Colour and texture of apples high pressure processed in pineapple juice

Innovative Food Science and Emerging Technologies 11 (2010) 39–46 Contents lists available at ScienceDirect Innovative Food Science and Emerging Technologies j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i f s e t Colour and texture of apples high pressure processed in pineapple juice Niranjala Perera a,b,⁎, T.V. Gamage b, L. Wakeling a, G.G.S. Gamlath c, C. Versteeg b a b c School of Science and Engineering, University of Ballarat, Mt. Helen Campus, Ballarat, VIC 3350, Australia CSIRO Food and Nutritional Sciences, Private Bag 16, Werribee, VIC 3030, Australia School of Exercise and Nutrition Science, Deakin University, Burwood, VIC 3125, Australia a r t i c l e i n f o Article history: Received 6 March 2009 Accepted 3 August 2009 Keywords: High pressure processing Minimal processing Enzymatic browning Texture Pineapple juice Apples a b s t r a c t Cubes of Granny Smith and Pink Lady apples were vacuum packed in barrier bags with 0% to 50% (v/v) pineapple juice (PJ) at 20°Bx and subjected to high pressure processing (HPP) at 600 MPa for 1–5 min (22 °C). The in-pack total colour change (ΔE) was observed over 4 weeks at 4 °C. Within <1 week of storage at 4 °C, texture, polyphenoloxidase, pectinmethylesterase activities, changes in ΔE and visual browning after opening the bags during air exposure (22 °C; 21% O2 ) for 5 h were also monitored. During the 4 weeks storage in bag visible colour changes were not observed. Texture and ΔE after 5 h air exposure were significantly affected by the apple variety, HPP time and % PJ used. The combined treatment significantly reduced residual PPO activity while PME activity was not affected in both varieties. Pineapple juice in combination with HPP could be used as a natural preservation system for minimally processed apples. Industrial relevance: Browning upon opening the packs and during air exposure can adversely affect the quality of fresh-cut fruits. Combined treatment of high pressure processing (HPP) and use of pineapple juice has the potential to prevent browning for several hours giving sufficient time for presentation and use in domestic and foodservice environment where high quality fresh-like fruit is required. © 2009 Elsevier Ltd. All rights reserved. 1. Introduction Consumers demand high quality, convenient minimally processed natural fruit products with fresh appearance, texture and flavour, which are free from preservatives and other additives. However, minimally processed fresh products have a relatively short shelf life due to wounding related to increased metabolism (King & Bolin, 1989; Joan & Kader, 1989; Watada, Abe & Yamuchi, 1990; Varoquaux, Mazollier & Albagnac, 1996; Paull & Chen, 1997) and microbial spoilage. Physiological and biochemical changes in such products occur at a faster rate than in intact fruits resulting in the rapid onset of enzymatic browning (Brecht, 1995; Buta & Abbott, 2000) and excessive tissue softening (Toivonen & DeEll, 2002). Enzymatic browning and the resultant discolouration of cut fruit products, upon exposure to air, is a major problem for the food industry impairing not only the colour of fresh-cut fruits but also the flavour and the nutritional quality (Rigal, Cerny, Richard-Forget & Varoquaux, 2001). Mainly the browning is developed due to enzymatic oxidation of phenols to quinones by polyphenoloxidase (PPO) in the presence of oxygen. Subsequently, these quinones condense and react non enzymatically with other substances such as phenolic compounds and amino acids to produce complex brown polymers. Sulfites are extensively used as PPO inhibitors to prevent ⁎ Corresponding author. School of Science and Engineering, University of Ballarat, Mt. Helen Campus, Ballarat, VIC 3350, Australia. Tel.: +61 3 9731 3367; fax: +61 3 9731 3201. E-mail address: niranjala.perera@csiro.au (N. Perera). 