Vanillin inhibits pathogenic and spoilage ...

Food Research International 39 (2006) 575–580 www.elsevier.com/locate/foodres

Vanillin inhibits pathogenic and spoilage microorganisms in vitro and aerobic microbial growth in fresh-cut apples 夽 H.P. Vasantha Rupasinghe a

a,¤

, Jeanine Boulter-Bitzer b, Taehyun Ahn c, Joseph A. Odumeru

b

Department of Environmental Sciences, Nova Scotia Agricultural College, P.O. Box 550, 50 Pictou Road, Truro, NS, Canada B2N 5E3 b Food Microbiology, Laboratory Services Division, University of Guelph, Guelph, Ont., Canada N1H 1J7 c Guelph Center for Functional Foods, Laboratory Services Division, University of Guelph, Guelph, Ont., Canada N1H 1J7 Received 14 September 2005; accepted 22 November 2005

Abstract The antimicrobial eVect of vanillin against four pathogenic or indicator organisms; Escherichia coli, Pseudomonas aeruginosa, Enterobacter aerogenes, and Salmonella enterica subsp. enterica serovar Newport and four spoilage organisms; Candida albicans, Lactobacillus casei, Penicillum expansum, and Saccharomyces cerevisiae that could be associated with contaminated fresh-cut produce, was examined. The minimal inhibitory concentration (MIC) of vanillin was dependent upon the microorganism and this ranged between 6 and 18 mM. When incorporated with a commercial anti-browning dipping solution (calcium ascorbate, NatureSeal™), 12 mM vanillin inhibited the total aerobic microbial growth by 37% and 66% in fresh-cut ‘Empire’ and ‘Crispin’ apples, respectively, during storage at 4 °C for 19 days. Vanillin (12 mM) did not inXuence the control of enzymatic browning and softening by NatureSeal. These results provide a new insight for vanillin as a potential antimicrobial agent for refrigerated fresh-cut fruits and vegetables. © 2005 Elsevier Ltd. All rights reserved. Keywords: Vanillin; Natural antimicrobial; MIC; Fresh-cut apple; Shelf life; Pathogenic and spoilage microorganisms

1. Introduction There has been a steady increase in consumer demand for convenient and nutritious minimally processed produce like fresh-cut apples (Gorny, 2003). Current fresh-cut processing technologies such as post-cut dipping in calcium ascorbate (NatureSeal™) to prevent enzymatic browning and softening allows a shelf life of up to 21 days for sliced apples (Chen, 1999; Rupasinghe, Murr, DeEll, & Odumeru, 2005). However, the fresh-cut produce industry is challenged with potential outbreaks of illness that could be associated with microbial growth during the extended shelf life of these prod-

夽 Mention of trade or Wrm names does not constitute an endorsement by the Nova Scotia Agricultural College and University of Guelph over others of a similar nature not mentioned. * Corresponding author. Tel.: +1 902 893 6623; fax: +1 902 893 1404. E-mail address: [email protected] (H.P.V. Rupasinghe).

0963-9969/$ - see front matter © 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2005.11.005

ucts (Alzamora & Guerrero, 2003). These current trends in the fresh-cut apple industry have led to a growing interest in investigating natural antimicrobial agents that are compatible with the chemical properties of post-cut dipping solutions of fresh-cut apples (Alzamora & Guerrero, 2003). Naturally occurring antimicrobials include compounds derived from biological materials (Brul & Coote, 1999). There are three classes of naturally occurring antimicrobial substances: (1) animal-derived enzymes (lysozyme, lactoperoxidase), other proteins (lactoferrin, lactoferricin, and ovotransferrin), and small peptides (histatins and magainins); (2) plant-derived secondary metabolites (phytoalexins, phenolics, and essential oils); and (3) microorganism-based bacteriocins (nisin and pediocin) (Beuchat & Golden, 1989; Brul & Coote, 1999; Gould, 1996). Recently, many plant extracts have been shown to possess antimicrobial activities against a wide range of microorganisms related to food spoilage and safety (Friedman, Henika, & Mandrell, 2002; Patrzykat & Douglas, 2003). Little is

