A Colored Avocado Seed Extract as a Potential

Natural Colorant
Deepti Dabas, Ryan J. Elias, Joshua D. Lambert, and Gregory R. Ziegler

C: Food Chemistry
Abstract: There is an increasing consumer demand for and scientific interest in new natural colorants. Avocado (Persea
americana) seed when crushed with water develops an orange color (λmax visible = 480 nm) in a time-dependent manner.
Heat treatment of the seed prevented color development, whereas the addition of exogenous polyphenol oxidase (PPO),
but not peroxidase restored color development. Color development was also inhibited by the addition of tropolone, an
inhibitor of PPO. Color formation resulted in a decrease in the concentration of polyphenols indicating utilization for
color formation. The orange color intensified as the pH was adjusted from 2.0 to 11.0, and these changes were only
partially reversible when pH was adjusted from 7.5 to 11.0 in the presence of oxygen, but completely reversible when
the pH was changed in the absence of oxygen. The color was found to be stable in solution at −18 ◦ C for 2 mo.
These results suggest that the avocado seed may be a potential source of natural colorant, and that color development is
PPO-dependent.
Keywords: avocado seed, natural color, Persea americana, polyphenol oxidase, polyphenols

Practical Application: There is growing public and scientific interest in the development of natural alternatives to synthetic
colorants in foods. Extracts of turmeric, paprika, and beets are examples of food-derived natural colorants. Avocado seeds,
which represent an under-utilized waste stream, form a stable orange color when crushed in the presence of air. Our data
indicate that avocado seed represents a potential source of new natural colorants for use in foods.

Introduction in the color segment will largely come from natural colorants,
Color plays a key role in determining the expectations and with the market expected to reach $773 million by 2014 (Rice
perceptions of consumers with respect to food (Delgado-Vargas 2010). The market for natural colorants was $250 million in 2006
and Paredes-Lopez 2003a, 2003b). It is one of the most obvious and had an estimated annual growth rate of 2.9% (Trim 2006).
characteristics of a food and, if not appealing, negatively impacts Whereas the synthetic colorants generally have similar proper-
consumer acceptance. Color is interrelated with flavor intensity ties, natural colorants, even if they are of the same shade, may
and sweetness and salinity sensations and may also indicate the differ in terms of chemistry and functionalities. For example an-
safety of a food. Although artificial colorants have a long history natto and turmeric differ in their functionalities. Annatto, as nor-
of use, and are easy to produce, stable, less expensive, and have bixin, will precipitate at pH below 5.0 whereas turmeric will not
better coloring properties than natural colorants, consumers have (foodproductdesign.com 2011). Thus it is crucial to understand
increasingly begun to consider synthetic colorants undesirable. the properties of any prospective natural colorant to decide about
Consequently, there has been increased effort to discover new its applications. The factors that deteriorate colorant stability and
natural alternatives (Socaciu 2008). efficacy include the presence of temperature, light, oxidizing and
By definition, a natural pigment is one that is synthesized by and reducing agents, acids or time of storage (Socaciu 2008). There
accumulated and/or excreted by living cells. It may also be synthe- is still a lack of natural colors to completely replace synthetic
sized by cells under stress or by dying cells (Hendry and Houghton colorants, and hence, efforts to search more options are needed
1996). Historically, natural pigments (e.g., saffron) were used to (Hazen 2011).
color food products. Currently, 26 natural colorants including an- Avocado (Persea americana Lauraceae) is an important tropical
thocyanins, curcumin, carminic acid, lycopene, betanin, paprika, crop which is rich in unsaturated fatty acids, fiber, vitamins B and
and saffron are permitted for use as exempt colorants in the United E, and other nutrients (Gomez Lopez 1998). The Hass variety is
States. Like their synthetic counterparts, natural pigments can be the most commonly grown. Total U.S. avocado production dur-
formulated as dyes (hydrophilic powders or lipophilic oleoresins) ing the 2004/05 season was 162721 tons with 90% originating in
or lakes (water insoluble forms, formulated by adsorbing dyes on California (World Horticultural Trade 2006). Mexico, the world’s
salts) (Socaciu 2008; Amiot-Carlin and others 2008). The growth largest producer, supplied 1.14 million tons in 2007 (McLeod
2008). The seed accounts for 16% of total avocado weight, and is
an under-utilized resource (Ramos-Jerz 2007). Ethnopharmaco-
logical studies of the Aztec and Maya cultures have reported the
MS 20110686 Submitted 5/31/2011, Accepted 8/24/2011. Authors are with
Dept. of Food Science, The Pennsylvania State Univ., Univ. Park, PA 16802,
use of decoctions of avocado seeds for the treatment of mycotic
U.S.A. Direct inquiries to author Lambert (E-mail: jdl134@psu.edu). and parasitic infections (Kunow 2003). Seeds have also been re-
ported for use against diabetes, inflammation, and gastrointestinal