1466-8564/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.ifset.2009.08.003 enzymatic browning in fruit products. However, sulfites are reported to induce adverse allergenic effects in certain sensitive individuals such as asthmatics (Sapers, 1993). Other additives including, 4-hexylresorcinol, cysteine, acidulants and chelating agents such as citric acid and phosphates are able to reduce enzymatic browning, but they are usually less effective than sulfites (Janovitz-Klapp, Richard & Nicolas, 1989). There is an increasing demand by consumers for substituting preservatives and any other additives with natural substances (Jang, Sanada, Ushio, Tanaka & Ohshima, 2002). Compounds of inherently natural origin would be widely accepted by consumers in the market. This consumer demand has stimulated the search for natural and safe antibrowning agents and processing methods that can result products with high quality, acceptable appearance, flavour and nutritional value in addition to the microbiological safety. High pressure processing (HPP) offers a natural, environmentally friendly alternative for pasteurisation and shelf life extension of a wide range of food products (Welti-Chanes et al., 2005). HPP in combination with packaging of good barrier properties can prevent browning in minimally processed products during storage in the sealed pack. However, due to only partial inactivation of the PPO enzyme it is not possible to prevent browning when the packs are opened and the products are exposed to air. The use of pineapple juice to inhibit enzymatic browning was investigated by others previously (Lozano-deGonzalez, Barrett, Wrolstad & Durst, 1993). It was reported that pineapple juice and ion-exchange fractions of pineapple juice were both equally effective as sulfite in the inhibition of enzymatic browning in fresh and dried apple rings. The best results were achieved in 40 N. Perera et al. / Innovative Food Science and Emerging Technologies 11 (2010) 39–46 browning prevention with the cation-exchanged fraction of pineapple juice. Wen and Wrolstad (1999) reported that a non volatile organic acid in pineapple juice was the major inhibitor of enzymatic browning in apple products. Labuza, Lillemo and Taoukis (1990) investigated the effect of proteolytic enzymes of plant origin on enzymatic browning inhibition. They found that ficin and papain were comparable to sulfite in prevention of browning in potatoes and apples. However, bromelain was effective only on apples during storage at 4 °C. McEvily (1991) reported that a ficin free extract prepared from the fig latex can inhibit enzymatic browning in apple and shrimp. There is no published research on the effectiveness of pineapple juice in combination with HPP as an inhibitory system for the prevention of enzymatic browning. This study was aimed to investigate the combined effect of HPP treatment and pineapple juice on some physicochemical parameters. Investigated were the treatment effects on total colour change during storage and upon exposure to air, textural changes and quality related enzymes (PPO and PME) in minimally processed Granny Smith and Pink Lady apples. 2. Materials and methods 2.1. Plant materials Granny Smith, Pink Lady apples and Smooth Cayenne pineapples were obtained from a local supermarket. Selected pineapples had a shell colour of half to three-fourths gold as described in Dole's fresh pineapple colour standard guide. Firmness was measured with a hand-held fruit pressure tester, (FT 327, Italy; 8 mm probe). Granny Smith and Pink Lady apples had a firmness of 3.5 and 4.5 kg respectively. The total soluble solids (Deta refractometer Bellingham and Stanley Ltd.) of the Granny Smith and Pink Lady apples were 13° and 15°Bx respectively. 2.2. Pineapple juice preparation Pineapples were peeled and the juice was extracted with a small bench top juice extractor (Nutrifaster Model Ruby 2000, USA). Single strength pineapple juice (12°Bx) was diluted with distilled water (v/v) to obtain 25% and 50% pineapple juice. The dissolved solids concentration of the 25 and 50% pineapple juices were adjusted to 20°Bx by the addition of food grade sucrose (CSR, Australia). The pH of the different concentrations of pineapple juice was maintained at 3.6. 