lable at ScienceDirect

Food Microbiology 38 (2014) 56e61

Contents lists avai

Food Microbiology

journal homepage: www.elsevier .com/locate/ fm

Use of the modified Gompertz equation to assess the Stevia rebaudianaBertoni antilisterial kinetics

Clara Miracle Belda-Galbis a, María Consuelo Pina-Pérez a, Josepa Espinosa a,Aurora Marco-Celdrán a, Antonio Martínez a,b, Dolores Rodrigo a,*

a Instituto de Agroquímica y Tecnología de Alimentos (IATA-CSIC), Carrer del Catedràtic Agustín Escardino Benlloch 7, 46980, Paterna, València, SpainbUnidad Asociada de Conservación y Seguridad de los Alimentos, Escuela Técnica Superior de Ingeniería Agronómica, Paseo Alfonso XIII 48,30203, Cartagena, Murcia, Spain

a r t i c l e i n f o

Article history:Received 14 February 2013Received in revised form27 June 2013Accepted 15 August 2013Available online 24 August 2013

Keywords:Food safetyNatural preservativesStevia rebaudiana BertoniListeria innocuaMathematical modellingKinetic parameters

* Corresponding author. Tel.: þ34 963 900 022-221E-mail addresses: lolesra@iata.csic.es, conbel@iata

0740-0020/$ e see front matter � 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.fm.2013.08.009

a b s t r a c t

In order to assess the antibacterial activity of Stevia rebaudiana Bertoni (Stevia), Listeria innocua growthwas characterized at 37 �C, in reference medium supplemented with a leaf infusion, a crude extract, anda steviol glycosides purified extract. Experimental data were fitted to the modified Gompertz model andthe antibacterial activity of Stevia was determined based on the lag time (l) and the maximum growthrate (mmax) reached, depending on the incubation conditions. As the leaf infusion showed the mostmarked elongation of l and the most marked mmax reduction, its antimicrobial effect was evaluated atdifferent concentrations, at 37, 22 and 10 �C. According to the results obtained, in general, the lower thetemperature or the higher the Stevia concentration, the longer the l and the lower the mmax, statisticallysignificant being the effect of reducing temperature from 37 or 22 to 10 �C, the effect of increasing Steviaconcentration from 0 or 0.5 to 1.5 or 2.5% (w/v), at 37 �C, and the elongation of l observed in presence of1.5 and 2.5% (w/v) of Stevia, at 22 �C. These results show that Stevia could be a bacterial growth controlmeasure if a cold chain failure occurs.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Food preservatives are used for their ability to inhibit or slowdown the multiplication of spoilage and/or pathogenic microor-ganisms in order to prolong food shelf life, ensuring quality andsafety (Ferrer et al., 2009).

Greater consumer awareness and concern regarding syntheticadditives, as well as the alarming incidence of new and re-emerging foodborne diseases, constitutes the main reason whyresearchers and food processors are looking for natural pre-servatives derived from plants, animals or microorganisms, whichpermit to obtain products that satisfy market requirements. In thisrespect, natural substances have been proved to be effective asantimicrobials when added directly as ingredients in food formu-lations (Pina-Pérez et al., 2009, 2012). Furthermore, there is anincreasing interest in the use of functional ingredients that avoidthe proliferation of microorganisms, increasing food value, with theadvantage that they are not considered additives. Prominentamong them are some of the substances present in the essential oils

8; fax: þ34 963 636 301..csic.es (D. Rodrigo).

All rights reserved.

of herbs and spices, which have bacteriostatic and bactericidalproperties (Rasooli, 2007), although their commercial application isquite difficult because of their volatile nature and their impact onthe organoleptic characteristics of products, specially taking intoaccount that their efficacy in food matrices requires the use of highconcentrations (Hyldgaard et al., 2012).

Stevia rebaudiana Bertoni (Stevia), also known as Stevia, sweetleaf, sweet herb of Paraguay, honey leaf and candy leaf (Jayaramanet al., 2008; Madan et al., 2010), is a perennial shrub native toParaguay, also present in neighbouring parts of Brazil andArgentina (Sivaram and Mukundan, 2003; Soejarto, 2002). It is oneof the 230 members of the genus Stevia and one of the two speciesof this genus that produce sweet steviol glycosides (Brandle andTelmer, 2007), which can be used as substitutes for sucrose, forcaries prevention and for the treatment of certain pathologies, suchas diabetes, obesity and hypertension (Chan et al., 2000; Das et al.,1992; Ghanta et al., 2007; Jeppesen et al., 2000). Notable amongthese steviol glycosides are stevioside and rebaudioside A. Both areextremely sweet (up to 300 and 450 times sweeter than sucrose,respectively) (Yadav and Guleria, 2012) and safe at the acceptabledaily intake (ADI) levels which range from 0 to 12 mg/kg bodyweight (bw)/day, in the case of rebaudioside A, and from 0 to10 mg/kg bw/day, in the case of stevioside, based on the JECFA ADI

C.M. Belda-Galbis et al. / Food Microbiology 38 (2014) 56e61 57

for steviol glycosides (expressed as steviol equivalents) of 0e4 mg/kg bw/day (JEFCA, 2009).

