JFS: Food Chemistry and Toxicology

Physicochemical Properties of Bread

Baked from Flour Blended with Immature
Wheat Meal Rich in Fructooligosaccharides
R. MUJOO AND P.K.W. NG
Food Chemistry and Toxicology

ABSTRACT: Grain of the soft white wheat cultivar Harus was harvested weekly from anthesis to maturity and
fructooligosaccharides (FOS) contents were determined by reversed-phase high-performance liquid chromatog-
raphy. Tests were carried out to determine the effect of adding immature wheat meal to a base flour of cultivar
Russ (hard red spring) on the quality characteristics of bread. FOS content was also analyzed in baked bread, and
the effect of transglutaminase in improving bread quality was examined. Marked decreases in FOS contents,
such as 1-kestose and nystose, were observed with grain maturation. The overall quality of bread appeared to be
acceptable, and the added FOS were retained after baking.
Keywords: fructooligosaccharides, FOS, immature wheat, transglutaminase, bread

Introduction pathogens, rebalancing of metabolic activ- ity of nondigestible fermentable carbohy-

F unctional foods have been described as

foods that, by virtue of physiologically
active food components, provide benefits
beyond basic nutrition and may prevent
disease or promote health (Thomas and Ear
1994). One functional ingredient that has
received much attention in the scientific lit-
erature is the fructooligosaccharides (FOS)
(Niness 1999). These are naturally occurring
sugars that can have beneficial effects as
food ingredients. FOS have been defined as
a combination of the 3 sugars 1-kestose (1-
kestotriose; GF2), nystose (1,1-kestotet-
raose: GF3), and 1F-␤-fructofuranosylnys-
tose (1,1,1-kestopentaose; GF4) (Lewis
1993). The units of glucose (G) and fructose
(F) are in linear chains, always with a single
glucose unit at the head, with short chains
of fructose units attached. The G-F linkage
is (1-2)-␣, the same as in sucrose (GF), and
subsequent F-F linkages are (2-1)-␤-glyco-
sidic in nature.
FOS occur naturally in many plants in-
cluding bananas, onions, wheat, barley, as-
paragus, and Jerusalem artichokes (Mitsuo-
ka and others 1987; Tashiro and others 1992;
Spiegel and others 1994). FOS have been
shown to exhibit beneficial health effects by
stimulating the growth of bifidobacteria in
the human colon, suppressing putrefactive
MS 20030276 Submitted 5/19/03, Revised 7/10/03,
Accepted 9/3/03. Authors Mujoo and Ng are with
the Dept. of Food Science and Human Nutrition,
Michigan State Univ., East Lansing, MI 48824-
1224. Direct inquiries to author Ng (E-mail:
ngp@msu.edu).
ities (such as lipid homeostasis), strength-
ening immune functions (immunostimula-
tion), and improving the bioavailability of
nutrients (Hidaka and others 1986; Mitsuo-
ka 1986; McKellar and Modler 1989; Tomo-
matsu 1994; Gibson and Roberfroid 1995;
Niness 1999; Roberfroid 1999). Secondary
health benefits associated with the effect
on intestinal microflora include a reduction
in constipation (Sano 1986; Takahashi
1986). Because of their physicochemical
properties, sweetening power, and low ca-
loric value of 1.5 kcal/g (Hosoya and others
1988), FOS have been added to pastry, con-
fectionery, and dairy products. They are
used worldwide to add fiber to food prod-
ucts (Roberfroid 1993; Niness 1999).
The safety of FOS has been documented
in various studies (Hidaka and others 1986;
Clevenger and others 1988; Tokunaga and
others 1989; Kolbye 1992; Coussement
1999). Results provided no evidence that
FOS possessed any genotoxic potential.
There is widespread and common knowl-
edge of the natural occurrence and con-
sumption of FOS in human food. Numerous
studies have been conducted in animals
and humans with little evidence of adverse
effects associated with FOS consumption.
Furthermore, Tokunaga and others (1986)
demonstrated no significant effects in rats
given doses up to 1.67 g/d. The only effect
noted was the occurrence of intestinal dis-
comfort, soft stools, or diarrhea after inges-
tion of large quantities of FOS (more than
5% of the diet in rats). Intestinal acceptabil-
drates differs from person to person. Many
people can consume more than 10 g/d with-
out noticeable undesirable effects, whereas
some people experience unacceptable in-
testinal discomfort after ingestion of even
small amounts of nondigestible ferment-
able carbohydrates (Coussement 1999). It
is, therefore, not possible to define a no-ef-
fect level for these substances.
Studies have shown that large quantities
of FOS are stored in the stems and grains of
wheat for much of its growing cycle. In spring
wheat kernels, FOS concentration was found
to be almost 20% of the dry matter at 9 d af-
ter anthesis (DAA), and the content per ker-
nel rapidly decreased thereafter (Escalada
and Moss 1976). Schnyder and others (1988)
reported that FOS made up 27% of the ker-
nel dry matter at anthesis. It increased from
0.3 mg per kernel at anthesis to a maximum
of 1.2 mg per kernel at 7 DAA and decreased
rapidly to 0.5 mg at 17 DAA. Therefore, it ap-
pears that maximum accumulation in wheat
occurs between the 2nd and 3rd week after
flowering, at the physiological stage termed
the milky phase. The high levels of fructose
polymers in immature wheat grains seem
particularly encouraging and allow for the
suggestion of harvesting of wheat at the
milky phase for the utilization of this material
as a functional food.
Even though immature wheat grain con-
tains high levels of FOS, which are benefi-
cial as functional foods, proteins, which give
strength to flour, are still in the early stages
of synthesis. Gliadins have been detected

