
1G
32
18
G3
.8(0
.4%
)1
5.8
G4
.90
.006
46
91
.65
G0
.21
0.2
4G
0.0
30
.2(0
.01
%)
aV
alu
esar
eg
iven
asm
ean
GS
Efr
om
nd
iges
tio
nex
per
imen
tp
erm
eal
typ
e.b
Val
ues
are
giv
enas
mea
nG
SE
(nZ
2)
of
du
pli
cate
anal
ysi
so
fd
ialy
sis
flu
id(D
F0–120)
fro
mtw
ose
par
ate
dig
esti
on
exp
erim
ents
per
po
rrid
ge
type.
cC
alcu
late
dto
tal
abso
rpti
on
fro
mth
em
eal
bas
edo
nth
eas
sum
pti
on
that
the
rela
tiv
eab
sorp
tion
isth
esa
me
for
DF
120–240
and
DF
240–360
asfo
rD
F0–120.
dV
alu
esar
eg
iven
asm
ean
GS
E(n
Z1
2)
of
two
dig
esti
on
exp
erim
ents
each
stu
die
din
six
sep
arat
ece
llex
per
imen
ts.
A.-K. Haraldsson et al. / Journal of Cereal Science 42 (2005) 243–254248
3.1.2. Content and molecular weight of b-glucan
The b-glucan content was 2.94% of dry matter for
untreated barley, 3.05% for malt A and 3.61% for malt B.
The molecular weight distribution was generally the same
for malt B compared to the raw material, while malt A
contained more b-glucan of lower molecular weight
(Fig. 2), resulting in a lower Calcofluor average molecular
weight ð �Mcf Þ in malt A (Table 3). However, all three
samples still contained b-glucan of high molecular weight
(O1.2!106) and thus the extent of b-glucan degradation
during the malting process was low.
The incubation step needed to remove sufficient phytate
risked a high degradation of b-glucan. To minimise this, the
incubations were made at 48 8C which lowers b-glucanase
activity (Rimsten et al., 2002). Only a small change
occurred in the molecular weight distribution of b-glucan
in malt B after the incubation and after preparation of
porridge B (Fig. 2). For malt A after incubation, much of the
b-glucan had been converted to the lower molecular weight
range and the molecular weight was reduced further by
porridge preparation. Thus the �Mcf for porridge A that was
almost three times lower than for the other two porridges
(Table 3), showing the effectiveness of the malting
conditions used for malt B in preserving the b-glucan
molecular weight.
3.2. Iron availability in porridges during digestion
3.2.1. Iron dialysability
Dialysable iron, defined here as the total amount of iron
that passed through the hollow fibre membranes of the
gastrointestinal model, and is thus considered not to be
associated with large complexes unavailable for absorption.
Table 1 shows that the amount of dialysable iron from
porridge meals varied between 0.4 and 7.9% of the total iron
content in the meal (5 mg). The iron content in the DF
samples collected during digestion of malt porridges with
less than 3 mmol IP6 per meal was significantly higher than
DF samples from the reference porridge with 324 mmol IP6
(85.7 mg phytate-P from IP3KIP6) per meal.
Even though there was virtually complete degradation of
IP6 in porridges made from both malt A and B, the amount
of dialysable iron was significantly different in these two
meals. Brune et al. (1992) found that all inositol phosphates,
at least IP6KIP3, impaired iron absorption to the same
extent in relation to their phosphorus content. Furthermore,
in a mixture of isolated inositol phosphates, IP3 and IP4 were
found to enhance the inhibiting effect of IP5 and IP6
(Sandberg et al., 1999). Less inositol phosphates (13.1 mmol
IP3KIP6 or 2.2 mg phytate-P) were detected in porridge A,
compared to malt B porridge (147 mmol or 14.7 mg phytate-
P) (Tables 1 and 2), which can explain the lower iron
dialysability in porridge B. The more efficient degradation
of inositol phosphates in malt A was probably caused by its
higher phytase activity. Different levels of modification of
the kernels, such as greater degree of cell wall degradation
Table 2
Distribution of inositol phosphates (IPi) in porridgesa and in dialysates (DF
samples)b taken out during digestion
Meal type IP6 IP5 IP4 IP3 SIP3KIP6
(mmol per meal or total volume)c
Porridge A
(nZ4)
!3 6.8G0.2 nd 7G0.6 13
DF0–120 nd nd nd 6 6
DF120–240 nd nd nd 7 8
DF240–360 nd nd nd 6 6
Porridge B
(nZ3)
!3 6G0.7 23G2.6 118G31 147
DF0–120 nd nd 9 48 57
DF120–240 nd nd 7 8 14
DF240–360 nd nd nd 7 7
Reference
(nZ3)
324G38 93G4.1 71G7.1 23G1.4 511
DF0–120 28 16 17 8 69
DF120–240 21 12 11 9 53
DF240–360 nd nd nd 7 7
Abbreviation used: nd, not detectable.a Values are given as meanGSE of n digestion experiments.b Values are from concentrated samples of one selected digestion
experiment per meal type.c Mean total volume per DF sample is 2.4 L.
