Innovative Food Science and Emerging Technologies · 2014-06-26 · Innovative Food Science and Emerging Technologies xxx (2011) xxx–xxx ⁎ Corresponding author. Tel.: +31 317

Innovative Food Science and Emerging Technologies xxx (2011) xxx–xxx

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Innovative Food Science and Emerging Technologies

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Comparing equivalent thermal, high pressure and pulsed electric field processes formild pasteurization of orange juice. Part I: Impact on overall quality attributes

R.A.H. Timmermans a,⁎, H.C. Mastwijk a, J.J. Knol a, M.C.J. Quataert a, L. Vervoort b, I. Van der Plancken b,M.E. Hendrickx b, A.M. Matser a

a Wageningen UR Food and Biobased Research, P.O. Box 17, 6700 AA Wageningen, The Netherlandsb Laboratory of Food Technology, Leuven Food Science and Nutrition Research Center, Department of Microbial and Molecular Systems, Katholieke Universiteit Leuven,Kasteelpark Arenberg 22 box 2457, 3001 Heverlee, Belgium

⁎ Corresponding author. Tel.: +31 317 481305; fax:E-mail address: [email protected] (R.A.H. Tim

1466-8564/$ – see front matter © 2011 Elsevier Ltd. Aldoi:10.1016/j.ifset.2011.05.001

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Article history:Received 3 March 2011Accepted 2 May 2011Available online xxxx

Keywords:Mild heat pasteurizationHigh pressure (HP) processingPulsed Electric Field (PEF) processingQuality parametersShelf lifeOrange juice

Mild heat pasteurization, high pressure processing (HP) and pulsed electric field (PEF) processing of freshlysqueezed orange juice were comparatively evaluated examining their impact on microbial load and qualityparameters immediately after processing and during two months of storage. Microbial counts for treatedjuices were reduced beyond detectable levels immediately after processing and up to 2 months of refrigeratedstorage. Quality parameters such as pH, dry matter content and brix were not significantly different whencomparing juices immediately after treatment and were, for all treatments, constant during storage time.Quality parameters related to pectinmethylesterase (PME) inactivation, like cloud stability and viscosity, weredependent on the specific treatments that were applied. Mild heat pasteurization was found to result in themost stable orange juice. Results for HP are nearly comparable to PEF except on cloud degradation, where alower degradation rate was found for HP. For PEF, residual enzyme activity was clearly responsible for changesin viscosity and cloud stability during storage.Industrial relevance: Development of mild processing technologies with a minimal impact on fruit juice can beconsidered as a true alternative of fresh fruit. The present work presents a fair comparison of mild heattreated, high pressure (HP) and pulsed electric field (PEF) processed orange juice as an alternative for thermalpasteurization. Orange juices were monitored during two months of storage.

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A.H., et al., Comparing equivalent thermal, hiative Food Science and Emerging Technologies

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1. Introduction

Nowadays, consumers choose more and more for minimallyprocessed foods with characteristics closer to those of fresh foodsusing minimal processing or reducing chemical preservatives (Allenda,Tomás-Barberán, & Gil, 2006; Oliveira & Oliveira, 1999). Juices are noexception, and there is, therefore, a strong tendency towards consump-tion of premium quality juices. These juices are directly obtained fromfruits (not from concentrate), are distributed through the refrigeratedchain and have a relatively short shelf life (Esteve, Frígola, Rodrigo,& Rodrigo, 2005). Traditionally, thermal pasteurization is used toinactivatemicro-organisms to prolong shelf life on the one hand, and toinactivate heat-stable pectinmethylesterase (PME) for preventing cloudloss on the other hand (Chen & Wu, 1998). Typically, for shelf stablejuices, processing times for thermal pasteurization are equivalent to90 °C for 1 min (Eagerman & Rouse, 1976). Thermal processing has anegative impact on the quality of orange juice such as loss of freshflavor (Braddock, 1999), degradation of ascorbic acid (Chen, Shaw, &

Parish, 1993), and discoloration (Arreola et al., 1991; Li, Sawamura,& Kusunose, 1988; Tatum, Nagy, & Berry, 1975). To avoid qualitydegradation of orange juice by thermal processing, non-thermal pres-ervation techniques suchashighpressure andpulsed electricfieldshavebeen studied during the last decade.