C 2011 Institute of Food Technologists
R

doi: 10.1111/j.1750-3841.2011.02415.x Vol. 76, Nr. 9, 2011 r Journal of Food Science C1335
Further reproduction without permission is prohibited
 Avocado seed extract as a colorant . . .

irregularity (Kunow 2003; Ramos-Jerz 2007). The powdered To measure L ∗ a∗ b∗ values, seeds were weighed, cut, and ground
form has been used for skin eruptions and to cure dandruff (Mor- in 1 vol deionized (DI) water in a Waring blender for 60 s at high
ton and Dowling 1987). speed. The ground paste was spread in a Petridish (CM-A128,
Avocado seeds have more antioxidant activity and polyphenol Minolta) and L ∗ a∗ b∗ values recorded at specified time intervals us-
content than the pulp (Soong and Barlow 2004; Wang and oth- ing a Konica Minolta chromameter CM-3500 d (Konica Minolta,
ers 2010). Phytochemical studies on avocado seeds have identified Ramsey, N.J., U.S.A.) in reflectance mode. The paste was mixed
various classes of natural compounds such as phytosterols, triter- thoroughly just before taking measurements. The conditions were
C: Food Chemistry

penes, fatty acids, furanoic acids, abscisic acid, proanthocyanidins, a ‘D’ illuminant with an 8◦ observer. Color difference (E) was
and polyphenols (Ding and others 2007; Leite and others 2009). calculated using the following formula:
Wang and others (2010) have reported the presence of catechin, 
epicatechin, and A- and B-type procyanidin dimers and trimers, E = (L − L 0 )2 + (a − a 0 )2 + (b − b 0 )2 ,
tetramers, pentamers, and hexamers in the seed.
Our laboratory has observed that avocado seed when ground where L 0 , a0 , and b0 are L, a, and b values, respectively, for the
with water and incubated in the presence of air produces a bright first time point measured.
orange color (previously unreported). The aim of the present work
was to characterize the color formation, to study the mechanism Role of PPO in color formation
of color formation and the color properties of the extract. Avocado seeds were prepared and ground as above. An addi-
Material and Methods tional 1.5 vol of DI water was added and mixed, and the paste
was kept at 100 ◦ C for 30 min to destroy endogenous enzyme
Reagents activity. The paste was cooled to 24 ◦ C in a water bath. To 25 g
Ripened avocado (Persea americana, Hass variety) were sourced of cooled paste 1000, 2500, or 5000 U of mushroom PPO (pre-
locally and stored at 4 ◦ C until used. No change in seed pared in phosphate buffered saline:NaCl, 136 mM; KCl, 2.7 mM,
color was observed during storage. Mushroom polyphenol ox- Na2 HPO4 , 10 mM; KH2 PO4 , 1.7 mM at pH 6.8) was added
idase (PPO) (tyrosinase), horseradish peroxidase, hydrogen per- and the mixture spread on a flat surface as described above. After
oxide, and tropolone were purchased from Sigma Chemical 35 min, methanol (3 vol) was added to the samples and they were
Co. (St. Louis, Mo., U.S.A.). DMSO (Dimethyl sulfoxide) was sonicated and centrifuged as above. Absorbance spectra were ob-
purchased from EMD Chemicals (Gibbstown, N.J., U.S.A.). Folin tained as described above. Positive control samples were prepared
Ciocalteu reagent was purchased from Fluka (Buchs, Switzerland). similarly but were not subjected to heat treatment. Negative con-
Gallic acid was purchased from Alfa Aeser (Lancaster, Pa., U.S.A.). trol samples were subjected to heat treatment but exogenous PPO
All other reagents were of the best commercial grade available. was not added.
