Food Research International 45 (2012) 3138

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Food Research International

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Use of enzymes to elucidate the factors contributing to bitterness in rye avour

Raija-Liisa Heini a,, Emilia Nordlund a, Kaisa Poutanen a, b, Johanna Buchert a
a
VTT Technical Research Centre of Finland, P.O. Box 1000 (Tietotie 2), FI-02044 VTT, Finland
b
University of Eastern Finland, Public Health and Clinical Nutrition, P.O. Box 1627, FI-70211 Kuopio, Finland

a r t i c l e i n f o a b s t r a c t

Article history: In spite of the health-benecial character of whole grain rye its use may be limited because of bitter taste. The
Received 3 June 2011 impact of non-volatile chemical compounds on the bitter taste of rye was analysed by the aid of enzymatic
Accepted 5 October 2011 hydrolysis, releasing potentially avour-active compounds from the rye matrix. Whole grain rye our
water suspension was treated with hydrolytic enzymes, whereafter portions of the rye suspensions were
Keywords:
baked into crackers, assessed for their sensory prole as well as solubilised hydrolysis products. Heat treat-
Rye
Whole grain
ment reduced the perceived bitterness. The treatment with enzyme preparation with high protease activity
Sensory evaluation increased the bitterness of rye and also wheat our both as suspension and as crackers. Other enzymes tested
Flavour (with high polygalacturonase, endo-glucanase, xylanase or amyloglucosidase activity) had no signicant im-
Bitterness pact on the perceived bitterness. Thus, small molecular weight peptides were considered to be a signicant
Enzymes contributor to the bitter note of rye.
Peptides 2011 Elsevier Ltd. All rights reserved.

1. Introduction
The current high interest in cereal whole grain foods derives from
epidemiological evidence suggesting their benecial role in health
maintenance in comparison with rened cereal foods. Epidemiologi-
cal studies show that intake of whole grain cereal foods reduces the
risk of type 2 diabetes (de Munter, Hu, Spiegelman, Franz, & van
Dam, 2007) and cardiovascular disease (Jensen et al., 2004; Mellen
et al., 2007). Health effects related to the consumption of whole
grain foods are assumed to be derived from dietary bre and phenolic
compounds present mostly in the outer layers of cereal grain (Arranz,
Saura-Calixto, Shaha, & Kroon, 2009; Okarter & Liu, 2010; Vitaglione,
Napolitano, & Fogliano, 2008).
Dietary recommendations and the consumption of whole grain
products do not meet due to several reasons. People do not necessar-
ily understand the health benets, they have difculties in identifying
whole grain foods (Adams & Engstr.om, 2000; Lang & Jebb, 2003; Seal,
Jones, & Whitney, 2006; Smith, Kuznesof, Richardson, & Seal, 2003,
2001; Smith, Smith, Richardson, & Seal, 2001), or they do not see
the recommendations personally relevant (Lappalainen et al., 1998).
Consumers tend to prefer rened wheat bread to whole wheat
bread, if equivalent ingredients and procedures are used (Bakke &
Vickers, 2007). Bakke and Vickers (2007) showed, however, that in-
gredient or processing modications can improve liking of whole
wheat bread to the level of rened bread.
Corresponding author. Tel.: + 358 20 722 5178; fax: + 358 20 722 7071.
E-mail addresses: raija-liisa.heinio@vtt. (R.-L. Heini), emilia.nordlund@vtt.
(E. Nordlund), kaisa.poutanen@vtt. (K. Poutanen), johanna.buchert@vtt. (J. Buchert).
Certain intrinsic avours of whole grain are often not regarded at-
tractive by consumers (Marquart et al., 2007). In particular avour of
whole grain rye is intensive and bitter, and may thus limit rye product
applications. People vary widely in their sensitivity to bitter com-
pounds, and several transduction mechanisms are suggested to exist
(Delwiche et al., 2001). However, the bitter taste of rye is not equal
with the cereal, rye-like avour (Heini, Liukkonen, Katina, Myllymki
& Poutanen, 2003). Bitterness has been shown to be localised in the
outer layers of the grain, mainly in the bran fraction (Heini, Liukkonen,
Katina, Myllymki & Poutanen, 2003). Volatile compounds in the air-
space of a product have traditionally been explained to be a reason for
avour formation of cereal products (Kirchhoff & Schieberle, 2001). It
is, however, obvious that non-volatile chemical compounds can bind
to the taste receptors and inuence taste perception, although their
role on cereal avour is not so well understood. The contribution of
phenolic compounds and/or small peptides and amino acids and/or
fatty acids on the bitterness of rye has been suggested (Hansen,
1995; Heini, Katina, et al., 2003; Heini et al., 2008; Liukkonen
et al., 2003; Schieberle, 1996; Shewry & Bechtel, 2001).
We have previously studied the impact of various processing tech-
niques on the avour of rye (Heini, 2003; Heini, 2006 and Heini,
Katina, et al., 2003; Heini, Liukkonen, Katina, Myllymki & Poutanen,
2003). By milling fractionation the kernel can be divided into frag-
ments with a distinct avour prole (Heini, Liukkonen, Katina,
Myllymki & Poutanen, 2003). The endospermic fraction of grain
has a very mild avour, resembling the avour of wheat, whereas
the bitter, intense avour and aftertaste are located in the bran
fraction. On the other hand the shorts, with high bioactivity, have a
cereal-like but not bitter avour (Heini, Liukkonen, Katina, Myllymki
& Poutanen, 2003). Sourdough fermentation followed by extrusion

0963-9969/$ see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodres.2011.10.006
32 R.-L. Heini et al. / Food Research International 45 (2012) 3138

