High hydrostatic pressure as a method to preserve fresh ...

Article

High hydrostatic pressure as a method to preservefresh-cut Hachiya persimmons: A structural approach

Jose Luis Vazquez-Gutierrez1, Amparo Quiles1, Erica Vonasek2,Judith A Jernstedt3, Isabel Hernando1, Nitin Nitin2,4 andDiane M Barrett4

AbstractThe ‘‘Hachiya’’ persimmon is the most common astringent cultivar grown in California and it is rich in tanninsand carotenoids. Changes in the microstructure and some physicochemical properties during high hydro-static pressure processing (200–400 MPa, 3 min, 25 �C) and subsequent refrigerated storage were analyzed inthis study in order to evaluate the suitability of this non-thermal technology for preservation of fresh-cutHachiya persimmons. The effects of high-hydrostatic pressure treatment on the integrity and location ofcarotenoids and tannins during storage were also analyzed. Significant changes, in particular diffusion ofsoluble compounds which were released as a result of cell wall and membrane damage, were followed usingconfocal microscopy. The high-hydrostatic pressure process also induced changes in physicochemicalproperties, e.g. electrolyte leakage, texture, total soluble solids, pH and color, which were a function of theamount 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 treatmentfor Hachiya persimmon. This treatment seems to improve carotenoid extractability and tannin polymerization,which could improve functionality and remove astringency of the fruit, respectively.

KeywordsHigh hydrostatic pressure, carotenoids, tannins, microstructure, shelf life

Date received: 9 December 2015; accepted: 8 March 2016

INTRODUCTION

Persimmons (Diospyros kaki L.) are widely grown inmany regions of the world and are a good sourceof phytochemicals such as tannins and carotenoids,which are biologically active compounds with significantnutritional value. According to Gu et al. (2008),high-molecular weight condensed tannins or pro-anthocyanidins, which are members of a specific groupof polyphenolic compounds and responsible for theastringency of the fruit, are also the major antioxidants

in persimmon pulp. Persimmons can be astringent ornon-astringent at harvest time, depending on the culti-var, the amount of accumulated soluble tannins in thefruit and their characteristics (Salvador et al., 2007).

1Group of Food Microstructure and Chemistry, Department ofFood Technology, Universitat Politecnica de Valencia, Valencia,Spain2Department of Biological and Agricultural Engineering, Universityof California-Davis, Davis, CA, USA3Department of Plant Sciences, University of California-Davis,Davis, CA, USA4Food Science and Technology Department, University ofCalifornia-Davis, Davis, CA, USA

Corresponding author:Jose Luis Vazquez-Gutierrez, Universitat Politecnica de Valencia,Camino de Vera s/n, Valencia 46022, Spain.Email: [email protected]

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‘‘Hachiya’’ persimmon is the most common astringentcultivar grown in California and besides containing alarge amount of condensed tannins, it is also rich incarotenoids (mainly b-cryptoxanthin, b-carotene andlycopene) and other phenolic compounds such as cat-echin and quercetin (Homnava et al., 1990; Karhanet al., 2003).

