International Journal of Food Microbiology 160 (2013) 273–281

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International Journal of Food Microbiology

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Combined effects of high pressure homogenization treatment and citral onmicrobiological quality of apricot juice

Francesca Patrignani, Giulia Tabanelli, Lorenzo Siroli, Fausto Gardini, Rosalba Lanciotti ⁎Dipartimento di Scienze degli Alimenti, Università degli Studi di Bologna, Cesena, Piazza Goidanich 60, 47521 Cesena (FC), Italy

⁎ Corresponding author. Tel.: +39 0547 338132; fax:E-mail address: rosalba.lanciotti@unibo.it (R. Lancio

0168-1605/$ – see front matter © 2012 Elsevier B.V. Allhttp://dx.doi.org/10.1016/j.ijfoodmicro.2012.10.021

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Article history:Received 16 May 2012Received in revised form 3 October 2012Accepted 8 October 2012Available online 8 November 2012

Keywords:High pressure homogenizationCitralSaccharomyces cerevisiaeApricot juice

High pressure homogenization (HPH) technique is able to significantly reduce spoilage microbiota in fruit juice.On the other hand, aroma compounds and essential oils can have a key role in the microbial stability of theseproducts. For this reason, the aim of this work was to evaluate the combined effects of an aroma compound(citral, used at a concentration of 50 mg/l) and HPH treatments (performed at 100 MPa for 1–8 successivepasses) on the inactivation dynamics of Saccharomyces cerevisiae SPA strain inoculated in apricot juices atlevel of about 4.5 log CFU/ml. Moreover, growth of surviving yeast cells was measured during the storageof the treated juice at 10 °C and pH, water activity, viscosity and volatile molecule profile of apricot juicewere studied. Since citral had been diluted in ethanol before the addition to juice, also samples with onlyethanol added at the same volume used to dissolve citral were considered. The results showed that yeastcell viability decreased with the increases of passes at 100 MPa and the relationship between yeast cellloads and number of passes at 100 MPa followed a linear trend.In addition, the effect of HPH treatment can be notably potentiated throughout the presence of citral and ethanol,increasing the time necessary to reach a spoilage threshold during storage. The volatile profiles of the juicesadded with citral showed a substitution by yeast metabolism of this aldehyde with molecule characterized by alower antimicrobial activity such as alcohols. The HPH treatments had also a significant effect on pH and viscosityof apricot juices while did not affect aw.

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

Fruit juices are perceived as “healthy” foods due to their low contentof sodium,minimal cholesterol, fat and richness in vitaminC, polyphenolsand flavonoids contributing to good antioxidant properties (Kumar et al.,2009; Patrignani et al., 2009). However, such products are susceptible tospoilage, thus having a limited shelf life (Lavinas et al., 2008). The spoil-age of fruit juices is primarily due to yeasts, responsible for the fermentedtaste and carbon dioxide production, lactic acid bacteria, which canproduce a buttermilk off-flavour, and moulds which contribute tothe spoilage by their surface growth (Tournas et al., 2006). Althoughfruit juices have always been considered as safe (due to the low pH),some studies have demonstrated that unpasteurized juices can vehiclefood-borne pathogens such as Salmonella spp. and Escherichia coliO157:H7 (Kriskó and Roller, 2005; Briñez et al., 2006b). To improvesafety and shelf life, commercial fruit juices are generally heat treatedand may contain preservatives (Jordan et al., 2001; Tribst et al., 2008).However, thermal processing causes some undesirable effects, such asnon enzymatic browning, off-flavour formation and vitamin loss(Polydera et al., 2003; Zhang et al., 2010; Calligaris et al., 2012). In

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recent times, the change in consumer habits and the increasing demandfor fresh juices free of chemical preservatives have stimulated the re-search of alternative processing technologies to produce foods with aminimum of nutritional, physicochemical, or organoleptic changesinduced by the technologies themselves. A wide literature showsthat fruit juices can be considered as an interesting and feasiblefield of application of HPH treatments, in order to achieve the reductionof themicrobial load while preserving the quality attributes of the freshproducts (Donsi et al., 2011). On the other hand, HPH is included amongthe not thermal technologies, because, despite the temperature increasedue to frictional heating in the homogenization valve (b1 °C/MPa), itwas widely shown that microbial inactivation during HPH treatments isonly marginally ascribable to thermal effects (Diels and Michiels, 2006;Donsi et al., 2011). A wide literature shows that HPH treatments areable to significantly reduce both naturally occurring anddeliberated inoc-ulated spoilagemicrobiota in fruit juices (Betoret et al., 2009; Briñez et al.,2007; Campos and Cristianini, 2007; Patrignani et al., 2009, 2010;Suarez-Jacobo et al., 2010; Tahiri et al., 2006; Bevilacqua et al., 2012). Inaddition, several studies have shown the potential of HPH treatmentsfor spoilage and pathogenic microorganisms inactivation as an alterna-tive to heat treatment for improving fruit juice safety and shelf-life(Briñez et al., 2006a,b, 2007; Patrignani et al., 2009, 2010). TheHPH treat-ment causes the modification of several physical characteristics of thejuices, such as the mean particle size of the suspended solids (Donsì et

274 F. Patrignani et al. / International Journal of Food Microbiology 160 (2013) 273–281

al., 2009; Grant, 1989; Stipp and Tsai, 1988), and juice viscosity (Betoretet al., 2009; Campos and Cristianini, 2007; Donsì et al., 2009; Patrignaniet al., 2010). In addition, the natural functional compounds of the juices,such as the flavonoids content in citrus juices (Betoret et al., 2009), aswell as the freshness and texture attributes (Lacroix et al., 2005) arebetter preserved by HPH treatments with respect to thermal ones.

Aroma compounds and essential oils can be an interesting alterna-tive to improve safety and shelf-life or fruit juices. Their antimicrobialpotential is well known (Burt, 2004; Fisher and Phillips, 2008; Holleyand Patel, 2005; Kalemba and Kunicka, 2003). A key role of essentialoils and their components in the microbial stability of beverages andjuices has been previously reported (Ndagijimana et al., 2004;Fitzgerald et al., 2004; Belletti et al., 2004, 2007, 2010; Sado Kamdemet al., 2011). The main limitations to an industrial use of these sub-stances as preservatives are their organoleptic impact and the variablecomposition of the essential oils (which can be reflected in their antimi-crobial activity) (Burt, 2004; Lanciotti et al., 2004; Belletti et al., 2008).The action of single constituents of the essential oils has been studiedto identify the most active molecules to balance the intrinsic variabilityof essential oils (Karatzas et al., 2000; Vázquez et al., 2001). Also, thecombination of essential oils and their bioactive compounds withother stabilizing treatments has been proposed to reduce their organo-leptic impact and to standardize the product safety and shelf-life re-quirements (Alzamora and Guerriero, 2003; Belletti et al., 2007, 2010;Espina et al., 2010; Donsi et al., 2011; Sado Kamdem et al., 2011;Bevilacqua et al., 2012).

