Article

Cite This: J. Agric. Food Chem. 2018, 66, 12353−12360 pubs.acs.org/JAFC

Targeted Inhibition of Enzymatic Browning in Wheat Pastry Dough

Linda Brütsch,† Seline Rugiero,† Stéphanie Spoerry Serrano,† Christian Städeli,‡ Erich J. Windhab,†
Peter Fischer,*,† and Simon Kuster†
†
Institute of Food Nutrition and Health, ETH Zürich, 8092 Zürich, Switzerland
‡
Jowa AG, 8604 Volketswil, Switzerland

ABSTRACT: Enzymatic browning primarily affects fruits and vegetables but also occurs in wheat-based food. Herein, the
browning behavior in wheat pastry dough was investigated aiming toward a targeted inhibitory treatment without influencing
the pastry dough properties such as workability or taste. Dough discoloration is attributed to several subsequent enzyme−
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substrate reactions, which can selectively be inhibited by food additives. In most cases, an effective and lasting inhibition is only
guaranteed by compounds acting upon multiple inhibition pathways. Despite their effectiveness, the unlimited use of
commercial inhibitors is nondesirable due to necessary labeling, thus sustainable and natural inhibitors usually occurring as
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conventional food ingredients are of interest. It is shown that white wine combined with lemon juice revealed itself as an ideal
combination for prevention of enzymatic browning in pastry dough.
KEYWORDS: enzymatic browning, wheat dough, targeted inhibition, shelf life, consumer acceptance

1. INTRODUCTION 1.10.3.1) and monophenol monooxygenase (EC 1.14.18.1)

Appearance, flavor, texture, and nutritional value are the main
attributes considered by the majority of consumers when
selecting food. Consequently, reactions and processes that
negatively affect one of these attributes are undesirable and
need to be avoided as best as possible. One process is
enzymatic browning, which is regarded as one of the leading
natural phenomena occurring during food processing and
storage influencing the quality of many products and
deteriorating customers’ acceptance.1−4 In certain foods
including dried fruits, tea, tobacco, and cocoa it improves or
contributes to the desired organoleptic properties being
essential for color and taste development. However, in most
cases enzymatic browning is negatively influencing the foods
quality or its perception. The prevention remains an ongoing
challenge driven by the quality loss of e.g. sensory and
nutritional aspects causing reduced consumer acceptance and
thus tremendous economic losses. The existing knowledge on
the structure of polyphenol oxidase (PPO), its mechanism of
action, and chemistry of enzymatic browning reaction in food
provides information on inhibiting the process to minimize the
discoloration.3−10 Enzymatic browning is generally associated
with the reaction catalyzed by PPO, and as enzymatic
browning is widely observed in plant products, it is not
surprising that they have been extensively studied and that the
enzyme was isolated from numerous fruits and vegeta-
bles.6−9,11−15 Browning in food products is generally related
to loss of cell integrity caused by mechanical or thermal
processes. This results in the disruption of membranes, cell
walls, and other cellular structures enabling interaction of
enzyme and its substrate. In the presence of oxygen, PPO is
then capable of catalyzing melanin formation, which is the
colored metabolic end product of enzymatic browning.
Under the trivial name PPO, two kinds of enzyme are
classified based on their substrate specificity. The first one
consists of two similar enzymes named catechol oxidase (EC
and the second class named laccase (EC 1.10.3.2), which is not
considered further. In the presence of oxygen, both mono-
phenol monooxygenase and catechol oxidase catalyze two
distinct reactions shown in Figure 1. The oxygen dependent
hydroxylation of monophenols into o-diphenols and sub-
sequently the oxidation of the o-diphenols into o-quinones and
water. The highly reactive o-quinones take part in various other
enzymatic and spontaneous nonenzymatic reactions (as
indicated with double arrows in Figure 1) forming intensely
colored polymerization products.6 However, also auto-
oxidation might play a major role in discoloration of dough.
As enzymatic browning can only take place if all the essential
reaction components are present, inhibitory approaches aim to
eliminate one or more of them. Possible target points are the
(i) elimination or chemical modification of the two substrates,
i.e. polyphenols and oxygen, (ii) the inactivation or
suppression of the enzymatic catalytic ability, or (iii) the
addition of compounds acting on the reaction products to
suppress enzymatic and nonenzymatic polymerization reac-
tions.5,7,15−18 Based on the different target points and
underlying inhibition mechanisms, all antibrowning agents
can be divided into six main categories to control enzymatic
browning: (i) substrate analogous, (ii) reducing agents, (iii)
complexing agents, (iv) acidulants, (v) quinone couplers, and
(vi) chelators.16,19 As shown in Figure 1, acidulants and
chelators are targeted toward PPO, substrate analogous,
reducing and complexing agents toward the substrates, oxygen
and polyphenols and the quinone couplers and reducing agents
toward the reaction products. Additionally, other enzymes
Received: August 17, 2018
Revised: October 18, 2018
Accepted: October 19, 2018
Published: November 7, 2018