Several studies have focused on improving qualityand extending shelf life of fresh-cut persimmonsthrough 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; Perez-Gago et al., 2005, 2009), and vacuum impregnation(Igual et al., 2008). High hydrostatic pressure (HHP)is a non-thermal processing technology that hasattracted interest because it adds value to fruits andvegetables. In most cases, the shelf life of fresh producecan be extended due to microbial and enzyme inactiva-tion, while the sensory characteristics are said to remainless altered than in products that have been thermallyprocessed (Ludikhuyze et al., 2002). However, variousquality changes after processing and during refrigeratedstorage of HHP-treated products have also beenreported. These include darkening in avocado slices(Woolf et al., 2013) and ‘‘Rojo Brillante’’ persimmon(Vazquez-Gutierrez et al., 2013) after HHP processing,firmness increases or decreases depending on the prod-uct (Gonzalez et al., 2010b; Vazquez-Gutierrez et al.,2011), and electrolyte leakage increases in HHP-treatedonion (Gonzalez et al., 2010a). Regarding alterationsduring storage, changes in color parameters have beenobserved in HHP-treated persimmons, onions, avocadopaste, and fruit juices during refrigerated storage(Barba et al., 2012; Cao et al., 2012; Jacobo-Velazquez and Hernandez-Brenes, 2010), and pH alter-ations in avocado paste and Rojo Brillante persimmoncubes have also been noted (Jacobo-Velazquez andHernandez-Brenes, 2010; Vazquez-Gutierrez et al.,2013). These alterations could be partly due to thefact that mechanisms of enzyme inactivation by highpressure treatment are very complex and depend onthe produce and the treatment conditions(Chakraborty et al., 2014; Eisenmenger and Reyes-De-Corcuera, 2009).

Some studies on HHP-treated Rojo Brillante persim-mons have also been carried out (Hernandez-Carrionet al., 2014; Plaza et al., 2012; Vazquez-Gutierrez et al.,2011; 2013). These studies showed a reduction ofbetween 32 and 44% in the total soluble tannin contentafter HHP treatment and a decrease of more than 90%in the total soluble tannins in HHP-treated persimmonafter seven days of refrigerated storage, compared tonon-treated persimmon. To our knowledge, no studieson the effect of HHP treatment on soluble tannins have

been carried out on other varieties of persimmon apartfrom Rojo Brillante persimmons.

Loss of cell integrity and subcellular compartmental-ization after processing may influence the extractabilityand the bioaccessibility of nutrients, which is the frac-tion of a nutrient that is released from the food matrixand available for intestinal absorption after digestion(Parada and Aguilera, 2007). Therefore, microstruc-tural characterization of food is essential to determin-ing whether food processing influences theextractability of potentially bioactive compounds as afirst step before studying their bioaccesibility. Somestudies have suggested that the bioaccessibility ofmicronutrients and phytochemicals in certain freshplant foods may be modified by HHP and that appli-cation of this novel technology may prove useful indeveloping healthier food products for consumers(McInerney et al., 2007). Promising results have beenobtained in the conservation of phenolic content andcarotenoid extractability after the application of HHPto tomato and carrot purees (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-Velazquez and Hernandez-Brenes, 2012). Moststudies on the effects of HHP processing and subse-quent refrigerated storage of fruit and vegetable prod-ucts have been carried out on juices and purees, andlittle is known about physicochemical changes in HHP-treated fresh-cut products during refrigerated storage.The aim of this work was to evaluate if HHP is aneffective method to preserve Hachiya fresh-cut persim-mons. For this purpose, changes in microstructure andphysicochemical properties after HHP processing andsubsequent refrigerated storage were studied. Locationand extractability of some bioactive compounds inHachiya persimmon are discussed as well.

MATERIALS AND METHODS

Sample preparation

Persimmon fruits (Diospyros kaki L. cv. Hachiya) wereharvested in Woodland, California and stored at 4 �C.Initial characteristics of persimmons, based on meas-urements from 12 different fruit, are given in Table 1.Cubes (15 mm3) were taken from the equatorial area ofthe fruit and placed in polyethylene bags; 20 to 24 cubeswere obtained per fruit and cubes from the same fruitwere sorted into eight different bags. Each bag con-tained approximately 80 g of sample from 20–22cubes. Each bag contained cubes from 6 to 8 differentfruit in order to guarantee representative samples. Thebags were gently compressed to partially evacuate theair without damaging the cubes, heat-sealed, and highpressure processed. The high pressure unit had a 2Lvessel and 900MPa maximum pressure level (Avure

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Technologies Inc, Kent, WA). The pressure transmit-ting medium was 1 part propylene glycol and 2 parts ofwater. The pressures employed in the treatments were200MPa and 400MPa for 3min at 25 �C. Samples wereanalyzed immediately after the HHP treatment andafter 7, 14, 21, and 28 days of storage at 4 �C. TheHHP treatments were carried out in triplicate and onebag of each batch was analyzed for each storage time.