has been shown to produce sour, intense avour and aftertaste to rye
(Heini, Katina, et al., 2003). During sourdough fermentation process
proteolysis takes place, resulting in increased peptide and amino acid
content (Gnzle et al., 2008; Loponen et al., 2009; Thiele et al., 2003).
The proteolysis is caused by endogenous enzymes present in grain, by
microbial enzymes from contamination of the grains and by enzymes
secreted by sourdough bacteria.
Enzymes have effectively been used to improve the technological
quality attributes of cereal products in general as reviewed by Tenkanen
et al. (2000) and Rastall (2007), but their effect on the perceived avour
is not so well understood. In this work different types of commercial
enzymes were used to modify the composition of rye our, where-
after the perceived rye avour was determined. Reasons for causes
for rye bitterness were subsequently suggested.
2. Material and methods
2.1. Enzymes
The enzymes used were commercial multienzyme preparations
containing several activities (Table 1). The key activities of the select-
ed enzyme preparations were determined as described below: poly-
galacturonase activity was measured according to Bailey and Pessa
(1989), endoglucanase activity according to IUPAC (1987), amylogly-
cosidase according to Bailey and Pessa (1989), endoxylanase accord-
ing to Bailey et al. (1992), -glucosidase and protease according to
Bailey and Linko (1990), and lipase according to Lowry et al. (1951).
Activity proling showed that the commercial enzyme prepara-
tions tested included several enzyme activities (Table 2). The activity
prole of Pectinex BE 3-L and Corolase 7089 was narrower than that
of other analysed preparations.
2.2. Flour
Medium coarse wholemeal rye our (Paakari) used in the experi-
ments was manufactured by Raisio Group, Finland. White rye our
(Mylly Matti) was manufactured by Helsingin Mylly Oy, Finland,
and special white wheat our (Sunnuntai) by Raisio Group, Finland.
2.3. Enzymatic treatments
The rye samples with or without enzymatic treatments were
prepared either as rye ourwater suspensions or after a heating pro-
cess as rye crackers. Rye ourwater suspensions (40 g rye our +
60 g water) were incubated with enzymes for 2 h at +40 C by mix-
ing occasionally with a spoon. The reference sample without enzyme
addition was incubated accordingly. Small rye crackers (approxi-
mately 3 cm in diameter) were prepared of the rye ourwater sus-
pension by using two teaspoons, and the surface of the crackers was
perforated with a fork. The crackers were baked for 15 min (for
2.5 min using steam and the rest 12.5 min with a convection mode)
at 175 C on a baking paper in a convection oven. The impact of
endogenous enzymes on rye avour was elucidated by analysing
both non-incubated and incubated (2 h + 40 C) rye ourwater
suspensions.
The enzyme dosages used in the screening trials are presented in
Table 1. In further experiments the enzymes were dosed according to
the selected activity as indicated in Table 2: 10 and 100 nkat activity/g
of rye our, except protease (Corolase 7089) was dosed 1 and
10 nkat/g rye our.
The impact of protease preparation (Corolase 7089) (5 nkat activity/g
our) was further elucidated using wholemeal rye, white rye and white
wheat ours with an attempt to verify the possible inuence of released
peptides on the perceived bitterness. The corresponding ours were
analysed also without enzymatic treatment. In sensory evaluation
both ourwater suspensions and baked crackers was studied.
2.4. Sensory evaluation
All sensory work was carried out at the sensory laboratory of VTT,
which fulls the requirements of the ISO standards (ISO, 1985, 1988).
The sensory panel consisted of ten trained assessors with proven
skills. All assessors of the internal sensory panel have passed the
basic taste test, the odour test and the colour vision test, and their
evaluation ability is routinely checked using individual control cards
for each assessor. The panel was particularly familiarized with the
sensory descriptors and the attribute intensities of rye and processed
rye in several pre-sessions prior to the evaluations with real samples,

Table 1
Commercial enzyme preparations used in the preliminary trials, activities and used dosages.

Commercial name Producer Reported main activities (main function examined in this study presented in bold) Main activity Enzyme dosage nkat/g our

Pectinex BE 3-L Novozymes Polygalacturonase, pectin lyase, pectin methylesterase 90,000 nkat PG/ml 1000
Econase CE AB Enzymes Xylanase, endo-glucanase and -glucosidase 16,600 nkat EG/ml 200
Veron CLX AB Enzymes -Amylase, transglutaminase, xylanase 4550 U AMY/g 50
Pentopan mono PG Novozymes Polygalacturonase, xylanase, endo-glucanase, amylase, phenolic acid esterase 361,000 nkat XYL/g 1000
Novozym 188 Novozymes -Glucosidase, -galactosidase, mannanase 5300 nkat B-GL/ml 200
Corolase 7089 AB Enzymes Endo-peptidase 177 nkat PRO/ml 10
Corolase LAP AB Enzymes Endo-peptidase 350 LAP/ml 2 3.5 LAP/g
Lipopan 50 BG Novozymes 1,3-Specic lipase 505,100 nkat LIP/g 1000
Biobake Fresh XL Quest Int. Amyloglucosidase 8500 nkat/g 100

nkat: nanokatal. 1 nanokatal is dened as the amount of enzyme required to raise the rate of reaction by 1 nmol/s under dened assay conditions.
PG, polygalacturonase; EG, endo-glucanase; AMY, -amylase; XYL, xylanase; B-GL, -glucosidase; PRO, endo-peptidase activity on azocasein; LAP, Leucine aminopeptidase activity;
LIP, lipase as measured as Kilo Lipase Units (KLU).

Table 2
Enzymatic activities in the commercial enzyme mixtures used in the treatments of the ryewater suspensions, where the main activity of the enzyme 100 or 10 nkat/ml (shown as bold).

Enzyme Endo-glucanase Polygalacturonase Xylanase Mannanase Protease Amyloglucosidase -Glucosidase

