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Bioactive compounds in rye flours with different extraction rates

Article  in  European Food Research and Technology · January 2007

DOI: 10.1007/s00217-006-0452-4

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Eur Food Res Technol
DOI 10.1007/s00217-006-0452-4
ORIGINAL PAPER
Bioactive compounds in rye flours with different extraction rates
Anna Michalska · Alicja Ceglińska · Henryk Zieliński
Received: 20 March 2006 / Revised: 3 July 2006 / Accepted: 29 July 2006


C Springer-Verlag 2006
Abstract Rye flours with extraction rate of 100% (whole-
meal flour), 95% (brown flour), 90% (brown flour) and
70% (light flour) were prepared in order to study the re-
lation between flour extraction rates and content of bioactive
compounds. The following compounds were analysed: total
phenolic compounds (TPC), total flavonoids (TF), inositol
hexaphosphate (IP6), reduced (GSH) and oxidized (GSSG)
glutathione, tocopherols (T) and tocotrienols (T3). The re-
duced/oxidized glutathione status (GSH/GSSG) of the flours
was examined as a potential index of flour resistance against
oxidative stress. The following observations were made in
relation to the flour extraction rates and bioactive compounds
contents: (a) milling process caused decrease in content in
TPC, TF, IP6, GSH and GSSG, T and T3, (b) the most re-
sistant against oxidation processes were suggested a brown
flours, then light and finally wholemeal flour, (c) the ratio
of tocotrienols to tocopherols (T3/T) was the highest in rye
flours with extraction rate of 100–90% whereas light flour
was the poorest source of tocopherols and tocotrienols. The
provided data support current trend to increase number of
rye products from wholemeal or brown flours.
Keywords Rye flours . Flour extraction rates . Bioactive
compounds . Glutathione status
A. Michalska · H. Zieliński (
)
Division of Food Science, Institute of Animal Reproduction and
Food Research of Polish Academy of Sciences,
Tuwima 10, P.O. Box 55, 10-718 Olsztyn 5, Poland
e-mail: haziel@pan.olsztyn.pl
A. Ceglińska
Warsaw Agricultural University,
02-787 Warsaw, Poland
Introduction
Rye (Secale cereale L.) has been considered to be a primi-
tive crop with low yield, long and weak straw, and problem-
atic behaviour regarding sprouting in the year. The positive
features in cultivation practices include low requirements re-
garding soil and fertilization, as well as a relatively good over
winter ability. The greatest rye producer used to be the for-
mer Soviet Union. Poland, which produces about 6 million
tonnes, and Germany, with almost 5 million tonnes last years,
are the largest producers nowadays [1]. Although the total
production of rye has diminished, its use as food for humans
has increased slightly over the 1990s. In the 2005 according
to the Faostat data the rye production in the Europe was about
14 million metric tonnes whereas wheat production was 15
times higher [2].
Nutritionists world wide recommend an increased con-
sumption of whole grain products and dietary fiber [3]. Now
that consumers are increasingly interested in health and their
knowledge of the relationship between diet and well-being
has raised, rye is likely, to gain interest and popularity [4].
Rye is second to wheat, the most commonly used grain in the
production of bread [5]. Rye grain like other cereal grains
contribute significant quantities of energy, protein, selected
micronutrients and non-nutrients to the human diet. The
whole grain contains a large variety of substances, espe-
cially those that are biologically active and demonstrate an-
tioxidant properties, which include free radical scavengers,
reducing agents, potential complexers of prooxidant metals
and quenchers of the formation of a singlet oxygen [6]. This
wide range of nutrients and bioactive compounds include
phytochemicals, such as lignans, phenolics acids, phytos-
terols, tocopherols and tocotrienols, inositol hexaphosphate
and other vitamins as well as water soluble low molecular
weight antioxidants [4]. These compounds are concentrated
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in the germ and in the outer layer of the kernel [7]. Since
mature rye grain such other cereal grains does not contain
ascorbic acid, the reduced glutathione represents important
component of the antioxidant defense system of the grain [8].
Moreover, the Haliwell–Asada cycle in which ascorbate and
glutathione are strictly linked, does not work in mature cereal
grains and other biochemical systems and synergism which
may occur between components of cereal grain antioxidant
screen, play an important role in establishing the balance
between prooxidant reactions and antioxidant mechanism of
cereal grains [9]. Therefore, the oxidised glutathione/reduced
glutathione ratio may also provide useful information about
the formation of mixed protein-disulfides (protein thiolation)
[8].
Rye is the only one among the grains with a wholegrain
