Postharvest Biology and Technology 35 (2005) 201–207

Reduction of internal browning of pineapple fruit

(Ananas comusus L.) by preharvest soil
application of potassium
Antonio Gomes Soaresa,∗ , Luiz Carlos Trugob,∗ , Neide Botrela ,
Luis Francisco da Silva Souzac
a Embrapa Agroindústria de Alimentos, Av. das Américas 29.501, Rio de Janeiro 23020-470, Brazil
b Instituto de Quı́mica, Laboratório de Bioquı́mica, Nutricional e de Alimentos, Universidade

Federal do Rio de Janeiro, Rio de Janeiro 21949-900, Brazil

c Embrapa Mandioca e Fruticultura, Rua Embrapa, s/n◦ , Bahia 44380-000, Brazil

Received 4 November 2003; accepted 10 July 2004

Abstract

The effect of preharvest soil application of potassium on the development of the physiological disorder called internal browning
(IB) in harvested pineapple fruit was studied. Potassium was applied as a soil dressing at 4–20 g of K2 O per plant in three split
applications at 8, 24 and 40 weeks after planting. Fruit were harvested 19 months after planting and were harvested at two
maturity stages, classified as color break (CB) or half ripe (HR) and stored at 7 ◦ C and 95% RH for 15 days followed by 5 days
at 25 ◦ C to simulate commercial handling. IB was more severe in HR than in CB fruit. Application of potassium reduced IB
in fruit of both maturities. Maximum response was achieved with 16 g of K2 O per plant. Phenolic content of CB and HR fruit
from plants treated with potassium was reduced up to 38 and 39%, respectively. Polyphenoloxidase (PPO) and phenylalanine
ammonia-lyase (PAL) activities varied according to the stage of maturity with higher activities in the ripe fruit. Potassium
treatment promoted a progressive and more accentuated decrease of PPO activities in HR when compared to CB fruit. Similarly,
PAL and POD activities were lower in fruit from plants treated with potassium as was the incidence of IB. This work showed
that soil applications of potassium to growing pineapple plants could improve fruit quality.
© 2004 Elsevier B.V. All rights reserved.

Keywords: Polyphenoloxidase; Peroxidase; Phenylalanine ammonia-lyase; Internal browning; Pineapple fruit

1. Introduction

∗ Corresponding author.
Pineapple fruit is a commercially important crop and
E-mail addresses: antonio.gomes@infolink.com.br a major export item for some countries. However, prob-
(A.G. Soares), lctrugo@iq.ufrj.br (L.C. Trugo). lems to maintaining adequate fruit quality standards,