Citral is a mixture of two isomers, geranial and neral, which areacyclic α, β-unsaturated monoterpene aldehydes naturally occurringin many essential oils from citrus fruits or other herbs or spices(Friedman et al., 2004; Tzortzakis and Economakis, 2007). The antimi-crobial action exerted by citral against yeasts and moulds in differentconditions has already been demonstrated (Belletti et al., 2007, 2008;Rivera-Carriles et al., 2005; Wuryatmo et al., 2003).

In this perspective this study evaluated the effect of HPH treatmentat 100 MPa in the presence of citral against Saccharomyces cerevisiae de-liberately inoculated in apricot juices after 1–8 successive passesthrough the homogenizer. Growth of surviving yeast cells was mea-sured during the storage of the treated juices at 10 °C. In addition, theeffects of the combined (50 mg/l citral and 100 MPa 1–8 passes) treat-ments on pH, water activity, viscosity and volatile molecule profile ofapricot juice were studied.

2. Materials and methods

2.1. Strain

Saccharomyces cerevisiae SPA, the strain used in thiswork, belongs tothe strain collection of the Department of Food Science of the Universityof Bologna andwas isolated from spoiled orangeade (Ndagijimana et al.,2004). The culture was maintained on slants of Sabouraud dextroseagar (SDA) (Oxoid Ltd., Basingstoke, Hampshire, United Kingdom).The suspension was prepared by inoculating a loop of the culture inSabouraud dextrose broth (SDB, Oxoid) and incubating it for 2 days at30 °C.

2.2. Inhibitory concentrations evaluation

The minimal inhibitory concentration (MIC) and the minimalbactericidal concentrations (MBC) for citral after 120 h were examinedby broth dilution method in 96-well, microtitre plates (M-medical,Milan, Italy). Wells inoculated with 4 log CFU/ml of S. cerevisiae SPAstrain were added with different concentrations of citral, solubilized ina constant amount of ethanol (1% v/v in wells). The microtiter plateswere incubated at 37 °C.

2.3. Juice preparation

Commercial apricot juices were purchased from a local supermarket.It was characterized by a pH value of 3.26, 14.0 °Brix, protein content of0.2%, and a water activity value of 0.996 (Patrignani et al., 2010). Thesamples were stored at refrigerated conditions (4 °C) before their useand warmed to 10 °C before inoculation with about 4.5 log CFU/ml ofthe tested strain. The juice was divided in three batches. The firstbatch was treated without addition of aroma compound (juice), thesecond batch was added with 50 mg/l of citral diluted in ethanol(Juice+EtOH+citral 50 mg/l) while the third batch was addedwith the same volume of ethanol used to dissolve the citral (1% v/vof the product)(Juice+EtOH).

2.4. High pressure homogenization (HPH) treatment

The juicewas subjected to a high pressure homogenization treatmentwith a continuous high-pressure homogenizer PANDA (Niro Soavi,Parma, Italy). Themachine, sterilized according to themanufacture's sug-gestions, was supplied with a homogenizing PS-type valve; the valve as-sembly includes a ball-type impact head made of ceramics, a stainlesssteel large inner diameter impact ring and a tungsten carbide passagehead. The inoculated fruit juices were subjected to HPH treatments at100 MPa for 1, 3, 5 and 8 passes. The inlet temperature of fruit juiceswas 10 °C and after each pass at 100 MPa, the fruit juices were cooledby using a thermal exchanger (Niro Soavi, Parma, Italy) because the in-crease rate of temperature was about 2.5 °C/10 MPa. The maximumtemperature reached by the samples did not exceed 40 °C. As controls,for each juice batch, 0.1 MPa treated inoculated juices were used. Thecollected samples were stored at 10 °C.

2.5. Microbiological analyses

The microbiological analyses were performed by appropriate sampleserial dilutions in sterile 0.9% (w/v) NaCl solution and by surface platingonto SDA agar (Oxoid Ltd., Basingstoke, Hampshire, United Kingdom).The plates were incubated at 28 °C for 48 h. Themicrobiological analyseswere performed immediately after homogenization treatments and after1, 3, 6, 8, 9, 10, 13, 15, 17 and20 days of storage at 10 °Cuntil the reachingof the yeast spoilage threshold (6.0 log CFU/ml). The data were modeledaccording to the Gompertz equation as modified by Zwietering et al.(1990). The spoilage threshold can be defined as the sum of k, corre-sponding to the initial level of yeast after homogenization treatment,and A, corresponding to the maximum cellular density increase with re-spect to initial cell load (k).

2.6. Physico-chemical analyses

The pH and water activity were measured immediately after treat-ments by using a pH-meter Basic 20 (Crison Instruments, Barcelona,Spain) and an Aqualab CX3-TE (Labo-Scientifica, Parma, Italy), respec-tively. Every measurement was made in triplicate. The viscosity(at 20 °C) was measured immediately after pressure treatments byusing a falling ball viscometer (Thermo Electron, Karlsruhe, Germany).The viscosity (Mpa/s)was calculated according to the following equation:

η ¼ K ρ1−ρ2ð Þ t

where η is the sample viscosity; K is the constant in relation to the usedsphere; ρ1 is the sphere density; ρ2 is the sample density (w/v); and t isthe time necessary to the sphere to run 10 cm (which is the valuerecorded with the viscometer).

y = 0.4164x - 0.0586R² = 0.945

y = 0.5437x + 0.0369R² = 0.9906

y = 0.5954x + 0.0893R² = 0.9789

0

1

2

3

4

5

0 1 2 3 4 5 6 7 8

|Log

N0/

N|

Number of passes at 100 MPa

JuiceJuice + EtOHJuice + EtOH + citral 50 mg/l

Fig. 1. Cell load reductions (Log10 CFU/ml) of Saccharomyces cerevisiae SPA in apricot juice,immediately after HPH, in relation to the severity of HPH treatment and the presence ofcitral and/or ethanol. N0: initial cell load before homogenization treatment. N: cell loadafter each homogenization treatment. Data are the mean of three different samples ofthree repeated experiments in different days. The variability coefficient ranged between5% and 7%.

