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

www.elsevier.com/locate/jnlabr/yjcrs

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

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