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LWT - Food Science and Technology 60 (2015) 86e94

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LWT - Food Science and Technology

journal homepage: www.elsevier .com/locate/ lwt

Effects of thermal and high pressure treatments in color and chemicalattributes of an oil-based spinach sauce

Ilce Gabriela Medina-Meza a, *, Carlo Barnaba b, Filippo Villani c,Gustavo V. Barbosa-C�anovas a

a Center for Nonthermal Processing of Food, Washington State University, Pullman, WA 99164-6120, USAb Department of Chemistry, Washington State University, Pullman, WA 99164-4630, USAc Dipartimento di Scienze e Tecnologie Alimentarie e Microbiologiche, Universit�a degli Studi di Milano, 20133 Milano, Italy

a r t i c l e i n f o

Article history:Received 5 May 2014Received in revised form29 August 2014Accepted 8 September 2014Available online 18 September 2014

Keywords:High pressure processingOil-based spinach saucePolyphenoloxidaseLipid oxidation

Chemical compounds studied in this article:Ascorbic acid (PubChem CID: 54670067)chlorophyll a (PubChem CID: 44602414)chlorophyll b (PubChem CID: 46926108)

* Corresponding author.E-mail addresses: ilce.medinameza@wsu.ed

(I.G. Medina-Meza).

http://dx.doi.org/10.1016/j.lwt.2014.09.0330023-6438/Published by Elsevier Ltd.

a b s t r a c t

We studied the effects of high pressure processing (HPP) on physico-chemical characteristics of an oil-based spinach sauce model system. Color, chlorophylls, ascorbic acid, polyphenoloxidase activity andlipid oxidation (measured as % oleic acid, peroxide value, and p-anisidine content) were evaluated.Multivariate analysis of variance (MANOVA) and principal component analysis (PCA) were used toelucidate the contribution of pressure and time on the observed changes. Both pressure and time had asignificant effect, with pressure be responsible for most of lipid oxidation. Color attributes were pre-served or even improved after HPP, with 500 MPa for 10 min the treatment with the best results. Higherrecovery of ascorbic acid content, as well as chlorophylls a and b was achieved; HPP was not so effectivein triggering lipid oxidation. A storage study for 21 days at 4 �C was performed in order to assess thelong-term effects of thermal and HPP. During storage, lipid oxidation was drastically inhibited in HPP-treated samples. The degradation of PPO via high pressure was found to be reversible, since partial ac-tivity was recovered after 7 days. These preliminary results demonstrate the reliability of HPP as analternative to thermal treatment to preserve physico-chemical characteristics of oil-based vegetablesauces.

Published by Elsevier Ltd.

1. Introduction

Vegetable sauces are characterized by pH and activity watervalues which allow their marketability for a short period of time, inrefrigerated conditions, or for a longer time if pasteurization orsterilization technologies are employed. Conventional thermalprocesses have been used to inactivate bacteria and enzyme ac-tivity in highly perishable vegetables, showing high efficiency inthe inactivation of spoilage and pathogenic microorganisms. At thesame time, thermal treatments can affect the concentration ofantioxidants, vitamins, carotenoids and flavonoids, as well as theorganoleptic properties of vegetables (Da Cruz, Fonseca Faria, IsaySaad, Andr�e Bolini, & Cristianini, 2010). Many semi-preservedfoods, including soft cheeses, sliced sausages, pasteurized milk,and vegetable sauces that are subjected to thermal treatment, are

u, medina.ilce@gmail.com

unable to guarantee commercial sterility, and consequently mustbe stored under refrigerated conditions for a limited time (Baiano,Tamagnone, Marchitelli, & Nobile, 2005).

On the other hand, nonthermal processing technologies forfood preservation and safety are gaining widespread acceptancethroughout the food industry. An example is high hydrostaticpressure processing (HPP), a technology which can be appliedalone or together with other preservation methods as additionalhurdles (Barrera, Blenkinsop, & Warriner, 2012; Jacobo-Vel�azquez& Hern�andez-Brenes, 2012). Food treated in this way has beenshown to retain its original freshness, flavor and taste, allowingmost foods to be preserved with minimal effects on appearanceand/or nutritional value (Balasubramaniam, Farkas, & Turek,2008).

