Z Lebensm Unters Forsch A (1997) 204: 6065 Springer-Verlag 1997

ORIGINAL PAPER
M. Pilar Cano

Begon a de Ancos

M. Gloria Lobo
Mariana Santos
Improvement of f rozen banana (Musa cavendi shi i , cv. Enana)
col our by bl anchi ng: rel at i onshi p bet ween browni ng, phenol s
and pol yphenol oxi dase and peroxi dase act i vi t i es
Received: 22 February 1996
Abstract Browning in banana (Musa cavendishii, cv.
Enana) processed products is a result of phenol oxida-
tion catalysed by polyphenol oxidase (PPO) and per-
oxidase (POD) or of other non-enzymatic reactions
(Maillard and Strecker mechanisms). Microwave and
steam blanching signicantly reduced PPO and POD
activities and phenol levels in banana esh, steam
blanching being the most eective method for enzyme
inactivation. Freezing/thawing processes produced
a signicant increase in phenol levels in all samples,
due to cellular breakdown. After microwave heating
browning processes occurred while steam-treated
samples did not exhibit a signicant colour change.
Extractable PPO and POD activities in all banana
samples increased as a consequence of freezing/thaw-
ing: steam-blanched slices exhibited lower residual ac-
tivities. High correlations occurred between phenols
and browning (r"0.86) in control samples. Blanched
samples (microwave or steam) only exhibited correla-
tions between PPO (r"0.80) and POD (r"0.80) ac-
tivities and browning.
Key words Browning reactions Banana processed
products Blanching Polyphenol oxidase Peroxidase
Int roduct i on
Enzymatic browning in fruit and vegetables can cause
undesirable quality changes during handling, process-
ing and storage. This reaction results mostly from poly-
phenol oxidase (PPO, EC 1.10.3.1) and peroxidase
(POD, EC 1.11.1.7) [14]. Both enzymes catalyse more
than one reaction and act on a number of substrates;
M.P. Cano ( ) B. de Ancos M.G. Lobo M. Santos
Plant Food Science and Technology Department, Instituto
del Frio (C.S.I.C.), Ciudad Universitaria, E-28040 Madrid, Spain
browning or darkening is the principal and more mani-
fest consequence, but discoloration, o-avours and
nutritional damage also occur [3, 5, 6]. However, the
browning potential of some plant products has been
reported to be also directly related to the phenol levels
[7], or a combination of both enzymatic activities
(PPO and POD) and phenols [8].
Bananas undergo rapid browning as a result of tissue
disruption and exposure to oxygen during the peeling
and slicing operations that take place prior to further
processing. The successful prevention of banana
browning by addition of sodium bisulphite has been
widely discussed [911], but blanching to inactivate
enzymes in whole peeled fruits has also been reported
[12, 13]. A process for banana puree preservation was
described by Garcia et al. [14], which required a mild
heat treatment plus addition of sodium bisulphite, cit-
ric acid and potassium sorbate. Other authors have
described the eect of some heat and chemical treat-
ments on the quality of banana puree stored at 30C
[15] or on intermediate-moisture banana products
[16].
To optimize the freezing process of banana slices, it is
necessary to study the eects of some mild heat treat-
ments and freezing on the enzymatic activities in the
fruit at dierent maturity levels. Cano et al. [12] de-
scribed the eects of some heat treatments on PPO and