In USA rebaudioside A is considered a GRAS compound (FDA,2008), so it is permitted in the formulation of beverages andsome foods, while in Europe the use of steviol glycosides as a foodadditives is accepted since 2011 (EC, 2011), after EFSA issued apositive opinion about their safety (EFSA, 2010).

The antimicrobial activity of Stevia and therefore its potentialuse as a natural preservative have recently been studied by variousresearchers (Debnath, 2008; Ghosh et al., 2008; Jayaraman et al.,2008; Tadhani and Subhash, 2006). So far it has been tested bymeasuring the inhibition zone diameter (IZD), using the discdiffusion technique. To our knowledge, until now no bacterial countreduction or kinetic studies have been conducted to test the anti-microbial capability of Stevia.

From a food safety point of view, mathematical modelling ofbacterial behaviour can be used to predict the contamination loadin foods over time as a function of various factors, by constructing amatrix of responses to a broad range of specific process and storageconditions (Ross and McMeekin, 1991; Scott et al., 2005; Whiting,1995). Consequently, the mathematical description of Stevia ef-fects from a kinetic point of viewwould be a very useful tool for thefuture step of benefit-risk assessment of the addition of Stevia as asucrose substitute and for the planning of an appropriate hazardanalysis critical control point (HACCP) system in the industry.

Listeria monocytogenes is a pathogen of great concern in mini-mally processed products because of its ubiquitous, psychrotrophicnature, and because of its ability to grow in acidic environments orwith low water activity values (Carpentier and Cerf, 2011), since allof these properties allow it to reach levels that jeopardize con-sumer health (Chan and Wiedmann, 2009).

For all these reasons, themain objectives of the present researchwork were (i) to evaluate the effect of different Stevia extracts onthe growth of Listeria innocua as a non-pathogenic surrogate ofL. monocytogenes (Char et al., 2010; Jadhav et al., 2013), in referencemedium, depending on temperature, and, (ii) to quantify the Steviaantimicrobial effect by means of mathematical models, from adeterministic point of view.

2. Material and methods

2.1. Culture preparation

Avials stock containing themicroorganism studied in stationaryphase (L. innocua, CECT 910) (6.5 �109 cfu/mL) was generated fromthe lyophilized pure culture provided by the Spanish Type CultureCollection, following the method described by Saucedo-Reyes et al.(2009).

2.2. Experimental design and description of substrates

Growth curves were obtained at 37 �C, in Tryptic Soy Broth (TSB;Scharlab Chemie S. A., Barcelona, Spain), in the presence of variousextracts, at various concentrations.

The extracts tested were the following: (i) a leaf prepared fromdried leaves (ANAGALIDE S. A., Huesca, Spain) and bottled water, (ii)a crude extract (GLYCOSTEVIA�-EP, ANAGALIDE S. A., Huesca,Spain) with a steviol glycosides content equal to or higher than 20%,and a purified extract of steviol glycosides (GLYCOSTEVIA�-95,ANAGALIDE S. A., Huesca, Spain) with a high percentage of rebau-dioside A (�80%, the product purity being greater than 95%).

The antibacterial activity of the most active product was thenevaluated at various concentrations (3 concentrations) and tem-peratures (37, 22 and 10 �C). With this aim, growth curves wereobtained in TSB (Scharlau Chemie S. A., Barcelona, Spain).

For each of the conditions studied, two bottles with 20 mL ofbroth and the Stevia extract at the desired concentration wereinoculated, the initial inoculum being ca. 1 � 105 cfu/mL. With thisaim, frozen vials of the available stock were defrosted at roomtemperature and diluted in 1& buffered peptone water (ScharlauChemie S. A., Barcelona, Spain) to achieve the desired inoculum sizeat time 0, taking into account the sample final volume and the cellaverage concentration of the vials in stock. After that, samples weretaken, diluted and seeded in duplicate at regular intervals until thestationary growth phase was reached, using 1& buffered peptonewater (Scharlau Chemie S. A., Barcelona, Spain) and Tryptic SoyAgar (TSA; Scharlau Chemie S. A., Barcelona, Spain), respectively, forthe dilution and seeded of the samples, in order to quantifiedmicroorganism growth by viable plate count.

