Innovative Food Science and Emerging Technologies 10 (2009) 457–462

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Innovative Food Science and Emerging Technologies

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High-pressure homogenization of orange juice to inactivate pectinmethylesterase

J. Welti-Chanes a, C.E. Ochoa-Velasco b, J.Á. Guerrero-Beltrán c,⁎a Departamento de Biotecnología e Ingeniería en Alimentos, Instituto Tecnológico y de Estudios Superiores de Monterrey, Av. Eugenio Garza Sada 2501 Sur, Col. Tecnológico, Monterrey,N. L. 64849, Mexicob Facultad de Ingeniería Química, Benemérita Universidad Autónoma de Puebla, Puebla, Puebla, Mexicoc Depto. Ingeniería Química y Alimentos, Universidad de las América-Puebla, Sta. Catarina Mártir, Cholula 72820, Puebla, Mexico

⁎ Corresponding author. Tel.: +52 222 2292126; fax:E-mail address: joseangel150@hotmail.com (J.Á. Gue

1466-8564/$ – see front matter © 2009 Elsevier Ltd. Adoi:10.1016/j.ifset.2009.05.012

a b s t r a c t

a r t i c l e i n f o

Article history:Received 3 March 2009Accepted 30 May 2009

Editor Proof Receive Date 24 June 2009

Keywords:Orange juiceHigh-pressure homogenizationHomogenizationMicrobial inactivationPectinmethylesterase inactivation

A homogenizer was used to treat orange juice at five pressures (0–250 MPa) and three initial temperatures(22, 35 and 45 °C). A maximum of five passes for the selected conditions were used to process orange juice.Pectinmethylesterase (PME) activity, microbial load, cloudy appearance, and vitamin C were evaluated in justsqueezed and homogenized orange juices. A reduction of 50.4, 49.4 and 37.8% of PME activity was observed injuice homogenized by one pass at 250 MPa at the initial temperatures of 22, 35, and 45 °C, respectively. Pectin-methylesterase activity in orange juice was reduced as passes number was increased. The final temperature ofthe five times homogenized orange juice was not beyond 28 and 37 °C after being treated at 100 and 250 MPa,respectively. More than 30 and 80% of enzyme activity was reduced after five passes at 100 and 250 MPa,respectively. Less that 8.7×102 and 1.85×103CFU/mL of mesophiles and yeasts plus molds, respectively, werecounted in orange juice treated five times at 100 MPa. The cloudy appearance of the homogenized orange juicewas maintained for 12 days under low temperature conditions.Industrial relevance: “Cold pasteurization” of orange juice, using a homogenizer as a high-pressure procedure,could be an alternative to thermal processing to avoid sensory, nutritional and physiochemical changes in juice.This processmaydeliver a pasteurized orange juicewith characteristics similar to just squeezed orange juice. Inaddition to reduce the microbial load, homogenization may reduce pectinmethylesterase enzyme, which maycause phase-separation in juice and consequently give an unwanted appearance that consumers dislike. Addi-tionally, homogenized orange juice appearance could be stable during several days before being brought to theconsumers' daily eating table.

© 2009 Elsevier Ltd. All rights reserved.

1. Introduction

Emerging technologies are being widely studied to deliver foodproducts with fresh like characteristics that consumers are demand-ing today. High pressure, static (high hydrostatic pressure, HHP) ordynamic (high-pressure homogenization, HPH), is one of the tech-nologies used mainly to inactivate microorganisms or enzymes whichmayalter or damage fresh foods products. The advantageof using thesenovel technologies is that some of them are also called nonthermaltechnologies because the processing temperature does not rise beyond40 °C (Guerrero-Beltrán, Welti-Chanes, & Martín-Belloso, 2007). Thisis important to obtain food productswith sensory and nutritional char-acteristics similar to fresh food products.