2448 JOURNAL OF FOOD SCIENCE—Vol. 68, Nr. 8, 2003 © 2003 Institute of Food Technologists
Further reproduction prohibited without permission
Physicochemical properties of bread . . .
at 10 DAA and high-molecular-weight glute-
nin subunits (HMW-GS) and low-molecular-
weight glutenin subunits (LMW-GS) at 13
DAA. The accumulation of these proteins
continues until physiological maturity is
reached (Ng and others 1990). It has long
been established that the product-making
quality of a wheat is influenced by protein
content and composition, with HMW-GS
having the largest effect on rheological and
baking properties of a flour (Payne and oth-
ers 1987).
Transglutaminase ( TG; protein-
glutamine ␥-glutamyl transferase, EC
2.3.2.13) catalyzes acyl transfer reactions,
introducing covalent cross-links in proteins
(Nonaka and others 1989). Cross-links form
between lysine and glutamine to form ⑀-
(␥Glu)-Lys bonds (Seguro and others 1996),
which help in the formation of new poly-
mers (Basman and others 2002a, 2002b)
and, in turn, help in strengthening the pro-
tein network, thereby modifying the rheo-
logical and baking properties of a flour.
In this study, contents of FOS in wheat
grains harvested at different stages of ma-
turity after anthesis were analyzed, the
bread-baking quality of flour supplement-
ed with immature wheat meal rich in FOS
was evaluated, and the use of TG for im-
proving that quality was also explored.
Materials and Methods
Collection of samples
The wheat cultivar Harus (soft white win-
ter) grown in the field in mid-Michigan in
the year 2000 was harvested at weekly inter-
vals starting from the seventh day of ap-
pearance of first anthers to maturity (43
DAA), altogether on 6 dates (7, 15, 22, 29, 36,
and 43 DAA). The samples were dried at
40 °C in a forced-air oven for 48 h, and dried
samples were stored in the freezer (–20 °C)
until analysis. The seeds used for FOS anal-
ysis and for blending in baking studies were
manually dehusked followed by grinding to
a fine powder in a coffee grinder. Grain of the
hard red spring wheat cultivar Russ of the
year 2001 (Agricultural Experiment Station,
NDSU, Fargo, N. Dak., U.S.A.), used for the
base flour of the bread baking study, was
milled into straight-grade flour with a Bu-
hler laboratory mill according to AACC Meth-
od 26-21A (AACC 2000).
Estimation of FOS contents
FOS contents of powdered samples from
grains harvested at different intervals and
of baked bread made from samples were
estimated using the procedure described
by Bancal and Gaudillere (1989) with some
modifications.
URLs and E-mail addresses are active links at www.ift.org
Sample preparation
Bread slices cut into pieces, freeze-dried,
and ground into powder were used for FOS
content estimation. Samples (500 mg) of
immature wheat meal or of bread powder
were extracted once in 10 mL boiling 25%
ethanol and then twice in 10 mL distilled
deionized water for 30 min. After each ex-
traction, the mixture was centrifuged at
10000 × g for 20 min at room temperature.
The extracts were combined, purified with a
C-18 cartridge (Sep-Pak; Millipore, Bedford,
Mass., U.S.A.), and then dried under vacu-
um at 70 °C to obtain dried residue.
Reversed-phase high-performance
liquid chromatography procedure
The dried residue was dissolved in 1 mL
water and analyzed by reversed-phase
high-performance liquid chromatography
(RP-HPLC) on a Millenium 2010 HPLC work
station consisting of a Waters 600E multisol-
vent delivery system (Waters, Milford,
Mass., U.S.A.). Separation of FOS was per-
formed using a constant flow (0.8 mL/min)
of distilled water and 2 250- × 4.6-mm ana-
lytical C-18 columns in series (Phenomenex
300 RP, Jupiter, 5 ␮m particle dia; Phenom-
enex, Torrance, Calif., U.S.A.). The carbohy-
drates were detected by both their molecu-
