Innovative Food Science and Emerging Technologies 11 (2010) 557–564

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Innovative Food Science and Emerging Technologies

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Effect of high pressure processing on the quality of acidified Granny Smith applepurée product

A. Landl a, M. Abadias b, C. Sárraga a, I. Viñas c, P.A. Picouet a,⁎a IRTA-TA, Food processing and Engineering, Finca Camps i Armet, 17121 Monells (Girona), Spainb IRTA, UdL-IRTA Centre, XaRTA-Postharvest, 191 Rovira Roure, 25198 Lleida, Catalonia, Spainc University of Lleida, UdL-IRTA Centre, XaRTA-Postharvest, 191 Rovira Roure, 25198-Lleida, Catalonia, Spain

⁎ Corresponding author. Tel.: +34 902 789 449x1450E-mail address: pierre.picouet@irta.es (P.A. Picouet)

1466-8564/$ – see front matter © 2010 Elsevier Ltd. Aldoi:10.1016/j.ifset.2010.09.001

a b s t r a c t

a r t i c l e i n f o

Article history:Received 26 March 2010Accepted 13 September 2010

Editor Proof Receive Date 8 October 2010

Keywords:Commercial-scale high pressure processingMild thermal pasteurizationGranny Smith apple puréeNutritional qualityVitamin C degradationMicrobial qualityStorageShelf life

The aim of the present study was the evaluation of the physicochemical, nutritional and microbial quality ofacidified Granny Smith (GS) apple purée processed on industrial-scale high pressure system during 3 weeks ofrefrigerated storage (5 °C±1 °C). Two commercially feasible pressure treatments (400 and 600 MPa/5 min/20 °C) and a mild conventional pasteurization at 75 °C/10 min, with pasteurization values of P70°C7.5 =8.15 min,were conducted and their effect on total vitamin C (total Vit C), ascorbic acid (AA) and total phenolic content(TPP), and on instrumental quality parameters (color, viscosity, soluble solids, titratable acidity and pH) werecomparatively studied. Inactivation of indigenousmicroorganisms (total aerobic mesophilic and psychrotrophiccounts and moulds and yeasts) of the apple product was also studied and monitored during storage. Total Vit Cand AA contents were unaffected by the 400 MPa and the mild pasteurization treatment. TPP content was notchanged during processing at 400 MPa, but was affected by the 600 MPa and also slightly by the pasteurizationtreatment. Experimental data on the loss of total Vit C during storage were described with a first-order reactionkinetic and times of half loss between 9.3 to 10.3 days could be estimated for the three studied processes. Storageprovoked loss of TPP content and color deterioration of pressurized GS puree samples, which was attributed toenzymatic browning reactions. Microbial counts were reduced by the different preservation techniques belowthe detection limit (50 cfu g−1) and storage revealed no further growth.Industrial relevance: This is one of the first studies applying commercial industrial-scale high pressure equipmentfor the pasteurization of an acidified apple purée product. The pressures of 400 and 600 MPa with 5 min holdingtime at ambient temperature render economically feasible processes with high throughput and productivity. Inthe European Union the most important fruits in terms of production are apples. Apple purée is a largelyconsumed preserve in many households and beside apple juice or cider is one of the most important appleproducts in the market. In contrast to traditional apple purée preparation, high pressure processing or mildthermal treatments could imply new opportunities for the apple processing industry in developing more fresh-like, value-added apple products with reasonable shelf life.

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1. Introduction

Nowadays, consumers are demanding new ready-to-eat products(e.g. apple desserts), which provide increased convenience, whilemaintaining fresh-like characteristics of flavour, texture, color,vitamin content, aroma and overall appearance, along with reason-able or extended shelf life. Shelf life and food quality are closelyrelated tomicrobial quality and other phenomena such as biochemicaland enzymatic reactions. To successfully address the new challengesfaced by the food industry interests in the research on minimalpreservation techniques, like high pressure processing (HPP) as well

as mild thermal treatments, on fruit and vegetable produce have beenpromoted.

The principles of high pressure processing of food have beenbroadly discussed by several authors since it was first reported byHite,1899. High pressure processing (HPP) at room temperature is nowknown as an alternative nonthermal food preservation method thataffects the viability of microbial cells and the structure of proteins/enzymes (Basak & Ramaswamy, 1998; Palou, López-Malo, Barbosa-Cánova, & Swanson, 2007; Palou, Lopez-Malo, Barbosa-Canova, Welti-Chanes, & Swanson, 1999; Rastogi, Raghavarao, Balasubramaniam,Niranjan, & Knorr, 2007), while leaving low molecular weight foodcompounds, such as vitamins, pigments, flavouring agents and othercompounds related to sensory, nutritional and health related qualitiesof the product, greatly unaffected (Butz et al., 2003, Fernández García,Butz, Bognàr, & Tauscher, 2001; Fernández García, Butz, & Tauscher,

558 A. Landl et al. / Innovative Food Science and Emerging Technologies 11 (2010) 557–564

2001; Nienaber & Shellhammer, 2001a,b; Oey, Lille, Van Loey, &Hendrickx, 2008). The effect of HPP on the quality characteristicsof food has been mainly attributed to the stability of covalent bondsto high pressure (Knorr, 1993). Unlike in thermal treatments, thetransmittance of pressure is instantaneous and uniform throughoutthe entire food, regardless of its size or shape (Palou et al., 2007;Rastogi et al., 2007). Moreover, thermal processes can often lead toquality changes in foods such as the destruction of vitamins,modifications to food texture and color and the development of off-flavours (Norton & Sun, 2008).