2.3. Preparation of apple cubes Apples were washed with a solution of 200 ppm chlorine (100 mL of Milton solution in 8 L potable water Milton, Australia). Washed apples were peeled and cored manually and mechanically diced into 1 cm3 pieces using a small bench top Anliker dicer (Anliker, Australia). The fruit pieces were held in a 0.1% (w/v) ascorbic acid solution (Robert Bryce and Company, Ltd., Australia) prior to packaging to prevent surface browning. Diced fruit pieces (200 g) were drained in a plastic strainer and packed into high oxygen barrier upright flexible retort pouches (Amcor, Germany; oxygen permeability: 2 cm3/1 m2/ 24 h package size 14 cm × 18.5 cm) with 100 mL 20˚Bx pineapple juice at concentrations of 0, 25 and 50% (v/v). Fruit pieces in 20˚Bx sugar solution were used as the control (0% pineapple juice). The pouches were vacuum-sealed at − 0.8 bar using the Webo-Matic E50G, vacuum packaging machine (Warner Bonk, Bochum). 2.4. High pressure processing Samples were treated in a 35 L high pressure vessel (Flow Pressure System QUINTUS® Food Press Type 35 L-600 sterilization machine Avure Technologies, Kent, WA, USA) at 600 MPa, at ambient temperature (18–22 °C) for 1, 3 and 5 min. The samples and the pressurising medium (water) were kept at a pre-determined initial temperature to obtain the expected processing temperature during compression. The initial temperature of the samples processed at 600 MPa and 22 °C were maintained at 7 °C. The compression rate was 4.2 MPa/s while the decompression rate was 40 MPa/s. Six hundred MPa pressure is considered to be economical and microbiologically safe at the pasteurisation level (Suthanthangjai, Kajda & Zabetakis, 2005). Pressure treated samples and untreated controls were stored at 4 °C for one month in styrofoam boxes, without exposure to light. 2.5. Total colour change (ΔE) The in-pack colour (L*, a* and b* values) of triplicate samples was measured through the transparent retort pouch at weekly intervals over 4 weeks using a Minolta Chroma Meter CR-300 (Minolta Corp., Osaka, Japan). Apple samples stored for <1 week at 4 °C were used for the air exposure study. The most prominent colour changes were observed at the end of the 5 h of air exposure. Therefore the total colour change after 5 h of air exposure (with triplicate samples) is discussed in this publication. The total colour change (ΔE) was calculated with the following equation (Hunter Lab, (1996)): ΔE=[(ΔL)2 +(Δa)2 + (Δb)2]½. 2.6. Visual browning score The visual browning score was developed based on the browning of the control samples (diced pieces) of each apple variety. The samples were scored for acceptability of colour with visual browning score system developed according to the 9-point hedonic scale ranging from 1 to 9 based on the severity of browning (visual browning score: 1—excellent, 2—highly acceptable, 3—acceptable, 4—moderately acceptable, 5—neither acceptable nor unacceptable, 6—moderately unacceptable, 7—unacceptable 8—highly unacceptable and 9—completely unacceptable). The visual browning score system was used to assess the samples during air exposure and visual ratings were allocated immediately after exposure and every hour over the 5 h period using triplicate of samples. 2.7. PPO extraction and assay PPO activity was determined from samples within <1 week of storage at 4 °C. The PPO enzyme was extracted from apples using the method described by Gauillard and Richard-Forget (1997) and Carbonaro and Mattera (2001) with some modifications. All the chemicals that were used in the extraction and assay of the enzymes were of analytical grade or higher degree of purity. Freeze-dried apple samples (6 g) were homogenized with 50 mL of McIlvaine citric acid phosphate buffer, (pH 6.5, containing 0.05 M sodium dodecyl sulphate, Sigma-Aldrich) using an Ultra Turrax T25 homogeniser (IKA Labotechnik, Germany) at 9500 rpm for 2 min. All subsequent steps were also performed at 4 °C. The zsuspension was centrifuged at 3000 × g for 15 min followed by 23,700 × g for 15 min. The supernatant was filtered through Whatman No. 4 filter paper and used as the enzyme extract to determine PPO activity. The PPO activity was determined in a reaction mixture (152.5 μL) containing 50 μL enzyme extract, 90 μL 0.1 M citric acid phosphate buffer (pH 6.5) and 12.5 μL caffeic acid (90 mg caffeic acid/100 mL citric acid phosphate buffer, pH 6.5) in a micro well plate. The absorbance of the mixture was measured at 420 nm for 10 min at 30 s intervals at 37 °C using a spectrophotometer (SpectraMax Plus384 Molecular Devices Corporation, Sunnyvale CA, USA). One unit of PPO activity was defined as a change in absorbance of 0.001 OD/min. The reaction rate was estimated from the initial linear portion of the plotted curve. The relative PPO activity of the samples was calculated by % PPO activity = At / Ao × 100, where At = PPO activity of high N. Perera et al. / Innovative Food Science and Emerging Technologies 11 (2010) 39–46 pressure treated fruit sample and Ao = PPO activity of untreated fruit sample. 