characterization of a wild strain of alicyclobacillus acidoterrestris: heat resistance and...

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Characterization of a Wild Strain ofAlicyclobacillus acidoterrestris: Heat Resistanceand Implications for Tomato JuiceAntonio Bevilacqua and Maria R. Corbo

Abstract: This article reports the characterization of a wild strain of Alicyclobacillus acidoterrestris and describes theimplications of the heat resistance of this microorganism in tomato juice. The strain (labeled as A. acidoterrestris γ 4)showed pH and temperature ranges for growth typical of the species (3.0 to 6.0 for the pH and 35 to 60 ◦C for thetemperature); heat resistance in tomato juice was as follows: DT values of 40.65, 9.47, and 1.5 min (at 85, 90, and95 ◦C, respectively) and z-value of 7 ◦C. A treatment at 70 ◦C for 15 min was found to be optimal for spore activation,whereas Malt Extract Agar, acidified to pH 4.5, showed good results for spore recovery. Concerning the implicationsof heat resistance of A. acidoterrestris on tomato juice, high temperatures required for spore inactivation determined ageneral decrease of the antioxidant activity (increase of the redox potential and reduction of the chain-breaking activity),but not the formation of brown compounds (namely, hydroxymethylfurfural), thus suggesting an effect on the secondaryantioxidants (carotenoids and ascorbic acid) rather than on lycopene.

Keywords: Alicyclobacillus acidoterrestris, heat resistance, nutritional value, tomato juice

Practical Application: Alicyclobacillus acidoterrestris is an emerging spore-forming microorganism, capable of causing spoilagein tomato juice. Due to their high thermal resistance, spores could be used as targets for the optimization of heat processing;this article reports on the assessment of thermal resistance of a wild strain of A. acidoterrestris, then focusing on the effectof the thermal treatment necessary to inactivate spores on the quality of tomato juice.

IntroductionAlicyclobacillus acidoterrestris is a Gram-positive, rod-shaped, and

spore-producing microorganism, usually isolated from soil (pri-mary source), plants, spoiled juices, tea, and equipments (sec-ondary sources) (Tokuda 2007). The main characteristics of thisspecies are the thermo-acidophilic behavior (that is, the abilityto grow at low pHs, 3.5 and 5, and high temperatures >35 ◦C),the presence in the membrane of ω-alicyclic fatty acids and theability to spoil juices and acidic products, by producing guaiacoland other halophenols (Wisotzkey and others 1992; Goto andothers 2007; Bevilacqua and others 2008; Walker and Phillips2008).

The first description of a spoilage incident attributable to ther-mophilic bacilli (later classified as A. acidoterrestris) was reportedby Cerny and others (1984), who described a spoilage event onpacked and pasteurized apple juice. Since then the impact of A.acidoterrestris has increased, as it has been isolated also in orangeand tomato juices and acidic drinks (Tokuda 2007) and nowadaysappears more widespread than in the past (Walker and Phillips2005).

MS 20100848 Submitted 7/27/2010, Accepted 12/3/2010. Authors are withDept. of Food Science, Faculty of Agricultural Science, Univ. of Foggia, Via Napoli25, 71100 Foggia, Italy. Authors are also with Food Quality and Health ResearchCentre (BIOAGROMED), Univ. of Foggia Via Napoli 25, 71100 Foggia, Italy.Direct inquiries to author Corbo (E-mail: [email protected]).

Tomato juices and other derivates are believed to have healthbenefits due to the antioxidant ability of their bioactive com-pounds, such as lycopene and vitamin C (Riso and others 2003);in fact, the consumption of tomato products has been associatedwith a lower risk of developing digestive tract and prostate cancers(Giovannucci and others 2002). This effect is probably attributableto lycopene and other antioxidant components, as they probablyprevent cell damage through synergistic interactions (Friedman2002; George and others 2004). Microflora of tomato and tomatojuices is mainly represented by moulds (Wade and Beuchat 2003a,2003b; Wade and others 2003), lactic acid bacteria (Bevilacquaand others 2007; Di Cagno and others 2009), spore-forming mi-croorganisms (Goto and others 2007; Tokuda 2007), and somepathogens (such as Salmonella sp.) (Mosqueda-Melgar and others2008).