All the experiments were performed in triplicate and includedthe obtainment of growth curves in TSB (Scharlau Chemie S. A.,Barcelona, Spain) without Stevia.

2.3. Growth modellization

The growth curves obtained for the various combinations ofStevia and temperature were fitted to the modified Gompertzequation (Gibson et al., 1988; Zwietering et al., 1990):

log10ðNtÞ ¼ Aþ C � e�e�B�ðt�MÞ(1)

where Nt represents the number of microorganisms (N) at time t(cfu/mL); A the lower asymptote value, i.e., the log10 of the initialcount (N0; log10 (cfu/mL)); C the difference between the curve as-ymptotes (log10 (cfu/mL)), that is, between Nmax and N0 (log10 (cfu/mL)); B the relative growth rate when t ¼M ((log10 (cfu/mL))/h); Mthe elapsed time until the maximum growth rate is reached (h);and e is Euler’s number, whose value is approximately equal to2.718.

This model (equation (1)) was used in accordance with studiespreviously carried out by other authors to model the effect ofnatural compounds on bacterial growth (Ferrer et al., 2009; Guillieret al., 2005; Pina-Pérez et al., 2009).

A, B, C and M were used to calculate the kinetic parameters lagtime (l, h) and maximum growth rate (mmax, (log10 (cfu/mL))/h)),employing the following equations (Gibson et al., 1988; McMeekinet al., 1993):

l ¼ M ��1B

�þ log10ðN0Þ � A

mmax(2)

mmax ¼ B� Ce

(3)

The data were fitted using the statistical software Statgraphics�

Centurion XV (Statpoint Inc., Virginia, USA).The accuracy of the fits was determined by means of the

adjusted determination coefficient (adjusted R2) and the meansquare error (MSE), whose mathematical expressions are asfollows:

Adjusted R2 ¼

26641�

n� 1��1� SSQregression

SSQtotal

�n� p

3775 (4)

MSE ¼ SSQresidualn� p

(5)

Table 1Modified Gompertz equation fit results for Listeria innocua growth at 37 �C, with/without the addition of different Stevia extracts.

C.M. Belda-Galbis et al. / Food Microbiology 38 (2014) 56e6158

where n represents the number of observations; p the number ofmodel parameters; and SSQ the sum of squares (Saucedo-Reyeset al., 2009).

Sample l (h) mmax ((log10(cfu/mL))/h)

adjustedR2

MSE

Control (without Stevia) 0.99 � 0.10a 0.64 � 0.04a 0.987 0.015Steviol glycosides

purified extract0.47 � 0.06a 0.58 � 0.05a 0.992 0.018

Crude extract 2.58 � 0.12b 0.31 � 0.04b 0.993 0.014Leaf infusion of 2.5% (w/v) 10.89 � 0.28c 0.25 � 0.01c 0.989 0.016

aee Different lowercase letters reflect significant differences between the l and mmax

values obtained in presence of different Stevia extracts.

2.4. Statistical data analysis

To evaluate the effects of temperature and Stevia concentration,an analysis of variance (ANOVA) was done. To determine whichlevels of each factor were significantly different (p � 0.05) a mul-tiple range test (MRT) was applied, using the Fisher distribution(LSD) to check equality of variances. All the statistical analyses werecarried out with Statgraphics� Centurion XV (Statpoint Inc., Vir-ginia, USA).

3. Results and discussion

3.1. Effect of different Stevia extracts on Listeria innocua growth

The Stevia effect on L. innocua growth was tested by obtaininggrowth curves of the microorganism in the presence of differentextracts: (i) a leaf infusion, (ii) a crude extract, and (iii) a purifiedextract, under optimal growth conditions, i.e., in referencemedium,at 37 �C (Rowan and Anderson, 1998) (Fig. 1).

Based on visual inspection of the curves obtained, and takinginto account the growth pattern of the bacterium studied in theabsence of Stevia, a marked elongation of the lag phase and agrowth rate decrease, reflected in the lower slope of the expo-nential phase, can be attributed to the infusion and the crudeextract.

The curves were fitted to the modified Gompertz model(Table 1) and the antimicrobial Stevia extracts effect was charac-terized based on kinetic parameters l and mmax.

No differences were detected between the controls and samplescontaining the purified extract, therefore no bacteriostatic/bacte-ricidal effect against L. innocua can be attributed to it (p > 0.05).However, an increase in l and a decrease in mmax were found for thecrude extract and the infusion (p � 0.05). The crude extract wasable to multiply l by two. With the infusion, the l value was 10times higher than the l value without Stevia. mmax was halved inboth cases, both in the presence of the crude extract and in thepresence of the infusion. Consequently, a statistically significant

Fig. 1. Listeria innocua growth curves obtained at 37 �C under the intervention ofdifferent Stevia extracts: control (>), purified extract (1%) (w/v) (-), crude extract(1%) (w/v) (6) and leaf infusion (2.5%) (w/v) (C). The dashed lines represent thegrowth curves obtained after fitting experimental data to the modified Gompertzequation.

antimicrobial effect can be attributed to these Stevia extracts(p � 0.05).