A.-K. Haraldsson et al. / Journal of Cereal Science 42 (2005) 243–254 249
and mobilisation of nutrients in malt A compared to malt B
and altered concentrations of other chelating agents, such as
organic acids and phenolic compounds (Maillard et al.,
1996; South, 1996), may also contribute to the differences in
iron availability.
3.2.2. Uptake of dialysable iron by Caco-2 cells.
In 1 h, 4.40 and 2.63 mg iron was calculated to be
absorbed from the DF0–120 samples for porridge A and
porridge B, respectively (Table 1). The Caco-2 cells were
used to reveal differences in iron availability between the
three porridges not detected by iron dialysability. However,
the differences in absorption level between the samples
seem to be influenced primarily by the iron content in the
dialysates, as shown by the similar percentage absorption of55Fe (1.61–1.95%) (Table 1).
Inositol phosphates significantly impair iron uptake by
Caco-2 cells (Glahn et al., 2002; Skoglund et al., 1999) and the
presence of inositol phosphates in the dialysates may thus
influence the iron uptake. The reference porridge contained
high amounts of phytate as well as a mixture of lower inositol
phosphates and some of these passed through the dialysis
membrane of the model (Table 2). Approximately 15% of the
IP6 of the reference porridge was detected in the dialysates. A
large percentage of IP3 from the meals was found in the
dialysates, indicating that IP3 was highly soluble during
digestion and readily dialysable. The relative uptake of
radioactive iron from DF0–120 from the reference porridge was
equal to that of porridge B, however, indicating that the low
amount iron absorbed from the reference sample (0.24 mg
Fe hK1) is a result of the low iron content in the DF0–120
sample applied to the cells rather than an inhibitory effect of
the inositol phosphates present in the dialysate. The slightly
higher percentage absorption of radioactive iron (1.95%) from
the dialysates collected during digestion of porridge A,
compared to the other two porridges (Table 1), may, however,
be caused by an almost complete absence of inositol
phosphates in the dialysate from this meal (Table 2).
Iron uptake by Caco-2 cells was studied using the DF0–
120 samples (Table 1), since the iron concentration was the
highest in these samples (213 mg Fe for porridge A and
163 mg for porridge B) compared to dialysates collected
later, suggesting that the level of available iron would be the
highest during this period. The content of dialyzable iron in
the DF120–240 samples was 116 mg Fe for porridge A and
80 mg for porridge B and in the DF240–360 samples it was
22 mg Fe for porridge A and 19 mg for porridge B,
respectively. In the case of the two latter dialysates of the
reference porridge, iron was undetectable. Since the
absorption of iron by the Caco-2 cells was influenced
chiefly by the iron contents in the samples, it may be
assumed that the percentage absorption of radioactive iron
would be the same for dialysates collected later in the
digestion experiments as for DF0–120. Based on this, the total
iron absorption from porridge A, porridge B and the
reference meal can be estimated to be 7.0, 4.3 and 0.2 mg
iron hK1, respectively (Table 1).
Measurements of dialysable iron have proven useful in
estimating iron availability (Forbes et al., 1989; Miller and
Berner, 1989; Reddy and Cook, 1991; Schricker et al.,
1981). In relation to the phytate content, a previous study
with the gastrointestinal model ranked iron dialysability in
meals in the same order as iron absorption in human studies
using a radioisotope technique (Salovaara et al., 2003).