Traditional apple purée preparation involves high temperatureprocessing during prolonged time and affects the nutritional quality ofthe raw material adversely. In industrial apple purée processing,trimmed and cored apples are chopped and precooked for 4–5 min atabout 96 °C and then, after finishing and filling, closed containers arepasteurized for 10–15 min in boiling water to ensure destruction ofspoilage organisms (Downing, 1989). The precooking process has toassure total inactivation of the endogenous polyphenol oxidase enzyme(PPO), the enzyme mainly responsible for the development ofundesirable oxidative browning especially in purée products (Chisari,Barbagallo, & Spagna, 2007; Cocci, Rocculi, Romani, & Dalla Rossa, 2006;Palou et al., 1999; Quiles et al., 2005; Rocha & Morais, 2002; Weemaes,Ludikhuyze, Van den Broeck, & Hendrickx, 1998;Weemaes et al., 1997).The effect of HPP on enzymes related to food quality was extensivelyreviewed by various authors (Ludikhuyze, Van Loey, Indrawati, Denys, &Hendrickx, 2002; Palou et al., 2007; Rastogi et al., 2007). To date,researchers have found that, upon pressurization, PPO may displayeither inactivation or enhanced catalytic activity (Guerrero-Beltrán &Barbosa-Cánovas, 2004; Palou et al., 2007). The pressure level needed forPPO inactivation at ambient temperature is strongly dependent on theplant origin, the pH of the medium and the environmental conditionssuchas thepresence of salts, sugars, or other additives (Ludikhuyzeet al.,2002; Weemaes et al., 1998). The surrounding environment andthe uniformity of the matrix, are also crucial for microbial inactivation(Palou et al., 2007; Rovere, 2002). Nowadays, the food industry uses avariety of techniques in combination with HPP (hurdle technology)to reduce or control enzyme activity during processing and storage,e.g. high pressure combined with low pH, refrigerated storage,oxygen exclusion or the addition of antibrowning agents, ratherthan one preservation method alone, to successfully preserve foodquality (Guerrero-Beltrán, Barbosa-Cánovas, Moraga-Ballesteros,Moraga-Ballesteros, & Swanson, 2006; Rovere, 2002). Acidification,e.g. by means of citric acid, could lead to an increase in the pressuresensitivity of the inactivation rate constant (Ludikhuyze et al.,2002).

The color of many fruit and vegetable products such as jams, fruitjuices and purée is generally preserved during HPP at ambienttemperature (Guerrero-Beltrán & Barbosa-Cánovas, 2004; Guerrero-Beltrán et al., 2006; Guerrero-Beltrán, Swanson, & Barbosa-Cánovas,2005; Ludikhuyze & Hendrickx, 2002) indicating its limited effect onpigments (e.g. chlorophyll, carotenoids, anthocyanins, etc.) responsiblefor the color of fruits and vegetables (Ahmed, Ramaswamy, &Hiremath,2005; Oey et al., 2008; Weemaes et al., 1999). In comparison,chlorophyll pigments are very susceptible to degradation at tempera-tures higher than 50 °C or during combined HP-thermal treatments(Oey et al., 2008;Weemaes et al., 1999). However during storage of HPPpurée products, certain quality constraints have been attributed toenzymatic browning reactions because of residual PPO activity(Guerrero-Beltrán & Barbosa-Cánovas, 2004; Palou et al., 1999;Polydera, Stoforos, & Taoukis, 2005).

A lot of researchwork in the field of HPP evaluated the effect of highpressure on the microbiological stability and on the retention ofnutritional quality attributes during processing and storage of acidfruit juices (Fernández García, Butz, Bognàr, et al., 2001; FernándezGarcía, Butz, & Tauscher, 2001; Lavinas, Miguel, Lopes, & ValenteMesquita, 2008; Nienaber & Shellhammer, 2001a,b; Polydera, Stoforos,

&Taoukis, 2003;Polydera et al., 2005; Sánchez-Moreno, Plaza, DeAncos,& Cano, 2003). The relative pressure sensitivity of vegetative cells,including yeasts and moulds, in combination with the intrinsicpreservation factor of low pH has made the preservation of acid foods(pH≤4.5), where most spoilage microorganisms are controlled andspore formers cannot grow, the most obvious application of HPP (Palouet al., 2007; Rastogi et al., 2007; Smelt, Hellemons, & Patterson, 2002).

HP treatment can however affect the rheological properties of foodproducts such as crushed fruits and vegetables, purée, pulp and juice.The observed effects are dependent on the conditions of the HPprocess and the type of fruit and vegetable (Oey et al., 2008).

The industrial relevance for the food processing industry wascontinuously growing over the last twodecades, stimulated also by thenumerous studies of different research groups, who pointed out thepotential use of this new processing technique. The first pressure-treated products (fruit jams) were introduced in the Japanese marketin 1991. Since then, the commercial use of HPP and the totalproduction of pressure- treated food products has been expanding,at the same time as equipment manufacturers have improved, inresponse to increase equipment productivity (Balasubramaniam,Farkas, & Turek, 2008; Rovere, 2002). To date, the most successfulapplications nowon themarket range from the in-pack preservation ofguacamole (avocado sauce), sliced meat products (salami, cookedham, cured ham), chicken or turkey breasts, orange juice, smoothies,and shellfish (oysters). Despite the increased commercial applications,to our knowledge limited data are available on the effect of industrial-scale HPP on the shelf life of fruit and vegetable products, as mostresearch groups focused their work on laboratory scale equipments.