POD enzymes, including a single experiment that in-
volved blanching the fruit in a microwave. Most re-
cently, Cano [ 17] reported the importance of selecting
the banana fruit to be frozen at the optimal level of
ripeness, using microwave treatment to obtain an
optimal quality in the nal product. Bananas with
a greenyellow peel colour were used to improve pro-
cessed fruit colour in terms of enzymatic browning in
frozen banana slices.
In this work an investigation was carried out into the
eects of microwave and steam blanching, at previously
selected conditions, on browning, phenol concentra-
tion, and PPO and POD activities in banana slices
(Musa cavendishii, var. Enana) during frozen storage.
The individual roles of these factors involved in the
biochemical processes of darkening have been tenta-
tively dened.
Mat eri al s and met hods
Plant material. Air-freight shipments of green bananas (M. caven-
dishii L. var. Enana), produced in the Canary Islands (Spain), were
obtained from a commercial source in Madrid. Undamaged fruits
free from infection were selected and stored at 14C and 8590%
relative humidity [18]. At the right stage of maturity for processing,
after 15 days of storage under the previously mentioned conditions,
fruits exhibiting a greenyellow peel colour [12] were removed from
storage and processed as follows.
Microwave treatment. Samples of peeled banana fruits were sliced
into 1.0-cm-thick pieces, selecting only the slices with homogeneous
diameters (3.03.2cm). Microwave treatment was carried out at
650 W for 30 s [ 17]. After blanching, the slices were packaged in
plastic bags and cooled at 8C with owing cold water (fruit slices
did not make contact with the cold water). Chilled microwave-
treated slices were vacuum-packed in plastic bags (PolyskinX12,
Cryovac) and frozen in an air-blast freezer at !40C for 30 min.
Steam blanching treatment. Samples of the peeled whole banana
fruits were steam blanched for 5 min in a conventional oven
(Rational, Combi-Master CM). Treated fruits were chilled and
frozen as stated in the previous section.
Control samples. Untreated banana slices were packed and frozen
under the same conditions as the treated samples. All frozen samples
were stored at !24C for a 12-month period.
Determination of browning. Browning of banana slices were deter-
mined by the procedure reported by Chan and Cavaletto [19]. Fruit
slices from each treatment and each storage period were freeze-dried
and then made into a homogeneous lyophilized powder for deter-
mination of darkening. Banana powder (2.5 g) was homogenized
with 15 ml of methanol for 3 min using external cooling. The mixture
was centrifuged at 6500 g at 5C for 10 min. The supernatant was
decanted into a 25-ml volumetric ask and made up to the correct
volume with additional methanol. The degree of browning was
determined by measuring the absorbance at 400 nm using a spectro-
photometer (Perkin-Elmer mod. Lambda 15). All analyses were
carried out in triplicate.
Determination of phenol concentration. One gram of lyophilized
powder of banana ( obtained as previously described) from each
treatment and storage period was extracted with 50 ml of deionized
water in an Omni-mixer, externally cooled and set at 4500 rpm for
3 min [17]. The homogenate was centrifuged at 12,000 g for 10 min
at 4C. The supernatant was vacuum ltered through a Whatman