2.8. PME extraction and assay PME was extracted from the apple samples within 1 week of storage at 4 °C following method proposed by Hagerman and Austin (1986) with some modifications. Freeze-dried apple samples (6 g) were homogenized in 100 mL of 8.8% (w/v) NaCl and Ultra Turrax (IKA—Werke, Staufen, Germany) at high speed (13,000 min− 1) for 15 s. The homogenate was stirred for 15 min and then centrifuged (J2MC, Beckman, USA) at 13,000 rpm for 25 min at 4 °C. The supernatant was assayed for PME activity by a titration of the free carboxylic groups produced from the pectin with 0.01 N NaOH using an automatic pH stat-titrator (Radiometer 854, titration Workstation, Lyon, France) at pH 7.5 and 30 °C (Duvetter et al., 2005). A 30 mL aliquot of a solution containing 0.15 M NaCl and 5% (w/v) apple pectin (70–75% esterification) was equilibrated to 30 °C and pH adjusted to 7.5. Following the addition of a 2–3 mL PME extract (depending on the remaining activity of the extract), the pH was quickly readjusted to 7.5 which was then maintained up to 20 min by the titration with 0.01 N NaOH. The volume of base VNaOH added was recorded as a function of time. All samples were measured in triplicate and the slope (S = VNaOH/t) of the initial linear part of the titration curve was determined covering a period of 5–10 min. The slope is directly proportional to the activity of PME per mL of the sample (activity) which could be obtained by Eq. (1) (Basak & Ramaswamy, 1997). þ Activityðμmol H min −1 mL −1 Þ = SðmL min −1 Þ × NNaOH ðμmol mL −1 Þ  VðmLÞ = VNaOH = t x NNaOH ðμmol mL  VðmLÞ −1 Þ ð1Þ Where, S is the slope of titration, VNaOH is the volume of standardized NaOH (μmol H+ mL− 1) solution used for titration; NNaOH is the concentration of the NaOH solution used. V is the volume of PME solution added into the reaction mixture and t is the reaction time in min. 2.9. Texture The texture of the apple cubes was measured using an Instron Universal Testing Instrument (model 4501, Instron Corp., Ohio, USA) with 100 N load cell. The firmness was determined by measuring the force required for an 8 mm cylindrical probe to penetrate into the fruit piece to a depth of 5 mm at a cross head speed of 2 mm/min. The texture of 10 apple cubes from triplicate packs was measured and the average values were calculated from 30 observations. All results were expressed as the maximum force (N) required for a compression of 5 mm. 41 residual enzyme levels of minimally processed apples. Three levels of each variable (HPP treatment time, concentration of pineapple juice) were chosen as given in (Table 1). The experiment was carried out according to a central composite face-centred design and additional replicates were used to allow the analysis of results by ANOVA (3 factor factorial design). The statistical model and graphical presentation were obtained using Design Expert 7.1.3 (Stat-Ease Inc., Minneapolis, MN, USA). Statistical analyses of total colour change during in-pack storage study was performed by applying three-way analysis of variance (ANOVA) and multiple comparisons of means conducted using the Least Significant Difference (LSD) at the confidence level of 99% using Genstat software Version 9 (Rothamsted Experimental Station, Harpenden, UK) by Statistical consultancy unit at University of Melbourne. 3. Results and discussion 3.1. In-pack total colour change (∆E) during storage Visually detectable colour differences were not observed in high pressure treated samples of either apple variety during in-pack storage for 4 weeks at 4 °C. Therefore, the visual browning scores are not reported here. However, the in-pack instrumentally measured ∆E values showed a statistically significant three factor interaction on variety× pineapple juice × storage time. Granny Smith apples showed minimal changes in ∆E (0.5 units) compared to Pink Lady samples (2 units) during the 4 weeks of storage (Fig. 1). In Pink Lady samples ∆E decreased up to week 3 and increased at the fourth week, whereas the