Journal of Food Engineering 79 (2007) 1196–1206

www.elsevier.com/locate/jfoodeng

Improvement in texture using calcium lactate and heat-shock

treatments for stored ready-to-eat carrots
D. Rico a, A.B. Martı́n-Diana a,*, J.M. Frı́as a, J.M. Barat b,
G.T.M. Henehan a, C. Barry-Ryan a
a
School of Food Science and Environmental Health, Postharvest Research Unit, Dublin Institute of Technology (DIT),
Cathal Brugha, Dublin 1, Ireland
b
Institute of Food Engineering for Development, Department of Food Technology, Universidad Politécnica,
Camino de Vera s/n, 46022 Valencia, Spain

Received 25 November 2005; accepted 11 April 2006

Available online 5 May 2006

Abstract

The use of calcium lactate solutions has been shown to be a healthy alternative to chlorine washing in order to maintain the shelf-life
of fresh-cut products. The aim of this research was to analyse the effects of calcium lactate (15 g L1) treatment at 25 C and 50 C (heat-
shock) on the textural properties of sliced carrots and to compare those with the chlorine treatment (120 mg L1) widely used in industry.
Several direct and indirect markers of textural changes in carrots during storage were used: Instron textural analysis, Cryo-SEM and
optical microscopic, sensory analysis, pectin methylesterase (PME) activity, calcium content and water activity. Samples treated with
calcium lactate maintained texture significantly (p < 0.05) better than samples treated with chlorine throughout storage. Calcium lactate
treatment produced a reduction in the water activity in sliced carrots and a higher firmness (Instron analysis) than chlorine treatment. In
addition, combined use of heat-shock and calcium lactate treatment increased PME activity significantly when compared to the other
treatments, results that were confirmed by sensory analysis. Cryo-SEM analyses showed that combined heat-shock and calcium lactate
treatment was more effective in maintaining the turgor of cortex tissue cells and reduced the extent of lignification at cutting-edge areas.
The use of calcium lactate combined with heat-shock is a promising washing method for fresh-cut carrots in order to preserve their tex-
ture and improve their nutritional value, avoiding the use of chlorine washing.
 2006 Elsevier Ltd. All rights reserved.

Keywords: Sliced carrot; Calcium lactate; Chlorine; Pectin methylesterase; Cryo-SEM; Texture; Water activity

1. Introduction Bord Glas http://www.bordglas.ie/facts/production.htm).

Recent growth in the ready-to-use vegetable industry
has been largely driven by increasing demand for conve-
nient, fresh and healthy foods. Minimally processed
(RTU) products typically involve peeling, slicing, dicing
or shredding prior to packaging and storage (Beuchat,
1999). In Ireland, current fruit and vegetable production
amounts to approximately €300 million per annum (An
*
Corresponding author. Tel.: +353 1402 4458; fax: +353 1402 4495.
E-mail address: anabelen.martindiana@dit.ie (A.B. Martı́n-Diana).
Carrot is one of the most important vegetables cultivated
in Ireland. It is an excellent raw snack due to its nutritional
value (high carotene content) and crispy texture (Lin,
Durance, & Scaman, 1998).
Increasing the quality retention and shelf-life of these
products is very important to cope with the demands from
both industry and consumers for long shelf-life products
that keep their freshness (Ohlsson, 1994). The marketing
of fresh-cut vegetables is limited by their short shelf life
and their decline in post-processing quality. This is due to
undesirable biochemical reactions associated with wounding
when compared to non-treated vegetables (Brecht, 1995).