lar weight and their internal structure and
detected by a refractive index detector (Wa-
ters 410 differential refractometer). Total
run time per sample was 40 min. Appropri-
ate dilutions of a solution containing each of
the sugar standards (1-kestose, nystose,
and 1F-␤-fructofuranosylnystose; Wako Pure
Chemical Industries Ltd, Osaka, Japan) were
used as the calibration standards. Chro-
matographic peaks were identified by com-
paring sample retention times to those of
known standard mixtures. Components
were quantified using Millenium software
by measuring peak areas and comparing
them to standard curves generated by plot-
ting area counts against concentration of
standards (0 to 30 ␮g). Linear regression was
used to calculate the calibration curve and
the correlation coefficient. All samples were
analyzed in duplicate.
Bread baking ingredients
Hard red spring wheat cultivar Russ flour
with 11.23% moisture and 12.72% protein
content (N × 5.7, 14% moisture basis) was
used as a base flour for the bread baking
test. Instant dry yeast (Red Star Yeast & Prod-
ucts, Milwaukee, Wis., U.S.A.) and Crisco all-
vegetable shortening (Procter & Gamble,
Cincinnati, Ohio, U.S.A.) were used. The ox-
idizing agent L-(+) ascorbic acid was from
Fischer (Fischer Scientific, Pittsburgh, Pa.,
U.S.A.).
Vol. 68, Nr. 8, 2003—JOURNAL OF FOOD SCIENCE
Baking procedure
Breads were prepared from 100 g flour fol-
lowing the procedure of AACC Approved
Method 10-10B (AACC 2000) with some mod-
ifications. Control bread was prepared with
100 g Russ wheat flour. Test bread contained
0.24% wt/wt FOS, added by substituting
Russ flour with 8.45 g of immature wheat
meal (7 DAA, 14% moisture). Another exper-
iment was conducted in which TG was added
at a 0.1% (wt/wt) level to the control and the
test samples to observe the effect of the en-
zyme on subsequent bread quality. The for-
Food Chemistry and Toxicology
mula also contained sugar (6.0 g), salt (1.5 g),
shortening (3.0 g), instant dry yeast (1.96 g),
ascorbic acid (5 mg), and water based on op-
timum water absorption from the Farino-
graph according to AACC method 54-21
(AACC 2000). The ascorbic acid solution was
prepared fresh each day before baking.
Breads were baked at 204 °C for 30 min.
Bread properties
The loaves were weighed and loaf vol-
umes were determined by the rapeseed dis-
placement procedure in a volume-measur-
ing apparatus 2 h after baking. Crumb
firmness was then determined as compres-
sion force value (10 mm compression with a
38-mm-dia plunger at a compression rate of
1.7 mm/s) measured using a TA.Hdi Texture
Analyzer ( Texture Technologies Corp.,
Scarsdale, N.Y., U.S.A.) according to AACC
Method 74-09 (AACC 2000). The compres-
sion firmness value (N) was calculated from
the curves, and the results were expressed
as the means of 2 measurements using 2
bread slices for each measurement (total
thickness of 2 slices was 25 mm).
Results and Discussion
FOS contents during grain
development
Figure 1 shows the HPLC elution profile of
the FOS of wheat meal obtained from grains
7 d after anthesis (DAA). Two of the 3 known
trisaccharides were detected in the extracts
of all samples: 1-kestose and nystose. A
third trisaccharide, 1F-␤-fructofuranosyl-
nystose, was not detected. The amount of 1-
kestose estimated by HPLC analysis was
maximum in grains at 7 DAA (2.08 g/100 g,
db), decreasing markedly thereafter (Table
1). It is unknown why no 1-kestose was de-
tected at 15 DAA. A number of extractions
were made, and the extracts were all ana-
lyzed. There could have been a sampling
error at 15 DAA. In any event, it is clear that
1-kestose was otherwise decreasing with
maturity. At maturity, namely, 43 DAA, 1-
kestose content had decreased to 0.88 g/
100 g. The amount of nystose also showed a
2449
 Physicochemical properties of bread . . .