Recently, attention has been paid to the effects of HPP on thecolor quality of green vegetables (Clariana, Valverde, Wijngaard,Mullen, & Marcos, 2011; Oey, Lille, Van Loey, & Hendrickx, 2008).HPP could increase the intensity of green characteristics, as aconsequence of cell disruption and subsequent leakage of

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pigments. At the same time, high pressure produces reversible and/or irreversible changes in the protein structure of plant enzymes.Peroxidase (POD) and polyphenoloxidase (PPO), both responsiblefor color loss in fruits and vegetables (Yamauchi & Watada, 1991),are resistant to pressures of 600e700 MPa and 25 �C. PPO activityafter high pressure treatments depends on the studied fruit orvegetable (Lopez-Malo, Palou, Barbosa-Canovas, Welti-Chanes, &Swanson, 1998; Terefe, Buckow, & Versteeg, 2014), but is generallylower than POD (Terefe et al., 2014).

All of these results indicate that HPP can be potentially useful forretaining quality characteristics of vegetables. However, few studieshave been performed to investigatewhether the post-effects of HPPon chlorophyll degradation and lipid oxidation. Meanwhile, it hasbeen reported that HPP helps to retain antioxidant activity of in-dividual fruits juices (Cao et al., 2012), but there are contradictoryresults regarding retention of ascorbic acid, antioxidant and poly-phenols contents in foods processed by high hydrostatic pressureagainst thermally processed samples (Keenan et al., 2010).

In this work we evaluate the effects of high pressure processingon physico-chemical characteristics of a vegetable sauce modelsystem consisting in an oil-based spinach sauce. Spinach (Spinaciaoleracea) provides not only texture and color to food preparations,but it is also a good source of vitamin C, vitamin A, dietary fiber, andminerals, especially iron (Toledo, Ueda, Imahori, & Ayaki, 2003).Among other qualities, we evaluated color, ascorbic acid content,PPO residual activity and chlorophylls content, as well as lipiddegradation (free fatty acids, peroxides and p-anisidine) in HPPtreated samples and compared those results with spinach saucesubmitted to a traditional thermal treatment. Multivariate analysisof variance (MANOVA) was used to study the effect of pressure andtime on chemical and color parameters. To visualize the similaritiesand differences in overall impact of the different processes onspinach sauce quality, all analyzed parameters were broughttogether in a principal component analysis (PCA). We chose thetreatments which showed the best results in color and chemicalattributes to perform a 21 d storage which simulated commercialstorage conditions.

2. Materials and methods

2.1. Sample preparation

All ingredients were purchased in a local market (Pullman, WA,USA). Spinach sauce was prepared as follows: 72 g/100 g freshspinach, 25 g/100 g commercial extra virgin olive oil, 2 g/100 g citricacid and 1 g/100 g salt were combined and mixed in an Osterizer™blender, then the physicochemical properties of the fresh saucewere analyzed. Samples (100 g) were vacuum sealed inside flex-ible 3 mil, 75 mm thickness, polypropylene pouches 15 cm �24 cm � 4 cm (Ultravac Solutions, Kansas City, MO, USA) for ther-mal and high-pressure processing, to avoid any contact betweenthe pressurization fluid and the samples. PPO assays, pH andcolor characterization were performed immediately after process-ing; an aliquot of each sample was in an upright freezer at �45 �C,before vitamin C, lipid oxidation and chlorophylls analyses wereperformed.

2.2. High pressure processing

The pouches were placed inside the cylindrical chambervessel (0.1 m internal diameter, 0.25 m internal height) andprocessed with a high hydrostatic pressure unit (EngineeredPressure Systems, Inc., Andover, MA, USA). The unit was operatedwith an electrohydraulic intensifier pump (Hochdruck- SystemeGmbH, AP 10-0670-1116, Sigless, Austria). Come up time was

0.8 min and the process conditions were 400, 500, and 650 MPafor 5 and 10 min at 20 �C (indicated as 400-5, 500-5, 650-5 and400-10, 500-10 and 650-10 in the rest of the paper). The pres-surization fluid was 5% Mobil Hydrasol 78 water solution.Depressurization time was less than 10 s, which was not includedin the process time. Three samples per cycle were treated. Thepressurization samples were cooled immediately in a cold waterbath to avoid further reaction.