High pressure processing (HP), when carried out around roomtemperature, is one of the non-thermal processes, that inactivatesbacterial cells, yeasts and molds without the use of heat, having aminimal effect on the sensory qualities associated with ‘fresh-like’attributes such as texture, color and flavor (Arroyo, Sanz, & Préstamo,1999; Bull et al., 2004; Hayashi, 1996). HP typically uses pressuresof 300–700 MPa for periods of 30 s up to a few minutes to destroypathogenic and spoilage bacteria. Bacterial spores may survive pres-sures above 1000 MPa at room temperature, but outgrowth can beprevented by temperature control (refrigerated storage) and lowpH conditions (e.g. acid products or acidification). In contrast to heatpasteurization, HP at room temperature showed no complete in-activation of pectinmethylesterase (PME) after pressure treatments(Basak & Ramaswamy, 1996; Goodner, Braddock, & Parish, 1998; Vanden Broeck, Ludikhuyze, Van Loey, & Hendrickx, 2000; Van denBroeck, Ludikhuyze, Van Loey, Weemaes, & Hendrickx, 1999). It hasbeen suggested that residual PME activity following high pressure

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2 R.A.H. Timmermans et al. / Innovative Food Science and Emerging Technologies xxx (2011) xxx–xxx

treatment is due to the persistence of a HP resistant PME fractionthat corresponds with the heat-stable PME fraction (Duvetter et al.,2009; Goodner et al., 1998; Van den Broeck et al., 2000). Althoughthere is some residual PME activity, the cloud of orange juice wasstable for more than 50 and 60 days at 4 °C, using conditions of 1 minat 700 MPa and 800 MPa respectively (Goodner et al., 1998; Nienaber& Shellhammer, 2001).

Pulsed electric field (PEF) processing, when carried out withlimited energy input, is another non-thermal preservation process.In a PEF process, food is treated in a chamber with two electrodesgiving short electric pulses to inactivate micro-organisms, in order toincrease the shelf life of treated food products with a minimal effecton product quality. Effects on enzyme inactivation largely dependon the thermal history of the sample during the PEF treatment.Application of short pulses of high electric fields with a duration ofmicroseconds and intensities in the order of 20–80 kV/cm minimizethe heating of food (Altunakar, Gurram, & Barbosa-Cánovas, 2007;Zhang, Qin, Barbosa-Cánovas, & Swanson, 1995), although the appliedpulse causes irreversible loss of the cell membrane functionality,leading to inactivation of microbial cells. Effectiveness of microbialinactivation by PEF depends on process parameters including electricfield strength, total treatment time, pulse width and pulse waveform,and onmedium related parameters such as conductivity, composition,temperature and microbial parameters (Aronsson & Rönner, 2001;Barsotti & Cheftel, 1999; Vega-Mercado et al., 1997; Wouters &Smelt, 1997). Concerning the inactivation of PME, it is suggested thata higher electric field strength and longer treatment time are moreeffective (Yeom, Streaker, Zhang, & Min, 2000a,b). Yeom, Zhang, andChism (2002) reported a 90% reduction in PME activity from orangejuice by applying 25 kV/cm for 250 μs. However, at these conditionsthe thermal load to the product is equivalent to 149 °C. Van Loey,Verachtert, and Hendrickx (2002) reported an inactivation of lessthan 10% at 35 kV/cm for 1000 pulses of 1 μs at temperatures whereinactivation of enzymes due to heat could be excluded.

There are many studies on the effect of mild preservation tech-niques on micro-organisms, quality and shelf life of food products.Variance in initial quality of the food, caused by use of differentspecies, maturity and storage conditions, and the use of differentprocess conditions make it difficult to compare the results obtained.The objective of this work was to compare the impact of mild heatpasteurization, high pressure processing and pulsed electric fieldprocessing of freshly squeezed orange juice on nutritional, qualityand sensorial parameters and consumer preference during 2 monthsof storage. Processing conditions were defined in such a way thatimmediately after processing equivalent products in terms ofmicrobial inactivation were obtained. In this research three partieswere involved, Katholieke Universiteit Leuven (Leuven, Belgium),Central Food Research Institute (Budapest, Hungary) and Wagenin-gen UR Food & Biobased Research (Wageningen, The Netherlands).This paper reflects part I of the work (Fig. 1). The impact on specificchemical and biochemical quality parameters is described in part II byVervoort et al. (under review) and the sensorial aspects, part III, aredescribed by Magyar-Horváth et al. (under review). The consumerstudy will be published in a separate publication.

2. Material and methods

2.1. Product

Orange juice was derived from a mixture of three varieties oforanges Valencia, Pera and Baladi, supplied by a commercial freshjuice producer located in The Netherlands. The oranges, present inequal proportions, were brushed, washed, hand sorted and washedagain one day before the trial by the supplier at 7 °C, and were storedin a clean plastic box at 4–6 °C.