Microstructural study

Confocal laser scanning microscopy (CLSM) and lightmicroscopy (LM) were used for the microstructuralstudy. A Vibratome 1000 Plus (The VibratomeCompany, St Louis, MO, USA) was used to obtainsections from the persimmon cubes parallel to the vas-cular tissue.

CLSM. 200 mm sections from the persimmon cubeswere stained with 200 mL of 0.1% Calcofluor White(Sigma-Aldrich, Inc.) for 15min to label cell walls ofthe persimmon tissue, after which the samples werewashed three times with 200 mL of deionized water.Each sample was mounted on a glass microscope slideand covered with a cover slip. Fluorescence confocalimages of stained persimmon tissue were acquiredusing an Olympus inverted microscope (Model IX71,Olympus, Tokyo, Japan). The microscope wasequipped with a spinning disk unit (Model IX2-DSU), which offers confocal images using an arclamp excitation and a charge coupled device (CCD)camera (Model C4742-80-12AG, Hamamatsu, Tokyo,Japan). Fluorescence of Calcofluor White was detectedusing an excitation filter (311 to 387 nm), a dichroicmirror (409 nm), and an emission filter (447 to460 nm, blue). The excitation and emission wavelengthsused for imaging carotenoid autofluorescence were 470/20 nm and 520/20 nm, respectively. Persimmon sampleswere imaged using a 10� (0.45NA) objective lens.

Twelve bit digital images in a TIFF format with animage size of 1344� 1024 pixels were acquired.Resulting fluorescence contrast from persimmonimages was false colored with cell walls as blue andcarotenoids as green.

LM. Unstained and neutral red stained 400 mm sectionswere observed with a light microscope. Neutral red wasused for tannin staining (Waisel et al., 1967). A 0.5%neutral red solution in acetone was prepared and fil-tered twice using Whatman No.1 paper. The filteredstock solution was diluted to 0.04% in an isotonic solu-tion of 0.95M mannitol/0.01M HEPES (N-[2-hydro-xyethyl] piperazine-N’-[2-ethane-sulfonic acid]) buffer,pH 7.8, and used as the staining solution. Sectionswere dipped in 600mL of diluted stain for a period of1 h, after which they were rinsed for 15min in the0.95M mannitol/0.01M HEPES buffer solution.Specimens were mounted on a microscope slide witha 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 illu-mination. A digital color camera (Olympus MicroFire,Japan) was attached to the microscope to captureimages (Olympus MicroFire software). Color photo-micrographs (800� 600 pixel resolution, white balancecorrected) were captured. For each sample, two differ-ent sections per cube were sampled from three differentcubes, each of which was obtained from a differentpolyethylene bag. Three micrographs from three differ-ent areas were obtained per section. In total, 18 micro-graphs per sample (2� 3� 3) were obtained and themost representative images were selected for the manu-script. Unstained sections (400 mm thick) were observedusing the same microscope to study the different carot-enoid structures.

Electrolyte leakage

For the determination of electrolyte leakage, four per-simmon cubes per sample were equilibrated at roomtemperature for 1 h. One cylindrical sample (5mm inheight and 5mm in diameter) per persimmon cube wasobtained with a cork borer from the center of the cubeand placed into 50-mL plastic tubes containing 20mLof an isotonic solution (0.95M mannitol) pre-equili-brated at 25 �C. Electrolyte leakage was measured aselectrical conductivity (s) in 0.95M mannitol solutionusing a conductivity meter (Accumet portable AP65,Fisher Scientific) over a 150min time period (ti) at25 �C in a shaking water bath system (ModelSWB1122A-1, Lindberg, USA) (Saltveit, 2002).Electrolyte leakage was calculated as a percentage oftotal electrical conductivity of the sample (equation

Table 1. Initial characteristics of Hachiya persimmon fruit

Characteristic Value

Diameter whole fruit (mm) 77.86 (3.38)

Weight whole fruit (g) 270.05 (31.80)

External color, unpeeled fruit L* 64.62 (4.59)

a* 17.88 (3.92)

b* 61.66 (5.13)

Total soluble solids (%) 21.17 (1.27)

pH 5.58 (0.10)

Flesh hardness (N) 10.63 (0.39)

Note: Means of at least three replicates are given, and values inbrackets correspond to the standard deviation.