0925-5214/$ – see front matter © 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.postharvbio.2004.07.005
202 A.G. Soares et al. / Postharvest Biology and Technology 35 (2005) 201–207
particularly the occurrence of internal browning, are
blunting the export potential of the producing coun-
tries. The quality of the fruit is dependent on many fac-
tors, such as planting and harvesting technologies and
postharvest procedures. Postharvest treatments such
as heat, waxing, atmosphere control and application
of 1-methylcyclopropene (Rohrbach and Paull, 1982;
Selvarajah and Herath, 1997; Selvarajah et al., 2001)
have been tested as alternatives to prevent internal
browning without success.
Adequate mineral plant nutrition is crucial for good
quality and productivity and the appropriate concentra-
tion of potassium in the soil appears to play a relevant
role in producing good quality pineapple. Adequate
levels of K in the soil appear to increase total solids
and produce fruit with larger diameter. However, if K
is too high, very acidic fruit with pale and rigid pulps
may be obtained (Dull, 1971).
There are some indications that adequate K in the
soil may also contribute to improved pineapple fla-
vor, to increased stalk diameter and to increased lev-
els of ascorbic acid. The latter may prevent some de-
gree of enzymatic browning by inhibition of polyphe-
noloxidase activity (Teisson, 1972; Teisson et al.,
1979).
The objective of the present work was to study the
effect of K levels in the soil on some characteristics
of pineapple fruit (Ananas comusus L.). The cultivar
Smooth Cayenne was used due to its importance in
the international fruit market. Foliar concentration of
potassium, browning incidence, phenolic contents and
enzymatic activities were evaluated.
2. Materials and methods
The experiment was carried out between April
2000 and November 2001 at the Experimental Sta-
tion of Embrapa-CNPMF, Cruz das Almas, Bahia
State, Brazil, with Smooth Cayenne pineapple as plant-
ing material. A standard salt mixture consisting of
1.450 kg ha−1 of calcium and 2 g of P2 O5 and 8 g
of N (as superphosphate and urea) per plant was ap-
plied just before planting. The calcium was applied
for pH adjustment of the soil. Potassium was applied
as KCl and expressed as K2 O. Nitrogen (8 g) as urea
was applied together with KCl in all treatments af-
ter planting at 8, 24, and 40 weeks with five repli-
cates as follows—T1 (control): 0 g K2 O/plant; T2: 4 g
K2 O/plant; T3: 8 g K2 O/plant; T4: 12 g K2 O/plant; T5:
16 g K2 O/plant; T6: 20 g K2 O/plant. The same amount
of nitrogen was also applied to the control experiment.
Phosphorus (P) was applied again at 24 weeks after
planting.
Twenty fruit from each treatment were harvested
in two maturity stages: color break (CB), when fruit
presented yellow coloration in the basal portion; and
half ripe (HR), when they showed yellow color up to
half of the length of the fruit peel. Five fruit replicates
from each potassium treatment at two stages of matu-
rity were used for all the analysis.
The normal temperature procedure used for pineap-
ple refrigeration in commercial chambers is 11 or
13 ◦ C. However, in wholesale, the refrigerated cham-
bers show 3 or 4 ◦ C variation. Consequently they may
go down to 7 ◦ C, the temperature considered to be the
low critical limit affecting fruit quality. A fungicide so-
lution (benomyl) was applied on the crown of the fruit
and they were stored at 7 ◦ C and 95% rh for about 15
days, which is the time usually taken to reach the re-
tailer. After this period fruit were kept for 5 days at
25 ◦ C which is the usual time for commercial sales and
than they were analyzed.
2.1. Foliar concentration of K
The evaluation of foliar concentration of K on leaf
material was made from “D” leaf, because it is consid-
ered as the one that represents the nutritional state of
the plant. They were collected on the eleventh month
after planting, at the beginning of flowering induction.
A pool of four leaves from each experiment were com-
bined and used as samples. The leaves were washed
in distilled water, dried at 65 ◦ C, ground and digested
according to Novozamsky et al. (1983). Concentra-
tion of K was measured by plasma emission spec-
trometry. A 2 g sample was transferred to an Erlen-
meyer flask and 15 mL of nitric acid, 10 mL of per-
chloric acid and 5 mL of sulfuric acid were added.
The mixture was gently boiled and kept under reflux
for 3 h. It was then cooled to room temperature and
filtered through a Whatman no. 1 filter paper into a
100 mL volumetric flask. The filtrate was used directly
to measure K by means of Spectroflame, ICP equip-
ment (Spectro Analytical Instruments, Model OES,
EUA).
 A.G. Soares et al. / Postharvest Biology and Technology 35 (2005) 201–207
2.2. Incidence of browning
All fruit of each treatment were cut longitudi-
nally into two halves. The incidence of browning
was determined by plotting the brown spots on plas-
tic sheets and measuring the discolored area using a
Delta Area Meter (ELE, USA). The area correspond-
ing to the discolored area was divided by the total
area. This ratio represents the proportion of internal
browning.
2.3. Polyphenoloxidase
The extraction of polyphenoloxidase (PPO) was car-
ried out following the method of Matsuno and Uritane
(1972) and the PPO activity was determined accord-
ing to the method of Teisson et al. (1979) and was ex-
pressed as unit kg−1 s−1 of fresh tissue (U kg−1 s−1 ).
A 20 g sample of fresh pulp tissue was homogenized
for 3 min in a Waring blender (IKA-T25, Labortechnik,
USA) with 50 mL 0.05M phosphate buffer, pH 7.0 at
4 ◦ C. The homogenate was then filtered through What-
man no. 1 filter paper and centrifuged at 9000 × g for
10 min. The supernatant was used for the PPO activity
assay. In the assay tube 1.0 mL of the enzymatic extract
was added to 3.6 mL of 0.1 M phosphate buffer, pH 7.0
and 0.1 mL of 10 mM catechol and was incubated for
30 min at 30 ◦ C. The reaction was stopped by the addi-
tion of 0.8 mL 2 N perchloric acid. The color was then
measured at 395 nm.
2.4. Peroxidase
The extraction of peroxidase (POD) was carried out
following the method of Matsuno and Uritane (1972)
as describe for PPO and the activity was expressed as
U kg−1 s−1 of fresh pulp. The supernatant (3.0 mL) was
added to 5.0 mL of 0.02M citrate–phosphate buffer, pH
5.0 and 0.5 mL of H2 O2 3% and 0.5 mL of guaiacol
following incubation for 5 min at 30 ◦ C. The reaction
was stopped by addition of 1.0 mL of sodium bissulfite
30%. The color was measured at 470 nm.
2.5. Phenylalanine ammonia-lyase
The extraction and determination of phenylalanine
ammonia-lyase (PAL) activity was carried out accord-
ing to the method of Rhodes and Wooltorton (1971)
203
and the activity was expressed as U kg−1 s−1 of pulp
fresh tissue.
Fruit pulp tissue (5.0 g) was homogenized with
50 mL of the extraction solution (Tris, EDTA, sucrose
and PVP in water solution, pH 8.0) for 3 min at 4 ◦ C.
The homogenate was then filtered through Whatman
no. 1 filtered and centrifuged at 10,000 × g for 10 min.
The pH of supernatant was adjusted to 8.9 with 2 M
solution of KOH. The supernatant (1.5 mL) was added
of 1.0 mL STO buffer (Tris) and 0.5 mL 60 mM of l-
phenylalanine. The substrate was incubated at 40 ◦ C
for 1 h and the reaction was stopped by using an ice
bath. The color was measured at 290 nm.
2.6. Phenolics compounds
Phenolics were extracted following the method of
Goldstein and Swaint (1963). Three successive cold ex-
tractions were performed, using 80% methanol as the
extraction solution. Total phenolics was determined by
the standard Follin–Denis method with color measure-
ment at 760 nm (AOAC, 1998).
3. Statistical analysis
All data were treated by analysis of variance
(ANOVA) considering the effects of K treatment, ma-
turity stage and the interaction between factors. Cor-
relation was checked using Pearson correlation. The
comparisons among the averages of the treatments
were accomplished by the least significance differ-
ence (LSD) test at the level of 1 and 5% of prob-
ability. All statistic analysis were performed using
the STATISTICA software, version 5.1, 98th edition
(StatSoft Inc., USA).
4. Results and discussion
Potassium levels in the leaves were increased signif-
icantly by soil application of the nutrient to the plants
(r = 0.74, P < 0.002) (Fig. 1A). Concentrations of up
to 28 g/kg were measured in the leaves from plants fer-
tilized with 16 and 20 g K2 O/plant (LSD = 1.92, P <
0.0002). This is considered to be adequate for plants
in the beginning of flowering induction (Lacoeuilhe,
1984). It thus seems that improved K supply in the soil
204 A.G. Soares et al. / Postharvest Biology and Technology 35 (2005) 201–207

