BIOACTIVITY OF GRAPEFRUIT SEED EXTRACT AGAINST PSEUDOMONAS SPP

BIOACTIVITY OF GRAPEFRUIT SEED EXTRACTAGAINST PSEUDOMONAS SPP

ANTONIO BEVILACQUA1,2,3, SONIA FICELO1, MARIA ROSARIA CORBO1,2

and MILENA SINIGAGLIA1,2

1Department of Food ScienceFaculty of Agricultural Science

Foggia UniversityVia Napoli 25, 71100, Foggia, Italy

2Istituto per la Ricerca e le Applicazioni Biotecnologiche per la Sicurezza e laValorizzazione dei Prodotti Tipici e di Qualità (BIOAGROMED)

Università di Foggia, Via Napoli 52-71100 Foggia, Italy

Accepted for Publication September 8, 2008

ABSTRACT

The effectiveness of grapefruit seed extract (GFSE) against three strains ofPseudomonas spp. (PSE 5 and PSE 8, two wild strains isolated from mozzarellacheese and identified as Pseudomonas fluorescens and Pseudomonas putida,respectively, and P. fluorescens DSMZ 50090) was studied. Biomass productionand viable cell count were evaluated. Data of biomass production were modeledthrough the modified Gompertz equation; the lag phase was assumed as the “nospoiling time” (NST), whereas the parameter A (maximum amount of biomassproduced) was used to quantify the inhibition exerted by the essential oil.Minimum inhibitory concentration (MIC) value was also evaluated.

The addition of 50–100 ppm of GFSE resulted in a prolongation of NST,whereas MIC values were 150 ppm for the wild strains and 200 ppm for P.fluorescens DSMZ 50090, respectively.

PRACTICAL APPLICATIONS

Pseudomonas spp. is a genus of great concern for the dairy industry,because of its spoiling ability both on milk and dairy products. The use ofthermal processing is the main technology to inhibit pseudomonads; however,it could not assure product quality when a post-contamination or the storage athigh temperatures occurs. Therefore, it is important to combine thermal pro-cessing with different means, able to preserve food quality.

3 Corresponding author. TEL: +39-0881-589232; FAX: +39-0881-589231; EMAIL: [email protected]

Journal of Food Processing and Preservation 34 (2010) 495–507.DOI: 10.1111/j.1745-4549.2008.00354.x 495© 2009 The Author(s)Journal compilation © 2009 Wiley Periodicals, Inc.

The use of natural compounds is considered as a promising approach andhas been proposed since the 1990s for different microorganisms (Nychas1995; Burt 2004); consumers, in fact, require friendly preservatives (i.e., nottoxic, natural and environmentally safe compounds).

In the light of this green consumerism, this paper could be considered asthe first step to propose the use of GFSE against Pseudomonadas spp. of dairyorigin.

INTRODUCTION

Psychrotrophic bacteria became an escalating problem for the dairyindustry, after the introduction of refrigerated storage of raw and processedmilk and dairy products (Smithwell and Kailasapathy 1995). The psychro-trophs from milk include both Gram-positive and Gram-negative bacteria(Munsch-Alatossava and Alatossava 2006), comprising Pseudomonas, Aero-monas, Enterobacter, Klebsiella and other coliforms, Bacillus, Clostridiumand Micrococcus (Shah 1994; Stepaniak 2000; Hayes and Boor 2001).

Among the cited genera, we could consider Pseudomonas (namelyPseudomonas fluorescens, Pseudomonas putida, Pseudomonas fragi,Pseudomonas putrefaciens and less frequently Pseudomonas aeruginosa) asthe most important one for its spoiling significance for milk and dairy prod-ucts, like mozzarella cheese, due to its proteolytic and lipolytic activity(Gilmour and Rowe 1990; Gunasekera et al. 2003; Michalac et al. 2003;Munsch-Alatossava and Alatossava 2006; Bevilacqua et al. 2007; Sinigagliaet al. 2008).

Significant contaminations by pseudomonads could occur due to theinadequately sanitized surfaces of milking, storage and transporting equipment(Munsch-Alatossava and Alatossava 2006); on the other hand, post-pasteurization contamination might happen at the filling operation (Mc Pheeand Gripphits 2002). In addition, even if cells are killed by heat treatment, theirenzymes are thermally stable and could contribute to the decay of dairyproducts (Frank 2001).