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(1)). Total conductivity of the samples was measuredafter freezing (�18 �C) and thawing them twice, result-ing in 100% ruptured cells.

Electrolyte leakage ð%Þ

¼ConductivityðtiÞ

Total conductivity� 100 ð1Þ

Texture analysis

A puncture test with a 5-mm diameter flat-tipped cylin-drical probe was performed on the persimmon cubeswith a universal testing machine (model TA.XT2 tex-ture analyzer, Stable Microsystems, Haslemere,England). The test was performed to a 90% strainusing a test speed of 1mm/s. The parameters studiedwere maximum force (N) or hardness and initial slopeor stiffness, calculated as the gradient of the line con-necting the origin of the curve to 20% maximum force(Nmm�1). One measurement per cube was performedand eight persimmon cubes per treatment were tested.

Total soluble solids and pH

Homogenate from three different cubes was preparedfor total soluble solids (TSS) and pH evaluation. Threereplicate homogenates per sample, each one from a dif-ferent polyethylene bag, were evaluated. TSS contentwas measured with an Atago 3840 PAL-a pocketrefractometer (Atago Co. Ltd., Japan) and expressedas �Brix.

Color evaluation

Color of persimmon samples was measured, in theCIELab system, using a Hunter colorimeter

(HunterLab, Reston, VA, USA). Measurements weretaken for L* (white to black or light to dark), a* (greento red), and b* (yellow to blue) parameters. The instru-ment was calibrated against a standard white tile.Measurements were individually taken on 10 persimmoncubes per treatment. Results were expressed as lightness(L*), chroma (C*) and hue angle (h).

Statistical analysis

One-way analysis of variance (ANOVA) with a 95%confidence interval was performed on the parametersusing Statgraphics Centurion (Statpoint Technologies,Inc., Warrenton, VA, USA). The LSD procedure (leastsignificant difference) was used to test for differencesamong the averages at the 5% significance level.

RESULTS AND DISCUSSION

Microstructural study

When sections of untreated persimmons were observedby CLSM, cell walls stained with Calcofluor Whitecould be seen in blue, while the autofluorescence emis-sion of carotenoids was registered as green (Figure1(a)). Carotenoids were typically located close to thecell walls, in the peripheral cytoplasm. When unstainedsections of the same sample were observed with brightfield microscopy (Figure 1(b)), carotenoids could beidentified by their characteristic yellow-orange color.The carotenoid compounds present in Hachiya persim-mon showed two different subcellular associations. Inone type, carotene-carrying lipid droplets or plastoglo-buli were found inside globular chromoplasts, similarto those previously described in Rojo Brillante persim-mon (Vazquez-Gutierrez et al., 2011). In the other,needle-shaped carotene crystals and smaller, round-shaped chromoplasts were observed inside the cellsas 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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The needle-shaped carotene crystals were similar tothose found in carrot roots (Vasquez-Caicedo et al.,2006), and the smaller, round-shaped chromoplastshave been previously described in papaya(Schweiggert et al., 2011).

Staining with neutral red allowed localization of tan-nins by their pink-red color inside what have beencalled tannin cells or tannic cells (Salvador et al.,2007; Yonemori et al., 1997). Overall, the tannin cellshad similar diameters (100–150 mm) but were longer

than the other cells in the parenchyma tissue ofHachiya persimmon (Figure 1(c)).