Thermal processing is conventionally used for the inactivation ofspoiling microorganisms and enzymes in tomato juice; however,strong treatments could affect significantly sensorial and nutri-tional qualities of this product (Goodman and others 2002). Infact, the volatile components and vitamin C of canned tomatojuice can be reduced by treatments at 100 ◦C for 10 min (Siesoand Crouzet 1977; Youssef and Rahman 1982). The color de-grades more rapidly with increasing temperature (Goodman andothers 2002; Sanchez-Moreno and others 2006). On the otherhand, thermal treatments would increase the overall antioxidantpotential of the tomato for the positive effect of the temperatureon the extractability of lycopene (Anese and others 1999, 2002;Sanchez-Moreno and others 2006). Besides, lycopene in tomato is

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Heat resistance of Alicyclobacillus acidoterrestris . . .

relatively resistant to thermal degradation, whereas other antiox-idants (ascorbic acid, tocopherol, and β-carotene) degrade morerapidly by thermal processing (Abushita and others 2000).

Although thermal treatments cause some undesirable changeson foods and some alternative approaches have been proposed,they still remain the most used methodology for the stabilizationof tomato juice. Moulds have been considered for a long timeas the target for the optimization of the thermal processing forjuices; however, the high thermal resistance of A. acidoterrestrisspores (Silva and Gibbs 2004; Ceviz and others 2009), along withthe increasing recovery of this microorganism in tomato-basedproducts (Tokuda 2007), suggests that it could be considered as anew test for designing thermal treatments for tomato juice.

Therefore, the main goals of this article were:1. To characterize a wild strain of A. acidoterrestris, isolated

from a spoilage incident in pear juice, in order to assess itsbehavior in relation to pH and temperature, as well as topoint out the optimal conditions (medium and activation)for spore recovery.

2. To study its thermal resistance in tomato juice and assess theimplications for the sensorial and nutritional quality (color,brown compounds, antioxidant activity).

Materials and Methods

StrainA strain of A. acidoterrestris, isolated from a spoiled pear juice

(A. acidoterrestris γ 4), was used throughout this study; it belongsto the Culture Collection of the Dept. of Food Science, Univ.of Foggia (Sinigaglia and others 2003). The strain was stored at4 ◦C on slants of Malt Extract Agar (MEA) (Oxoid, Milan, Italy),acidified to pH 4.5 through a sterile solution of citric acid (1:1,w/w).

Spore preparationSporulation was achieved on acidified MEA; after the incuba-

tion at 44 ◦C for 7 d, spores were washed from the surface ofthe plates through cold sterile distilled water and centrifuged at1000 g for 10 min; then, the pellet was washed 2 times, suspendedin distilled water and heat-processed at 80 ◦C for 10 min to de-stroy the vegetative cells. The number of spores was determinedby plate counting on acidified MEA (incubated at 44 ◦C for 48h).

Biochemical profileThe biochemical profile of the strain γ 4 was assessed through

the miniaturized system API 50 CH, using the suspension medium50 CHB (Biomeriux, Marcy L’Etoile, France). The API test wasincubated at 45 ◦C for 48 h.

Critical pHs for spore germinationpH values for spore germination were assessed on MEA plates,

adjusted to pHs ranging from 2.0 to 7.0; the plates were incubatedwith 100 μL of spore suspension (the concentration of the sus-pension was ca. 7 log spores/mL) and incubated at 45 ◦C for 5 d.Spore germination (that is, the formation of visible colonies ontothe surface) was assessed every day.

Effect of temperature on spore germinationThe effect of temperature on spore germination was assessed on

MEA plates, acidified to pH 4.5 and inoculated as reported before.The plates were incubated at 35, 45, 55, and 60 ◦C for 5 d, andspore germination was assessed every day.

Spore activation and media for the optimal recovery ofspores

Aliquots of tomato juice (9 mL, pH 4.4, 12◦Bx) were addedwith 1 mL of spore suspension (ca. 7 log spores/mL), in orderto achieve a final concentration of 6 log spores/mL, and heattreated in a water bath at 60 ◦C/30 min, 60 ◦C/60 min, 70 ◦C/15min, 70 ◦C/30 min, 80 ◦C/5 min, 80 ◦C/10 min, and 80 ◦C/20min. After each treatment, the viable count of A. acidoterrestris wasevaluated on 3 different media:

Bacillus acidocaldarius medium (BAM, medium n. 402)(DSMZ 2010)Orange serum agar (OSA) (Orr and Beuchat 2000)MEA, acidified to pH 4.5.All the media were incubated at 45 ◦C for 5 d.