Another topic of great concern is the interest toward green consumerism,increased significantly in this decade (Nychas 1995; Burt 2004). Consumers,in fact, require more and more friendly compounds for prolonging the shelflife of foods, i.e., natural molecules, economical, not toxic and easily found onsale. One possibility, e.g., those reported in the papers of Burt (2004) andNarasimhan et al. (2008), is the use of essential oils.

This paper focused on the grapefruit seed extract (GFSE), known also ascitrus seed extract; it is an essential oil obtained from the seeds, the pulp andthe white membranes by grinding the grapefruit seed and juiceless pulp andmixing them with glycerin (van Woedtke et al. 1999).

496 A. BEVILACQUA ET AL.

GFSE has been shown to possess antibacterial, antiviral, antifungal andantiparasitic properties (Reagor et al. 2002; Xu et al. 2007); in addition, there isgood evidence that the extracts of grapefruit show low levels of antioxidants(Giamperi et al. 2004). Chemical analyses and gas chromatography massspectrometry (GC-MS), in fact, pointed out in the past that the main constituentsof seed extract and pulp are flavonoids (Tirillini 2000), ascorbic acid, toco-pherols, citric acid (Armando et al. 1997), limonoids and sterols (Braddock andBryan 2001).

Finally, some papers suggested that the consumption of bioactive com-pounds of GFSE could prevent the development of colon cancer (Vanamalaet al. 2006) and that the flavonoid naringin, found in GFSE, is an excellentplasma lipid lowering agent (Gorinstein et al. 2005).

GFSE was proposed to prevent the growth of different microorganisms;however, little is known on its impact on the Gram-negative spoiling micro-organisms of dairy origin. Therefore, this study was aimed to investigate theuse of GFSE for the inhibition of pseudomonads, as the first step to propose anew preservative for the inhibition of these foodborne microorganisms in realsystems.

MATERIALS AND METHODS

Strains

This study focused on three different strains of Pseudomonas: two wildstrains, named PSE 5 and PSE 8, isolated from mozzarella cheese, phenotyp-ically identified, respectively, as P. fluorescens and P. putida, through the API20 NE miniaturized system and belonging to the Culture Collection of AppliedMicrobiology Laboratory (Department of Food Science, Foggia University,Italy), and P. fluorescens DSMZ 50090, purchased from a Public Collection(Deutsche Sammlung von Mikroorganismem und Zellkulturen’s collection,DSMZ, Braunschweig, Germany).

The strains were maintained on Plate Count Agar slants (Oxoid, Milan,Italy) at 4C and monthly transferred.

Strain Revitalization and Cocktail Preparation

Before each assay, the strains were grown in Plate Count broth (PC broth)(glucose, 1 g/L; yeast extract, 2.5 g/L; tryptone, 5.0 g/L), and incubated at 25Cfor 72 h; then, the cultures (primary cultures) were serially diluted (1:10) in asterile saline solution (NaCl 0.9%) four times, to achieve a final concentrationof approximately 5 log cfu/mL (single suspensions).

497PSEUDOMONAS INHIBITION

The microbial cocktail was prepared by mixing 5 mL of the primarycultures of each strain, thus preparing a mix with the same volumes of thethree isolates (1:1:1); then, this suspension was serially diluted four times inthe saline solution, to achieve a population number of ca. 5 log cfu/mL(cocktail).

Antimicrobial Solutions

GFSE was purchased from Biocitro Probena s.l. (Zaragoza, Spain) as awater-soluble essential oil; stock solutions (0–100,000 ppm) were freshly pre-pared in water before each use and sterilized by filtering through membranes(0.2 mm, Millipore, Milan, Italy).

Evaluation of Antimicrobial Activity of GFSE

The bioactivity of GFSE against Pseudomonas spp. was evaluatedthrough a modified micro-dilution method, proposed by Falcone et al. (2005)and modified as follows. Sterile tubes, containing 20 mL of PC broth andadded with 200 mL of the different stock solutions of GFSE, in order to attainan amount of active compound ranging from 0 to 1,000 ppm (0, 50, 100, 150,200, 250, 300, 500, 700, 900 and 1,000 ppm), were inoculated separatelywith 200 mL of the single suspensions of the three strains or with 200 mL ofthe cocktail. The initial cell number in the samples was approximately 3 logcfu/mL.