culture, and the consumption of rye should be increased in the
light of this benefit. Nowadays, its use is limited mainly as a
result of the problem arising from its flavour [10]. However,
rye consumption in Europe might increase if ingredients
were produced with the specific rye-like flavour modified to
a slightly milder one, without significantly decreasing the
contents of bioactive compounds in the rye.
Milling and baking are the most common techniques used
in grain processing for human food. The principal nutritional
benefit of processing is to increase the bioavailability of the
nutrients present in the grain. Essentially this is brought
about by making the cereal grain a better substrate for di-
gestive enzymes. This is achieved at both a physical and/or
a chemical level. In the milling process, the grains may be
fractionated into different types of flour. The proportion of
the original rye that is ultimately converted to flour is referred
to as the extraction rate. Typical values for white flours are
between 72 and 80%, between 85 and 98% for brown flours
and 100% for wholemeal flour. Wholemeal flour is the most
popular rye flour for baking, however rye flour with an ex-
traction rate of about 80% is also widely used. In addition
to the traditional use of different types of rye flour, various
types of rye flakes, breakfast cereals with rye contents up
to 55% are also available [11]. With decreasing extraction
rates in milling, more and more of the outer grain layers are
removed. At present, little is known about relationship be-
tween the extraction rates and bioactive compounds contents
in different type of rye flours. Recently, the only information
about losses in dietary fiber and associated bioactive com-
pounds such as vitamins B group, tryptophan and lignans
were reported [7, 12].
For this reason, the aim of this work to determine the rela-
tionship between the extraction rates and total phenolic com-
pounds, total flavonoids, inositol hexaphosphate, reduced
and oxidized glutathione, tocopherols and tocotrienols. The
reduced/oxidized glutathione status (GSH/GSSG) of the
flours was examined as a potential index of flour resistance
against oxidative stress. For this purpose a four types of
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Eur Food Res Technol
rye flours, namely wholemeal flour, two kinds of brown rye
flours and light rye flour. The reduced/oxidized glutathione
status (GSH/GSSG) of the flours was examined as a potential
index of flour resistance against oxidative stress.
Materials and methods
Reagents
Glutathione (γ -glutamyl-cysteinyl-glycine; GSH), inositol
hexaphosphoric acid (dodecasodium salt) from corn, oxi-
dized glutathione (GSSG), ( ± ) catechin, ferulic acid were
purchased from Sigma (Sigma Chemical Co., St. Louis, MO,
USA). Standards of tocopherols (α-T, β-T, γ -T, δ-T) and to-
cotrienols (α-T3, β-T3, γ -T3) were obtained from Merck
and Sigma. Folin–Ciocalteu Reagent (FCR) as well as all
other reagents of reagent-grade quality were from POCh,
Gliwice, Poland.
Materials
Rye grains of the cultivar Amilo were selected from breeding
materials grown in central Poland (DANKO, Plant Breed-
ing Co., Laski) in 2004. Samples were tempered to 14.0%
moisture and milled on a Quadrumat Senior laboratory mill
(Brabender) to obtain a straight grade flour with extraction
rates of 100, 95, 90 and 70%, respectively. Samples from
three replications were chosen for analysis. Flour samples
were stored at 4 ◦ C and the chemical analysis were carried
out within the first week of the storage.
Rye flour quality tests
Flour was characterized with the following analyses: mois-
ture, ash, protein and starch content as well as Amylograph
(Brabender) and Falling Number. Protein content was mea-
sured following AACC method using Foss Tecator apparatus
whereas starch content was determined by the polarymetric
method [13]. Moisture and ash content of flours were anal-
ysed according to AOAC 15.950.01 and 15.955.03, respec-
tively [14]. Falling Number values were determined with a
Falling Number apparatus (Perten, Sweden) using AACC
method 56-81B [13]. All analyses were made in triplicate.
Preparation of phosphate-buffered saline and 80%
methanol flour extracts
The flour was extracted in triplicate with phosphate-buffered
saline pH 7.4 PBS (15 mL per 1 g of flour) or with 80%
aqueous methanol (1/10, w/v) (10 mL per 1 g of flour) for
2 h of shaking at 37 ◦ C. Then samples were centrifuged
at 12000 × g for 15 min in a Beckman GS-15 R centrifuge
Eur Food Res Technol
(Beckman Instruments, Inc., Palo Alto, CL, USA). The fresh
extracts were used to determine total phenolic compounds
and total flavonoid content.
Analysis methods
The content of total phenolic compounds (TPC) was de-
termined in PBS and 80% methanol extracts according to
Shahidi and Naczk [15]. Exactly 0.25 mL aliquot of the re-
spective extract was mixed with 0.25 mL Folin–Ciocalteu
Reagent (previously diluted with water 1:1, v/v) and 0.5 mL
saturated sodium carbonate (Na2 CO3 ) solution and 4 mL
