COMBINED EFFECTS IN PREVENTING ENZYMATIC

BROWNING REACTIONS IN MINIMALLY PROCESSED FRUIT

M.C. NICOLI’. M. ANESE and C. SEVERINF

Dipartimento di Scienze degli Alimenti,

University of Udine
via Marangoni 97, 33100 Udine, Italy

Accepted for Publication November 22, 1993

ABSTRACT

Fresh sliced apples were dipped in ethanol or cysteine solutions and/or

packaged in air or modified atmosphere in order to retard oxidative browning,
Modified atmosphere packaging was effective in inhibiting darkening for at least
nine days at refrigerated storage conditions. The combination ethanol dip/modi-
fied atmosphere packaging resulted as a suitable method in maintaining the
original color in apple slices even after exposure to air,

INTRODUCTION

In the last few years great interest has been shown in the production of
ready-to-use packs of fresh fruit and vegetables. The fruit or vegetables in
question are presented for sale conveniently peeled, cored or sliced in prepacked
packages (King and Bolin 1989; O’Beirne 1989; Stringer 1990; Marchetti et al.
1992). However, these minimally processed products are affected by several
enzymatic degrading reactions which lead to the loss of their typical fresh color
and appearance. Storage in refrigerated conditions can only reduce the rate of
these degrading reactions and thus suitable treatments are necessary to slow
tissue decay and to extend the product’s shelf-life. The prevention of enzymatic
browning cannot be achieved using traditional methods; in fact, blanching can
cause marked textural changes and the use of sulfites is limited because of their
toxicity (Iyengar and McEvily 1992). Among the great number of substances
that have recently been proposed as potential inhibitors of enzymatic browning,
L-cysteine and ethanol seem to be practical alternatives to sulfites. In fact,
cysteine proved as effective as sodium sulfite in preventing browning in various

‘To whom correspondence should be sent.

2Present address: Dipartimento di Protezione e Valorizzazione Agroalimentare, University of
Bologna, Via San Giacomo 7, 40126 Bologna, Italy.

Journal of Food Quality 17 (1994) 221-229. All Rights Reserved.

Copyright 1994 by Food & Nutrition Press, Inc., Trumbull, Connecticut. 22 1
222 M.C. NICOLI. M. ANESE and C. SEVERINI

processed fruit and vegetables (Montgomery 1983; Dudley and Hotchkiss 1989;
Molnar-Per1 and Friedman 1990a,b; Nicoli et al. 1991; Richard et al. 1991). As
regards ethanol, although it is generally considered a weak inhibitor of
polyphenoloxidase activity (Valero et al. 1990; Nicoli et al. 1991), it could be
proposed for minimally processed vegetables on account of its ability to reduce
water activity and to act as a "hurdle" for microbial growth (Lerici et al. 1983;
Ingram and Buttke 1984; Labuza and Breene 1989). Oxidative enzymatic
reactions can also be successfully minimized by the exclusion of oxygen from
the package, as observed by Barth et al. (1993) and Molnar-Per1 and Friedman
(1990b) in the case of color retention of broccoli, sliced apples and potatoes.
Here we present the results of research into the combined effects of ethanol
or L-cysteine treatments with modified atmosphere packaging to prevent
enzymatic browning in refrigerated fresh apple slices.