© 2018 American Chemical Society 12353 DOI: 10.1021/acs.jafc.8b04477

J. Agric. Food Chem. 2018, 66, 12353−12360
Journal of Agricultural and Food Chemistry Article

Figure 1. Schematic representation of known possibilities to chemically inhibit enzymatic browning reactions. General accepted enzymatic
browning reaction starting with tyrosinase activity from a para-phenolic compound to a 3,4-polyphenol followed by mainly enzymatic PPO
activities to yield the corresponding ortho-quinone derivative. Colored compounds are marked with an asterisk, whereas the color gradient
indicated the increase in discoloration with progression of the reaction. The inhibition of the reaction can be targeted toward the substrates:
Oxygen or polyphenols (bold i.e. substrate analogous), the reaction products (bold and italic i.e. quinone couplers), or the enzyme (gray i.e.
acidulant). The polyphenol oxidase PPO is represented by its active center containing two copper ions bond to six histidine residues. Adapted from
Nicolas et al.20

Table 1. Overview of the Selected Anti-Browning Agents To Investigate Their Inhibitory Effect Targeted toward Decreasing
Enzymatic Browning in Pastry: The Anti-Browning Agents, Suppliers, and Modes of Action Are Listed below
Antibrowning agent Supplier Mode of action
Citric acid (monohydrate) Sigma-Aldrich Acidulant, chelator
L-Ascorbic acid Sigma-Aldrich Reducing agent, acidulant
N-Acetyl L-cysteine Merck Suchard OHG Acidulant, Reducing agent, chelator
Thiourea Merck Suchard OHG Quinone coupler
Ferulic acid Merck Suchard OHG Substrate analogous
trans-Cinnamic acid Sigma-Aldrich Substrate analogous
Cinnamaldehyde Sigma-Aldrich Substrate analogous
Kojic acid Sigma-Aldrich Tyrosinase inhibitor
Chitosan Molekula Quinone coupler and weak chelator
Ethylenediamine tetraacetic acid ACROS Chelator
L-Cysteine SAFC Sigma-Aldrich Quinone coupler and chelator
Sodium acetate Fluka Analytical
L-Phenylalanine Merck KGaA Substrate analogous
L-Lysine Merck KGaA Chelator and quinone coupler
White Grape juice (G) (polyphenols, malic and tartaric acid) JaMaDu - Coop Acidulant, reducing agent, chelator, substrate analogous
Lemon juice (L) (ascorbic and citric acid) Freshly squeezed Acidulant, reducing agent, chelator
White Wine (W) (polyphenols, malic and tartaric acis, alcohol) Swiss Fendant − Coop Acidulant, reducing agent, chelator, substrate analogous

modifying either the reaction products or PPO itself can thus
be used to prevent enzymatic browning.16,20
The consequences of enzymatic browning reaction are
mainly characterized for fruits and vegetables although the
same phenomenon is observable in wheat-based products such
as pastry dough.21−23 All wheat-based products acquire their
share of PPO and substrate with the wheat flour, which
contains aleuron and bran cells. PPO and its substrates are
highly concentrated in the aleuron, a single-cell layer between
bran and endosperm. Contamination of the flour and
acquisition of PPO results during milling. Due to the
multistage milling process and grain morphology a complete
separation of bran and endosperm cannot be ensured while
staying economically viable. Especially with increasing
extraction rate, i.e. using a higher percentage of the endosperm,
the amount of bran and aleuron increases.15 Additionally, the
mechanical impact during milling disrupting the cell structure
might also lead to enzyme and substrate damage.20,24−27
Enzymatic browning is initiated during dough formation and
remains a major challenge for fresh-pastry goods as
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deterioration influences the optical dough properties and
thus decreases consumer acceptance. To overcome the
browning and produce high quality wheat-based dough and
batters, commonly extra white flours are utilized. These flours
consist of only the innermost endosperm but also leave for
lower quality flours to be used elsewhere. Herein, the goal was
to replace the extra white flour (type 380) with standard white
flour (type 550), which is prone to undergo enzymatic
browning during storage. To minimize the discoloration during
a four-week shelf life of the fresh pastry dough different
inhibitory treatments were elucidated. The inhibiting additives
are selected based on their reported impact on product flavor,
taste, color, and texture, inhibitory potential, innocuousness,
cost, and legal admission.14,28 In a first step, the extent of
enzymatic browning was assessed in flour-water mixtures by
quantitative color measurements as well as optical inves-
tigation. Second, the inhibitory potential of a selection of
antibrowning agents was tested in pastry dough during a shelf-
life of 4 weeks. Finally, a set of natural alternatives providing
DOI: 10.1021/acs.jafc.8b04477
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Table 2. Overview on the Different Dough Samples and Their Composition