nkat/ml nkat/ml nkat/ml nkat/ml nkat/ml nkat/ml nkat/ml

Pectinex BE 3-L 1 100 4 2 0 2 0

Econase CE 100 19 294 14 0 0 3
Novozym 188 5 210 72 36 0 2287 100
Corolase 7089 0 6 4 2 10 9 0
Biobake Fresh XL 0 476 41 35 0 100 0
 R.-L. Heini et al. / Food Research International 45 (2012) 3138
and using verbal denitions describing the ends of the intensity scales
of the various attributes. The blind-coded samples were presented to
the trained assessors in random order, and water and toast wheat
bread were provided for cleansing the palate between the samples.
The panel was instructed to sniff each sample prior to tasting in the
given order, and to keep a one minute break between each sample
while tasting. The assessors were instructed not to swallow the sam-
ples. The scores were recorded and collected using a computerized
data system (Compusense Five, CSA, Computerized Sensory Analysis
System, Compusense Inc., Guelph, ON, Canada).
In the rst phase the impact of the enzyme preparations (Table 1)
on the perceived bitterness of rye ourwater suspensions and rye
crackers was evaluated by trained assessors (n = 7) by using the dif-
ference from control method on a scale 5 + 5 (5 = clearly less
bitter than the reference, +5 = clearly more bitter than the reference,
the bitterness of the reference without the enzymatic treatment
being 0). The bitterness of the rye ourwater suspensions and of
the crackers was evaluated from white 8 cl plastic containers with a
lid in separate sessions. The reference sample was served rst.
In the second phase the sensory proles of the ve enzyme-treated
(Table 2) rye our-water suspensions were determined by a trained
panel (n = 9) using descriptive analysis with nine sensory attributes
in two replicate sessions (Heini, 2003; Lawless & Heymann, 1999;
Meilgaard et al., 1999) (Table 3). The vocabularies of the sensory attri-
butes were developed by describing the differences between the sam-
ples, using a ve-member expert panel, specialized in cereal products
in round-table sessions. The attribute intensities were rated on contin-
uous unstructured, graphical intensity scales. The scales were 10 cm in
length and verbally anchored at each end, the left side of the scale
Fig. 1. The impact of the enzyme preparates with hydrolytic main activities on the
perceived bitterness of (a) rye ourwater suspensions and (b) rye crackers, measured
as the difference from the reference without any enzyme treatment on the scale
5 +5 (n = 7).
33
corresponding to the lowest intensity (value 0) and the right side to
the highest intensity (value 10) of the attribute. The rye samples, ap-
proximately 10 g each, were presented to the assessors as ourwater
suspensions from three-digit coded plastic containers, 75 ml, covered
with lids in random order. The closed containers were served immedi-
ately after the enzymatic treatments. Each assessor was provided with
six our samples (ve samples treated with the enzyme and one refer-
ence without the enzymatic treatment) to be evaluated in one session.
In the third phase the impact of protease preparation (Corolase
7089, 5 nkat/g our) on the bitterness of different our types
wholemeal rye, white rye and white wheat served as suspensions
and crackers, was studied by using difference from control method
on a scale 5 +5 in duplicate sessions by trained assessors
(n = 10). The bitterness of the rye ourwater suspensions and of
the crackers was evaluated from white 8 cl plastic containers with a
lid in separate sessions.
2.5. Chemical analyses
The enzymatically treated rye-our suspensions were centrifuged
by using an ultra-centrifuge (15,000 rpm, Sorvall Instruments Dupont
RC5C for 10 min), and the amounts of reducing sugars, soluble pro-
teins and total phenolic compounds were analysed as two replicates
from the supernatants, except as three replicates for the reducing
sugars in Table 5. Reducing sugars were determined spectrophoto-
metrically (Shimadzu UV-Visible Recording Spectrophotometer UV-
260) by the dinitrosalicylic acid (DNS) method (Bernfeld, 1955).
The amount of soluble proteins was measured by the Lowry method
(Lowry et al., 1951) and total phenolic compounds by the Folin
Ciocalteau method (Singleton & Rossi, 1965). The content of free
ferulic acid was analysed without replicates from the supernatants
by HPLC (Bartolom & Gmez-Cordovs, 1999).
2.6. Statistical analysis
Means of the sensory proling raw data obtained were calculated.
The signicance of each descriptive attribute in discriminating between
the samples was analyzed using analysis of variance (ANOVA), and
Tukey's Honestly Signicant Difference (HSD) test (signicance of
differences at p b 0.05). A two-way ANOVA was applied as the gener-
al linear model (GLM) procedure for the samples by using the SPSS
software (SPSS, SPSS Inc.). ANOVA was used to test statistical differ-
ences in sensory attributes between the samples, and the statistical
difference between the sessions. When the difference in ANOVA
among the samples was statistically signicant, pair-wise compari-
sons of these samples were analyzed using Tukey's test. The session
effect was not statistically signicant in sensory assessments. The
chemical data was analysed as well statistically by ANOVA and
Tukey's test by using the SPSS software.
3. Results
3.1. Screening the impact of enzymes on the perceived bitterness of rye
Most of the nine commercial hydrolytic enzyme preparations
(Table 1) increased the perception of bitterness of rye (Fig. 1a
and b). The perceived bitterness was increased most by the Corolase
7089 treatment followed by Pectinex BE 3-L and Econase CE. Novozym
188 and Biobake Fresh XL also increased the bitterness to some extent,
especially when analyzed as waterour suspensions (Fig. 1a). Effects
of Veron CLX, Pentopan mono PG, Corolase LAP and Lipopan 50 BG on
the rye avour were minor when compared to the reference. Regard-
less of the sample format (raw our-water suspension or cracker), the
results were rather equivalent, although the effects of the enzymes on
the perceived bitterness were smaller in the crackers.
34 R.-L. Heini et al. / Food Research International 45 (2012) 3138

O-FRESH (F=5.30 p<0.001)

10,0

AFTERTASTE (F=3.37 p<0.01) 8,0 O-RYELIKE (F=1.73 ns)

6,0

4,0

F-BITTER (F=2.52 p<0.05) 2,0 O-MALT (F=3.65 p<0.01)

0,0

F-CHEMICAL (F=12.41 p<0.001) T-THICKNESS (F=40.39 p<0.001)

F-SWEET (F=0.41 ns) F-RYELIKE (F=4.35 p<0.001)

Pectinase Cellulase B-glucosidase Protease Amyloglucosidase Reference

Fig. 2. Sensory proles of enzyme-boosted (100 nkat/g our, except for protease 10 nkat/g our) ryewater suspensions (n = 2 9). Commercial enzyme preparates used in the
experiment: Pectinex BE 3-L (pectinase), Econase CE (cellulase/hemicellulase), Novozym 188 (-glucosidase), Corolase 7089 (protease) and Biobake Fresh XL (amyloglucosidase).