Heat resistanceHeat resistance was assessed as follows: 9 mL of tomato juice and

1 mL of spore suspensions (approximately 107 spores/mL) weresealed in glass vials (external diameter, 8.0 mm; height, 75 mm;thick glass walls, 1.0 mm); aliquots of 9 mL of tomato juice, addedwith 1 mL of a saline solution (0.9% NaCl) were used as referencesamples for the evaluation of the changes time/temperature.

The samples, both the inoculated and the reference ones, wereheat treated at 85, 90, and 95 ◦C (Table 1) in a water bath withagitation. The evolution of the temperature inside the vials wasmonitored through a thermocouple and a datalogger linked to acomputer (Datalogger SHP, Parma, Italy). After each treatment,samples were immediately cooled under running water to simulatean industrial treatment. The viable count of A. acidoterrestris wasassessed on MEA, acidified to pH 4.5 and incubated at 45 ◦C for2 d.

For each temperature, data were modeled through a linear equa-tion, reading as follows:

log N = log N0 − b · t,

where log N and log N0 are, respectively, the viable count at thetime t and the initial count (log CFU/mL); b is a fitting parameterand t the time (min). The fitting parameter b was assumed as thedecimal reduction time, that is, the time to attain a reduction inspore number of 1 log CFU/mL.

Then, D values were plotted as a function of the temperaturewith a linear regression procedure; the slope of the line was as-sumed as the z-value (◦C).

Color determinationA tristimulus colorimeter (Chromameter-2 Reflectance, Mi-

nolta, Osaka, Japan) equipped with a CR-300 measuring headwas used; the apparatus was standardized with a white tile beforemeasurement and the data expressed in the CIE L∗, a∗, and b∗Hunter scale parameters.

Table 1 –Combinations of time/temperature for the evaluation ofspore heat resistance.

Temperature (◦C) Time (min)a

85 0 70 140 210 28090 0 15 30 45 6095 0 2.5 5 7.5 10aReal values, without the time needed for heating and cooling.

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Five measurements were carried out; from a∗ and b∗ values,the chroma [(a ∗2 + b ∗2)1/2] and hue angle [tan−1(b ∗/a ∗)] wereevaluated: hue is the name of the color (red, yellow, blue, green,and so on) and chroma is a measure of color saturation or purity.

Finally, the effect of the various heat treatments was evaluatedas total color difference:

�E∗ = (�L∗2 + �a ∗2 + �b ∗2)1/2

.

Determination of brown compoundsThe amount of uncolored and brown compounds (hy-

drodymethylfurfural), produced throughout heat treatment, wasassessed as follows: 20 mL of heat-processed tomato juices werecentrifuged at 10000 rpm for 20 min at 20 ◦C; the pellet wasdiscarded and the aqueous fraction used for the analytical deter-mination.

Brown and uncolored compounds were evaluated throughabsorbance measurement at 294 and 420 nm by means of a UV-visible spectrophotometer (Beckman DU 640, Beckman Instru-ments, Fullerton, Calif., U.S.A.).

Antioxidant activity: redox potential and chain-breakingactivity

The antioxidant activity of tomato samples was assessed throughthe redox potential and the chain-breaking activity, evaluated asfollows:

1. Redox potential. An aliquot of 20 mL of tomato aqueousfraction was placed in a tree-neck flask; the oxygen was re-moved from the sample flushing nitrogen for 10 min. Redoxmeasurements were performed through a voltmeter appa-ratus equipped with a platinum electrode and a Ag/AgCl,Cl−sat reference electrode (Hanna Instruments, mod. 8417,Singapore, Singapore). The electrodes were soaked in thesamples and the change of redox potential with a frequencyof 1 min was monitored for 30 min.

2. The chain-breaking activity was evaluated according to themethod of Brand-Williams and others (1995), modified byManzocco and others (1998). The breaking rate of a stablefree radical 2,2-diphenyl-1-picryhydrazyl (DPPH·) (Sigma-Aldrich, Milan, Italy) at 515 nm was monitored. In its radicalform, DPPH· adsorbs at 515 nm, but upon reduction by anantioxidant or a radical species, its adsorption disappears.A volume of 2.9 mL of 6.0 × 10−5M DPPH methanolsolution was used; the reaction was started by adding 100μL of the samples, and the bleaching of DPPH· at 515 nmwas followed at 25 ◦C for at least 40 min. The followingequation was used to achieve the reaction rate (k):

1

Abs3− 1

Abs30= −3kt,

where Abs0 and Abs are the initial and the absorbance value at thetime t. In this way, the chain-breaking activity was evaluated as− Abs −3

t per gram of dry matter or mg of lycopene equivalents.