MATERIALS AND METHODS

Trials were carried out using apples (Golden Delicious) from a local
foodstore. We cored and sliced the apples 1 cm thick. Samples were subjected
to different procedures: (a) packaging in low permeability pouches filled with
air or nitrogen/carbon dioxide (80:20 v h ) ; (b) dipping for 10 min in solutions
of 2.5% (w/w) (corresponding to 0.54 M) ethanol and packaging in low
permeability pouches filled with air or nitrogenkarbon dioxide (80:20 v/v); (c)
dipping for 10 min in solutions of 0.1 % (w/v) (corresponding to 8.25 mM)
L-cysteine and packaging in low permeability pouches filled with air or
nitrogedcarbon dioxide (80:20 v/v).
In the case of ethanol and L-cysteine treatments, control samples were
dipped for 10 min in distilled water and packed in low permeability pouches
filled with air. The low permeability pouches consisted of polyethylene
terephtalate (PET) film supplied by AMB (Udine, Italy) having the following gas
permeability: 2.6 ml O? m-' day-' atm-I at 25C. The packaged fruits were then
stored at OC for 9 days. The enzymatic browning was evaluated by colorimetric
measurements on the samples taken at different storage times. In order to
simulate homehandling procedures, the enzymatic browning of six-day stored
samples was followed for at least three days after pouch removal. After the
packages were opened, samples were kept at 20C.
The changes in color were followed by colorimetric measurements using a
tristimulus colorimeter (Chromameter-2 Reflectance Minolta, Japan) equipped
with a CR-200 measuring head. The instrument was standardized against a white
tile before each measurement. Color was expressed in L*, a*, b* Hunter scale
parameters and a* and b* were used to compute hue angle (tan-' b*/a*) (Little
1975; Clydesdale 1978; Mastrocola and Lerici 1991). L* values were plotted
 PREVENTING BROWNING OF PROCESSED FRlJIT 223

-*, 110
1
\
#I 105
n
c
I
wl
L

A
100
-
m
0

2 95
a
3
I
so I I I I I I I I I 1

0 1 2 3 4 5 6 1 8 9

Time (days)

FIG. 1 CHANGES IN HUE ANGLE VALUES DURING

STORAGE AT OC OF APPLE SLICES SEALED IN A LOW
PERMEABILITY BAG WITH AIR OR N2/C02(80:20 v l v )

against log time and the slopes of linear portion of these curves were obtained
by linear regression (Sapers and Douglas 1987).
The final ethanol concentration in apple slices dipped for 10 min in the
2.5% (w/w) ethanol solution was determined using an enzymatic method
(Boehringer Mannheim GMBH ,Germany). Ethanol concentration was expressed
as g alcoho1/100 g of sample.
Data are the average of at least five repetitions. Measurements were carried
out at different points of the slice surface. The variability coefficients for color
measurements were found to be less than 10 % .

RESULTS AND DISCUSSION

The effects of modified atmosphere (MA) on the enzymatic browning

reaction of apple slices during storage at OC are shown in Fig. 1. It can be noted
that the N,/CO, atmosphere packaging was very effective in preventing
224 M.C. NICOLI. M. ANESE and C. SEVERINI

A
m
\
* 105 -
rn air

* 110 1 B

-I

=wl 100

90
0 1 2 3 4 5 6 7 8 9

Time (days)
FIG. 2. CHANGES IN HUE ANGLE VALUES OF APPLE SLICES DIPPED FOR 10 MIN IN
2.5% (w/w) ETHANOL SOLUTION (A) OR IN 0.1 % (w/v) CYSTEINE SOLUTION (B) AND
PACKAGED UNDER AIR OR N,/CO, (80:20 v/v)
Samples dipped for 10 min in distilled water and packed in air taken as control.
Storage temperahue: OC.

browning reaction for at least 9 days of storage; in the case of aerobic

packaging, however, hue angle values greatly decreased within the first few
hours of storage. Browning of apple slices, previously dipped either in 2.5%
ethanol or 0.1 % L-cysteine solutions and packaged in air or N,/CO, atmosphere,
was followed during cold storage as shown in Fig. 2. Samples dipped for 10
min in distilled water and packed in air were taken as control. Ethanol, the
concentration of which in the dipped apple slices was found to be 0.33 gh00 g,
was effective in retarding enzymatic browning even for aerobic packaged
samples (Fig. 2A), while L-cysteine pretreatments protected the air packaged
 PREVENTING BROWNING OF PROCESSED FRUIT 225

apple slices from darkening only during the first 24 h of storage (Fig. 2B). As
reported by Valero el af. (1990), the inhibiting action of ethanol on polyphenol-
oxidase activity could be attributed to the ability of this substance to bind the
enzyme or to compete with the phenolic substrates. Consequently the presence
of oxygen in the package would not easily induce color changes of the
ethanol-dipped apple slices. On the contrary, the poor inhibiting effect of L-
cysteine observed for samples packaged in aerobic conditions suggests that the
loss of cysteine antibrowning properties could be attributed to the oxidation of
this amino acid by atmospheric oxygen. A decrease of the inhibiting effect of
L-cysteine during storage time was previously observed by Montgomery (1983)
for a pear juice concentrate: the author correlated the increase in the browning
rate of the product with the consumption of the amino acid during storage.

