High hydrostatic pressure as a method to preserve fresh

Article
High hydrostatic pressure as a method to preserve
fresh-cut Hachiya persimmons: A structural approach
José Luis Vázquez-Gutiérrez1, Amparo Quiles1, Erica Vonasek2,
Judith A Jernstedt3, Isabel Hernando1, Nitin Nitin2,4 and
Diane M Barrett4
Abstract
The ‘‘Hachiya’’ persimmon is the most common astringent cultivar grown in California and it is rich in tannins
and carotenoids. Changes in the microstructure and some physicochemical properties during high hydrostatic pressure processing (200–400 MPa, 3 min, 25 C) and subsequent refrigerated storage were analyzed in
this study in order to evaluate the suitability of this non-thermal technology for preservation of fresh-cut
Hachiya persimmons. The effects of high-hydrostatic pressure treatment on the integrity and location of
carotenoids and tannins during storage were also analyzed. Significant changes, in particular diffusion of
soluble compounds which were released as a result of cell wall and membrane damage, were followed using
confocal microscopy. The high-hydrostatic pressure process also induced changes in physicochemical
properties, e.g. electrolyte leakage, texture, total soluble solids, pH and color, which were a function of the
amount of applied hydrostatic pressure and may affect the consumer acceptance of the product.
Nevertheless, the results indicate that the application of 200 MPa could be a suitable preservation treatment
for Hachiya persimmon. This treatment seems to improve carotenoid extractability and tannin polymerization,
which could improve functionality and remove astringency of the fruit, respectively.
Keywords
High hydrostatic pressure, carotenoids, tannins, microstructure, shelf life
Date received: 9 December 2015; accepted: 8 March 2016
INTRODUCTION
Persimmons (Diospyros kaki L.) are widely grown in
many regions of the world and are a good source
of phytochemicals such as tannins and carotenoids,
which are biologically active compounds with significant
nutritional value. According to Gu et al. (2008),
high-molecular weight condensed tannins or proanthocyanidins, which are members of a specific group
of polyphenolic compounds and responsible for the
astringency of the fruit, are also the major antioxidants
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DOI: 10.1177/1082013216642049
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in persimmon pulp. Persimmons can be astringent or
non-astringent at harvest time, depending on the cultivar, the amount of accumulated soluble tannins in the
fruit and their characteristics (Salvador et al., 2007).
1
Group of Food Microstructure and Chemistry, Department of
Food Technology, Universitat Politècnica de València, Valencia,
Spain
2
Department of Biological and Agricultural Engineering, University
of California-Davis, Davis, CA, USA
3
Department of Plant Sciences, University of California-Davis,
Davis, CA, USA
4
Food Science and Technology Department, University of
California-Davis, Davis, CA, USA
Corresponding author:
José Luis Vázquez-Gutiérrez, Universitat Politècnica de València,
Camino de Vera s/n, Valencia 46022, Spain.
Email: [email protected]
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‘‘Hachiya’’ persimmon is the most common astringent
cultivar grown in California and besides containing a
large amount of condensed tannins, it is also rich in
carotenoids (mainly b-cryptoxanthin, b-carotene and
lycopene) and other phenolic compounds such as catechin and quercetin (Homnava et al., 1990; Karhan
et al., 2003).