The aimof the presentworkwas to compare the effectiveness of twoeconomically feasible highpressure processes at 400 MPa and 600 MPa/5 min/20 °C with industrial-scale equipment (Hyperbaric Wave 6500/120, N.C. Hyperbaric, S.A., Burgos, Spain) on acidified Granny Smithapple purée product containing ascorbic acid with a desired shelf life of2–3 weeks at refrigeration conditions. The effect of HPP was evaluatedon physicochemical (color, texture, soluble solids, titratable acidity andpH) and nutritional quality parameters (ascorbic acid and total phenoliccontent, TPP) and compared with a mild conventional pasteurizationtreatment (P70°C7.5 =8.15 min)asa functionof treatmentand storage time(21d/5 °C±1 °C). Inactivation of indigenous microorganisms (totalaerobic count of mesophilic and psychrotrophic microorganisms,mouldsandyeasts) of theappleproductwas also studied andmonitoredover the storage period at 5 °C.

2. Materials and methods

2.1. Raw material

Granny Smith (GS) apples were purchased from a local fruitpackinghouse (Girona Fruits SCCL, Girona, Spain) and stored at 3±1 °C until processing. The cultivar GS was chosen because of itsavailability at a regular quality throughout the whole year. GS applelots were characterized according to fruit firmness (fruit pressuretester, Model FT 327, Effegi, Italy), soluble solids content (SSC), andpH. Initial fruit characterization carried out at least on three fruits intriplicate gave the average characteristics of 8.3±0.6 kg cm−2

firmness, 11.2±0.3 °Brix and pH 3.41±0.06.

2.2. Purée product preparation

Apples were washed, peeled, cut and cored before blending(Thermomix, Vorwerk, Wuppertal, Germany) with acerola C juicepowder (0.25% w/w; Obipectin, Bischofszell, Switzerland) and freshlemon juice (2.5% w/w) as natural antibrowning substances andglucose-fructose syrup (3% w/w; Roquette Laisa España S.A., Barce-lona, Spain). Acerola C juice powder served as a strong natural sourceof vitamin C (16–18% according to Obipectin) in the formulation and

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the lemon juice as a natural acidulant. Puréewas immediately vacuumpacked in composite pouches containing a metallic layer (PET/MET+PE, Sacoliva S.L., Barcelona, Spain) in portions of 100 g. The oxygenpermeability of the bagswas b1.5 cm3 m−2 24 h (ASTMD3985). Threereplicates of apple purée were prepared for each combination oftreatment and storage time and stored under refrigeration condition(2–4 °C) overnight until HPP and conventional heat pasteurization.Three control samples were prepared in the same manner and storedunder the same conditions.

2.3. Preservation treatments

2.3.1. High pressure processingIndustrial HPP was performed on commercial-scale high pressure

system (Hyperbaric Wave 6500/120, N.C. Hyperbaric, S.A., Burgos,Spain) with a maximum capacity of 120 L and a potential maximumpressure of 650 MPa. Purée samples were pressurized at 400 MPa and600 MPa for 5 min with water at 20 °C as the pressure-transmittingmedium. Pressurization rate for the 400 MPa cycle was 204 MPa/minand 220 MPa/min for the 600 MPa cycle. Time for decompression was≤10 s. Initial temperature of the purée was between 9–10 °C and theaverage temperature after pressurization was about 16.0 °C. Aftertreatments apple product was stored at 5±1 °C during 21 days andanalyzed as described below.

2.3.2. Conventional pasteurization processSample pouches were submersed in a circulated water bath

adjusted to 75 °C and held at this temperature during 10 min. Theheating regime was validated in preliminary studies by monitoringthe temperature profile of the product inside the packages by meansof a datalogger (PicoVACQ 1TC, TMI-Orion, Montpellier, France). Thepasteurization process was equivalent to a pasteurization valueP70°C7.5 =8.15 min with a maximum temperature of 73.4 °C±0.3 °C

achieved. After removal from the water bath, samples wereimmediately placed in refrigeration conditions during 21 days andanalyzed as described below.

2.4. Physicochemical and physical analysis

Three samples were analyzed periodically for each combination ofstorage time×treatment at day 0, 7, 14 and 21 of cold storage (5 °C)and compared to raw control purée at day 0.

2.4.1. Color measurementThe color change of apple purée was measured with a Konica

Minolta chromameter (Model CR-410 HS, Minolta, Tokyo, Japan). Theequipment was set up for illuminate D65 (2°observer angle) andcalibrated using a standard white reflector plate. Readings wereobtained applying the standard CIE L*a*b* (1976) color system, whereL* is the lightness value, a* indicates hue on a green (−) to red (+)axis, and b* indicates hue on a blue (−) to yellow (+) axis. For themeasurement, purée was poured into a cylindrical sample cup andmeasured. Color differences (ΔL*Δa*Δb*) were calculated by sub-tracting mean control readings of the raw purée (L0*a0*b0*) fromindividual L*a*b* readings after treatment. To illustrate the degree incolor change the total color difference ΔΕ*=(ΔL*2+Δa*2+Δb*2)0.5 atday 21 of storage was computed.

2.4.2. Soluble solids content and pHSoluble solids content was measured with a portable refractom-

eter (Quick-BrixTM 90; Mettler Toledo GmbH, Giessen, Germany).Apple purée was squeezed through a piece of filter paper (10 μm poresize, Ahlstrom Barcelona, S.A., Barcelona, Spain). One drop was placedon the refractometer glass prism and the SSC was obtained as Brix.

PH was measured with a Crison pH meter (Crison PH 25, CrisonInstruments S.A., Barcelona, Spain) in combination with a Crison

puncture electrode (Crison pH 5053, Crison Instruments S.A.,Barcelona, Spain), which was calibrated prior to each measurementwith phosphate buffers at pH 4 and 7.

2.4.3. ViscosityA rotational viscometer (Visco Star plus, Fungilab, S.A., Barcelona,

Spain), interfaced to a computer,was used for rheologicalmeasurementand data acquisition. Samples were temperature-equilibrated at 25 °Cin a water bath and filled in a concentric cylindrical cup. A combinationof spindle R6 and velocity 5 rpmwas set up for all analysis. Each samplewas analyzed in threefold replication and results were expressed asmean values.