High hydrostatic pressure was applied for the first time to treatfoods, especially milk, by Hite (1899). He also reported the effect ofhigh hydrostat pressure on microbial load in fruits and vegetables(Hite, Giddings, & Weakley, 1914). Chlopin and Tammann (1903) sug-gested that high pressure altered the vital microbial functions in sucha way that their biological functions become injured; later on, they

+52 222 2292727.rrero-Beltrán).

ll rights reserved.

observed that microorganisms recovered their biological functions.Timson and Short (1965) treated raw milk using high pressure toreduce the microbial load. Since then, much research has been con-ducted using high hydrostatic pressure to accomplish several pur-poses, mainly microbial and enzyme inactivation. In the 1990s and2000s numerous reports have been published about nonthermal tech-nologies. Research related to the effects of high pressure on vegetativemicroorganisms (Yuste, Capellas, Fung, & Mor-Mur, 2004), microbialspores (Estrada-Girón, Guerrero-Beltrán, Swanson, & Barbosa-Cánovas, 2007; Heinz & Knorr, 2001), activation or inactivation offood enzymes (Ludikhuyze, Indrawati, Van den Broeck,Weemaes, &Hendrickx, 1998; Ludikhuyze, Van Leoy, Indrawati, Denys, &Hendrickx, 2001; Tedjo, Eshtiaghi, & Knorr, 2000; Asaka & Hayashi,1991, Guerrero-Beltrán, Barbosa-Cánovas, & Swanson, 2004), andchemical reactions influencing quality (Ludikhuyze&Hendrickx, 2001)in fruit and vegetable products have been reported elsewhere.

Regarding food enzymes, pectinmethylesterase and polygalac-turonase, found in citrus fruit products, react with pectic substancescleaving methyl esters to render poly-D-galacturonic acid (Whistler &Daniel, 1985) which modify the physical appearance of citrus juices.If these enzymes are not inactivated, they eventually will destroythe cloudy stability in citrus fruit juices (Haard, 1985). The ordinary

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process to inactivate these enzymes is by pasteurization; however,citrus fruit juices are very sensitive to heat leading to flavor changes injuice during heat processing. Thus, some studies have been conductedby high pressure to study its effect on sensory, microbial and enzymecharacteristics of foods. Goodner, Braddock, and Parish (1998) pointedout that high pressure (500–600 MPa) inactivated the heat labile pec-tinesterase in orange and grapefruit juices. The cloudy stability inorange juice was preserved after 10 min of processing at pressures inthe range of 500 to 900 MPa (Goodner, Braddock, Parish, & Sims,1999).Nienaber and Shellhammer (2001) found Dp-values ranged from 4.6to 117.5 min to inactivate pectinmethylesterase in orange juice treatedat pressures in the range of 400 to 600 MPa. Ogawa, Fukuhisa, Kubo,and Fukumoto (1990) pointed out that pectinesterase was completelyinactivated after pressurization at 300 or 400 MPa for 10 min in Sat-suma mandarin juice. All of those studies have been carried out usinghigh hydrostatic pressure machines. Nevertheless, a few studies havebeen conducted using homogenizers for microbial and, or enzyme in-activation purposes. Taylor, Roach, Black, Davidson, and Harte (2007),for instance, observed a significant reduction of Escherichia coli K-12in sodium chloride solutions (0.9%) in the range of 100 to 250 MPausing a high-pressure homogenizer. Pathanibul, Taylor, Davidson, andHarte (2007), on the other hand, using similar pressure conditions,reported an inactivation of around 5.5 and 6 log10 (CFU/mL) of E. coliK-12 and Listeria innocua, respectively, inoculated in apple and carrotjuices. Briñez, Roig-Sagués, Hernández-Herrero, and Guamis-López(2006) studied the inactivation effect of an HPH system on E. coliinoculated in whole and skim milk treated at 300 MPa; they found aninactivation range of 3.9–4.3 log10 (CFU/mL)units after 2 hof treatment.

The aim of this research was to treat just squeezed orange juice byhigh-pressure homogenization to inactivate pectinmethylesterase andnatural microbial flora.