However, it is possible that the total iron fraction is not
readily available for absorption, and a combination of in
vitro digestion with uptake studies using Caco-2 cells
provides a tool to study both passive diffusion and active
absorption of iron (Au and Reddy, 2000; Gangloff et al.,
1996; Glahn et al., 1996). Based on the data obtained from
the gastrointestinal model and from uptake from Caco-2
cells in the present study, the iron availability of the three
porridge meals can be ranked in decreasing order as
porridge AOporridge B[reference.
3.3. Changes in b-glucan during digestion
For practical reasons only two experiments for each meal
type were selected for b-glucan analyses. It is notable that
very similar molecular weight distributions were generally
obtained between replicates (Figs. 2 and 3), showing that
effects on b-glucan during incubation, porridge preparation
and digestion in the model were repeatable.
3.3.1. Characteristics of b-glucan in ileal delivery samples
The extraction yield of b-glucan (extractable b-glucan as
percentage of dry sample weight) generally increased when
60 min
Ref
eren
ce
n n n
Mal
t A
104
105
106
Mal
t B
104
105
106
Molecular weight (g/mol)
104
105
106
104
105
106
Fig. 3. Molecular weight distribution of b-glucan from replicates of ileal deliveries (ID samples) at 60 min and during time periods 0–120, 120–240 and 240–
360 min for reference, porridge A and porridge B. Full and dotted lines represent duplicate runs. Note that ileal delivery from the reference at 60 min contains
only one sample.
A.-K. Haraldsson et al. / Journal of Cereal Science 42 (2005) 243–254250
samples were digested in the model (Table 3). This increase
might be due to an increased concentration of b-glucan
caused by removal of dialysable material or to b-glucan
becoming more easily extractable the longer the time spent
in the model. For the ID240–360 samples, the yield of
extracted b-glucan decreased further, which might be a
result of a more extensive breakdown of b-glucan after
a longer time spent in the model. Since only b-glucans with
a molecular weight larger than 10,000 Da are detected by
the Calcofluor method (Jørgensen and Aastrup, 1988),
a substantial degradation would make a large part of the
b-glucan undetectable causing an apparent decrease in the
extraction yield.
The concentration of b-glucan in the collected intestinal
contents (mg b-glucan per gram of wet sample) varied from
0.08 in ID240–360 from porridge A to 1.74 in ID120–240 from
the reference porridge (Table 3). Between 120 and 240 min
of digestion, a large part of the meal had reached the ileum
and passed through the ileo-ceacal valve, and a large sample
volume was collected during this period. This can at least
partly explain the high recovery found in these samples.
For all three meal types, the amount of b-glucan
recovered from the model was much lower than the amount
introduced. This is because not all b-glucan is extractable
and there is degradation of b-glucan into low molecular
weight components during digestion.
After 60 min digestion, a 1 ml sample (ID0–60) was
drawn from the continuously collected ID0–120 sample. In
the reference porridge and porridge B, the molecular weight
distribution in the ID0–60 sample was similar to that found in
the porridges prior to digestion (Figs. 2 and 3), with similar�Mcf values (Table 3). Furthermore, all samples of ID0–120
contained more b-glucan of lower molecular weight than the
sample at 60 min, with a �Mcf that was almost half of that
found in ID0–60. This would indicate that, in the beginning,
b-glucan of higher molecular weight may pass more rapidly
through the model and/or that the degradation of the
b-glucan gradually increases with time spent in the model.
3.3.2. Fate of b-glucan throughout the gastrointestinal
model
During digestion, samples were also taken from the
duodenal (Duo1⁄2), jejunal (Jej1⁄2
) and ileal (Ile1⁄2) compart-
ments at the time when approximately 50% of the meal had
passed through the different parts of the small intestine, to
detect changes in the �Mcf of b-glucan throughout the model
(Fig. 4). �Mcf for the reference porridge and porridge A
decreased during passage in the small intestine, where as it
remained constant in porridge B. This decrease in molecular
weight was most obvious for the larger molecular weights
b-glucan components, as seen for the p50 and p90
percentiles. For all samples the yield of extracted b-glucan
increased with time spent in the model, although the levels
were very different.