The impact of endogenous enzymes on rye avour was also
assessed by comparing both non-incubated and incubated (2 h +
40 C) rye our-water suspensions. Incubation decreased signicantly
only the thickness (F(3,39) = 33.8; p b 0.001) without having any
effect on the odour or avour attributes (data not shown).
3.2. Impact of selected enzymes on sensory prole and chemical
compounds of rye
Corolase 7089 (protease), Pectinex BE 3-L (pectinase), Econase CE
(cellulase/hemicellulase), Novozym 188 (-glucosidase) and Biobake
Fresh XL (amyloglucosidase) were further examined to evaluate their
effects on perceived bitterness and other sensory attributes, chemis-
try and structure of the rye our matrix. The rye ourwater suspen-
sions were incubated with the enzyme preparations at one enzyme
addition level (100 nkat; protease 10 nkat), the reference being with-
out enzyme addition. Corolase 7089 (protease) treatment again pro-
duced the most signicant (F(5108) = 2.5; p = 0.03) increase in the
perceived bitterness of the rye suspension as compared to the refer-
ence without the enzyme addition, whereas the other tested enzymes
(Pectinex BE 3-L, Econase CE, Novozym 188 and Biobake Fresh XL)
had no clear effect on bitterness (Fig. 2, Table 4). Statistically signi-
cant differences after Corolase treatment were detected also in
several other sensory characteristics: in fresh and malty odour, in
thickness, and in rye-like, chemical and bitter avour (Table 4). Cor-
olase treatment caused least fresh and malty odour, and least rye-
like but most chemical-like avour. The most intense aftertaste was
perceived in the Corolase-treated suspension, and the least intense
in Pectinex- and Biobake Fresh-treated suspensions as well as in the
reference suspension. Econase addition decreased signicantly the
thickness of the suspension, indicating efcient hydrolysis of the ce-
real polysaccharides, whereas the thickness of suspensions after Pec-
tinex, Novozym and Biobake Fresh additions was similar to the
reference (Table 4).
The reaction products obtained in the enzymatic treatments were
analysed from the supernatants after centrifugation of rye our
water suspensions (Table 5). The cellulase/hemicellulase preparation
Econase CE and -glucosidase preparation Novozym 188 degraded
the carbohydrates of the cell walls, detected as an increase in the
amount of the reducing sugars of the supernatant. Novozym 188 con-
tains both -glucosidase and amyloglucosidase activity, and it seems
that the increased content of reducing sugars in the supernatant is
due to the hydrolysis of starch and its degradation products by
these enzymes. In the case of Econase the hydrolysis is expected to
be caused by endo-glucanase and xylanase activities. Econase in-
creased the reducing sugar content by 187% and Novozym 188 by

Table 3
The attributes, their abbreviations, denitions and anchors used in the descriptive analysis.

Attribute Abbreviation Denition Anchors

Odour
Freshness O-FRESH Intensity of fresh odour in the headspace of the sample Not at all freshfresh
Rye-like O-RYELIKE Intensity of rye-like odour (e.g. fresh whole grain rye bread) Not at all rye-likerye-like
in the headspace of the sample
Malty O-MALT Intensity of malty odour (e.g. dark beer or fermented grains) Not at all maltyMalty
in the headspace of the sample

Texture
Thickness T-THICKNESS Degree of thickness when evaluated how quickly the Not at all thickthick
suspension ow from the spoon

Flavour
Rye-like F-RYELIKE Intensity of rye-like avour (e.g. fresh whole grain rye bread) Not at all rye-likeRye-like
Sweetness F-SWEET Intensity of sweetness Not at all sweetSweet
Chemical F-CHEMICAL Intensity of chemical avour (e.g. solvent) Not at all chemicalChemical
Bitterness F-BITTER Intensity of bitterness Not at all bitterBitter
Aftertaste AFTERTASTE
intensity INTENSITY Flavour (e.g. pungent, bitter, sweet) staying after tasting WeakStrong
 R.-L. Heini et al. / Food Research International 45 (2012) 3138 35

Table 4
Effect of enzyme additions on the sensory proles of the rye our-water suspensions. Mean descriptive analysis ratings on a 010 scale (N = 9, two replicates).

Enzyme O-Fresh O-Ryelike O-Malty Thickness F-Ryelike F-Sweet F-Chemical F-Bitter Aftertaste intensity

Reference 7.1b 7.1 2.3a 8.5c 6.9b 3.3 2.1a 5.6a 5.7a
Pectinex BE 3-L 6.9b 6.6 2.3a 7.6c 7.3b 3.4 2.1a 6.1ab 5.9a
Econase CE 7.0b 6.7 2.3a 3.7a 6.6b 3.5 2.7a 6.5ab 6.4ab
Novozym 188 7.3b 6.7 2.1a 8.1c 6.9b 3.0 2.0a 6.6ab 6.4ab
Corolase 7089 5.1a 5.6 4.3b 6.0b 4.8a 3.6 6.3b 7.6b 7.8b
Biobake Fresh XL 6.5b 6.5 2.5a 7.4c 6.5b 4.0 2.8a 6.2ab 6.1a
a-c
Means in each column followed by a different letter signify that the samples are statistically signicantly different in respect of that attribute (Tukey's HSD test; p b 0.05).

Table 5
The effect of enzyme treatments on the formation of reducing sugars, soluble proteins and phenolic compounds in rye ourwater suspensions (40:60) and their free ferulic acid
concentrations.

Enzyme Enzyme dose nkat/g our Reducing sugars g/l Solubilised protein g/l Total phenolic compounds g/l Free FA g/g
a a ab
Reference 15.9 5.14 18.2 1.56 0.30 0.03 16.0
Pectinex BE 3-L 10 17.7 2.81a 18.1 0a 0.59 0.06cd 12.0
100 18.4 2.10a 17.7 0.64a 0.39 0.04abc
Econase CE 10 21.1 2.37ab 17.9 0.35a 0.52 0.05 bcd 45.5
100 29.8 3.76c 19.9 0.21a 0.72 0.07d
Novozym 188 10 19.9 3.39ab 20.1 1.20a 0.32 0.03ab 15.0
100 28.1 3.85 bc 20.5 0.85a 0.35 0.03abc
Corolase 7089 10 18.5 4.22a 36.0 2.97 b 0.70 0.07d 25.0
100 19.3 2.40ab 41.0 0.14c 0.72 0.07d
Biobake Fresh XL 10 19.9 1.65ab 18.7 1.84a 0.25 0.02a 12.9
100 23.2 0.57abc 22.6 0.64a 0.30 0.03ab
ad
Means in each column followed by a different letter signify that the samples are statistically signicantly different in respect of that variable (Tukey's HSD test; p b 0.05).
As measured by Lowry method (Lowry et al., 1951).
As measured by FolinCiocalteau method (Singleton & Rossi, 1965).
Free FA = free ferulic acid.