2.5. Chemical analysis

Purée samples for chemical analysis were frozen stored at –80 °C. Allthe analyses were carried out in duplicate of each packaged puréesample. All the chemicals used were of analytical reagent grade andwaterwas obtainedwith aMilli-Qpurification system(Branstead, USA).

2.5.1. Determination of ascorbic acid (AA) and total vitamin C (total Vit C)Themethod applied to determine total vitamin C (total Vit C=AA+

dehydroascorbic acid, DHAA) was based on the procedure of López,Montaño, Garcia, and Garrido (2005) with some modifications. Onegram of frozen stored (−80 °C) purée samples was weighed incentrifugation tubes followed by the addition of chilled extractionbuffer containing 60 g L−1 metaphosphoric acid (Sigma-Aldrich) and1 mMEDTA to 10 g of final weight. After gently stirring for 5 min at 4 °Csamples were centrifuged for 10 min at 11000×g at 4 °C (Beckman J2-M2, Beckman Coulter, Inc. Fullerton, CA, USA). The supernatant wasfiltered through a nylon syringe filter (0.45 μm) (Teknokroma,Barcelona, Spain) and analyzed immediately by HPLC, giving the AAcontent. For determination of the total vitamin C, 0.5 mL aliquot of thefiltratewasmixedwith 0.3 mLof 2 g L−1 dithiotreithol solution in 0.2 Msodium phosphate buffer pH 7 and 0.15 mL 45% (w/v) dipotassiumhydrogen phosphate. Samples were incubated 10 min at roomtemperature in the dark. Reduction was stopped with 0.3 mL 2 M o-phosphoric acid and the samples were filtered as described above. Tenmicroliters of the samples was injected on an HP 1100 system (AgilentTechnologies, Waldbronn, Germany) equipped with a quaternarypump, a DAD and a Zorbax SB-Aq column (150 mm×3.0 mm, 3.5 μm)(Agilent Technologies, Waldbronn, Germany). The mobile phaseconsisted of ultrapure water adjusted to pH 2.3 with o-phosphoricacid. The flow rate was 0.45 mL/min. The detectionwas done at 244 nmand AA was quantified according to a standard curve obtained byinjecting known amounts of ascorbic acid solutions (0–300 ng) (Sigma,Madrid, Spain). Resultswere expressed asmgAA(total Vit C) kg−1 freshweight sample (mg AA kg−1 f.w.). The DHAA content was calculatedby subtracting the initial AA content from the total Vit C content.

2.5.2. Total polyphenols (TPP)Two grams of stored (−80 °C) purée samples was weighed, and

20 mL of solvent (100% methanol) was added and mixed thoroughly.Samples were centrifuged at 12,100×g for 10 min at 4 °C (BeckmanJ2-M2 Centrifuge, Beckman Coulter, Inc. Fullerton, CA, USA) andaliquots of clear supernatantwere collected and stored at−20 °C untilanalysis. The amount of TPP in extracts was determined according tothe Folin-Ciocalteu procedure (Singleton & Rossi, 1965). A total of0.125 μL of sample extract or gallic acid (GA) standard solutions wasmixed with 625 μL Folin-Ciocalteu reagent (diluted 1:10 with H2O).Samples were incubated 3 min and then 500 μL of tempered (45 °C)sodium carbonate (7.5%w/v) was added. The mixture was incubated15 min at 45 °C and absorbance was measured at 750 nm using a UV–Vis spectrophotometer (UV-1603, Shimadzu, Kyoto, Japan). Resultswere expressed as mg gallic acid equivalents (GAE) kg−1 fresh weightsample (mg GAE kg−1 f.w.).

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2.5.3. Titratable acidity (TA)Titratable acidity was determined by titrating 5 g homogenized

purée sample diluted in 50 mL water with 0.1 N NaOH to an end pointof pH 8.1 using a potentiometric titrator system (785 DMP Titrino,Metrohm AG, Herisau, Switzerland) (AOAC, 1990, Part 942.15).Results were expressed as g malic acid equivalents (MAE) kg−1

purée fresh weight (g MAE kg−1 f.w.).

2.6. Microbial determination of indigenous microbiota

Total aerobic mesophilic and psychrotrophic count and mouldsand yeasts were analyzed before (raw control purée) and aftertreatments and at regular intervals on samples stored at 5±1 °Cthroughout the storage period. A sample of purée (10 g) was dilutedwith 90 ml of saline peptone (SP, 1 g L−1 peptone, 8.5 g L−1 NaCl) andhomogenized in a Stomacher (Model 400, Seward, London, UK) atregular speed for 2 min. To determine total aerobic mesophilic (TAM)and psychrotrophic (TAP) counts of purée samples, homogenateswere serially diluted and plated on Plate Count Agar (PCA, BiokarDiagnostics, Beauvois, France) followed by incubation at 30±1 °C for3 days and at 6.5±1 °C for 10 days, respectively. Moulds and yeasts(M&Y) were determined by plating the homogenates in GlucoseChloramphenicol Agar (GCA, Biokar Diagnostics) followed by incu-bation at 25±1 °C for 5 days. After incubation, plates were countedand the results expressed as logarithm of colony forming units pergram (log cfu g−1). Determinations were carried out in triplicate (3packaged samples). Detection limit was 50 cfu g−1 (1.70 log cfu g−1).

2.7. Statistical analysis

To evaluate the effect of the individual treatments duringprocessing on quality parameters compared to the raw purée, one-way analysis of variance (ANOVA) was performed. General linearmodel (GLM) with interaction storage time×treatment was appliedto assess the effect of treatment and storage time during shelf life.Both analyses were carried out using the SAS Enterprise Guide3.0.2.414 and means were compared for both analyses according toTukey (HSD) with a level of significance at Pb0.05.