2. Materials and methods

2.1. Orange juice preparation

Oranges (Citrus sinensis), Valencia variety, were purchased at theCentral Market Supplier in Puebla City. Oranges were washed, cut inhalves and pressed by hand using a domestic rotary orange juiceprocessor. The orange juice pulp was discarded by sieving.

2.2. High-pressure treatment

The orange juice was high pressure treated using a continuousFPG7400H:350 Hi-Drive homogenizer (Stansted Fluid Power, Ltd.,Essex, UK) at a constant flow rate of 7 L/h. Juice was warmed up atthree temperatures (22, 35 and 45 °C) just before homogenizationby re-circulating juice into the system. Afterward, juice was treatedat five pressures (50, 100, 150, and 250 MPa) and selected number ofpasses (0–5). The homogenizer has a cooling system at the end ofthe tubing arrangement to cool down any increase of temperatureof the processed liquid. The juice temperature did not increase beyond19, 27, or 37 °C after homogenization at 22, 35, or 45 °C, respectively.The homogenized orange juice was analyzed for pectinmethylesteraseactivity and natural microbial flora. All processing of orange juicewereperformed in duplicate.

2.3. Physicochemical analysis

Total soluble solids (TSS, °Bx)weremeasured using a refractometer(Atago USA, Inc. Bellevue, WA). pH was measured using an Orion pHmeter model 420 (Orion Research Inc., Boston, MA). Titratable acidity(percentage of citric acid) was assessed by the 22.058 AOAC method(1984). Vitamin C was determined by titration with 2,6-dichlorphe-nolindophenol using the 43.064 AOAC method (1984). Each test wasperformed in triplicate.

2.4. Pectinmethylesterase assay

Pectinmethylesterase activity was evaluated using the Rouse,Atkins, and Huggart (1954) method with modifications. Ten millilitersof previously warmed up (30 °C) citric pectin solution (CPS) (1%) wasplaced into a double wall beaker. Afterward, 10 mL of orange juice and0.1755 g of sodium chloride were added to the CPS and mixed forhomogenization. The system was heated again until reaching 30 °Cby circulating water into the walls of the beaker. Sodium hydroxidesolution (0.1 or 0.5 N)was added to themixture of CPS and juice (CPS–juice) until reaching pH 7.5. After that, titration of the CPS–juice mix-ture was performed by adding sodium hydroxide solution (0.02 N)to maintain pH at 7.5 as a function of time. The pectinmethylesteraseactivity (meq/mLmin)was calculated using themilliequivalent (meq)of sodium hydroxide (VNaOH*NNaOH), sample volume (mL) and the re-action time (min). A Cole-Parmer (Vernon Hills, Illinois) re-circulationbathwas used for controlling temperature. Each test was performed intriplicate.

2.5. Cloudy appearance

Fifty mL of just squeezed or homogenized orange juice was placedinto plastic centrifugal tubes and then stored at low temperature (4 °C)during 12 days. The cloudy appearance of the juice was evaluated bysimple observation. The percentage of juice separation was evaluatedmeasuring the settling of particles in juice.

2.6. Microbial counts

Mesophiles and yeasts plus molds were counted using the pourplate method. Nutrient and potato dextrose agars were used to eval-uate the survivals of mesophiles and yeasts plus molds, respectively,in juice. After pour plating, Petri dishes were placed into ovens at 37(24–48 h) and 30 °C (5 days), respectively.

2.7. Microbial and enzyme inactivation modeling

A first order modeling was used to analyze the microbial load in-activation:

LogN = −mP + b

where N is the residual microbial load (CFU/mL), P is pressure (MPa)or number of passes, m is the slope (1/MPa or 1/number of passes),and b is the intercept (−).

A linear relationship between residual enzyme activity (%) andpressures (MPa)was used tomodel the effect onpectinmethylesterase.

2.8. Statistical analysis

Experimental data were statistically analyzed performing an anal-ysis of variance (ANOVA) using a Minitab 14 program. A p≤0.05 wasused as the criterion value to determine significant differences withintreatments.

