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Browning in apples: Exploring the biochemical basis of an easily‐observable

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DOI: 10.1002/bmb.21083

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Laboratory Exercise
Browning in Apples: Exploring
the Biochemical Basis of an
Easily-Observable Phenotype
From the Department of Biology, Creighton University, Omaha,
Nebraska 68178
Abstract
Many fruits and vegetables undergo browning when they
are cut and the tissue is exposed to the air. This is due to the
activity of the enzyme polyphenol oxidase (PPO, EC
1.14.18.1) with endogenous substrates. In this laboratory
experiment, students prepare slices of different varieties of
apples and assess the rate of browning. They make a simple
extract of the apple tissue and measure the activity of PPO
using 3,4-dihydroxy-L-phenylalanine (L-DOPA) as substrate.
They determine the protein concentration of the extract with
the Bradford Coomassie Blue reagent and calculate the spe-
cific activity of PPO. Finally, the students measure the total
Keywords: Apples; melanin formation; phenolic metabolites;
polyphenol oxidase
Introduction
The expression of genetic information leads from DNA to
RNA to protein. Proteins are the major mediators of function
in all cells and are ultimately responsible for the determina-
tion of most of an organism’s visible traits or phenotypes.
Some proteins act as enzymes and catalyze individual chemi-
cal reactions. Others facilitate transport across cellular
membranes, serve as components of the cytoskeleton, func-
tion as receptors for signaling molecules, or regulate expres-
sion of the DNA. The relationship between a protein and an
observable phenotype, however, is often complex and
depends on that protein’s concentration and activity as well
Volume 00, Number 00, Month/Month 2017, Pages 00–00
*To whom correspondence should be addressed. Tel.: 1(480)-202-
1735. Email: Charles.Deutch@asu.edu
Received 2 June 2017; Revised 14 July 2017; Accepted 30 July 2017
Present address of Charles E. Deutch: School of Mathematical and
Natural Sciences, Arizona State University at the West campus, 4701
W. Thunderbird Road, Glendale, AZ 85306
DOI 10.1002/bmb.21083
Published online 00 Month 2017 in Wiley Online Library
(wileyonlinelibrary.com)
Biochemistry and Molecular Biology Education
*
Charles E. Deutch
concentration of the potential substrates for PPO with the
Folin–Ciocalteau phenol reagent using a gallic acid standard
curve. By comparing the tendency of the apples to turn
brown, the specific activity of PPO, and the concentration of
potential substrates, they can assess the biochemical basis
of the browning phenotype. This experiment can be done as
a series of weekly laboratory exercises, as an intensive 1-
week laboratory project, or as the basis of an extended stu-
dent research investigation. V C 2017 by The International
Union of Biochemistry and Molecular Biology, 00:000–000,
2017.
as its interactions with other proteins and molecules in the
cell. Undergraduate students in biochemistry, genetics, and
cell biology courses often have trouble understanding the
process by which a particular phenotype is formed. The
objective of this short laboratory project is to study the role
of the enzyme polyphenol oxidase (PPO, EC 1.14.18.1) and its
substrates in causing certain fruits and vegetables to turn
brown when they are exposed to the air. The reddish-brown
melanin pigments formed through enzyme activity are often
seen as a sign of spoilage. As part of this project, students (1)
test the tendency of different varieties of apples to turn
brown; (2) measure the specific activity of the enzyme poly-
phenol oxidase with 3,4-dihydroxy-L-phenylalanine (L-DOPA)
as the substrate; and (3) determine the total concentration of
phenolic compounds with the Folin–Ciocalteau phenol
reagent using a gallic acid standard curve. The project is
based on several papers from the primary literature [1–4]
and goes beyond simpler studies of apple browning which
have been described as science fair projects [5, 6] or other
undergraduate laboratory exercises [7–11]. Depending on
the instructor’s preferences and institutional constraints,
this project can be done as a series of two to four weekly
laboratory exercises, as a one-week intensive laboratory
experiment, or as a more open-ended student research
project.
1
 Biochemistry and
Molecular Biology Education

