ORIGINAL PAPER

Inhibition of Pichia membranifaciens by Homogenisationand Antimicrobials

Antonio Bevilacqua & Maria Rosaria Corbo &

Milena Sinigaglia

Received: 23 February 2010 /Accepted: 4 October 2010 /Published online: 16 October 2010# Springer Science+Business Media, LLC 2010

Abstract This paper reports on the investigation of the useof citrus extract (0–3 ppm), sodium benzoate (0–300 ppm)and pressure homogenisation (PH) (0–90 MPa) againstPichia membranifaciens. The centroid approach was usedto combine the three variables and point out their individualand interactive effects. The assays were performed in alaboratory medium (YPD broth), and the samples werestored at 25 °C for 6 days; the viable count of P.membranifaciens was evaluated, and the data were mod-elled through the Gompertz equation, to assess thereduction of the initial contamination just after thehomogenisation (RN) and the prolongation of the lag phase(I1). The results of this paper suggested that citrus extractcould be a suitable alternative for the inhibition of P.membranifaciens in acidic drinks, as a low amount of thiscompound (3 ppm) increased the lag phase by 60–70%. Inaddition, homogenisation (90 MPa) was able to reducesignificantly the initial cell number.

Keywords Pichia membranifaciens . Homogenisation .

Citrus extract . Benzoate . Centroid approach

Introduction

Spoiling microflora of juices is composed mainly by yeasts,moulds and acid-tolerant bacteria (Elez-Martinez et al.2005; Tournas et al. 2006). Pichia, Candida, Saccharomy-ces and Rhodotorula are the yeasts generally isolated inspoiled juices (Las Heras-Vazquez et al. 2003; Tournas etal. 2006); in particular, Saccharomyces and Pichia produceethanol from sugars or form biofilm (Rates et al. 2001).

Pichia membranifaciens is widespread and is consideredas the target microorganism for the optimisation of thermaltreatments of juices. It is able to grow at low temperaturesand to spoil chilled foods; the maximum temperature for itsgrowth is 32–37 °C (Boekhout and Robert 2003). More-over, P. membranifaciens is resistant to heat and tomoderate amounts of salt, SO2, sorbic, benzoic and aceticacids (Boekhout and Robert 2003). Concerning the effect ofbenzoic acid, at 1 mM, this compound decreased thegrowth rate of yeast, while it determined the completeinhibition at 3 mM (Veiga and Madeira-Lopes 2000).

Traditional juice pasteurisation is usually designed toinactivate pectinmethylesterase and reduce the levels ofspoiling microorganisms (Char et al. 2010b); however, it iswell-known that thermal treatments affect perceived quality(Berry and Veldhuis 1977; Attaway et al. 1988). Non-pasteurised juices have some desirable characteristics in termsof vitamin C concentration, color and overall quality, but theyhave a short shelf life due to microbial and enzymatic spoilage(Winniczuk and Parish 1997). Some promising approacheshave been proposed (like high hydrostatic and homogenisa-tion pressure, pulsed electric fields and ultrasound, theaddition of essential oils and other natural compounds).

Pressure homogenisation (PH) has been proposed andintroduced in the pharmaceutical, cosmetic, chemical and foodindustry for the preparation or stabilisation of emulsions and

A. Bevilacqua :M. R. Corbo :M. SinigagliaDepartment of Food Science, Faculty of Agricultural Science,University of Foggia,Via Napoli 25,71122 Foggia, Italy

A. Bevilacqua (*) :M. R. Corbo :M. SinigagliaFood Quality and Health Research Center (BIOAGROMED),University of Foggia,Via Napoli 25,71122 Foggia, Italye-mail: a.bevilacqua@unifg.it

A. Bevilacquae-mail: abevi@libero.it

Food Bioprocess Technol (2012) 5:1061–1067DOI 10.1007/s11947-010-0450-1

suspensions or to create physical changes in products (Floury etal. 2002). Moreover, homogenisation has been used as a non-thermal approach for the inactivation of spoiling micro-organisms (Bevilacqua et al. 2007, 2009; Corbo et al. 2009;Diels and Michiels 2006; Pereda et al. 2007).

In particular, Bevilacqua et al. (2009) used homogenisa-tion to inactivate some spoiling microorganisms of fruitjuices, like Bacillus coagulans, Lactobacillus plantarum,Lactobacillus brevis, P. membranifaciens, Rhodotorulabacarum and Saccharomyces bayanus. The results pointedout that P. membranifaciens was the most resistant yeast,with the complete inactivation occurring only at 130 MPa(Bevilacqua et al. 2009).