Involvement of PPO was examined by use of tropolone, an in-
Preparation of colored extract hibitor of PPO. Tropolone (58 μM) was added during grinding
Avocado seeds were separated from fruit, washed and peeled. of the seed, after which the paste was spread on a flat surface
Seeds were weighed, cut with a knife, and ground in 0.7 vol of for 35 min and processed as above. For studies with exogenous
deionized (DI) water in a Waring blender for 60 s at high speed. PPO, tropolone were added to the inactivated paste prior to
The resulting paste (pH 6.4) was spread to 1 cm thickness, on the addition of PPO and the reaction was carried as outlined
a flat surface (to enable uniform exposure to air) and incubated above.
at 24 ◦ C for 35 min. The paste was mixed with a spatula 2 to
3 times during this time. The colored paste was transferred to a
Role of peroxidase in color formation
beaker, an equal volume of methanol was added, and the mixture
Avocado seeds were prepared, ground and heat inactivated as
was sonicated for 20 min in a Branson 3510 sonicating water bath
above. To 25 g of cooled paste, 1000, 2500, or 5000 U horseradish
(Danbury, Conn., U.S.A.). An additional 2 vol of methanol was
peroxidase (in phosphate buffered saline, pH 6.8) and 1.9 g H2 O2
added, and the mixture centrifuged at 1200 × g for 10 min. The
per 1000 U were added. The paste was incubated and processed
supernatant was collected and dried under vacuum to remove the
as described for exogenous PPO experiments.
methanol. Residual water was then removed by freeze drying, and
the resultant powder was stored in a desiccator at −20 ◦ C.
Phenolic content of the colored extract
Kinetics of color formation To the freeze-dried extract, 50 vol hexane was added and the
The kinetics of color formation was studied using 2 methods: mixture was shaken for 30 min in an orbital shaker at 600 rpm to
paste was incubated for different time intervals, the color extracted remove residual lipids. Hexane was removed by centrifugation at
and absorbance of the extract measured or paste was incubated and 1200 × g for 10 min. The marc was then extracted with 50 vol
CIE (International Commission on Illumination) L ∗ a∗ b∗ values of methanol:ethyl acetate (1:1, v/v) for 1 h in the orbital shaker
were read at different time points. at 600 rpm. The supernatant was separated by centrifugation, and
For the absorbance measurement, pastes were prepared as de- dried under vacuum at 24 ◦ C. The marc was then extracted with
scribed above with the modification that 10 g samples were in- 50 vol of DI water at 600 rpm for 1 h, centrifuged and the aque-
cubated for 0.5, 2, 5, 10, 15, 20, 25, 30, and 35 min after which ous supernatants dried under vacuum at 24 ◦ C for 12 h. Both the
they were combined with methanol (3 vol), and then sonicated methanol:ethyl acetate and water fractions were used for determi-
and centrifuged as above. Visible absorbance spectra were recorded nation of phenolic content by the modified method of Singleton
immediately (λ = 380 nm to 700 nm) using an Agilent 8453 spec- and Rossi (1965). Aliquots of the samples of appropriate dilutions
trophotometer (Agilent Technologies, Santa Clara, Calif., U.S.A.) were prepared in methanol and were combined with 790 vol of
by placing samples in disposable 1.5 mL cuvettes (Plastibrand, DI water and 5 vol of Folin–Ciocalteu reagent. The solution was
Wertheim, Germany). mixed and 15 vol of 15% sodium carbonate solution was added to