Sample Flour Salt Citric acid Inhibitor Natural Inhibitor Ice water Margarine
Reference 1 280 g flour type 550 4 g 0.5 g 80 mL 155 g
Reference 2 280 g flour type 380 4 g 0.5 g 80 mL 155 g
Low inhibitor conc. 280 g flour type 550 4 g 1g 80 mL 155 g
High inhibitor conc. 280 g flour type 550 4 g 5g 80 mL 155 g
Low natural inhibitor conc. 280 g flour type 550 4 g 1 gdm 74 mL 155 g
Low natural inhibitor conc. 280 g flour type 550 4 g 5 gdm 50 mL 155 g
High natural inhibitor conc. 280 g flour type 550 10 gdm 20 mL 155 g

Figure 2. Color change in (A) flour−water mixtures and (B) pastry dough due to enzymatic browning. The discoloration was assessed
colorimetrically and optically. In (A) the colometrically measured discoloration over a period of 24 h using the CIE L*a*b* color space is
presented (measured in triplicates for both flour types). Flour type 380 (black symbols) did not demonstrate optically determinable changes going
in line with constant brightness and redness. While flour type 550 (gray symbols) showed a distinct discoloration resulting from the enzymatic
browning. The enzymatic browning resulted in a decreasing brightness and increasing redness of the flour−water mixtures. In (B) the pastry
dough’s made from flour type 380 and 550 after two months of storage represent the discoloration and the appearance of shell particles in flour
type 550.

the same inhibitory potential is proposed for the envisaging
natural products.
2. MATERIAL AND METHODS
2.1. Flour Types. Flour type 550 IPS (standard white flour) was
obtained from Meyerhans Mühlen AG (Weinfelden, Switzerland).
Flour type 380 S (extra white flour) was purchased from Group
Minoteries, Bruggmühle Goldach (Goldach, Switzerland). Wheat
(Triticum aestivum L.) flour type 550 consists of inner, peripheral
endosperm and parts of the aleuron layer using an extraction rate of
72%. For flour type 380 only the innermost endosperm is used,
resulting in an extraction rate of approximately 30%. The different
extraction rates employed give rise to flour types with compositional
and nutritional differences. This in turn affects their dough forming
and viscoelastic properties. From an economical point of view, flour
type 550 is preferably utilized for pastry dough production but shows
enzymatic browning during processing and storage. Therefore,
suitable inhibitory treatments of the browning need to be found
allowing for its industrial applicability.
2.2. Food Additives. The inhibitory potential of a variety of food
additives listed in Table 1 was elucidated in dough aiming at
improving the pastry quality. The additives were selected and
classified according their inhibitory effect (Figure 1). Despite of
their effectiveness the necessity of labeling them induced the search
for natural alternatives. Thus, lemon juice, grape juice, and white wine
were investigated as potential natural alternatives to chemical
antibrowning agents. Their richness in acids, polyphenols, and other
antibrowning agents make them highly potential enzymatic browning
inhibitory.
2.3. Investigation of the Flour Browning Discoloration. To
assess the extent of discoloration independently of the effect of fatty
acids or other ingredients added during dough formation, a flour−
water mixture was studied. Flour type 380 or type 550 was mixed with
water to achieve a moisture content of 40%wb. The mixture was filled
into Petri dishes forming a 2 mm thick layer mimicking the final
dough. The appearance of the flour−water mixtures was judged
optically and the color change was determined colorimetrically in
triplicate (CR300 Chroma Meter, Konica Minolta Sensing Singapore
Pte Ltd.) using the CIE L*a*b* color space over a period of 24 h
until leveling out.
2.4. Pastry Dough Preparation and Inhibitory Treatment.
The browning behavior and inhibitory effectiveness of the
antibrowning agents was analyzed in pastry dough samples. Flour
type 380 and type 550 served as references illustrating the targeted
and occurring browning, respectively. The inhibitory potential of the
browning agents was solely studied in samples made from flour type
550. All pastry dough samples were prepared according to the
following recipe: 280 g flour and 4 g salt were mixed with a
KitchenAid KSM175 (Whirlpool Corporation, USA) on level 1 for 1
min at room temperature. For the reference samples 0.5 g of citric
acid were dissolved in 80 mL of ice water and added to the flour
mixture. Ice water was used to control the temperature during dough
formation, i.e. counteract the temperature increase due to the mixing
process and thus prevent sticky dough. To assess the effectiveness of
inhibitors in pastry dough made from flour type 550, citric acid was
replaced by 1 or 5 g of the selected additives. For the natural