Please cite this article as: Timmermans, R.A.H., et al., Comparing equivmild pasteurization of orange juice..., Innovative Food Science and Emerg

2.2. Selection of process conditions

Process conditions were selected to result in an equivalentprocessing impact, with respect to microbial safety and spoilagemicro-organisms (Matser, Mastwijk, Bánáti, Vervoort, & Hendrickx,2010). The Food and Drug Administration recommends a 5-logreductionof the “pertinentmicro-organism”,which is themost resistantmicro-organism, in orange juice E. coli O157:H7 (FDA, 2004).

As a guideline FDA recommends a minimum temperature–timeequivalent for juice of 71.1 °C for 3 s for products with a pH in therange of 3.6–4.0. However, this specific temperature–time is insuffi-cient to inactivate spoilage organisms.

Furthermore, these minimum conditions have to be translated inprocess conditions for large-scale heat exchangers as used in this study,concerning limitations in heat penetration in large scale systems. Forparticulate and more viscous products both the average temperatureand processing time must be increased to guarantee that a minimumsufficient degree of processing has occurred. The industrial conditionsfor heat pasteurization of premium juices are equivalent to 90 °C for2 s or 84 °C for 20 s (Mazzotta, 2001). However, these processingconditions by far exceed the criterion for microbial inactivation andat these temperatures the quality of treated orange juice is far toomuch compromised for the scope of this comparative study. In thisstudy, 72 °C for 20 s was used as a reference treatment for mild heatpasteurization of orange juice.

For high pressure treatment, literature reports 1–5 min at 350–500 MPa to inactivate E. coli in orange juice (Damar, Bozoglu, Hizal,& Bayindirh, 2002; Erkmen & Dogan, 2004; Linton, McClements,& Patterson, 1999). Industrial conditions used are 1–2 min at 500–600 MPa to show optimal safety and quality. For this study therefore1 min at 600 MPa was used at an industrial HP production line.

For pulsed electric field processing, conditions recommended toinactivate E. coli and other pathogens are 25–35 kV/cm during 20–300 μs (Alvarez & Heinz, 2007; Wouters, Dutreux, Smelt, & Lelieveld,1999). However, for this technology, actual design of the equipment,especially the treatment chamber, strongly influences the effective-ness of the treatment and the temperature reached during the PEFtreatment itself (Mastwijk et al., 2007). PEF conditions were usedthat were in line with the literature data and that shown in previousstudies to result in safe and stable orange juices (Matser, Schuten,Mastwijk, & Lommen, 2007).

2.3. Preparation and processing

Juice from 1300 kg of oranges was extracted in a single batchusing an automatic juice extractor (Master Zumoval, Valencia, Spain)to obtain 650 L of orange juice with pulp. After pressing, the juicewas sieved (mesh 3.5 mm) and transported via a cooling vessel to astorage tank (4–6 °C), and stirred until filling. The juice was sub-jected to different treatments: fresh/untreated, mild heat treatment,high pressure processing (HP) and pulsed electric field (PEF), seeFig. 1. Orange juice was bottled under hygienic conditions in 250 mLPET bottles and sealed with PE caps. The bottles and caps werepre-sterilized by gamma irradiation at 15 kGy (Isotron, Ede, TheNetherlands).

2.3.1. Mild heat pasteurizationHeatingwas performed using a pilot-scale, continuousflow tubular

heat exchanger (inner diameter 10 mm). Orange juice was sieved fora second time prior to pasteurization because of technical parametersof the tubular heat exchanger. The juice was preheated to 45 °C, thenheated to 72 °C and subsequently held at this temperature for 20 s.Directly after the holding tube, the juice was cooled down to 2 °C andmanually filled under hygienic conditions in a laminar flow cabinetinto the PET bottles.

alent thermal, high pressure and pulsed electric field processes foring Technologies (2011), doi:10.1016/j.ifset.2011.05.001


Part I

Valencia oranges

Pera oranges

Baladi oranges

Brushing & washing

Handsorting

Washing

Extraction

Juice

HP Bottling Bottling

Untreated orange juice

HP treated orange juice

Heat treated orange juice

PEF treated orange juice

PEF treatmentHeat pasteurizationBottling

Distribution

Quality aspects Sensorial aspects

Central Food Research Institute

(Budapest)

Nutritional aspects

Consumer study

Katholieke Universiteit

Leuven

(Leuven)

Katholieke Universiteit

Leuven

(Leuven)

Food & Biobased Research

(Wageningen)


(Wageningen)

Microbiology


(Wageningen)

Part II Part IIISeparate

publication

Fig. 1. Overview of the production, handling and analysis of orange juice samples and dissemination of publications.