3. Results and discussion

3.1. Physical and general physicochemical analysis

The effects of HPP and conventional pasteurization on physical andphysicochemical properties of GS purée product were compared withunprocessed raw control purée (Table 1). The evolution of the

Table 1Physicochemical properties of the different treated purée products and raw purée stored fo

Treatment Day SSC (°Brix) pH Viscosity (mPa

Raw 0 14.2±0.1 3.20±0.01 82,922±39400 14.2±0.1 3.22±0.01a 10,7341±23667 14.4±0.1xy 3.12±0.01c 81,106±2645

400 MPa 14 14.1±0.1xy 3.16±0.01b 95,276±860321 14.3±0.1y 2.97±0.00d,y 135,068±89780 14.0±0.1 3.21±0.01a 93,395±71617 14.0±0.2y 3.16±0.01b 104,630±19,61

600 MPa 14 13.7±0.1y 3.15±0.01b 93,678±20,2121 13.9±0.1y 2.99±0.02c,xy 116,267±49890 13.8±0.0c 3.21±0.01a 88,341±34777 14.5±0.2ab,x 3.13±0.00b 109,460±10,08

Past. 14 14.2±0.1b,x 3.17±0.00b 72,711±415321 14.9±0.3a,x 3.03±0.01c,x 96,850±12,58

aValues are means±standard deviation, n=3.Values with abc superscripts within a column and treatment indicate significant differencessignificant differences (Pb0.05) between treatments for the same storage day. Values with

parameters was also monitored during the shelf life period of 21 days.The raw purée had a soluble solid content of 14.2±0.1°Brix, pH of3.20±0.01 and the titratable acidity was 7.8±0.3 g kg−1. Comparedwith fresh apples (11.2°Brix and pH of 3.41) SSC increased in thepurée due to the addition of glucose-fructose syrup and was moreacid because of the addition of citric acid. Means of SSC, pH and TAdid not significantly change by the different treatments and duringrefrigerated storage. The consistency of the purée product expressedas viscosity (mPa s) was altered by processing (Table 1). HPprocessing raised viscosity, but only the 400 MPa treatment wasfound significantly different compared with control purée. Duringstorage no clear tendency could be found concerning the viscositystability of the purée product also due to the high variations foundwithin replicate samples. Viscosity was found positively correlatedwith HPP for many of fruit and vegetable products. Ahmed et al.(2005) reported that the viscosity of mango pulp increased aftermoderate HP treatments at 100 or 200 MPa (15 or 30 min at 20 °C)while a decreasing trend was found at higher pressure levels (300–400 MPa). HP treated tomato purée (400 MPa/15 min/25 °C) showedthe highest consistency compared with conventionally thermaltreated purées (Sánchez-Moreno, Plaza, De Ancos, & Cano, 2006).Navel orange juice treated at 600 MPa for 4 min at 40 °C resulted in ahigher viscosity than thermal treated one (Polydera et al., 2005). Theincrease in viscosity due to pressure was attributed to an increasein the linearity of cell walls and volumes of particles due to thepermeabilization of cell walls (Oey et al., 2008; Sánchez-Moreno et al.,2003).

Initial CIE L*a*b* parameters of the raw purée were L0*=58.3,a0*=−8.0 and b0*=24.2 (Table 1). The mild thermal treatment had asignificant effect (Pb0.05) on the chromaticity coordinates a* and b*,indicating non-enzymatic browning. Parameter a*was shifted tomorepositive (reddish) values and yellowness (value b*) decreased.Discoloration and non-enzymatic browningdue to thermal treatmentscan result from several reactions, including Maillard condensation,caramelisation and destruction of pigments (Ibarz, Pagán, & Garza,2000). Due to the maximum temperatures achieved in our work(≤75 °C), pigment destruction is most likely the responsible factor fordiscoloration of the purée. HPP increased insignificantly the L* valuesright after processing indicating a lightening of the purée surface. Thisphenomenon was also observed in tomato based products (Sánchez-Moreno et al., 2006) and might be caused by cell disruption duringHP treatment. Chromaticity coordinates a* and b* remained constantduring HP treatment. Ahmed et al. (2005) observed that color qualityparameters remained almost constant after HP treatment of mangopulp indicating pigment stability. These findings were also supportedby Guerrero-Beltrán and Barbosa-Cánovas (2004) and Guerrero-

r 21 days at 5°Ca.

s) L* a* b* TA (g kg−1)

y 58.3±0.8 −8.0±0.3x 24.2±1.0x 7.8±0.3x 59.5±0.5a −7.9±0.2a,x 22.7±0.7xy 7.8±0.2

54.9±1.2b,y −2.6±1.0b,y 22.4±0.2 7.6±0.350.8±1.6c,y 1.4±1.2c,y 22.4±0.7 7.7±0.147.5±0.8c,y 4.0±0.4d,y 21.3±0.3 8.1±0.3

y 59.9±1.1a −8.3±0.1a,x 24.0±1.1a,x 7.8±0.32 55.7±1.5b,y −3.8±1.1b,xy 21.6±0.6b 7.6±0.62 48.8±0.8c,y 2.6±1.2c,y 22.2±0.3ab 7.7±0.2

46.2±0.6c,y 4.7±0.4c,y 22.0±0.6ab 8.1±0.7y 58.0±0.8 −4.8±0.2y 20.7±0.5y 7.5±0.16 59.5±0.7x −4.9±0.1x 21.4±0.4 7.8±0.2

58.4±0.8x −4.5±0.1x 21.1±0.6 7.8±0.20 57.7±0.8x −4.4±0.2x 20.6±0.5 7.6±0.4

(Pb0.05) as a function of the storage period. xyz superscripts within a column indicateout superscripts are not significantly different.