C1336 Journal of Food Science r Vol. 76, Nr. 9, 2011

Avocado seed extract as a colorant . . .

the sample. Samples were mixed again and the absorbance deter- Results
mined at 765 nm after 2 h. Gallic acid was prepared in methanol
and was used as the standard. The total amount of phenolic com- Kinetics of color formation
pounds was expressed as gallic acid equivalent, GAE (mg/g). Un- The kinetics of color formation in avocado seed were followed
colored extract, prepared in a method analogous to the colored by measuring changes in visible absorbance intensity and by de-
except that methanol was added immediately after grinding the termining L ∗ a∗ b∗ values and color difference (E, Figure 1).
seeds, was also tested for phenolic content for comparison. Figure 1A shows visually how the orange color intensity increased

C: Food Chemistry
with time. The λmax of the color was 480 nm (Figure 1B) and
absorbance increased over the time course of the experiment
in an exponential fashion approaching an asymptote by 20 min
Effect of pH on color of extract
(Figure 1C). A similar time-dependent increase in E values was
Phosphate buffered saline at pH 2.0, 4.0, 6.0, 7.5, 9.0, and 11.0
observed (Figure 1D). Within 0.5 min there was noticeable dif-
was prepared in 20% aqueous methanol and combined with freeze
ference in color compared to water.
dried extract at a final concentration of 2 mg/mL. The samples
were vortexed and then centrifuged at 1200 × g for 10 min. Vis- Role of PPO in color development
ible absorbance spectra were recorded as described above. Hunter We hypothesized that the color development in crushed avo-
Lab values were determined using a Konica Minolta chromameter cado seed was enzyme dependent and observed that heat treat-
CR400 by placing the sample in a 1 mm path length rectangu- ment prevented color development (negative control, Figure 2A)
lar quartz cuvette (Fisherbrand, Pittsburgh, Pa., U.S.A.) against a compared to untreated seed (positive control, Figure 2A). Since
white background. A ‘C’ illuminant and an 2◦ observer were used. PPO is involved in color production in number of fruits and
For the determination of E at different pH values, L 0 , a0 , and vegetables (Munoz and others 2007), and is present in avocado
b0 values were that of a blank measured by placing DI water in the (Gómez-López 2002), we investigated whether addition of ex-
cuvette. ogenous PPO would restore color formation. Addition of in-
To determine if the effect of pH on the color of the extract was creasing amounts of mushroom PPO to heat-inactivated crushed
reversible, extract at a concentration of 2 mg/mL in phosphate seed resulted in restoration of color development (Figure 2A).
buffered saline containing 20% aqueous methanol (pH 7.5) was To further demonstrate the role of PPO in color formation, re-
prepared as described above. The visible absorbance spectra were actions were also carried out in the presence of tropolone. The
determined. The pH of the solution was adjusted to 11.0 by the addition of tropolone prevented the formation of color when
addition of NaOH and the spectra determined again. The pH added during grinding and likewise inhibited color formation
was readjusted to 7.5 by the addition of HCl and the visible ab-
sorbance spectra were recorded a final time. A similar experiment
was conducted by adjusting the pH from 7.5 to 2.0 and back to
7.5. Both sets of experiments were also conducted while purging
nitrogen through the samples at a rate of 5 mL/s. This was done
to assess the role of oxygen on the effects of pH on color. The
amount of dissolved oxygen after N2 flush was determined using
a Thermo-scientific portable DO meter, Orion 087003 (Orion,
Barrington, Ill., U.S.A.) and found to be reduced to 0.15 ± 0.01
mg/L. The addition of acid and base to change the pH changed
the final volume of the solutions by less than 2%.