3R.A.H. Timmermans et al. / Innovative Food Science and Emerging Technologies xxx (2011) xxx–xxx

2.3.2. High Pressure Processing (HP)Untreated juice packed in PET bottles with PE caps was subjected

to HP treatment.An industrial unit, WAVE 6000/55 high pressure unit (NC Hyper-

baric, Burgos, Spain) was used to treat the orange juice at 600 MPa for1 min. Water of 17 °C was used as pressure fluid, where it took 3 minto come up to the desired pressure, and less than 5 s to depressurize.The temperature rise during compression was 3 °C/100 MPa.


2.3.3. Pulsed Electric Field (PEF)For PEF processing a continuous flow pilot plant described in more

detail by Mastwijk (2006) and Lelieveld, Notermans, and De Haan(2007) was used. Prior to processing, the entire pilot plant systemwas cleaned (CIP) and pre-sterilized (SIP 120 °C for 20 min). Thetemperature of the juice in the storage tank was 11±2 °C. First, thejuice was heated to 38 °C in a preheating section. Then PEF treat-ment was applied using monopolar pulses of 2 μs duration at nominal




electric field strength of 23 kV/cm. The treatment device was acolinear chamber with a 20 mm gap. The outlet temperature was58 °C at a repetition rate of 90 Hz and a flow rate of 130 L/h. Pulseenergy yields 31±3 J per pulse, specific energy input was 76±4 J/mLand number of pulses was 18±2. Directly after PEF treatment thejuice was cooled back to 14 °C and samples were manually bottled in250 mL PET bottles under hygienic conditions in a laminar air flowcabinet.

2.4. Storage and sampling

Immediately after filling bottles were stored at 4 °C, with tem-peraturemeasurements taken at 30 min intervals using calibrated tem-perature data loggers (I-button DS1921G, Maxim Direct, Dallas, U.S.A.).Fresh/untreated samples were also stored frozen below −40 °C as areference sample for sensorial analysis. The samples were analyzed intriplicate formicrobiological, chemical and physical properties after 1, 2,9, 20, 28 and 58 days of storage.

2.5. Microbial analysis

Samples were taken before and after filling, from the raw anddifferent types of treated orange juice. These samples were analyzedin triplicate on total plate count, Enterobacteriaceae, E. coli, Lactic acidbacteria, yeast and molds following methods ISO 4833, ISO 21528–2,ISO 16649–2, ISO 15214, and ISO 7954 respectively. During storage ofthe orange juice at 4 °C, samples were taken at indicated times andanalyzed in triplicate.

2.6. Color

Colormeasurements were executed at 22±1 °C, using a HunterlabColorFlex (Hunterlab, Reston, Virgina, U.S.A.). This spectrophotometeruses an illuminant of D65 and a 10° angle observer as reference. Theinstrument was standardized each time with a black and whiteceramic plate. Samples were filled in glass cuvets, and Hunter valuesa* (redness), b* (yellowness), and L* (lightness) were measured intriplicate. Color difference, ΔE, was calculated from L*, a*, and b*parameters, using Hunter-Scotfield's equation ΔE=(Δa2+Δb2+ΔL2)1/2 where a=a–a0, b=b–b0 and L=L–L0. The subscript ‘0’

indicates initial color, and the y-intercept of linear regression modelswas taken for this number. ΔE was calculated for each treatment andzero-values for each treatment were used as comparison. Dependingon the value of ΔE the development in color difference during storagetime could be estimated as not noticeable (0–0.5), slightly noticeable(0.5–1.5), noticeable (1.5–3.0), well visible (3.0–6.0) and great (6.0–12.0) (Cserhalmi, Sass-Kiss, Tóth-Markus, & Lechner, 2006).

2.7. Brix

The °brix of centrifuged samples of orange juice (10 min at 360 xg)was measured by a digital hand-held refractometer at 22 °C±1 °C(PR-1, Atago, Japan).

2.8. pH

The pH measurements of orange juice (22°±1 °C) were per-formed with a pH meter (Metrohm, 826 pH mobile) with a glass-electrode (Metrohm) and calibrated before every measurement at pH4 and 7.

2.9. Dry matter content

Moisture in 10 g orange juice was evaporated from the sample byoven drying, 4 h at 70 °C followed by 20 h at 105 °C. Total dry matter


content was determined gravimetrically as residue remaining afterdrying. Samples were cooled in a desiccator prior to weighing.

2.10. Cloud stability

Cloud stability was investigated by measurements of sedimenta-tion rate and scoring the opacity of the cloud. The sedimentation ratewas characterized by recordings of the height of the interface of thesediment and cloud in bottles that were left undisturbed duringrefrigerated storage time. In addition, the opacity of the cloud in thebottles was scored on a 1–5 scale (1 transparent to 5 opaque) by visualappearance to have an indication on the rate of changes from opaqueto transparent.