Although the use of medicinal plants as sources of natural an-timicrobials is booming (Hammer et al., 1999; Radulovi�c et al.,2007), to our knowledge only a few studies have reported theantimicrobial capability of Stevia (Bader et al., 2007; Debnath,2008; Seema, 2010; Sivaram and Mukundan, 2003; Tadhani andSubhash, 2006), and so far no research work has proposed amathematical model to describe bacterial growth behaviour inpresence of different Stevia extracts.

Differences in antimicrobial capability between the various ex-tracts studied must be due to their different composition. In thepresent study, the least processed material, the infusion, showedthe highest antimicrobial capability against L. innocua, followed bythe crude extract. The purified extract, which was the most pro-cessed of the three, did not seem to affect the growth pattern ofL. innocua under the conditions studied, probably because the in-gredients that contribute antimicrobial capability to Stevia aredegraded in the process of purification.

It is well understood that correlating antimicrobial activity withthe phytochemicals present in an extract is a complex task (Ferreret al., 2005; Nobmann et al., 2009), because the exact composi-tion of any mixture of ingredients determines its antimicrobialcapability, and there may even be differences between mixturesthat in principle are the same, if they are not obtained at the sametime and from the same rawmaterial. For example, it is known thatthe antimicrobial activity of essential oils depends on the compo-sition of the oil, which in turn depends on the individual plant, theenvironmental conditions inwhich it grew, the part fromwhich theoil was obtained and, of course, the extraction process (Karakayaet al., 2011). In general, the process of obtaining purified extractsentails the loss of some potentially antimicrobial ingredients(Nobmann et al., 2009).

Steviol glycoside compounds have been described as diterpenesresponsible for the natural sweetness of Stevia. Marketed purifiedextracts from its leaves mainly contain stevioside (>80%) andrebaudioside A (>90%). In the present study, the purified extract,which has no apparent antimicrobial activity, contains more than95% of steviol glycosides, whereas the crude extract possesses aminimum concentration of 20%. The steviol glycoside concentra-tion of Stevia leaves has been reported as varying between 4 and20% (Gardana et al., 2010; Wöelwer-Rieck, 2012). It might bethought that the antimicrobial nature of Stevia depends on in-gredients other than steviol glycosides. The phytochemicals con-tent of Stevia leaves has been reported to be rich in flavonoids,alkaloids, chlorophylls, xanthophylls, hydroxycinnamic acids (caf-feic acid, catechin, epicatechin, cinnamic acid), non-glycosidediterpenes, saponins, sterols and terpenes (Markovi�c et al., 2008;Tadhani and Subhash, 2006). Because of the terpenic chemicalstructure of steviol glycosides, it cannot be ruled out that they mayhave antimicrobial properties per se and/or combined with other

C.M. Belda-Galbis et al. / Food Microbiology 38 (2014) 56e61 59

compounds (Brandle and Telmer, 2007). Phenolic compounds havebeen extensively reported to be antimicrobials. According toMuanda et al. (2011), water extract (WE) from Stevia leaves had anantimicrobial effect against Staphylococcus aureus, Bacillus subtilis,Escherichia coli, Pseudomonas aeruginosa and Candida albicans.Muanda et al. (2011) compared the effectiveness of Stevia leaf WE,methanol WE (50:50 (v/v)) and essential oil, revealing that the leafWE showed the highest antimicrobial effect. They determined thecompounds identified in each extract, the leaf WE being rich inprotocatechin, catechin, rutin, quercetin glycosyl and quercetindehydrate. Most of the compounds identified by Muanda et al.(2011) are flavonoids. Therefore, according to Choi et al. (2006),the antibacterial activity of Stevia extracts could mainly be due toflavonoids, aromatic acids, terpenoids and their ester contents.

In any case, the results obtained show that the Stevia crudeextract and the infusion studied have antimicrobial properties, sothey could be used as free-calorie sweeteners able to extend foodshelf life.

Fig. 2. Listeria innocua growth curves at different temperatures (37 �C (a), 22 �C (b)and 10 �C (c)), in presence of different Stevia infusion concentrations: 0% (w/v) (>),0.5% (w/v) (-), 1.5% (w/v) (6) and 2.5% (w/v) (C). The solid lines represent the growthcurves obtained after fitting experimental data to the modified Gompertz equation.