Table 1Gompertz parameters of Saccharomyces cerevisiae SPA recovery dynamics equation injuice during storing at 10 °C in relation to homogenization pressure severity and thepresence of citral and/or ethanol.

Samples Number of passesat 100 MPa

K A μmax λ R Time

Juice 0 4.2 4.1 0.6 0.6 0.992 3.81 4.0 4.4 0.6 1.0 0.999 4.43 3.3 5.1 0.7 1.8 0.993 6.05 1.7 6.4 0.5 1.4 0.994 11.18 1.2 7.4 0.5 5.9 0.994 15.7

Juice+EtOH+citral50 mg/l

0 4.6 3.8 0.4 5.9 0.997 9.61 4.0 4.3 0.4 6.0 0.994 10.63 2.7 6.0 0.3 4.2 0.979 14.85 1.0 7.5 0.4 4.2 0.996 18.28 0.6 7.9 0.6 6.7 0.992 16.4

Juice+EtOH 0 4.8 3.6 0.6 2.7 0.990 4.81 4.3 4.3 0.5 1.2 0.992 4.93 3.1 5.8 0.4 0.8 0.989 8.45 1.3 6.7 0.5 3.1 0.993 13.88 0.6 8.1 0.4 2.9 0.992 16.9

Data are the mean of three different samples of three repeated experiments in differentdays. The variability coefficient ranged between 5% and 7%.Time: the time (days) necessary to reach the cell load of 6.0 log cfu/ml chosen asspoilage threshold.k: initial level of yeast (log CFU/ml) after homogenization treatment.A:maximum cellular density increasewith respect to the initial cell load (k) (log CFU/ml).μmax: maximum specific growth rate (log (CFU/ml)/days).λ: latency time (lag time) (days).R: correlation coefficient.

275F. Patrignani et al. / International Journal of Food Microbiology 160 (2013) 273–281

2.7. Aroma profile

Three 10 ml vials sealed by PTFE/silicon septa and containing 5 mlof juice immediately after the treatments and after the reaching of theyeast spoilage threshold (6.0 log CFU/ml) were prepared for headspacevolatile compound analysis by GC-MS coupled with a solid phasemicroextraction (GC–MS-SPME) technique. The samples were pre-heated 10 min at 45 °C. An SPME fiber covered by 75 mm CarboxenPolydimethyl Siloxane (CAR/PDMS StableFlex) (Supelco, Steiheim,Germany) was exposed to each sample at 45 °C for 40 min, and finally,the adsorbed molecules were desorbed in the GC for 10 min. For peakdetection, an Agilent Hewlett–Packard 6890 GC gas-chromatographequipped with a MS detector 5970 MSD (Hewlett–Packard, Geneva,Switzerland) and aVarian (50 m×320 μm×1.2 μm) fused silica capillarycolumn were used.

Volatile compoundswere separated under the following conditions:helium carrier gas (1 mL/min), initial column temperature 50 °C for1 min, heated to 65 °C at 4.5 °C/min, followed by heating to 230 °C at10 °C/min, maintained for 25 min. Injector, interface, and ion sourcetemperatures were 250, 250, and 230 °C, respectively. Compoundswere identified by computer matching of mass spectral data withthose of compounds contained in the Agilent Hewlett–Packard NIST98 and Wiley vers. 6 mass spectral database.

2.8. Statistical analysis

For each sample, themicrobiological and physico-chemical results areexpressed as the mean of the three different samples of three repeatedexperiments in different days. The analyseswere performed immediatelyafter HPH treatment and after 1, 3, 6, 8, 9, 10, 13, 15, 17 and 20 days ofstorage at 10 °C. The data were statistically analyzed using the one-wayANOVA procedure of Statistica 6.1 (StatSoft Italy srl, Vigonza, Italy). Thedifferences between mean values were detected by the HSD Tukey'stest and evaluations were based on a significance level of P≤0.05.

For microbiological analyses, the variability coefficients were calcu-lated as percentage ratios between standard deviations and meanvalues.

3. Results

3.1. Inactivation of Saccharomyces cerevisiae SPA in relation to thecombined treatments applied

In order to study the combined effects of citral and HPH on the inac-tivation dynamics of Saccharomyces cerevisiae SPA inoculated in apricotjuices, 50 mg/l of citral was used. This concentration was significantlylower than MIC and MCB, having, with an inoculum level of about 4log CFU/ml, values of 200 and 210 mg/l, respectively. This low level ofcitral was chosen to better evaluate its eventual interactive effect withHPH treatment without detrimental effects on the sensorial propertiesof apricot juice. Since citral was diluted in ethanol before the additionto apricot juice, also samples with only ethanol added at the samevolume used to dissolve citral and samples not supplemented withethanol and citral were considered. The different juices were subjectedto 100 MPa for 1, 3, 5 and 8 passes and compared with samples homog-enized at atmospheric pressure.

Data relative to the cell load reductions of S. cerevisiae SPA in apricotjuice, immediately after HPH, in relation to the severity of HPH treatmentand the presence of citral and/or ethanol are shown in Fig. 1. The yeastcell viability decreasedwith the increases of passes at 100 MPa, indepen-dent of the supplementation with citral and/or ethanol. The relationshipbetween yeast cell loads and the number of passes at 100 MPa followeda linear trend.

After 8 passes at 100 MPa, cell loads of 1.2, 0.5 and 0.3 log CFU/mlwere detected in the controls, samples supplemented with ethanoland samples with citral and ethanol, respectively.

3.2. Recovery dynamics of Saccharomyces cerevisiae SPA in relation tothe combined treatments applied

To study the fate of surviving cells in relation to the treatmentsapplied, yeast cell loads were recorded during the storage at 10 °C.The data weremodeled according to the Gompertz equation as modifiedby Zwietering et al. (1990).