water. The mixture was allowed to stand at room tempera-
ture for 25 min and then it was centrifuged at 2000 × g for
10 min. Supernatant absorbance was measured at 725 nm
using a spectrophotometer (UV-160 1PC, Shimadzu, Japan).
The data were calculated on ferulic acid equivalents.
Total flavonoid content was determined with a spectropho-
tometric method [16]. Briefly, 0.25 mL of the PBS or 80%
methanol extract was diluted with 1.25 mL of distilled wa-
ter. Then 75 µL of a 5% NaNO2 solution was added, and
the mixture was allowed to stay at room temperature. After
6 min, 150 µL of a 10% AlCl3 × 6 H2 O solution was added,
and the mixture was allowed to stand for another 5 min. Af-
ter that, 0.5 mL of 1 M NaOH was added. The solution was
well mixed, and the absorbance was measured immediately
against the prepared blank at 510 nm using a spectropho-
tometer (UV-160 1PC, Shimadzu, Japan) in comparison with
the standards prepared similarly with known ( ± ) catechin
concentrations. Then the results were expressed as mg of
( ± ) catechin equivalents. Data were reported as means and
standard deviation for at least three replications.
Inositol hexaphosphate (IP6) was determined as follows:
flours were extracted with 20 mL 0.5 M HCl for 5 h us-
ing a BM1 magnetic stirrer. The extract was centrifuged at
3500 × g for 40 min (Centrifuge MPW-360) and the super-
natants were decanted, frozen overnight (−18 ◦ C), thawed at
room temperature and recentrifuged at 3500 × g for 40 min.
The supernatants (15 mL) were vacuum evaporated to dry-
ness at 40 ◦ C and dissolved in 15 mL of 0.025 M HCl. The
samples were then transferred to the mini-columns filled with
Dowex AG 1-X8 resin, from which the inositol phosphates
were eluted using 2 M HCl (5 × 4 mL). After the solvent had
been removed by evaporation, the dry residue was dissolved
in a mobile phase. Then the samples were analysed with
HPLC using a Shimadzu chromatograph (LC-10 AD pump,
refractometric detector RID-6A, CTO 6A column oven) and
a Nova-Pak C18 column [17, 18]. The mobile phase was a
mixture of methanol and 0.05 M formic acid (51:49, v/v) and
1.5 mL/100 mL tetrabutylammonium hydroxide (TBA-OH).
The flow rate was 0.4 mL/min. Inositol hexaphosphoric acid
(dodecasodium salt) from corn was the external standard and
injections were made with a 20-µL loop.
Reduced (GSH) and oxidized glutathione (GSSG) was
extracted from the samples according to Smith et al. [19].
Both forms of glutathione were determined according to
the spectrofluorometric method [20]. The detailed procedure
was described previously [8]. The assays were performed
using a Perkin-Elmer LS 50 B Luminescence Spectrometer.
The data were calculated on gram of dry matter basis of the
respective type of flour.
Tocopherols (α-T, β-T, γ -T, δ-T) and tocotrienols (α-
T3, β-T3, γ -T3) were extracted with methanol (0.5 g of
flour/7 mL) and then evaporated extracts were redissolved
in n-hexane. The tocols were separated by HPLC on Lichro-
spher Si 60 5-µm particle size, 4 mm × 250 mm column,
according to the method described by Paterson and Qureshi
[21]. Twenty microlitres of each sample was injected into
column. The HPLC systems consisted of a Shimadzu model
LC pump series 10 AD, and a Shimadzu RF-535 fluores-
cence spectrometer. The mobile phase was 0.5% isopropanol
in hexane. Flow rate was 1 mL/min, and the peaks were de-
tected using an excitation wavelength of 295 nm and emis-
sion wavelength of 330 nm. The contents of tocols were
calculated from the peak areas using standard curves of to-
copherols (α-T, β-T, γ -T, δ-T) and tocotrienols (α-T3, β-T3,
γ -T3).
Statistical analysis
Data were subjected to multifactor analysis of variance
(ANOVA) using the least-squared difference test with the
Statgraphic 5.0 Program (Statistical Graphic, Rockville, MD,
USA) and multiple correlation using Statistica 5.1 Program
(Statsoft, Tulsa, Okla, USA) for Windows using a PC-
Pentium.
Results and discussion
The physicochemical properties of rye flour with extraction
rate of 100, 95, 90 and 70% are compiled in Table 1. The flour
quality tests showed clearly the effect of extraction rate on
protein, starch and ash content as well as on starch viscosity
and falling number value. The wholemeal flour (extraction
rate of 100%) contained the highest content of proteins and
ash whereas the light flour (extraction rate of 70%) contained
the highest amount of starch with maximal viscosity. All
these findings were statistically significant differ. There was
also a clear impact of grain milling on flour’s falling number
value. The for kind of flours appeared falling number higher
than 250 whereas flours with falling number ranged from 125
to 200 are the most required for baking. The data provided
in this study indicate for the lowest amylolitic activity of
whole meal flours since falling number was decreasing due
to the grain milling into the light flour (extraction rate of
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 Eur Food Res Technol