CLSM and LM images of sections of HHP-treatedsamples are presented in Figure 2. Cell wall emissionsof samples following HHP treatment at 200MPa (D0)were similar to those of the untreated sample (Figure1), while the carotenoid autofluorescence was moreintense (Figure 2(a)). The fluorescence intensity of cellwalls decreased with higher pressure treatment(400MPa) and became more irregular (Figure 2(g)).

D0

200

MP

a/3

min

400

MP

a/3

min

D14 D28

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 28days of storage. Arrows: carotenoids.


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Differences in the autofluorescence emission ofcarotenoids could also be observed, depending on thepressure applied. In tissue treated with 200MPa, carot-enoids still seemed to be associated with cell mem-branes and chromoplasts (Figure 2(a)). This wascorroborated when unstained sections were observedwith the light microscope (Figure 2(d)). Following the400MPa treatment, carotenoid autofluorescence wasstill detected inside the cells (Figure 2(g)), but caroten-oids were observed inside shriveled masses centrallylocated in cells (larger, darker structures in Figure2(j)). The microstructural differences observed in theHHP-treated samples could have implications in carot-enoid extractability. Moderate high pressure (up to400MPa) has been reported to cause damage or break-down of subcellular organelle membranes (Kato andHayashi, 1999). HHP processing altered the subcellularstructures where the carotenoids were initially located,thus potentially improving their release and solubiliza-tion. Carotenoid autofluorescence emission wasbrighter in both treatments after 14 days of storage,while fluorescence emission of cell walls was less intenseafter storage than at previous stages (Figure 2(b) and(h)). After 14 days of storage, carotenoids seemed tostill be associated with the cell walls in the samplestreated at 200MPa (Figure 2(e)), whereas in the sam-ples treated at 400MPa, most of the carotenoids werelocated towards the center of the cells (Figure 2(k)).After 28 days of storage, carotenoid autofluorescenceemission decreased regardless of the pressure applied

and the same occurred with the fluorescence emissionof cell walls (Figure 2(c) and (i)). Carotenoids and cellwalls of the samples treated at 200MPa were less dis-tinguishable at this stage (Figure 2(f)). Cell walls of thesamples treated at 400MPa were also less distinct thanat previous stages, whereas carotenoid structures weredarker and more aggregated and less homogeneouslydispersed than before (Figure 2(l)). Decreases in theintensity and uniformity of cell wall fluorescence withincreasing levels of HHP treatment and storage timemay indicate cell wall degradation (Murdoch et al.,1999; Wallwork et al., 1998). According to these results,the application of 200MPa might alter the permeabilityof the chromoplast membrane, leading to partial releaseof carotenoids, which spread throughout the cytoplasmduring storage but were still predominantly located inareas close to the plasmalemma (Figure 2(a) to (f)).When 400MPa was applied, the chromoplast mem-branes underwent greater damage, as has been previ-ously observed in HHP-treated persimmon ‘‘RojoBrillante’’ (Vazquez-Gutierrez et al., 2011), and thecarotenoids accumulated in one large structure insidethe cell (Figure 2(g) to (l)). The progressive retraction ofthe vacuolar membrane (tonoplast) and plasmalemmamembrane due to the diffusion of solutes to the apo-plast would favor the aggregation of carotenoids andcytoplasm content towards the center of the cell.

Figure 3 shows micrographs of neutral red stained,HHP-treated Hachiya persimmons and provides infor-mation on the localization of the tannins within the

D0

200

MP

a/3

min

400

MP

a/3

min

D14 D28

Figure 3. Bright field light microscopy. Observation of tannins in the parenchyma tissue (flesh) of HHP treated Hachiyapersimmon stained with neutral red after 0, 14, and 28 days of storage.