Color stability
Freeze-dried extract was dissolved in phosphate buffered saline
in 20% aqueous methanol at pH 7.5 at a concentration of
2 mg/mL. The samples were centrifuged at 1200 × g for 10
min to remove any undissolved solids. The supernatant was trans-
ferred to new tubes, sealed and kept at −18 ◦ C, 4 ◦ C, 24 ◦ C,
and 40 ◦ C. Aliquots of samples were periodically removed and the
absorbance at 480 nm and E values measured.

Statistical analysis
Results are shown as mean of 3 independent determinations.
Error bars represent the standard error of mean (SEM) or the stan-
dard deviation (SD) as indicated in the figure legends. Differences
between means were tested for significance by one-way analysis of
variance (ANOVA) with Dunnett’s posttest significance or two- Figure 1–Kinetics of color formation in avocado seed paste. (A)
way ANOVA with Bonferroni posttest as appropriate. Significance A visual change in color of the paste was observed with the
time of incubation. Representative results of 3 independent experi-
was achieved at P < 0.05. All statistical analyses were performed ments. (B) Spectrophotometric analysis at 35 min showed a λmax at
using Graphpad Prism software (Graphpad Software, San Diego, 480 nm. (C) Absorption at 480 nm (mean ±SD of 3 replicates) and (D) E in-
Calif., U.S.A.). creased with the time of incubation (mean of 2 independent experiments).

Vol. 76, Nr. 9, 2011 r Journal of Food Science C1337

 Avocado seed extract as a colorant . . .

in PPO-supplemented heat-inactivated paste formation of color whereas E greater than 5 represents a readily observable change
(Figure 2A). (Obón and others 2009).
Since the enzyme peroxidase can also be involved in oxidative To determine if the effect of pH on color was reversible, the
reactions in fruits and vegetables (Chisari and others 2007), its pH of the colored avocado seed extract in solution was increased
role was explored. The addition of exogenous peroxidase and from 7.5 to 11.0 and then reduced back to 7.5. As the pH was
H2 O2 to the inactivated paste did not result in significant increases increased in the presence of air, an increase in absorbance intensity
in absorbance at 480 nm compared to heat-inactivated control in the visible region was observed as was the appearance of a new
C: Food Chemistry

samples (Figure 2B). Visually, the peroxidase paste had a slight maxima (λ = 393 nm, Figure 5A). When the pH was reduced back
brown color, but did not develop the orange color characteristic to 7.5, absorbance intensity in the near UV range was reversed, but
of PPO-treated samples. larger differences remained in the visible portion of the spectrum
(Figure 5A). When similar experiments were conducted under a
Phenolic content nitrogen atmosphere, the shape of absorbance spectra at pH 11.0
In order to assess the impact of color production on was different from the spectrum at the same pH under air, and the
seed phytochemistry, the total polyphenol content of the ex- only changes occurred in the near UV. Changes in the spectrum
tracts were determined. The phenolic content of the col- induced by increasing the pH to 11.0 under nitrogen were also
ored extract and the uncolored extract was found to be completely reversed when the pH was reduced back to 7.5. Much
219.4 ± 4.5 mg/g GAE and 283.2 ± 5.8 mg/g GAE, respec- smaller changes in absorbance spectra were observed when pH was
tively (Figure 3). A higher concentration of phenolics was found reduced from 7.5 to 2.0 (Figure 5B). A complete reversal of the
in the methanol:ethyl acetate fraction than in the water fraction changes was observed both in the presence and absence of oxygen
for both the colored and uncolored extracts (Figure 3). when pH was increased back to 7.5.