Background Information
Many fruits and vegetables undergo browning when they are
cut and exposed to the atmosphere. This is often a problem
with apples, which are the most widely-consumed fruits in
the United States [12]. Apples (Malus domesticus) are grown
around the world, and there are many cultivars or varieties
of apples including Braeburn, Cortland, Red, and Golden
Delicious, Fuji, Gala, Granny Smith, Honey Crisp, Jonathan,
McIntosh, Pacific Rose, Pink Lady, and Wealthy. The varie-
ties available depend on the geographic location and time of
year. Apples are often harvested in the late summer and fall
but then stored in the cold for distribution at other times of
the year. While apple production has traditionally been
based on selective breeding or hybridization, an Italian-led
consortium announced in 2010 that they had decoded the
complete genome of the apple in collaboration with horticul-
turalists at Washington State University using the Golden
Delicious variety [13]. The genome has about 57,000 genes,
the highest number of any plant genome studied to date and
more genes than the human genome (about 27,000). This
new understanding of the apple genome will help scientists
identify genes and gene variants that contribute to resistance Metabolic pathway for the formation of melanin
FIG 1 from L-tyrosine. The first two steps are catalyzed
to disease and drought or that lead to other desirable
by polyphenol oxidase, which is also called
characteristics.
tyrosinase.
Exposure of apple tissue to the air leads to the oxidation
of compounds called polyphenols that are naturally found in
the fruit and to the gradual formation of melanins that are Experimental Procedures
reddish-brown in color. The amount of browning is often General Procedures
expressed using a color scale, many of which are available This project involves a number of basic laboratory proce-
online [14]. The biochemical pathway of melanin formation dures including the use of top-loading balances, the trans-
is shown in Figure 1, starting with the amino acid tyrosine. fer of small volumes of liquids with micropipetters, the
The initial steps in melanin formation are catalyzed by poly- measurement of light absorbance at specific wavelengths,
phenol oxidases (PPO, EC 1.14.18.1), a large family of and the preparation of appropriate graphs. I have done this
copper-containing enzymes that are also called tyrosinase, experiment with undergraduate students ranging from
monophenol monooxidase, or catechol oxidase depending on sophomores to seniors. To ensure that students can do the
their source and substrate specificity [15]. Recent studies experiments efficiently, I have usually included a simple
indicate that the Golden Delicious variety has 10 genes micropipetting exercise at the beginning of the term in
encoding polyphenol oxidase distributed among three chro- which students transfer varying volumes of methylene blue
mosomes [4]. The enzymes act on several related compounds and water to a series of tubes to give the same final vol-
that have one or more hydroxyl groups attached to a ben- ume, measure the absorbance of the solutions at 600 nm,
zene ring. Figure 2 shows some of the polyphenolic com- and then construct a standard curve. I have routinely used
pounds found in apples which are substrates for PPO. They 13 3 100 mm glass tubes which fit directly into the cuvette
are responsible for the color, astringency, and flavor associ- holder of a simple spectrophotometer like a Thermo Spec-
ated with the fruit. The activities of polyphenol oxidase and tronic Genesys 20 and had the students make their graphs
the concentrations of polyphenolic substrates vary among using Excel. Other instructors may prefer to use different
different apple cultivars and are related to the degree of instruments and regular cuvettes and to make graphs with
browning [1, 3, 16]. While all natural varieties of apples are
other software. However, this preliminary exercise is very
susceptible to browning when they are cut or damaged, sev-
helpful in getting students who have relatively little labora-
eral non-browning “arctic” varieties have been made with
tory experience ready to do the project.
reduced expression of PPO by gene silencing (http://www.
arcticapples.com/how-did-we-make-nonbrowning-apple/). Browning in Different Varieties of Apples
These apples are not yet widely distributed in North America The objective of this part of the experiment is to assess the
because they are GMOs (genetically modified organisms) but phenotype of browning in different varieties of apples. An
may be available in 2017. assortment of apples purchased from a local grocery store