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Beltrán et al. (2005), who demonstrated that peach purée with andwithout antibrowning agents treated between 103 and 517 MPa (5–25 min) showed a bright yellow color equivalent to the freshlyprepared purée. Color compounds of processed fruits can howeverchange during storage due to incomplete inactivation of enzymesandmicroorganisms, which can result in undesired chemical reactionsin the food matrix (Oey et al., 2008).

Table 2 shows the color data during storage represented as colordifferences (ΔL*, Δa* and Δb*). During storage, discoloration ofpressurized samples occurred, which was indicated by the reductionof the lightness variable L* and the increase of the chromaticityvariable a*. Color deterioration was of the same magnitude and nostatistically significant differences could be observed between the 600and the 400 MPa treatment. The initial green-yellow color of the GSpurée deteriorated between day 7 and 14 of cold storage. From day 14to day 21, color deterioration slowed down and no significantdifference was detected as a function of time concerning the lightnessvalues for any of the treatments. Color coordinate b* (yellowness)remained constant after treatment and during the shelf life period. Incontrary, pasteurized samples maintained initial L*a*b* values overtime and no significant differences could be detected duringrefrigerated storage. ΔΕ* values of the HP processes were 16.4±0.8and 17.7±0.8 for the 400 and 600 MPa respectively versus 5.3±0.4as the ΔE* for the mild pasteurization. Similar results were obtainedby Guerrero-Beltrán, Barbosa-Cánovas, Moraga-Ballesteros, Moraga-Ballesteros and Swanson (2006) who treated standardized mangopurée (pH 3.5 and 500 ppm AA) at 552 MPa during 5 min. The totalchange in color ΔE* reached amaximum of 16.6±0.3 after 1 month ofcold storage at 3 °C. Although not analyzed in this work, the datasuggest that the polyphenoloxidase (PPO) enzyme, which is mainlyresponsible for enzymatic browning in fruit based products, was justpartially inactivated by the HP treatments (Chisari et al., 2007; Cocciet al., 2006; Guerrero-Beltrán & Barbosa-Cánovas, 2004; Guerrero-Beltrán et al., 2006; Guerrero-Beltrán et al., 2005; Palou et al., 1999;Rocha & Morais, 2002; Weemaes et al., 1998; Weemaes et al., 1997).Rocha and Morais (2002) obtained high correlations between colorparameters and PPO activity for minimally processed ‘Jonagored’apple cubes (R2=0.71 for L* value, R2=0.97 for a* value). Guerrero-Beltrán and Barbosa-Cánovas (2004) reported that addition of1000 ppm ascorbic acid to natural peach purée led to 14.67%reduction of PPO activity, while increased holding time minimallyenhanced the inhibition of PPO at the same pressure. More recentlyGuerrero-Beltrán et al. (2005) demonstrated the good color stabilityand increased shelf life of peach puree treated at 517 MPa (5 min)containing ascorbic acid (500 ppm) and storage at refrigeratedtemperature (3 °C) up to 21–30 days. Good color stability over thestorage period for the mild thermal treated product indicatessufficient inactivation of the heat sensitive PPO enzyme. Normally,heat treatments of 70–90 °C significantly destroy the catalytic activityof the PPO (Chisari, Barbagallo and Spagna, 2007; Chutintrasri &Noomhorm, 2006; Weemaes et al., 1997). The data suggest that themaximum temperatures of 73.4±0.3 °C achieved during the mild

Table 2Least square means of ΔL*=(L*−L0*), Δa*=(a*−a0*) and Δb*=(b*−b0*) color differenc

Treatment ΔL* Δa*

day 0 day 7 day 14 day 21 day 0 d

400 MPa 1.2a −3.4b,y −7.5c,y −10.8c,y 0.1d,y 5600 MPa 1.6a −2.6b,y −9.4c,y −12.1c,y −0.3c,y 4Past. −0.3 1.3x 0.1x −0.6x 3.2x 3

Means with abc superscripts within a row are significantly different (Pb0.05).Means with xyz superscripts within column are significantly different (Pb0.05).Means without superscripts are not significantly different.

pasteurization in combination with the low pH, refrigerated storage,and the barrier properties of the pouches inhibited the PPO enzymeactivity of the purée product.