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H.P.V. Rupasinghe et al. / Food Research International 39 (2006) 575–580

known about the resistance mechanisms of microorganisms against these naturally occurring antimicrobial compounds, although many, such as vanillin, benzaldehyde, ferulic acid, estragole, guaiacol and eugenol, are hydrophobic and contain aromatic structures similar to the ones found in classical preservatives such as benzoic acid (Fig. 1) (Brul & Coote, 1999; Cerrutti & Alzamora, 1996). Vanillin (4-hydroxy-3-methoxybenzaldehyde) is the predominant phytochemical that occurs in vanilla beans and is a generally regarded as safe (GRAS) Xavoring compound used widely in ice cream, beverages, biscuits, chocolate, confectionary, desserts, etc. (Beuchat & Golden, 1989; Hocking, 1997; Ramachandra Roa & Ravishankar, 2000). Vanillin, present in the essential oil fraction of the vanilla bean, is structurally similar to eugenol (2-methoxy-4-(2-propenyl)phenol) from cloves and is known to be antimycotic (Beuchat & Golden, 1989) and bacteriostatic (Fitzgerald et al., 2004a). Inhibitory action of vanillin at MIC was found to be bacteriostatic in contrast to the more potent phenolic antimicrobials such as carvacrol and thymol (Fitzgerald et al., 2004a) that are bactericidal (Friedman et al., 2002; Ultee, Bennik, & Moezelaar, 2002). Based on the studies conducted using Escherichia coli, Lactobacillus plantarum, and Listeria innocua, the inhibitory activity of vanillin resides primarily in its ability to detrimentally aVect the integrity of the cytoplasmic membrane, with the resultant loss of ion gradient, pH homeostasis and inhibition of respiratory activity (Fitzgerald et al., 2004a). Recently, it has been shown extensively that vanillin is eVective in inhibiting yeast and moulds in vitro (Cerrutti & Alzamora, 1996; Fitzgerald, Stratford, & Narbad, 2003; López-Malo, Alzamora, & Argaiz, 1995, 1997, 1998; Matamoros-León, Argaiz, & López-Malo, 1999) and in fruit puree or juice (Castañón, Argaiz, & López-Malo, 1999; Cerrutti & Alzamora, 1996; Cerrutti, Alzamora, & Vindales, 1997; Fitzgerald, Stratford, Gasson, & Narbad, 2004b). Vanillin (12 mM) inhibited the growth of four food spoilage yeasts, Saccharomyces cerevisiae, Zygosaccharomyces rouxii, Debaryomyces hansenni, and Zygosaccharomyces bailii, in culture media and apple puree for 40 days storage at 27 °C (Cerrutti & Alzamora, 1996). Incorporation of vanillin (3–7 mM) into fruit-based agars inhibited the growth of four Aspergillus species for 2 months (LópezMalo et al., 1995). When combined with 2 mM potassium sorbate, 3 mM vanillin could inhibit the growth of three

CHO

CH 2 CH=CH 2

COOH

Penicillium species: Penicillium digitatum, Penicillium glabrum, and Penicillium italicum, grown in potato dextrose agar (pH 3.5, aw 0.98) for 1 month (Matamoros-León et al., 1999). Fitzgerald et al. (2004b) reported that two yeast strains, S. cerevisiae and Candida parapsilosis, inoculated at a level of »104 cfu/ml in apple juice and peach-Xavored soft drink were inhibited by vanillin at 20 and 10 mM concentrations, respectively, over an 8-week storage period at 25 °C. Therefore, an investigation of antimicrobial properties of vanillin when incorporated in calcium ascorbate (NatureSeal™), a commercial post-cut dipping solution, could oVer new opportunities for extending the shelf life of fresh-cut fruits. The objectives of this study were to determine: (i) dose-dependent eVectiveness of vanillin as a natural antimicrobial agent against selected pathogenic, indicator, and spoilage organisms and (ii) eVectiveness of vanillin (12 mM) when incorporated with NatureSeal to suppress the total aerobic microbial growth on fresh-cut ‘Empire’ and ‘Crispin’ apples. 2. Materials and methods 2.1. Microorganisms and culture conditions All microorganisms used in this study were obtained from the American Type Culture Collection (ATCC), Manassas, VA. The pathogenic and indicator strains include: E. coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Enterobacter aerogenes (ATCC 13048), Salmonella enterica subsp. enterica serovar Newport (ATCC 6962), and the four spoilage organisms were Candida albicans (ATCC 10231), S. cerevisiae (ATCC 9763), Penicillum expansum (ATCC 7861), Lactobacillus casei (ATCC 7469). For the two yeast species, malt extract broth (MEB) growth medium was used, while Man Rogosa and Sharpe (MRS) agar was used for L. casei, and Trypticase soy broth (TSB) was used for the remaining cultures. These culture media were purchased from Oxoid Inc., Nepean, Ont., Canada. 2.2. Preparation of vanillin DiVerent vanillin concentrations (0, 1.5, 3, 6, 12, and 18 mM) were prepared in triplicate by adding the appropriate amounts of vanillin (Ashland Canada Inc., Mississauga, Ont., Canada) into Xasks containing corresponding sterile growth medium and heating for 2 min using a microwave. The medium was then dispensed into 10 ml aliquots in sterile glass tubes. 2.3. Susceptibility testing

OCH 3 OH Vanillin

OCH 3 OH Eugenol

Benzoic acid

Fig. 1. The chemical structure of vanillin in comparison to eugenol and commercial preservative benzoic acid.