2 Polyphenol oxidase, substrates, and browning in apples

 Polyphenolic compounds found in apple tissue that are potential substrates for polyphenol oxidase.
FIG 2
or farm stand is made available, but each group is encour-
aged to bring in their own sample. Only one medium-sized
apple is needed for this experiment. The apple is washed
and peeled to remove most of the pigmented outer coating.
With a sharp knife, two 5 mm (0.5 cm) thick sections are
cut from the middle part of the apple outside of the core
and placed in a plastic petri dish. Students monitor the for-
mation of color on the apple surface during the lab period.
The students then take the petri dish home and continue to
monitor color formation at 12 h intervals over the next
2 days. They use a simple color scale [14] as a guide and
note both the color and the fraction of the surface of the
fruit that has turned brown with time.
Preparation of an Extract of Apple Tissue
The objective of this part of the experiment is to prepare
an extract of the apple tissue that can be used for enzyme
and protein assays and for the determination of total phe-
nolic compounds. The rest of the apple that was used for
the browning experiment is chopped into fine pieces with a
knife. Apple tissue (50 g) is weighed out on a top-loading
balance and transferred to a mortar along with 10 g of
purified white quartz sand (Sigma Aldrich). 10 mL of 0.1M
sodium phosphate buffer, pH 6.8 are added and the tissue
is ground for 5–10 min with a pestle to make a slurry. The
slurry is added to an 8-in. square of double-thick cheese-
cloth on top of a 250 mL beaker. The cheesecloth is pulled
up around the apple pulp and squeezed to express as much
of the liquid as possible. The liquid is transferred to two
15 mL screw cap plastic centrifuge tubes and centrifuged
at about 3,000 rpm in a clinical centrifuge for 5 min. The
Deutch
supernatant liquid is transferred to a clean 50 mL plastic
centrifuge tube and kept on ice while the students are
doing the enzyme assays.
Enzyme Assay for Polyphenol Oxidase
The objective of this part of the experiment is to measure
the activity of the polyphenol oxidase (PPO) in the apple
extract using 3,4-dihydroxy-L-phenylalanine (L-DOPA) as
the substrate. This compound is oxidized first to dopamine,
which then forms the colored product dopachrome. Stu-
dents are provided with a buffer solution (0.1M sodium
phosphate, pH 6.8), a substrate solution (20 mM L-DOPA in
0.1M sodium phosphate, pH 6.8), and a simple spectropho-
tometer such as a Thermo Spectronic Genesys 20 instru-
ment. The limit of solubility of L-DOPA is close to 20 mM so
it may be necessary to adjust the pH of the solution with a
small amount of concentrated HCl to produce a clear
solution.
The students first measure the rate of product forma-
tion in the spontaneous reaction of L-DOPA with oxygen,
that is, in the absence of any extract. 3.8 mL of 0.1M
sodium phosphate buffer, pH 6.8 is added to a
13 3 100 mm test tube along with 0.2 mL (200 mL) of the
20 mM L-DOPA substrate. The solution mixed vigorously on
a Vortex mixer for 5 sec to introduce as much oxygen as
possible. The tube is put into the cuvette holder of the spec-
trophotometer, the instrument is set to zero absorbance at
a wavelength of 475 nm, and the absorbance is read at
15 s intervals for 3 min. To calculate the rate, students
make a graph in which they plot A475 as a function of time.
I have them do this using Excel, but they can also do it
3