Table 1—Fructooligosaccharide (1- Table 2—Loaf weight, volume, texture, and fructooligosaccharide content of
kestose and nystose) contents in bread samplesa
wheat kernels at different stages of
maturitya Fructan (mg/serving
[35 g]) dry basis
Weight Vo lu me Firmness
Days 1-Kestose Nystose
(g) (cm 3 ) (N) 1-Kestose Nystose
after (dbb g/100 g; (dbb g/100 g;
anthesis mean ± SD) mean ± SD) Ctrl 130.3 ± 3.6 875 ± 49 2.06 ± 0.15 — —
7 2.08 ± 0.08 0.99 ± 0.03 Ctrl + TG 132.6 ± 2.9 785 ± 25 2.22 ± 0.27 — —
15 — 0.12 ± 0.005 Ctrl + IWM 130.6 ± 2.7 750 ± 46 3.08 ± 0.01 25 ± 2.8 2 ± 0.0
22 0.60 ± 0.06 0.05 ± 0.006 Ctrl + IWM + TG 132.5 ± 1.0 720 ± 28 3.38 ± 0.09 28 ± 5.6 2 ± 0.0
29 0.80 ± 0.08 0.03 ± 0.006 a Baked from control flour (Ctrl) or control flour plus immature wheat meal (IWM), with or without added