no. 4 lter. The solid residue obtained by centrifugation was hom-
ogenized again with 25 ml of deionized water and centrifuged as
above. The second supernatant was also ltered and the two ltrates
were combined in a 100-ml volumetric ask. The combined ltrates
were made up to 100 ml with deionized water. Of this aqueous
extract, 1 ml was added to 7.5 ml deionized water, and 0.5 ml Folin
and Ciocalteau Phenol Reagent (Sigma) was added. The sample was
then mixed and, after 5 min, 1 ml of saturated sodium carbonate
(Merck) solution was added and then the mixture was shaken. The
optical density of the solution at 670 nm was measured after 1 h. The
amount of total phenols was calculated from a standard curve of
dopamine prepared at the same time.
Preparation of enzyme extract. A crude enzyme extract was pre-
pared by taking 10 g of raw or thawed (at 4C for 2 h) banana
samples and homogenizing then in an Omni-mixer at 16,000 g with
50 ml of phosphate buer (pH 7.0) containing 10 g l of insoluble
polyvinylpyrrolidone (PVPP). Homogenization was carried out
with external cooling for 3 min at 30-s intervals. The homogenate
was centrifuged at 16,000 g at 4C for 15 min. The supernatant was
ltered through a nylon cloth and the volume obtained was meas-
ured carefully.
Polyphenoloxidase (PPO) assay. The enzyme activity was deter-
mined by measuring the rate of increase in absorbance at 420 nm
(A
"`"
) and 25C using a Lambda 15 double beam spectro-
photometer (Perkin-Elmer). The reaction mixture contained 2.9 ml
of 0.07 M of catechol solution in 0.05 M phosphate buer (pH 7.0)
and 100 l of diluted [1: 1, v: v, 0.2 M phosphate buer (pH 7.0)] or
undiluted enzyme extract. The activity was calculated on the basis of
the slope of the linear portion of the curve of A
"`"
plotted against
time (up to 3 min). Enzyme activity was expressed as A
"`"
min mg protein. Residual PPO activity was expressed as a ra-
tio of values measured for the treated or/and frozen sample versus its
control.
Peroxidase (POD) assay. The enzyme activity was determined by
measuring the rate of increase in absorbance at 485 nm (A
"``
) and
25C of a mixture containing 2.7 ml of 0.05 M phosphate buer
(pH 7.0), 200 l of 10 g l p-phenylenediamine in distilled water (H>
donor), 100 l of 15 ml l of hydrogen peroxide (oxidant) and 25 l
of diluted or non-diluted enzyme extract (total reaction volume
"3.0 ml). The enzyme activity was calculated on the basis of the
slope of the linear portion of a plot of A
"``
against time (up to
3 min). Enzyme activity was expressed as A
"``
min mg pro-
tein. Residual POD activity was expressed as a ratio of measure-
ments from treated and/or frozen sample versus an untreated
sample.
Protein content. Protein concentration in all extracts was deter-
mined using a Bio-Rad kit for the Bradford reaction [20].
Objective colour measurement. Triplicate samples (one slice each)
were placed in a 5-cm-diameter covered plastic dish. The sample
dish was placed on the light port of a Hunter Lab Model M25-9
colorimeter. Standard colour plate no. c2-19952 , with reectance
values of "77.65, a"!1.51 and b"21.41, was used as
reference. The sample dish was covered to avoid stray light. The ,
a and b values were recorded, and the derived functions for hue
(h), saturation (C), and total colour dierence (E) were calculated:
h"arctan (b/a); C"(a`#b`)`; E"[(