3. Results and discussion

3.1. Physiochemical characteristics

Table 1 shows the physicochemical characteristics of orange juicebefore being homogenized. It can be observed that pulp content wasthe only difference between juice containing and noncontaining pulp.The reason of that was because pulp separation in juice was carried outby sieving before analysis. The reduction of pulp in the juicewas impor-tant to avoid blocking of the high-pressure system during processing.

Table 1Just squeezed orange juice physicochemical characteristics.

Characteristic Juice

Containing pulp Noncontaining pulp

pH 4.0±0.30 4.0±0.27TSS (% w/w) 10.2±0.80 10.0±1.00Citric acid (%) 1.2±0.5 1.1±0.40Pulp (%) 16.0–18.0 6.0–7.0TSS/acid ratio 8.5±0.6 8.6±1.0

Table 2Regression parameters for PME activity in pressurized orange juice at selected initialtemperatures.

Temperature (°C) Slope (%/MPa) Intercept (%) R2

22 −0.192 104.3 0.95335 −0.195 104.3 0.93945 −0.262 98.6 0.950

Fig. 2. Passes number effect on PME inactivation in orange juice treated at 100 (■) and250 (□) MPa.

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On the other hand, pH, TSS, citric acid content and TSS/acid ratio weresimilar in the two types of juices.

3.2. Pressure and initial temperature effect on PME

The initial PME activity [(1.787±0.006)×10−4meq/mL min] inorange juice was reduced to 50.4, 49.4 and 37.8% after one single passof juice through the HPH system (250 MPa) at the initial temperaturesof 22, 35, and 45 °C, respectively. Fig. 1 illustrates the remaining PMEactivity in orange juice processed by one single pass as a functionof pressure. A linear tendency was observed for PME inactivation inorange juice (R2N0.939). It can be also observed that, the higher thetemperature to process orange juice, themore inclined the slope goingdownward; this means that lower PME activity was observed as tem-perature and pressure increased. A significant difference (pb0.05)wasobserved among temperatures, being the PME activity more reducedwhen orange juice was processed at 45 °C. No significant difference(pN0.05) was observed in PME activity when juice was homogenizedat 22 and 35 °C. A significant difference (pb0.05) was observed withinpressures. Table 2 shows the linear regression parameters for PMEactivity, in orange juice, after pressurization at the three initial temper-atures. Linear regression coefficients higher that 0.939 were obtained.Therefore, PME inactivation, at the selected temperatures, was inacti-vated linearly as pressure increased.

3.3. Passes number effect on orange juice PME

Fig. 2 illustrates the effect of number of passes and pressure (100and 250 MPa) on PME activity in orange juice (22 °C). A significantdifference (pb0.05) was observed for PME activity in orange juice forboth pressure and number of passes. No significant difference (pN0.05)was observed in PME activity in orange juice homogenized from 1 to5 passes when processing at 100 MPa. It can also be observed thatPME activity in juice was dramatically reduced when processing at250 MPa; however, no significant difference (pN0.05) was observedin PME activity in juice between 1 and 2 passes or among 3, 4 and 5passes. Therefore, at 250 MPa, the inactivation of PME was higher asthe circulation number increased. Therefore, one or two passes maygenerate orange juice with less than 50% of the initial PME.

Fig. 1. Pressure and initial temperature (22,♦; 35,■; 45 °C,▲) effect on PME inactivationin orange juice.