2.11. Viscosity

The apparent viscosity of the juices was measured after centrifu-gation (10 min at 360 xg) using a Brookfield viscometer (model LVDV-E, Brookfield Engineering Laboratories, Stoughton, USA). Avolume of 16 mL of the juice was placed in the UL-adapter, withcorresponding spindle. Viscosity was determined at temperaturesin the range of 21.5–22.5 °C. Measurements were performed intriplicate.

2.12. Statistical analysis

Changes in the quality parameters (color, brix, pH, dry matter,viscosity, cloud stability) for the different treatments (untreated, heat,HP, PEF) were quantified. Initial changes that were observed directlyafter processing were determined together with small changes thatwere observed during storage by linear regression. A single sidedt-test was used to quantify the differences between the various treat-ments. Differences in the initial level and during storage were con-sidered significant when the confidence level (CL) was larger than95% (Pb0.05).

3. Results and discussion

3.1. Microbiological analysis

The batch of samples that was produced (n=12) was evaluatedwithin 24 h after processing. All samples in the batch complied withEC regulation 2073/2005, which describes microbial criteria forfoodstuffs. Microbial assessment during storage aimed to characterizethe effects of processing, hygiene and spoilage (Table 1). Duringstorage at 4 °C microbial counts were analyzed on the days specified.For each day of sampling, three PET bottles were randomly chosenand analyzed. Untreated orange juice showed to have low microbialcounts until t=9 days, where at t=20 days growth was observed.High quality oranges were used in this study, since one normallycould expect initial populations of viable aerobic bacteria in raw juicebetween 103 and 105 cfu/mL even up to 108. For yeast and molds,counts of 103 cfu/mL up to 105 cfu/mL could be found, dependingon species, maturity and storage conditions (Bull et al., 2004; Elez-Martínez, Soliva-Fortuny, &Martín-Belloso, 2006). All samples treatedby mild heat, HP and PEF showed to be under detection limit after2 months of storage at 4 °C. The latter corresponds to the expecta-tions, since all process conditions were selected on equivalentprocessing, with respect to microbial food safety and spoilage.

3.2. Color

Color differences between untreated and the different types oftreated orange juice were determined at different time-intervalsduring storage. Hunter L*, a* and b* values were measured, and resultsof regression kinetics and t-test are shown in Tables 2 and 3.



Table 2Regression of kinetic parameters for frozen untreated, untreated, mild heat treated, high pr

Untreated frozen Untreated

Color L Slope −0.04±0.01t=0 57.00±0.04 57.22±0.08

Color a Slope −4.53 E-03±4.21E-03t=0 1.94±0.02 1.95±0.02

Color b Slope −0.05±0.02t=0 63.51±0.09 61.84±0.11

Brix Slope 1.32E-03±6.27E-03t=0 11.73±0.06 11.59±0.03

pH Slope −8.60E-03±1.80E-03t=0 3.34±0.01 3.37±0.01

Dry matter content Slope −0.03±0.01t=0 9.48±0.01 10.09±0.05

Viscosity Slope −2.67E-03±2.46E-03t=0 1.56±0.02 1.57±0.01

Table 1Microbial counts evaluated at different times during storage of untreated, mild heattreated, high pressure processed (HP) and pulsed-electric field (PEF) treated orangejuice.

9 days2 days1 days0 days 20 days 28 days 58 days

E.Coli (ISO 16649-2) cfu/g

Untreated

Heat pasteurized

HP

PEF

<10 n.d.

Enterobacteriaceae (ISO 21528-2) cfu/g

Untreated

Heat pasteurized

HP

PEF

<10

Total aerobic plate count (ISO 4833) cfu/g

Untreated 1.4*105 1.4*105 n.d.

Heat pasteurized

HP

PEF

<1000 <1000

Yeast (ISO 7954) cfu/g

Untreated 910 990 420 420 6.2*104 1.1*105 n.d.

Heat pasteurized

HP

PEF

<10

Mould (ISO 7954) cfu/g

Untreated 17 12 32011 19 4700 n.d.

Heat pasteurized

HP

PEF

<10

Lactic acid bacteria (ISO 15214) cfu/g

Untreated 1000 810 640 530 4.7*104 1.2*105 n.d.

Heat pasteurized

HP

PEF

<100

n.d. = not determined.



The overall trend for the color of orange juices during storagetime showed that lightness (L* value) decreased, redness (a* value)increased, and yellowness (b* value) decreased, which is consistentwith research of Nienaber and Shellhammer (2001) (data not shown).There are no significant differences in color parameters between HPtreated and untreated orange juice, where mild heat pasteurized andPEF treated orange juice showed differences for all L*, a* and b* valuesdirectly after production.

Mild heat pasteurized orange juice was significantly lighter thanHP and untreated orange juice, having less red color and more yellow-ness. PEF treated samples showed the opposite: significantly darker incolor than untreated, HP and heat, with significantly more red and lessyellow tints.