Inoculum for each of the microorganisms was prepared to a concentration of approximately 1 £ 108 cfu/ml using McFarland standards (Med-Ox Diagnostics Inc., Ottawa, Ont., Canada) and 100 l of inoculum was used to inoculate each of the prepared tubes containing culture media with


six vanillin concentrations. S. cerevisiae, P. expansum, and C. albicans inoculated tubes were grown at 28 °C for 18 h on a shaker at 200 rpm. E. coli, P. aeruginosa, E. aerogenes, and S. Newport were grown at 37 °C for 18 h on a shaker at 200 rpm. L. casei was grown at 30 °C in 5% CO2 for 18 h on a shaker at 200 rpm. Growth of microorganisms was determined by turbidity readings at 600 nm using a spectrophotometer (Baxter Diagnostics Inc., DeerWeld, IL, USA). For cultures that exceeded the range of accurate measurements of the spectrophotometer, cultures were diluted 10-fold with the corresponding culture medium. Standard growth curves with six data points were established for each of the eight microorganism and used to determine the viable cells from the OD600 values. The experiment was conducted in triplicate for each vanillin concentration and was repeated independently 2 times. 2.4. Treatment of fresh-cut apples with vanillin Two apple cultivars, ‘Empire’, and ‘Crispin’, were harvested at commercial maturity from a commercial orchard in Simcoe, Ont., Canada in 2002. The Xesh Wrmness (Lb), total soluble solid content (%), and starch index (Cornell generic starch staining test) at the harvest of ‘Empire’, and ‘Crispin’ were 17.6, 11.5, 2.6 and 18.1, 12.4, 3.1, respectively. Apples were stored in standard controlled atmosphere (CA, 2.5% O2 + 2.5% CO2, 0 °C, >95% RH) for 6–8 months. Upon removal from CA storage, apple slices were prepared within 2 h at room temperature. The apples were washed in an antimicrobial solution (100 l/l sodium hypochlorite, pH adjusted to 6.5 using citric acid, and chilled to 4–6 °C) for 2 min in a fume hood under aseptic conditions. They were air dried for 10–15 min, then cored and sliced into 10 equal slices (wedges) with skin using a sanitized, sharp, stainless steel apple slicer (The Pampered Chef™, Markham, Ont., Canada). Immediately after slicing, apple slices were dipped either in an aqueous solution of 6% (w/v) calcium ascorbate (NatureSeal™, Mantrose-Haeuser Co. Inc., Westport, CT, USA) with or without 12 mM vanillin chilled to 4–6 °C, or in chilled (4–6 °C) water for 3 min. The excess liquid on slices was allowed to drain. Each treatment was conducted in triplicate using separate containers. Six slices from a pool of 100 slices were randomly selected and packed in polyethylene bags (oxygen transmission rate of 220 cm3/m2/24 h, 12 £ 17 cm in size). A bag of six slices was considered as an experimental unit or replicate. The bagged apple slices were held at 4 °C and removed on days 1, 5, 9, 14, and 19 for testing aerobic colony counts. Another set of bags was prepared for assessing the fresh-cut quality (Wrmness and color) and the samples were removed from 4 °C storage at 0, 4, 8, 13, and 18-day intervals.