Then, the samples were incubated at 25C; biomass production and cellnumber were evaluated after 0, 4, 8, 12, 16, 24, 30, 48, 72, 96 and 168 h ofincubation.

Biomass Production

The production of biomass was evaluated through absorbance measure-ment at 600 nm, by using a UV-Vis spectrophotometer (Beckman DU 600,Fullerton, CA).

Cell Number Evaluation

Cell number was evaluated through the traditional standard plate countmethod; the samples were serially diluted in sterile saline solution and plated(spread plate method) onto the surface of Pseudomonas Agar Base (Oxoid),added with Pseudomonas CFC selective supplement (Oxoid), incubated at 25Cfor 48–72 h. Data were expressed as log cfu/mL.


Statistical Analysis

The analyses were performed on three different samples for each GFSEamount and repeated twice (n = 6); data were submitted to one-way analysis ofvariance and Tukey’s test through the software Statistica for Windows (Stat-soft, Tulsa, OK) (P < 0.05).

Modelling the Data of Biomass Production: The No Spoiling Time

As absorbance values (Abs) followed the classical sigmoidal trend (lag,exponential and stationary phases), they were modeled through the Gompertzequation, as modified by Zwietering et al. (1990):

y k A t A= + × − ×( )× −( ) +[ ]{ }exp exp .maxμ λ2 7182 1

where y is the absorbance (dependent variable); k and A are, respectively, theinitial and the maximum level of biomass (Abs600nm); mmax is the increase ofbiomass per hour (Abs600nm/h); l, the lag phase (i.e., the time when the biomassdid not increase significantly), labeled in this paper as “no spoiling time”(NST) and t the time (h) (independent variable), respectively.

Inhibition Index

The parameter A was used to estimate the inhibitory effect, exerted byeach amount of GFSE on the production of biomass by Pseudomonas spp.,through the inhibition index (Chaibi et al. 1997; Bevilacqua et al. 2008),calculated as follows:

I IA A

Ac a

c

. . =−( )

×100

where Ac and Aa are the values of parameter A for the control and the samplecontaining the essential oil, respectively.

This parameter was used as a standardized measure of GFSE bioactivity,when pseudomonads attained the stationary phase.

Minimum Inhibitory Concentration Evaluation

For each strain, the minimum inhibitory concentration (MIC) was evalu-ated as the lowest amount of GFSE that resulted both in a constant cell numberfor at least 96 h and in NST > 168 h (running time).


RESULTS AND DISCUSSION

The use of essential oils for the inhibition of pathogens and spoilingmicroorganisms could be considered a promising way for the stabilization ofperishable products. In the present paper, this approach was used to control thegrowth of pseudomonads of dairy origin through the addition of GFSE to asynthetic medium.

Data of biomass production were modeled through the Gompertz equa-tion, as they showed a sigmoidal trend. The high regression coefficients(R2 > 0.995) (data not shown) underlined the adequacy and the high signifi-cance of the proposed approach.

Table 1 reports the lag phase values (parameter l of the Gompertz equa-tion) estimated for the three strains and the cocktail, as a function of theamount of GFSE in the medium. As reported in Materials and Methods, the lagphase could be regarded also as NST. It is well known, in fact, thatpseudomonads are responsible for off-flavor and off-odor in milk and milk-based foods, because of the production of extracellular enzymes (proteases andlypases) (Frank 2001). Sufficient enzyme to cause defects is usually presentwhen the population of pseudomonads and psychrotrophs attains the criticalvalue of 6–7 log cfu/mL (or 6 log cfu/g for solid foods). In a preliminary phase,we had observed that a significant increase of biomass (i.e., an increase of Absat 600 nm) occurred when the cell number attained a threshold of 6 log cfu/mL(data not published); therefore, the lag phase, obtained through the absorbancemeasurement, could be used as a tool to quantify the time to attain this criticalbreakpoint.