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inhibitors (lemon juice, grape juice, and white wine) three different
concentrations were tested: 1, 5, and 10 g dry matter corresponding
to 6, 30, and 60 g of liquid, respectively. The liquid addition was
compensated by reducing the amount of ice water added. After 1 min
of mixing on level 1, 155 g of baking margarine (Mifa AG,
Frenkendorf, Switzerland) was added and kneaded on level 3 until a
smooth, viscoelastic dough was formed. The dough was flattened to a
thickness of 3 mm. Thereof three A5 sized rectangles were cut,
wrapped in plastic bags, and put into storage at 8 °C for 4 weeks. An
overview on the different pastry dough samples and their composition
is given in Table 2.
2.5. Optical Pastry Dough Evaluation. Browning behavior and
inhibitory potential were determined based on the induced changes in
optical pastry dough properties. The appearance of the dough was
examined by (i) optical inspection including counting of aleuron shell
particles and (ii) quantitative color measurements using the CIE
L*a*b* color space. In combination, both methods provide an
objective pastry dough assessment as well as an indication if
differences are perceivable and acceptable by the consumer. The
optical inspection consisted of counting and classifying the amount of
shell particles on the dough surface and judging the dough color. Shell
particles visible from a viewing distance of 30 cm and diameter equal
to 1 mm or larger were considered in the counting. Samples with 0 to
1 visible particle per cm2 are regarded as ideal. Samples with 1 to 5
particles per cm2 dough were in the acceptable range. Exceeding 5
particles per cm2 gave rise to nontolerable dough. The particle
counting and optical judgment was carried out every 2 days for the
first 2 weeks of storage and every 3 days for the remaining 2 weeks. All
particles on the A5 sizes dough samples were counted followed by
calculating the number of particles per cm2. Colorimetric measure-
ments were performed four times for the freshly prepared dough, i.e.
directly after production and after 1, 2, and 4 weeks of storage. All
experiments have been conducted in triplicates on different days and
different batch order. Reference dough made from flour type 380 and
type 550 were prepared with every pastry dough series for direct
comparison and evaluation of the differences between the different
batches. The individual samples were examined in six different spots.
The average of the values with corresponding standard deviation was
calculated.
3. RESULTS AND DISCUSSION
The inhibitory potential of several additives to suppress the
enzymatic browning and to improve the optical pastry dough
properties was investigated in flour−water mixtures (Section
3.1) and dough under simulated shelf-life conditions for 4
weeks (Section 3.2). Industrially applicable and clean label
solution using natural ingredients such as lemon juice, grape
juice, and white wine are discussed in Section 3.3.
3.1. Characterization of the Discoloration in Flour−
Water Mixtures. The color change of the flour−water
mixtures over 24 h was investigated by optical and colorimetric
means intending to characterize the induced alterations
(Figure 2). The flour−water mixtures prepared from flour
type 380 did not demonstrate distinct, optically perceivable
alterations. Also, no increase in amount of shell particles on the
pastry dough surface was observed. In contrast, the sample
made from flour type 550 indicated two specific changes. The
base color turned reddish-brown and the amount of shell
particles increased from 0−1 to over 10 particles per cm2 on
the sample surface. The colorimetric evaluation presented in
Figure 2A confirmed the findings of the optical evaluation. For
the flour water mixture made of flour type 380, the brightness
L* (84.1 ± 0.9) and redness a* (−2.7 ± 0.1) value remained
constant over 24 h (Figure 2A, gray symbols). The b* value
(yellowness) increased from 10.6 after 1 h to 22.8 after 12 h
and remained constant thereafter. Progression of the b* value
for the mixtures of flour type 550 followed the same trend
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increasing from 11.1 to 22.3 (Figure 2A, black symbols). The
samples additionally demonstrated a lower initial brightness
and increased redness. The brightness L* decreased from 81.3
to 64.4, while the redness a* increased from −1.7 to 1.4 during
the 24 h storage. Consequently, enzymatic browning induced