Another interesting approach for the stabilisation ofjuices is the use of natural antimicrobials, in particularessential oils (EOs). EOs are aromatic oil liquids obtainedmostly from plant material. They exhibit antiviral, antibac-terial, antimycotic, antitoxigenic, antiparasitic and insecti-cidal properties, and their use is allowed in European Union(EU) and USA (21 CFR 182.60) in food (as flavouring,either extracted from plant material or synthetically manu-factured), perfumes and pharmaceuticals (Burt 2004; USFood and Drug Administration 2010).

One of the most promising EOs is the citrus extract,proposed by Fisher and Phillips (2008) as “the possibleanswer for the use of EOs in foods”. Citrus extract is usedas a flavouring agent for cosmetics, soap and many otherproducts (Cadwallader et al. 1992; Filho et al. 2003; Trytekand Fiedurek 2005) and was proposed as a suitablepreservatives in foods (e.g. fish, meat, chicken, dairyproducts, fruit and vegetables and confectionary goods) asan ingredient or as a volatile compound in the head space ofthe package (Fisher and Phillips 2008; Char et al. 2010a).The bioactivity of citrus extract against the spoiling micro-organisms of juices was studied by Bevilacqua et al.(2010); thus, they found that a concentration of 3 ppmcontrolled the growth of P. membranifaciens.

Therefore, this paper was aimed to investigate thecombined effect of PH, citrus extract and a traditionalchemical (sodium benzoate) on the growth of P. membra-nifaciens. The assays were performed in a model system,and the methodology of centroid was used, to study thepossibility of reducing the amount of traditional chemicals(e.g. sodium benzoate and the other salts of benzoic acids)in juices.

Materials and Methods

Microorganism

A strain of P. membranifaciens (DSMZ 70169), pur-chased from a public collection (DSMZ, Deutsche

Sammlung von Mikroorganismen und Zellkulturen,Braunschweig, Germany), was used throughout this study.The microorganism was maintained on yeast potatodextrose agar (YPD) slants (bacteriological peptone,20 g/l; yeast extract, 10 g/l; glucose, 20 g/l; agar, 12 g/l)at 4 °C.

Before each experiment P. membranifaciens wasgrown in YPD broth, adjusted to pH 4.0 through 2.0 NHCl, and incubated at 25 °C for 72 h. Then, the brothculture was centrifuged at 1,000×g for 10 min, washedwith saline solution (0.9% NaCl) and adjusted to about8 logcfu/ml. Yeast cell number was assessed through platecount on YPD.

Antimicrobials

The antimicrobial compounds used throughout this researchwere citrus extract (BiocitroLIQUID®; Quinabra, Probena,Spain) and sodium-benzoate (JT Baker; Milan, Italy).

The supplier reported the following composition forcitrus extract: ascorbic acid and ascorbates (vitamin C),linked with citrus bioflavonoids, 4–7.20%; hydratedglycerin linked with other traces of citrus polyphenols,carbohydrates, bio-flavoproteins, pectin, citrus sugars,citric acid, 30.80–36.60%; water, 6.00–11.00%; andstabiliser and inert carrier, 50%. Chemical analysesrevealed that the concentration of ascorbic acid andcitrus bioflavonoid was about 56,000 ppm; among thebioflavonoids, naringin was present at a minimum of6,500 ppm. Finally, the limonene content was 30,000–50,000 ppm.

Both citrus extract (0.5–3.0 ppm) and sodium benzoate(50–300 ppm) were added directly to the laboratorymedium, due to their high water solubility.

Design of Experiments

The individual and interactive effects of homogenisation,sodium benzoate and citrus extract were assessed throughthe centroid approach. A centroid is a mixture design, i.e. isparticular kind of design of experiment in which the factorsare ingredients or components of a mixture. Mixtures aredifferent from other types of experimental design becausethe proportions of the constituents must add up to 100%.Increasing the level of one constituent necessarily reducesthe level of the others.

Each variable of the design can assume a standardisedvalue ranging between 0 and 1; the standardised valuesused in a mixture design are usually 0, 0.17, 0.33, 0.50,0.67 and 1 (Table 1). The centroid could be representedgraphically as an equilateral triangle, where the threevariables represent the vertices and the black points thecombinations of the design (Fig. 1).