3.3.3. b-Glucanase activity during digestion
To test whether there was b-glucanase activity in the
porridges and whether it increased during passage through
Table 3
Extraction yield of b-glucan (% of sample), concentration of extractable b-glucan in intestinal fluid (mg/g), recovery of b-glucan (%) and Calcofluor average
molecular weight (g/mol) of extractable b-glucan
Sample Properties of b-glucan Properties of arabinoxylan
Extraction
yielda (%)
Content/digestab
(mg/g)
Re-coveryc
(%)ð1= �Mcf !105Þd
(g/mol)
F 90 (% of F 60 in F 90)e
Arabinose (%) Xylose (%) Galactose (%)
Porridge A
Untreated 0.51 – – 1181 – – –
Incubated 0.34G0.05 – – 378G53 – – –
Porridge 0.40G0.01 – – 305G46 – – –
ID0–60 0.90G0.19 – – 361G52 – – –
ID0–120 0.48G0.21 0.17 1.44 216G6.1 0.40 (82.4) 0.48 (90.6) 0.12 (61.5)
ID120–240 1.34G0.20 0.39 4.31 124G1.1 0.80 (73.1) 0.97 (83.6) 0.25 (37.5)
ID240–360 0.40G0.12 0.08 0.94 99G0.61 0.61 (68.0) 0.73 (79.0) 0.26 (31.7)
Porridge B
Untreated 0.63 – – 1353 – – –
Incubated 0.67G0.02 – – 914G52 – – –
Porridge 0.78G0.14 – – 979G1.3 – – –
ID0–60 1.61G0.13 – – 759G29 – – –
ID0–120 2.03G0.07 0.98 5.30 448G20 0.36 (88.9) 0.42 (98.3) 0.10 (60.4)
ID120–240 5.07G0.62 1.52 22.2 184G1.8 0.69 (77.2) 0.74 (94.4) 0.24 (40.9)
ID240–360 3.67G0.25 1.00 13.7 137G5.4 0.49 (79.3) 0.50 (99.6) 0.29 (38.3)
Reference
Untreated 0.63 – – 1496 – – –
Porridge 0.96G0.06 – – 958G23 – – –
ID0–60 0.89G0.29 – – 850G46 – – –
ID0–120 1.43G0.07 0.49 9.48 450G6.8 0.36 (85.4) 0.40 (99.5) 0.12 (48.8)
ID120–240 4.89G0.13 1.74 18.6 208G13 0.70 (80.8) 0.76 (95.1) 0.21 (41.7)
ID240–360 4.61G0.25 0.87 9.31 103G9.4 0.71 (53.6) 0.75 (65.2) 0.26 (33.8)
Arabinoxylans are shown as content of arabinose, xylose and galactose (% of sample) in a fraction precipitated at 90% EtOH (F 90) and % of a fraction
precipitated at 60% EtOH (F 60) in F 90 from ileal delivery samples.a Given as percent of dry matter of collected sample. Values are of two digestion experiments meanGSE (nZ2).b Given as milligram b-glucan per gram intestinal content collected (wet weight of ileal delivery samples).c Given as percent of b-glucan introduced to the model.d Values are means of two selected digestion experiments, meanGSE (nZ2).e Given as percent of sample dry matter. Values are mean of duplicate analyses from one selected digestion experiment (deviation less than 4% from mean).
A.-K. Haraldsson et al. / Journal of Cereal Science 42 (2005) 243–254 251
the model, extracts were analysed for b-glucanase activity
using an extremely sensitive assay. The inverse molecular
weight of b-glucan was plotted against time after mixing
where enzyme activity is given by the slope (Fig. 5). Little
activity was found in the reference porridges and porridge A,
and no detectable activity in porridge B. Notable levels of
b-glucanase activity were found in the ID240–360 samples
from the reference porridge and porridge A while only
a small activity was detected in porridge B. The activity of
b-glucanase increased during the passage through the
model, which could explain the decrease in the molecular
weight in the reference porridge and porridge A samples
during digestion, but does not explain the low molecular
weight in ID240–360 of porridge B. Thus other factors must be
involved, such as a dependence of the molecular weight on
the transit rate through the model. The increase in activity
also explains the low extraction yield of detectable b-glucan
in porridge A. The b-glucanase may originate from
microorganisms or from endogenous enzymes in the malt.
A higher total count of microorganisms was found in malt A
(1.3!106 cfu) as compared to malt B (1.0!103 cfu).