Statistical analysesFor each experiment, 2 different batches were prepared; for

each batch, the analyses were performed twice. The differencesamong the different samples were analyzed through the 1-wayanalysis of variance (1-way ANOVA); it works in a very different

way from t-test. In fact, 1-way ANOVA does not examine thedifference among means directly, but it looks at the variabilityof the data and compares the variability among the groups andwithin each group. The statistic test is the F-test, set at a P-level of0.05 and the null hypothesis (H0) is that the groups have the samemeans.

After the basic ANOVA test, Tukey’s test was run. ANOVA,in fact, tells us if there are some differences among the differentgroups, but not which group is different. This information can beobtained through a post hoc test. The main purpose of this articlewas to compare each group with all others; therefore Tukey’s testwas used (P-level, 0.05).

Statistical analyses were performed through the statistical pack-age Statistica for Windows (Tulsa, Okla., U.S.A.).

Results and Discussion

Strain characterization: biochemical profile, pH,temperature, spore activation, and recovery

Biochemical characterization of the strain was performedthrough the miniaturized system API 50 CHB; the results showedthat A. acidoterrestris γ 4 was able to grow and acidify in presenceof maltose, lactose, and esculin and grow in presence of rhamnose.The assay was negative for the other substrates of the miniaturizedsystems.

These data appeared quite different from the biochemical pro-file reported by Goto and others (2007) for the type strain ATCC49025; this isolate, in fact, was able to grow and acidify also inpresence of glycerol, erythritol, L-arabinose, D-galactose, inositol,mannitol, sorbitol, cellobiose, sucrose, and threalose. This differ-ence highlights the strong variability among the different strains ofA. acidoterrestris, probably because of the primary isolation source.Moreover, the strain under investigation appeared quite differentalso from the other species of the genus Alicyclobacillus, whichgenerally are able to use more carbohydrates that the strain herebystudied (Goto and others 2007).

Other key elements for the characterization of A. acidoterrestrisis the assessment of pH and temperature ranges for growth. Con-cerning pH, our results were in agreement with those reported inthe literature; Goto and others (2007), Walker and Phillips (2008),and Bevilacqua and others (2008), in fact, reported that A. aci-doterrestris growth was observed in a range 3.0 to 6.0 (2.0 to 7.0for some strains isolated from soil) with optimal value around 4.0.Alicyclobacillus acidoterrestris γ 4 spores were able to germinate be-tween 3.0 and 6.0, with an optimal recovery between 4.0 and 4.5;outside this range (4.0 to 4.5), spore germination was observed,but recovery appeared significantly lower (1 log units or more atpH 3.5, 5, and 5.5, 2 to 3 log at pH 3.0 and 6.0) (Table 2).

Focusing on the effect of the temperature (Table 3), as expected,the optimal temperature for spore recovery was 45 ◦C, with a

Table 2 –Effect of the pH on the recovery of the spores of Alicy-clobacillus acidoterrestris γ 4 on MEA plates, incubated at 45 ◦C.

pH log CFU/mL∗

3.0 5.6a

3.5 5.9a,b

4.0 7.2c

4.5 7.5c

5.0 6.2b

5.5 6.2b

6.0 4.6d

∗Values with different superscripts are significantly dif-ferent (one-way ANOVA and Tukey’s test) (P < 0.05).

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difference of 1 log at 35 and 55 ◦C; the effect of the temperatureappeared strong at 60 ◦C.

Generally described as a thermophilic species (Goto and others2007; Walker and Phillips 2008), A. acidoterrestris can grow in alarge range of temperature. The data of the present article con-firmed the thermophilic behavior of the strain γ 4; however, thehigh spore recovery at 35 ◦C can have a strong practical implica-tion. This temperature is often registered in Southern Italy duringsummer; therefore, spoilage by alicyclobacilli can represent a greatthreat for food industry, in particular for fresh juices produced anddistributed by local vendors.