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Sample Preparation

Aliquots of YPD broth, acidified to pH 4.0, were addedwith sodium benzoate (50–300 ppm) and citrus extract(0.5–3.0 ppm), as reported in Table 2, and homogenised at15–90 MPa through a homogeniser Panda 2K (PANDA 2Khomogeniser, Niro Soavi s.p.a., Parma, Italy) for 2 ms. Thehomogeniser was equipped with a ball valve, suggested forthe disruption of cells (Diels and Michiels 2006). Beforeeach experiment, the circuits of the equipment were cleanedwith NaOH (3%) and HNO3 (0.5%), as suggested by theproducer; the use of chlorine is not advisable, as it couldcorrode plastic sheathings. Then, circuits were washedwith sterile and distilled water (70 °C) and cooled withsterile distilled water (20 °C). The exit temperature of thesamples was about 35–40 °C at 90 MPa; just after thehomogenisation, samples were distributed into 100-mlsterile tubes and immediately cooled to 20 °C in a waterbath.

An aliquot of acidified YPD broth, neither homogenisednor containing the antimicrobials, but inoculated with P.membranifaciens, was used as control (Table 2).

The samples were stored at 25 °C for 6 days; the viablecount of P. membranifaciens was evaluated everyday onYPD plates, incubated at 25 °C for 4 days.

Statistical Analyses and Data Modeling: Primary Models

The analyses were performed in triplicate. Data weremodelled through the Gompertz equation, as modified byZwietering et al. (1990) by the software Statistica forWindows (Statsoft, Inc., Tulsa OK, USA):

y ¼ k þ A� exp � exp mmax � 2:7182ð Þ � l� tð Þ A= þ 1½ �f gð1Þ

where y is the viable count (dependent variable) (logcfu/ml); kand A are respectively the initial cell count and its maximumincrease attained in the stationary phase (logcfu/ml); μmax isthe maximal growth rate [log(cfu/ml)/day]; 1 is the lag phase(day) and t the time (day).

The differences amongst the kinetic parameters in thedifferent combinations of the design were revealedthrough the one-way analysis of variance and Tukey’stest (p<0.05).

The parameters k (initial cell count) and 1 (lag phase)were used for the evaluation of two standardised indices,labelled respectively as RN (reduction of cell concentrationafter homogenisation) and I1 (increase of the lag phasewithin the storage) and evaluated as follows:

RN ¼ NC � NS

NCð2Þ

Table 1 Coded values of the independent values of centroid

Coded values Citrus extract (ppm) Benzoate (ppm) PH (MPa)

0 0 0 0

0.17 0.5 50 15

0.33 1.0 100 30

0.5 1.5 150 45

0.67 2.0 200 60

1 3.0 300 90

Fig. 1 Graphical representation of a centroid through a ternary plot.Each corner represents a combination where a variable is set to 100%and the other to 0%

Table 2 Combinations of the design

Combinations Coded values Real values

Citrusextract

Benzoate PH Citrus extract(ppm)

Benzoate(ppm)

PH(MPa)

1 1 0 0 3 0 0

2 0 1 0 0 300 0

3 0 0 1 0 0 90

4 0 0.5 0.5 0 150 45

5 0.5 0 0.5 1.5 0 45

6 0.5 0.5 0 1.5 150 0

7 (centroid) 0.33 0.33 0.33 1 100 30

8 0.67 0.17 0.17 2 50 15

9 0.17 0.67 0.17 0.5 200 15

10 0.17 0.17 0.67 0.5 50 60

Control 0 0 0 0 0 0

Food Bioprocess Technol (2012) 5:1061–1067 1063

I l ¼ lS � lClS

ð3Þ

where N and 1 are the cell counts and the lag phase and thesubscripts C and S are referred to the control and the 10combinations of the design.

Secondary Model: Building the Polynomial Equation

The standardised indices RN and I1 were used as the inputvalues to build a polynomial equation, through the softwareStatistica for Windows:

y ¼X3

i¼1

bixi þX3

i�

X3

j

bijxixj þX3

i�

X3

j�

X3

k

bijkxixjxk þ "

ð4Þwhere βi, βij and βijk are the coefficients of the individual(xi) and interactive effects (xixj−xixjxk) of the independentvariables (homogenisation, citrus extract and sodiumbenzoate); ε is the standard error of the model.

Results and Discussion

Table 3 shows the viable count of P. membranifaciens justafter the homogenisation (parameter k of Gompertz equa-tion) and the lag phase in the 10 combinations of thedesign, along with the data of the control. Focusing on thecombinations 1, 2 and 3 (i.e. the samples were a variablewas set to 1 and the other two to 0), homogenisation at90 MPa (combination 3) reduced the initial cell count by1 logcfu/ml; otherwise, the antimicrobial prolonged the lag

phase of the population from 0.40 days to 1.93 and1.02 days (for the citrus extract and benzoate, respectively)(combinations 1 and 2).