177%. Pectinex, Corolase and Biobake Fresh also increased the amount
of reducing sugars in the supernatant but less than Econase and
Novozym. The protease preparation Corolase duplicated the amount
of soluble protein in the supernatant, whereas the other enzymes
inuenced the protein concentration only little (Table 5). Cellulase
(Econase), protease (Corolase) and pectinase (Pectinex) increased
the content of phenolic compounds of the supernatant as measured
by the FolinCiocalteau method (Table 5). However, the amount of
free ferulic acid was highest in the Econase-treated suspension
(Table 5). When the share of free ferulic acid of total ferulic acid
was determined, 65% of ferulic acid was found to be bound to the ma-
trix (data not shown). The free ferulic acid concentration increased
56% by the Corolase treatment and 184% by the Econase treatment,
respectively. The total amount of free ferulic acid was increased by
the Econase treatment, probably because the extraction of ferulic
acid improved by the enzymatic treatment. Corolase and Econase
treatments also caused slight increase in the amount of diferulic
acid (data not shown). The impact of soluble proteins and phenolic
compounds on the perceived bitterness of enzyme-treated rye our
is presented graphically in Fig. 3.
bitterness of all ours: from +2.4 of wholemeal rye to + 3.1 of
white wheat our (Table 6). The impact of the Corolase treatment
varied depending on the our type of the crackers: it increased the
perceived bitterness of white wheat signicantly less than the bit-
terness of the two rye crackers (Fig. 4). The bitterness was found
to diminish signicantly during the heat-treatment, i.e. baking to
crackers.
The content of water-soluble protein of the waterour suspen-
sions incubated without enzyme addition was clearly highest in the
wholemeal rye (Table 6). The Corolase treatment increased the solu-
ble protein content of all our samples, however, the highest increase
(over 7-fold) was in wheat our. Without enzymatic treatment the
content of phenolic compounds in the supernatant of wholemeal
rye our was 3-fold when compared to the corresponding superna-
tants of white rye and white wheat our (Table 6). The Corolase

3.3. Verication of the impact of protease on the perceived avour and

amounts of chemical compounds

The impact of the protease preparation Corolase 7089 (5 nkat/g

our) on wholemeal rye, white rye and white wheat ours was fur-
ther examined with an attempt to verify the possible inuence of re-
leased peptides on the perceived bitterness. In sensory evaluation
the samples were compared to the sample made of wholemeal rye
our, which was also presented as one of the samples to the panel.
White wheat our was detected signicantly less bitter than whole-
Fig. 3. Perceived bitterness, soluble proteins and phenolic compounds of rye our
meal rye our; the bitterness of white wheat our was 1.8, while water suspensions treated with different enzyme preparations. Perceived bitterness
the bitterness of whole grain rye was 0.1 on the scale 5 + 5 evaluated on the intensity scale 010 (0 = not bitter, 10 = very bitter). Soluble pro-
(Fig. 4, Table 6). The Corolase treatment increased signicantly the teins, Total phenolics.
36 R.-L. Heini et al. / Food Research International 45 (2012) 3138

Fig. 4. Bitterness of rye ourwater suspensions and crackers after Corolase 7089 treatments (5 nkat/g our) (n = 2 10). The bitterness on the x-scale is presented by using a dif-
ference from control scale 5 + 5 ( 5 = clearly less bitter than the reference, + 5 = clearly more bitter than the reference). The letters indicate statistical differences between
the our-water suspension samples (ac) and between the cracker samples (AC). Columns marked by a different letter signify that the rye samples are statistically signicantly
different in respect of bitterness (Tukey's HSD test; p b 0.05).

treatment increased the content of phenolic compounds in all sam-
ples and mostly in the white wheat sample; 2-fold in wholemeal
rye, 3-fold in white rye and 6-fold in white wheat. The amount of
peptides correlated with the perceived bitterness, whereas the
amount of phenolic compounds did not. For example, the total
amount of phenolic compounds of wholemeal rye and Corolase-
treated white rye was approximately similar, although they deviated
signicantly in their bitterness.
4. Discussion
Flavour of whole grain rye is often considered intensive and bitter,
and may thus limit consumption of rye as food products. In the pre-
sent work compounds causing the bitter taste of whole grain rye
were studied by treating rye our with enzyme preparations having
different specicities. Treatment of the ourwater suspensions
with ve hydrolytic enzyme mixtures was found to cause statistically
signicant differences in several sensory attributes, i.e. in fresh and
malty odour, thickness, rye-like, chemical avour, bitter taste and af-
tertaste intensity. Addition of Corolase (protease) caused most in-
tense changes in the avour. Polysaccharide hydrolyzing enzyme
preparates Pectinex and Biobake Fresh as well as the rye suspensions
without any enzyme treatment had the weakest aftertaste, whereas
the Corolase-treated suspension was perceived as having the stron-
gest aftertaste. Cellulasehemicellulase mixture (Econase) produced
the least thick texture in the suspension, as expected after hydrolysis
of cell wall polymers, whereas Pectinex (pectinase), Novozym (-
glucosidase) and Biobake Fresh (amyloglucosidase) treatments resulted
in texture similar to the reference sample indicating limited action on rye
polysaccharides. Protease (Corolase) also caused some decrease in the
perceived thickness of the suspension.
While increasing signicantly the perceived bitterness as com-
pared to the reference (F(5108) = 2.5; p = 0.03), protease (Corolase)
treatment of rye concurrently also decreased the fresh odour, thick-
ness and rye-like avour, whereas malty odour, chemical-like avour
and aftertaste intensity were increased. The non-proteolytic enzyme
preparations containing e.g. cellulase, pectinase or amyloglycosidase
activities did not affect the bitterness of the ryewater suspensions,
despite of the fact that the availability of the phenolic compounds
was increased especially with the cellulase/hemicellulase preparation
(Econase). Thus, the increase in the perceived bitterness can be sug-
gested to be linked to increased amount of peptides and amino
acids. The partial proteolysis, where small peptides are released,
could be visualized in increased amount of soluble protein in the
samples. The amount of soluble proteins was doubled as a result of
the protease treatment. The results obtained strongly support the
reported impact of proteolysis on enhancing the perceived avour
during sourdough fermentation of cereals (Gnzle et al., 2008).
Incubated (2 h +40 C) and non-incubated rye ourwater sus-
pensions were also compared, aiming to solve the impact of the en-
dogenous enzymes of rye on the perceived bitterness. Incubation
decreased signicantly the thickness of the suspension (F(3,39) =
33.8; p b 0.001), without having any effect on the odour or taste attri-
butes. Endogenous cell-wall degrading enzyme activities, such as cel-
lulase and xylanase, are well-known to exist in grains (Dornez et al.,
2009), and the enzymatic solubilisation of cell wall polymers de-
creased viscosity. Endogenic proteolytic activity could not be detected
as plain water treatment did not increase the perceived bitterness.

Table 6
The impact of soluble proteins and total phenolic compounds on the perceived bitterness of different our-water suspensions treated with Corolase.