In the controls, pseudomonads experienced an NST value of 13.22–14.15 h. The addition of 50 ppm of GFSE resulted in an increase of this

TABLE 1.NO SPOILING TIME (h) EVALUATED FOR PSEUDOMONAS SPP. IN VITRO

(PC BROTH ADDED WITH GRAPEFRUIT SEED EXTRACT)

GFSE amount (ppm)

0 50 100 150 200

PSE 5 (Pseudomonas fluorescens) A13.22aB39.97b

C51.78cB>168*d

B>168*d

PSE 8 (Pseudomonas putida) A13.74aB41.97b

D119.82cB>168*d

B>168*d

P. fluorescens DSMZ 50090 A14.15aA26.40b

A30.48b,cA41.32d

B>168*e

Cocktail A13.75aA28.76b

B37.60cA49.50d

B>168*e

a,b Values in a line with different symbols are significantly different (P < 0.05) (one-way ANOVA andTukey’s test).A,B Values in a column with different symbols are significantly different (P < 0.05) (one-way ANOVAand Tukey’s test).* Lag phase > running time (168 h).


parameter; some differences, however, were pointed out among the threestrains. The increase, in fact, was more pronounced for the wild strains, whichexperienced a NST of approximately 40 h. On the other hand, the prolongationof this kinetic parameter appeared to be moderate-to-low for the collectionstrain (26.40 h).

In PC broth + 100 ppm of GFSE, P. fluorescens DSMZ 50090 showed anNST similar to the value observed for the lowest amount of the essential oil.On the contrary, the increase in the amount of the active compound resulted ina further increase in the NST of the two wild strains and the antimicrobialeffect was very strong for P. putida, which exhibited a value of this parameterof 119.82 h.

When the amount of GFSE in the broth was 150 ppm, the wild strains(PSE 5 and PSE 8) appeared to be completely inhibited (NST > 168 h). On theother hand, P. fluorescens DSMZ 50090 and the cocktail experienced values41.32 and 49.50 h, respectively. The statistical analyses revealed that thedifference between the single P. fluorescens culture and cocktail was notsignificant (P > 0.05).

Higher amounts of GFSE (>200 ppm) inhibited all the strains for theentire running time (NST > 168 h).

The inhibitory effect of GFSE against the three strains and the cocktailwas confirmed by the parameter A. The parameter A was modeled as inhibitionindex, thus highlighting the inhibitory effect exerted by the essential oil on thebiomass production by pseudomonads.

It is important to underline that the parameter A is the maximum valueof biomass, attained by the three strains in the stationary phase. The micro-organisms attained the stationary phase in different incubation times(depending on the strain and on the amount of GFSE); therefore, the inhi-bition index could not be related to a single time analysis, as reported else-where (Bevilacqua et al. 2008). In this paper, it pointed out the inhibitionexerted by the essential oil when the microbial targets attained somethingsimilar to a steady state (i.e., for a short time no changes were observed inthe system).

Inhibition index values for the different strains and for the cocktail arereported in Fig. 1. It is interesting to underline that at the lowest amounts of theessential oil (50–100 ppm) P. fluorescens DSMZ appeared to be more suscep-tible than the wild strains, as it experienced a higher inhibition (22.97 and46.55% at 50 and 100 ppm, respectively). Otherwise, the inhibition indexevaluated for the wild strains at 100 ppm was approximately 30–33%. Forhigher amounts of the essential oil, the results seemed quite different; at150 ppm, in fact, the strains PSE 5 and PSE 8 were completely inhibited (thevalue of the index, in fact, was ca. 100%), whereas the complete inhibition ofP. fluorescens DSMZ 50090 was observed only at 200 ppm.


Table 2 reports the data of the viable count. In the control samples, thepopulation attained a critical value of ca. 9 log cfu/mL after 72 h of incubation.On the other hand, the addition of GFSE to the medium resulted in a signifi-cantly lower cell level, depending both on the contact time cells-essential oiland on the amount of the antimicrobial in the broth.

Cell load data and NST values were used to point out MIC, defined as theminimum amount of GFSE able to inhibit the increase of either the biomassand cell number for the entire running time.

MIC values were 150 ppm for the wild strains (PSE 5 and PSE 8) and200 ppm for P. fluorescens DSMZ 50090. On the basis of the NST and MICvalues, we could suggest a susceptibility hierarchy of the studied strains asfollows:

P putida P fluorescens. . wild strain PSE 8 wild strain P( ) > SSE 5

DSMZ 50090

( ) > P

fluorescens

.