It is interesting to underline that the reduction due to PHtreatment is lower than that reported for the same strainelsewhere; in fact, Bevilacqua et al. (2009) found that insaline solution, P. membranifaciens DSMZ 70169 wasreduced by 2.72 logcfu/ml at 90 MPa. This difference couldbe due to at least two different reasons:

1. The different medium: it is well-known, in fact, that themedium could affect in a strong way the effectivenessof homogenisation; in particular, fluid reology plays afundamental role, as reported by many authors (Corboet al. 2010; Kleinig et al. 1995; Miller et al. 2002).

2. The physiological state of cells: Harrison et al. (1991)reported that actively growing cells of Alcaligeneseutrophus could be disrupted at 60 MPa by a singlepass, whereas two or three passes were required toinactivate cells under stationary phase. Bevilacqua et al.(2009) used cells of P. membranifaciens at the beginningof the stationary phase; otherwise, in this research, theexperiments were performed on cells adapted to acidicconditions and in the late stationary phase. Probably, thecultivation under a slight stress could induce a multi-stress response; however, this hypothesis needs to beconfirmed on a larger number of samples.

In the other samples of the design (runs 4–10), theprolongation of the lag phase, as well as the viable countjust after PH treatment, relied upon the combination of thethree variables; for example, the maximum value of the lagphase (1.86 days) was recovered in combination 8,containing 2 ppm of citrus extract and 50 ppm of benzoateand homogenised at 15 MPa.

Neither the maximal growth rate (μmax) nor the increaseof population attained in the stationary phase (A) wasinfluenced by homogenisation, citrus extract or benzoate(data not shown).

Due to the significance of the effects of the threevariables on the parameters k and 1, the reduction in theviable count just after homogenisation (RN) and theprolongation of the lag phase (I1) were used as input datato build a polynomial equation.

The extent of the reduction of the viable count just afterthe homogenisation (RN) could be expressed through thefollowing equation:

RN ¼ 4:11 � citrus½ � þ 6:18 � benz½ � þ 84:00 � PH½ �þþ28:47 � citrus½ � � benz½ � � 54:37 � citrus½ � � PH½ � � 18:78 � benz½ � � PH½ �

ð5ÞIn particular, the parameter RN was affected both by the

individual effects of citrus extract, benzoate and homoge-

Table 3 Viable count after the homogenisation (parameter k of theGompertz equation) and lag phase of P. membranifaciens throughoutthe storage

Combination k (log cfu/ml) 1 (day)

1 6.18±0.08a 1.93±0.15a

2 6.06±0.07a 1.02±0.26b

3 5.03±0.11c 0.14±0.01e

4 5.84±0.12a,b 0.76±0.05c

5 5.87±0.09a,b 0.81±0.04c

6 6.04±0.08a 0.55±0.03d

7 5.91±0.09a,b 1.15±0.13b

8 6.08±0.09a 1.86±0.07a

9 5.81±0.07a,b 0.76±0.08c

10 5.69±0.05b 0.56±0.05d

Control 6.06±0.04a 0.19±0.02e

The values are accompanied by their standard error. Superscriptedletters indicate the statistical differences amongst the differentcombinations of the design (one-way ANOVA and Tukey’s test, p<0.05)

1064 Food Bioprocess Technol (2012) 5:1061–1067

nisation and by their interactions (citrus/benzoate, citrus/homogenisation and benzoate/homogenization). However,the different terms of the equations showed differentweights, as reported in the Pareto chart of standardisedeffects (Fig. 2a). Bars represent the standardised effect ofeach term of the equation. The vertical line is thesignificance breakdown (p<0.05). Pareto chart highlightedthat the parameter RN was affected only by PH, asindividual term.

The influence of the three independent variables on theparameter RN can be evidenced better through the ternaryplot (Fig. 2b). This figure shows at the vertices the threevariables (citrus extract, benzoate and PH); the black pointsrepresent the 10 combinations of the centroid.

The ternary plot highlighted the importance on thehomogenisation pressure on RN, which increased to 80%at the maximum value of pressure (90 MPa).

A polynomial equation was obtained also for theincrease of the lag phase (I1); the equation reads as follows:

I l ¼ 63:54 � citrus½ � þ 22:10 � benz½ � þ 58:97 � HPH½ � ð6ÞIn this case, the secondary model contained only the

individual terms of the independent variables; moreover,Pareto chart of the standardised effects (Fig. 3a) revealedthat the effect of benzoate was not significant. The mostsignificant terms was the citrus extract, followed by thehomogenisation. Finally, the ternary plot (Fig. 3b) high-lighted that the lag phase could be increased by 60–70%with 3 ppm of citrus extract added and homogenising thesamples at 90 MPa.