Flour Enzyme dosage Bitterness ( 5+5) Soluble proteins mg/ml Total phenolic compounds g/l

Wholemeal rye (reference) 0 nkat/g our 0.1 0.42b 23.2 3.68b 0.62 0.01b
Wholemeal rye 5 nkat/g our 2.4 1.35a 49.8 2.26d 1.28 0.04d
White rye 0 nkat/g our 0.6 1.14bc 12.5 0.14a 0.25 0a
White rye 5 nkat/g our 2.6 1.02a 36.8 0.92c 0.71 0. 04a
White wheat 0 nkat/g our 1.8 1.66c 10.5 0.07a 0.28 0a
White wheat 5 nkat/g our 3.1 1.14a 77.1 4.52e 1.76 0.01e
ae
Means in each column followed by a different letter signify that the samples are statistically signicantly different in respect of that variable (Tukey's HSD test; p b 0.05).
 R.-L. Heini et al. / Food Research International 45 (2012) 3138
Proteases are classied to endopeptidases or exopeptidases
depending on their mode of action (Raksakulthai & Haard, 2003). En-
dopeptidases hydrolyse specic peptide bonds within the polypeptide
chain, whereas exopeptidases catalyse the formation of free amino
acids or small peptides from the ends of the polypeptide chain. Protein
hydrolysis increases solubility and digestibility and decreases viscosi-
ty without diminishing nutritional value of proteins, but protein hy-
drolysates have frequently bitter taste caused by certain peptides.
Many L-amino acids (arginine, proline, leusine, phenylalanine, trypto-
phane and isoleusine) are bitter-tasting, phenylalanine, tryptophane
being the most bitter (Raksakulthai & Haard, 2003). In addition, main-
ly hydrophobic peptides may also have a bitter taste (Raksakulthai &
Haard, 2003). The perceived bitterness of a peptide depends consider-
ably on its amino acid composition, which in turn affects the hydro-
phobicity. The specic sequence of the amino acids does not
inuence on the bitterness of a protein taste. Tripeptides are more bit-
ter than dipeptides, which are again more bitter than free amino acids
(Raksakulthai & Haard, 2003).
Exopeptidases can reduce bitterness by producing avour precur-
sors and avour-active compounds. Protein hydrolysates, which com-
monly are perceived as bitter, can be rendered less bitter by
enzymatic treatment applying proline specic exo- and endopepti-
dases (FitzGerald & O'Cuinn, 2006). The protein-hydrolysing enzymes
tested in this work were protease Corolase 7089 and peptidase Coro-
lase LAP, which are known have endoprotease and exoprotease as
main activities, respectively. The exopeptidase nature of Corolase
LAP may explain why it did not increase the bitterness, since probably
it could not release peptides from proteins, but rather modied the
peptides already present. This was also suggested by Gilmartin and
Jervis (2002) with muscle proteins tests with Corolase LAP. Respec-
tively, Corolase 7089 on the other hand released peptides from the
cereal proteins. Corolase 7089 has also been reported to not contain
aminopeptidase (exopeptidase) side activities (Gilmartin & Jervis,
2002; Spellman et al., 2009) that could subsequently de-bitter the
peptides formed.
In addition to the role of peptides, the impact of phenolic com-
pounds on bitter avour has been suggested (Heini, Liukkonen,
Katina, Myllymki & Poutanen, 2003). In the present work, the prote-
ase preparation Corolase 7089 and cellulase preparation Econase CE
increased mostly the amount of phenolic compounds, analysed as
total phenolic content or as free ferulic acid content. Interestingly,
the increase in bitterness of rye was not as obvious after Econase
treatments as after Corolase treatment, although the cellulase treat-
ment liberated higher amounts of phenolic compounds, especially
ferulic acid from the rye matrix. The cellulase preparation Econase
and -glucosidase preparation Novozym 188 hydrolyzed carbohy-
drates of the rye matrix increasing clearly the concentration of reduc-
ing sugars in the supernatant, which might have assisted the release
of cell wall-associated phenolic compounds from matrix. It is also
possible that some phenolic compounds are covalently bound to pro-
teins, and as a result of partial proteolysis their solubility is increased,
thus contributing to increased bitterness. The protease preparation
Corolase 7089 contained also some carbohydrate-degrading activities
(e.g. xylanase, polygalacturonase and amyloglucosidase) which
might have resulted into the release of cell wall associated phenolic
compounds.
The formation of bitter-tasting compounds was further evaluated
by studying the impact of protease preparation Corolase 7089 on dif-
ferent grain ours, wholemeal rye, white rye and white wheat our.
The impact of Corolase on the bitterness varied depending on the
our type. Corolase treatment increased signicantly the bitterness
of all ours further, conrming the role of certain peptides in per-
ceived bitterness. The bitterness of endosperm rye and wheat ours
decreased during the heat-treatment (baking of crackers), whereas
with wholemeal rye such effect was not observed. The Corolase
treatment increased the soluble protein content of all our types;
37
however, the highest increase was detected in wheat our. Thus,
protease seems to have easier access to wheat proteins than to rye
proteins. Wheat and rye proteins are known to be different, mainly
since rye does not contain high molecular weight proteins capable
of disulde bonding (i.e. gluten) (Khler & Wieser, 2000). Different
particle size of the our types was not taken into account in this
study. It might also have a slight effect on the results presumably
white wheat our having the smallest particle size, while medium
coarse rye our had the largest.
Corolase treatment also affected the amount of phenolic compo-
nents solubilised as measured by the FolinCiocalteau method. With-
out the Corolase treatment a 3-fold amount of phenolic compounds
was released into the water phase from wholemeal rye compared to
the white rye and white wheat our. The result was expected, be-
cause a considerable amount of phenolic compounds is known to be
located in the outer layers of the grain (Liu, 2007). The bran fraction
contains also more free amino acids than other fractions of rye
(Mustafa et al., 2007) and these amino acids might also have con-
tributed to the phenolic assay. The Corolase treatment increased
the content of phenolic compounds in all samples and mostly in
the white wheat sample. Again, it is possible that the high amount