The final step for the preliminary characterization of thestrain γ 4 was the study of the effects of different combinationsof time/temperature on spore activation (Table 4). No signif-icant differences were recovered among the 3 different media(P > 0.05); moreover, the effect of the different combinationsof time/temperature on spore recovery were quite similar, al-though at 80 ◦C on BAM medium, lower counts were observed.Due to the long times of treatment at 60 ◦C, the combination 70◦C/15 min could be considered as a good compromise for sporeactivation.

Concerning the effect of the different media for spore recovery,the results hereby reported appeared of great concern. The Intl.Fruit Union (IFU) suggested in the past BAM as the optimalmedium for spore recovery in fruit juice (Bevilacqua and others2008); however, the preparation of this medium is quite difficult,as it is necessary to prepare 2 solutions (solution A, containingyeast extract, glucose, and some macro minerals, and solution B,containing some micronutrients as trace amounts) and then mixthem. On the other hand, OSA medium was proposed by Orrand Beuchat (2000) for an optimal recovery of spores; it containssome classical ingredients of microbiological media (tryptone andyeast extract) and 20% of orange juice and is adjusted to pH3.5 with a solution of malic acid. Despite its results in termsof spore enumeration and recovery, it appears too complex fora routine determination. This is the reason why we proposed a

Table 3 –Effect of the temperature on the recovery of spores of A.acidoterrestris γ 4 on MEA plates, acidified at pH 4.5.

Temperature log CFU/mL∗

35 5.84a

45 6.92b

55 5.83a

60 2.3c

∗Values with different superscripts are significantly dif-ferent (1-way ANOVA and Tukey’s test) (P < 0.05).

Table 4 –Spore recovery (log CFU/mL) of A. acidoterrestris γ 4 inlaboratory media. Spore was activated through heat shock at differenttemperatures and times.

Combination MEA∗ OSA BAM

60 ◦C to 30 min 6.47aA 6.52a

A 6.47aA

60 ◦C to 60 min 6.31aA 6.39a

A 6.17aA

70 ◦C to 15 min 6.30aA 6.00a

A 6.17aA

70 ◦C to 30 min 6.17aA 6.17a

A 6.00aA

80 ◦C to 5 min 5.93aA 5.70a

B 5.00bB

80 ◦C to 10 min 5.97aA 5.84a

B 5.90aA

80 ◦C to 20 min 5.97aA 5.73a

B 5.17bB

∗MEA = Malt Extract agar; OSA = Orange Serum Agar; BAM = Bacillus acidocaldariusmedium.a,bValues in a line with different superscripts are significantly different (1-way ANOVAand Tukey’s test) (P < 0.05).A,BValues in column with different capital letters are significantly different (1-way ANOVAand Tukey’s test) (P < 0.05).

simple medium (acidified MEA) for alicyclobacilli determination;the results of this article suggested that it showed similar (or higherin some cases) performance values if compared to the classicalmedia. Therefore, it can be proposed as a suitable alternative.Further investigations are required to validate these results on alarger number of strains.

Thermal resistanceSilva and Gibbs (2004) proposed A. acidoterrestris as the tar-

get to design thermal treatments for fruit juices, since it appearedmore heat resistant than other spoiling microorganisms. Heat resis-tance of spores is greatly variable and relies upon various elements(the strain, the pH, and kind of medium, the conditions attainedthroughout sporulation); Silva and Gibbs (2001) reported that D-value at 95 ◦C in juices could range from 0.06 to 5.3 min andz-value varied from 7.2 and 12.9 ◦C.

The strain γ 4 showed D-values at 85, 90, and 95 ◦C of, re-spectively, 40.65, 9.47, and 1.5 min; otherwise, z-value was 7 ◦C(Table 5). The data recovered highlighted the high thermal resis-tance of the spores of A. acidoterrestris, thus posing the problemof the real effectiveness of the traditional thermal processing em-ployed by juice producers. It is a common practice, in fact, use athermal treatment at 90 to 95 ◦C from seconds to some minutes(Bates and others 2001); however, the data aforementioned un-derlined that a 5 D reduction of A. acidoterrestris spores, consideredby many authors as the main goal for juice safety and stability(Vieira and others 2002), could be obtained at 95 ◦C for 7.5 to9 min.

Effect of thermal treatment on tomato juiceThe final step of this research focused on the effect of thermal

treatments on nutritional and qualitative characteristics of tomatojuice (color, production of brown compounds, namely, hydrox-ymethylfurfural, and antioxidant activity).