3.4. Microbial reduction

3.4.1. Pressure and initial temperature effectsFig. 3 depicts the effect of pressure and initial temperature on

microbial load in orange juice. Both, the initial temperature and pres-sure had an effect on the microbial load in orange juice. The higher re-duction of the microbial load was around 1.5 Log cycles. A fist ordermodelwas used to examine themicrobial load inactivation as pressureincreased (Fig. 3, Table 3). Small values of the correlation coefficientswere obtained for microbial reductions in the processed juice at aninitial temperature of 22 °C; however, no significant difference (pN0.05)was observed for mesophiles and yeast plus molds in orange juiceheated at 22 and 35 °C and treated at the selected pressures. A signif-icant difference (pb0.05)was observed formesophiles and yeasts plusmolds in juice previously heated at 45 °C in comparisonwithmicrobialload in juice heated at 22 and 35 °C. No significant difference (pN0.05)was observed within 200 and 250 MPa for the two types of microor-ganisms in orange juice. Table 3 shows the linear regression param-eters after plotting the Lognumberof survivals (CFU/mL)versuspressure(MPa) data. It is observed, in Table 3, the corresponding amount of pres-sure (1/slope), at the selected temperatures, to reduce one Log cycle ofmesophiles and yeasts plus molds. Therefore, more pressure is requiredto inactivate one Log cycle of both types of microorganisms (mesophilesandmolds plus yeasts) in orange juicewhen homogenizing at low initialtemperatures and vice versa.

Fig. 3. Initial temperature effect on mesophiles (M) and yeasts plus molds (YM) inorange juice homogenized one time (one pass) at selected pressures.

Table 3Linear regression parameters for mesophiles (M) and yeasts plus molds (YM) inactiva-tion in orange juice homogenized one time (one pass) at different initial temperatures.

Temperature(°C)

Microorganismtypes

Slope(1/MPa)

Pressurea

(MPa)Intercept R2

22 M −0.0021 476 3.20 0.620YM −0.0019 526 3.54 0.750

35 M −0.0032 312 3.55 0.900YM −0.0023 435 3.89 0.886

45 M −0.0050 200 3.81 0.952YM −0.0053 189 3.93 0.926

a Pressure to inactivate one Log cycle.

Table 4Linear regression parameters for mesophiles (M) and yeasts plus molds (YM) in orangejuice (22 °C) homogenized at 100 and 250 MPa.

Pressure (MPa) Microorganism types Slope (1/MPa) Intercept R2

100 M −0.220 3.97 0.965YM −0.186 4.12 0.933

250 M −0.257 4.24 0.956YM −0.218 3.93 0.975

Fig. 5. Storage time (4 °C) effect on PME activity in homogenized (one pass) orangejuice (22 °C).

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3.4.2. Passes number effectFig. 4 illustrates the passes number effect on the microbial load

in the pressurized (100 and 250 MPa) orange juice (22 °C). Both, thepasses number and pressure had an effect on the microbial load in thehomogenized orange juice. Themicrobial load decreased as the passesnumber increased. The microbial counts in orange juice homogenizedfive times were 870 and 950 mesophiles/mL and 1850 and 700 moldsplus yeasts/mL for 100 and 250 MPa of treatment, respectively. A sig-nificant difference (pb0.05) was observed, for microbial load inacti-vation in orange juice, within passes number. However, no significantdifference (pN0.05) was observed for microbial load inactivation injuice among 1, 2, and 3 passes or between 4 and 5 passes. Also, nosignificant difference (pN0.05) was observed for yeasts plus molds inorange juice homogenized at 100 and 250 MPa. Table 4 shows thelinear parameters for microbial inactivation. Correlation coefficientvalues higher than 0.933 were observed for mesophiles and yeastsplus molds inactivation.

3.5. PME activity and cloudy stability in homogenized orange juiceduring storage

Fig. 5 illustrates the storage (4 °C) time effect on PME activity inhomogenized (one pass) orange juice (initial temperature of 22 °C). Itcan be observed that PME activity, in just squeezed orange juice, wasreduced as pressure treatment increased (0 day). Afterward, the PMEactivity was increased during the storage time. More than 100% of PMEactivity was achieved in orange juice after twelve days of storage. Theincreased PME activity could be due to isoenzymes arising (Richard-son & Hyslop, 1985) throughout the storing of orange juice. Severalresearchers have reported increasing of enzyme activity due to high-pressure treatment to fruit products (Asaka & Hayashi,1991, Guerrero-Beltrán et al., 2004). They pointed out that high pressure may splitisoenzymes that could react later on in the food system. Therefore, thesame phenomenon could have occurred after orange juice process-ing by HDP. A significant difference (pb0.05) was observed for PMEactivity in orange juice within pressures and storage times. Compar-able behavior, regarding PME activity, was observed in just squeezedorange juice previously heated at 45 °C (Fig. 6). However, lower PME

Fig. 4. Passes number effect on mesophiles (▲, 100 MPa; ■, 250 MPa) and yeasts plusmolds (♦, 100 MPa; ●, 250 MPa) in orange juice (22 °C) treated at selected pressures.

activationwas observed during the storage period in this type of treatedjuice. This behavior in orange juice, previously heated at 45 °C, con-cerning PME activity activation could be probably due to the inacti-vation effect of initial temperature on the enzyme.