Yeom et al. (2000a) and Min, Jin, Min, Yeom, and Zhang (2003)reported a significant lighter appearance of PEF treated juice over heattreatment.

Despite the significant differences in L* a* b* color attributes ofsamples, it ismorevaluable fromapractical pointof view tocompare theDE values of orange juice. DE values, indicated in Fig. 2, are the sum of L*a* and b* values, which is more associated to consumer perception thansingular L* a* or b* values, since consumers do not judge each particularattribute, but the combination of them (Cserhalmi et al., 2006).

Based on the observed DE values, it was found that all types oforange juice showed a noticeable (DE 0.5–1.5) difference in colorcompared to its color on day 1. There were no noticeable colordifferences between the different treatments.

3.3. Brix

In citrus juices, °brix is used to indicate the percentage of solublesugars and is one of the most important factors for grading the qualityof a citrus juice (McAllister, 1980). °Brix varies for juices obtainedfrom different species of oranges and during seasons, and it is there-fore not easy to compare °brix values with other studies. PEF treatedorange juice gave a slightly lower °brix after processing (Fig. 3).However, during storage no significant changes were observed in thesamples (for none of the processing methods).

3.4. pH

The effect of mild heat treatments on pH of orange juice is shownin Fig. 4. No significant differences between untreated and differenttypes of treated juice were found. There was no variation during

essure processed (HP) and pulsed-electric field (PEF) treated orange juice.

Pasteurized HP PEF

−8.68E-03±1.47E-03 −1.02E-02±1.23E-03 −1.94E-02±8.54E-0457.60±0.04 57.10±0.03 56.31±0.02

3.02E-03±6.22E-04 3.30E-03±4.61E-04 3.19E-03±3.70E-041.83±0.02 1.98±0.01 2.07±0.01

−0.05±0.00 −0.04±0.00 −0.05±0.0062.51±0.07 61.93±0.05 61.61±0.05

1.07E-03±7.86E-04 6.70E-04±8.17E-04 2.22E-03±7.06E-0411.54±0.02 11.59±0.02 11.39±0.02

−2.96E-04±4.25E-04 −3.94E-04±3.70E-04 −9.32E-05±3.95E-043.35±0.01 3.35±0.01 3.35±0.01

3.34E-03±1.35E-03 3.52E-03±1.70E-03 2.42E-03±1.57E-039.96±0.04 9.92±0.05 9.83±0.04

3.11E-04±2.91E-04 1.24E-03±5.52E-04 2.73E-03±2.95E-041.53±0.01 1.60±0.02 1.46±0.01



Table 3Differences in quality parameters between various groups given by the confidence level after a one-sided t-test. Statistically significant differences with CLN95% are indicated in bold.

untr.—past.⁎ untr.—HP⁎ untr.—PEF⁎ past.—HP⁎⁎ past.—PEF⁎⁎ HP—PEF⁎⁎

Color L Slope 99.2% 98.9% 94.5% 78.8% 99.9% 99.9%y-intercept 99.9% 91.9% 99.9% 99.9% 99.9% 99.9%

Color a Slope 96.4% 97.1% 96.4% 65.5% 57.9% 57.9%y-intercept 99.9% 88.5% 99.9% 99.9% 99.9% 99.9%

Color b Slope 53.9% 65.5% 53.9% 97.7% 53.9% 99.4%y-intercept 99.9% 75.8% 97.1% 99.9% 99.9% 99.9%

Brix Slope 50.0% 53.9% 55.7% 52.3% 86.3% 91.8%y-intercept 90.3% 57.9% 99.9% 91.9% 99.9% 99.9%

pH Slope 99.9% 99.9% 99.9% 57.9% 65.5% 72.6%y-intercept 91.9% 86.4% 91.9% 61.8% 53.9% 61.8%

Dry matter Slope 99.9% 99.9% 99.9% 53.9% 65.5% 69.1%y-intercept 97.7% 99.4% 99.9% 75.7% 98.6% 90.3%

Viscosity Slope 88.5% 94.3% 98.6% 93.2% 99.9% 99.1%y-intercept 99.7% 90.2% 99.9% 99.9% 99.9% 99.9%

Cloud sedimentation Slope 86.7% 91.0% 99.7% 93.9% 99.0% 99.8%y-intercept 83.5% 68.4% 86.3% 93.5% 93.9% 92.9%

⁎ Evaluated for the first 9 days.⁎⁎ Evaluated over the entire period of 58 days.


storage time, except for the untreated orange juice, in which pHdecreases significantly over the first 9 days. This is mainly caused bythe limited number of measurements during storage included in dataanalysis for the untreated samples (spoiled after day 9) compared tothe treated samples (data available until day 58).