Several studies have focused on improving quality
and extending shelf life of fresh-cut persimmons
through the application of modified atmospheres
(Ghidelli et al., 2010; Murakami et al., 2012;
Palmer-Wright and Kader, 1997), edible coatings
(Ghidelli et al., 2010; Ergun and Ergun, 2010; PérezGago et al., 2005, 2009), and vacuum impregnation
(Igual et al., 2008). High hydrostatic pressure (HHP)
is a non-thermal processing technology that has
attracted interest because it adds value to fruits and
vegetables. In most cases, the shelf life of fresh produce
can be extended due to microbial and enzyme inactivation, while the sensory characteristics are said to remain
less altered than in products that have been thermally
processed (Ludikhuyze et al., 2002). However, various
quality changes after processing and during refrigerated
storage of HHP-treated products have also been
reported. These include darkening in avocado slices
(Woolf et al., 2013) and ‘‘Rojo Brillante’’ persimmon
(Vázquez-Gutiérrez et al., 2013) after HHP processing,
firmness increases or decreases depending on the product (Gonzalez et al., 2010b; Vázquez-Gutiérrez et al.,
2011), and electrolyte leakage increases in HHP-treated
onion (Gonzalez et al., 2010a). Regarding alterations
during storage, changes in color parameters have been
observed in HHP-treated persimmons, onions, avocado
paste, and fruit juices during refrigerated storage
(Barba et al., 2012; Cao et al., 2012; JacoboVelázquez and Hernández-Brenes, 2010), and pH alterations in avocado paste and Rojo Brillante persimmon
cubes have also been noted (Jacobo-Velázquez and
Hernández-Brenes, 2010; Vázquez-Gutiérrez et al.,
2013). These alterations could be partly due to the
fact that mechanisms of enzyme inactivation by high
pressure treatment are very complex and depend on
the produce and the treatment conditions
(Chakraborty et al., 2014; Eisenmenger and ReyesDe-Corcuera, 2009).
Some studies on HHP-treated Rojo Brillante persimmons have also been carried out (Hernández-Carrión
et al., 2014; Plaza et al., 2012; Vázquez-Gutiérrez et al.,
2011; 2013). These studies showed a reduction of
between 32 and 44% in the total soluble tannin content
after HHP treatment and a decrease of more than 90%
in the total soluble tannins in HHP-treated persimmon
after seven days of refrigerated storage, compared to
non-treated persimmon. To our knowledge, no studies
on the effect of HHP treatment on soluble tannins have
been carried out on other varieties of persimmon apart
from Rojo Brillante persimmons.
Loss of cell integrity and subcellular compartmentalization after processing may influence the extractability
and the bioaccessibility of nutrients, which is the fraction of a nutrient that is released from the food matrix
and available for intestinal absorption after digestion
(Parada and Aguilera, 2007). Therefore, microstructural characterization of food is essential to determining whether food processing influences the
extractability of potentially bioactive compounds as a
first step before studying their bioaccesibility. Some
studies have suggested that the bioaccessibility of
micronutrients and phytochemicals in certain fresh
plant foods may be modified by HHP and that application of this novel technology may prove useful in
developing healthier food products for consumers
(McInerney et al., 2007). Promising results have been
obtained in the conservation of phenolic content and
carotenoid extractability after the application of HHP
to tomato and carrot pureés (Patras et al., 2009),
orange juice (de Ancos et al., 2002; Plaza et al., 2011),
blueberry juice (Barba et al., 2012), and avocado paste
(Jacobo-Velázquez and Hernández-Brenes, 2012). Most
studies on the effects of HHP processing and subsequent refrigerated storage of fruit and vegetable products have been carried out on juices and purees, and
little is known about physicochemical changes in HHPtreated fresh-cut products during refrigerated storage.
The aim of this work was to evaluate if HHP is an
effective method to preserve Hachiya fresh-cut persimmons. For this purpose, changes in microstructure and
physicochemical properties after HHP processing and
subsequent refrigerated storage were studied. Location
and extractability of some bioactive compounds in
Hachiya persimmon are discussed as well.
MATERIALS AND METHODS
Sample preparation
Persimmon fruits (Diospyros kaki L. cv. Hachiya) were
harvested in Woodland, California and stored at 4 C.
Initial characteristics of persimmons, based on measurements from 12 different fruit, are given in Table 1.
Cubes (15 mm3) were taken from the equatorial area of
the fruit and placed in polyethylene bags; 20 to 24 cubes
were obtained per fruit and cubes from the same fruit
were sorted into eight different bags. Each bag contained approximately 80 g of sample from 20–22
cubes. Each bag contained cubes from 6 to 8 different
fruit in order to guarantee representative samples. The
bags were gently compressed to partially evacuate the
air without damaging the cubes, heat-sealed, and high
pressure processed. The high pressure unit had a 2 L
vessel and 900 MPa maximum pressure level (Avure
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Table 1. Initial characteristics of Hachiya persimmon fruit
Characteristic
Value
Diameter whole fruit (mm)
Weight whole fruit (g)
External color, unpeeled fruit
77.86
270.05
64.62
17.88
61.66
21.17
5.58
10.63
Total soluble solids (%)
pH
Flesh hardness (N)
L*
a*
b*
(3.38)
(31.80)
(4.59)
(3.92)
(5.13)
(1.27)
(0.10)
(0.39)
Note: Means of at least three replicates are given, and values in
brackets correspond to the standard deviation.