0260-8774/$ - see front matter  2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jfoodeng.2006.04.032
 D. Rico et al. / Journal of Food Engineering 79 (2007) 1196–1206
Chlorine solutions (50–200 ppm) are widely used to
wash fruits and vegetables as well as fresh-cut produce in
order to reduce microbial growth (Beuchat, 1992, 1999).
The possible formation of carcinogenic chlorinated com-
pounds in water (chloramines and trihalomethanes) has
called into question the use of chlorine for this purpose.
Future regulatory restrictions on the use of chlorine for
washing of ‘‘ready-to-eat’’ vegetables are likely and will
require the development of functional alternatives (Ahve-
nainen, 1996; Cherry, 1999; Fan, Toivonen, Rajkowski,
& Sokorai, 2003; Gould, 1995; Graham, 1997; Seymour,
1999; Wiley, 1994).
There have been many attempts to find alternative
decontamination treatments to chlorine. These include
the use of organic acids, organic salts, irradiation, ozone
and whey permeate among others (Ahvenainen, 1996;
Martı́n-Diana et al., 2005a, 2005b; Martı́n-Diana et al.,
in press-a,b; Tapia et al., 2003; Wiley, 1994; Xu, 1999).
Most of this research has focused on microbiological
safety and few have studied the effects on texture and
the possible improvement of textural properties. However,
some studies of textural properties have been carried out
including: studies on vacuum impregnation (Grass, Vidal,
Betoret, Chiralt, & Fito, 2003), preheating (Vu et al.,
2004), electric pulses (Giner et al., 2000) and irradiation
(Han, Gomes-Feitosa, Castell-Perez, Moreira, & Silva,
2004).
In recent years the use of physiologically active com-
pounds (PAC) has attracted interest to consumers and
industry (minerals, probiotics, etc.) (Alzamora et al.,
2005). Public recommendations have supported the idea
that health can be enhanced if calcium intake is increased
and balanced with an adequate intake of phosphorus
(USA Institute of Medicine, 1998). Moreover, the calcium
content of the diet is critical in most stages of life (Grass
et al., 2003). To give consumers the opportunity to increase
their calcium intake without resorting to supplementation,
the industry has been encouraged to fortify ready-to-eat
products (Cerklewski, 2005). In recent years the use of
physiologically active compounds (PAC) has been of huge
interest to consumers and industry (minerals, probiotics,
etc.) (Alzamora et al., 2005).
Calcium is reported to maintain vegetable firmness by
cross-linking with both cell wall and middle lamella pectin
(Grant, Morris, Rees, Smith, & Thom, 1973). Thus, fruit
and vegetables treated with calcium are generally firmer
than controls during storage (Camire, Ismail, Work, Bush-
way, & Halteman, 1994; Luna-Guzman, Cantwell, & Bar-
rett, 1999; Lester & Grusak, 1999; Suutarinen, Anakainen,
& Autio, 1999). Calcium lactate 5–30 g/L has been used as
a firming agent for fruits such as cantaloupes, strawberry
and carrots among others (Main, Morris, & Wehunt,
1986; Martı́n-Diana et al., 2005a; Morris, Sistrunk, Sims,
Main, & Wehunt, 1985). It has been reported that calcium
lactate is a good alternative to calcium chloride because it
avoids the bitterness or off-flavours associated with the
chloride salt (Luna-Guzman & Barrett, 2000).
1197
Heat-shock treatments, alone or combined with cal-
cium, have also been used to prevent browning reactions
and maintain texture in various vegetables and fruits
(Hisaminato, Murata, & Homma, 2001; Loaiza-Velarde,
Tomas-Barberan, & Salveit, 1997). Heat treatment resulted
in tissue firming in potatoes (Bartolome & Hoff, 1972) and
tomatoes (Floros, Ekanayake, Abide, & Nelson, 1992).
Firming effects obtained from heat treatments alone or
combined with calcium treatments have been attributed
to the action of heat-activated pectin methyl esterase
(PME) and/or to increased calcium diffusion into tissues
at higher temperatures (Bartolome & Hoff, 1972; Garcia,
Herrera, & Morilla, 1996). Previous work in this labora-
tory showed that calcium lactate combined with heat-shock
maintained texture during storage of fresh-cut lettuce
(Martı́n-Diana et al., 2005b, Martı́n-Diana et al., 2005).
The objective of this study was to analyse the effect of
heat-shock and calcium lactate on the texture of a root
vegetable (carrot) since this treatment showed good results
with a leafy vegetable. The evaluation of texture was car-
ried out by means of direct (Cryo-SEM, optical micros-
copy, texturometry and sensory analysis) and indirect
measurements (water activity, calcium content and PME)
during storage for 10 days.
2. Materials and methods
2.1. Sample preparation
Carrots (Daucus carota sp.) grown in Spain, were pur-
chased in a local supermarket and stored at 4 C before
processing. Three washing treatments were conducted in
parallel, prepared from the same batch of product. Carrots
were rinsed briefly (1 min with water at 20–25 C) in order
to remove soil contamination during peeling. They were
hand peeled, in one direction using a manual peeler, remov-
ing a minimal amount of surface tissue. The carrots were
then sliced manually with a sharp knife into discs approx-
imately 5 mm in thickness. The temperature in the pilot
processing plant was 25 C.
Washing treatments of carrots were performed by
immersion of the sliced carrots in each treatment solution.
The samples were sanitised with chlorinated water
(120 ppm) and calcium lactate (15 g L1) at room (25 C)
and 50 C temperature. Chlorinated water was prepared
by adding sodium hypochlorite solution (120 g L1 avail-
able chlorine) to distilled water to obtain 120 mg L1 free
chlorine (pH  8). Calcium lactate (Sigma, USA) was
diluted to 15 g L1 (pH = 6.5). For all treatments the solu-
tions were prepared using distilled water stored at 25 C.
Each treatment was carried out in different baskets
(200 g carrot product L1) and immersed in the corre-
sponding washing solution for 1 min with agitation and
subsequently dried for 5 min using an automatic salad
spinner.
Processed carrots were pooled, mixed and subsequently
packaged in bags (200 · 320 mm) of 35 lm oriented
1198 D. Rico et al. / Journal of Food Engineering 79 (2007) 1196–1206
polypropylene (OPP) (Amcor Flexibles Europe-Brighouse,
United Kingdom). Each package contained 200 g of
product. The packages were chilled in a blast freezer at
0 C for 2 min before heat-sealing under atmospheric con-
ditions and stored at 4 C for 10 days. Four independent
batches were carried out.
2.2. Direct textural measurements
2.2.1. Cryo scanning electron microscopy (Cryo-SEM)
Cryo-SEM was used to observe the effects of wounding
stress. Samples were cut in rectangular pieces 4 · 5 mm.
Two different orientations were used: (i) orientation 1,
showing the peeler-wounded area and (ii) orientation 2,
which shows the knife-cut area. Orientations 1 and 2 both
show cortex areas, but in the first case, the external part of
the cortex is shown, and in the second, inner parts of the
cortex, closer to the xylem and cambium, are shown
(Fig. 1). 200·, 500· and 1000· magnifications were used.
The samples were frozen by immersion in Slush Nitro-
gen (210 C). After that, the samples were fractured,
etched (at 94.5 C, 105 Torr vacuum, for 15 min), gold
coated and viewed in the cold-stage scanning electron
microscope (JEOL JSM-5410). Using this technique, the
fractured surface of the frozen sample was viewed directly
Fig. 1. Cryo-SEM analysis. Orientation (1) is referred to as the peeler-wounded area and orientation (2) is referred to as the knife-wounded area.
while being maintained at 150 C or lower (Bomben &
King, 1982). Micrographs at day 1 (after treatment) and
after 10 days of storage at 4 C were analysed. Three differ-
ent magnifications were analysed (·200, ·500 and ·1000).
Two samples per treatment were used and 10 micro-
graphs per sample were obtained.
2.2.2. Optical microscopy
An optical microscope (Olympus, Model BH2, Minne-
sota, USA) was used to analyse the structure of sliced car-
rots at day 1 and after 10 days of storage in fresh samples.
The microscope image was captured with an attached dig-
ital camera (Canon Power Shot, CCD 3.1 MPixels, Japan).
Three different magnifications were analysed (100·, 200·
and 400·).
2.2.3. Textutomer analysis
Texture properties of carrots were assessed using a tex-
ture analyser Aname (TA.XT.PLUS, Surrey, UK). A
500 N load cell was attached. A puncture test was per-
formed using a 0.5 cm diameter aluminium probe with cir-
cular-shape section. The speed setting for the experiment
was 120 (mm/min) and maximum load was reported (N).