3.2. Effect of temperature and Stevia concentration on microbialgrowth

On the basis of the previous results and because of the antimi-crobial effect shown against L. innocua, the Stevia infusion wasselected for a deeper study of Stevia concentration and incubationtemperature interaction on the antimicrobial capability observed.Stevia leaf infusion was prepared at 0, 0.5, 1.5 and 2.5% (w/v) andincubated at 37, 22 and 10 �C to test the impact of the combinationsstudied on L. innocua growth (Fig. 2). To quantify both effects,experimental data were fitted to the modified Gompertz model.Table 2 provides the l and mmax values obtained in each case as wellas the goodness of each fit (adjusted R2 and MSE).

In view of the results obtained, L. innocua growth was affectedby temperature in the range [37e10 �C], with a significant elon-gation of l (10 times) and a significant reduction of mmax (5 times)when the temperature was reduced to 10 �C, in the absence ofStevia (control) (p � 0.05). Equations (7) and (8) show the depen-dence between growth kinetic parameters and temperature.

lcontrol ¼ �0:228� T þ 8:998; R2 ¼ 0:999 (7)

mcontrol ¼ þ0:017� T � 0:107; R2 ¼ 0:999 (8)

With the infusion, the result was similar. Temperature reductionfrom 37 or 22 �C to e10 �C entailed an elongation of l and areduction of mmax, significantly being the difference between thevalues obtained at 10 �C and the values obtained at higher tem-peratures in presence of 0.5 and 1.5% (w/v) of Stevia, as well as thedifference between the values obtained at 10 and at 22 �C inpresence of 2.5% (w/v) of Stevia.

With regard to the antimicrobial concentration effect, Fig. 2ashows the comparative results for different Stevia infusions at 37 �C.At this temperature, a significant influence of Stevia concentrationon l and mmax values was observed (p � 0.05). Stevia leaf infusionwith a concentration of 2.5% (w/v) significantly increased l value byup to 9 times at 37 �C (Table 2), while the mmax values decreased byup to 3 times at 37 �Cwhen Stevia leaves were prepared at the sameconcentration, so that, at optimum growth temperature this naturalproduct was able to slow down the growth of the microorganismstudied at concentrations equal or higher than 1.5% (w/v).

Table 2Modified Gompertz equation fit results for Listeria innocua growth at 37, 22 and10 �C, with/without leaf infusion at different concentrations (0.5,1.5 and 2.5% (w/v)).

Temperature(�C)

% Stevia(w/v)

l (h) mmax ((log10(cfu/mL))/h)

adjustedR2

MSE

37 0 0.984 � 0.175a,A 0.625 � 0.043a,A 0.991 0.0200.5 1.498 � 0.116a,C 0.593 � 0.033a,D 0.992 0.0181.5 3.840 � 1.025b,E 0.420 � 0.085b,G 0.990 0.0222.5 9.432 � 0.276c,GH 0.229 � 0.000c,I 0.993 0.011

22 0 2.926 � 0.63d,A 0.325 � 0.007d,B 0.990 0.0260.5 3.647 � 0.629d,C 0.295 � 0.021d,E 0.994 0.0121.5 5.667 � 0.890e,E 0.318 � 0.012d,G 0.994 0.0142.5 8.193 � 0.579f,G 0.262 � 0.049d,I 0.990 0.016

10 0 10.784 � 5.642g,B 0.128 � 0.036e,C 0.989 0.0260.5 11.295 � 1.680g,D 0.099 � 0.017e,F 0.979 0.0361.5 12.017 � 0.960g,F 0.122 � 0.019e,H 0.989 0.0292.5 12.365 � 1.806g,H 0.086 � 0.006e,J 0.988 0.029

aee Different lowercase letters reflect significant differences between the the l andmmax values obtained in presence of different concentrations of Stevia according totemperature.AeD Different uppercase superscript letters reflect significant differences betweenthe the l and mmax values obtained at different storage temperatures for a deter-mined Stevia concentration.