Technologies Inc, Kent, WA). The pressure transmitting medium was 1 part propylene glycol and 2 parts of
water. The pressures employed in the treatments were
200 MPa and 400 MPa for 3 min at 25 C. Samples were
analyzed immediately after the HHP treatment and
after 7, 14, 21, and 28 days of storage at 4 C. The
HHP treatments were carried out in triplicate and one
bag of each batch was analyzed for each storage time.
Microstructural study
Confocal laser scanning microscopy (CLSM) and light
microscopy (LM) were used for the microstructural
study. A Vibratome 1000 Plus (The Vibratome
Company, St Louis, MO, USA) was used to obtain
sections from the persimmon cubes parallel to the vascular tissue.
CLSM. 200 mm sections from the persimmon cubes
were stained with 200 mL of 0.1% Calcofluor White
(Sigma-Aldrich, Inc.) for 15 min to label cell walls of
the persimmon tissue, after which the samples were
washed three times with 200 mL of deionized water.
Each sample was mounted on a glass microscope slide
and covered with a cover slip. Fluorescence confocal
images of stained persimmon tissue were acquired
using an Olympus inverted microscope (Model IX71,
Olympus, Tokyo, Japan). The microscope was
equipped with a spinning disk unit (Model IX2DSU), which offers confocal images using an arc
lamp excitation and a charge coupled device (CCD)
camera (Model C4742-80-12AG, Hamamatsu, Tokyo,
Japan). Fluorescence of Calcofluor White was detected
using an excitation filter (311 to 387 nm), a dichroic
mirror (409 nm), and an emission filter (447 to
460 nm, blue). The excitation and emission wavelengths
used for imaging carotenoid autofluorescence were 470/
20 nm and 520/20 nm, respectively. Persimmon samples
were imaged using a 10 (0.45 NA) objective lens.
Twelve bit digital images in a TIFF format with an
image size of 1344 1024 pixels were acquired.
Resulting fluorescence contrast from persimmon
images was false colored with cell walls as blue and
carotenoids as green.
LM. Unstained and neutral red stained 400 mm sections
were observed with a light microscope. Neutral red was
used for tannin staining (Waisel et al., 1967). A 0.5%
neutral red solution in acetone was prepared and filtered twice using Whatman No.1 paper. The filtered
stock solution was diluted to 0.04% in an isotonic solution of 0.95 M mannitol/0.01 M HEPES (N-[2-hydroxyethyl] piperazine-N’-[2-ethane-sulfonic acid]) buffer,
pH 7.8, and used as the staining solution. Sections
were dipped in 600 mL of diluted stain for a period of
1 h, after which they were rinsed for 15 min in the
0.95 M mannitol/0.01 M HEPES buffer solution.
Specimens were mounted on a microscope slide with
a drop of deionized water, covered with a cover slip,
and immediately observed with a light microscope
(Olympus System Microscope, Model BHS, Tokyo)
with 4.0 objective magnification with bright field illumination. A digital color camera (Olympus MicroFire,
Japan) was attached to the microscope to capture
images (Olympus MicroFire software). Color photomicrographs (800 600 pixel resolution, white balance
corrected) were captured. For each sample, two different sections per cube were sampled from three different
cubes, each of which was obtained from a different
polyethylene bag. Three micrographs from three different areas were obtained per section. In total, 18 micrographs per sample (2 3 3) were obtained and the
most representative images were selected for the manuscript. Unstained sections (400 mm thick) were observed
using the same microscope to study the different carotenoid structures.