Granny Smith apple samples showed a fairly stable ∆E throughout the storage period. The statistical analysis on ∆E values also showed another significant (p <0.001) three factor interaction: variety× HPP time× % pineapple juice (Fig. 2). In general the ∆E was high in the Pink Lady samples compared to the Granny Smith samples at different HPP treatment times. Significant differences in the ∆E of Pink Lady samples were observed only in 25% pineapple juice containing samples HP-treated for 1 min (Fig. 2). Granny Smith samples HPP for 3 and 5 min treated with 25% and 50% pineapple juice showed a significantly lower ∆E than the control. The colour of HP-treated products depends on the inhibition of browning related enzymes and the stability of the pigments as affected by HPP treatment. In-pack browning of the HP-treated products also depends on the packaging material, its oxygen permeability after HPP, and storage conditions. While the products inside the pack are under oxygen free environment and no visible browning is observed and the measured colour changes may be attributed to the changes occurred in pigments due to the HP treatment. Ahmed and Ramaswamy (2006) 2.10. Experimental design Response surface methodology was used to investigate the combined effect of HPP treatment time and concentration of pineapple juice (%) on total colour change, browning, texture and Table 1 Variables and levels used in central composite response surface design. Independent variables % Pineapple juice (v/v) HPP time (min) Levels −1 0 1 0 1 25 3 50 5 − 1—lowest level; 0—medium level; 1—highest level. Fig. 1. Interaction of different concentrations of pineapple juice and HPP time on inpack total colour change of apples during four weeks of storage at 4 °C ■ (GS, 0% PJ), ● (GS, 25% PJ), ▲ (GS, 50% PJ), □ (PL, 0% PJ), ○ (PL, 25% PJ), ∆ (PL, 50% PJ). 42 N. Perera et al. / Innovative Food Science and Emerging Technologies 11 (2010) 39–46 Fig. 2. Interaction of variety and HPP time on total colour change of apples during inpack storage after four weeks at 4 °C ■ (GS, 0% PJ), ● (GS, 25% PJ), ▲ (GS, 50% PJ), □ (PL, 0% PJ), ○ (PL, 25% PJ), ∆ (PL, 50% PJ). reviewed literature on the effect of HPP on the colour of fruit products and concluded that minimal colour changes are caused by HPP treatments. The majority of the studies on the colour/pigment of fruits demonstrated that insignificant changes occur during HPP, but a decrease in colour is reported during storage (Ahmed et al., 2005). Ahmed, Ramaswamy and Hiremath (2005) observed that there was no visual colour change in mango pulps after high pressure treatments at Fig. 4. Response surface curves for visual browning scores of apples treated with different concentrations of pineapple juice after 5 h air exposure (a) Granny Smith and (b) Pink Lady. 100–400 MPa/20 °C/15–30 min. They observed that colour parameters of mango pulps remained constant after high pressure treatment indicating pigment stability. Our observations may indicate that carotenoids may be contributing to the in-pack colour stability of Pink Lady samples which has a more yellowish flesh than the Granny Smith apples. Butz et al. (2003) found that there were no significant differences in carotenoid content between the pressure treated and the control samples of fruit juices. The characteristic light greenish colour of the Granny Smith apple is due to the presence of chlorophyll pigment (Bartram, 1986). Lopez-Malo, Paloue, Barbosa-Canovas, Welti-Chanes and Swanson (1998) reported that HPP helped to preserve colour in avocado puree, by preventing browning and facilitating retention of green colour. Green beans, HP-treated at 500 MPa for 1 min at ambient temperature, have been shown to maintain a more intense bright green colour on the vegetable's surface (Krebbers, Matser, Koets & Van den Berg, 2002). However, Van Loey, Weemaes, Vanden Broek, Ludikhuyze and Indrawati (1998) reported that elevating pressure from 200 to 800 MPa accelerates the degradation of chlorophyll in broccoli. 