!
`
)`#(a

!a
`
)`
#(b

!b
`
)`]`, where

, a

and b

are the colour parameters

of the control sample and
`
, a
`
and b
`
, the colour parameters of
each treated sample.
Statistical analysis. A factorial in a completely randomized design
with three replications was used. There are two treatments (micro-
wave and steam), and ve frozen storage periods ( just-frozen, 3, 6,
9 ad 12 months). The data were analysed using the INSTAT pro-
gramme. To determine whether any of the variables were correlated,
the CORR procedure of INSTAT was used to calculate Pearsons
correlation coecients (r) [21].
Resul t s and di scussi on
The eect of blanching treatments on the browning
index is shown in Table 1. The control sample showed
no changes in browning due to freezing and frozen
storage after 12 months. Steam blanching of whole
peeled bananas for 5 min resulted in a minimum
61
Table 1 Eect of blanching
treatments on the browning
index in frozen banana slices
during storage (!18C)
Blanching Storage time (months)
treatment
Raw Just frozen 3 6 9 12
Browning
index
Control 12.17 Aa 12.32 Aa 10.48 ABa 10.28 ABa 10.78 ABa 6.42 Ba
Microwave 7.85 Ab 11.60 Ab 9.56 Aa 7.71 Ab 11.85 Ab 9.24 Ab
Steam 12.03 Aa 11.84 Ab 8.25 Aa 8.08 Ab 10.32 Aa 8.52 Ab
(abc) Means within column not followed by the same letter dier ( P40.05)
(AB) Means within row not followed by the same letter dier ( P(0.05)
Raw is the unfrozen samples (treated or not)
Control is the unblanched frozen samples. Standard errors are 0.27 for control and raw samples and
0.49 for all others
All data are the average of three independent experiments
Table 2 Eect of blanching
treatments on phenol levels
frozen banana slices during
storage (!18C)
Blanching Storage time (months)
treatment
Raw Just frozen 3 6 9 12
Phenols (mg/100g sample)
Control 17.37 Aa 13.22 Ba 10.29 Cb 11.05 Db 11.10 Da 6.75 Ea
Microwave 5.06 Ac 4.94 Ac 7.30 Ba 9.61 BCa 9.50 BCb 8.27 Cb
Steam 10.01 Ab 10.52 Ab 12.72 Bc 14.47 Bc 11.33 Aa 15.84 Bc
Phenols were expressed as milligrams of dopamine per 100 g of wet tissue
(abc) Means within column not followed by the same letter dier ( P40.01)
(ABCD) Means within row not followed by the same letter dier (P40.01)
Raw, as per Table 1
Control samples as in Table 1. All data are the average of three independent experiments
browning in raw (unfrozen) and frozen products
throughout the whole storage period. However, micro-
wave treatment produced a signicant reduction
( P40.05) in the browning index in raw banana slices,
indicating that proper microwave blanching conditions
could improve product appearance. Freezing of micro-
wave-treated samples signicantly increased the
browning in just-frozen samples, but during frozen
storage this index decreased, and samples exhibited an
appearance that was similar to that of the steam-blanc-
hed for the samples same periods of frozen storage.
Blanching signicantly changed phenol levels
(Table 2). Microwave-treated and steam-treated sam-
ples showed a signicant ( P40.01) decrease in their
phenol level as a consequence of thermal treatment.
Control samples showed a continuous diminution
of phenol levels during frozen storage. In micro-
wavetreated samples, an insignicant increase of
phenol levels occurred after 3 months of frozen storage.
Steam-treated samples also showed this eect; the
phenol content of these samples was 15.84 mg/100f.w.
(twice the control value) after 12 months.
The freezing process also produced a 37% increase in
residual PPO activity (Table 3). Control frozen samples
exhibited the highest PPO residual activities during
almost the rst 9 months of storage. Only those frozen
samples stored for 12 months showed a lower PPO
activity with respect to the raw sample (unfrozen). This
fact justies the necessity of carrying out a prefeeezing
treatment, such as blanching, in order to diminish this
enzymatic activity. Steam blanching strongly aects the
PPO activity under the conditions used. Raw samples
treated with steam for 5 min showed a 96% reduction
of PPO activity and a continuous decrease during
frozen storage. However, microwave-blanched samples
showed a residual activity of PPO equal to 49%. This
last deactivation seemed to be reversible, because
frozen microwaved samples exhibited higher PPO re-
sidual activity compared with just-treated unfrozen
samples. In this sense, only microwave-treated frozen
samples stored for a 12-month period showed a re-
sidual PPO activity similar to that of the microwave-
treated unfrozen samples.
The POD activity in processed bananas was very