Fig. 1. Effect of potassium fertilization on foliar concentration of K (A), on PPO activity (B), POD activity (C) and PAL activity (D). Values
are average of five replicates and the error bars are standard errors of the means.

is a good technique to increase K concentration in the
plant.
The incidence of browning was higher in most cases
in HR than in CB fruit (P < 0.001). Crop treatment
with potassium showed a marked decreased in brown-
ing. The control treatment presented around 50% of
browning incidence which decreased to 3% in HR and
10% in CB when the higher potassium level was applied
to the soil (Table 1). Pineapple fruit is sensitive to low
storage temperatures, developing physiological disor-
der caused by chilling injures. The main undesirable in-
jury is the internal browning (Swete Kelly and Bagsha,
1993; Zhou et al., 2003). Internal browning may happen
in two phases. Browning starts during storage in chill-
ing temperatures but it does not present evident symp-
toms. The second phase occurs when fruit are removed
from the refrigeration temperature to 20 or 25 ◦ C. In
this phase the symptoms become visible (Teisson et
al., 1979; Paull and Rohrbach, 1985). Akamine (1976)
verified that internal browning was minimized by ex-
posing pineapple fruit to 38 ◦ C for 24 h. The problem
is that at this temperature inadequate acidity, low lev-
els of ascorbic acid and total sugars may be developed
in the fruits. Abreu (1995) suggested the use of plastic
package bags to modify the fruit atmosphere in order to
reduce browning. However, the incidence of browning
was still high with this procedure.
In this work, it was observed that high K levels in the
soil dramatically reduced this disorder. It was also ver-
ified that maturity stages have a strong influence on the
incidence of internal browning of pineapple fruit. These
results are consistent with previous reports that indi-
cate K as an important mineral for quality of pineapple
fruit (de Carvalho et al., 1994). The use of potassium
produced also a significant decrease of phenolic com-
pounds in the pulp with 38% reduction for CB fruits
and 39% for HR (LSD = 16.17, P < 0.001) (Table 1).
Phenolics are the main substrate of browning enzymes
and consequently grown with increased K treatments
become less susceptible to internal browning.
PPO activity varied according to the stage of matu-
rity with HR fruit showing higher enzymatic activity
 A.G. Soares et al. / Postharvest Biology and Technology 35 (2005) 201–207 205

Table 1
The effect of potassium on the internal browning and phenolic compounds of pineapple pulp
K2 O/plant (g) Internal browning (%) Two-way ANOVA (P)

Color break Half ripe Treatment Maturity stage Interaction

0 40.9 a 51.4 a
4 17.6 b 44.2 a
8 10.0 c 24.7 b 0.001 0.001 0.001
12 16.5 b 15.3 b
16 7.5 c 6.0 c
20 9.9 c 3.0 c
Phenolic compounds (mg/100 g)