Electrolyte leakage
For the determination of electrolyte leakage, four persimmon cubes per sample were equilibrated at room
temperature for 1 h. One cylindrical sample (5 mm in
height and 5 mm in diameter) per persimmon cube was
obtained with a cork borer from the center of the cube
and placed into 50-mL plastic tubes containing 20 mL
of an isotonic solution (0.95 M mannitol) pre-equilibrated at 25 C. Electrolyte leakage was measured as
electrical conductivity (s) in 0.95 M mannitol solution
using a conductivity meter (Accumet portable AP65,
Fisher Scientific) over a 150 min time period (ti) at
25 C in a shaking water bath system (Model
SWB1122A-1, Lindberg, USA) (Saltveit, 2002).
Electrolyte leakage was calculated as a percentage of
total electrical conductivity of the sample (equation
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(1)). Total conductivity of the samples was measured
after freezing (18 C) and thawing them twice, resulting in 100% ruptured cells.
Electrolyte leakage ð%Þ
Conductivityðti Þ
¼
100
Total conductivity
ð1Þ
(HunterLab, Reston, VA, USA). Measurements were
taken for L* (white to black or light to dark), a* (green
to red), and b* (yellow to blue) parameters. The instrument was calibrated against a standard white tile.
Measurements were individually taken on 10 persimmon
cubes per treatment. Results were expressed as lightness
(L*), chroma (C*) and hue angle (h).
Statistical analysis
Texture analysis
A puncture test with a 5-mm diameter flat-tipped cylindrical probe was performed on the persimmon cubes
with a universal testing machine (model TA.XT2 texture analyzer, Stable Microsystems, Haslemere,
England). The test was performed to a 90% strain
using a test speed of 1 mm/s. The parameters studied
were maximum force (N) or hardness and initial slope
or stiffness, calculated as the gradient of the line connecting the origin of the curve to 20% maximum force
(Nmm1). One measurement per cube was performed
and eight persimmon cubes per treatment were tested.
Total soluble solids and pH
Homogenate from three different cubes was prepared
for total soluble solids (TSS) and pH evaluation. Three
replicate homogenates per sample, each one from a different polyethylene bag, were evaluated. TSS content
was measured with an Atago 3840 PAL-a pocket
refractometer (Atago Co. Ltd., Japan) and expressed
as Brix.
Color evaluation
Color of persimmon samples was measured, in the
CIELab system, using a Hunter colorimeter
One-way analysis of variance (ANOVA) with a 95%
confidence interval was performed on the parameters
using Statgraphics Centurion (Statpoint Technologies,
Inc., Warrenton, VA, USA). The LSD procedure (least
significant difference) was used to test for differences
among the averages at the 5% significance level.
RESULTS AND DISCUSSION
Microstructural study
When sections of untreated persimmons were observed
by CLSM, cell walls stained with Calcofluor White
could be seen in blue, while the autofluorescence emission of carotenoids was registered as green (Figure
1(a)). Carotenoids were typically located close to the
cell walls, in the peripheral cytoplasm. When unstained
sections of the same sample were observed with bright
field microscopy (Figure 1(b)), carotenoids could be
identified by their characteristic yellow-orange color.
The carotenoid compounds present in Hachiya persimmon showed two different subcellular associations. In
one type, carotene-carrying lipid droplets or plastoglobuli were found inside globular chromoplasts, similar
to those previously described in Rojo Brillante persimmon (Vázquez-Gutiérrez et al., 2011). In the other,
needle-shaped carotene crystals and smaller, roundshaped chromoplasts were observed inside the cells
as well as close to cell walls (Figure 1(a) and (b)).
Figure 1. Confocal laser scanning microscopy (Calcofluor White stained) (a) and bright field light microscopy
(b) (unstained) and C (neutral red stained) images of untreated parenchyma tissue of Hachiya persimmon.
gb: globular chromoplast; cr: needle-shaped carotene crystal; rs: small, round-shaped chromoplast; t: tannin cells.
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Vázquez-Gutiérrez et al.
The needle-shaped carotene crystals were similar to
those found in carrot roots (Vásquez-Caicedo et al.,
2006), and the smaller, round-shaped chromoplasts
have been previously described in papaya
(Schweiggert et al., 2011).