Effect of pH on color Stability of the colored extract

The intensity and the hue of the colored avocado seed extract in At pH 7.5, the color of the extract in 20% aqueous methanol
solution were increased by increasing the pH of the solution. In- was unstable at 40 ◦ C and a sharp increase in absorbance at
creases were observed across the visible spectrum, and at pH 11.0, 480 nm was observed during the first 12 h of storage
a second visible absorbance maxima (λ = 440 nm) and a max- after which the intensity decreased (Figure 6A). Samples
ima in the near UV portion of the spectra developed (Figure 4A). stored at 24 ◦ C showed similar changes although the kinet-
Absorbance at 480 nm increased slightly between pH 2.0 and 9.0, ics were slower. An increase in absorbance occurred until day
reaching a point of inflection, followed by a rapid increase to pH 10 followed by a subsequent decrease. At both 4 ◦ C and
11.0 (Figure 4B). −18 ◦ C, the absorbance of the samples increased at much
E also increased as a function of increasing pH (Figure 4B). slower rates than at the higher temperatures. No significant
The change in E values was largely due to decreased lightness, change was observed until day 60 in the samples stored at
L (from 89.1 to 80.0), and increased b (yellowness) (from 2.0 to −18 ◦ C. The E values of the extract stored at pH 7.5
24.8) from pH 2.0 to 11.0. The value of a (redness) also increased showed similar trends to those observed spectrophotometrically
but to a lesser extent (from −0.1 to 1.8). E less than 1.5 does (Figure 6B). For samples stored at −18 ◦ C at pH 7.5, the E value
not represent a significant change in color compared to baseline was 1.65 and no visible change in color was discerned. However,

Figure 2–Role of PPO and peroxidase in the

development of color in avocado seed paste.
(A) The absorbance at 480 was determined in
heat-inactivated avocado paste following
addition of increasing amount of exogenous
PPO. Tropolone (marked ‘T’ in the figure) was
added to determine if selective inhibition of
PPO prevented color formation. (B) The
absorbance at 480 nm was determined after
the addition of exogenous peroxidase and H2 O2
to heat-inactivated paste. The data represent
the mean ± SD of 2 independent experiments.

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Avocado seed extract as a colorant . . .

samples stored at higher temperatures (4 ◦ C, 24 ◦ C, and 40 ◦ C) exogenous mushroom PPO restored color formation. PPO from
had E values greater than 5 over the time period of storage study avocado pulp has been found to be very active and resistant to
and had noticeable changes in color. treatments including heat, making avocado products very prone
to enzymatic browning (Gómez-López 2002), but there is scant
Discussion information on PPO from avocado seed. The orange color formed
In the present study, we characterized the color production in in the seed may have resulted from a particular substrate present
avocado seed upon maceration and exposure to air, the mech- in the avocado seed or inhibition of PPO by the products formed

C: Food Chemistry
anism of color formation and the color properties of resultant during an intermediate stage of the reaction.
extract. A stable orange color was produced when avocado seeds PPO catalyzes both the oxygen-dependent hydroxylation of
were ground and incubated at 24 ◦ C. The color production oc- monophenols to their corresponding O-diphenols and the oxida-
curred rapidly with a noticeable change produced as early as tion of O-diphenols to their cognate O-quinones (Lin and oth-
0.5 min of incubation. Kinetic analysis showed that intensity of the ers 2010). The quinones then may polymerize into red, brown,
color formation increased in an exponential fashion and began to or black pigments depending on conditions like the nature and
approach an asymptote at 20 min. Such kinetics of formation are amount of endogenous phenolic compounds, the presence of
typical of an enzyme catalyzed reaction. For example, Arias and oxygen, reducing substances, or metallic ions, the pH and tem-
others found that pear (Pyrus communis) PPO-mediated oxidation perature, and the activity of the PPO (Dogan and others 2006).
of dihydroxyphenylalanine (DOPA) resulted in a similar trend in Browning reactions in fruits and vegetables occur when tissues
absorbance at 420 nm (Arias and others 2007). We observed that are damaged and PPO is released. Here, we observed that the
heat treatment prevented color formation, whereas the addition of orange colored extract was stable during observation window

Figure 3–Comparison of the phenolic content of

the colored and the uncolored avocado seed
extract. Higher total phenolic concentrations
were observed in the uncolored extract. This
difference was statistically significant
(P < 0.05). Mean ± SEM of 3 independent
experiments.