alterations affected the brightness and redness of the dough.
The induced alterations in yellowness may derive from
different discoloration reactions including oxidation being
independent of the flour type. The discrepancy in initial dough
color and browning behavior between the two flour types
originated from constituents such as polyphenols and
polyphenol oxidase, which are present in larger quantities in
flour type 550 than in type 380. This in turn derived from the
differing milling extraction rates producing flours with
compositional variations. With increasing extraction rate the
amount of aleuron, acquisition of polyphenols and polyphenol
oxidase generally rises. Their presence in larger quantities gives
rise to more pronounced and accelerated browning during
storage.
Pastry dough made from flour type 380 and type 550
demonstrated comparable changes in optical properties as the
flour−water mixture (Figure 2B). The browning behavior of
mixtures and pastry dough solely differed in reaction speed,
which was considerably longer for the later one. The gluten
network in dough and additional ingredients may have
hindered the enzymatic browning reaction resulting in a
retarded progression. For dough made from flour type 550 first
changes became apparent a few days after preparation. It took
up to 4 weeks to reach the full extent of discoloration. During
storage a decrease in brightness L* values from 79.8 to 69.1,
slight increase in redness (a* = 1.5), and increased amount of
shell particles (≫10/cm2) was recorded. The increase in b*
value (yellowness) derived from nonenzymatically induced
reactions occurring in both sample types. Pastry dough from
flour type 380 demonstrated only slight alterations over storage
time. A decrease in L* value was measured after 4 weeks
storage from 84.4 to 81.8, which cannot be perceived by the
human eye. Similar discoloration was observed by Jukanti et
al.,29 Fuerst et al.,30 and Kruger et al.,31 who investigated the
behavior in wheat noodles. They reported a decrease in
brightness L* and increase in yellowness b* during storage.
Discoloration of durum wheat (Triticum durum) products
might lead to altered yellowness while hard wheat (Triticum
aestivum) products demonstrate changes in redness.
3.2. Chemical Inhibition of the Enzymatic Browning.
Production of pastry dough with standard flour type 550 makes
prevention of the occurring discoloration indispensable.
Inhibition or retardation of the browning is achieved by
physical (packaging, thermal treatment) and chemical (pH
reduction, addition of inhibitors) methods. In this work,
chemical inhibitors were investigated as they do not require
changes in the pastry dough making process in contrast to
physical methods: (i) Lemon juice and citric acid were added
in different concentrations lowering the pH to elucidate the
effect of acidulation on the browning reaction and the pH
sensitivity of the enzyme, (ii) a wide range of chemicals and
(iii) natural ingredients (Section 3.3) targeting toward the
enzyme, its substrates (oxygen or polyphenols), their reaction
products, or combinations thereof were examined (Table 1).
3.2.1. Inhibition by pH Reduction. Acidification by lemon
juice and citric acid is commonly used in affected foods such as
apples and offered a viable solution to decrease PPO activity by
pH reduction. To ensure inhibition of enzymatic browning, i.e.
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Figure 3. (A) Average color changes of dough samples after 4 weeks of storage. Low values of brightness L* indicated darker, more colored dough
samples, while increasing Hue a* reflected more reddish appearance of the dough samples. The color change of the reference samples made from
flour type 380 (light gray circles, dough image in Figure 3B) and 550 (black circles, freshly made dough image in Figure 3D, aged dough image in
Figure 3F) as well as the inhibitory potential of selected antibrowning agents is presented (dough images 3C, 3E, and 3F). A decrease in L* and
increase in a* value was observed for all investigated samples. The amount of shell particles on the dough is indicated by the circle size. With
increasing circle size higher amounts of shell particles were counted. The tolerance range, in which the color of the dough is acetated by the
consumer, is marked with the red square in the upper left corner. Samples lying in the tolerance range demonstrate a remarkable improvement of
the optical properties of the dough compared to the sample made from flour type 550 without additives.