with graph paper. They fit a line to as many points as pos-
sible. Students are told that it may not be possible to
include all of the readings since enzyme reactions often
slow down as a substrate is exhausted (either L-DOPA or O2
in this case) or as an inhibitory product is formed. They
express the rate of the reaction as a change in absorbance
with time (DA475 min21).
The students then try to determine the PPO activity in
their apple extract by setting up a new reaction in which
they add 3.6 mL of 0.1M sodium phosphate buffer, pH 6.8
to a clean tube along with 0.2 mL (200 mL) of the apple
extract and 0.2 mL (200 mL) of the 20 mM L-DOPA substrate
solution. After mixing the solution vigorously on a vortex
mixer for five (5) s, the tube is put back into the spectro-
photometer and the absorbance at 475 nm is measured at
15 s intervals for 3 min. A new rate plot is made using
either Excel or graph paper as before. Students are asked
to look at the results carefully and to make adjustments to
the volume of the apple extract as necessary. They can use
a smaller volume of the extract (25, 50, or 100 mL) if the
rate is too fast or a larger volume of the extract (300, 400,
600, or 1,000 mL) if the rate is too slow. The amount of
buffer is adjusted so that the total volume is always 4.0 mL
(4000 mL). Once they have found a volume of the apple
extract that gives a good rate of reaction, the students
repeat the assay so that they have three replicate values
with the same amount of extract. They subtract the rate
for the control (spontaneous) reaction to get the net PPO
activity (DA475 min21) and then calculate the average rate
of reaction as DA475 min21 mL21 of the extract. It does not
take very long to run the reactions, so I usually have them
average the rates with two different volumes of the extract
to get the best estimate of its activity. The apple extract
then is stored in the freezer at 2208C until the next lab
session.
Measurement of Protein Concentrations
The objectives of this part of the experiment are to prepare
a protein standard curve using bovine serum albumin
(BSA) as the protein standard and the Bradford Coomassie
Blue reagent [17] and to use this standard curve to deter-
mine the protein concentration of the apple extract. The
students set up a series of 13 3 100 mm tubes with varying
amounts of 1 mg mL21 BSA and water to give a total sam-
ple volume of 0.1 mL (100 mL). They then add 3 mL of the
Bradford Coomassie Blue protein reagent to each tube.
They mix the solutions by inversion, and after 10 min at
room temperature, measure the absorbance at 595 nm in
the spectrophotometer. By plotting the number of micro-
grams (lg) of BSA on the X-axis and absorbance on the Y-
axis, they generate a standard curve.
Since they do not know the protein concentration of
their apple extract, they then need to repeat the assay with
several different volumes of their sample. I have had them
test volumes from 5 to 100 mL in duplicate, using water to
4 Polyphenol oxidase, substrates, and browning in apples
Biochemistry and
Molecular Biology Education
make up the total sample volume to 0.1 mL (100 mL). They
again add 3 mL of the Bradford Coomassie Blue protein
reagent and mix the solutions by inversion. After 10 min at
room temperature, they read the absorbance at 595 nm.
They use the slope of their standard curve to convert the
absorbance values of those samples that fall within the
range of the standards to an amount of protein, and then
calculate the protein concentration of the extract in
mg mL21. Finally, they calculate the specific activity of the
polyphenol oxidase activity in their apple extract as a D475
min21 (mg protein)21 by dividing the activity value by the
protein value. The apple extract contains many other pro-
teins besides PPO, but the higher the specific activity, the
greater the concentration of this particular enzyme.
Measurement of Total Phenolic Compounds
in the Apple Extract
The objective of this part of the experiment is to determine
the concentration of phenolic compounds in the apple
extract. Because each type of apple contains a different
mixture of compounds, I have had the students just mea-
sure the total concentration of these compounds using the
Folin–Ciocalteau reagent [18]. This reagent reacts with aro-
matic compounds that have one or more hydroxyl or thiol
groups. Because the Folin–Ciocalteau reagent also reacts
with amino acids (and is often used for protein assays), it is
necessary to remove macromolecules from the apple
extract. The students first clarify some of their extract by
adding 1.0 mL to a 1.5 mL microcentrifuge tube and
centrifuging the sample in a microcentrifuge at 13,000 rpm
for 5 to 20 min. They then transfer 500 mL of the superna-
tant to a Pall Nanosep 3K centrifugal spin column with a
3000 Dalton cutoff membrane. This membrane will retain
almost all the proteins and peptides in the extract but let
the smaller molecules including the phenolic compounds
through. They centrifuge the sample for 20 min at
13,000 rpm in a microcentrifuge. Even after the initial clar-
ification, some extracts still clog up the membrane filter
rapidly and it may be possible to obtain only 100 to 200 mL
of filtrate.
As with the protein assay, it is first necessary to make
a standard curve for the phenolic compounds and the
Folin–Ciocalteau reagent. They are provided with a stock
solution of gallic acid at a concentration of 1 mg mL21 and
a solution of 2% Na2CO3. They are also provided with the
Folin–Ciocalteau reagent (Sigma Aldrich) that has been
diluted 1/2 with water.
To make the standard curve, they add 3.0 mL of 2%
Na2CO3 to a series of 13 3 100 mm tubes. They then add
varying volumes of 1 mg mL21 gallic acid and water to give
a total sample volume of 0.1 mL (100 mL). Finally, they add
0.1 mL (100 mL) of the diluted Folin–Ciocalteau reagent.
The solutions are mixed on a vortex mixer and allowed to
sit at room temperature for 30 min. The absorbance of the
 Summary of apple characteristics
TABLE 1