Table 1 Physicochemical properties of rye flours with different extraction rates

Starch
Flour extraction Moisture content % Protein content % Ash content % Starch content % Max temp. Max viscosity Falling Number (s)
rate (%) gelation (◦ C) (UA)

100 11.5 ± 0.04 a 8.65 ± 0.01 a 1.60 ± 0.02 a 55.7 ± 0.14 a 81.0 a 1450 ± 42 a 397 ± 7 a
95 11.7 ± 0.02 be 8.31 ± 0.15 bd 1.43 ± 0.01 b 55.8 ± 0.28 a 79.0 a 1470 ± 28 a 335 ± 1 b
90 11.7 ± 0.02 ce 8.38 ± 0.24 ad 1.48 ± 0.01 c 55.8 ± 0.28 a 82.0 a 1500 ± 28 a 351 ± 1 c
70 12.0 ± 0.01 d 6.87 ± 0.20 c 0.69 ± 0.01 d 57.4 ± 0.14 b 81.0 a 1560 ± 28 b 316 ± 5 d

Data expressed as mean ± standard deviation (n = 3). Within each column, means with the same letter are not significantly different (P ≤ 0.05).

2 pounds [e.g., vitamin C, Cu(I), etc.]. Despite the undefined

chemical nature of FCR, the total phenols assay by FCR is
1,6 convenient, simple, and reproducible. As a result, a large
body of data has been accumulated, and it has become a
routine assay in studying phenolics antioxidants [35].
1,2
The content of total phenolic compounds (TPC) was anal-
T3/T