No b-glucanase activity was detected in any of the enzyme
solutions used in the model, except for the lipase solution,
which showed a limited activity. This, although it was not
ideal, had probably had no significant influence on the
results, since only the reference porridge and porridge A
samples showed a large increase in b-glucanase activity.
Significant degradation of b-glucan during passage
through the upper gastrointestinal tract of ileosomists has
been shown by Sundberg et al. (1996). Comparing intake to
excretion revealed a notable loss (13–64%), with depoly-
merisation of the b-glucan dependent on both the food
preparation and transit time through the tract. The
degradation of viscous fibre (e.g. b-glucan and arabino-
xylan) in the small intestine probably has an important effect
in the ileal digestibility of nutrients, the re-absorption of
compounds such as bile acids and on intestinal function.
3.4. Extractability of arabinoxylan
Two fractions of extractable arabinoxylan were collected
by precipitation at 60 and 90% EtOH to obtain one fraction
0
100
200
300
400
500
600
700
800
Mol
ecul
ar w
eigh
t (g/
mol
)
0
1
2
0
100
200
300
400
500
600
700
800
Mol
ecul
ar w
eigh
t (g/
mol
)
0
1
2
3
4
0
100
200
300
400
500
600
700
800
Duo 1/2 Jej 1/2 Ile 1/2
Duo 1/2 Jej 1/2 Ile 1/2
Duo 1/2 Jej 1/2 Ile 1/2
Mol
ecul
ar w
eigh
t (g/
mol
)
0
1
2
3
4
5
6
7
8
9
10
(a)
(b)
(c)
Fig. 4. Calcofluor average molecular weight (black bars) and percentiles
p10 (white bars), p50 (light grey bars) and p90 (dark grey bars) and
extraction yield of b-glucan (C) in samples from porridge A (a), porridge
B (b) and reference porridge (c), collected from different compartments of
the intestine (Duo1⁄2 , Jej1⁄2and Ile1⁄2
).
0
0.5
1
1.5
2
2.5
3
0 4 8 12 16 20 24Time (h)
1/M
cf*1
05
Fig. 5. The inverse of Calcofluor average molecular weight ð1= �Mcf !105Þ
of purified b-glucan as a function of time after mixing with enzyme extracts
from reference (B), porridge A (,), porridge B (6) and from ileal
delivery samples collected between 240 and 360 min (ID240–360) from
porridge A (&), porridge B (:) and reference (C). An increasing curve
shows a decrease in molecular weight with time.
A.-K. Haraldsson et al. / Journal of Cereal Science 42 (2005) 243–254252
with only large molecules (F 60) and another with both
small and large molecules (F 90). It was found that most of
the arabinoxylan was of a large size in the ID0–120 samples,
since the differences in the content of arabinose and xylose
between the fractions were small (Table 3). For the
reference porridge and porridge A, the proportion of high
molecular weight arabinoxylan decreased over time. The
ratio between arabinose and xylose remained constant,
however, indicating that no structural changes had occurred.
The decrease in the molecular weight of arabinoxylan was
not as extensive as for b-glucan. The content of extractable
galactose increased with time spent in the model for all three
types of porridges, indicating an accumulation of extrac-
table arabinogalactan (Andersson et al., 2003).
3.5. Conclusion
The overall aim of this work was to prepare foods that
combined the nutritional aspects of iron availability and
specific polysaccharides in barley. The results show that,
by high temperature steeping with lactic acid and with
appropriate subsequent treatment, a malted product can be
obtained that has improved iron availability and retained a
b-glucan of relatively high molecular weight throughout
simulated upper gastrointestinal digestion. The pilot scale
malted barley exhibits properties similar to those obtained
in laboratory scale malting. A soaking step subsequent to
malting virtually completely removed phytate from the
malts, leading to improvement in iron dialysability after
in vitro digestion and iron uptake in a cell model. The rank
of the iron availability in the meals, in decreasing order, is
porridge AOporridge B[reference porridge, whereas
ranking of the molecular weight distribution of b-glucan
during digestion was porridge BRreferenceOporridge A.
It was not possible using the malted products to combine a
high phytase activity with an inhibited b-glucanase
activity. The gastrointestinal model, used for the first
time to study changes in fibre components, showed good
repeatability.