Each test was performed on a single slice of carrot (at least
25 slices per treatment were analysed).
 D. Rico et al. / Journal of Food Engineering 79 (2007) 1196–1206
2.2.4. Sensory analysis
The sensory analysis of the sliced carrots was performed
for over 10 days of storage by a panel with an age range of
25–35 years. Texture (1) and the overall visual quality (2)
(OVQ) of a blind sample were scored on a hedonic scale
of 1–5. The texture was evaluated by the fracture of differ-
ent pieces of carrots with the fingers. The 15 member sen-
sory panel was selected from among the members of the
department and the evaluation was carried out in the sen-
sory evaluation laboratory. The hedonic scales were as
follows:
(1) Texture: 0 = Soft, 3 = limit of saleability, 5 = fresh-
crispy.
(2) OVQ: 0 = not fresh appearance–very poor appear-
ance, 3 = limit of saleability, 5 = excellent fresh
appearance.
Data analysis was carried out with Compusense Five
software (Release 4.4, Ontario, Canada).
2.3. Indirect textural measurements
2.3.1. Pectin methylesterase activity (PME) E.C.3.1.1.11
PME activity was measured using the method described
by Kimball (1991). Ten grams of tissue was diluted in an
extraction solution (0.2 M sodium phosphate buffer, pH
7.5 containing 1 M sodium chloride and 10 mM dithiothre-
itol) and homogenised at 4 C for 2 min at 5500 rpm. The
macerate was incubated at 4 C for 30 min with agitation
and centrifuged at 12,500g for 30 min at 4 C. One millili-
ters of this extract was mixed with 40 mL of substrate solu-
tion (0.1% pectin). The solution was adjusted to pH 7.0
with 1.0 M NaOH, and the pH of the solution was re-
adjusted to pH 7.5 with 0.05 M NaOH. After the pH
reached 7.5; 0.2 mL of 0.05 N NaOH was added. The time
required to return to pH 7.5 was recorded. Activity was
quantified as carboxyl groups formed by the hydrolysis
of methyl esters of pectin and was measured tritrimetrically
using a pH electrode to monitor the production of H+. The
enzymatic activity can be described by Eq. (1)
PME activity ¼ ð0:05 N NaOHÞ  ðX mL extractedÞ
 ð0:2 mL NaOHÞ
 106 =ð1 mL sampleÞ  ð10 g sampleÞ
 ðTime in minÞ ð1Þ
Three macerates per treatment and day were prepared.
Triplicates of the enzymatic activity were analysed.
2.3.2. Water activity
The water activity of the treated samples was measured
with a fast water activity meter (GBX scientific FA-st/1,
Cédex, France). A slice of carrot in a small plastic cup
and was placed onto the base of the air tight test chamber.
The measuring head enclosed the sample and formed an
1199
airtight seal with the base. At least 15 samples were mea-
sured per treatment.
2.3.3. Calcium analysis
A Metrohm advanced ion chromatography system
equipped (Herisau, Switzerland) with Model 761 IC Pumps,
Metrohm Spark Triathlon autosampler and 817 Bioscan
Pulsed amperometric detector was used for the analysis.
Data acquisition and processing were performed with Metr-
ohm IC-Net 2.3 software. Bioscan temperature was con-
trolled at 25 C. The column employed for analysis was a
Metrosep C2 150 analytical column (250 mm · 4 mm).
The injection volume was 20 lL. The mobile phase was
aqueous Tartaric acid (4.0 mM L1) and dipicolinic acid
(0.75 mM L1). The flow rate used was 1 mL/min. Calcium
content was expressed in ppm.
2.4. Statistical analysis
Differences among the treatment and storage samples
were tested by multi analysis of variance (ANOVA). Differ-
ences are reported as significant to 95% LSD interval. Stat-
graphics software (version 2.1; Statistical Graphics Co.,
Rockville, USA) was used to analyse of the data. Three
independent trials or batches were carried out.
3. Results and discussion
Shelf-life can be defined as the length of time the vegeta-
ble can maintain the appearance, safety and nutritional
value that appeals to the consumer (Delaquis, Stewart,
Toivonen, & Moyls, 1999). Previous sensory studies
showed sliced carrots treated with calcium lactate were
acceptable to consumers up to 10 days of storage (Mar-
tı́n-Diana et al., 2005a). Textural quality of the samples
at day 1 and 10 was analysed in order to compare the three
treatments (chlorine, calcium lactate at 25 C and calcium
lactate at 50 C). Data at day 1 were studied to evaluate
the short term effect of the treatments on the product. Dur-
ing storage, the carrots, as any standard processed product,
suffered changes in quality, including texture. Measure-
ments at day 10 were used to evaluate the influence of
the treatments on these changes.
3.1. Direct measurements
3.1.1. Cryo scanning electron microscopy (Cryo-SEM)
Microscopic observations of the parenchyma tissue were
carried out to obtain an understanding of the different
effects caused by the treatments in the texture properties.
Cryo-SEM micrographs were analysed after treatment on
day 1 (Fig. 2) and at day 10, the end of the storage period
(Fig. 3) for sliced carrots treated with chlorine (120 ppm),
with calcium lactate (15 g L1) at 25 C and with calcium
lactate (15 g L1) at 50 C.
At day 1 the parenchyma tissue showed a compact struc-
ture consisting of cells, intercellular spaces or ‘‘pores’’ and
1200 D. Rico et al. / Journal of Food Engineering 79 (2007) 1196–1206
Fig. 2. Cryo-SEM micrographs of sliced carrot tissues in the peeler-wounded area (orientation 1) day 1 in samples treated with chlorine (120 mg L1),
calcium lactate (15 g L1) at 25 C and calcium lactate at 50 C.
Fig. 3. Cryo-SEM micrographs of sliced carrot tissues in the peeler-wounded area (orientation 1) at day 10 in samples treated with chlorine (120 mg L1),
calcium lactate (15 g L1) at 25 C and calcium lactate at 50 C.
capillaries. The micrographs showed the cells had a highly
compact structure. The intercellular spaces referred as
‘‘pores’’ play an important role in the penetration of micro-
organism and their size increases during the senescence
state (Lapsley, Escher, & Hoehn, 1992). At day 1 no
‘‘pores’’ were observed in any of the samples (Fig. 2) per-
haps due to their small size during early storage. In all sam-
ples, internal cellular features were observed as a dispersed
network in agreement with other author results (Roy, Wat-
ada, Conway, Erbe, & Wergin, 1994). This network corre-
sponds to membrane and cell wall fragments which are
dispersed after the sublimation process used for sample
preparation.
During the preparation of the samples for Cryo-SEM
microscopy, a fracture in the tissue was produced. Differ-
ences in the way the tissues broke were observed, with a
higher amount of cells detached instead of broken into
halves in the case of chlorine treated samples (Fig. 2). This
type of rupture, due to cell-to-cell debonding (fracture of
middle lamella) (Anino, Salvatori, & Alzamora, in press),
can be favoured by the presence of weaker intercellular
bonds and has been associated with dry textures or chill-
ing-injured stone fruits (Harker, Stec, Hallett, & Bennett,
1997). Cell rupture driven tissue failure is associated with
crispiness (Harker et al., 1997). This phenomena mostly
occurred in calcium lactate treated samples, with a low
number of detached cells, perhaps due to stronger middle
lamella, reinforced by the interactions of calcium with
pectic acid.
In fresh produce, cell adhesion is presumed to be a func-
tion of three factors: strength of the middle lamella, the
area of cell to cell contact and the extent of plasmodesma-
tal connections (Harker et al., 1997).
Other difference observed at day 1 was that in the
wounded areas (Fig. 2) samples treated with chlorine the
shape of the cells was more elongated, while cells in calcium
treated samples were more rounded. This loss in the
rounded shape could result from a loss in turgor due to a
higher water loss caused by the stress produced by the min-
imal processing treatment (Roy et al., 1994). Sliced carrots
treated with calcium lactate showed compact and rounded
shapes regardless of the temperature of the treatment.
After 10 days of storage (Fig. 3) the differences observed
in the wounded areas at the beginning of the storage are
more evident. Samples treated with chlorine and also with
calcium lactate at 25 C showed a reduction in cell size and
a flat appearance compared to those treated with calcium
lactate at 50 C. Moreover, in the cut areas, an incipient
plasmolysis and/or shrinkage of the cytoplasm (gaps
between cell membrane and cell wall) was observed in sam-
ples treated with chlorine and calcium lactate at 25 C.
Rojas, Gerschenson, and Marangoni (2001) have suggested
 D. Rico et al. / Journal of Food Engineering 79 (2007) 1196–1206 1201