3.2. Total colour change (∆E) and visual browning after air exposure Fig. 3. Response surface curves for total colour change of apples treated with different concentrations of pineapple juice after 5 h air exposure (a) Granny Smith and (b) Pink Lady. The two varieties showed different patterns in their ∆E values with reference to % pineapple juice used and HPP treatment times (Fig. 3a and b). However, for both apple varieties the samples treated with the highest concentration of pineapple juice 50% and the longest HPP treatment time 5 min recorded the lowest ΔE and the least browning score after 5 h of air exposure. The lowest ΔE value for Granny Smith apples was lower than that of the Pink Lady apples and this same N. Perera et al. / Innovative Food Science and Emerging Technologies 11 (2010) 39–46 43 quinones produced by PPO (Richard-Forget, Goupy & Nicolas, 1992). Prevention of enzymatic browning using sulphur containing amino acids and peptides on apples has been demonstrated by Friedman and Molnar-Perl (1990). Palou, Lopez-Malo, Barbosa-Canovas, WeltiChanes and Swanson (1999) reported that the initial colour of banana puree could be preserved by HPP at 500–700 MPa, for 10 min at 21 °C. This treatment also reduced the browning rate significantly. In our study both HPP and pineapple juice additively contributed to minimise the total colour change and visual browning in both apple varieties during (5 h) air exposure. 3.3. PPO activity Fig. 5. Influence of HPP time and different concentrations of pineapple juice on residual polyphenoloxidase activity (a) Granny Smith and (b) Pink Lady. trend was observed with visual browning scores (Fig. 4a and b ). The observed differences in the two apple varieties may be due to the differences in the susceptibility to browning of the apple varieties (Lozano-de-Gonzalez et al., 1993; Rocha & Morias, 2001; Ye, Heng, Yuan-peng, Feng & Shu-wei, 2007; Joshi et al., 2007), PPO activities and perhaps differences in polyphenol composition (Joshi, Rupasinghe, Pitts & Khanizadeh, 2007). In HPP (100–1000 MPa/−20 °C to 60 °C), cell walls and membranes are prone to disruption (Michel & Autio, 2001; Prestamo & Arroyo, 1998; Van Buggenhout, Messagie, Van Loey, & Hendrickx, 2005). This leads to the mixing of enzymes and substrates in the disrupted plant tissue. Browning is enhanced when such products are exposed to air. The inhibitory effect of fresh pineapple juice could be due to the presence of bromelain enzyme, sulfhydryl compounds or by organic acids (malic and citric acids). The use of pineapple juice to inhibit enzymatic browning in apples has been reported by (Lozano-de-Gonzalez et al., 1993; Labuza et al., 1990). The effectiveness of pineapple juice on prevention of browning was also reported by (Chaisakdanugull et al., 2007) on the cut surface of banana slices. This study suggested that browning inhibition could be achieved with pineapple juice to similar extent with 8 mM ascorbic acid but less than with 4 mM sodium metabisulfite. In addition, sulfhydryl compounds present in pineapple juice are reported to be effective as antibrowning agents (Bennion, 1990; Collins, 1960; Wrolstad & Wen, (2001)). It is also generally accepted that thiol compounds present in pineapple juice control the enzymatic browning reaction by the formation of colourless compounds with o- The degree of inactivation of PPO in Granny Smith and Pink Lady apples were significantly (p < 0.05) affected by 50% pineapple juice and 5 min HPP treatment time. HPP treatment time had the greatest effect on the activity of PPO in both apple varieties (Fig. 5a and b) and was significant at p < 0.05 (Table 2). In Granny Smith apples with the increase of HPP treatment time, the PPO activity reduced drastically than in Pink Lady apples. The interaction between HPP treatment time and pineapple juice concentration also had significant effect on the activity of PPO in Granny Smith apples. The highest decrease in PPO activity was observed at 50% pineapple juice concentration and with the 5 min HPP treatment time (Fig. 5a). Combined treatment of 5 min HPP treatment time and 50% pineapple juice inactivated about 40% of PPO in Granny Smith apples, compared to 19% and 21% inactivation in 5 min HPP only treatment and pineapple juice only treatments respectively (Fig. 5a). The same trend was shown by the pineapple juice and HPP treatments on inactivation of PPO in Pink Lady apples (Fig. 5b). Combined treatment of 5 min HPP treatment time and 50% pineapple juice significantly (p < 0.05) inactivated about 30% of PPO in Pink Lady apples compared to 8% and 