Figure 4. Overview illustrating the improvement of the optical properties of pastry dough made from flour type 550 (A) treated with antibrowning
agents. Antibrowning agents improved the amount of shell particles (B), the dough color (D), or both parameters (C). Chemicals reported in bold
demonstrated a significant improvement of the indicated property, whereas the ones in regular only had a slight beneficial effect. All pictures were
taken of dough samples that were stored for 4 weeks at 8 °C.

to render the enzyme inactive during the storage time of 4
weeks, a pH value of 4 or lower is required.19,32 A pH between
4 and 5.5 retarded the browning, enabling storage without
quality decrease for 2 weeks. Values larger than pH 5.5 lead to
a comparable browning as the untreated standard pastry dough
made from flour type 550. Exceeding pH 7 accelerated the
discoloration. Consequently, a satisfying inhibition was only
achieved in very acidic dough affecting the organoleptic
properties including taste and smell. Moreover, lowering the
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pH interfered with Maillard and caramelization reactions
during baking. Below a pH of 6.5 the baking time increased
with decreasing pH. At pH 4 solely the edges of the pastry
dough turned brown after more than 25 min of baking and no
puffing was observed. Acidification proved itself as potent
inhibitory treatment for enzymatic browning in dough but
applicability is restricted due to the impairment of taste, smell,
and baking behavior.
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3.2.2. Inhibitory Effect of Antibrowning Agents in Dough.
Aiming at decreasing browning during storage, antibrowning
agents with differing inhibitory mechanisms were elucidated.
The inhibitory potential of the compounds was evaluated
based on color alterations (L* and a* value) and the amount
of shell particles as summarized in Figure 3. The effect is
studied by comparing the treated doughs to those made from
flour type 550. Pastry dough made from flour type 550
demonstrated the most pronounced discoloration (Figure 3A,
black circles and Figure 3D (freshly made dough) and Figure
3F (dough after 4 weeks of storage)) with decrease in
brightness L* and increase in redness a*. Pastry dough made
from flour type 380 demonstrated the targeted optical
properties (Figure 3A, light gray circles and Figure 3B (freshly
made and aged dough)). The inhibitory effect on the amount
of shell particles was best illustrated for citric acid (Figure 3A,
blue circles). With addition of 5 or 1 g of citric acid the
amount of shell particles was decreased to 0−1 and 3−4 from
over 5 per cm.2 The particles visible on the dough surface are
about 1 mm in diameter and the color did not intensified
during storage or turned reddish. Addition of thiourea and
DTPA (Figure 3A, red and dark gray circles) among others did
not influence the base color of the initial dough when freshly
prepared but reduced the discoloration during storage resulting
in brighter dough. Thiourea and DTPA were not capable of
impacting the amount of shell particles on the dough surface.
Out of all chemicals investigated only few including N-acetyl-L-
cysteine were capable of beneficially influencing both quality
aspects. N-Acetyl L-cysteine was effective in decreasing the
amount of shell particles to 0−1 or 2−4 per cm2 if 5 or 1 g was
added (Figure 3A, green and dark green circles and Figure
3C). It further improved the initial dough color. Nevertheless,
it remained darker than the reference pastry dough made from
flour type 380. Moreover, no significant decrease in brightness
was measured in samples treated with N-acetyl-L-cysteine
during storage.
Due to their inhibitory potential presented in Figure 3 the
antibrowning agents were classified into three groups
according to their inhibition mechanism as illustrated in
Figure 4, with Figure 4A showing the target dough color. The
first group (Figure 4B) with citric acid demonstrating a
positive effect on the amount of shell particles consisted of
kojic acid, sinapic acid, ferulic acid, and trans cinnamic acid.
This group includes many acidulants, which shift the PPO
activity outside its natural active pH range and thus reduces
significantly its activity. However, in the concentration added
the pH reduction was nonsufficient. Hence, the reported
inhibitory effect must originate mainly from other inhibitory
pathways. Citric acid inhibited the reaction due to its
additional chelating properties capable of sequestering the
copper atom at the active center of the enzyme. This disabled
or decreased the catalytic activity of the enzyme and reduced
browning around the shell particles.15 Kojic acid successfully
inhibited the enzyme due to its similarity with the common
reaction products. It irreversibly bound to the active center
decreasing the catalytic ability. The inhibition of cinnamic,
sinapic, and ferulic acid originated from their ability to act as
substrate analogous. They were oxidized instead of the
polyphenols hindering the formation of colored reaction
products. The second group of added ingredients contains
N-acetyl-L-cysteine, L-cysteine, ascorbic acid, EDTA, and
chitosan (Figure 4C). The multipathway inhibition of the