C.M. Belda-Galbis et al. / Food Microbiology 38 (2014) 56e6160

To define the dependence relationship between growth kineticparameters (l and mmax values) and Stevia concentration ([0e2.5%(w/v)]), the following mathematical equations were obtained:

l37 �C ¼ þ3:019� ½Stevia� þ 0:008; R2 ¼ 0:959 (9)

m37 �C ¼ �0:150� ½Stevia� þ 0:593; R2 ¼ 0:998 (10)

At 22 �C, the presence of Stevia also increased l and reducedmmax, significant being the increment of l observed in presence of1.5 and 2.5% (w/v) of Stevia. At 10 �C, however, no statistically sig-nificant differences were observed between the control samplesand the ones containing Stevia (p > 0.05). This implies that theantimicrobial nature of the infusion declines as the temperaturedecreases, or else that the bacterium is more resistant to its effectsat low temperatures. It is well known that microorganisms whichsurvive or adapt to a given stress often gain resistance to others(Wesche et al., 2009). When bacteria are grown at low tempera-tures, they modify their membrane composition to increase theircold tolerance; these changes could also increase Stevia resistance(Rattanachaikunsopon and Phumkhachorn, 2010; Veldhuizen et al.,2007). In any case, it seems that Stevia could slow down the growthof L. monocytogenes if a cold chain failure occurs or if contaminatedfoods are kept incorrectly.

The followings surface response models (Equations (11) and(12)) describe the effect of the two factors studied on l and mmax,either alone and in combination (p� 0.05). Both were developed inview of the results obtained, i.e., taking into account that both thetemperature and the Stevia concentration affect significantly thegrowth pattern of the microorganism studied.

l ¼ �20:932� 1:205 T þ 0:017� T2 þ 0:086� T � ½Stevia�;R2 ¼ 0:909

(11)

mmax ¼�0:084þ0:019�Tþ0:066�½Stevia��0:001�T�½Stevia�;R2 ¼ 0:951

(12)

4. Conclusions

In view of the potential industrial applicability of Stevia inthe search for natural, healthy sweetness sources, the antimi-crobial description of different Stevia products has become animportant subject for study because of the lack of knowledge inthis area and the requirement for the industry to be costeffective. The mathematical modelling of Stevia antibacterialcapability under different conditions (temperatures and con-centrations) is a first step on the industrial pathway to futureStevia incorporation not only as a sweetener but also as a pre-servative. The current use of this natural product to sweetenfoodstuffs and beverages could be reassessed in the light of theadditional preservative properties shown during the shelf life offood products if a cold chain failure occurs or if foods are keptincorrectly, especially against psychrotrophic pathogen micro-organisms such as L. monocytogenes, which are of great impor-tance in pasteurized beverages.

Acknowledgements

The authors are grateful to the Ministry of Economy andCompetitiveness for providing financial support by means of CYCITproject AGL2010-22206-C02-01 ALI. Clara Miracle Belda-Galbis is

grateful to the CSIC for providing her with a JAE-predoctoral grantin 2010.

References

Bader, A., Panizzi, L., Cioni, P.L., Flamini, G., 2007. Achillea ligustica: composition andantimicrobial activity of essential oils from the leaves, flowers and some pureconstituents. Cent. Eur. J. Biol. 2, 206e212.

Brandle, J.E., Telmer, P.G., 2007. Steviol glycoside biosynthesis. Phytochemistry 68,1855e1863.

Carpentier, B., Cerf, O., 2011. Review e persistence of Listeria monocytogenes in foodindustry equipment and premises. Int. J. Food Microbiol. 145, 1e8.

Chan, P., Tomlinson, B., Chen, Y.J., Liu, J.C., Hsieh, M.H., Cheng, J.T., 2000. A double-blind placebo-controlled study of the effectiveness and tolerability of oralstevioside in human hypertension. Br. J. Clin. Pharmacol. 50, 215e220.

Chan, Y.C., Wiedmann, M., 2009. Physiology and genetics of Listeria monocytogenessurvival and growth at cold temperatures. Critic. Rev. Food Sci. Nutr. 49, 237e253.

Char, C.D., Guerrero, S.N., Alzamora, S.M., 2010. Mild thermal process combinedwith vanillin plus citral to help shorten the inactivation time for Listeria innocuain orange juice. Food Bioprocess Technol. 3, 752e761.

Choi, Y.M., Noh, D.O., Cho, S.Y., Suh, H.J., Kim, K.M., Kim, J.M., 2006. Antioxidant andantimicrobial activities of propolis from several regions of Korea. Lwt-Food Sci.Technol. 39, 756e761.

Das, S., Das, A.K., Murphy, R.A., Punwani, I.C., Nasution, M.P., Kinghorn, A.D., 1992.Evaluation of the cariogenic potential of the intense natural sweeteners ste-vioside and rebaudioside A. Caries Res. 26, 363e366.

Debnath, M., 2008. Clonal propagation and antimicrobial activity of an endemicmedicinal plant Stevia rebaudiana. J. Med. Plants Res. 2, 45e51.

EC, 2011. Commission Regulation (EU) No. 1131/2011of 11 November 2011amending annex II to Regulation (EC) No. 1333/2008 of the European Parlia-ment and of the Council with regard to steviol glycosides. Off. J. Eur. Un. L 295,205e211.