2.3. Thermal treatment

Pouch samples were subjected to thermal heating (TT in the restof the paper) by immersing in a water bath (Memmert GmbH,WB22, Schwabach, Germany) for 30e45 s at which time they hadachieved a core temperature of 70 �C. They were held at thistemperature for 10 min. The temperature of the sauce during theheat process was monitored using K-type thermocouples con-nected to a datalogger (Jenco, Model 7000 APL) inserted through aseptum glued onto the outer surface of the pouches. Then thesamples were taken out and immediately immersed in ice water inorder to preserve the residual activity of the enzymes.

2.4. pH and color

The pH was determined by direct immersion of the electrodewith a potentiometer (Orion Research Inc., Boston, Massachusetts,USA).

Color determination was carried out using a CR-200 spectro-photometer (Minolta Camera Co., Osaka, Japan) equipped with astandard illuminant D65. The instrument was calibrated using awhite color tile standard. L* (lightness), a* (redness), b* (yellow-ness), C* (chroma, 0 at the center of the color sphere), and H� (hueangle, red ¼ 0�, yellow ¼ 90�, green ¼ 180�, blue ¼ 270�) werequantified on each sample using a 10� position of the standardobserver (CIE, Paris, France, 1978). Ten measurements were con-ducted on random points on at least 3 samples.

The net color difference (DE*) was determined using L*, a* and b*values, comparing them with the value of an unprocessed sample:

DE* ¼ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiðDL*Þ2 þ ðDa*Þ2 þ ðDb*Þ2

q(1)

Hue angle (H�) was determined using the followingrelationship:

H� ¼ tan�1

�b*

a*

�(2)

and the chroma or saturation index (C*) was evaluated using theequation:

C* ¼ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiða*Þ2 þ ðb*Þ2

q(3)

2.5. Extraction of fat fraction and lipid oxidation analyses

A sample (10 g) was placed in a flask with 50 mL of n-hexaneand mixed using a magnetic stir for 5 min. The organic fractionwasseparated from the spinach leaves by vacuum filtration with aWhatman No. 41 paper filter. Then the filtered organic fraction wasrecovered and filtered again on anhydrous sodium sulfate toremove water traces. After filtration, the organic solvent wasevaporated using a Rotavapor (model Buchi 461). The extracted oilwas subjected to the following analyses:

Table 1Multivariate analysis of the effect of single and combined variables (time andpressure) on each color parameter considered. Values represent probability (p); ap < 0.05 (in bold) is considered significant.

L* a* b* H* C*

Time 0.000 0.000 0.000 0.000 0.000Pressure 0.086 0.002 0.000 0.000 0.000Pressure*Time 0.000 0.000 0.000 0.014 0.000

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a) Acidity was determined by titrationwith 0.1 mol equi/L NaOH inthe presence of phenolphtalein to evaluate the free fatty acidscontent; acidity was expressed as grams of oleic acid per 100 g ofoil (ECC, 1991).

b) Peroxide value (POV) was determined by titration with 0.01 Nsodium thiosulphate in the presence of potassium iodide toevaluate hydroperoxides formation (primary oxidation);peroxide value was expressed as milliequivalents (mEq) ofactive oxygen per kilogram of oil; according to ECC 1991 withslight modifications. After the extraction, 25 mL of an aceticacid: chloroform (3:2 mL/mL) solution was added in a flask tothe sample along with 0.5 mL of saturated solution of potassiumiodide. Then the mix was gently shaken and kept in the dark for5 min. After that, 75 mL of distillated water was added alongwith 2 mL of 1 g/100 mL starch solution.

c) p-Anisidine value (PAV) was measured according to AOCS Offi-cial Methods (AOCS, 1993) in order to evaluate the secondaryoxidation (hydroperoxides decomposition); measurementswere made by dissolving the oil samples in 2,2,4-trime-thylpentane and reading the absorbance at 350 nm before andafter the addition of the p-anisidine reagent.

2.6. Determination of ascorbic acid (vitamin C)

The vitamin C content in spinach sauce was determined bydirect titration with iodine according to (Keenan, R€oßle, Gormley,Butler, & Brunton, 2012) with slight modifications.