Staining with neutral red allowed localization of tannins by their pink-red color inside what have been
called tannin cells or tannic cells (Salvador et al.,
2007; Yonemori et al., 1997). Overall, the tannin cells
had similar diameters (100–150 mm) but were longer
D14
D28
400 MPa/3 min
200 MPa/3 min
D0
than the other cells in the parenchyma tissue of
Hachiya persimmon (Figure 1(c)).
CLSM and LM images of sections of HHP-treated
samples are presented in Figure 2. Cell wall emissions
of samples following HHP treatment at 200 MPa (D0)
were similar to those of the untreated sample (Figure
1), while the carotenoid autofluorescence was more
intense (Figure 2(a)). The fluorescence intensity of cell
walls decreased with higher pressure treatment
(400 MPa) and became more irregular (Figure 2(g)).
Figure 2. Confocal laser scanning microscopy (top row, blue) and bright field light microscopy (bottom row).
Observation of carotenoids in parenchyma tissue of Hachiya persimmon treated at 200 and 400 MPa after 0, 14, and 28
days of storage. Arrows: carotenoids.
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Differences in the autofluorescence emission of
carotenoids could also be observed, depending on the
pressure applied. In tissue treated with 200 MPa, carotenoids still seemed to be associated with cell membranes and chromoplasts (Figure 2(a)). This was
corroborated when unstained sections were observed
with the light microscope (Figure 2(d)). Following the
400 MPa treatment, carotenoid autofluorescence was
still detected inside the cells (Figure 2(g)), but carotenoids were observed inside shriveled masses centrally
located in cells (larger, darker structures in Figure
2(j)). The microstructural differences observed in the
HHP-treated samples could have implications in carotenoid extractability. Moderate high pressure (up to
400 MPa) has been reported to cause damage or breakdown of subcellular organelle membranes (Kato and
Hayashi, 1999). HHP processing altered the subcellular
structures where the carotenoids were initially located,
thus potentially improving their release and solubilization. Carotenoid autofluorescence emission was
brighter in both treatments after 14 days of storage,
while fluorescence emission of cell walls was less intense
after storage than at previous stages (Figure 2(b) and
(h)). After 14 days of storage, carotenoids seemed to
still be associated with the cell walls in the samples
treated at 200 MPa (Figure 2(e)), whereas in the samples treated at 400 MPa, most of the carotenoids were
located towards the center of the cells (Figure 2(k)).
After 28 days of storage, carotenoid autofluorescence
emission decreased regardless of the pressure applied
D14
D28
400 MPa/3 min
200 MPa/3 min
D0
and the same occurred with the fluorescence emission
of cell walls (Figure 2(c) and (i)). Carotenoids and cell
walls of the samples treated at 200 MPa were less distinguishable at this stage (Figure 2(f)). Cell walls of the
samples treated at 400 MPa were also less distinct than
at previous stages, whereas carotenoid structures were
darker and more aggregated and less homogeneously
dispersed than before (Figure 2(l)). Decreases in the
intensity and uniformity of cell wall fluorescence with
increasing levels of HHP treatment and storage time
may indicate cell wall degradation (Murdoch et al.,
1999; Wallwork et al., 1998). According to these results,
the application of 200 MPa might alter the permeability
of the chromoplast membrane, leading to partial release
of carotenoids, which spread throughout the cytoplasm
during storage but were still predominantly located in
areas close to the plasmalemma (Figure 2(a) to (f)).
When 400 MPa was applied, the chromoplast membranes underwent greater damage, as has been previously observed in HHP-treated persimmon ‘‘Rojo
Brillante’’ (Vázquez-Gutiérrez et al., 2011), and the
carotenoids accumulated in one large structure inside
the cell (Figure 2(g) to (l)). The progressive retraction of
the vacuolar membrane (tonoplast) and plasmalemma
membrane due to the diffusion of solutes to the apoplast would favor the aggregation of carotenoids and
cytoplasm content towards the center of the cell.
Figure 3 shows micrographs of neutral red stained,
HHP-treated Hachiya persimmons and provides information on the localization of the tannins within the
Figure 3. Bright field light microscopy. Observation of tannins in the parenchyma tissue (flesh) of HHP treated Hachiya
persimmon stained with neutral red after 0, 14, and 28 days of storage.