that since the plasmalemma has little mechanical resistance
it is the osmotic pressure exerted on the cell wall that
accounts for the turgor pressure-induced elasticity of cells
and tissues.
Fig. 4 shows lignin production in the wounded area in
samples treated with chlorine, and to a lesser degree in
samples treated with calcium lactate. Lignification is a met-
abolically costly process that requires large quantities of
carbon skeletons and reducing equivalents. Plants do not
possess a mechanism to degrade lignin, so any carbon
invested in lignin biosynthesis is not recoverable. These
results could be interpreted as a higher stress in samples
treated with chlorine which would be reflected in a higher
lignification and consequent textural changes.
At day 10 a beneficial effect of the use of heat-shock
(50 C) combined with calcium lactate, compared to the
use of calcium at 25 C, was observed. Sliced carrots trea-
ted with heat-shock showed higher cellular turgor and a
lower degree of shrinkage. An hypothesis to explain this
phenomena is that a higher temperature wash increased
the solubility of calcium lactate and/or enhanced the diffu-
sion of calcium through the parenchymal tissue (Bartolome
& Hoff, 1972; Garcia et al., 1996). Moreover, the use of
temperature at 50 C might have expanded the air within
the carrot tissues, creating a permanent deformation of
the cells, and the subsequent cooling and contraction of
this air might have impregnated the surrounding water into
the carrot therefore maintaining the moisture and turgor of
the cells better. While the effect of the treatments were evi-
dent in the wounded areas, that was not the case in the
internal tissue; probably due to the thickness of the tissue.
3.1.2. Optical microscopy
Optical microscopy was used to analyse the effect on the
texture of chlorine, calcium and calcium combined with
heat-shock.
At day 1, the tissue in sliced carrots did not show signif-
icant differences between treatments (p < 0.05) (Fig. 5I). In