Spinach sauce (10 g) was transferred into a 250 mL Erlenmeyerflask. Then 25 mL of sulfuric acid 2 mol equi/L was added, mixedand diluted with 100 mL of distilled water. The mixture was vac-uum filtered with Whatman No. 1 filter paper and then 3 mL of 1 g/100 mL starch solution was added as indicator. The solution was

Chla�mg

�g dry matter

�¼ ½ð12:7ÞðAbs663Þ � ð2:6ÞðAbs645Þ�ðml AcetoneÞ

mg dry matter

Chlb�mg

�g dry matter

�¼ ½ð12:7ÞðAbs645Þ � ð2:6ÞðAbs663Þ�ðml AcetoneÞ

mg dry matter

directly titrated with 0.1 mol equi/L iodine solution previouslystandardized. A blank titration was performed prior to titration ofeach sample (n ¼ 3). Each mL of 0.1 mol equi/L iodine is equivalentto 8.806 mg ascorbic acid.

2.7. Polyphenoloxidase (PPO) enzymatic activity

Enzyme activity of polyphenol oxidase (PPO) was assessed ac-cording to the method of Jung, Lee, Kim, and Ahn (2013) with slightmodifications. Spinach sauce (10 g) was mixed with 40 mL of0.2 mol/L phosphate sodium buffer (pH 6.5) for 30 min at 4 �C. Thesupernatant was collected to determine PPO activity by centrifu-gation at 13,000 � g for 20 min at room temperature. The collectedsupernatant (0.1 mL) was reacted with 2 mL of standard buffer (pH7.0) containing 2 mL of 0.1 g/100 mL catechol and 0.1 mL enzymeextract. Catechol was oxidized by PPO present in the extract,forming the brownish compound o-quinone. The absorbance was

measured at 420 nm for 2 min at 25 �C using a DU 640B spectro-photometer (Beckman, USA). The specific activity PPO wasexpressed as the rate of linear change in absorbance over reactiontime (A/min). The residual activity (RA) of PPO was estimated usingthe following equation:

RAð%Þ ¼ AAcontrol

� 100

where Acontrol and A are the specific enzyme activities in the control(raw sample) and the treated sample, respectively.

2.8. Chlorophylls content

Chlorophylls content was determined according to the methoddescribed by Albanese et al. (Albanese, Russo, Cinquanta, Brasiello,& Di Matteo, 2007) with slight modifications. 5 g of sample werehomogenized in 10 mL of 80 mL/100 mL acetone solution at 4 �Cwith a shaker bath for 20 min and centrifuged at 13,500 � g (Bio-fuge-Primo, Italy). The supernatant was filtered and diluted to10 mL with 80 mL/100 mL acetone. Absorbance (A) was measuredat 645 and 663 nm using a DU 640B spectrophotometer (Beckman,USA). Chlorophyll a and b concentrations were calculated asfollows:

2.9. Statistical analyses

All analyses were carried out in triplicate, except for colormeasurements where six replicates were performed. Means andstandard deviations were calculated with SPSS (Version 22.0, SPSSInc., Chicago, IL) statistical software. SPSS was used to performmultivariate analysis of variance (MANOVA) and PrincipalComponent Analysis (PCA). Tukey's honest significant differencetest (HSD) at a 95% confidence level (p < 0.05) was used todiscriminate among different samples.

3. Results and discussion

3.1. Effect of HPP and thermal treatments

Color is an important quality characteristic and a major indexaffecting sensory perception and consumer acceptance of foods,

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especially for fresh or processed green vegetables (Steet & Tong,1996). However, as the most widely distributed green vegetablepigments, chlorophylls are highly susceptible to enzymatic or non-enzymatic degradation during processing and storage (Monreal, DeAncos, & Cano, 1999; Yamauchi & Watada, 1991), and the degra-dation pathway is regulated by the peroxidase-hydrogen peroxidepathway, which opens the chlorin ring, thus resulting in a colorlesscompound (Yamauchi & Watada, 1991).

In our experiments, color assessment was obtained throughCIELAB scale analysis. L*, a* and b* values were used to obtain hue(H

�) and chroma (C*), as well as DE, which permitted rapid com-

parison between different treatments. It is known that HPP treat-ments have limited effect on pigments, but this could be amplifiedduring storage, due to incomplete inactivation of enzymes andmicroorganisms (Oey et al., 2008).