antibrowning agents improves both the amount of shell
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particles and the dough color. Attacking on different stages
along the reaction mechanism allowed for a long-lasting
inhibition and should therefore be favored. In case of N-acetyl-
L-cysteine the inhibition potential derived from the ability to
act as reducing agent, quinone coupler, and PPO inhibitor
through complex formation.15 Ascorbic acid functioned as
acidulant and reducing agent while L-cystein acted as quinone
coupler and chelator. Chitin is capable of chelating the copper
atom and acting as quinone coupler. The third group of
antibrowning agents (Figure 4D) included thiourea, sodium
acetate, cinnamaldehyde, L-phenylalanin, and L-lysine benefi-
cially influencing the dough color. This group consisted of
quinone couplers, reducing agents, and substrate analogous.
Quinone couplers and reducing agents both interact with the
colored quinones. Quinone couplers form colorless reaction
products when interacting with the quinones. Reducing agents
reverse the progressing discoloration by reducing the formed
quinones to less intensely colored compounds. Moreover, they
are capable of reacting with the oxygen decreasing the amount
available for the polyphenol oxidase. Substrate analogous
hindered or decreased the formation of colored reaction
products binding to the enzyme instead of the polyphenols.
Consequently, they did not improve the initial dough color but
positively affected the dough during storage.
The results indicated that the action of reducing agents and
quinone couplers was responsible for an improved pastry
dough color. The decrease in shell particles was linked to
modification of the catalytic ability of the enzyme. Pairing of
different chemicals or employing inhibitors targeting several
reaction pathways hence provided the most potent inhibition
of enzymatic browning in pastry dough. Even though the
optical dough properties were improved by addition of the
investigated antibrowning agents no treatment was sufficient to
reach the brightness of the referenced pastry dough
manufactured with flour type 380 (Figure 3A and Figure 3B
for fresh and aged dough). Achieving a sufficient and enduring
inhibition would require the addition of higher antibrowning
agents’ concentration, which is nondesirable from a labeling
point.
3.3. Natural Inhibition of the Enzymatic Browning.
Taking the results of the previous section into account, the
inhibitory potential of natural inhibitors was investigated
aiming toward lasting, natural dough. Lemon juice, grape juice,
and white wine were selected as potent inhibitors based on
their composition. Freshly pressed lemon juice was expected to
affect the browning similarly as a combination of the
chemically produced citric and ascorbic acid. Pastry dough
samples based on flour type 550 containing 5 g of lemon juice
showed brighter initial base color (see Figure 3A, orange
circles, Figure 3G for dough image). The discoloration during
shelf life is shown in Figure 5 (pink line). On the initial dough
no shell particles were observed, but during storage the L*
value decreased and the amount of visible shell particles
increased. The improved initial optical properties of the freshly
prepared pastry dough with lemon juice in comparison to
reference dough made from flour type 550 (Figure 5, black
line) most likely derived from the inhibitory potential of citric
and ascorbic acid acting as chelator and reducing agent. The
color alteration at later stages during storage was attributable
to depletion of the inhibitors. Grape juice was used due to its
polyphenol and acid content, but it demonstrated a low
inhibitory potential independent of concentration. The weak
inhibition potential is shown by the decrease in brightness L*
DOI: 10.1021/acs.jafc.8b04477
J. Agric. Food Chem. 2018, 66, 12353−12360
Journal of Agricultural and Food Chemistry
Figure 5. Changes in brightness L* of dough samples over a storage
time of 4 weeks. Reference samples 380 and 550 are presented in gray
and black, respectively. Dough based on flour type 550 treated with
grape juice (blue), with lime juice (purple), and treated with white
wine (red) show similar browning as the untreated 550 dough
(black). Dough based on flour type 550 with addition of white wine
and lemon juice (green) showed minimum discoloration similar to
the untreated 380 dough (gray). The average color of the measured
dough showed an RGB plot transformed by Adobe RGB1998.icc from
the CIE L*a*b* color space. All data points were measured in real
triplicate, and the error bars indicate standard deviation.
value (79.0 to 71.0) in Figure 5 (blue line) at a concentration
of 5 g. Compared to the reference dough made from flour type
550, the pastry dough treated with grape juice is slightly
brighter and less reddish after 4 weeks of storage. The amount
of shell particles remained unaffected by grape juice giving
similar counts as for the reference flour type 550. White wine
contains alcohol in addition to the polyphenols and acid
present in lemon and grape juice. Already concentrations as
low as 1 g of white wine improved the optical dough properties