Fig. 4. Lignification observations in Cryo-SEM micrographs of sliced carrot tissues in the peeler-wounded area (orientation 1) at day 10 in samples treated
with chlorine (120 mg L1), calcium lactate (15 g L1) at 25 C and calcium lactate at 50 C.

Fig. 5. Optical micrographs of sliced carrots after the treatment (I, day 1) and at the end of storage (II, day 10) for sliced carrots treated with chlorine
(120 mg L1), calcium lactate (15 g L1) at 25 C and calcium lactate at 50 C.
1202 D. Rico et al. / Journal of Food Engineering 79 (2007) 1196–1206
all the cases the cells appeared well defined. However after
10 days of storage, samples treated with calcium lactate at
50 C had better cell wall definition than samples treated
with chlorine or calcium lactate at room temperature
(Fig. 5II). This better cell definition may be due to: better
diffusion of calcium lactate into the tissue at 50 C; the
effect of warm water in the cells ability to retain water
and/or the activation of PME.
3.1.3. Texturometry
Significant differences (p < 0.05) in the force–distance
curves using puncture test were not observed between treat-
ments (data not shown). All the treatments showed an
increase in load before rupture followed by a rapid reduc-
tion in force after rupture.
The resistance of the sliced carrots to the puncture probe
was measured after treatment (day 1) and at the end of
storage (day 10) (Fig. 6). Using mechanical fracture, signif-
icant differences (p < 0.05) between treatments were
observed. Higher maximum load was needed to break
sliced carrots treated with calcium lactate than for samples
treated with chlorine (day 1 and 10) (Fig. 6). While the use
of calcium had a significant effect on the maximum load
compared to chlorine, the temperature of the calcium
washing solution did not affect the maximum load. The
mechanism of fracture using a puncture probe can be
related to cell rupture or cell-to-cell debonding (rupture
of the middle lamella) (Anino et al., in press). Samples trea-
ted with calcium need a higher force to be fractured and
perhaps the cell turgor was higher in these samples or the
middle lamella stronger.
3.1.4. Sensory evaluation
Sensory analyses (5 point rating scale) were carried out
immediately after the treatment and at the end of storage
(days 1 and 10) (Fig. 7). Although the study is focused
on texture, overall visual quality (OVQ) was also
evaluated.
Fig. 6. Texturometer analysis (maximum load, N) after treatment (day 1) and at the end of the storage (day 10) in sliced carrots treated with chlorine
(120 mg L1), calcium lactate (15 g L1) at 25 C and calcium lactate at 50 C.
The sensory panel observed significant (p < 0.05) differ-
ences in texture between samples during storage (Fig. 7I).
The scores on the day after the treatment were lower than
at the end of the storage, as expected. The panel could not
find differences between treatments at day 1. However, at
the end of storage, carrots washed with calcium lactate at
50 C showed higher scores for texture than the others
treatments (calcium lactate at 25 C or chlorine). At day
10, none of the samples scored above the limit of saleability
(3), though samples treated with calcium at 50 C gave sig-
nificantly higher scores for texture (p < 0.05).
The OVQ significantly decreased during storage (p <
0.05) regardless the treatment (Fig. 7II). Visual appearance
was better at the beginning of storage than at the end of
storage. Heat-shock combined with calcium lactate scored
better for appearance than the use of calcium at 25 C or
chlorine.
At the end of the shelf-life (day 10), only the samples
treated with calcium lactate at 50 C scored significantly
better than the other two samples for saleability. These dif-
ferences might be associated with the control of browning.
Heat-shock may have caused an inhibition of browning-
related enzymes that is reflected in higher visual appear-
ance scores. These findings were in agreement with others
studies where the use of heat-shock was successful in
extending the quality of fresh-cut lettuce (Baur, Klaiber,
Hammes, & Carle, 2004; Martı́n-Diana et al., 2005b;
Martı́n-Diana et al., 2005; Zhang, Zhaoxin, Zhifang, &
Xiang, 2005).
3.2. Indirect measurements
3.2.1. Pectin methylesterase activity (PME) E.C.3.1.1.11
PME activity (Mol COO · min1 · g1) in sliced car-
rots was studied at day 1 and day 10 for the different trea-
ted samples in cortex and vascular tissues (Figs. 8I and II).
Significant differences (p < 0.05) were observed between tis-
sues. The extracts from the vascular tissue had significantly
 D. Rico et al. / Journal of Food Engineering 79 (2007) 1196–1206 1203

Fig. 7. Sensory analyses after the treatment (day 1) and at the end of the storage (day 10) in sliced carrots treated with chlorine (120 mg L1), calcium
lactate (15 g L1) at 25 C and calcium lactate at 50 C. Sensory texture (I) and OVQ (II) were scored on a hedonic scale. The dashed line indicates
acceptable texture for saleability.

Fig. 8. Pectin methylesterase (PME) was measured after treatment (day 1) and at the end of storage (day 10) in different regions of sliced carrots treated
with chlorine (120 mg L1), calcium lactate (15 g L1) at 25 C and calcium lactate at 50 C. Tissue samples were analysed from the cortex (I) and vascular
(II) regions.