Acknowledgements
The study was financially supported by NUTEK/
VINNOVA (the Swedish Board for Industrial and Technical
Development/the Swedish Agency for Innovation Systems,
project number P11492), the SNF Swedish Nutrition
Foundation and Wilhelm and Martina Lundgren’s Science
Foundation.
A.-K. Haraldsson et al. / Journal of Cereal Science 42 (2005) 243–254 253
References
Andersson, A.M.M., Merker, A., Nilsson, P., Sorensen, H., Aman, P., 1999.
Chemical composition of the potential new crops Barbara vulgaris,
Barbara verna and Lepidium campestre. Journal of the Science of Food
and Agriculture 76, 179–186.
Andersson, A.A.M., Courtin, C.M., Delcour, J.A., Fredriksson, H.,
Schofield, J.D., Trogh, I., Tsiami, A.A., Aman, P., 2003. Milling
performance of north European hull-less barleys and characterization of
resultant millstreams. Cereal Chemistry 80, 667–673.
Au, A.P., Reddy, M.B., 2000. Caco-2 cells can be used to assess human iron
bioavailability from a semipurified meal. Journal of Nutrition 130,
1329–1334.
Bergman, E.-L., Autio, K., Sandberg, A.-S., 2000. Optimal conditions for
phytate degradation, estimation of phytase activity, and localization of
phytate in barley (cv. Blenheim). Journal of Agricultural and Food
Chemistry 48, 4647–4655.
Brune, M., Rossander-Hulthen, L., Hallberg, L., Gleerup, A., Sandberg, A.-S.,
1992. Iron absorption from bread in humans: inhibiting effects of cereal
fiber, phytate and inositol phosphates with different numbers of phosphate
groups. Journal of Nutrition 122, 442–449.
Carlsson, N.-G., Bergman, E.L., Skoglund, E., Hasselblad, K.,
Sandberg, A.-S., 2001. Rapid analysis of inositol phosphates. Journal
of Agricultural and Food Chemistry 49, 1695–1701.
Cavallero, A., Empilli, S., Brighenti, F., Stanca, A.M., 2002. High
(1/3),(1/4)-b-glucan barley fractions in bread making and their
effect on human glycemic response. Journal of Cereal Science 36,
59–66.
Davidsson, M.H., Dugan, L.D., Burns, J.H., Bova, J., Story, K.,
Drennan, K.B., 1991. The hypocholestrolemic effects of b-glucan in
oatmeal and oat bran. Journal of the American Medical Association
265, 1833–1839.
Forbes, A.L., Adams, C.E., Arnaud, M.J., Chichester, C.O., Cook, J.D.,
Harrison, B.N., Hurrell, R.F., Kahn, S.G., Morris, E.R., Tanner, J.T.,
Whittaker, P., 1989. Comparison of in vitro, animal, and clinical
determinations of iron bioavailability: international nutritional anemia
consultative group task force report on iron bioavailability. American
Journal of Clinical Nutrition 49, 225–238.
Fredlund, K., Bergman, E.-L., Rossander-Hulthen, L., Isaksson, M.,
Almgren, A., Sandberg, A.-S., 2003. Hydrothermal treatment and
malting of barley improved zinc absorption but not calcium absorption
in humans. European Journal of Clinical Nutrition 57, 1507–1513.
Fredrikson, M., Carlsson, N.-G., Almgren, A., Sandberg, A.-S., 2002.
Simultaneous and sensitive analysis of Cu, Ni, Zn, Co, Mn, and Fe in
food and biological samples by ion chromatography. Journal of
Agricultural and Food Chemistry 50, 59–65.
Gangloff, M.B., Glahn, R.P., Miller, D.D., van Campen, D.R., 1996.
Assessment of iron availability using combined in vitro digestion and
Caco-2 cell culture. Nutrition Research 16, 479–487.
Glahn, R.P., Gangloff, M.B., van Campen, D.R., Miller, D.D., Wein, E.M.,
Norvell, W.A., 1995. Bathophenanthrolene disulfonic acid and sodium
dithionite effectively remove surface-bound iron from Caco-2 cell
monolayer. Journal of Nutrition 125, 1833–1840.
Glahn, R.P., Wien, E.M., van Campen, D.R., Miller, D.D., 1996. Caco-2
cell iron uptake from meat and casein digests parallels in vivo studies:
use of a novel in vitro method for rapid estimation of iron
bioavailability. Journal of Nutrition 126, 332–339.