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fruit tissue. Wounding stresses result in metabolic acti-vation in plant tissues and, in some cases, ethylene pro-duction (Varoquaux and Wiley, 1994), which maytrigger the formation of insoluble tannin polymers.The mechanical effect resulting from tannin polymer-ization inside tannin cell vacuoles has been hypothe-sized to cause greater degradation of the tonoplast inthese cells as compared to the rest of the parenchymacells of ‘‘Rojo Brillante’’ persimmons (Salvador et al.,2007). Additional degradation of the tonoplast after thepolymerization of tannins allows the neutral red stainto penetrate more easily and therefore accumulate to agreater extent, which permits correlation of stainingintensity with degree of polymerization inside tannincells. The tannin cells of the samples treated at400MPa (Figure 3(d)) stained less intensely than thetannin cells of the untreated or 200MPa treated sam-ples at the beginning of storage (Figures 3(a) and 1(c)).Tannin cells of untreated samples remained less stainedthan the HHP-treated samples throughout the entirestorage time (data not shown), while tannin cells ofthe HHP-treated samples darkened during storage(Figure 3(b), (c), (e), and (f)). The sections of samplestreated at 200MPa (Figure 3(b) and (c)) showed agreater number of stained tannin cells, which werealso darker in color than those observed in theuntreated or 400MPa-treated samples. This is a signof greater accumulation of neutral red and thereforegreater degradation of the tonoplast of the tannincells of the samples treated at 200MPa than in theother samples. This would indicate a greater level oftannin polymerization in the samples treated at200MPa than in the other samples, which could con-tribute to loss of astringency in these samples.

Electrolyte leakage

The untreated, unprocessed samples showed no vari-ation in electrolyte leakage values during 28-day refri-gerated storage (Figure 4). The electrolyte leakagevalues of the samples treated at 200MPa progressivelyincreased during the first 14 days of storage and main-tained significantly higher values (P< 0.05) than theuntreated samples towards the end of the storageperiod despite significantly decreased leakage(P< 0.05) during the final 14 days of storage.Regarding the samples subjected to 400MPa, theyshowed a significantly higher electrolyte leakage value(P< 0.05) than both the untreated samples and thosesubjected to 200MPa immediately after the HHP treat-ment (day 0). In addition, the 400MPa treatment hadthe highest electrolyte leakage values during the entire28-day period of storage. The immediate increase in theelectrolyte leakage observed after the application of400MPa may indicate severe damage to the membranes

and changes in membrane permeability, as suggested bythe microstructural studies. These results agree withGonzalez et al. (2010a) in their study on HHP andthermally treated onions.

Texture

Hardness (calculated as maximum force in Newtons) ofthe untreated persimmons decreased during the first 14days of storage, then experienced a significant increase(P< 0.05) during the last two weeks of storage (Figure5(a)). The application of either 200 or 400MPa byHHP triggered a significant immediate (day 0) decrease(P< 0.05) in hardness, compared to the untreated sam-ples. Hardness values of the HHP-treated samplesremained significantly lower (P< 0.05) than the valuesof the untreated samples during the whole 28-day stor-age period. There were significant differences (P< 0.05)between the samples treated at 200 and 400MPa at thebeginning of the storage period, but they disappearedas of the second week of storage.

The initial slope values followed similar trends as themaximum force ones (Figure 5(b)), with the HHPapplication at either 200 or 400MPa resulting in a sig-nificant decrease in slope. However, there were no sig-nificant differences (P> 0.05) in the initial slopebetween the 200 versus 400MPa treated samplesduring the whole storage period.

Maximum force has been related to hardness andresistance to failure and initial slope related to the stiff-ness of the material under the load, or turgor pressure(Bourne, 2002). The samples treated at 400MPa alsohad the lowest values of maximum force and initialslope on day 0, which may indicate a loss of turgorpressure caused by HPP-induced rupture of membranesand cell wall degradation. The untreated sample, whichthe microstructural study indicated had intact

120

100

80

60

Ele

ctro

lyte

leak

age

(%)

40

20

00 7 14

Days at 4 °C

Untreared 200 MPa 400 MPa

21 28

Figure 4. Percentage of electrolyte leakage of untreatedand HHP-treated persimmons during 28 days of storagewith LSD intervals.