MANOVA on color characteristics showed that time and pres-sure, as well as their interaction, had significant effects on colorchanges (p < 0.001). Considering each response separately, the ef-fect of pressure on L* valuewas not significant (p¼ 0.086), but it didin the other parameters considered (Table 1). As shown in Fig. 1A,after thermal and high-pressure treatments the green componentof color became less intense (decrease in L*), indicating that saucecolor was changing from light green to dark green. The onlyexception is represented by 500-10, in which no significant

Fig. 1. Color parameters value for treated and untreated spinach sauce: A) luminosity(L*), a* and b* values; B) DE values. * ¼ p < 0.05, ** ¼ p < 0.01; aef, AeE: identicalletters indicate that the samples are not statistically different, according to an analysisof variance and Tukey's multiple mean comparison test (n ¼ 3, p < 0.05).

differencewas observed (p > 0.05). At the same time, the significantincrease in b* observed for 500-5 and 650-5 samples indicated acorresponding increase in the yellow component. In order to decidewhich of the performed treatments would be used for storage ex-periments, we calculated the total color difference (DE), which is acombination of L*, a* and b* values obtained (Fig. 1B) through theapplication of Equation 1. As shown, only 400-10, 500-10 and 650-10 showed lower differences compared with a pasteurized sample,whereas 500-5 was the treatment which caused greater degrada-tion in color. In Fig. 2, H

�and C* values are plotted: thermal treated

samples showed lower tonality, again indicating a shift to yellow,compared to fresh samples. This is due to heat that catalyzes thedegradation of chlorophylls to pheophytins (Van Loey et al., 1998).Among high pressure treatments, the deviation from freshappearance is due to a synergistic effect of both pressure and timeof processing (as from MANOVA analysis), resulting in a greaterdifference for the sample treated at 500 MPa for 10 min. However,when saturation values among samples is compared, the applica-tion of 500 MPa for 10 min results in a slight increase of thischaracteristic, indicating that is comparable with a fresh sample.

Fig. 2. Hue angle (H� , top) and chroma value (C*, bottom) of treated and untreatedspinach sauce.

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The interpretation of the spatial distribution of the PCA scoresseems coherent with the results obtained. The biplot for the firsttwo principal components (accounting for 96.94% of the totalvariance) is shown in Fig. 3A. TT sample is separated from the

Fig. 3. PCA plot for color and chemical determination in treated and untreated spinachsauce: A) color analysis; B) ascorbic acid, chlorophylls a and b, PPO residual activity; C)lipid oxidation.

others and opposite to Control. Samples 650-5, 650-10 and 400-10are clustered together, whereas 500-5 and 650-5 are scattered inrespect to Control. 500-10 and Control sample although not clus-tered, are closer in the PCA plot.

Regarding physico-chemical analyses, thermal treatment wasthe most aggressive for all the considered parameters (Table 2). Aslight increase in pH was observed in the high pressure processedsamples (data not shown), probably due to cell rupture that causedthe liberation of organic acids and/or to a shift in chemical equi-librium due to a pressure-induced effect. However, the increase inpH values cannot be attributed to ascorbic acid content. In fact, thecontent of vitamin C decreased dramatically in the thermal treatedsample, although a discrete reduction was observed in pressure-treated samples. Several authors (Patras, Brunton, Da Pieve, &Butler, 2009; Sanchez-Moreno, Plaza, de Ancos, & Cano, 2006) re-ported that pressure above 500 MPa improved the retention ofascorbic acid in vegetables. In our study, ascorbic acid retentionwasbetween 70 and 80% in HP treated samples, with the exception of400MPae5min, where no significant degradation of vitamin C wasobserved. Conversely, only the 30% of ascorbic acid was retained inthermal treated spinach sauce. Recently, it has been found that HPPcould increase the ascorbic acid content in spinach leaves, although500 MPa caused a significant decrease in ascorbic acid content,suggesting that there is a matrix effect on the degradation of thisvitamin (Jung et al., 2013).

Our results with spinach sauce during thermal treatment anddifferent pressures showed stability of chlorophylls at lower pres-sures, as previous reported (Butz et al., 2002). As Table 2 shows, anincrease of chlorophyll a content at low pressure can be observed(400 MPa), attributable to pressure-induced extraction, whereas atstronger treatment the degradative/accumulative effects compen-sated and the pigment content was stable. Contrarily, chlorophyll bwas stable under high-pressure conditions. Hence, high-pressuretreatments reduced chlorophylls degradation and consequentlygreen color losses of processed vegetable sauces. It is interesting tonotice that the significant decrease in chlorophyll a for 500-5 iscorrelated to the observed color degradation (Fig. 1). This pressure-resistance of chlorophylls can be attributed to the stability of thepigment structure to high pressure due to the negligiblecompressibility of covalent bonds (Tauscher, 1995; Weemaes et al.,1999).