The Gompertz parameters in relation to homogenization pressureseverity and the presence of citral and/or ethanol are shown inTable 1, which reports also the time necessary to reach the value of6.0 log CFU/ml, resulting from the sum of k and A, chosen, accordingto literature data as spoilage threshold (Patrignani et al., 2010).,Asexpected, the k values, corresponding to the initial level of yeast afterhomogenization treatment, decreased with the increase of passes at100 MPa. The presence of ethanol and citral further decreased the kvalues. The latency time in the absence of ethanol and citral increased(of about 10-fold) with the number of passes at 100 MPa passingfrom 0.56 days (sample without citral and ethanol and HPH untreated)to 5.89 days (sample without citral and ethanol and subjected to

0

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7

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9

0 2 4 6 8 10 12 14 16 18 20

log

CF

U/m

l

Time (days)

0

1

2

3

4

5

6

7

8

9

0 2 4 6 8 10 12 14 16 18 20

log

CF

U/m

l

Time (days)

0

1

2

3

4

5

6

7

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9

0 2 4 6 8 10 12 14 16 18 20

log

CF

U/m

l

Time (days)

A

B

C

0 passes

1 passes

3 passes

5 passes

8 passes

0 passes

1 passes

3 passes

5 passes

8 passes

0 passes

1 passes

3 passes

5 passes

8 passes

Fig. 2. Experimental data points of Saccharomyces cerevisiae SPA recovery in relation tothe combined treatment applied and lines obtained from Gompertz model fitting.A: Control Juice. B: Juice+EtOH+citral 50 mg/l. C: Juice+EtOH. Data are the meanof three different samples of three repeated experiments in different days. The variabil-ity coefficient ranged between 5% and 7%.

276 F. Patrignani et al. / International Journal of Food Microbiology 160 (2013) 273–281

8 pushed at 100 MPa). However, themaximum λ values were observedin the HPH samples supplemented with citral.

In addition, the presence of citral increased significantly (P≤0.05)the time necessary to attain 6.0 log CFU/ml at 10 °C. The unpressurizedcontrol samples without any supplementation reached the criticalspoilage value after 3.8 days of storage at 10 °C while those containingcitral spoiled after 9.5 days. Also the supplementation of ethanol to theunpressurized samples delayed, although in minor extent, the spoilagetime of 1 day with respect to the controls. The application of HPH in-creased the timenecessary to attain the spoilage threshold independentof the presence of ethanol and citral. However, at a given number ofpasses at 100 MPa (up to 5 passes), the presence of the terpene, aug-mented the time necessary to attain 6.0 log CFU/ml (A+k) of6–8 days with respect to the control samples. The effects of citral addi-tion on spoilage time increases were less marked (less than 1 day withrespect to the samples without any supplementation and subjected to8 passes at 100 MPa) in the samples treated 8 passes at 100 MPa,where probably prevailed the HPH disrupting effects.

Experimental data points of Saccharomyces cerevisiae SPA recoveryin relation to the combined treatment applied and lines obtainedfrom Gompertz model fitting are shown in Fig. 2. This figure showsalso the instantaneous cell load reduction due to the different HPHtreatments with respect to initial inoculum.

3.3. GC-MS profiles in relation to the combined treatments applied

Immediately after the combined treatments applied and after thereaching of the spoilage threshold (corresponding to a cell level of 6.0log CFU/ml), the different samples were analyzed using a GC-MS-SPME to obtain specific volatile molecule profiles in relation to sanitizingstrategy (HPH; citral-ethanol-HPH; ethanol-HPH) and spoilage patterns.Though the results obtainedwith this technique do not necessarily reflectthe quantitative ratio of all the compounds detected (due to the affinitybetween molecules and fiber and the adsorption conditions), they canprovide interesting information about relative changes in aroma compo-sition of food products. The data have been reported in Tables 2 and 3 andare expressed as percentage of the peak area of each compound with re-spect to the total area (which is reported in the tables). In the tables onlythe main components of each aroma profile are reported. Neverthelesstheir presence represented at least the 95% of the total area in all thejuices. The profiles of the juices without ethanol and citral can be as-sumed as representative of the apricot juiceflavor (Table 2) and the com-pounds detected has been already described as components of apricotfruit aroma or apricot juice flavor (Guillot et al., 2006; Aubert et al.,2010; Gokbulut and Karabulut, 2012). The most important class of com-pounds was represented by aldehydes (more than 80% of the total peakarea), mainly constituted by furfural, deriving from the thermal treat-ments, to which the commercial fruit juice used for these trials wassubjected. The percentage of aldehydes derived from lipoxigenasepathway (hexanal, heptanal, nonanal, (E)-2-octenal) decreasedmarkedly when the juices were subjected to the higher number ofHPH passes. By contrast, the concentrations of benzaldehyde and4-methyl-benzaldehyde, as well as terpineol among alcohols, increasedunder the same conditions. Also acids showed the samebehavior,mainlydue to the acetic acid increase. The concentration of the remaining com-pounds remained quite constant independent of the number of HPHpasses. The addition of citral and ethanol, obviously, reduced the per-centage of compounds deriving from the fruit juice. In particular, in thesample added with ethanol and citral, the two constituents of citral(neral and geranial) were responsible for the 45–50% of the total peakarea, with a significant diminution of this content at the higher HPHpasses; ethanol accounted for 16% of the total area in the notHPH treatedjuice and its concentration increasedwith the number of passes. Furfuralaccounted for about 20% of the total area of the identified molecules inthe samples homogenized at environmental pressure and increased itslevels as the pressure treatment severity increased. The presence of

geranyl acetate and terpinolene at very low levels is probably the conse-quence of impurity of the citral used in the trial. Also cyclohexanonewasdetected at concentration of about 1% only in these juices. Similar to con-trol juice, acetic acid concentration increased with the number of HPHpasses. The presence of ethyl decanoate (not present in the controljuice) was probably the result of a chemical reaction between the

Table 2Effects of various passes through a high-pressure homogenizer at 100 MPa on volatile aroma compounds (% peak area) of apricot juices deliberately inoculated with Saccharomycescerevisiae, added or not with ethanol and/or citral (50 mg/l) immediately after the combined treatments applied.