3.2. Changes in ascorbic acid and total polyphenolic content

Vit C is very susceptible to oxidation under certain environmentalconditions like heat, aw, presence of oxygen, heavy metal ions andalkaline pH (Perera, 2007) degrading their biological activity. Hence,Vit C loss provides a useful index of oxidative deterioration and istherefore used to define the shelf life, e.g. of minimally processedfruits and vegetables as well as fruit juices (Perera, 2007; Polyderaet al., 2003, 2005). Initial total Vit C content was 354±34 mg kg−1,whereas 138±100 mg kg−1 was present as AA, resulting in apercentage of 61% DHAA of the raw control purée (Table 3). Thisinitial concentration was mainly attributed to the addition of theacerola juice powder to the purée formulation and to some lesserextent by the fresh lemon juice. After pressurization treatments totalVit C concentration was 331±31 mg kg−1 at 400 MPa and fellsignificantly (Pb0.05) to 278±17 mg kg−1 at 600 MPa. Expressedas the percentage of Vit C retention it was found that 93.5% and 78.5%of the total Vit C retained immediately after treatment. Mildpasteurization had no influence on the total Vit C content, as 100%of the total Vit C was retained after the treatment. AA contentshowever decreased more substantially during the treatments. Themost noticeable decrease of initial AA content was found for the600 MPa cycle, where only 14.5% of residual content was detected incomparison to 57% of residual AA content found for the 400 MPaprocess. Retention of initial AA for the pasteurization was 61.5%.Picouet, Landl, Abadias, Castellari, and Viñas (2009) found recently,that for the same purée formulation heated by using microwaves,100% total Vit C and 71.3% of residual AA content could be retainedsubsequent treatment. It was generally reported that oxygen and thefood matrix influence the AA stability during HPP and subsequentstorage. For example, higher Vit C loss was found in fruit juicecompared to buffer solutions because of the existence of endogenouspro-oxidants such as metal ions and enzymes (Oey et al., 2008). ForHPP (400 MPa/25 °C/15 min) tomato purée it was found 39% and 30%decreases in the initial content of AA and total Vit C respectively(Sánchez-Moreno et al., 2006). In a former study, Sánchez-Morenoet al. (2003) observed that between several combination treatmentsof HP and heat, HP (100 MPa–400 MPa) with higher temperatures(60 °C) showed a higher decrease in the content of Vit C. Duringstorage the analysis of the Vit C content revealed strong deteriorationfor all treatments. This deterioration can be explained in a first step bythe oxidation of AA to DHAA, which is then further irreversiblyconverted into 2,3-diketogulonic acid (Davey et al., 2000; Perera,2007). During shelf life a strong decline of the AA contents concerningall treatments could also be revealed. At day 21, decrease in AAcontent was in a range of 79% for the pasteurization process comparedto 97% and 94.5% in case of the 400 and 600 MPa cycle.

es according to the interaction storage time×treatment.

Δb*

ay 7 day 14 day 12 day 0 day 7 day 14 day 12

.4c 9.4b,x 12.0a,x −1.5xy −1.8 −1.9 −3.0

.2b 10.6a,x 12.7a,x −0.2a,x −2.7b −2.1ab −2.2ab

.1 3.5y 3.6y −3.5y −2.9 −3.2 −3.7

Table 3Nutritional quality of the different treated purée products and raw purée stored for21 days at 5 °Ca.

Treatment Day Total Vit C(mg kg−1)

Ascorbic acid(mg kg−1)

DHAA(mg kg−1)

TPP(mg kg−1)

Raw 0 354±34x 138±100x 216±116 1180±23x

0 331±31a,x 79±19xy 265±47a 1179±46a,x

7 201±2b,x 17±6 184±7ab 966±48b,x

400 MPa 14 116±1c 7±0 109±2b 486±47c,z

21 69±1c 4±1 66±2b 462±11c,z

0 278±17a,y 20±13y 265±19a 888±143a,z

7 136±17b,y 15±2 111±20b 816±100a,y

600 MPa 14 97±9bc 8±0 93±3b 685±89b,y

21 61±2c 9±3 52±5b 642±47b,y

0 335±44a,x 85±17xy 289±91a 1028±49ab,y

7 188±20b,x 61±26 127±9ab 1063±28ab,x

Past. 14 128±7c 40±9 88±7ab 1029±20b,x

21 79±8c 29±11 51±11b 1137±39a,x

aValues are means±standard deviation, n=3.Values with abc superscripts within a column and treatment indicate significantdifferences (Pb0.05) as a function of the storage period. xyz superscripts within acolumn indicate significant differences (Pb0.05) between treatments for the samestorage day. Values without superscripts are not significantly different.

Table 4Experimental vitamin C loss rates, k (days−1) and their respective times of half loss, t1/2(days), of high pressurized and thermally pasteurized GS apple purée.

Treatment Vit C loss rate Time of half loss

k (days−1) R2 t1/2 (days)

400 MPa −0.075±0.002a 1.00 9.3600 MPa −0.070±0.004 0.98 10.0Past. −0.068±0.003 0.99 10.3

a Standard error.

562 A. Landl et al. / Innovative Food Science and Emerging Technologies 11 (2010) 557–564

The experimental data on the loss of total Vit C could also bedescribed by using the standard equation of a first-order reactionkinetic given below:

−dCdt

= kC ð1Þ

ln C = lnC0 −ktð Þ ð2Þ

Where C is the concentration at time t (days), C0 the concentra-tion of the treated purée at time zero, k the first-order rate constant(day−1) and t the storage time (day). Experimental data of theremaining Vit C concentration defined as ln(C/C0) were plottedagainst storage time (Fig. 1) and the rate constant kwas calculated asthe slope of the curves. The calculated k values together with thecorrelation coefficient R2 are given in Table 4. Lower Vit C loss rateswere found for the conventionally pasteurized apple product com-pared to the respective values for high pressure treated product.Vit C degradation rate k for the 600 MPa treatment was foundslightly lower than for the 400 MPa process despite the high initialloss determined during the 600 MPa pressurization (Table 3). Someauthors described that the AA degradation fitted better to a zeroorder model (C=C0−kt) than to a first-order reaction kinetic.Zulueta, Esteve, and Frígola (2010) reported recently that the AAdegradation rate during storage of orange juice-milk beverage was

Fig. 1. Plot of remaining Vit C concentration ln(C/C0) against storage time (days) treatedat HPP 400 MPa (◊), HPP 600 MPa (□) and conventional pasteurization (Δ).

adjusted to a zero order model. Polydera et al. (2003) found twostages for ascorbic acid degradation during storage of HPP recon-stituted orange juice with the latter stage fitting more appropriateto a zero order kinetic. However, our experimental data showedgood fit to the model given above (Eq. (1)) with correlation coeffi-cients of 0.98–1.00. Shelf life of the acidified apple purée productcould be estimated as the time period in which 50% of the Vit C contentwas lost (C=0.5 C0). Shelf life was therefore calculated through Eq. (2)and depicted in Table 4 as times of half loss (t1/2). According the Vit Closs rates shelf life in a range of 9.3, 10.0 and 10.3 days were estimatedfor the 400 and 600 MPa process and for the mild pasteurizationprocess respectively. However, even if vitamin C was lost duringstorage, its content in purée samples was higher than that of freshapple flesh, which is reported to be different between cultivars,seasons, harvest date and some other factors. Values between 29 and120 mg/kg have been reported in different cultivars (Molina-Delgado,Larrigaudiere, & Recasens, 2009; Planchon, Lateur, Dupont, & Lognay,2004; Vilaplana, Valentines, Toivonen, & Larrigaudiere, 2006).