Poliphenoloxidase (PPO) activity results in enzymatic browningof damaged fruits and vegetables, resulting in changing in theappearance and organoleptic properties (Hendrickx, Ludikhuyze,Van den Broeck, & Weemaes, 1998). Thus, PPO inactivation ishighly desirable and HPP can be used as an alternative to thermaltreatment. The residual activities of PPO in spinach sauce afterthermal processing and HPP were estimated and compared to thecontrol (Table 2). Thermal treatment had a higher impact on theactivity of this enzyme, with about 45% of residual activity. AmongHPP treatments, higher processing time caused enzyme activationin 500 and 650 MPa treated samples (61% and 59%, respectively), inagreement with results reported by other studies in vegetables andfruits treated with pressure above 500 MPa (Garcia-Palazon,Suthanthangjai, Kajda, & Zabetakis, 2004; Gomes & Ledward,1996).

The barostability of enzymes is variable, depending on source,time and value of applied pressure; exhaustive reviews of highpressure enhancement of enzymes was provided by Eisenmengeret al. (Eisenmenger & Reyes-De-Corcuera, 2009) and more recentlyby Terefe et al. (Terefe et al., 2014). Peroxidase, catalase, phospha-tase and PPO are resistant to pressures of 600e700 MPa at 25 �C,and in PPO appreciable differences in pressure resistance aredependent on the plant source. The enhancement in activity washypothesized to be from pressure-induced changes in interactions

Table 2Physico-chemical characteristics of fresh (control), thermal (TT) and high-pressure treated oil-based spinach sauce.

Control TT 400-5 400-10 500-5 500-10 650-5 650-10

pH 5.5 ± 0.01a 5.5 ± 0.02a 4.9 ± 0.04bc 4.9 ± 0.02cde 4.8 ± 0.01de 4.8 ± 0.03e 5.0 ± 0.02b 4.9 ± 0.02bcd

Ascorbic acid (mg/100 g) 30.7 ± 2.0a 9.38 ± 0.18d 30.77 ± 2.6a 24.25 ± 0.8bc 25.9 ± 0.7b 20.8 ± 2.3c 23.0 ± 2.1bc 23.5 ± 0.8bc

PPO (% residual activity) 100a 44.6 ± 5.0cd 51.9 ± 4.6bc 48.7 ± 2.3bc 34.2 ± 1.1cd 61.1 ± 1.0b 46.0 ± 5.7c 59.2 ± 2.0b

chlorophyll a (mg/g) 1.6 ± 0.1cd 1.4 ± 0.2cd 2.5 ± 0.3a 2.5 ± 0.2ab 1.8 ± 0.4c 1.8 ± 0.2bc 0.9 ± 0.4cd 1.2 ± 0.2cd

chlorophyll b (mg/g) 0.9 ± 0.2a 1.4 ± 0.0a 1.4 ± 0.6a 1.2 ± 0.3a 1.1 ± 0.1a 1.2 ± 0.2a 0.7 ± 0.4a 0.6 ± 0.4a

Acidity (% oleic acid) 4.3 ± 1.1a 3.7 ± 0.7a 3.7 ± 1.7a 5.4 ± 2.4a 3.9 ± 0.3a 2.1 ± 0.3a 2.5 ± 0.3a 3.0 ± 0.3a

POV (meqO2/kg) 3.5 ± 0.7e 4.8 ± 0.7cd 7.9 ± 0.3a 5.9 ± 0.5bc 6.9 ± 0.2ab 6.5 ± 0.2bc 6.5 ± 0.4b 4.0 ± 0.1de

p-Anisidine 1.3 ± 0.2d 8.8 ± 0.4a 9.2 ± 1.9a 6.2 ± 0.6bc 6.6 ± 0.7b 4.7 ± 0.4bc 4.7 ± 0.2bc 3.8 ± 0.2bc

Values are expressed as the mean ± standard deviation.a-d: identical letters within a single raw indicate that the samples are not statistically different, according to an analysis of variance and Tukey's multiple mean comparison test(n ¼ 3, p < 0.05).