36 1.07 ± 0.01 — transglutaminase (TG).

b Analyses were carried out in duplicate.
43 0.88 ± 0.06 — All data are mean ± SD.
a Analyses were carried out in quadruplicate.
Food Chemistry and Toxicology

b db = dry basis.

most likely due to loss of FOS from the peri- was observed when immature wheat grain
decrease from 0.99 g/100 g at 7 DAA to 0.03 carp. Degradation of the FOS in the pericarp (7 DAA) meal was added to control flour
g/100 g at 29 DAA. Nystose could not be occurs during the period when parenchyma yielding a total of 0.24% FOS. However, a
detected in grains harvested after 29 DAA and tube cells of the pericarp are known to slight increase in loaf weight was noted
using the procedures described in this degenerate and when most nutrients enter- when TG was added to both control flour
study. ing the kernel are deviated to the rapidly and to flour blended with immature wheat
Total FOS concentration has been report- growing endosperm. meal ( Table 2). The mean loaf volume of
ed to be almost 20% of the dry matter at 9 baked bread was lower for samples blended
DAA with the amount per kernel decreasing Quality characteristics of baked with immature wheat meal (750 cm3) than
rapidly thereafter (Escalada and Moss bread prepared from flour those made from control flour (875 cm3). TG
1976). Schnyder and others (1988), on the supplemented with immature also appeared to lower the loaf volume,
other hand, observed that FOS made up wheat meal rich in whether added to control flour (785 cm3) or
27% of the kernel dry matter at anthesis. It fructooligosaccharides to the flour blended with immature wheat
increased from 0.3 mg per kernel at anthesis No effect on the loaf weight of the bread grain (720 cm3) (Table 2; Figure 2). Crumb
to a maximum of 1.2 mg per kernel at 7 DAA
and decreased rapidly after 10 DAA. Only
0.5 mg FOS was found per kernel at 17 DAA
(Schnyder and others 1988). The published
data reported the total amount of FOS but
not the amounts of each of the individual
components, as were determined in the
present study. However, the highest FOS
content at around 7 DAA and the rapid de-
crease thereafter, reported by the above
authors, was similar to results observed in
the present study. Thus, FOS metabolism
appears to be very active only during the
first 2 or 3 wk after anthesis, with rapid net
synthesis occurring in the first week and
rapid net degradation occurring between 10
and 17 DAA (Schnyder and others 1988).
Figure 1—High-performance liquid chromatography (HPLC) separation of fruc-
At anthesis, the prevailing structure of tans from immature wheat grain (7 d after anthesis).
the kernel is the pericarp (Evers 1970). Briarty
and others (1979) reported an endosperm
volume of only about 7 mm3 at 10 DAA when
kernel fresh weight was 25 mg. Schnyder and
others (1988) observed that the pericarp
fraction made up 47% of the total kernel dry
matter at 10 DAA when kernel fresh weight
was 37 mg. It is thus clear that a high propor-
tion of the nutrients imported into the kernel
during the first week post-anthesis is parti-
tioned to the pericarp. Active synthesis of FOS
in the pericarp, therefore, seems to be relat-
ed to the rapid growth of the pericarp and
Figure 2—Effect of incorporating immature wheat grain meal rich in fructans
associated with significant partitioning of
and of transglutaminase enzyme (TG) on external appearance of bread. 1 =
imported nutrients to this tissue. The marked control; 2 = control + TG; 3 = control + immature wheat grain meal; 4 = con-
decrease in the kernel FOS after 10 DAA is trol + immature wheat grain meal + TG.