similar to that of PPO (Table 4). POD activity was
higher in just-frozen and frozen stored samples com-
pared with control (unblanched samples). Steam-blan-
ched samples showed very low or zero POD activity.
These samples exhibited great deactivation and no re-
generation of POD during frozen storage. However,
62
Table 3 Eect of blanching
treatments on residual
polyphenol oxidase (PPO)
activity in frozen banana slices
during storage (!18C). (A
"`"
Change in absorbance at
420 nm)
Blanching Storage time (months)
treatment
Raw Just frozen 3 6 9 12
Residual PPO activity (%)
Control 100 Aa 137 Ba 158 BCa 128 Ba 138 Ba 62 Ca
(39.1) (56.1) (64.3) (52.1) (56.5) (25.4)
Microwave 49 Ab 88 Bb 114 Cb 69 Bb 157 Da 52 CEa
Steam 4 Ac 2 Bc 1 BCc 0.2 Cc 0.2 Cb 0 Cb
Residual PPO activity is expressed as a percentage of the value for the raw (untreated and unfrozen)
sample
PPOactivity values are expressed in A
"`"
min mg protein. Numbers in parentheses indicate the
actual PPO acivities in control samples
(abc) Means within column not followed by the same letter dier ( P(0.01)
(ABC) Means within row not followed by the same letter dier ( P(0.05)
Raw and control samples, respectively, are the same as per Table 1. All data are the average of
three independent experiments
Table 4 Eect of blanching
treatments on residual
peroxidase (POD) activity in
frozen banana slices during
storage (!18C). (A
"``
Change in absorbance at
485 nm)
Blanching Storage time (months)
treatment
Raw Just frozen 3 6 9 12
Residual POD activity (%)
Control 100 Aa 156 Aa 200 Ba 146 Aa 121 Aa 97 Aa
(42.6) (69.8) (89.1) (65.2) (54.2) (43.2)
Microwave 53 Ab 110 Bb 104 Bb 105 Bb 67 Ab 52 Ca
Steam 2.4 Ac 4 Bc 0.5 Cc 0.2 Cc 0 Cb 0 Cc
Residual POD activity is expressed as a percentage of the value for the raw (untreated and unfrozen)
sample
POD activity values are expressed in A
"``
min mg protein. Numbers in parentheses indicate
the actual POD activities in control samples
(abc) Means within column not followed by the same letter dier ( P(0.05)
(ABC) Means within row not followed by the same letter dier ( P(0.05)
Raw and control samples, respectively, are the same as per Table 1. All data are the average of
three independent experiments
microwave treatment has a dierent eect on POD
activity in banana slices; it produced a 47% decrease in
POD activity. This eect was overcome during freezing
and storage. Just-frozen samples showed a slightly
higher POD activity than the control (raw) sample and
a 35% lower activity than the untreated just-frozen
sample. The residual activity of POD of microwave-
treated banana slices was close to that of the raw
sample (100%, 42.6 A
"``
min mg protein) after
6 months of frozen storage. Frozen microwavetreated
samples stored for 9 and 12 months showed 67% and
52% residual activity of POD, respectively.
Steam-blanched banana slices showed the greatest
total colour dierence compared to the control (un-
treated) samples (Table 5). Microwave-treated and
steam-blanched samples had signicantly dierent
( P40.05) objective colorimeter values. Unblanched
samples showed similar values for the rst 3 months,
but from then on the frozen samples exhibited a signi-
cantly lower total colour dierence than in the rst
stages of storage. Steam-blanched frozen samples main-
tained the total colour dierence for 6 months, however
these samples had a signicantly reduced value of this
parameter at the end of the storage period. Microwave-
treated frozen samples showed higher total colour dif-
ference values compared with steam-blanched samples,
but the values were lower than control measurements
for the rst 6 months of storage.
Relationships between browning, phenolic levels
and PPO and POD activities
To determine whether any of the variables were statist-
ically interrelated, Pearsons correlation coecients (r)
were calculated by treating the analytical data from
each triplicate as separate observations (Table 6). In
control, the only signicant ( P40.05) correlation
with browning was the phenol level (r"0.86), which
indicates that, at the substrate and enzyme levels
63
Table 5 Eect of blanching
treatments on total colour
dierence in frozen banana slices
during storage (!18C)
Blanching Storage time (months)
treatment
Raw Just frozen 3 6 9 12
Total colour dierence
Control 0 9.03 Aa 8.02 Aa 5.60 Ba 4.95 BCa 3.83 Ca
Microwave 2.37 Aa 4.24 BCb 6.60 Db 8.39 Cb 3.90 Bb 5.02 Cb
Steam 3.54 Ab 4.59 Bb 3.72 ABc 4.22 ABb 2.80 Cc 2.96 Cc
Total colour dierence is expressed as (E"[(