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Electrolyte leakage
The untreated, unprocessed samples showed no variation in electrolyte leakage values during 28-day refrigerated storage (Figure 4). The electrolyte leakage
values of the samples treated at 200 MPa progressively
increased during the first 14 days of storage and maintained significantly higher values (P < 0.05) than the
untreated samples towards the end of the storage
period despite significantly decreased leakage
(P < 0.05) during the final 14 days of storage.
Regarding the samples subjected to 400 MPa, they
showed a significantly higher electrolyte leakage value
(P < 0.05) than both the untreated samples and those
subjected to 200 MPa immediately after the HHP treatment (day 0). In addition, the 400 MPa treatment had
the highest electrolyte leakage values during the entire
28-day period of storage. The immediate increase in the
electrolyte leakage observed after the application of
400 MPa may indicate severe damage to the membranes
120
Electrolyte leakage (%)
fruit tissue. Wounding stresses result in metabolic activation in plant tissues and, in some cases, ethylene production (Varoquaux and Wiley, 1994), which may
trigger the formation of insoluble tannin polymers.
The mechanical effect resulting from tannin polymerization inside tannin cell vacuoles has been hypothesized to cause greater degradation of the tonoplast in
these cells as compared to the rest of the parenchyma
cells of ‘‘Rojo Brillante’’ persimmons (Salvador et al.,
2007). Additional degradation of the tonoplast after the
polymerization of tannins allows the neutral red stain
to penetrate more easily and therefore accumulate to a
greater extent, which permits correlation of staining
intensity with degree of polymerization inside tannin
cells. The tannin cells of the samples treated at
400 MPa (Figure 3(d)) stained less intensely than the
tannin cells of the untreated or 200 MPa treated samples at the beginning of storage (Figures 3(a) and 1(c)).
Tannin cells of untreated samples remained less stained
than the HHP-treated samples throughout the entire
storage time (data not shown), while tannin cells of
the HHP-treated samples darkened during storage
(Figure 3(b), (c), (e), and (f)). The sections of samples
treated at 200 MPa (Figure 3(b) and (c)) showed a
greater number of stained tannin cells, which were
also darker in color than those observed in the
untreated or 400 MPa-treated samples. This is a sign
of greater accumulation of neutral red and therefore
greater degradation of the tonoplast of the tannin
cells of the samples treated at 200 MPa than in the
other samples. This would indicate a greater level of
tannin polymerization in the samples treated at
200 MPa than in the other samples, which could contribute to loss of astringency in these samples.
Untreared
200 MPa
400 MPa
100
80
60
40
20
0
0
7
14
Days at 4 °C
21
28
Figure 4. Percentage of electrolyte leakage of untreated
and HHP-treated persimmons during 28 days of storage
with LSD intervals.
and changes in membrane permeability, as suggested by
the microstructural studies. These results agree with
Gonzalez et al. (2010a) in their study on HHP and
thermally treated onions.
Texture
Hardness (calculated as maximum force in Newtons) of
the untreated persimmons decreased during the first 14
days of storage, then experienced a significant increase
(P < 0.05) during the last two weeks of storage (Figure
5(a)). The application of either 200 or 400 MPa by
HHP triggered a significant immediate (day 0) decrease
(P < 0.05) in hardness, compared to the untreated samples. Hardness values of the HHP-treated samples
remained significantly lower (P < 0.05) than the values
of the untreated samples during the whole 28-day storage period. There were significant differences (P < 0.05)
between the samples treated at 200 and 400 MPa at the
beginning of the storage period, but they disappeared
as of the second week of storage.
The initial slope values followed similar trends as the
maximum force ones (Figure 5(b)), with the HHP
application at either 200 or 400 MPa resulting in a significant decrease in slope. However, there were no significant differences (P > 0.05) in the initial slope
between the 200 versus 400 MPa treated samples
during the whole storage period.