Color break Half ripe

0 103.9 a 93.0 a
4 93.7 a 83.8 a
8 91.7 a 85.6 a 0.001 0.01 NS
12 78.0 b 68.1 b
16 67.2 c 54.5 b
20 64.5 c 56.0 b

Same letters in the same column means no statistical difference for LSD test (P < 0.001).

than CB fruit (LSD = 8.25, P < 0.001). Ripe pineap-
ples are usually more susceptible to internal brown-
ing and higher levels of oxidative enzymes contribute
to this process (Selvarajah et al., 2001). A strong in-
verse correlation was observed between PPO activity
and K treatment (r = −0.93 for CB fruit and r = −0.96
for HR fruit, with LSD = 8.03 (P < 0.05)). Although
PPO activity was always higher in HR fruit, the potas-
sium treatment promoted a progressive and more ac-
centuated decrease when compared with the CB fruit
(Fig. 1B).
Although showing a tendency toward lower values
for POD activity in HR fruit than in the CB fruit, with
average levels of 1338 and 1405 U kg−1 s−1 , respec-
tively, this difference in the averages was not signif-
icant. However, treatment with potassium decreased
the POD activity compared with the control (Fig. 1C).
The reduction reached 30% for the HR fruit and 20%
for the CB fruit, LSD = 10.34 (P < 0.001). Zhou
et al. (2003) verified that POD activity is present in
pineapple fruit during the whole postharvest period and
not only internal browning is present Several authors
showed that POD activity is also related to enzymatic
browning of fruits and vegetables (Nicolas et al., 1994;
Williams et al., 1985; Teisson et al., 1979). POD en-
zyme oxidizes phenolics to quinines, which are con-
densed to form browning polymers in the presence of
H2 O2 (Robinson, 1991). Those reactions cause unde-
sirable changes in the products affecting their quality
(flavor, aroma and color). Although the average POD
activity was lower in the HR fruit internal browning
were higher in the control and in treatments 3 and 4
when compared with CB fruit. This is an indication that
POD may be not a key enzyme in the internal browning
process.
It was also observed that the average PAL activity
was higher in the HR than in the CB fruit, with av-
erage levels of 1133 and 795 U kg−1 s−1 , respectively,
with significant differences, LSD = 8.55 (P < 0.001).
It is also noted that there was a significant effect of
potassium in the decrease of PAL activity in both treat-
ments, LSD = 8.22 (P < 0.001), however, no significant
interaction was observed between the stages of matu-
rity and treatment (Fig. 1D). PAL activity increases
in several vegetables including pineapple, induced by
cold temperature (Graham and Paterson, 1972; Paull
and Rohrbach, 1985; Zhou et al., 2003; Vukomanovic,
1988; Abreu, 1995). In the present work the induc-
tion of PAL activity was more accentuated in the HR
fruit. However, in both CB and HR stages of maturity
studied the effect of soil treatment with potassium pro-
moted a significant decrease reaching 33 and 27%, re-
spectively, with the highest K concentration (Fig. 1D).
Data from literature indicate that PAL is a key enzyme
206 A.G. Soares et al. / Postharvest Biology and Technology 35 (2005) 201–207
for the biosynthesis of polyphenols (Ke and Saltveit,
1989). This is in accordance with our results since the
decrease of PAL with the treatments was strongly cor-
related with the decrease in the internal browning inci-
dence (r = −0.93 for CB and −0.88 for HR fruit; LSD
= 11.15, P < 0.05).
The concentration of potassium used for planting
in the present experiment promoted increased levels
of intracellular potassium with a consequent reduc-
tion of vacuole pH as a result of the pathways in-
volved in the proton pumps (Forgac, 1989; Futai et al.,
1987). These intracellular conditions contribute to the
decrease in the activities of oxidative enzymes with a
consequent reduction in the internal browning of the
fruit.
In conclusion, different K levels in the soil pro-
moted dramatic effects on PPO, PAL and POD activi-
ties with significant differences among treatments. PPO
and PAL activities were higher in ripe fruit than in the
color break fruit for all the treatments, when compared
with control. PPO and PAL enzymes were more im-
portant in the browning process but their effects were
minimized with the increase of potassium in the plant-
ing soil. The results of this work clearly show that ap-
plication of relatively high levels of potassium in the
soil is a simple and low cost procedure to decrease the
incidence of browning and to improve the commercial
qualities of pineapple.
Acknowledgements
The authors would like to acknowledge the financial
support from MCT-CNPQ, FAPERJ and EMBRAPA,
Brazil. Author LCT is recipient of a research fellowship
from CNPQ.
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