lower (p < 0.05) PME activity values than extracts from the
cortex tissue, regardless of the treatment and storage times
(Figs. 8I and II). These results are in agreement with Niel-
sen and Christensen (2002), who found extracts from peel
showed 2- to 5-fold higher levels of PME activity when
compared to extracts from fruit flesh (for orange, lemon,
lime, grapefruit and clementine fruits).
Fig. 8 shows the changes in PME activity during storage
(day 1–10). Significant differences (p < 0.05) were observed
again between carrots treated with calcium lactate at 25 C
and calcium lactate at 50 C. Samples washed with chlorine
did not change significantly from day 1 to day 10. No dif-
ferences during storage between tissues (cortex and vascu-
lar) PME profiles were observed. Thus, it seems that the
synthesis of PME and its activity respond to the same influ-
ences, regardless of the location of the enzyme. Differences
between treatments were observed, samples washed with
chlorine had significantly lower PME activity values
(p > 0.05) than carrots washed with calcium lactate.
The results showed an activation of PME after treat-
ment with calcium lactate. The firming effect observed
(Fig. 6) might be due to a PME-related increase in the
cross-linking between carboxyl groups in pectin molecules
and their interaction with endogenous calcium (Ni, Lin,
1204 D. Rico et al. / Journal of Food Engineering 79 (2007) 1196–1206
Fig. 9. Water activity (Aw) after treatment (day 1) and at the end of storage (day 10) in sliced carrots treated with chlorine (120 mg L1), calcium lactate
(15 g L1) at 25 C and calcium lactate at 50 C.
& Barret, 2005). This enhanced PME activity was also
increased when washing at higher temperature: 50 C
washed sliced carrots showed higher PME values than
those washed at 25 C, which is consistent with other the
findings of authors (Lee & Kader, 2000), who found
50 C to be the optimum temperature to stimulate PME
activity. However, other authors (Tijskens, Waldron, Ng,
Ingham, & Dijk, 1997) suggested that temperatures above
70 C were needed to stimulate the PME activity while
Ni et al. (2005) found that 60–70 C was required to acti-
vate this activity.
It is clear from this data that heat-shock is the most
important factor in raising PME activity. The beneficial
effect on texture of mild temperature treatments and cal-
cium solutions has usually been explained in terms of the
activation of pectin methyl esterase (PME) (Bartolome &
Hoff, 1972). PME is responsible for cleaving the methoxyl
groups from methylated pectic substances, generating free
pectic acids (Belitz & Grosh, 1986), which contain newly
available carboxyl groups. Endogenous and added calcium
can consequently make plant tissues firmer by binding to
the carboxylate moiety (Stanley, Bourne, Stone, & Wismer,
1995).
These results were supported by the sensory analysis
data (Fig. 6), since panelists were only able to observe sig-
nificant differences between samples treated with calcium at
50 C and samples treated at 25 C (chlorine or calcium
lactate).
3.2.2. Water activity
Water activity levels were measured in samples treated
with calcium at 25 C and 50 C and samples treated with
chlorine. Significantly lower water activity values
(p < 0.05) were found in samples treated with calcium lac-
tate at day 1 and day 10, regardless of the treatment tem-
perature (Fig. 9). These findings are in agreement with
Shelef (1994) who suggested that the use of calcium lactate
reduced water activity.
Water activity affects the textural properties of fresh-cut
vegetables during storage, e.g. the crispness and overall
hedonic texture (Katz & Labuza, 1981).
Enzyme and protein stability may be influenced by
water activity. Most enzymes and proteins must maintain
conformation to remain active. Therefore, maintaining
critical water activity levels to prevent conformational
changes is important to food quality. These water activity
data were consistent with texturometer measurements, sen-
sory analyses and Cryo-SEM, where samples treated with
calcium lactate had the highest crispiness values.
3.2.3. Calcium content
Samples treated with calcium lactate showed signifi-
cantly higher levels of calcium than those treated with chlo-
rine (p < 0.05). Comparing treatments with calcium lactate,
the combination with 50 C increased the calcium lactate of
the samples, compared with 25 C, though not significantly
(Table 1).
These results suggested the effect of temperature in
diffusion of the calcium throughout the tissues. Perhaps
longer exposure times would be necessary in order to
observe a significant effect of temperature on the solubility
and permeability of calcium lactate as reported by oth-
ers authors (Bartolome & Hoff, 1972; Garcia et al.,
1996).
Table 1
Calcium concentration (mg 100 g1) after washing treatment in sliced
carrots treated with chlorine (120 mg L1), calcium lactate (15 g L1) at
25 C and calcium lactate at 50 C
Treatments Calcium (mg/100 g)
Calcium lactate 25 C 0.162a
Calcium lactate 50 C 0.191a
Chlorine 0.122b
Values designated by the same letter are not significantly different
(p > 0.05).
 D. Rico et al. / Journal of Food Engineering 79 (2007) 1196–1206
4. Conclusions
The use of calcium lactate in the washing treatment
maintained the texture of fresh sliced carrots better than
chlorine as measured using a variety of indicators. Calcium
lactate combined with mild temperature (50 C) inhibited
lignification and plasmolysis processes at the end of storage
better than calcium at 25 C. Direct measurements (sensory
analysis and texturometer measurements) corroborated
these findings. This improvement could be associated with
the activation of PME. The results showed that the use of a
calcium lactate wash at a warm temperature (50 C) was
more effective in preventing softening during storage. The
treatment at 50 C may have caused expansion of air pock-
ets within the carrot tissues allowing the infiltration of the
calcium solution. This data indicates that calcium lactate