TABLE 1.
SLOPE VALUES FOR THE LINEAR PORTION OF L* VS. LOG TIME CURVE

Dip composition Packaging Slope rz

none AIR -4.18a 0.99

none Nz /coz -0.42a 0.89

0 .l% L-cysteine AIR -1.18b 0.92

0.1% L-cysteine N z /co2 0.49h 0.79

2.5% EtOH AIR 0.03b 0.63

2.5% EtOH Nz /COz 0.07b 0.69

a: p<O.O1
b: p t 0 . 0 5

T h e initial p e r c e n t a g e s of Nz and COz in t h e p a c k a g e s were respectively 80 a n d 20 (v/v)

In Table 1 the slope values of the linear portion of L* values vs. log time
curve of the apple slices dipped and/or packaged in air or in modified
atmosphere are reported. It can be observed that the treatments are very
effective in preventing darkening, except the L-cysteine dipping and/or aerobic
packaging. In fact, as previously observed for the hue values, in the last case the
extent of browning became severe after only a few days of cold storage.
226 M.C. NICOLI, M. ANESE and C. SEVERINI

0 0.5 1 1.5 2

Time (hours)
FIG. 3. CHANGES IN HUE ANGLE VALUES, AT 20C, OF APPLE SLICES,
PREVIOUSLY PACKAGED IN N2/C0, (80:20 v/v) AND STORED AT OC FOR 6 DAYS,
AFTER POUCH OPENING
Data compared with those of a fresh-prepared sample exposed to air.

-*, 110
1
*
*
\

n
100
EtOH -Nz/COz
EtOH-air
.-
I
wl
s 90
-Q
0,

4' 80
0
3
I
70 I I I I

0 1 2 3

Time (days)
FIG. 4.CHANGES IN HUE ANGLE VALUES, AT 20C, OF APPLE SLICES,
PREVIOUSLY DIPPED IN SOLUTIONS OF 0.1% (w/v) CYSTEINE OR 2.5%
(w/w) ETHANOL, RESPECTIVELY, PACKAGED IN AIR OR N,/CO, (80:20 v/v)
AND STORED AT OC FOR 6 DAYS, AFTER POUCH OPENING
Data compared with those of a freshly prepared sample exposed to air.
 PREVENTING BROWNING OF PROCESSED FRUIT 227

A rapid development of browning in sliced apples, previously packed in MA

and stored at 0C for 6 days, was observed when the packages were opened, as
shown in Fig. 3. It can also be noted that the color changes of samples held in
MA packages were faster than those of a fresh-prepared apple slice taken as
control. Although enzymatic browning can be stopped by the exclusion of
oxygen from pouch head-space, it should be pointed out that other alterative
reactions, such as pectin degrading and plant cell breakdown, can only be
slightly influenced by MA packaging (Bolin and Huxoll 1989); thus the
enhanced browning observed when the packages were opened might be due to
the increased contact of oxidative enzymes and substrates as a consequence of
textural damages or to a change in the redox system of the tissue. The
effectiveness of the pretreatments in retarding darkening of apple slices after the
opening of the PET pouches is shown in Fig. 4.In all cases browning was lower
than that observed for the fresh-prepared apple slices; however, the ethanol
dip/MA packaging combination produced only slightly increased browning
compared with that observed in the freshly sliced samples.

CONCLUSIONS

Results reported in the present study show that enzymatic browning can be
successfully inhibited for long storage times by application of modified
atmosphere packaging. Nevertheless, an enhanced browning rate was observed
as the packages were opened: the combination of ethanol dip with MA
packaging was effective in slowing down darkening even after the pouches were
opened.

ACKNOWLEDGMENTS

This work was supported by a research grant of the National Research

Council of Italy, No. 93.02431 .CT06.

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