Table 3Multivariate analysis of the effect of single and combined variables (time and pressure) on each chemical parameter considered. Values represent probability (p); a p < 0.05 (inbold) is considered significant.

Ascorbic acid PPO Chlorophyll a Chlorophyll b Acidity POV p-Anisidine

Time 0.041 0.000 0.452 0.679 0.960 0.106 0.040Pressure 0.079 0.304 0.000 0.049 0.071 0.035 0.029Pressure*Time 0.417 0.000 0.546 0.775 0.043 0.639 0.680

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with other constituents in the extract or from the release ofmembrane-bound enzymes. However, it is recognized that pres-sures higher than 700 MPa are needed to completely inactivatePPO.

Lipid oxidation under high hydrostatic pressure has beenscarcely studied; however, during the past few years, the impor-tance of oxidative degradation in qualities and organoleptic prop-erties of processed food have received increased attention (Medina-Meza, Barnaba, & Barbosa-C�anovas, 2014). Oil-based sauces aresusceptible to auto-oxidation or, if subjected to heat processing, tothermo-oxidation, and the extent of these reactions could be

Fig. 4. Color evaluation of spinach sauce during 21 days of storage: luminosity (L*, A), a* valu400 MPa for 10 min (400-10); , 500 MPa for 10 min (500-10); 600 MPa for 1

mitigated or triggered by the presence of antioxidants or pro-oxidants, respectively. In this study, no difference in free fattyacids content was observed, but a significant increase in POVindicated that different processes promoted the first stages of lipidoxidation. As expected, the thermal treated sample had higherperoxides value; however, when spinach sauce was subjected tolower pressure, an appreciable increase of primary oxidationproducts was observed (Table 2). As previously reported, highpressure did not promote triglycerides hydrolysis, since bondscission results in volume expansion that is thermodynamically notfavored in HPP (Medina-Meza et al., 2014; Tauscher,1995). Focusing

es (B); hue angle (H� , C), and chroma values (D): , thermal treatment (TT); ,0 min (600-10).

Fig. 5. Chemical parameters of spinach sauce during 21 of storage: ascorbic acid (A),chlorophyll a (B) and chlorophyll b (C): , thermal treatment (TT); , 400MPa for10min(400-10); , 500MPa for10min(500-10); , 600MPa for10min (600-10).

I.G. Medina-Meza et al. / LWT - Food Science and Technology 60 (2015) 86e9492

only in HPP-treated samples, MANOVA analysis showed that timeand pressure had a different effect on the considered chemicalvariables. From Table 3, it could be evinced that chlorophylldegradation and lipid oxidation are pressure-dependent, whereasthe loss of ascorbic acid and the denaturation of polyphenoloxidaseare most likely time-dependent. Finally, the slight increase in oleicacidity observed could be attributed to a synergic effect betweenthe two processing variables. As from PCA analysis, more drastictreatments are clustered together (Fig. 3B), whereas lower pressureprocessing seemed to be less relevant to chlorophylls and vitamin Ccontent. Regarding lipid oxidation, a similar pattern was observed:beside TT, the application of 650 MPa is correlated to an increase insecondary oxidation products (PAV), whereas lower pressure (400-5 and 400-10) increased hydroperoxides formation.

3.2. Effects of HPP and thermal treatments during storage

Considering the preliminary results described above, wedecided to realize a time-dependent evaluation of qualitative andphysico-chemical parameters, maintaining the treated samples at4 �C for 21 days.

Color differences between conventional and pressure-processedspinach sauce were determined during storage. High-pressuretreatments scarcely affected color in spinach sauces during stor-age, preserving the green characteristics of the samples. The mainchanges were observed in the L*, a*, hue angle (H�) and chromavalues (C*) (Fig. 4). The yellow component (b* value) of spinachsauce remained almost constant during storage (data not shown),as previously reported for guacamole sauce (Lopez-Malo et al.,1998) and beans (Krebbers, Matser, Koets, & Van den Berg, 2002)processed by hydrostatic pressure. The reduction in the L* value inthe 500 MPa test is less important compared to 400 and 650 MPa(Fig. 4A). The increase of a* value (from �17 to �8) in the thermaltreated sample confirmed the loss of green characteristics, turninginto an olive-green color that was stable until the end of the storageperiod (Fig. 4B). However, among pressure treatments, no signifi-cant variation in tonality was observed, indicating that HPP dras-tically improved overall color stability in spinach sauce. Asexpected, color vividness decrease was more important in thethermal treated sample; however, a significant difference amongpressure treatments was found, being again at 500 MPa, the onethat better preserved this characteristic.