washing solutions at 50 C have a beneficial effect on min-
imally processed carrots during storage.
Acknowledgements
This research was supported by a Technological Sector
Research grant (2002–2006) and International Collabora-
tion Award Scheme (ICAS) (2005–2006). The authors
would like to thank the laboratory facilities and the elec-
tron microscopy service at University Politécnica of Valen-
cia and to Amcor Flexibles (Europe-Brighouse, United
Kingdom) for provided the packaging material.
References
Ahvenainen, R. (1996). New approaches in improving the shelf life of
minimally processed fruit and vegetables. Trends in Food Science and
Technology, 7, 179–187.
Alzamora, S. M., Salvatori, D., Tapia, M. S., Lopez-Malo, A., Welti-
Chanes, J., & Fito, P. (2005). Novel functional foods from vegetable
matrices impregnated with biologically active compounds. Journal of
Food Engineering, 67, 205–214.
An Bord Glas (2004). <http://www.bordglas.ie/facts/production.htm>
Accessed 4.04.04.
Anino, S. V., Salvatori, D. M., & Alzamora, S. M. (in press). Changes in
calcium level and mechanical properties of apple tissue due to
impregnation with calcium salts. Food Research International.
Bartolome, L. G., & Hoff, J. E. (1972). Firming of potatoes: Biochemical
effect of preheating. Journal of Agriculture and Food Chemistry, 20,
266–270.
Baur, S., Klaiber, R., Hammes, W. P., & Carle, R. (2004). Sensory and
microbiological quality of shredded, packaged iceberg lettuce as
affected by pre-washing procedures with chlorinated and ozonated
water. Innovative Food Science and Emerging Technologies, 5, 45–55.
Belitz, H. D., & Grosh, W. (1986). Food chemistry (second ed.). New-
York: Springer-Verlag, pp.774.
Beuchat, L. R. (1992). Surface disinfection of raw produce. Dairy, Food
and Environmental Sanitation, 12, 6–9.
Beuchat, L. R. (1999). Survival of Escherichia coli O157:H7 in bovine
faeces applied to lettuce and ineffectiveness of chlorinated water as a
disinfectant. Journal of Food Protection, 62, 845–849.
Bomben, J. L., & King, C. J. (1982). Heat and mass transport in the
freezing of apple tissue. Journal of Food Technology, 17, 615–632.
Brecht, J. K. (1995). Physiology of lightly processed fruits and vegetables.
Horticultural Science, 301, 8–22.
1205
Camire, E. M., Ismail, S., Work, T. M., Bushway, A. A., & Halteman, W.
A. (1994). Improvements in canned lowbush blueberry quality. Journal
of Food Science, 59, 394–398.
Cerklewski, F. L. (2005). Calcium fortification of food can add unneeded
dietary phosphorus. Journal of Food Composition and Analysis, 18,
595–598.
Cherry, J. P. (1999). Improving the safety of fresh produce with
antimicrobials. Food Technology, 53, 54–59.
Delaquis, P. J., Stewart, S., Toivonen, P. M. A., & Moyls, A. L. (1999).
Effect of warm, chlorinated water on the microbial flora of shredded
iceberg lettuce. Food Research International, 32, 7–14.
Fan, X., Toivonen, P. M., Rajkowski, K., & Sokorai, K. (2003). Warm
water treatment in combination with modified atmosphere packaging
reduces undesirables effects of irradiation on the quality of fresh-cut
Iceberg lettuce. Journal of Agriculture Food Chemistry, 51, 1231–1236.
Floros, J. D., Ekanayake, A., Abide, G. P., & Nelson, P. E. (1992).
Optimization of postharvest dips in calcium chloride on strawberry.
Journal of Agricultural and Food Chemistry, 4, 30–33.
Garcia, J. M., Herrera, S., & Morilla, A. (1996). Effects of postharvest dips
in calcium chloride on strawberry. Journal of Agricultural and Food
Chemistry, 44, 30–33.
Giner, J., Gimeno, V., Espachs, A., Elez, P., Barbosa-Canovas, G. V., &
Martin, O. (2000). Inhibition of tomato (Licopersicon esculentum
Mill.) pectin merhylesterase pulsed electric fields. Innovation in Food
Science and Emergency Technologies, 1, 57–67.
Gould, G. W. (1995). Homeostatic mechanisms during food preservation
by combined methods. In G. V. Barbosa-Canovas & J. Welti-Chanes
(Eds.), Food preservation by moisture control (pp. 397–410). Lancaster,
PA: Technomic Publishing Co., Inc.
Graham, N.J.D. (1997). The role of ozone and potassium permanganate
in drinking water treatment. In 3rd International workshop on drinking
water quality management and treatment technology, 5–6 March.
Taipei, Taiwan (ROC).
Grant, G. T., Morris, E. R., Rees, D. A., Smith, P. J. C., & Thom, D.
(1973). Biological interactions between polysaccharides and divalent
cations: The egg-box model. FEBS Letters, 32, 195–198.
Grass, M. L., Vidal, D., Betoret, N., Chiralt, A., & Fito, P. (2003).
Calcium fortification of vegetables by vacuum impregnation interac-
tions with cellular matrix. Journal of Food Engineering, 56, 279–284.
Han, J., Gomes-Feitosa, C. L., Castell-Perez, E., Moreira, R. G., & Silva,
P. F. (2004). Quality of packaged romaine lettuce hearts exposed to
low-dose electron beam irradiation. LWT Food Science and Technol-
ogy, 37, 705–715.
Harker, F. R., Stec, M. G. H., Hallett, I. C., & Bennett, C. L. (1997).
Texture of parenchymatous plant tissue: a comparison between tensile
and other instrumental and sensory measurements of tissue strength
and juiciness. Postharvest Biology and Technology, 11, 63–72.
Hisaminato, H., Murata, M., & Homma, S. (2001). Relationship between
enzymatic browning of cut lettuce and phenylalanine ammonialyase
activity, and prevention of browning by inhibitors of polyphenol
biosynthesis. Bioscience, Biotechnology and Biochemistry, 65,
1016–1021.
Institute of Medicine (1998). Dietary references intake (DRI) for calcium,
phosphorus, magnesium, vitamin D and fluoride (p. 432). Food and
Nutrition Board, Standing Committee on the Scientific Evaluation of
Dietary References Intakes. Wahington, DC: National Academic
Press.
Katz, E. E., & Labuza, T. P. (1981). Effect of water activity on the sensory
crispness and mechanical deformation of snack food products. Journal
of Food Science, 46, 403–409.
Kimball, D. A. (1991). Citrus processing-quality control and technology.
New York: Van Nostrand Reinhold.
Lapsley, K. G., Escher, F. E., & Hoehn, E. (1992). The cellular structure