Total polyphenolic compounds were affected significantly (Pb0.05)by pasteurization and pressurization at 600 MPa and remainedunchanged during processing at 400 MPa in comparison with the rawapple product (Table 3). The retention of TPP content after 600 MPaprocess was 75%, whereas in case of the pasteurization 87% of thephenolics were retained in the purée product. The data show that therewas no loss of phenolic compounds found for the 400 MPa. Storagerevealed that mild pasteurization preserved higher levels of phenolicsthan pressure-treated samples. There was no major loss of TPP contentconcerning the pasteurized purée samples during storage. Comparingthe 400 MPa and the 600 MPa treatments, it was found that despitethe adverse affect on the initial TPP content of the product, towardsthe end of the storage period (days 14 and 21) the 600 MPa cycleretained significantly higher contents of TPP (Pb0.05) than the400 MPa. Regarding the 400 MPa treatment, levels of TPP contentdecreased sharply between day 7 and day 14. Data suggest that levelsof phenolic compounds are related to residual PPO activity aftertreatment, reflecting the efficacy of the treatments in preserving thenutritional quality of the purée.

3.3. Microbiological analysis

Initial counts of mesophilic microorganisms were low being 3.31log cfu g−1 and 3.22 log cfu g−1 for TAM and M&Y, respectively, andbelow thedetection limit (50 cfu g−1, 1.7 log cfu g−1) for TAP (data notshown). High microbial reductions were obtained by all treatments,withmicrobial counts below the detection limit after processing (bothHPP treatments and pasteurization). Moreover, microbial countsremained below the detection limit for the whole storage period at5 °C, and no growth was observed except for the pasteurized samplesafter 21 days of storage, with final M&Y population of 2.03 log cfu/g.The combination of low temperature and acid pH (3.2) and absence ofO2 in the vacuum bag packaged samples made this product micro-biologically stable. Similarly, Guerrero-Beltrán et al. (2006) did notobserve growth of microorganisms (b10 cfu/g) in mango puréetreated with HPP at 552 MPa and stored at 3 °C for 27 days. In thecase of peach purée with or without antioxidants and treated at

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517 MPa for 5 min, no microbial growth of total count or yeasts andmolds (b10 cfu g−1) was observed after storage at 3 °C (Guerrero-Beltrán et al., 2005). Lavinas et al. (2008) investigated the effect of HPPon the survival of natural microbiota of cashew apple juice treated byHPP and they found that after treatment at 350 MPa for 7 min or400 MPa for 3 min, aerobicmesophilic bacteria decreased froma rangeof 4.6 to 5.9 cfu/mL to undetectable levels (10 cfu/mL). They also didnot observemicrobial growth after 8 weeks of storage at 4 °C. Krebberset al. (2003) showed that HPP treatment of tomato puree at 700 MPafor 2 min reduced thenaturalmicrobiota (3.6 log cfu total aerobic platecount at 30 °C/mL) to a level below the detection limit (b1.5 log cfu/mL) and no outgrowth of themicroorganisms occurred during storageof 8 weeks. In a recent study, Picouet et al. (2009), observed a slightincrease of TAM count during storage at 5 °C for the same puréeformulation treated by microwaves, but under different packagingconditions.

4. Conclusions

This is one of the first studies dealing with the evident potential ofnon-thermal high pressure pasteurization of acidified GS apple puréewith industrial-scale equipment. It was evidenced that unlike the600 MPa treatment, the 400 MPa pressurization and the mildconventional pasteurization did not affect adversely the initialinstrumental and nutritional quality of the acidified Granny Smithpurée. During storage however none of the treatments prevented fullyundesirable changes during a shelf life of 3 weeks. The time of halfloss of total Vit C at 5 °C was in a range of 9.3 to 10.3 days. Colordeterioration occurred especially for the pressurized purées betweenday 7 and 14 of cold storage. Quality constraints could be overcome byusing another apple cultivar with lower browning tendency or byincorporating othermore powerful antibrowning agents. In this sense,‘Shampion’ apple cultivar was suggested for the production of light-colored purées, due to its lower chlorogenic acid content and PPOactivity (Oszmiański, Wolniak, Wojdylo, & Wawer, 2008). From themicrobiological point of view, apple purée obtained by HP processingwas stable when stored at 5 °C and comparable with the mildconventionally pasteurized purée. In conclusion, a commercialmicrobiological shelf stable HP processed apple product could beobtained, which retained physicochemical and high levels of nutri-tional quality parameters for at least 7 days at 5 °C. More studies arenecessary to successfully address the browning problems and toextend shelf life.

Disclaimer

The views and opinions expressed in this publication are purelythose of the writers and may not in any circumstances be regarded asstating an official position of the European Commission.

Acknowledgements

Authors are grateful to the European Commission (ISAFRUIT Projectfunded under the Thematic Priority 5-Food Quality and Safety of the 6thFramework Programme of RTD (Contract no. FP6-FOOD-CT-2006-016279) for their financial support and to the Spanish government forINIA research contract CTE/3597/2003 (BOE 23/12/2003).

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