The use of essential oils for pseudomonads inhibition is extensively reported inthe literature. The data, however, deal with P. aeruginosa inhibition throughgrape extracts (Vitis vinifera L.) (Baydar et al. 2004), oils produced fromMetasequoia glyptostroides (Bajpai et al. 2007), Achillea extract (Stojanovicet al. 2005), coconut extract (Chakraborty and Mitra 2008), thymol and car-

0

20

40

60

80

100

120

50 ppm 100 ppm 150 ppm 200 ppm

Inh

ibit

ion

Ind

ex (

%)

P. fluorescens (wild strain, PSE 5)

P. putida (wild strain PSE 8)

P. fluorescens DSMZ 50090

cocktail

FIG. 1. EFFECTIVENESS OF GFSE AGAINST PSEUDOMONAS SPP.: INHIBITION EXERTEDBY THE ESSENTIAL OIL ON THE PRODUCTION OF BIOMASS. BARS REPRESENT

THE STANDARD ERROR


vacrol (Lambert et al. 2001). Unfortunately, none of the cited papers andothers that could be easily found in the literature focused on the inhibition ofPseudomonadaceae of dairy origin.

Xu et al. (2007) used GFSE, alone or in combination with nisin, citricacid, sodium lactate and potassium sorbate, to prolong the shelf life of mini-mally processed cucumber and lettuce, showing that GFSE could be able tocontrol the growth kinetic of Gram-negative aerobes, mainly represented byPseuodmonas, Erwinia and Serratia (King et al. 1991), and suggesting that itmight be a promising approach for pseudomonads inhibition.

The results of this work confirmed this idea, and supported the hypothesisof Reagor et al. (2002) and Xu et al. (2007), who suggested that GFSEappeared to have a greater inhibitory effect on Gram-positive organisms thanon Gram-negative ones. In a preliminary phase, in fact, we found that anamount of GFSE of approximately 40–50 ppm was enough to inhibit thegrowth of Lactobacillus plantarum, L. brevis and Bacillus coagulans (data notyet published).

Regarding the mode of action of GFSE, it could be attributed to thephenolic compounds, such as catechins, epicatechin, epocatechin-3-O-gallate,

TABLE 2.VIABLE CELL COUNT (LOG cfu/mL) OF PSEUDOMONAS SPP. FOR SOME SELECTED

COMBINATIONS OF EXPERIMENTAL DESIGN

Time (h) Control GFSE 50 ppm GFSE 100 ppm GFSE 150 ppm

P. fluorescens (wild strain, PSE 5)48 8.93a 7.23b –* –72 9.12a 8.74b – –96 9.20a 8.87b 6.2c 3.7d

Pseudomonas putida (wild strain PSE 8)48 8.90a 7.32b – –72 9.10a 8.27b – –96 9.21a 8.93b 3.5c 3.6c

P. fluorescens DSMZ 5009048 8.87a 8.11b 7.34c –*72 9.21a 8.41b 7.56c –96 9.16a 8.46a,b 8.18b 7.42d

Cocktail48 8.92a 7.85b 6.96c –*72 9.15a 8.43b 6.78c –96 9.21a 8.18b 8.06b 7.40d

Values in a line with different letters are significantly different (P < 0.05) (one-way ANOVA andTukey’s test). Data are the mean (n = 6).* Not evaluated.GFSE, grapefruit seed extract.


dimeric, trimeric and tetrameric procyanidins (Saito et al. 1998) or todifferent molecules (triclosan and methyl-p-hydroxy benzoate) (Sakamotoet al. 1996).

In conclusion, our results showed that an amount of GFSE of approxi-mately 150–200 ppm could be used successfully to control pseudomonadsgrowth and prolong the NST up to 168 h (7 days). Moreover, the antimicrobialeffect was bacteriostatic rather than bactericidal, and seemed species- andstrain-dependent. P. putida, in fact, was more sensible than P. fluorescens anda different susceptibility was recovered for the two strains of P. fluorescensused in this paper. Further investigations are proposed to validate these data infoods (i.e., milk or liquid dairy products), using GFSE alone or combined withdifferent hurdles.

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BIOACTIVITY OF GRAPEFRUIT SEED EXTRACT AGAINST PSEUDOMONAS SPP

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