The secondary models (Eqs. 5 and 6) showed someinteresting results and suggested that PH could be used asan initial hurdle to reduce the initial contamination by P.membranifaciens; otherwise, citrus extract acted as an

Fig. 2 Effects of PH, citrus extract and sodium benzoate on theparameter RN (reduction of the viable cell count just after thehomogenisation; percentage referred to the control). a Pareto chart;b ternary plot (the values on the axis represent the coded values ofvariables)

Fig. 3 Effects of PH, citrus extract and sodium benzoate on theparameter I1 (increase in the lag phase; percentage referred to thecontrol). a Pareto chart; b ternary plot (the values on the axis representthe coded values of variables)

Food Bioprocess Technol (2012) 5:1061–1067 1065

hurdle throughout the storage. The effect of the benzoatewas not significant; in fact, the highest concentration(300 ppm) did not affect the growth of the yeast. The USFDA approve the use of sodium benzoate at concentrationlower than 0.1%; otherwise, the amounts allowed in EU arebetween 0.15% and 0.5% (US Food and Drug Administra-tion 2010; European Union 2010). Therefore, an increase inbenzoate concentration would be possible, as in this paper,a low amount of the compound was used (300 ppm, i.e.0.03%), if compared to the critical threshold. However, anincrease in benzoate is not advisable, as recent surveysreport cases of intoxication by benzene due to juices andother acidic drinks, and it has been suggested that benzenecould be formed by benzoic acid and its salts (ConsumersUnion 2010).

Concerning the effects of the other two hurdles used inthis research, a preliminary investigation by Bevilacqua etal. (2010) revealed that low amounts of citrus extract, likethose proposed in this paper, did not produce any negativeconsequence both on the nutritional value and organolepticcharacteristics of juices, thus suggesting that it could beused as a friendly antimicrobial compound to prolong juiceshelf life. A similar effect (i.e. prolongation of the shelflife), different from the simple reduction of the initial cellcount, was observed, as homogenisation acted also on theincrease of the lag phase. This effect was clearly indicatedby Bevilacqua et al. (2007) for the vegetative forms of astrain of Alicyclobacillus acidoterrestris, due probably to akind of reversible stress. Diels et al. (2005) reported thatafter homogenisation, a cell could be dead or alive and theonly evidence of a possible stress was a transientpermeabilisation of the membrane of bacteria. No data areavailable for yeasts; however, a similar mechanism could besuggested for P. membranifaciens.

Another interesting result was the lack of significantinteractive effects of the independent variables on thereduction of the initial contamination as well as on theprolongation of the lag phase.

These results confirmed the gamma hypothesis, sug-gested by Zwietering et al. (1992) and confirmed byLambert and Bidlas (2007a, b). These authors stated thatdifferent hurdles combine together independently, thus nosynergistic effect could be recovered and that each hurdlecan act primarily on its target at cell level (Corbo et al.2009).

The gamma hypothesis was confirmed by Lambert andBidlas (2007a, b) against Aeromonas hydrophila andEnterobacter sakazakii, inoculated in a laboratory medium,added with NaCl and some antimicrobials and adjusted todifferent pHs. Gamma hypothesis is in contrast with thetheory of the hurdle approach proposed by Leistner (1978),who stated that hurdles/variables of a system could act in aninteractive way and strengthen each other.

A possible synthesis between the theory of Leistner andthe gamma hypothesis was proposed by McMeekin et al.(2000), who stated that the different variables/hurdles of asystem act mainly as independent elements; however, thereare some situations (e.g. in the growth/no growth interface)when it is possible to see a synergistic action amongst them.

In the conditions of the present paper, P. membrani-faciens was far from the interface growth/no growth;therefore, homogenisation and citrus extract acted inde-pendently.

Conclusions

The results of this paper suggested that the combination ofPH with some antimicrobials could be used successfully tocontrol the growth of P. membranifaciens in acidic drinks.In particular, it is possible to highlight two key features:

1. Although sodium benzoate has been considered forlong time as the preservative to inhibit the growth ofspoiling microorganisms in foods, the results of thepresent paper suggest that it cannot be used to prolongthe lag phase and/or control the growth of P. mem-branifaciens. Citrus extract could be a suitable alterna-tive to benzoate; low concentration of this essential oil,in fact, prolonged significantly the lag phase of P.membranifaciens. Moreover, citrus extract, if used atlow concentrations (like in this paper), did not affectthe organoleptic characteristics of juices.

2. The combination of PH/citrus extract could be pro-posed as a suitable alternative for the stabilisation ofjuices and acidic drinks: homogenisation would reducethe initial contamination by spoiling microorganisms,while citrus extract could improve product stabilityduring the storage.

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