The cloudy appearance in orange juice, just after homogenization(0–250 MPa; 22 °C), was opaque and homogenous. No differencewas observed by simple inspection between homogenized and non-homogenized orange juices. However, after 6 h of storing at 4 °C, thecloudyappearanceof thenon-treatedorange juicedisappeared; therefore,the juice was turning transparent. This means that pectinmethylester-ase chemically reacted with pectic substances (pectindemethylation)leading to loss of the cloudy appearance (Haard,1985)wanted in thesetypes of citrus juices. However, the cloudy appearance of the homog-enized (one pass at the selected pressures 50–250 MPa) orange juicestored at refrigeration temperature remained stable for 12 days (Fig. 7).The staying of the cloudy appearance in the orange juice could also bedue to the reduction of the particle size of the remaining pulp in juiceafter squeezing and sieving.

Fig. 8 illustrates the storage time (4 °C) effect on PME activity ofthe homogenized (100 MPa, 1–5 passes, 22 °C) orange juice. A signif-icant difference (pb0.05) was observed for PME activity in orange juicewithin storage time and passes number. No significant difference(pN0.05) was observed for PME activity in orange juice between zero

Fig. 6. Storage time (4 °C) effect on PME activity in homogenized (one pass) orangejuice (45 °C).

Fig. 7. Stability of the cloudy appearance in orange juice after 12 days of storage at 4 °C.

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and four days of storing or between eight and 12 days of storing. Thesame behavior was observed for PME activity in orange juice whenhomogenization was performed at 250 MPa (Fig. 9). However, using250 MPa of pressure, to treat orange juice, the PME inactivation wasmore effective as the passes number increased. The cloudy appearanceof the homogenized orange juice remained stable after 12 days ofstoring at 4 °C (Fig. 7).

3.6. Vitamin C

The vitamin C content (57±7 mg/100 mL in just squeezed juice)in orange juice homogenized (50–250 MPa, 22 °C) by one pass was

Fig. 8. Storage time (4 °C) effect on PME activity in homogenized (100 MPa, 1–5 passes,22 °C) orange juice.

Fig. 9. Storage time (4 °C) effect on PME activity in homogenized (250 MPa, 1–5 passes,22 °C) orange juice.

not affected after pressurization (56±16 mg/100 mL). Vitamin C wasbarely affected by number of passes (1–5) after processing at 100(66±3 mg/100 mL) or 250 MPa (54±3 mg/100 mL). As a result, thevitamin C content remained stable after homogenization at differentpressures and passes number.

4. Conclusions

A linear behavior was observed in the PME inactivation in orangejuice homogenized at the selected pressures and temperatures. Nosignificant difference was observed in PME activity in orange juicewarmed up at 22 or 35 °C before processing. A maximum of 50% ofPME inactivation was observed after a single pass of the orange juicethroughout the homogenizer at 250 MPa; however, its cloudy appear-ance was maintained for 12 days of storing under refrigeration (4 °C).Thirty one and 80% of PME inactivation in orange juice was observedafter five passes at 250 MPa; the cloudy appearance of this processedjuice was also maintained for 12 days under refrigeration. The micro-bial counts in orange juice homogenized five times at 100 and 250 MPawere less that 3.0 and 3.3 Log cycles for mesophiles and yeasts plusmolds, respectively.

Acknowledgment

Author C.E. Ochoa-Velazcowould like to thank the National Councilof Science and Technology (CONACyT) in Mexico for the economicalsupport provided for the completion of his Master's Degree studies.

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