PPO activity PPO sp. act. Polyphenols

Browning (DA475 min21 Protein (DA475 min21 (mg GA PPO sp. act.
Apple variety rate mL 21) (mg mL21) mg21) equiv. mL21) 1Polyphenols

Red Delicious 3 0.0555 0.282 0.197 0.430 0.627

Granny Smith 3 0.0778 0.263 0.296 0.135 0.431
Honey Crisp 1 0.0264 0.214 0.123 0.150 0.273
Gala 2 0.0400 0.212 0.189 0.275 0.464
Golden Delicious 2 0.0478 0.248 0.192 0.182 0.374
Fuji 2 0.0320 0.215 0.149 0.370 0.519

solutions is then read at 750 nm in a spectrophotometer three (Gala, Golden Delicious, Fuji) more slowly, and one
and a standard curve prepared as for the protein assay. (Honey Crisp) even more slowly. Assessing the rate of brow-
To determine the total phenolic concentration of their ning was relatively difficult in that there were often spots or
apple extract, they test volumes from 5 to 100 mL in the areas of a cut slice that turned brown faster than others. The
same way, making up the total sample volume to 0.1 mL apple browning scale noted above [14] is easy to use, but is
with water. The students then use the standard curve to missing # 5. There are other color scales available online for
determine the number of “gallic acid equivalents” or total beer colors, wall paints, and human skin pigmentation that
phenolic compounds per mL of their extract. As with the could be used instead. As an alternative to using slices of
protein assay, they use as many of the absorbance values apple tissue, one can coarsely grate the tissue with a hand
that fall within the range of the standard curve as possible. grater [3]. This exposes more of the tissue to the air and
They can average all of the usable concentrations to obtain allows for somewhat more consistent analysis. While color
a single value for their apple extract. formation can be assessed by eye, it can also be quantita-
tively measured by color reflectance [3]. However, the instru-
Compilation of Data
ments needed for this (e.g. a Minolta CM-700d spectropho-
The results are then compiled into a large table. For each
tometer) are relatively expensive and less often available in
variety of apple, the students record the rate of browning,
an undergraduate laboratory.
the specific activity of the polyphenol oxidases in the apple
extract as D475 min21 (mg protein)21, and the total phenolic
compound concentration as gallic acid equivalents mL21 of
the apple extract. Since the potential for forming melanin
should reflect both the specific activity of the enzyme and
the concentration of the possible substrate, students can
add the specific activity value to the phenolic compound
concentration value to get a better estimate of the melanin-
forming potential. They can then rank the different apples
according to this value and compare this ranking to the
observed browning phenotype.

Results and Discussion

This project was initially devised for use in a typical under-
graduate laboratory course with several sections in which 24
students worked six groups of four. A set of representative
data is shown in Table 1. Six varieties of apples commonly
FIG 3
available in supermarkets in the United States were analyzed
as described above. Of these six varieties, two (Red Delicious
and Granny Smith) tended to turn brown relatively rapidly,
Rate plots of polyphenol oxidase activity with L-
DOPA as the substrate. Control reaction (䊉),
600 mL of Red Delicious extract (䊏), 600 mL of
Granny Smith extract (䉱), and 600 mL of Honey
Crisp extract (䉬).