Maximum force has been related to hardness and
resistance to failure and initial slope related to the stiffness of the material under the load, or turgor pressure
(Bourne, 2002). The samples treated at 400 MPa also
had the lowest values of maximum force and initial
slope on day 0, which may indicate a loss of turgor
pressure caused by HPP-induced rupture of membranes
and cell wall degradation. The untreated sample, which
the microstructural study indicated had intact
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(a)
(b)
12
Untreared
200 MPa
400 MPa
Slope (Nmm–1)
Fmax (N)
9
6
3
0
8
Untreared
200 MPa
400 MPa
6
4
2
0
0
7
14
21
28
0
7
14
Days at 4 °C
21
28
Days at 4 °C
Figure 5. Maximum force (a) and initial slope (b) of untreated and HHP-treated persimmons during 28 days of storage
with LSD intervals.
(a)
(b)
25
Untreared
200 MPa
400 MPa
6.5
Untreared
200 MPa
400 MPa
6
pH
TSS (°Brix)
22.25
20
5.5
17.5
5
15
0
7
14
Days at 4 °C
21
28
0
7
14
Days at 4 °C
21
28
Figure 6. Total soluble solids content (a) and pH (b) of untreated and HHP-treated persimmons during 28 days of
storage with LSD intervals.
semipermeable plasma membranes and cell walls,
thereby allowed cellular turgor pressure to be maintained throughout storage, would be expected to have
the highest hardness and stiffness. Correspondingly,
these samples had the highest values of maximum
force and initial slope, as well as the lowest electrolyte
leakage values.
Treatment at 200 MPa was correlated with a
decrease in hardness and stiffness but, unlike the treatment at 400 MPa, did not result in an immediate
increase in electrolyte leakage. In addition, the cell
walls of the samples treated at 200 MPa did not show
signs of severe degradation at the beginning of the storage time (Figure 2(a)). Moreover, maximum force,
which is related to cell wall integrity (Fuentes et al.,
2014), was higher in the 200 MPa treated samples
than those treated at 400 MPa. All of this may indicate
that the cell walls in the samples treated at 200 MPa
were not as damaged after the HHP treatment, compared to the 400 MPa treatment.
TSS and pH
The initial TSS content of the untreated samples was
21.17%, similar to the values provided by Ergun and
Ergun (2010). The TSS had significantly decreased in
the untreated samples by the end of the 28-day storage
period (Figure 6(a)). The application of HHP caused a
decrease in TSS content immediately after treatment
(day 0) regardless of the pressure level. TSS content
of HHP-treated samples reached a maximum after 7
days of storage (only significant at P < 0.05 in the
case of the samples treated at 400 MPa) and decreased
progressively during the remaining 21 days of storage
time. Both treated and untreated samples had similar
TSS content at the end of the storage period. As
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Color
Lightness (L*), chroma (C*), and hue angle (h) values
decreased progressively during storage in the untreated
(a)
70
Untreared
200 MPa
400 MPa
L*
60
50
40
30
0
(b)
7
60
14
Days at 4 °C
Untreared
21
28
200 MPa
400 MPa
C*
45
30
15
0
0
(c)
7
90
14
Days at 4 °C
Untreared
21
28
200 MPa
400 MPa
85
h
previously discussed, electrolyte leakage increased in
both HHP-treated samples during the first week of storage, likely due to alterations in the cellular and subcellular structures caused by the pressure treatments, as
observed in the microstructural studies. Greater and
faster diffusion of electrolytes took place in the samples
treated at 400 MPa which was associated with more
initial membrane and cell wall damage. This would
explain the higher electrolyte leakage and the increase
in TSS at day 7 in these samples.
pH increased towards the end of the storage time in
the untreated samples (Figure 6(b)). Samples treated at
200 MPa had significantly higher (P < 0.05) pH values
than the untreated or 400 MPa treated samples on day
0 following treatment and until the end of the storage
time. Samples treated at 400 MPa had similar pH
values to the untreated samples at day 0. However,
pH decreased progressively in the 400 MPa samples
and it was significantly lower than the pH of the
other samples during the whole storage period. The
polymerization of tannins, which have an acidic character, would explain the progressive decrease in TSS
and the increase in pH in samples treated with
200 MPa, which also had the highest number of and
darkest tannic structures after neutral red staining
(Figure 3(b) and (c)). The greater structural damage
observed in the 400 MPa treated samples may have
favored the diffusion of soluble tannins and hindered
their interaction and subsequent precipitation. This
would also explain their lower pH values and weaker
neutral red staining compared to the 200 MPa treated
samples. During the last weeks of storage, neutral red
staining in persimmons treated at 400 MPa was more
intense than on day 0 (Figure 3(e) and (f)), which may
indicate a higher level of tannin precipitation and
would agree with the decrease in TSS in these samples
at the same storage time.