Glahn, R.P., Lee, O.A., Yeung, A., Goldman, M.I., Miller, D.D., 1998.
Caco-2 cell ferritin formation predicts nonradiolabeled food iron
availability in an in vitro digestion/Caco-2 cell culture model. Journal
of Nutrition 128, 1555–1561.
Glahn, R.P., Wortley, G.M., South, P.K., Miller, D.D., 2002. Inhibition of
iron uptake by phytic acid, tannic acid and ZnCl2: studies using an in
vitro digestion/Caco-2 cell model. Journal of Agricultural and Food
Chemistry 50, 390–395.
Hallberg, L., Brune, M., Rossander, L., 1989. Iron absorption in man:
ascorbic acid and dose-dependent inhibition by phytate. American
Journal of Clinical Nutrition 49, 140–144.
Haraldsson, A.-K., Rimsten, L., Larsson Alminger, M., Andersson, R.,
Andlid, T., Aman, P., Sandberg, A.-S., 2004. Phytate content is reduced
and b-glucanase activity suppressed in malted barley steeped with lactic
acid at high temperature. Journal of the Science of Food and Agriculture
84, 653–662.
Hidalgo, I.J., Raub, T.J., Borchardt, R.T., 1989. Characterization of the
human colon carcinoma cell line (Caco-2) as a model system for
intestinal epithelial permeability. Gastroenterology 96, 736–749.
Hurrell, R.F., Reddy, M.B., Juillerat, M.-A., Cook, J.D., 2003. Degradation
of phytic acid in cereal porridges improve iron absorption by human
subjects. American Journal of Clinical Nutrition 77, 1213–1219.
Jacobs, D.R., Meyer, K.A., Kushi, L.H., Folsom, A.R., 1998. Whole-grain
intake may reduce the risk of ischemic heart disease death in
postmenopausal women: the Iowa women’s health study. American
Journal of Clinical Nutrition 68, 248–257.
Jenkins, A., Jenkins, D.J.A., Zdravkovic, U., Wursch, P., Vuksan, V., 2002.
Depression of the glycemic index by high levels of b-glucan fiber in two
functional foods tested in type-2 diabetes. European Journal of Clinical
Nutrition 56, 622–628.
Jørgensen, K.G., Aastrup, S., 1988. Determination of b-glucan in
barley, malt, wort and beer, in: Linkens, H.F., Jackson, J.F. (Eds.),
Modern Methods of Plant Analysis VII. Springer/LLC, New York,
pp. 88–108.
Larsson, M., Rossander-Hulthen, L., Sandstrom, B., Sandberg, A.-S., 1996.
Improved zinc and iron absorption from breakfast meals containing
malted oats with reduced phytate content. British Journal of Nutrition
76, 677–688.
Larsson, M., Minekus, M., Havenaar, R., 1997. Estimation of the
bioavailability of iron and phosphorus in cereals using a dynamic
in vitro gastrointestinal model. Journal of the Science of Food and
Agriculture 74, 99–106.
Liu, S., Stampfer, M.J., Hu, F.B., Giovannucci, E., Rimm, E., Manson, J.E.,
Hennekens, C.H., 1999. Whole-grain consumption and risk of coronary
heart disease: results from the nurses’ health study. American Journal of
Clinical Nutrition 70, 412–419.
Maillard, M.-N., Soum, M.-H., Boivin, P., Berset, C., 1996. Antioxidant
activity of barley and malt: relationship with phenolic content.
Lebensmittel-Wissenschaft und Technologie—Food Science and
Technology 29, 238–244.
McCleary, B., Sheeran, H., 1987. Measurement of cereal a-amylase: a new
assay procedure. Journal of Cereal Science 6, 237–251.
Meyer, K.A., Kushi, L.H., Jacobs, D.R., Slavin, J.L., Sellers, T.A.,
Folsom, A.R., 2000. Carbohydrates, dietary fiber, and incident type-2
diabetes in older women. American Journal of Clinical Nutrition 71,
921–930.
Miller, D.D., Berner, L.A., 1989. Is solubility in vitro a reliable
predictor of iron bioavailability? Biological Trace Element Research
19, 11–24.