22% inactivation in 5 min HPP only treatment and pineapple juice only treatments respectively (Fig. 5b). These observations suggest that HPP and pineapple juice seems to have an additive influence on the inactivation of PPO. Therefore, the reduction of both browning score and ∆E in apples after exposure to the air may be a result of the partial inactivation of PPO enzyme with 50% of pineapple juice and 5 min HPP treatments. Pink Lady apples also followed a similar trend; however, the relationships were not as strong as in Granny Smith apples. This may be due to the varietal differences in PPO activity. Apple PPO has been intensively studied because of its importance to the processing industry. PPO activity in apples varies with cultivar, tissue type (Janovitz-Klapp et al., 1989), storage conditions (SolviaFortuny et al., 2001; Rocha & Morias 2001), and fruit maturity (Murata et al., 1995). A number of studies from selected sources have been conducted to establish methods to inhibit PPO by means of HPP. At 1 min HPP treatment time apple PPO in cell free extracts showed activation at 200 MPa and inactivation at 900 MPa respectively (Anese, Micoli, Dall-Aglio & Lerici, 1995). Residual activity of purified apple (cv. Golden Delicious) PPO was determined after HPP treatment at 800 MPa for 30 min (Weemaes, Ludikhuyze, Van Den Broeck & Hendrickx, 1998). HPP inhibition of PPO in diced apples (cv. Golden Delicious and Granny Smith) was promoted by immersion in 4-hexylresorcinol for 15 min, where as immersion in ascorbic acid or citric acid solutions for 15 min did not result PPO sensitization due to HPP (Ibarz et al., 2000). The highest level of PPO inhibition (54.1%) was shown by the organic acid fraction of the pineapple juice, which confirms the important role of organic acids (malic and citric acid) in inhibiting enzymatic browning in cut surfaces of banana slices with pineapple juice (Chaisakdanugull et al., 2007). Moreover, the pineapple juice fraction that had the highest bromelain activity showed the lowest level of PPO inhibition (27.7%) (Chaisakdanugull et al., 2007). Therefore, it is unlikely that bromelain in pineapple juice was contributed to PPO inhibition. Labuza et al. (1990) also reported that bromelain did not inhibit PPO activity of 44 N. Perera et al. / Innovative Food Science and Emerging Technologies 11 (2010) 39–46 Table 2 Analysis of variance and coefficients of the response surface models describing the effect of combined high pressure-% pineapple juice on the browning score, total colour change and residual and PPO activity of Granny Smith samples after eliminating non-significant terms. Source Browning Score Estimated coefficient Model Intercept A-% pineapple juice B-HPP time A×B A2 B2 R2 Total colour change p-value Estimated coefficient <0.0031 4.7 0.005 0.0012 − 0.015 0.0193 Residual PPO activity p-value Estimated coefficient Texture 0.0005 7.24 0.08 0.22 − 0.003 0.9471 <0.0001 0.0146 0.0074 0.9342 Estimated coefficient p-value 0.0004 113.97 0.415 − 20.71 p-value 0.0021 13.65 0.0484 <0.0001 2.04 0.0306 0.9031 − 0.91 0.0014 − 0.10 0.9263 0.0473 The ANOVA for PME activity is not shown in this table since none of the treatment variables had significant effects on this parameter. The residual activity of PPO and PME were calculated as the ratio of the activity of the enzyme in the treated samples to that in the untreated sample. mushroom in an aqueous model system, but it was effective in inhibiting browning of refrigerated apples. 3.4. Texture Texture of Granny Smith apples was significantly (p < 0.05) affected by HPP treatment time (Fig. 6a) and (Table 2). The texture of Pink Lady apples was not affected by the any of the treatment parameters (Fig. 6b). The lowest firmness was observed with 0% pineapple juice and 1 min HPP time and the greatest retention of firmness was detected in the apples treated with 0% pineapple juice and with the longest (5 min) HPP time. The observed apparent firming effect may be attributed to the activation of the endogenous PME activity with HPP and the longer HPP treatment time. The higher the PME activity, demethylation of pectin, which increases the probability that two adjacent polygalacturonic polymer chains form an ‘egg box’ structure in the presence of divalent cations such as calcium leading to an apparent increase in firmness (Van Dijk & Tijskens, 2000). 