ysed in PBS and 80% methanol extracts. In this study, the

0,8
0,4
0 spite of their significant role in protection of grain against
100% 95% 90% 70%
Fig. 1 The effect of flow extraction rate on the ratio of tocotrienols to
tocopherols (T3/T)
70%). In sourdough baking, low values for falling number
are preferred because high amylolytivc activity ensures a
strong and rapid start for fermentation and development of a
characteristic flavour [22].
Among various plant components, phenolic compounds
are a group highly abundant in cereals [23–25]. Phenolic
compounds are located in the outermost aleuron layers and
bran and in the germ of the grains and their content in differ-
ent kind of flours will be depend on the milling process. They
are mostly covalently bound to cell wall polymers and can be
released after alkali, acid or enzymatic treatment of samples
prior extraction [26–28]. However, the experimental condi-
tions commonly used to detect bound phenolics acids by
alkaline hydrolysis result in loss of several phenolics acids,
partially dihyroxy-derivatives, and addition of ascorbic acid
or a metal chelator is needed to for totally prevent the loss of
these compounds [29]. In ground rye grain most of phenolics
compounds exist in the bound form with other grain com-
ponents that were realized upon enzymatic hydrolysis [30].
A fraction of soluble phenolic compounds is also present in
cereals and can be extracted with a mixture of solvents of
various polarities [23, 27, 31–34].
In this study, soluble phenolics compounds of rye flour
were determined by Folin–Ciocalteu Reagent (FCR) in a
routine assay. Obviously, FCR is nonspecific to phenolics
compound as it can be reduced by many nonphenolic com-
term total phenolic compounds (TPC) covered all pheno-
lics present in flours whereas total flavonoids (TF) included
only determination of flavonoids. The phenolic compounds
are localized mainly in the outer layers of the grain and de-
pathogens and pests, their content in different kind of flours
will be depend on the milling process. Results indicate a
higher concentration of TPC in PBS extracts than in 80%
methanol extracts (Table 2). It can be suggested that PBS
is a better solvent for extraction of TPC when compared to
the 80% methanol. The content of TPC in rye flours with
extraction rates of 100–90%, based on PBS extracts, ranged
from 2.43 to 2.31 mg/g d.m. and it was higher by average
47% when evaluation was based on the data provided by
80% methanol extracts. The content of TPC in flour with
extraction rate of 70% was approximately two-fold lower in
respect to the flours with extraction rates of 100–90%, for
both solvents used. It was also found that total flavonoids
content (TF) ranged only from 3 to 7% of TPC extracted by
PBS and from 30 to 32% of TPC extracted by 80% methanol.
TF in flours was two- to three-fold higher for 80% methanol
extracts than in PBS extracts (Table 2). Rye flours with ex-
traction rate range from 100 to 90% had similar level of TF,
whereas extraction rate of 70% decreased content of these
compounds. These findings confirm that milling process into
light flours removes outer layers of the grain, in which phe-
nolic compounds are mostly concentrated. Thus, it can be
concluded that milling process is negligible process with re-
spect to content of TPC and TF in rye flours with extraction
rates of 100–90%.
Currently, the role of phenolic compounds is much more
significant, especially in food technology since they may
have influence on colour, flavour and nutritional value. The
basic phenolics occurring in cereals are mainly phenolic
acids, after that a small amounts of tannins and flavonoids

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Eur Food Res Technol

Table 2 The content of total

phenolic compounds (TPC) and TPCa (mg/g d.m.) TFb (µg/g d.m.)
total flavonoids (TF) in PBS and Flour extraction rate (%) PBS 80% MeOH PBS 80% MeOH
80% methanol extracts of
milling products of whole grain 100 2.31 ± 0.06 a 1.43 ± 0.05 a 148 ± 15 a 431 ± 13 a
of rye cultivar Amilo 95 2.31 ± 0.09 a 1.33 ± 0.01 b 165 ± 4 a 400 ± 5 b
90 2.43 ± 0.24 a 1.29 ± 0.01 c 167 ± 6 a 414 ± 5 a
70 1.89 ± 0.01 b 0.75 ± 0.01 d 57 ± 2 b 330 ± 9 c

Data expressed as mean ± standard deviation (n = 3). Within each column, means with the same letter are
not significantly different (P ≤ 0.05).
a
The data were calculated as mg of ferulic acid equivalents.
b
The data were calculated as µg of ( ± ) catechin equivalents.