2450 JOURNAL OF FOOD SCIENCE—Vol. 68, Nr. 8, 2003 URLs and E-mail addresses are active links at www.ift.org
Physicochemical properties of bread . . .
firmness of bread samples increased from
2.06 N for control bread, and to 3.08 N for
bread in which control flour was partially
blended with immature wheat meal. Addi-
tion of TG resulted in further firming effects
on the bread containing immature wheat
meal; these values of firmness increased
from 3.08 N to 3.38 N ( Table 2). Further-
more, the crumb structure of the bread sam-
ples baked with immature wheat meal ap-
peared to be slightly tighter than those of
the control flour breads (Figure 3).
Although the quality characteristics of
bread baked from control flour containing
immature wheat meal are lower than those
of bread baked from 100% control flour, the
decrease in quality is minor and the bread
appears to be of acceptable quality and is
nutritionally rich in FOS. Because it was dif-
ficult to separate bran from the immature
wheat grains, the flour used for substitution
was high in bran content as well. Studies
have shown that incorporating wheat bran
into a breadmaking system results in many
major changes in dough properties and
bread quality characteristics (Dubois 1978;
Lai and Hoseney 1989; Gan and others
1992). Bread made from flour containing
bran usually has increased water absorp-
Figure 3—Effect of incorporating im-
mature wheat grain meal rich in fruc-
tans and of transglutaminase enzyme
(TG) on internal appearance of bread.
1 = control; 2 = control + TG; 3 = con-
trol + immature wheat grain meal; 4 =
control + immature wheat grain meal
+ TG.
URLs and E-mail addresses are active links at www.ift.org
tion, decreased loaf volume, impaired
crumb structure, darker crumb color, and
reduced softness. These are in general
agreement with our findings for bread sam-
ples that contained immature wheat meal.
Published results also suggest that the loaf
volume-depressing effect of fibrous mate-
rials is the result of reduced gas retention
rather than reduced gas formation (Pomer-
anz and others 1977). In the present study,
TG did not appear to have any improving
effect on the bread quality, in contrast to a
previous report (Gerrard and others 1998).
However, it has also been observed that the
positive effect of TG seems to be more pro-
nounced with flour samples of lower quali-
ty (Basman and others 2002b). The flour
quality of the Russ wheat variety, which was
used as a base flour for the experiments in
the present study, is considered good and
strong. This could explain why no improving
effect on the bread quality was observed
upon addition of TG to the flour samples.
Fructooligosaccharide content in
the baked bread
Tests on retention of the 0.24% FOS con-
tent incorporated into the flour used for
baking bread showed the presence of both
1-kestose and nystose in bread after bak-
ing, as detected by HPLC analysis (Table 2).
No FOS were detected in bread baked from
the control flour alone. The amount of 1-
kestose detected in the bread supplement-
ed with immature wheat meal was 93 mg/
loaf of bread (130.6 g), which was somewhat
lower than 160 mg, the amount incorporat-
ed per loaf. On the other hand, nystose con-
tent detected in baked bread (8 mg/130.6 g)
was substantially below the incorporated
amount (70 mg/130 g): almost a 9-fold de-
crease. The decreases may be due to inter-
actions of these sugars with other compo-
nents of the flour during bread baking.
Nonetheless, almost 25 mg of 1-kestose and
2 mg of nystose per serving of bread (35 g)
were detected, both of which are significant
quantities considering no FOS were detect-
ed in the bread made from control flour.
The average daily intake of FOS from
common food items has been estimated to
be approximately 806 mg/d (Spiegel and
others 1994). The levels at which FOS were
present in baked bread substituted with
immature wheat meal (27 mg/serving) will
not raise the daily consumption of FOS to
unacceptable levels. With the retained
amount of FOS detected in this study, daily
intake levels of even 10 slices of FOS-en-
riched bread per day will be far below the
no-observed-effect limit in rats (2170 mg/
kg/d) (Tokunaga and others 1986) and be-
low the levels found to produce adverse ef-
Vol. 68, Nr. 8, 2003—JOURNAL OF FOOD SCIENCE
fects (soft stools/diarrhea) in humans (44 g)
(Spiegel and others 1994).
Incorporation of FOS from immature
wheat grain into bread gives it high added
value and provides an additional source of
FOS in our diet. Moreover, this innovative
destination redirects the use of potential
surplus wheat crop, and allows the growing
of alternative plants in the cultivation area
after early harvest of the immature wheat
crop, economically benefiting the growers.
Conclusions
Food Chemistry and Toxicology
T he data reported here show that FOS
accumulation in wheat grains is maxi-
mum during the first 2 wk after anthesis.
Blending strong wheat flour with immature
wheat meal rich in FOS produced bread of
considerably acceptable quality. Even
though the quality of bread prepared from
the control flour alone was slightly better,
bread with immature wheat meal contained
FOS, which are known for their health ben-
efits. Therefore, the wheat crop could be
utilized in an alternate way, with the produc-
tion of FOS-rich bread providing a viable
market for the immature harvested grain.
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