3.5. Dry matter content

There is no change in dry matter content during storage for anytreatment (Fig. 5). The dry matter content of untreated orange juices

0

1

2

3

4

5

0 10 20 30 40 50 60

Time (days)

DE

val

ue

well visible

noticeable

slightly noticeable

not noticeable

Fig. 2. Total color difference (DE-value) for untreated ( ), mild heat pasteurized ( ),high pressure processed (HP) (●) and pulsed electric field (PEF) ( ) processed orangejuice. (For interpretation of the references to color in this figure legend, the reader isreferred to the web version of this article.)

11.0

11.2

11.4

11.6

11.8

12.0

0 10 20 30 40 50 60

Time (days)

Bri

x (°

)

Fig. 3. °Brix of serum of centrifuged orange juice (10 min 360 xg) for untreated ( ),mild heat pasteurized ( ), high pressure processed (HP) (●) and pulsed electric field(PEF) ( ) processed orange juice during storage at 4 °C.


showed a slight decrease in the first 9 days, but this decrease is onlynoticed because a small number of data points during storage wereused for regression. Similar dry matter values were found at t=1, 2and 9 days for the treated juices, however the treated juices wereanalyzed for a longer storage time, and therefore the small decreaseduring the first days of storage did not contribute to the overall trendin their regression lines.

3.6. Cloud stability

Cloud loss is considered as a quality defect in shelf stable citrusjuices derived from concentrate and it is one of the main reasons for

3.2

3.3

3.4

3.5

0 10 20 30 40 50 60

Time (days)

pH

Fig. 4. pH for untreated ( ), mild heat pasteurized ( ), high pressure processed (HP)(●) and pulsed electric field (PEF) ( ) processed orange juice during storage at 4 °C.

9.4

9.6

9.8

10.0

10.2

10.4

10.6

0 10 20 30 40 50 60

Time (days)

Dry

mat

ter

(%)

Fig. 5. Dry matter content for untreated ( ), mild heat pasteurized ( ), high pressureprocessed (HP) (●) and pulsed electric field (PEF) ( ) processed orange juice duringstorage at 4 °C.



Fig. 6. Observed sedimentation and cloud loss of untreated, mild heat pasteurized, high pressure pasteurized (HPP) and pulsed electric field (PEF) processed orange juice bottlesduring the first 115 days of storage at 4 °C.

5


the level of heating in heat pasteurization, where 90–100% of PME isinactivated (Goodner et al., 1998, 1999; Goodner, Braddock, Parish, &Sims, 1999; Irwe & Olsson, 1994). However, it is doubtful to considercloud loss as a quality defect in fresh juice, since consumers acceptthe degradation (Parish, 1998). Moreover, modern transparentpackaging actively shows the extent of cloud degradation, referringto the natural degradation process in fresh squeezed orange juice.Nevertheless, it is an interesting parameter to evaluate and comparethe different preservation techniques.

Cloud stability was measured evaluating the degree and rate ofsedimentation, by recording the height of the interface of the sedi-ment and cloud from bottles shown in Fig. 6. Degree of sedimentationwas calculated as percentage, where the height of the sediment wasexpressed as a function of the total height. Sedimentation of 0%corresponds to a completely stable orange juice, having no cloud loss.During storage time, the orange juices are sedimented according toa non-linear response (Fig. 7). To compare the cloud stability ofdifferent treatments, a direct evaluation of the observed differencesbetween different treatments during storage was conducted. Sincethe point-wise differences in height are sufficiently small, linear

0

20

40

60

80

100

0 20 40 60 80 100 120Time (days)

Sedi

men

tatio

n of

clo

ud (

%)

Fig. 7. Sedimentation of cloud observed from untreated ( ), mild heat pasteurized ( ),high pressure processed (HP) (●) and pulsed electric field (PEF) ( ) processed orangejuice stored at 4 °C.


regression could be applied to the data of the differences (Squires,1988). After a storage time of 2 months, all treatments showed toreach an equilibrium, having the same level of sedimentation.Therefore, it is more interesting to evaluate the rate of sedimentation.Samples with a high degree of sedimentation during the first days ofstorage were considered to have a high sedimentation rate, having alow cloud stability. PEF treated orange juice had the highest degree ofsedimentation (P≤0.05), followed by HP, mild heat pasteurizationand untreated orange juice, respectively (Fig. 7).

The opacity of the cloudwas scored by visual observation to obtainan indication which mild treatment gives the fastest transition ofopaque to transparent (scores from 5 to 1). This is a qualitativemeasure of the cloud loss, caused by activity of the endogenous PME.Results shown in Fig. 8, showed that untreated orange juice is thefastest in getting transparent, which suggests that it has the highestenzyme activity, followed by PEF, HP and heat pasteurization.