Minekus, M., Marteau, P., Havenaar, R., Huis in’t Veld, J.H.J., 1995. A
multicompartimental dynamic computer-controlled model simulating
the stomach and small intestine. Alternatives to Laboratory Animals 23,
197–209.
Newman, R.K., Lewis, S.E., Newman, C.W., Boik, R.J., 1989. Hypocho-
lesterolemic effect of barley foods on healthy men. Nutrition Reports
International 39, 749–760.
Newman, R.K., Klopfenstein, C.F., Newman, C.W., Guritino, N.,
Hofer, P.J., 1992. Comparison of the cholesterol-lowering properties
of whole barley, oat bran and wheat red dog in chicks and rats. Cereal
Chemistry 69, 240–244.
Pereira, M.A., Jacobs, D.R., Pins, J.J., Raatz, S.K., Gross, M.D.,
Slavin, J.L., Seaquist, E.R., 2002. Effect of whole grains on insulin
sensitivity in overweight hyperinsulimic adults. American Journal of
Clinical Nutrition 75, 848–855.
A.-K. Haraldsson et al. / Journal of Cereal Science 42 (2005) 243–254254
Reddy, M.B., Cook, J.D., 1991. Assessment of dietary determinants of
nonheme-iron absorption in humans and rats. American Journal of
Clinical Nutrition 54, 723–728.
Reddy, N.R., Sathe, S.K., Salunkhe, D.K., 1982. Phytates in legumes and
cereals. Advances in Food Research 28, 1–92.
Rimsten, L., Haraldsson, A.-K., Andersson, R., Alminger, M.,
Sandberg, A.-S., Aman, P., 2002. Effect of malting on b-glucanase
and phytase activity in barley grain. Journal of the Science of Food and
Agriculture 82, 904–912.
Rimsten, L., Stenberg, T., Andersson, R., Andersson, A., Aman, P., 2003.
Determination of b-glucan molecular weight using SEC with Calcofluor
detection in cereal extracts. Cereal Chemistry 80, 485–490.
Salovaara, S., Larsson Alminger, M., Eklund-Jonsson, C., Andlid, T.,
Sandberg, A.-S., 2003. Prolonged transit time through the stomach and
small intestine improves iron dialyzability and uptake in vitro models.
Journal of Agricultural and Food Chemistry 51, 5131–5136.
Sandberg, A.-S., Svanberg, U., 1991. Phytate hydrolysis by phytase in
cereals; effects on in vitro estimation of iron availability. Journal of
Food Science 56, 1330–1333.
Sandberg, A.-S., Brune, M., Carlsson, N.-G., Hallberg, L., Skoglund, E.,
Rossander-Hulten, L., 1999. Inositol phosphates with different numbers
of phosphate groups influence iron absorption in humans. American
Journal of Clinical Nutrition 70, 240–246.
Sandstrom, B.M., Sandberg, A.-S., 1992. Inhibitory effects of isolated
inositol phosphates on zinc absorption in humans. Journal of Trace
Elements and Electrolytes in Health and Disease 6, 99–103.
Schricker, B.R., Miller, D.D., Rasmussen, R.R., Van Campen, D., 1981. A
comparison of in vivo and in vitro methods for determining availability
of iron from meals. American Journal of Clinical Nutrition 34,
2257–2263.
Skoglund, E., Lonnerdal, B., Sandberg, A.-S., 1999. Inositol phosphates
influence iron uptake in Caco-2 cells. Journal of Agricultural and Food
Chemistry 47, 1109–1113.
Slavin, J.L., Martini, M.C., Jacobs, D.R., Marquart, L., 1999. Plausible
mechanisms for the protectiveness of whole grains. American Journal
of Clinical Nutrition 70, 459S–463S.
South, J., 1996. Changes in organic acid levels during malting. Journal of
the Institute of Brewing 102, 161–166.
Sundberg, B., Wood, P., Lia, A., Andersson, H., Sandberg, A.-S.,
Hallmans, G., Aman, P., 1996. Mixed-linked b-glucan from bread of
different cereals is partly degraded in the human ileosomy model.
American Journal of Clinical Nutrition 64, 878–885.
Theander, O., Aman, P., Westerlund, E., Andersson, R., Petterson, D.,
1995. Total dietary fibre determined as neutral sugar residues, uronic
acid residues, and klason lignin (the Uppsala method): collaborative
study. Journal of AOAC International 78, 1030–1044.