Juice Juice+EtOH+Citral 50 mg/l Juice+EtOH

Number of passes at 100 MPa Number of passes at 100 MPa Number of passes at 100 MPa

Compounds C1 1 3 5 8 C 1 3 5 8 C 1 3 5 8Thiophene-2-octyl 1.03 1.03 0.27 0.11 0.35 0.26 0.21 0.25 0.25 0.40 0.52 0.49 0.50 0.23 0.31Hydrocarbons 1.03 1.03 0.27 0.11 0.35 0.26 0.21 0.25 0.25 0.40 0.52 0.49 0.50 0.23 0.312-pentanone 1.14 1.18 1.24 0.67 0.84 −2 – – – – – – – – –

3-penten-2-one – – – – – 0.20 0.26 0.27 0.25 0.32 0.56 0.57 0.56 0.58 0.555-hepten-2-one, 6-methyl 0.65 0.70 1.04 1.26 1.18 0.52 0.55 0.42 0.47 0.61 0.86 0.89 1.03 1.09 0.99Cyclohexanone – – – – – 1.47 1.39 1.24 0.98 0.84 – – – – –

Ketones 1.79 1.88 2.28 1.92 2.02 2.19 2.20 1.93 1.70 1.78 1.42 1.46 1.59 1.67 1.54Geranyl-acetate – – – – – 0.12 0.13 0.12 0.14 0.23 – – – – –

Ethyl-9-decanoate – – – – – 2.41 2.37 2.12 1.96 2.08 4.31 4.77 4.83 3.39 3.92Esters – – – – – 2.53 2.50 2.24 2.10 2.31 4.31 4.77 4.83 3.39 3.92Hexanal 4.56 4.79 2.20 1.38 1.18 0.44 0.43 0.46 0.42 0.55 0.56 0.68 1.40 0.86 0.50Heptanal 3.31 3.22 1.63 1.05 0.59 0.23 0.23 0.30 0.42 0.60 0.45 0.51 0.88 1.13 0.34Nonanal 1.17 1.24 1.18 1.04 0.90 0.34 0.34 0.43 0.36 0.33 0.22 0.21 0.19 0.18 0.252-octenal (E) 4.15 3.05 0.69 1.01 0.97 0.19 0.21 0.20 0.11 0.29 0.26 0.25 0.43 0.45 0.21Furfural 63.14 62.62 66.23 68.16 67.27 19.89 19.48 19.11 21.03 20.67 37.88 37.01 38.06 39.19 40.74Benzaldehyde 0.59 0.61 0.99 1.27 2.10 0.44 0.45 0.45 0.44 0.52 0.73 0.78 0.66 0.82 0.774-methyl benzaldehyde 0.67 0.54 1.94 1.99 1.89 0.11 0.12 0.16 0.21 0.26 0.26 0.23 0.27 0.31 0.292-furancarboxaldehyde, 5-methyl 0.73 0.72 0.81 0.92 0.83 0.10 0.11 0.13 0.21 0.23 0.28 0.32 0.38 0.43 0.142,4-Heptadienal 5.37 5.16 6.18 4.92 5.62 2.31 2.61 3.05 3.21 3.46 6.05 5.88 5.74 4.40 5.20Neral – – – – – 22.01 21.96 22.25 21.22 20.18 – – – – –

Geranial – – – – – 27.41 27.68 26.86 25.01 23.11 – – – – –

Aldehydes 83.70 81.96 81.86 81.74 81.36 73.47 73.61 73.42 72.65 70.20 46.69 45.87 48.02 47.77 48.44Ethyl alcohol 16.41 16.27 17.43 17.42 19.55 37.81 38.29 37.24 37.08 37.801-octenol 0.60 0.63 0.78 0.67 0.76 0.71 0.74 0.51 0.47 0.46 0.21 0.20 0.20 0.28 0.25Terpineol 1.97 1.98 2.75 3.35 3.96 0.79 0.59 0.58 0.76 1.04 2.28 2.04 2.02 2.25 2.03Alcohols 2.57 2.60 3.53 4.03 4.73 17.91 17.60 18.51 18.65 21.04 40.30 40.53 39.46 39.60 40.08Acetic acid 4.29 4.28 6.04 5.36 6.87 0.76 0.75 0.83 1.10 1.28 1.51 1.55 1.60 3.08 2.40Butanoic acid – – – – – 0.29 0.28 0.22 0.23 0.21 – – – – –

Hexanoic acid – – – – – – – – – – 0.14 0.13 0.14 0.15 0.16Octanoic acid 0.90 0.91 0.84 0.99 0.87 0.31 0.29 0.21 0.32 0.33 0.44 0.36 0.34 0.61 0.48Decanoic acid 2.49 2.41 2.56 3.12 2.78 – – – – – – – – – –

Acids 7.68 7.59 9.44 9.47 10.52 1.36 1.32 1.27 1.65 1.83 2.09 2.04 2.08 3.84 3.03Terpinolene – – – – – 0.75 0.62 0.43 0.39 0.34 – – – – –

1-3-8 menthatriene – – – – – 0.36 0.37 0.25 0.19 0.21 – – – – –

Other terpenes – – – – – 1.11 0.99 0.68 0.58 0.56 – – – – –

% 96.86 95.06 97.38 97.27 98.98 98.83 98.43 98.30 97.58 98.12 95.33 95.16 96.42 96.50 97.32Total area 3 3409 3900 3474 3272 3884 9466 9060 8317 8261 7850 5207 5085 4561 4292 4073

Data are the mean of three different samples of three repeated experiments in different days. The variability coefficient ranged between 5% and 7%.1 Sample treated at 0.1 MPa.2 Under detection limit.3 Arbitrary units (x100000).

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added ethanol and the decanoic acid present in the control juice andabsent in these samples. This hypothesis was confirmed by the sameresults obtained in the juices inwhich only ethanol was added. In thislatter case ethanol represented the 31–38%of the total peak area. Also inthese samples the increasing trend of acetic acid concentration at thehigher number of HPH passes was confirmed.

After reaching the spoilage threshold (corresponding to yeast con-centration of 6.0 log CFU/ml), the total peak area increased markedlywith respect to the same samples before spoilage (Table 3), as a resultof yeast metabolism. The volatile profiles showed obviously a generalincrease of molecules deriving from yeast activities such as ethanol,3-methyl-1-butanol (amyl alcohol), 2-methy-1-propanol (isobutylalcohol), phenethyl alcohol, 3-methy-1-butanol (isoamylic alcohol),1-butanol, ethyl acetate, fatty acids and their ethyl esters. Ethanol wasthe main compound detected and increased its percentage in the con-trols (35–39%), but also in the juices added with ethanol (44–47%)with respect to the not spoiled samples. This increase was less marked(19–22%) in the juices containing citral, possibly due the weight onthe total percentage of the terpenes derived from citral. In addition,high amounts of ethyl octanoate (up to 20%) were found in the controlsand in the samples addedwith ethanol; the same ester was produced inamuch lower percentage in the presence of citral (below2%), indicating

that the esterase activity was less active in this case towards thissubstrate.