3.5. PME activity None of the treatment parameters had significant effect on the activity of PME in both apple varieties. However, some variability was observed in the residual PME activity. For instance 51% reduction in PME activity was observed in Granny Smith apples with 25% pineapple juice and 3 min HPP treatment time whereas apparent activation was observed with 0% pineapple juice and 5 min HPP treatment time (118% residual activity). In Pink Lady apples 98% of residual PME activity was observed with 25% pineapple juice and 3 min HPP treatment time and 198% activation was observed with 0% pineapple juice and 5 min HPP treatment time (results not shown). The best retention of PME activity was observed for both varieties of apples with samples HP-treated for 5 min regardless of the pineapple juice concentration. The greater retention of PME activity and the HPP induced activation may contribute towards the firming effect observed in apple samples with 5 min HPP treatment time. Although Table 3 Analysis of variance and coefficients of the response surface models describing the effect of combined high pressure-% pineapple juice on the browning score and residual PPO activity of Pink Lady apples. Source Browning Score Estimated coefficient Model Intercept A-% pineapple juice B-HPP time A×B A2 B2 R2 Fig. 6. Response surface curves for texture of apples treated with different concentrations of pineapple juice (a) Granny Smith and (b) Pink Lady. Residual PPO activity p-value Estimated coefficient <0.0001 5.04 − 0.05 1.0 <0.0001 0.0079 0.0024 p-value 0.0007 11.72 − 19.75 <0.0001 − 7.27 0.0415 − 0.2 0.9107 0.8893 The ANOVA for total colour change, texture and residual PME activity are not shown in this table since none of the treatment variables had significant effects on these parameters. N. Perera et al. / Innovative Food Science and Emerging Technologies 11 (2010) 39–46 the variety Pink Lady showed a higher range of PME activity than Granny Smith apples, the firming effect was not quite obvious in the variety Pink Lady. Thus the HP-induced effect on activity of PME depends not only on the HPP conditions and the type of fruit but also the detailed biochemical composition of the PMEs present. Denes, Baron and Drilleau, (2000), reported that PME purified from apple was pressure stable in the range of 100–650 MPa at 25 °C. However, PME activity in orange juice was inhibited by more than 88% after HPP treatment at 600 MPa and 25 °C for 5 min (Nienaber & Shellhammer 2001; Bull et al., 2004). Similarly, significant inhibition of orange PME after HPP treatment at 600 MPa and ambient temperatures for 5 to 5.8 min was also reported by Truong, Boff, Min, and Shellhammer (2002). Our results also demonstrated the presence of stable PME activity in apples under the treatment conditions of 600 MPa tested in this study. 4. Conclusions Results of this study showed that combined high pressure and pineapple juice gave a significant reduction of the out of pack browning during air exposure and thus could be used for the prevention of browning in HP-treated apples. Processing with 50% pineapple juice and 5 min HPP time resulted in the best quality retention in both apple varieties Granny smith and Pink Lady. This study shows that combinations of HPP and pineapple juice are more effective than the same conditions used in isolation on the prevention of browning when products are exposed to air. These same conditions also led to 40% and 30% inactivation of PPO in Granny Smith and Pink Lady apples respectively. Increase in HPP time had a firming effect on apples, which is postulated to be due to enhanced PME activity under pressure. HPP at ambient conditions with vacuum packaging in 50% pineapple juice as processing media in high barrier bags and refrigerated storage results in high quality product with relatively good storage stability over at least 4 weeks and delayed browning after opening. The product will remain highly attractive for several hours after opening, giving sufficient time for presentation in domestic and food service environments where high quality freshlike fruit products such as fruit salads (e.g. apple pieces in pineapple juice) are required. 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