!
`
)`#(a

!a
`
)`#(b

!b
`
)`]` (Hunter
Lab values).

, a

and b

are the control sample values

(abc) Means within column not followed by the same letter dier ( P(0.05)
(ABC) Means within row not followed by the same letter dier (P(0.05)
Raw is the unfrozen samples (treated or untreated)
Control is as per Table 1. All data are the average of three independent experiments
Table 6 Pearsons correlation
coecients (r) between
darkening or non-enzymatic
browning index and phenols,
PPO and POD activities in
frozen banana slices during
storage (!18C)
Blanching Pearsons correlation coecient (r)
treatment
Darkening Non-enzymatic browning index
Phenols PPO POD Phenols PPO POD
Control 0.74 0.70 0.81 0.86* 0.61 0.28
Microwave 0.36 !0.40 !0.21 !0.03 0.73 0.69
Steam !0.30 0.73 0.72 !0.87* 0.79 0.80
Pearsons correlation coecients (r) were calculated by using the CORR procedure as described in
Materials and methods
Darkening is the corresponding calculated total colour dierence (E"[(

!
`
)`#
(a

!a
`
)`#(b

!b
`
)`]` (Hunter Lab values)
Non-enzymatic browning index analysed as described in Materials and methods
* Signicant correlation at P40.05
investigated, the substrate level was the major factor
contributing to darkening. However, when the slices
were steam blanched, the browning index was negative-
ly correlated with this parameter. No correlation was
found to occur between browning and phenol levels
in microwave-treated slices. Enzymatic activities, i.e.
those of PPO and POD, only exhibited a signicant
correlation ( P40.05) with browning (r"0.79 and
r"0.80, respectively) in steam-blanched samples,
showing that the PPO and POD deactivation obtained
by steam treatment signicantly inhibited the brown-
ing process.
Relationships between total colour dierence,
phenolic levels and PPO and POD activities
In control samples the most signicant ( P40.05) cor-
relation (r"0.81) was found to occur between total
colour dierence and POD activity. The correlation
between POD activity and the phenol level or PPO
activity decreased such that r"0.74 and r"0.70,
respectively. There was no correlation between any of
the parameters studied for the microwave-treated sam-
ples, indicating that, in addition to the enzymatic
browning, other non-enzymatic reactions could take
place in the product as a consequence of the blanching
and freezing process. Steam-blanched banana slices
only showed a low level of correlation (r"0.73 and
0.72) between enzymatic activities of PPO and POD
and the total colour dierence calculated by Hunter
Lab parameters.
Enzymatic browning in bananas has been studied
extensively [15, 14, 22, 23]. However, the relationships
between phenol levels PPO and POD activities and
browning and the total colour dierence in blanched
bananas prior to freezing have not been elucidated.
Also, there are no published reports available that
describe the use of microwave or steam blanching to
prevent enzymatic browning in order to produce com-
mercially acceptable frozen banana slices. Blanching
partially inactivates PPO and POD activities to certain
low levels, depending on the treatment, and it provides
a new approach to study the individual roles of phenol,
PPO and POD in enzymatic browning and the pro-
duction of colour dierences. This approach had been
employed by Ma et al. [24] in an investigation of this
relationship in frozen sweet potatoes.
64
Results show that, in the main, the phenol level is
critical in the browning process. In unblanched con-
trols, browning is more dependent on the original
amount of PPO and POD activities in the substrate,
because these enzymes are present in such high
amounts relative to substrate levels so that, at equilib-
rium, most of the substrate has been oxidized to brown
pigments [7].
In contrast, in steam-blanched samples, PPO and
POD activities decreased dramatically and the amount
of substrate diminished (31%). In this situation, enzy-
matic activities became the limiting factors together
with the phenol level. Also, we can observe that, in all
frozen samples, the phenol level increased as a conse-
quence of the cellular disruption produced by freezing.
However, in microwave-blanched samples the only lim-
iting factors were the residual enzymatic activities pres-
ent in the tissue. It can also be expected that when PPO
and POD are completely inactivated, no enzymatic
browning can occur. However, this fact could only be
true for microwave-treated samples, because steam-
blanched samples showed relatively high browning
values indicating a near-complete inactivation of these
oxidative enzymes.
The total colour dierence, as calculated using
Hunter Lab colour parameters, only partially reects
the enzymatic browning of banana samples. Other
mechanisms of darkening occurred in banana pro-
cessed products which also aect the sample colour.
Microwave-treated samples with partially inactivated
PPO and POD showed large total colour dierence
values. In these samples, there were no correlations
between phenol level, PPO or POD activities and
total colour dierence, indicating the presence of other
non-enzymatic mechanisms which aected the sample
colour.
In summary, prevention of enzymatic browning in
frozen banana products (without the addition of chem-
icals) by blanching is the result of the dramatic of PPO
and POD activities and, depending on the type of
blanching, a reduction in phenol levels. The browning
potential was correlated with phenol levels in control
samples, but with residual PPO and POD activities in
blanched and frozen samples. Microwave blanching
aects the colour of the banana samples, not only in
terms of enzymatic browning, since other non-enzy-
matic mechanisms contribute to the colour dierence
of these samples as compared with control.
Acknowledgement This research was supported by the Comisio n
Interministerial de ciencia y Tecnolog a through the project no.
ALI95-0105
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65