In addition to this behavior, in the juices containing citral it is inter-esting to observe the fate of neral and geranial. In fact, the percentage ofthese twomolecules drastically decreased after spoilage, accounting foronly 3% of the compounds detected. By contrast, high percentage of thecorresponding alcohols, nerol and geraniol, were detected (about 40% ifconsidered together). In addition, high amount of β-citronellol, com-prised between 10 and 15%, was found, and minor amounts of otherterpene alcohol, i.e. (Z)-isogeraniol (a geraniol isomer) and linalool,accounting for about 1 and 2%. Part of nerol and geraniol was esteri-fied with ethanol (2–3% of the total percentage). Finally also severalocimene isomers, probably deriving from citral metabolism, weredetected (up to 4%). High concentration of other terpenes, such aslimonene, α-pinene and β-pinene were detected in these juices;these molecules were not found in the spoiled control and in thejuice containing only ethanol.

The application of HPH treatment induced further quali-quantitativemodifications of volatile molecule profiles whose entities depended ofthe number of passes. In all the samples, an increase of total ketonesand aldehydes was observed in relation to the application of successiveHPH passes.

Table 3Effects of various passes through a high-pressure homogenizer at 100 MPa on volatile aroma compounds (% peak area) of apricot juices deliberately inoculated with Saccharomycescerevisiae, added or not with ethanol and/or citral (50 mg/l), after the reaching of the spoilage threshold (6 log CFU/ml).

Juice Juice+EtOH+Citral 50 mg/l Juice+EtOH

Number of passes at 100 MPa Number of passes at 100 MPa Number of passes at 100 MPa

Compounds C1 1 3 5 8 C 1 3 5 8 C 1 3 5 8Hexadecane 0.65 2.99 1.08 2.26 0.09 0.19 1.01 0.5 0.35 0.16 0.72 2.15 0.62 0.46 0.23Hydrocarbons 0.65 2.99 1.08 2.26 0.09 0.19 1.01 0.5 0.35 0.16 0.72 2.15 0.62 0.46 0.232-butanone, 3-hydroxy −2 – – – 0.75 – – – 0.12 0.19 0.32 0.32 0.75 0.74 0.422-propanone, 1-hydroxy – – – – 0.99 – – – – 0.16 – – – 1.91 0.892,3-butanedione – – – – 0.98 – – – – 0.94 – 0.23 0.58 1.05 1.892 H (pyran)-2,6 (3 H) dione – – – – 0.89 0.21 – 0.12 0.29 0.16 0.26 0.22 0.24 0.5 0.223-penten-2-one (E) – 0.46 0.48 – – 0.25 – 0.09 0.52 – 0.3 0.52 0.47 0.4 0.115-hepten-2-one, 6-methyl 0.53 0.62 0.66 0.78 0.93 0.2 0.12 0.31 0.74 0.52 0.77 0.87 0.37 0.23 0.88Ketones 0.53 1.08 1.14 0.78 4.54 0.66 0.12 0.52 1.67 1.97 1.65 2.16 2.41 4.83 4.41Ethyl acetate 1.23 0.81 0.34 1.17 0.67 0.16 0.41 1.12 0.28 0.33 1.1 0.83 0.63 0.32 0.831-butanol, 3-methyl acetate 0.56 0.51 0.19 0.88 0.34 – – – – – 0.5 0.34 0.27 0.18 0.35Ethyl,9-decenoate 1.71 3.28 5.46 2.11 1.09 0.74 2.93 1.11 0.72 0.54 1.71 1.51 2.69 1.63 2.18Hexanoic acid, ethyl ester 5.31 5.96 4.15 2.09 0.29 1.03 2.35 0.7 0.53 0.41 4.05 3.25 2.87 1.9 0.86Octanoic acid, ethyl ester 19.76 21.51 20.12 22.99 22.55 1.08 1.11 0.86 0.08 0.79 20.54 18.78 19.4 19.69 17.02Decanoic acid, ethyl ester 5.78 4.16 4.49 3.39 3.59 0.85 2.74 0.25 0.85 1.86 5.05 5.21 4.17 3.6 2.93Neryl acetate – – – – 0.74 0.71 0.19 0.25 0.34 – – – – –

Geranyl acetate – – – – 2.49 1.86 0.45 0.6 0.67 – – – – –

Esters 34.35 36.23 34.75 32.63 28.53 7.09 12.11 4.68 3.31 4.94 32.95 29.92 30.03 27.32 24.17Hexanal 0.25 0.35 0.90 1.29 0.84 0.26 0.17 0.43 – – 0.11 0.18 – – 0.11Nonanal 0.23 1.43 1.86 0.95 0.91 0.51 1.16 0.23 0.52 0.15 0.19 0.36 0.35 0.76 0.45Furfural 0.38 0.33 0.19 0.96 2.74 0.5 0.11 0.54 0.96 0.57 0.56 0.29 0.79 1.09 1.52Benzaldehyde 0.33 0.66 0.56 0.43 0.45 0.28 – – 0.39 – 0.75 0.61 0.45 0.13 0.412-furancarboxaldehyde, 5-methyl – – – – 0.92 – – – 0.23 1.09 0.31 0.25 0.64 2.21 1.94Neral – – – – – 0.97 1.15 0.88 1.81 2.35 – – – – –

Geranial – – – – – 1.21 0.97 2.14 3.14 1.12 – – – – –

Aldehydes 1.19 2.77 3.51 3.63 5.86 3.73 3.56 4.22 7.05 5.28 1.92 1.69 2.23 4.19 4.43Ethyl alcohol 39.31 35.7 35.92 39.93 38.47 19.39 20.74 19.13 20.73 22.99 45.79 45.28 45.46 44.53 47.631-propanol-2-methyl 6.56 3.82 2.28 3.23 2.16 – – – – – 0.73 0.31 0.35 0.44 0.471-hexanol 1.15 1.07 1.8 0.86 0.48 0.11 0.12 0.08 0.11 0.17 0.21 0.25 0.12 0.51 0.49Heptanol – 0.16 1.27 0.24 0.18 – – – – – 0.2 0.28 0.32 0.27 0.461-octenol – – 0.99 0.97 0.13 0.09 0.09 – 0.19 0.12 0.11 0.35 0.32 0.14 0.122-furanmethanol 1.07 0.56 0.51 1.61 1.66 1.4 1.24 1.34 1.38 1.75 1.27 1.33 1.79 2.12 1.82Phenylethyl alcohol 1.87 1.07 0.92 0.69 0.91 0.16 0.12 0.14 0.09 0.27 1.86 1.34 1.41 1.01 0.47α-terpineol 1.55 0.83 0.89 1.97 1.99 1.42 0.96 1.28 1.48 1.11 1.39 1.32 1.31 1.18 1.371-butanol,3-methyl 1.32 3.12 2.73 2.5 3.75 0.91 0.45 0.32 0.45 1.77 2.46 4.6 3.27 2.14 5.76Linalool – – – – – 2.35 1.91 2.5 2.33 1.62 – – – – –