Due to the significant differences already observed after thetreatments, data regarding chemical parameters were normalizedfor the values corresponding to time 0. Fig. 5 shows the kineticbehavior of ascorbic acid and chlorophyll a and b during 21 daysstorage. Comparing high-pressure treatments, ascorbic acid re-covery was higher when lower pressure was applied (z80% ofinitial value at the end of the storage period), contrary to resultsfound by other authors in beans (Krebbers et al., 2002). In highpressure-treated samples, degradation is marked during the firstweek, followed by a plateau. Ascorbic acid degradation after HPPhas previously been reported and the high retention observed hasbeen attributed to the pressure-mediated inactivation of endoge-nous pro-oxidative enzymes (Oey et al., 2008).

Regarding chlorophylls, a progressive decrease of pigmentscontent was observed in all samples, probably due to a coupledlipid auto-oxidation that could justify the conversion of chloro-phylls to pheophytins (Van Loey et al., 1998; Yamauchi & Watada,1991). It seems unlikely to attribute chlorophyll degradation toenzymatic activity (primarily lipoxygenase), due to the pressure-induced inactivation of these enzymes. High retention of chloro-phylls agreed with tonality retention observed in color analysis.

Lipid oxidation during storage has been extensively reported forthermal processed foods, being the most important factor in

organoleptic deterioration ofmeat and fish products (Medina-Mezaet al., 2014). Vegetable sauces are generally prepared using higholeic vegetables oils, such as olive and sunseed oils, which are verysusceptible to radical attack, followed by oxygen incorporation and

I.G. Medina-Meza et al. / LWT - Food Science and Technology 60 (2015) 86e94 93

subsequent formation of primary and secondary products ofoxidation. In our study, we performed a time-course analysis ofperoxides (POV) and secondary products (reported as p-anisidinevalue) in order to assess the potential of HPP in reducing

Fig. 6. Peroxides content (POV, A), p-anisidine value (B) and polyphenoloxidase (PPO)residual activity (C) of spinach sauce during 21 days of storage:fx1, thermal treatment(TT); fx2, 400 MPa for 10 min (400-10); fx3, 500 MPa for 10 min (500-10); fx4,600 MPa for 10 min (600-10).

organoleptic degradation of oil-based spinach sauce. In all treat-ments, peroxide degradation was constant after 3 days of storage(Fig. 6A), probably due to the depletion of oxygen in the sealedpackages. However, considering secondary products (primarilymalonaldehyde and other short-chain derivatives), lower pressureswere not capable to enhance their formation (þ40% in 400-10 and500-10 at 21 days of storage), whereas 650-10 caused an increase ofabout 80% from the initial content, but not in the same extent as TT(þ130%). This result confirmed the opportunities offered by highhydrostatic pressure in oil-based sauces preservation.

The inactivation of PPO seemed to be partially reversible whenobtained through the application of hydrostatic pressure. As shownin Fig. 6C, residual activity after thermal treatment was about 4%after 1 day of treatment and was maintained constant through thestorage period. However, in HPP-treated samples, a significant in-crease (p < 0.01) in activity was observed after 7 day of storage,indicating that PPO could recover its catalytic potential overtime.This behavior could be attributed to HPP-induced stabilization orchanges in interactions with other constituents in the extracts, asobserved for mushroom, avocado and tomato, among others(Eisenmenger & Reyes-De-Corcuera, 2009).

4. Conclusions

The results obtained in this work demonstrate the reliability ofhigh hydrostatic pressure treatments as an alternative to thermalprocessing to preserve physico-chemical characteristics of a vege-table oil-based sauce, namely spinach sauce. Color attributes weremaintained or even improved after HPP, with 500 MPa for 10 minbeing the treatment with the best results. Higher recovery ofascorbic acid content, as well as chlorophylls, was obtained; at thesame time, pressures below 650 MPa were not effective in trig-gering lipid oxidation. The degradation of PPO via HPP was found tobe reversible, since partial activity was recovered after 7 days ofstorage. Further research is needed to evaluate the effectiveness ofHPP in microbiological safety, as well as in preserving organolepticproperties through sensorial analysis.

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