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semipermeable plasma membranes and cell walls,thereby allowed cellular turgor pressure to be main-tained throughout storage, would be expected to havethe highest hardness and stiffness. Correspondingly,these samples had the highest values of maximumforce and initial slope, as well as the lowest electrolyteleakage values.

Treatment at 200MPa was correlated with adecrease in hardness and stiffness but, unlike the treat-ment at 400MPa, did not result in an immediateincrease in electrolyte leakage. In addition, the cellwalls of the samples treated at 200MPa did not showsigns of severe degradation at the beginning of the stor-age time (Figure 2(a)). Moreover, maximum force,which is related to cell wall integrity (Fuentes et al.,2014), was higher in the 200MPa treated samplesthan those treated at 400MPa. All of this may indicatethat the cell walls in the samples treated at 200MPa

were not as damaged after the HHP treatment, com-pared to the 400MPa treatment.

TSS and pH

The initial TSS content of the untreated samples was21.17%, similar to the values provided by Ergun andErgun (2010). The TSS had significantly decreased inthe untreated samples by the end of the 28-day storageperiod (Figure 6(a)). The application of HHP caused adecrease in TSS content immediately after treatment(day 0) regardless of the pressure level. TSS contentof HHP-treated samples reached a maximum after 7days of storage (only significant at P< 0.05 in thecase of the samples treated at 400MPa) and decreasedprogressively during the remaining 21 days of storagetime. Both treated and untreated samples had similarTSS content at the end of the storage period. As

Fm

ax (

N)

Slo

pe (

Nm

m–1

)

Days at 4 °C Days at 4 °C

Untreared12 8

6

4

2

0

(a) (b)

9

6

3

0

200 MPa 400 MPa Untreared 200 MPa 400 MPa

0 7 14 21 28 0 7 14 21 28

Figure 5. Maximum force (a) and initial slope (b) of untreated and HHP-treated persimmons during 28 days of storagewith LSD intervals.

TS

S (

°Brix

)

pH

Days at 4 °C

Untreared(a)

25 6.5

6

5.5

5

22.25

20

17.5

15

(b)200 MPa 400 MPa Untreared 200 MPa 400 MPa

0 7 14 21 28

Days at 4 °C

0 7 14 21 28

Figure 6. Total soluble solids content (a) and pH (b) of untreated and HHP-treated persimmons during 28 days ofstorage with LSD intervals.


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previously discussed, electrolyte leakage increased inboth HHP-treated samples during the first week of stor-age, likely due to alterations in the cellular and subcel-lular structures caused by the pressure treatments, asobserved in the microstructural studies. Greater andfaster diffusion of electrolytes took place in the samplestreated at 400MPa which was associated with moreinitial membrane and cell wall damage. This wouldexplain the higher electrolyte leakage and the increasein TSS at day 7 in these samples.

pH increased towards the end of the storage time inthe untreated samples (Figure 6(b)). Samples treated at200MPa had significantly higher (P< 0.05) pH valuesthan the untreated or 400MPa treated samples on day0 following treatment and until the end of the storagetime. Samples treated at 400MPa had similar pHvalues to the untreated samples at day 0. However,pH decreased progressively in the 400MPa samplesand it was significantly lower than the pH of theother samples during the whole storage period. Thepolymerization of tannins, which have an acidic char-acter, would explain the progressive decrease in TSSand the increase in pH in samples treated with200MPa, which also had the highest number of anddarkest tannic structures after neutral red staining(Figure 3(b) and (c)). The greater structural damageobserved in the 400MPa treated samples may havefavored the diffusion of soluble tannins and hinderedtheir interaction and subsequent precipitation. Thiswould also explain their lower pH values and weakerneutral red staining compared to the 200MPa treatedsamples. During the last weeks of storage, neutral redstaining in persimmons treated at 400MPa was moreintense than on day 0 (Figure 3(e) and (f)), which mayindicate a higher level of tannin precipitation andwould agree with the decrease in TSS in these samplesat the same storage time.