β-citronellol – – – – – 13.31 15.74 11.49 9.58 13.6 – – – – –

Nerol – – – – – 18.45 15.7 19.73 19.96 17.38 – – – – –

(Z)-isogeraniol – – – – – 1.19 1.13 1.34 1.02 0.52 – – – – –

Geraniol – – – – – 21.74 15.83 18.78 21.48 19.67 – – – – –

Alcohols 52.83 46.33 47.31 52.00 49.73 80.52 74.03 76.13 78.80 80.97 54.02 55.06 54.35 52.34 58.59Acetic acid 2.18 0.47 0.75 1.43 3.42 0.54 0.34 0.67 0.94 0.9 0.86 0.97 1.3 2.22 1.6Hexanoic acid 2.14 2.79 2 1.88 0.95 – – – – – 2.11 1.73 1.72 0.61 1.21Octanoic acid 1.48 1.77 0.9 1.01 1.16 0.53 0.11 0.22 0.45 0.83 0.25 0.93 1.26 2.77 2.73Decanoic acid 1.04 0.92 1.06 1.24 1.35 – – – – – 0.45 0.26 0.33 0.18 0.24Acids 6.84 5.95 4.71 5.56 6.88 1.07 0.45 0.89 1.39 1.73 3.67 3.89 4.61 5.78 5.78Limonene 0.31 0.22 0.17 0.22 0.19 1.52 1.37 1.28 0.98 0.55 0.15 0.31 0.38 0.11 0.12β-pinene – – – – – 1.01 2.53 3.95 2.33 0.8 – – – – –

α-pinene – – – – – – – 0.52 0.46 0.37 – – – – –

β-(E)ocimene – – – – – 0.69 1.78 3.73 1.16 0.69 – – – – –

Ocimene – – – – – 1.18 – – – – – – – – –

β-(Z)ocimene – – – – – 0.23 0.19 0.56 – – – – – – –

Neo.allo-ocimene – – – – – 0.4 0.33 0.72 0.5 0.52 – – – – –

Other terpenes 0.31 0.22 0.17 0.22 0.19 5.03 6.2 10.76 5.43 2.93 0.15 0.31 0.38 0.11 0.12% 96.70 95.57 92.67 97.08 95.82 98.29 97.48 97.70 98.00 97.98 95.08 95.18 94.63 95.03 97.73Total area 3 9657 11167 12427 10344 9539 12553 15974 13122 12193 11983 9211 8759 9551 9362 9279

Data are the mean of three different samples of three repeated experiments in different days. The variability coefficient ranged between 5% and 7%.1 Sample treated at 0.1 MPa.2 Under detection limit.3 Arbitrary units (x100000).

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3.4. Changes in physic-chemical features in relation to the combinedtreatments applied

The effects of the different combined treatments onpH,water activity(aw) and viscosity of the treated samples were studied (Table 4). TheHPH treatment itself had a significant effect on the pH and viscosity ofapricot juices while did not affect aw. In fact, the pH values of apricotjuices decreased by increasing the severity of the HPH treatments,while the viscosity increased after the first pass at 100 MPa, and while

it slightly decreased with further passes. The addition of citral and/orethanol before HPH treatment did not modify the pH, aw and viscositybehaviors observed in relation to 100 MPa passes applied.

4. Discussion

The cell lod decreases observed are in agreement with the hypothesisthat, in the multipass treatment, the effect of each pass is additive and,therefore, each homogenization pass causes the same reduction of the

Table 4pH, water activity and viscosity of apricot juices deliberately inoculated with Saccharomycescerevisiae, added or not with ethanol and/or citral (50 mg/l) in relation to the high pressionhomogenization treatments at 100 MPa.

Samples Number ofpasses at100 Mpa

pH Water activity Viscosity(Mpa/s)

Juice C1 3.32±0.01 A 0.989±0.001 A 6.5±1.1 A

1 3.24±0.02 B 0.987±0.002 A 10.8±0.9 B

3 3.22±0.01 B 0.985±0.002 A 9.9±0.5 B

5 3.19±0.01 BC 0.988±0.001 A 9.2±1.2 BC

8 3.15±0.02 C 0.987±0.003 A 8.3±0.7 C

Juice+EtOH+Citral(50 mg/l)

C 3.31±0.01 A 0.986±0.001 A 6.2±0.8 A

1 3.22±0.03 B 0.989±0.002 A 10.4±0.4 B

3 3.19±0.02 B 0.987±0.002 A 9.6±0.8 BC

5 3.17±0.01 B 0.988±0.001 A 8.7±0.6 BC

8 3.16±0.02 B 0.987±0.003 A 8.0±0.7 C

Juice+EtOH C 3.29±0.03 A 0.987±0.002 A 6.8±0.6 A

1 3.26±0.01 A 0.989±0.002 A 11.1±0.4 B

3 3.21±0.02 B 0.986±0.001 A 10.1±0.8 B

5 3.20±0.01 B 0.985±0.003 A 9.4±1.2 BC

8 3.14±0.01 C 0.988±0.001 A 8.3±0.3 C

For each considered sample and for each column, valued with the same superscriptletters are not statistically different (P>0.05).

1 Sample treated at 0.1 MPa.

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microbial load (Diels and Michiels, 2006; Patrignani et al., 2010).However, the literature data concerning the HPH cell inactivation areconflicting. In fact, Donsì et al. (2007) and Donsì et al. (2009) observeda non additive trend for multiple passes processes at a given pressurelevel on some spoilage and pathogenic species attributing this trendmainly to the physiological diversity within a population and to the ex-istence of resistant cells of the initial microbial population able to sur-vive the passes at the pressure applied. Also Patrignani et al. (2009)showed that the inactivation of Saccharomyces cerevisiae 635 in apricotand carrot juices was not linearly related to the number of passes at100 MPa. In fact, the inactivation level achieved after each pass at100 MPawas lower than that of the previous one and tended to a plateauwhose valuewas affected by both the initial inoculums level and the juicetype.

The increase of the time necessary to reach the spoilage thresholdincreased with the number of passes at 100 MPa independent of citraland ethanol supplementation due to the reduction of inoculated yeastpopulation and to the modification of microstructure that in its turnsaffects nutrient diffusion (Stecchini et al., 1998).