Deutch 5

Gallic acid standard curve with the Folin–Ciocal-
FIG 4
teau phenol reagent. The dotted line is a smooth
curve fitted to the data points. The solid line is a
best-fit linear regression line which gave the
equation and R2 value shown in the graph.
Measurement of polyphenol oxidase activity in the
extracts of apple tissue using L-DOPA as the substrate was
relatively easy. A typical set of rate plots is shown in Figure
3. The control reaction without any apple extract showed
no change in absorbance. The rates for the Red Delicious,
Honey Crisp, and Fuji extracts were noticeably different.
The rate of the reaction for a given extract increased pro-
portionally with the volume. In most cases, volumes of 600
or 1000 mL gave the best results. Replicate assays of the
same extract with a particular volume varied by less than
5%. The bovine serum albumin standard curve showed
good linearity up to 20 mg of protein. The individual
extracts varied somewhat in protein concentration but vol-
umes of 10, 20, and 50 mL usually fell with the range of the
standard curve. As indicated in Table 1, the Granny Smith
extract had the highest specific activity and the Honey Crisp
extract had the lowest specific activity. The homogenization
of the apple tissue with a mortar and pestle using sand was
easy but could be done using another abrasive such as alu-
mina. It could also be done on a larger scale with a
blender, mill, or apple press.
Quantitation of the phenolic compounds in the apple
extracts was accomplished using the Folin–Ciocalteau phe-
nol reagent [18]. Gallic acid was used to make a standard
curve, an example of which is shown in Figure 4. As
increasing amounts of gallic acid were added to 2%
Na2CO3, the solutions became more and more yellowish
green. The resulting standard curve with the phenol
reagent showed some curvature and deviation from linear-
ity. The best fit straight line gave a R2 value of 0.9812,
which was lower than that found with the BSA protein
standard curve. Similar deviations from linearity have been
observed before [19] and may reflect the fact that each gal-
lic acid molecule has three potentially-reactive hydroxyl
groups. As a result, the best results were obtained using
6 Polyphenol oxidase, substrates, and browning in apples
Biochemistry and
Molecular Biology Education
small amounts (5, 10, 20 mL) of the apple extracts which
gave absorbance values in the lower part of the standard
curve. This was actually desirable because relatively small
of volumes of filtrate were obtained with some of the
extracts from the Pall Nanosep centrifugation devices. In
this data set, Red Delicious and Fuji had the highest pheno-
lic concentrations and Granny Smith and Honey Crisp the
lowest concentrations. In previous studies in which individ-
ual phenol compounds were quantitated by HPLC [3], Fuji
apples were also found to have very high concentrations.
In general, the rate of browning was related both to
the specific activity of PPO and to the phenolic compound
concentration. In some cases like Red Delicious and Granny
Smith, the enzyme activity appeared to be a better indica-
tor of the tendency to brown. In others like Gala and Fuji, a
higher phenolic compound concentration increased brow-
ning for a mid-range enzyme activity. For an apple like
Honey Crisp that showed a slow rate of browning, both the
PPO activity and phenolic compound concentration were
low.
There are many ways this project could be varied.
While there are good reasons to compare different apple
cultivars, there are also some advantages to having all the
students use the same variety of apple. This would allow
them to double-check their calculations, to test the repro-
ducibility of the experiment, and to determine the natural
variation from apple to apple. This experiment involves
more sequential calculations than many introductory labo-
ratory exercises, and some students need additional help in
keeping them all straight. Alternatively, the rate of brow-
ning for the same type of apple at different temperatures
(since apples are often stored in the cold) and to relate this
to the activity of the enzyme as measured at different tem-
peratures. The measurement of PPO activity could be done
with standard cuvettes and more advanced spectrophotom-
eters that have thermostatically-controlled cuvette holders
which allow for automatic recording of absorbance at dif-
ferent temperatures. The assays could also be done in
microtiter plates with a microtiter plate reader connected
to a computer.
Because there are multiple genes for PPO in apples, it
is likely that the activity measured in the extracts repre-
sents several different isoenzymes. These might be sepa-
rated and analyzed by gel electrophoresis [11]. Gel electro-
phoresis could also be used to determine the total protein
profile of the apple extracts and to see where in this profile
the PPO occurs. Because of the importance of browning in
apples, there have been many studies on potential inhibi-
tors of polyphenol oxidase [20–22]. As an extension of this
project, students could test an assortment of natural and
synthetic chemicals as inhibitors of the enzyme using L-
DOPA as the substrate. They could then test the effect of
these compounds on browning in apple slices or grated
apple tissue. This would more directly reinforce the rela-
tionship between enzyme activity and the observable
 phenotype. Finally, if instruments for the quantitative anal-
ysis of phenolic compounds by HPLC were available, this
would improve the analysis of potential substrates. The
Folin–Ciocalteau phenol does react with other compounds
such as the amino acids tyrosine and cysteine, so the values
seen with the apple extracts may include other metabolites.
HPLC analysis would allow a determination of the specific
substrates involved in browning in different apple cultivars.
There are almost no limits to the possible uses of this sim-
ple experimental system.
References
[1] Amiot, M. J., Tacchini, M., Aubert, S., Nicolas, J. (1992) Phenolic com-
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