Concerning color, the studied combinations of time/temperature did not affect L∗, a∗, and b∗ parameters, thus it sug-gested that the thermal treatments did not start nonenzymaticbrowning reactions (data not shown). This result was confirmedby absorbance values at 294 nm (which gives a quantitative indica-tion of the formation of the intermediate, not colored compoundsby nonenzymatic browning reactions) and 420 nm (synthesis ofthe final-colored compounds of nonenzymatic browning reac-tions) (Vaikousi and others 2008; Sahin and others 2009). Theseparameters, in fact, did not change significantly following thermaltreatments (data not shown).

This result (that is, none effect on color and browning) wasnot strange, as some authors reported in the past that lycopene ap-peared quite stable to thermal degradation (Nguyen and Schawartz1999). On the other hand, the effects of thermal treatments ontomato juice was evident on the antioxidant activity, as evidencedby redox potential and chain-breaking activity. Figure 1 reports theredox potential of tomato juice, treated at different temperatures(85, 90, and 95 ◦C); it is clear that temperature caused a significantincrease of this parameter, if compared to the control, proba-bly because of a partial inactivation/destruction of heat-sensible

Table 5–DT and z values of A. acidoterrestris γ 4 in tomato juice.

Temperature DT (min) z (◦C)

85 ◦C 40.6590 ◦C 9.47 7.0095 ◦C 1.5


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antioxidants (ascorbic acid and some carotenoids). However,Nicoli and others (2004), Anese and others (2002), and Man-zocco and others (1998) reported that redox potential is a ther-modynamic measure, without any indication of the rate of the

redox reaction. This information could be obtained through thechain-breaking activity (Figure 2), which decreased significantlyafter the thermal treatments, thus it confirmed the effects of thetemperature on the heat-sensitive antioxidants of tomato juice.

0

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Figure 1–Redox potential (mV) of tomato juice samples inoculated with A. acidoterrestris and heat treated at various temperature/time combinations.Letters indicate significant differences (1-way ANOVA and Tukey’s test; P < 0.05).


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Figure 2–Chain-breaking activity of tomato juice samples inoculated with A. acidoterrestris and heat treated at various temperature/time combinations.Letters indicate significant differences (1-way ANOVA and Tukey’s test; P < 0.05).


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ConclusionsThe optimization of a thermal treatment is a balancing between

the inactivation of the targets (spoiling microorganisms and/orpathogens or enzymes in juices) and retaining the sensorial andnutritional properties of foods.

Concerning tomato juice, A. acidoterrestris could be consideredas a suitable target to design a preserving treatment; an importantissue in this field is the great variability of the strains of this species.The strain hereby characterized showed some typical characteris-tics of alicyclobacilli:

1. The thermoacidophilic behavior, with growth ranges of 3.0to 6.0 (for the pH) and 35 to 60 ◦C (for the temperature);

2. The heat resistance in tomato juice (D90◦C and z-values of9.45 min and 7 ◦C, respectively).

Other peculiar characteristics of the strain were:1. The biochemical profile, with a pattern of sugar utilization

different from those reported for other alicyclobacilli;2. MEA as a suitable medium for spore recovery.In the 2nd phase, the main topic was to assess the effect of

thermal treatments on tomato juice; the results showed that:� High temperature did not induce the degradation of lycopene,

as inferred by color and absorbance at 294 and 420 nm.� However, the antioxidant activity of the juice was compromised,

with a general increase of redox potential and a decrease of thechain-breaking activity, thus suggesting a possible degradation ofthe secondary antioxidants of tomato (carotenoids and ascorbicacid).The implication of this results is strong: assuming that a 5 D

reduction of alicyclobacilli spores should be achieved for the stabi-lization of juice and considering the DT and z-values of spores, atreatment at 95 ◦C for at least 7.5 min should be performed. Thiscombination should be labeled as a minimal treatment, becauseheat resistance of alicyclobacilli is greatly variable and it is wellknown that some strains showed higher values of DT and z thanthose evaluated for the strain γ 4.

A possible perspective could be the validation of the results ofthis article with more strains of A. acidoterrestris to assess strainvariability and the minimal combinations of time/temperature forthermal processing of tomato juice.

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characterization of a wild strain of alicyclobacillus acidoterrestris: heat resistance and...

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