The molecules supplemented in this study are both endowed withantimicrobial activity (Belletti et al., 2008, 2010). However, ethanol wasused at concentration (1%) not able to significantly influence S. cerevisiaegrowth and citral at a concentration corresponding to the 25% of theMIC.On the other hand, this study showed that their antimicrobial activity canbe notably potentiated throughout their combination with HPH treat-ment. In fact, a significant increase of the time necessary to reach thespoilage threshold was obtained after the supplementation of ethanoland, particularly, of citral.

The cumulative damages caused by the HPH treatment and thepresence of terpenes injured the cells causing a major inhibition ofS. cerevisiae SPA growth in the samples subjected to the combinedstrategy adopted. In fact, althoughDiels andMichiels (2006) postulatedthe “all or nothing”mechanism for HPH cell disruption, some literaturereports support the hypothesis of sub-lethal damages induced by theprocess (Vannini et al., 2004; Iucci et al., 2007; Donsì et al., 2007,2009; Taylor et al., 2007; Patrignani et al., 2009.)

The cavitation phenomena and the sublethal temperature rise duringthe treatment (the maximum temperature reached by the samples after8 passes did not exceed 40 °C due to the thermal exchanger) increasedthe vapor pressure of ethanol and/or citral and, in turn, enhanced themolecule bioactivity, augmenting their possibility to dissolve in theyeast cell membrane. The importance of vapour pressure in the

expression of toxicity against microorganisms has been pointed out byseveral authors (Walker et al., 1975; Lanciotti and Guerzoni, 1993;Guerzoni et al., 1994; Caccioni et al., 1997; Gardini et al., 1997; Lanciottiet al., 1999, 2004; Belletti et al., 2010). On the other hand, a wide litera-ture indicates the use of sublethal temperatures as a tool to increase theantimicrobial activity of several bioactive compounds including citral,linalool and β-pinene, S-carvone, carvacrol, cymene, cinnamom, vanillin,(E)-2-hexenal, and citron essential oil (Karatzas et al., 2000; Knight andMcKellar, 2007; Periago et al., 2004; Belletti et al., 2007, 2010; Char etal., 2009).

The aroma profile of the juice without ethanol and citral can beconsidered representative of the apricot aroma and the compoundsdetected have already been described by several authors in the apricotfruits and juices (Gokbulut and Karabulut, 2012; Guillot et al., 2006;Aubert et al., 2010). The application of successive HPH passes deter-mined the reduction of thepercentage of the aliphatic aldehydes respon-sible for “green” or “herbal” notes and increased the concentration ofother substances important for their organoleptic impact, such asbenzaldehyde (almond odour) and terpineol (lilac odour). In addition,furfural, the main component of apricot juice aroma as a result of thethermal treatments to which the juice was subjected, increased itsconcentration after the application of 3–8 passes at 100 MPa. Thesame behavior was observed also in the juices containing citral andethanol. Although HPH is regarded as a non thermal technology, duringtreatment temperature increases due to frictional heating in the ho-mogenization valve (Diels and Michiels, 2006; Donsi et al., 2011). Thetemperature rise depends on several factors (inlet temperature, pres-sure level, number of passes, matrix, valve geometry, and temperatureexchanger). In the present work the use of a thermal exchanger limitedthe temperature rise and after 8 passes the outlet temperature did notexceed 40 °C. However, the repeated passes at 100 MPa seem to inducea matrix thermal stress able to increase the level of furfural and otheroxidation markers. In addition, the application of 3–8 passes at 100 MPaincreased the total alcohol amount and acetic acid attributable to thestripping action of HPH from the inoculated yeast cells due to cavitationphenomena (Guerzoni et al., 1999).

After reaching the spoilage threshold (6.0 log CFU/ml), the volatileprofiles of the samples of juice not supplemented with ethanol and/orcitral showed an increase (with respect to the sample analyzed immedi-ately after the HPH treatment) of molecules deriving from yeast metab-olism such as ethanol, 3-methyl-1-butanol, 1-butanol, esters, ketones,acids etc. The application of repeated passes at 100 MPa determined anaccumulation of ketones and aldheydes. The involvement of ketones inthe responsemechanisms of cells to stresses is documented while estersare regarded as yeast signaling molecules (Kocsis and Weselake, 1996;Isakoff et al., 1996).

The aroma profile of the juice containing ethanol was similar tothat observed for apricot juice alone, with the exclusion of ethanolconcentration which further increased with respect to the samplebefore fermentation.

In the juice containing citral, the two original aldehydes (neral andgeranial)were almost completely substituted by yeastmetabolismwithmolecule characterized by a presumably lower antimicrobial activity,among which alcohols were the most important. It has been demon-strated that the reduction of the aldehydes to nerol and geraniol is thefirst step of citral biotransformation by penicilli which can successivelybring to compounds partially detected also in this experiment, such as lin-alool, α-terpineol, limonene, α- and β-pinene (Esmaeili and Tavassoli,2010). A similar detoxifying mechanism, i.e. reduction to the respectivealcohols, was shown for six carbon aliphatic aldehydes (Patrignani et al.,2008).

The increase of viscosity in apricot juice in the HPH treated samples,was previously observed and attributed to the changes during HPHtreatments of macromolecules such as proteins and polysaccharides,and mainly those involving pectin (Patrignani et al., 2009, 2010). Inparticular these authors attributed the changes observed to the ability

280 F. Patrignani et al. / International Journal of Food Microbiology 160 (2013) 273–281

of HPH treatments to enhance the activity of pectin methylesterase,with consequent increase of the exposure of pectin side chains, the in-teraction between protein and pectin molecules as well as the pectingelling ability. A consistent residual pectin methylesterase E activity inthermally stabilized industrial preparation of peach, pear, apple, apricot,red grapefruit andpineapple has been reported and it iswell documented(Castaldo et al., 1997; Riener et al., 2009). In addition, HPH-induced po-tentiating effect on the activity of several enzymes is documented(Vannini et al., 2004; Iucci et al., 2007, 2008).

In conclusion, the multipass HPH treatment applied was able topotentiate the antimicrobial activity of natural antimicrobials such as eth-anol and citral used at sub-lethal concentrations, increasing significantlythe juice shelf-life. In addition, it induced appealing texture modificationassociated to interesting changes of volatile molecule profile character-ized by the reduction the molecules responsible for the “green” or“herbal” notes and the increase of those conferring “almond” and“lilac” notes. Consequently, the HPH treatment, associated to the useof natural antimicrobials, can have a potential of diversifying themarketof fruit juices without detrimental effects on product safety andshelf-life and of satisfying the consumer needs for the reduction of tra-ditional preservatives.

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