EFSA, 2010. Scientific opinion on the safety of steviol glycosides for the proposeduses as a food additive. EFSA J. 8, 1537e1621.

FDA, 2008. GRN No. 253. FDA, p. 99.Ferrer, C., Ramón, D., Muguerza, B., Marco, A., Martínez, A., 2009. Effect of olive

powder on the growth and inhibition of Bacillus cereus. Foodborne Pathog. Dis.6, 33e37.

Ferrer, M., Soliveri, J., Plou, F.J., López-Cortés, N., Reyes-Duarte, D., Christensen, M.,Copa-Patino, J.L., Ballesteros, A., 2005. Synthesis of sugar esters in solventmixtures by lipases from Thermomyces lanuginosus and Candida antarctica B,and their antimicrobial properties. Enzyme Microb. Technol. 36, 391e398.

Gardana, C., Scaglianti, M., Simonetti, P., 2010. Evaluation of steviol and its glyco-sides in Stevia rebaudiana leaves and commercial sweetener by ultra-high-performance liquid chromatographyemass spectrometry. J. Chromatogr. A1217, 1463e1470.

Ghanta, S., Banerjee, A., Poddar, A., Chattopadhyay, S., 2007. Oxidative DNA damagepreventive activity and antioxidant potential of Stevia rebaudiana (Bertoni)Bertoni, a natural sweetener. J. Agric. Food Chem. 55, 10962e10967.

Ghosh, S., Subudhi, E., Nayak, S., 2008. Antimicrobial assay of Stevia rebaudianaBertoni leaf extracts against 10 pathogens. Int. J. Biol. 2, 27e31.

Gibson, A.M., Bratchell, N., Roberts, T.A., 1988. Predicting microbial growth: growthresponses of salmonellae in a laboratory medium as affected by pH, sodiumchloride and storage temperature. Int. J. Food Microbiol. 6, 155e178.

Guillier, L., Pardon, P., Augustin, J.C., 2005. Influence of stress on individual lag timedistributions of Listeria monocytogenes. App. Environ. Microbiol. 71, 2940e2948.

Hammer, K.A., Carson, C.F., Riley, T.V., 1999. Antimicrobial activity of essential oilsand other plant extracts. J. Appl. Microbiol. 86, 985e990.

Hyldgaard, M., Mygind, T., Meyer, R.L., 2012. Essential oils in food preservation:mode of action, synergies, and interactions with food matrix components.Front. Microbiol. 3.

Jadhav, S., Shah, R., Bhave, M., Palombo, E.A., 2013. Inhibitory activity of yarrowessential oil on Listeria planktonic cells and biofilms. Food Control 29, 125e130.

Jayaraman, S., Manoharan, M.S., Illanchezian, S., 2008. In-vitro antimicrobial andantitumor activities of Stevia rebaudiana (Asteraceae) leaf Extracts. Trop. J.Pharm. Res. 7, 1143e1149.

JEFCA, 2009. Safety Evaluation of Certain Food Additives. WHO Library Cataloguing-in-Publication Data, India.

Jeppesen, P.B., Gregersen, S., Poulsen, C.R., Hermansen, K., 2000. Stevioside actsdirectly on pancreatic beta cells to secrete insulin: actions independent of cyclicadenosine monophosphate and adenosine triphosphate-sensitive Kþ-channelactivity. Metabol. Clin. Exp. 49, 208e214.

Karakaya, S., El, S.N., Karagözlü, N., Sahin, S., 2011. Antioxidant and antimicrobialactivities of essential oils obtained from oregano (Origanum vulgare ssp. hirtum)by using different extraction methods. J. of Med. Food 14, 645e652.

Madan, S., Ahmad, S., Singh, G.N., Kohli, K., Kumar, Y., Singh, R., Garg, M., 2010. Steviarebaudiana (Bert.) Bertoni e a review. Indian J. Nat. Prod. Resour. 1, 267e286.

Markovi�c, I.S., Ðarmati, Z.A., Abramovi�c, B.F., 2008. Chemical composition of leafextracts of Stevia rebaudiana Bertoni grown experimentally in Vojvodina.J. Serb. Chem. Soc. 73, 283e297.

McMeekin, T.A., Olley, J.N., Ross, T., Ratkowsky, D.A., 1993. Predictive Microbiology -Theory and Application. Research Studies Press Ltd., Somerset (UK).

C.M. Belda-Galbis et al. / Food Microbiology 38 (2014) 56e61 61

Muanda, F.N., Soulimani, R., Diop, B., Dicko, A., 2011. Study on chemical compositionand biological activities of essential oil and extracts from Stevia rebaudianaBertoni leaves. Lwt-Food Sci. Technol. 44, 1865e1872.