when freshly prepared (Figure 5, red). During storage L*
decreased following the progression of lemon and grape juice.
Increasing concentrations of wine delayed the decrease in
brightness and increase in redness. The amount of shell
particles followed a similar trend, i.e. increase in number and
size with storage time and lower concentrations of wine added.
The more pronounced inhibitory potential of lemon juice
compared to grape juice or white wine is linked to their
stronger chelating effect: Citric acid and ascorbic acid tented to
bind stronger to the active center than tartaric or malic acid
present in grape juice and wine.
For improvement of the browning inhibition the natural
ingredients were combined aiming at interfering at several
reaction points of the browning (see Figure 1). Lemon juice
and white wine were paired because of their whitening effect
and the ability to reduce the amount of browning shell
particles. Addition of 1 g dry mass each was not sufficient in
achieving lasting inhibition. Increasing the concentration to 5 g
dry mass of both components reduced the pH to 5.5. The
pastry dough exhibited a similar brightness as the reference
dough made from flour type 380 (see Figure 5, gray line). An
initial but small decay in brightness L* (82.0 to 80.0) was
monitored in the first week of storage and then remained
constant during the remaining storage time following the
progression of flour type 380. Dough samples made with flour
type 380 and the one treated with wine and lemon juice were
comparable in terms of color after 4 weeks of storage. The
reference pastry dough (flour type 380, Figure 5, gray line)
showed L*, a*, and b* values of 83.8, −2.3, and 16.6 while the
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pastry dough with wine and lemon juice showed values of 82.0,
−1.1, and 15.4 (freshly prepared) and 80.3, 0.7, and 14.4 (shelf
life 4 weeks) as also depicted in Figure 3A (light gray and
orange circle) and Figure 3G. The most distinct color
differences were found for a* values, indicating a greenish
tone for flour type 380 and reddish for the wine and lemon
juice treated dough. Moreover, the count of shell particles was
reduced; that is, no particles were visible in the freshly
prepared dough, and no increase in size, color intensity, or
number occurred during storage. In summary, addition of
white wine and lemon juice enabled browning inhibition and
yielded dough samples with improved optical properties such
as color and reduced amount of discolored shell particles. The
alcohol content in combination with the lower pH value is seen
as the main reason for the determined prolongation. Addition
of natural ingredients such as a combination of lemon juice and
white wine to pastry dough based on flour type 550 was
capable of inhibiting enzymatic browning during storage.
This work aimed at replacing extra white flour type 380 with
standard white flour type 550 without impacting the pastry
dough quality. The replacement allows for increased milling
extraction rate so that flour type 550 instead of flour type 380
can be used for pastry doughs. However, utilization of flour
type 550 resulted in enzymatic browning of pastry dough
during shelf-life. An investigation of the enzymatic browning
pathways, transient browning behavior and inhibitory
possibilities was carried out for dough to decrease the
deterioration thereof during storage. Discoloration of the
dough can be attributed to the darkening of the dough base
color and an increase in size, amount, and color intensity of
aleuron shell particles. This darkening is observed in a decrease
of brightness L* and increase in the a* value. Antibrowning
agents were added targeting toward the individual inhibition
possibilities of the enzymatic browning and were generally able
to increase the brightness of the dough or decrease the amount
and intensity of the shell particles. However, to reach a lasting
and extensive inhibition, high amounts of inhibitors are
required, which increases the number of food additives and
moves away from a natural product. Therefore, natural food
additives were selected based on the trials with the individual
chemicals and tested for their inhibitory potential. Especially
acidulants, chelators, quinone couplers, and reducing agents
have been proven as viable inhibitors. All of them are found in
considerable concentrations in grape juice, lemon juice, and
white wine. Lemon juice demonstrated the highest inhibitory
potential while white wine revealed itself as ideal additive for
prevention of enzymatic browning and mold formation. In
summary, a combination of lemon juice and white wine yield
pastry dough (flour type 550) with optical properties
comparable to those made form flour type 380.
■
AUTHOR INFORMATION
Corresponding Author
*E-mail: peter.fischer@hest.ethz.ch.
ORCID
Peter Fischer: 0000-0002-2992-5037
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
This research was supported and funded by Jowa AG and
Swiss Food Research. We acknowledge the assistance of Horst
DOI: 10.1021/acs.jafc.8b04477
J. Agric. Food Chem. 2018, 66, 12353−12360
Journal of Agricultural and Food Chemistry Article

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