Overall, TSS values progressively decreased in the
HHP-treated samples during storage despite the signs
of cell wall solubilization observed in the microstructural study (Figure 2(b), (c), (h), and (i)). This could
indicate that either pectins interacted with other compounds to form insoluble complexes or their solubilization is compensated by precipitation of other
compounds such as tannins. Soluble tannins can form
a complex with water-soluble pectin during fruit softening (Taira et al., 1997). In the same way, solubilization
of pectins from the cell wall triggered by the HHP treatments could favor the formation of these complexes
and subsequent insolubilization of tannins.
80
75
70
0
7
14
Days at 4 °C
21
28
Figure 7. Color parameters (L*, C*, and h) of untreated
and HHP-treated persimmons during 28 days of storage
with LSD intervals.
samples, most markedly during the first two weeks of
storage (Figure 7). The application of HHP triggered a
significant immediate decrease in L* and C* values
which continued during the entire storage period, compared to the untreated samples (Figure 7(a) and (b)).
Significant differences (P < 0.05) appeared in the values
of L* and C* between the two HHP-treated samples as
of the third week of storage. These parameters followed
an increasing trend in the samples treated at 200 MPa,
as opposed to a decreasing trend in the samples treated
at 400 MPa. The same trend was observed in the hue
angle values. However, h values of HHP-treated samples were similar to those of untreated persimmons in
the first 7 days of storage (Figure 7(c)). Hue angle
9
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Food Science and Technology International 0(0)
decreased abruptly in the 400 MPa treated samples
after 14 days of storage; however, it increased at this
point in the samples treated at 200 MPa to values similar to those of untreated samples for the remainder of
the storage time.
The microstructural study also revealed changes in
color of the carotenoid fraction after the HHP treatments (Figure 2(d) and (j)), in particular between HHPtreated and untreated samples. Low values of L* in the
HHP-treated persimmon Rojo Brillante have been previously attributed to activation of browning enzymes
such as polyphenoloxidase (Hernández-Carrión et al.,
2014; Vázquez-Gutiérrez et al., 2013). Carotenoid
structures observed at the end of storage in the
400 MPa treated samples were darker and larger than
those observed in either other samples or earlier stages
of storage and could explain the lower values of L*, C*,
and h registered in these samples.
CONCLUSIONS
Significant changes in tissue structure and physicochemical properties took place in fresh-cut Hachiya
persimmon during HHP treatment and subsequent
storage, which were overall greater as higher pressure
was applied. Modifications of cell walls and membranes led to mechanical disruption of the food
matrix, which may improve the extractability of
some compounds. HHP treatment at 200 MPa appears
to be a suitable treatment to preserve fresh-cut
Hachiya persimmon for 28 days. This treatment
keeps the best physicochemical properties, preserves
the integrity of the carotenoids, and triggers tannin
polymerization in fresh-cut Hachiya persimmon,
which could decrease the astringency of the product.
Further microbiological and sensory analyses should
be carried out in order to determine the stability and
acceptability of the product by the consumers and
whether astringency in Hachiya persimmon is removed
by HHP processing.
ACKNOWLEDGEMENTS
The authors wish to thank Stan Tufts and Darryl Mack for
supplying and processing of persimmon fruit, respectively.
DECLARATION OF CONFLICTING INTERESTS
The author(s) declared no potential conflicts of interest with
respect to the research, authorship, and/or publication of this
article.
FUNDING
The author(s) wish to acknowledge the Spanish Ministry of
Science and Innovation for the PhD and mobility grants
(AP2007-03748) awarded to J.L. Vázquez Gutiérrez.
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