Nobmann, P., Smith, A., Dunne, J., Henehan, G., Bourke, P., 2009. The antimicrobialefficacy and structure activity relationship of novel carbohydrate fatty acidderivatives against Listeria spp. and food spoilage microorganisms. Int. J. FoodMicrobiol. 128, 440e445.

Pina-Pérez, M.C., Silva-Angulo, A.B., Rodrigo, D., Martínez-López, A., 2009. Syner-gistic effect of pulsed electric fields and CocoanOX 12% on the inactivation ki-netics of Bacillus cereus in a mixed beverage of liquid whole egg and skim milk.Int. J. Food Microbiol. 130, 196e204.

Pina-Pérez, M.C., Silva-Angulo, A.B., Rodrigo, D., Martínez López, A., 2012.A preliminary exposure assessment model for Bacillus cereus cells in a milkbased beverage: evaluating high pressure processing and antimicrobial in-terventions. Food Control 26, 610e613.

Radulovi�c, N., Mi�sic, M., Aleksi�c, J., Ðokovi�c, D., Pali�c, R., Stojanovi�c, G., 2007. Anti-microbial synergism and antagonism of salicylaldehyde in Filipendula vulgarisessential oil. Fitoterapia 78, 565e570.

Rasooli, I., 2007. Food preservation e a biopreservative approach. Food 1, 111e136.Rattanachaikunsopon, P., Phumkhachorn, P., 2010. Assessment of factors influ-

encing antimicrobial activity of carvacrol and cymene against Vibrio cholerae infood. J. Biosci. Bioeng. 110, 614e619.

Ross, T., McMeekin, T.A., 1991. Predictive microbiology. Applications of a square rootmodel. Food Aust. 43, 202e207.

Rowan, N.J., Anderson, J.G., 1998. Effects of above-optimum growth temperatureand cell morphology on thermotolerance of Listeria monocytogenes cells sus-pended in bovine milk. Appl. Environ. Microbiol. 64, 2065e2071.

Saucedo-Reyes, D., Marco-Celdrán, A., Pina-Pérez, M.C., Rodrigo, D., Martínez-López, A., 2009. Modeling survival of high hydrostatic pressure treated

stationary- and exponential-phase Listeria innocua cells. Innov. Food Sci. Emerg.Technol. 10, 135e141.

Scott, V.N., Swanson, K.M.J., Freier, T.A., Pruett Jr., W.P., Sveum, W.H., Hall, P.A.,Smoot, L.A., Brown, D.G., 2005. Guidelines for conducting Listeria mono-cytogenes challenge testing of foods. Food Prot. Trends 25, 818e825.

Seema, T., 2010. Stevia rebaudiana: a medicinal and nutraceutical plant and sweetgold for diabetic patients. Int. J. Pharm. Life Sci. 1, 451e457.

Sivaram, L., Mukundan, U., 2003. In vitro culture studies on Stevia rebaudiana.In Vitro Cell. Develop. Biol.-Plant 39, 520e523.

Soejarto, D.D., 2002. Botany of Stevia and Stevia rebaudiana. In: Kinghorn, A.D. (Ed.),Stevia: the Genus Stevia. Taylor & Francis, London (UK), pp. 18e39.

Tadhani, M.B., Subhash, R., 2006. In vitro antimicrobial activity of Stevia rebaudianaBertoni leaves. Trop. J. Pharm. Res. 5, 557e560.

Veldhuizen, E.J.A., Creutzberg, T.O., Burt, S.A., Haagsman, H.P., 2007. Low tempera-ture and binding to food components inhibit the antibacterial activity ofcarvacrol against Listeria monocytogenes in steak tartare. J. Food Prot. 70, 2127e2132.

Wesche, A.M., Gurtler, J.B., Marks, B.P., Ryser, E.T., 2009. Stress, sublethal injury,resuscitation, and virulence of bacterial foodborne pathogens. J. Food Prot. 72,1121e1138.

Whiting, R.C., 1995. Microbial modeling in foods. Crit.Rev. Food Sci. Nutr. 35, 467e494.

Wöelwer-Rieck, U., 2012. The leaves of Stevia rebaudiana (Bertoni), their constitu-ents and the analyses thereof: a review. J. Agric. Food Chem. 60, 886e895.

Yadav, S.K., Guleria, P., 2012. Steviol glycosides from Stevia: biosynthesis pathwayreview and their application in foods and medicine. Critic. Rev. Food Sci. Nutr.52, 988e998.

Zwietering, M.H., Jongenburger, I., Rombouts, F.M., van’t Riet, K., 1990. Modeling ofthe bacterial growth curve. Appl. Environ. Microbiol. 56, 1875e1881.