of peptides in the sample interfered with the analysis of total pheno-
lic compounds by FolinCiocalteau method, and thus, gave a too
high value of phenolics. However, the positive impact of Corolase
protease preparation on release of phenolic compounds could also
be detected by the ferulic acid analysis of rye samples (Table 5).
The amount of peptides correlated with the perceived bitterness,
whereas the amount of phenolic compounds did notTable 6. Al-
though the peptide content correlated with the bitterness, also the
quality of the peptides affects the bitter avour. For example, al-
though the white wheat and white rye had relatively same peptide
content, they differed in their perceived bitterness. Total phenolic
compounds consist of various phenolic compounds, from which
some have impact on avour, whereas others do not. For example,
the total amount of phenolic compounds of wholemeal rye our
and Corolase-treated endosperm rye our was approximately simi-
lar, although they deviated signicantly in their bitterness. This sug-
gests only a minor role of phenolics on rye bitterness formation
compared to that of the peptides. However, this does not exclude
the fact that some of the phenolic compounds may contribute the
bitterness, since in addition to ferulic acid, the detailed phenolic
composition was not analysed in this study. We have previously
shown that certain phenolic compounds particular phenolic
acids, alkylresorcinols and lignans may positively correlate with
the bitterness of cereals (Heini, Liukkonen, Katina, Myllymki &
Poutanen, 2003). Thus, not only the amount of chemical compounds
is relevant to the perceived avour, but also their quality.
5. Conclusions
The results of the current work clearly showed that especially
small molecular peptides resulting from partial proteolysis of rye pro-
tein generate the bitter avour of rye our suspensions. However,
the role phenolic compounds as contributors to the bitter note of
rye cannot be excluded. Ferulic acid, the most abundant phenolic
acid of rye but appearing mainly in a bound form, was not found to
increase the bitterness. Naturally the impact of other compounds,
e.g. lipid-derived compounds such as fatty acids may contribute to
the perceived intense and bitter taste of rye, but at least in this
work the hydrolysis of lipids by lipase was not found to increase the
bitterness. Heat treatment seemed to reduce the perceived bitterness
of rye our suspension. Further studies are, however, needed to gain
more understanding on the chemical background of bitterness. This
knowledge is needed for the development of tailored methods for
avour design of healthy rye foods.
38 R.-L. Heini et al. / Food Research International 45 (2012) 3138
Acknowledgements
The work has been carried out as a part of the FLAVENZ project
funded by Tekes the Finnish Funding Agency for Technology
and Innovation. Financial support of Fazer Leipomot Oy, Linkosuon
Leipomo Oy, Polttimo Yhtit Oy, Primulan Leipomot Oy, Ravintoraisio
Oy and Vaasan & Vaasan Oy is also acknowledged. Funding from
Academy of Finland to Kaisa Poutanen is gratefully acknowledged.
References
Adams, J. F., & Engstr.om, A. A. (2000). Dietary intake of whole grain vs. recommenda-
tions. Cereal Foods World, 2, 7578.
Arranz, S., Saura-Calixto, F., Shaha, S., & Kroon, P. A. (2009). High contents of nonextract-
able polyphenols in fruits suggest that polyphenol contents of plant foods have
been underestimated. Journal of Agricultural and Food Chemistry, 57, 72987303.
Bailey, M. J., & Pessa, E. (1989). Strain and process for production of polygalacturonase.
Enzyme and Microbial Technology, 12, 266271.
Bailey, M. J., & Linko, M. (1990). Production of -galactosidase by Aspegillus oryzae in
submerged bioreactor cultivation. Journal of Biotechnology, 16, 5766.
Bailey, M. J., Biely, P., & Poutanen, K. (1992). Interlaboratory testing of methods for
assay of xylanase activity. Journal of Biotechnology, 23, 257270.
Bakke, A., & Vickers, Z. (2007). Consumer liking of rened and whole wheat breads.
Journal of Food Science, 72(7), S473S480.
Bartolom, B., & Gmez-Cordovs, C. (1999). Barley spent grain: Release of hydroxycin-
namic acids (ferulic and p-coumaric acids) by commercial enzyme preparations.
Journal of Agricultural and Food Chemistry, 79, 435439.
Bernfeld, P. (1955). Amylases and . In S. P. Colowick, & N. O. Kaplan (Eds.), Methods
in enzymology, Vol 1. (pp. 149157)New York: Academic Press.
de Munter, J. S. L., Hu, F. B., Spiegelman, D., Franz, M., & van Dam, R. M. (2007). Whole
grain, bran, and germ intake and risk of type 2 diabetes: A prospective cohort study
and systematic review. PLoS Medicine, 4, 13851395.
Delwiche, J. F., Buletic, Z., & Breslin, P. A. S. (2001). Covariation in individuals' sensitivities to
bitter compounds: Evidence supporting multiple receptor/transduction mechanisms.
Perception & Psychophysics, 63(5), 761776.
Dornez, E., Gebruers, K., Delcour, J. A., & Courtin, C. M. (2009). Grain-associated xyla-
nases: Occurrence, variability, and implications for cereal processing. Trends in
Food Science & Technology, 20, 495510.
FitzGerald, R. J., & O'Cuinn, G. (2006). Enzymatic debittering of food protein hydroly-
sates. Biotechnology Advances, 24, 234237.
Gilmartin, L., & Jervis, L. (2002). Production of cod (Gadus morhua) muscle hydrolysates.
Inuence of combinations of commercial enzyme preparations on hydrolysate pep-
tide size range. Journal of Agricultural and Food Chemistry, 50, 54175423.
Gnzle, M. G., Loponen, J., & Gobbetti, M. (2008). Proteolysis in sourdough fermenta-
tions: Mechanisms and potential for improved bread quality. Trends in Food Science
and Technology, 19, 513521.
Hansen, A. (1995). Flavour compounds in rye sourdough and rye bread. In K. Poutanen, &
K. Autio (Eds.), International rye symposium: Technology and products (pp. 160168).
Helsinki, Finland: VTT symposium.
Heini, R.-L. (2003). Inuence of processing on the avour formation of oat and rye. VTT
Biotechnology, Espoo. VTT Publications 494. 72 p. + app. 48 p. ISBN 951-38-6042-
6; 951-38-6043-4 (URL: http://www.vtt./inf/pdf/) (Dissertation)
Heini, R. -L., Katina, K., Wilhelmson, A., Myllymki, O., Rajamki, T., Latva-Kala, K., Liukkonen,
K. -H., & Poutanen, K. (2003). Relationship between sensory perception and avour-