0

1

2

3

4

1 2 9 20 28 58 85 115

Time (days)

Judg

emen

t by

visi

on

Fig. 8. Judgement of color of cloud serum of untreated ( ), mild heat pasteurized ( ),high pressure processed (HP) ( ) and pulsed electric field (PEF) ( ) processed orangejuice during storage at 4 °C from 1 (transparent) to 5 (opaque) to have an indication forcloud stability. (For interpretation of the references to color in this figure legend, thereader is referred to the web version of this article.)



1.2

1.4

1.6

1.8

2.0

0 10 20 30 40 50 60

Time (days)

Vis

cosi

ty (

cP)

Fig. 9. Viscosity of serum of centrifuged (10 min at 360 xg) untreated ( ), mild heatpasteurized ( ), high pressure processed (HP) (●) and pulsed electric field (PEF) ( )processed orange juice during storage at 4 °C.


In part II of this study (Vervoort et al., under review), PMEinactivation levels of 85, 92 and 34% were measured at day 1 for heat,HP and PEF pasteurization respectively. This high enzyme activity ofPEF treated orange juice corresponds to the results shown in Figs. 7and 8, where the PEF treated juice showed to have to least stablecloud of treated products. Although the PME activity of the HP treatedjuice was slightly more reduced than heat pasteurized juice (Vervoortet al., under review), more cloud was retained for heat pasteurizedjuice than HP treated orange juice. This disagreement is difficult tointerpret. Possibly it may be related to the existence of a heat-labileand heat-stable fraction of PME. In addition, it should be mentionedthat the juice for heat pasteurization contained pulp particles ofsimilar size compared to the juice for HP pasteurization because ofsieving.

3.7. Viscosity

Viscosity of the serum of centrifuged orange juice was measuredduring storage time. The observed changes in viscosity were small(Fig. 9, Table 2). Just after processing, the viscosity was significantlydifferent for mild heat pasteurized and PEF treated orange juice.During storage, viscosity of mild heat pasteurized juice remainedstable, whereas the viscosity of HP and PEF treated juice increased.This effect was observed by Cserhalmi et al. (2006) and Polydera,Stoforos and Taoukis (2005) as well, but considered to be moremathematically different than practically relevant.

The increase of viscosity during storage is likely induced by activityof PME. This enzyme demethoxylates pectin substances which cancrosslink with divalent cations and precipitate (Ting & Rouseff, 1986).

4. Conclusion

Comparative evaluation of a single batch freshly squeezed orangejuice, using mild preservation techniques with equivalent processinglevels regarding microbial inactivation, showed no microbial counts(below detection limit) in all processed orange juices during storageof 2 months.

Quality parameters related to microbial shelf life, brix, pH and drymatter content showed to be constant during storage for all mildlytreated orange juices. Color changes are not noticeable up to onemonthof storage, and no differences between treatments are perceptible.

The most relevant differences were found for HP and PEF inviscosity and cloud stability due to residual enzyme activity. Thesignature of cloud loss can be seen as a characteristic of orange juiceclose to fresh juice. If this degradation of cloud stability during storageis desirable, it is advised to use PEF or HP as a preservation technique,if a more stable product is desirable it is advised to apply heatpasteurization. Depending on the degree of cloud degradation one canchoose for PEF for faster sedimentation or HP for slower.


With this study it was shown that HP and PEF are good novelpreservation techniques to increase microbial shelf life of orange juicefrom 9 to 20 days up to two months when stored at 4 °C, maintainingthe fresh character. As equivalent processing was chosen with respectto food safety, it seems that mild heat pasteurization is a goodalternative as well, since it resulted in a comparable quality. However,it can be expected, that when more realistic industrial processingconditions are chosen, the quality of heat pasteurized orange juice willbe lower. HP and PEF conditions used in this research are also used inthe limited applications of these technologies on industrial scale, andtherefore HP and PEF are considered to be the best options to enlargeshelf life based on results of quality parameters. As shown in Fig. 1,other parameters are described in part II (Vervoort et al., underreview) and part III (Magyar-Horváth et al., under review).

Acknowledgements

This study has been carried out with financial support from theCommission of the European Communities, Framework 6, Priority 5“Food Quality and Safety”, Integrated Project Novel Q FP6-CT-2006-015710.

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http://www.fda.gov/Food/GuidanceComplianceRegulatoryInformation/GuidanceDocuments/Juice/ucm072557.htm

http://www.fda.gov/Food/GuidanceComplianceRegulatoryInformation/GuidanceDocuments/Juice/ucm072557.htm