[36]. These compounds may be at least partially responsible
for the beneficial effects associated with cereal consump-
tion. Epidemiological studies strongly suggest that phenolics
compounds, including phenolics acids and flavonoids, have
protective effect against coronary diseases and possess free
radical scavenging properties from which antibacterial, an-
ticarcinogenic, anti-inflammatory and antiallergic properties
may origin [4, 36, 37] Moreover, their role is much more no-
ticeable due to the fact of reduce risk of some cancers [38].
It was reported that rye was a good source of hydroxycinna-
mates, especially ferulic acid and ferulic acid dehydrodimers
[39] and for this reason, the milling process of rye grains us-
ing extraction rates of 100–90% may stored these compounds
in flours.
The results of quantitative analyses of inositol hexaphos-
phate (IP6) and glutathione (GSH, GSSG, GSH/GSSG ratio)
in rye flours are shown in Table 3. The grain milling process
initially caused statistically significant increase in IP6 con-
tent in brown flours, and then almost completely lack of IP6
in light flour with extraction rate of 70% when compared
to wholemeal flour. The increase in IP6 content by 13%
was noted in brown flours while IP6 found in rye light flour
with extraction rate of 70% was only 1% of IP6 found in
wholemeal flour. These data, similarly to other reports [40],
showed a negative effect of milling process on IP6 content in
different kind of rye flours. Phytic acid is a main phosphorus-
storing compound in cereal grains. Regarding human health,
IP6 is important to prevent against cancer, heart diseases
and formation of renal stones [41, 42]. Adverse effects of
IP6 however have been reported as its excess may reduces
the bioavailability of inositol, phosphorus, iron and other
essential minerals [43].
Breadmaking degrades phytic acid to myoinositols with a
lower number of phosphate groups [44]. These myoinositol
may also have partly similar beneficial effects like those of
IP6, as it has been found that myoinositols are involved in cell
signalling in mammalian cells [45]. The role of phytic acid as
well as its lower forms formed during dough preparation and
baking, is significant because of the prevention from colon
cancer and protection against inflammatory diseases resulted
from chelating activity and suppressing iron-catalysed redox
reactions [46].
The level of GSH and GSSG noted in the four kinds of
rye flour is shown in Table 3. The milling process caused
increase of GSH content by 11 and 7% in brown flours
with extraction rate of 95 and 90%, respectively when com-
pared to the content in wholemeal flour. In contrast, the
grain milling process into the light flour with extraction
rate of 70% caused a decrease of GSH content by 8% and
GSSG by 16%. There is little known about the role of glu-
tathione in rye flours. Even though the level of GSH is low
in rye flours (300–400 nmol/g), this compound is consider
to play crucial role in redox reactions in flour and baking
technology. It is generally accepted that disulphide bonds
(SS) formed by oxidation of sulphydryl groups (SH) are
important in stabilising the polymer structure of glutenin.
Moreover, content of glutathione in flour was found to be
related to the rheological properties of dough [47]. The
calculated GSH/GSSG ratio showed the highest values for
flours with extraction rate of 95 and 90% whereas the low-
est was found for wholemeal flour (Table 3). This finding
may indicate that the most resistant against oxidation pro-
cesses during flour storage are brown flours with extraction

Table 3 Content of inositol hexaphosphate (IP6), reduced (GSH) and oxidised (GSSG) glutathione, and GSH/GSSG ratio in rye flours

Flour extraction rate (%) IP6 (µmol/g d.m.) GSH (µmol/g d.m.) GSSG (µmol/g d.m.) GSH/GSSG

100 10.85 ± 0.18 a 0.412 ± 0.012 a 0.201 ± 0.007 a 2,05 ± 0,02 a

95 12.23 ± 0.14 b 0.455 ± 0.008 b 0.186 ± 0.007 be 2,46 ± 0,14 bef
90 12.23 ± 0.14 b 0.441 ± 0.008 c 0.184 ± 0.006 ce 2,40 ± 0,03 ceg
70 0.09 ± 0.01 d 0.378 ± 0.009 d 0.169 ± 0.010 d 2,25 ± 0,08 dfg

Data expressed as mean ± standart deviation (n = 3). Within each column, means with the same letter are not significantly different (P ≤ 0.05).