active volatile compounds of germinated, sourdough fermented and native rye following
the extrusion process. Food Science and Technology, 36, 533545.
Heini, R. -L., Liukkonen, K. -H., Katina, K., Myllymki, O., & Poutanen, K. (2003). Milling
fractionation of rye produces different sensory proles of both our and bread.
Food Science and Technology, 36, 577583.
Heini, R. -L. (2006). Sensory attributes of bakery products. In Y. H. Hui, H. Corke, I. De
Leyn, W. -K. Nip, & N. Cross (Eds.), Bakery products: Science and technology
(pp. 285298). Ames, Iowa, USA: Blackwell Publishing ISBN 0-8138-0187-7.
Heini, R. -L., Liukkonen, K. -H., Myllymki, O., Pihlava, J. -M., Adlercreutz, H., Heinonen,
S. -M., & Poutanen, K. (2008). Quantities of phenolic compounds and their impacts
on the perceived avour attributes of rye grain. Journal of Cereal Science, 47,
566575.
ISO 6658 (1985). Sensory analysis. Methodology. General guidance. (14 pp.).
ISO 8589 (1988). Sensory analysis. General guidance for the design of test rooms. (9 pp.).
IUPAC (International Union of Pure and Applied Chemistry) (1987). Measurement of
cellulase activities. Pure and Applied Chemistry, 59, 257268.
Jensen, M. K., Koh-Banerjee, P., Hu, F. B., Franz, M., Sampson, L., Grnbk, M., & Rimm,
E. B. (2004). Intakes of wholegrains, bran, and germ and the risk of coronary heart
disease in men. American Journal of Clinical Nutrition, 80, 14921499.
Kirchhoff, E., & Schieberle, P. (2001). Determination of keyaroma compounds in the
crumb of a three-stage sourdough rye bread by stable isotope dilution assays and
sensory studies. Journal of Agricultural and Food Chemistry, 49(9), 43044311.
Khler, P., & Wieser, H. (2000). Comparative studies of high Mrsubunits of rye and
wheat, III. Localisation of cysteine residues. Journal of Cereal Science, 32, 189197.
Lang, R., & Jebb, S. A. (2003). Who consumes whole grains, and how much? In Cam-
bridge University Press. Proceedings of the Nutrition Society, 62, 123127.
Lappalainen, R., Kearney, J., & Gibney, M. (1998). A pan EU survey of consumer atti-
tudes to food, nutrition and health: An overview. Food Quality and Preference, 9,
467478.
Lawless, H. T., & Heymann, H. (1999). Descriptive analysis. Sensory evaluation of food
Principles and practices (pp. 341378). Gaithersburg, Maryland, USA: Chapman &
Hall/Aspen Publishers, Inc 827 p.
Liu, R. H. (2007). Whole grain phytochemicals and health. Journal of Cereal Science,
46(3), 207219.
Liukkonen, K. H., Katina, K., Wilhelmson, A., Myllymki, O., Lampi, A. M., Kariluoto, S.,
Piironen, V., Heinonen, S. M., Nurmi, T., Adlercreutz, H., Peltoketo, A., Pihlava, J. M.,
Hietaniemi, V., & Poutanen, K. (2003). Process-induced changes on bioactive com-
pounds in whole grain rye. Proceedings of the Nutrition Society, 62. (pp. 117122):
Cambridge University Press.
Loponen, J., Kanerva, P., Zhang, C., Sontag-Strohm, T., Salovaara, H., & Gnzle, M.
(2009). Prolamin hydrolysis and pentosan solubilization in germinated-rye sour-
doughs determined by chromatographic and immunological methods. Journal of
Agricultural and Food Chemistry, 57, 746753.
Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951). Protein measurement
with the Folin phenol reagent. Journal of Biological Chemistry, 193, 265275.
Marquart, L., Miller Jones, J., Cohen, E. A., & Poutanen, K. (2007). The future of whole
grains. In L. Marquart, D. R. Jacobs, G. H. McIntosh, K. Poutanen, & M. Reicks
(Eds.), Whole grains and health (pp. 315). Ames, Iowa, USA: Blackwell Publishing
Professional.
Meilgaard, M., Civille, G. V., & Carr, B. T. (1999). Sensory evaluation techniques (3rd ed.).
New York, USA: CRC Press 387 p.
Mellen, P. B., Liese, A. D., Tooze, J. A., Vitolins, M. Z., Wagenknecht, L. E., & Herrington,
D. M. (2007). Whole-grain intake and carotenoid artery atherosclerosis in a multi-
ethnic cohort, the Insulin Resistance Atherosclerosis Study. American Journal of Clinical
Nutrition, 85, 14951502.
Mustafa, A., man, P., Andersson, R., & Kamal-Eldin, A. (2007). Analysis of free amino
acids in cereal products. Food Chemistry, 105, 317324.
Okarter, N., & Liu, R. H. (2010). Health benets of whole grain phytochemicals. Critical
Reviews in Food Science and Nutrition, 50, 193208.
Raksakulthai, R., & Haard, N. F. (2003). Exopeptidases and their application to reduce
bitterness in food: A review. Critical Reviews in Food Science and Nutrition, 43(4),
401445.
Rastall, R. (2007). Novel enzyme technology for food applications. Cambridge: Woodhead
Publishing Ltd. 336 p.
Schieberle, P. (1996). Intense aroma compoundsUseful tools to monitor the inuence
of processing and storage on bread aroma. Advances in Food Science (CMTL), 5(6),
237244.
Seal, C. J., Jones, A. R., & Whitney, A. D. (2006). Whole grains uncovered. Nutrition Bulletin,
31, 129137.
Shewry, P. R., & Bechtel, D. B. (2001). Morphology and chemistry of the rye grain. In W.
Bushuk (Ed.), Rye: Production, chemistry and technology (pp. 69127). (second ed.).
St. Paul, Minnesota, USA: American Association of Cereal Chemists.
Singleton, V. L., & Rossi, J. A. (1965). Colorimetry of total phenolics with phosphomo-
lybdiphosphotungstic acid reagents. American Journal of Enology and Viticulture,
45, 44524455.
Smith, S., Smith, A., Richardson, D. P., & Seal, C. J. (2001). Regional variations in con-
sumer knowledge and purchasing of whole grain foods. Proceedings of the Nutrition
Society, 60. (pp. 218A): Cambridge University Press.
Smith, A. T., Kuznesof, S., Richardson, D. P., & Seal, C. J. (2003). Behavioural, attitudinal
and dietary responses to the consumption of wholegrain foods. Proceedings of the
Nutrition Society, 62, 2. (pp. 455467): Cambridge University Press.
Spellman, D., O'Cuinn, D. G., & FitzGerald, R. J. (2009). Bitterness in Bacillus proteinase
hydrolysates of whey proteins. Food Chemistry, 114, 440446.
Tenkanen, M., SalmenkallioMarttila, M., & Poutanen, K. (2000). Baking with enzymes:
What makes it happen. : The World of Food Ingredients May/June 3841.
Thiele, C., Gnzle, M. G., & Vogel, R. F. (2003). Fluorescence labeling of wheat proteins
for determination of gluten hydrolysis and depolymerization during dough proces-
sing and sourdough fermentation. Journal of Agricultural and Food Chemistry, 51,
27452752.
Vitaglione, P., Napolitano, A., & Fogliano, V. (2008). Trends in Food Science and Technology,
19, 451463.