Figure 4–Effect of pH on the color of the avocado

seed extract. (A) Absorption spectra at different
pH values show a λmax at 480 nm at
pH 7.5, 9.0, and 11.0; at pH 11.0 an additional
λmax at 440 nm was observed. Representative
results of 3 independent experiments. (B)
Absorption at 480 nm and E values were seen
to increase as a function of pH. Mean ± SD of 3
replicates.

Vol. 76, Nr. 9, 2011 r Journal of Food Science C1339

 Avocado seed extract as a colorant . . .

explored and did not apparently produce highly polymerized extract. The phenolic compounds present in the colored extract
melanoidins. may contribute to functional attributes. The reduction in pheno-
The total phenolic content of methanol:ethyl acetate and wa- lic content during color development may be due to oxidation
ter extracts in freeze-dried colored extract was calculated to be of phenolic compounds by the PPO. During processing of fresh
219.4 mg/g. Soong and Barlow (2004) used ethanol:water at high grapes to raisins, most of the 2 major hydroxycinnamic acids and
temperature for extraction of phenolics of avocado seed and cal- all of the procyanidins and flavan-3-ols were lost. The reasons
culated the phenolics as 88.2 mg GAE/g. Wang and others (2010) for this loss were likely to be both enzymatic and nonenzymatic
C: Food Chemistry

used acetone:water:acetic acid (70:29.7:0.3, v/v/v) and found (Karadeniz and others 2000).
the values to be 51.6 mg/g GAE for the seed of Hass variety Although often associated with undesirable browning reactions,
of avocado, whereas the corresponding values for pulp was only PPO has also shown to produce attractive colors in some systems.
4.9 mg/g GAE. In the present study, the phenolic content of the PPO-catalyzed conversion of catechins to theaflavins in the pro-
colored extract was 22.5% lower than the content in uncolored duction of black tea is responsible for the characteristic orange
color of that beverage (Balentine and others 1997). A yellow-
colored product was synthesized through oxidation of ferulic acid
with laccase, an enzyme functionally similar to PPO. It was found
found that in a biphasic system containing ethyl acetate a stable
yellow-colored product is formed whereas in an aqueous system
browning occurred. This difference was attributed to the incom-
plete activity of PPO in the medium containing ethyl acetate

Figure 5–Effect of pH change and the presence or absence of air on the

rehydrated avocado seed extract. (A) When the pH was increased from
7.5 to 11.0, in the presence of oxygen, the absorption intensity increased
and 2 more absorbance maxima emerged at pH 11.0. When pH was de-
creased back to 7.5, only partial restoration of color character was ob-
served. When pH was increased to 11.0 under a nitrogen atmosphere, an
increase was observed in near UV range. On reducing the pH back to 7.5,
complete reversal of absorbance intensities was observed. Representa-
tive results of 3 independent experiments. (B) When pH was reduced from Figure 6–Effect of storage on color of the rehydrated avocado seed extract.
(A) Absorbance intensity at 480 nm and (B) E were determined during
storage for 60 d at pH 7.5 at −18 ◦ C ( ), 4 ◦ C ( r), 24 ◦ C (䉱), and 40 ◦ C
7.5 to 2.0 and adjusted back to 7.5, similar effects were observed under
both air and nitrogen atmospheres. Representative results of 3 indepen-
dent experiments. (䉲). Mean ± SD of 3 replicates.

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Avocado seed extract as a colorant . . .

(Mustafa and others 2005). Catechin was oxidized at pH 7.5 us- Acknowledgment
ing PPO and it was observed that the absorbance maxima of the This study was supported in part by NIH grant AT 004678
product was 430 nm which corresponded to a yellow-colored (to JDL).
product (Jiménez-Atiénzar and others 2004). A yellow dye from
phlorodzin, a flavonoid specific to apples, was synthesized in References
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phenolic content; the colored extract may have additional func-
tional attributes which should be explored.

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