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Table 4 The content of tocopherols and tocotrienols in rye flours (µg/g d.m.)
Tocopherols (T)
Flour extraction rate (%) α β γ δ
100 4.69 ± 0.83 1.43 ± 0.12 0.08 ± 0.01
95 3.70 ± 0.56 1.64 ± 0.16 0.09 ± 0.01
90 3.84 ± 0.63 1.59 ± 0.03 0.09 ± 0.01
70 1.96 ± 0.17 1.06 ± 0.09 0.06 ± 0.01
Data expressed as mean ± standart deviation (n = 3). Within each column, means with the same letter are not significantly different (P ≤ 0.05).
rate of 95 and 90%, then light flour and finally wholemeal
flour.
In this study, the tocopherols and tocotrienols profile of
the four kind of rye flours was investigated. The reason for
selection these compounds among others was connected with
a fact that bakery industries mainly prefer rather light flours
than wholegrain flour and that is why supplementation of α-
tocopherol or other bioactive compounds is usually necessary
[48, 49].
In this paper, simple methanol extraction was performed
according to the method of Peterson and Quaresi [21]. The re-
sults indicate the highest level of tocopherols and tocotrienols
in rye flours with extraction rate of 100%. It was found that
α-tocopherol was the major fraction of tocopherols whereas
α-tocotrienol and β-tocotrienol formed the main pool of to-
cotrienols (Table 4). Milling process decreased contents of
these bioactive compounds. Among the total tocopherols a
statistically significant decrease by 50 and 44% was found
for light flour when compared to the wholemeal and brown
flours, respectively. Milling process caused a higher losses
in tocotrienol contents and all the differences noted between
wholemeal, brown and light flours were statistically signifi-
cant. Cereal grains are unique group because of the fact that
they contain more tocotrienols than any other food products
[38]. Our data showed that ratio of tocotrienols to tocopherols
(T3/T) was higher than one in rye flours with extraction rate
of 100–90%. The light flour with extraction rate of 70% was
the poorest source of tocopherols and tocotrienols (Table 4).
As lipophilic substances, tocols are intimately associated
with lipid components of the sample matrix. For this rea-
son, the sample preparation methods for tocopherols and
tocotrienols analysis fall into two categories: nonsaponifica-
tion (solvent extraction) and saponification methods (alka-
line hydrolysis). More recently, the use of supercritical fluid
extraction has been proposed as an alternative method for the
determination of vitamin E from food [50]. This method was
recently compared to hot saponification followed by solvent
extraction and extraction without saponification in cereals by
Panfili et al. [51]. It was shown that the percent of tocopherols
and tocotrienols recovery was higher after hot saponification
followed by solvent extraction when compared to methanol
extraction. For this reason, the presented data express only
those tocols extractable by methanol.
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Eur Food Res Technol
Tocotrienols (T3)
Total α β γ Total
– 6.19 ± 0.90 a 4.96 ± 1.00 4.82 ± 0.50 – 9.78 ± 1.43 a
– 5.43 ± 0.58 a 2.84 ± 0.35 4.08 ± 0.45 – 6.92 ± 0.53 b
– 5.53 ± 0.64 a 2.77 ± 0.46 3.75 ± 0.07 – 6.52 ± 0.46 b
– 3.08 ± 0.19 b 0.49 ± 0.01 1.93 ± 0.15 – 2.42 ± 0.15 c
The role of tocopherols and tocotrienols in diet is essential
because of the multiprotective effect of these bioactive com-
pounds. They destroy nitrite, a component which presence
in food chain is associated with some type of stomach can-
cers [38]. Moreover, tocotrienols are inhibitors of cholesterol
synthesis [4].
Concluding remarks
The growing public awareness of the role of rye products
containing nutrients, bioactive compounds as well as antiox-
idants in diet could support consumption of this crop. It can
be suggested that in the nearest future more and more rye
products will be available on market, especially those orig-
inated from wholemeal or brown flours [52]. The provided
data in this study support this idea.
Acknowledgements We gratefully acknowledge research grant no.
PBZ-KBN-094/P06/2003/13 from the Polish State Committee for Sci-
entific Research. This article is a part of the Ph.D. of A. Michalska.
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