Overall, TSS values progressively decreased in theHHP-treated samples during storage despite the signsof cell wall solubilization observed in the microstruc-tural study (Figure 2(b), (c), (h), and (i)). This couldindicate that either pectins interacted with other com-pounds to form insoluble complexes or their solubiliza-tion is compensated by precipitation of othercompounds such as tannins. Soluble tannins can forma complex with water-soluble pectin during fruit soften-ing (Taira et al., 1997). In the same way, solubilizationof pectins from the cell wall triggered by the HHP treat-ments could favor the formation of these complexesand subsequent insolubilization of tannins.

Color

Lightness (L*), chroma (C*), and hue angle (h) valuesdecreased progressively during storage in the untreated

samples, most markedly during the first two weeks ofstorage (Figure 7). The application of HHP triggered asignificant immediate decrease in L* and C* valueswhich continued during the entire storage period, com-pared to the untreated samples (Figure 7(a) and (b)).Significant differences (P< 0.05) appeared in the valuesof L* and C* between the two HHP-treated samples asof the third week of storage. These parameters followedan increasing trend in the samples treated at 200MPa,as opposed to a decreasing trend in the samples treatedat 400MPa. The same trend was observed in the hueangle values. However, h values of HHP-treated sam-ples were similar to those of untreated persimmons inthe first 7 days of storage (Figure 7(c)). Hue angle

L*C

*h

Days at 4 °C

Untreared(a)

(b)

(c)

70

60

45

30

15

0

90

85

80

75

70

60

50

40

30

200 MPa 400 MPa



0 7 14 21 28

Days at 4 °C0 7 14 21 28

Days at 4 °C0 7 14 21 28

Figure 7. Color parameters (L*, C*, and h) of untreatedand HHP-treated persimmons during 28 days of storagewith LSD intervals.


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decreased abruptly in the 400MPa treated samplesafter 14 days of storage; however, it increased at thispoint in the samples treated at 200MPa to values simi-lar to those of untreated samples for the remainder ofthe storage time.

The microstructural study also revealed changes incolor of the carotenoid fraction after the HHP treat-ments (Figure 2(d) and (j)), in particular between HHP-treated and untreated samples. Low values of L* in theHHP-treated persimmon Rojo Brillante have been pre-viously attributed to activation of browning enzymessuch as polyphenoloxidase (Hernandez-Carrion et al.,2014; Vazquez-Gutierrez et al., 2013). Carotenoidstructures observed at the end of storage in the400MPa treated samples were darker and larger thanthose observed in either other samples or earlier stagesof storage and could explain the lower values of L*, C*,and h registered in these samples.

CONCLUSIONS

Significant changes in tissue structure and physico-chemical properties took place in fresh-cut Hachiyapersimmon during HHP treatment and subsequentstorage, which were overall greater as higher pressurewas applied. Modifications of cell walls and mem-branes led to mechanical disruption of the foodmatrix, which may improve the extractability ofsome compounds. HHP treatment at 200MPa appearsto be a suitable treatment to preserve fresh-cutHachiya persimmon for 28 days. This treatmentkeeps the best physicochemical properties, preservesthe integrity of the carotenoids, and triggers tanninpolymerization in fresh-cut Hachiya persimmon,which could decrease the astringency of the product.Further microbiological and sensory analyses shouldbe carried out in order to determine the stability andacceptability of the product by the consumers andwhether astringency in Hachiya persimmon is removedby 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 withrespect to the research, authorship, and/or publication of this

article.

FUNDING

The author(s) wish to acknowledge the Spanish Ministry ofScience and Innovation for the PhD and mobility grants

(AP2007-03748) awarded to J.L. Vazquez Gutierrez.

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