of selected apple varieties. Food Structure, 11, 339–349.
Lee, S., & Kader, A. (2000). Preharvest and postharvest factors influenc-
ing vitamin C content of horticultural crops. Postharvest Biology and
Technology, 20, 207–220.
1206 D. Rico et al. / Journal of Food Engineering 79 (2007) 1196–1206
Lester, G. E., & Grusak, M. A. (1999). Postharvest application of calcium
and magnesium to honeydew and netted muskmelons: effects on tissue
ion concentrations, quality, and senescence. Journal of the American
Society for Horticultural Science, 124, 545–552.
Lin, T. M., Durance, T. D., & Scaman, C. H. (1998). Characterization of
vacuum microwave, air and freeze dried carrots slices. Food Research
International, 31, 111–117.
Loaiza-Velarde, J. G., Tomas-Barberan, F. A., & Salveit, M. E. (1997).
Effect of intensity and duration of heat shock treatment on wound-
induced phenolic metabolism in Iceberg lettuce. Journal of American
Society for Horticultural and Science, 122, 873–877.
Luna-Guzman, I., & Barrett, D. M. (2000). Comparison of calcium
chloride and calcium lactate effectiveness in maintaining shelf stability
and quality of fresh-cut cantaloupe. Postharvest in Biology and
Technology, 19, 61–72.
Luna-Guzman, I., Cantwell, M., & Barrett, D. M. (1999). Fresh-cut
cantaloupe: Effects of CaCl2 dips and heat treatments on firmness and
metabolic activity. Postharvest in Biology Technology, 17, 201–213.
Main, G. L., Morris, J. R., & Wehunt, E. J. (1986). Effect of pre-
processing treatment on the firmness and quality characteristics of
whole and sliced strawberries after freezing and thermal processing.
Journal of Food Science, 51, 391–394.
Martı́n-Diana, A. B., Rico, D., Barry-Ryan, C., Frias, J. M., Mulcahy, J.,
& Henehan, G. T. M. (2005a). Comparison of calcium lactate with
chlorine as a washing treatment for fresh-cut lettuce and carrots:
quality and nutritional parameters. Journal of the Science of Food and
Agriculture, 85, 2260–2268.
Martı́n-Diana, A. B., Rico, D., Barry-Ryan, C., Frias, J. M., Mulcahy, J.,
& Henehan, G. T. M. (2005b). Effect of calcium lactate concentration
and temperature washing treatments on quality retention of salad-cut
Iceberg lettuce. Food Research International, 38, 729–740.
Martı́n-Diana, A. B., Rico, D., Frias, J. M., Barry-Ryan, C., Mulcahy, J.,
& Henehan, G. T. M. (2005). Effect of heat-shock on browning-related
enzymes in minimally processed iceberg lettuce and crude extracts.
Bioscience, Biotechnology and Biochemistry, 69(8), 213-1–213-9.
Martı́n-Diana, A. B., Rico, D., Frias, J., Henehan, G. T. M., Barat, J. M.,
Mulcahy, J., et al. (in press-a). Effect of calcium lactate and heat-shock
on texture in fresh-cut lettuce during storage. Journal of Food
Engineering (doi:10.1016/j.jfoodeng.2005.08.037).
Martı́n-Diana, A. B., Rico, D., Mulcahy, J., Frı́as, J., Henehan, G. T. M.,
& Barry-Ryan, C. (in press-b). Whey permeate as bio-preservative for
shelf-life maintenance of fresh-cut vegetables. Innovative Food Science
& Technologies (doi:10.1016/j.ifset.2005.08.002).
Morris, J. R., Sistrunk, W. A., Sims, C. A., Main, G. L., & Wehunt, E. J.
(1985). Effects of cultivar, postharvest storage, pre-processing dip
treatments and style of pack on the processing quality of strawberries.
Journal of American Society for Horticultural and Science, 110,
172–177.
Ni, L., Lin, D., & Barret, M. D. (2005). Pectin methylesterase catalyzed
firming effects on low temperature blanched vegetables. Journal of
Food Engineering, 70, 546–556.
Nielsen, J. E., & Christensen, T. M. I. E. (2002). Distribution of pectin
methyl esterase and acetylesterase in the genus Citrus visualized by
tissue prints and chromatography. Plant Science, 162(5), 799–807.
Ohlsson, T. (1994). Minimal processing-preservation methods of the
future – an overview. Trends in Food Science and Technology, 5,
341–344.
Rojas, A. M., Gerschenson, L. N., & Marangoni, A. G. (2001).
Contributions of cellular components to the rheological behaviour of
kiwifruit. Food Research International, 34, 189–195.
Roy, S., Watada, A. E., Conway, W. S., Erbe, E. F., & Wergin, W. P.
(1994). Low-temperature scanning electron microscopy of frozen
hydrated apple tissue and surfaces organism. HortScience, 29,
305–309.
Shelef, L. A. (1994). Antimicrobial effects of lactates: a review. Journal of
Food Protection, 57, 445–450.
Seymour, I. J. (1999). Review of current industry practice on fruit and
vegetable decontamination (Review no. 14, pp. 1-38). Campden &
Chorleywood Food Research Association, Gloucestershire, UK.
Stanley, D. W., Bourne, M. C., Stone, A. P., & Wismer, W. V. (1995).
Low temperature blanching effects on chemistry firmness and structure
of canned green beans and carrots. Journal of Food Science, 60,
327–333.
Suutarinen, J., Anakainen, L., & Autio, K. (1999). Comparison of light
microscopy and spatially resolved Fourier transform infrared (FT-IR)
microscopy in the examination of cell wall components of strawberries.
Lebensmittel Wissenschaft und Technologie, 31, 595–601.
Tapia, M. S., Schulz, E., Gomez, V., Lopez-Malo, A., & Welti-Chanes,
J., 2003. A new approach to vacuum impregnation and functional
foods: Melon impregnation with calcium and zinc. In IFT annual
meeting book of abstracts, 2003, July 12–16, Chicago, IL, USA
(Abstract no. 60D-2, pp.158). Chicago, IL: Institute of Food
Technologist.
Tijskens, L. M. M., Waldron, K. W., Ng, A., Ingham, L., & Dijk, C. V.
(1997). The kinetics of pectin methyl esterase in potatoes and
carrots during the bleaching. Journal of Food Engineering, 34, 371–
385.
Vu, T. S., Smout, C., Sila, D. N., Ly Nguyen, B., Van Loey, A. M. L., &
Hendrickx, M. E. G. (2004). Effect of preheating on the thermal
degradation kinetics of carrot texture. Innovative Food Science and
Emerging Technologies, 5, 37–44.
Wiley, R. C. (Ed.). (1994). Minimally processed refrigerated fruits and
vegetables. New York, USA: Chapman & Hall.
Zhang, L., Zhaoxin, L., Zhifang, Y., & Xiang, G. (2005). Preservation of
fresh-cut celery by treatment of ozonated water. Food Control, 16,
279–283.