577

0.1% peptone for 1 min using a stomacher (BagMixer 400, Interscience, St. Nom, France), and preparing 10-fold serial dilutions (1 ml in 9 ml of 0.1% peptone) as required. Aliquots (1 ml) of diluted sample preparations were plated in duplicate onto ACC PetriWlm™ (3M Microbiology Products, St. Paul, MN, USA) and incubated at 37 °C for 48 h. PetriWlm™ having counts ranging from 20 to 200 cfu were considered to be within the countable range. 2.6. Firmness of the cut surface The Wrmness of the cut-surface of each apple slice was determined by a hardness tester with a test anvil (a ball) of 5.0 mm diameter interfaced to a software system (Model HHP-2001-FV, Bareiss Prüfgerätebau GmbH, Germany). The principle of the hardness tester is based on the penetration principle, which involves pressing a speciWed shape of test stamp into a test specimen at a level of spring force deWned by standard value. The measurements were done by pressing the test stamp onto the cut-surface of apple slices manually until the acoustic signal to indicate the measurement. Measuring time was 3 s and unit of force was Newton (N). Three readings were recorded for each apple slice around the mid point area between endocarp and skin (two readings on one side and one reading on the other side). 2.7. Color intensity of the cut-surface The color of each apple slice was determined using a colorimeter (model CR-300; Minolta, Japan), which had been calibrated with a standard white plate (Y D 92.30, x D 0.3162, y D 0.3323). Three readings of ‘L’ (lightness), ‘a’ (green chromaticity) and ‘b’ (yellow chromaticity) co-ordinates were recorded for each slice around the mid point area between endocarp and skin (two readings on one side and one reading on the other side). Observations were recorded for each of six slices per replicate in triplicate at each time interval. A decrease in ‘L’ value indicates a loss of whiteness, a more positive ‘a’ value means progressive browning and a more positive ‘b’ value indicates more yellowing. The whiteness index [Whiteness Index D 100 ¡ ((100 ¡ L)2 + a2 + b2)1/2] was calculated using the above three values as described by Bolin and Huxsoll (1991). 2.8. Statistical analysis Statistical analysis was conducted on data using a factorial ANOVA, the general linear model, with SAS System version 8e for Windows. Mean separations were examined using Tukey’s Studentized range test (t-test). 3. Results

2.5. Total aerobic colony counts

3.1. Antimicrobial activity of vanillin in vitro

Total aerobic colony counts (ACC) of apple slices were obtained by homogenizing 25 g of apple slices in 225 ml of

The dose-dependent inXuence of Wve concentrations (0, 1.5, 3, 6, 12, 18 mM) of vanillin on the growth of eight micro-

578


organisms was evaluated by measurement of optical density (turbidity) in culture medium. The growth of microorganisms displayed an inverse relationship with increasing concentrations of vanillin (Table 1). The dose response eVect of vanillin was dependent on the microorganism. For example, among the four pathogenic bacteria, 73% of growth inhibition was observed for E. coli at 6 mM vanillin, while the other three organisms demonstrated lower inhibitory eVect (45% or less) at the same level of vanillin. Similarly, among the spoilage microorganisms, 91% of growth inhibition was observed for P. expansum at 3 mM vanillin but similar growth reduction for L. casei occurred at 18 mM vanillin. In general, most microorganisms tested in this study showed MIC between 6 and 18 mM vanillin. However, the MIC of P. expansum seems to be slightly beyond the highest concentration (18 mM) used in this study. 3.2. Antimicrobial eVect of vanillin on fresh-cut apples In general, total aerobic colony counts of untreated fresh-cut apple slices increased by approximately 4.3 log cfu/g fresh weight from day 1 to day 19 under 4 °C conditions (Table 2). In comparison, the increments of total microbial loads of NatureSeal + 12 mM vanillin treated apple slices was on average only 1.6 log cfu/g fresh weight. The percent inhibition of total microbial growth during the 19-day post-cut storage as a result of the incorporation of vanillin in the post-cut dipping solution were 37% and 66% in fresh-cut ‘Empire’ and ‘Crispin’ apples, respectively. The suppression of microbial growth was signiWcant (P < 0.01) in both apple cultivars during days 5 to 19 of the post-cut storage period. NatureSeal alone decreased the microbial growth of ‘Empire’ apple slices on days 5, 9, and 14 of post-

Table 2 Total microbial load of fresh-cut ‘Empire’ and ‘Crispin’ apples treated with or without vanillin Cultivar Post-cut treatment

Empire

Post-cut storage at 4 °C (days) 1

5

9

14

19

Total aerobic colony counts (log cfu/g) 2.07a 3.25a 4.45a 4.61a 0.23a 0.54a 0.99b 2.71b 3.95b 4.47a

Water NatureSeal

0.63a

NatureSeal + vanillin

1.02b

1.39c

2.32c

2.65c

Mean 2.92a 2.53b 1.60c

T*, ENS, D* (n D 3, d.f. D 82, SEM D 0.36) Crispin

Water NatureSeal

0.67a 0.93a

1.85a 1.77a

3.05a 3.36a

4.39a 3.93a

5.01a 4.35a

2.99a 2.86a


0.77a

0.98b

1.25b

1.33b

1.96b

1.26b

T*, ENS, D* (n D 3, d.f. D 82, SEM D 0.43) DiVerent superscripts in each column indicate signiWcant diVerences in the mean at D 0.05. T, treatments; E, experiments; D, days at 4 °C storage; NS, not signiWcant; ¤ , signiWcant at p < 0.01.

cut storage when compared to that of water treated slices, possibly due to it’s ability to retain the cellular integrity at cut surface of apple. 3.3. Flesh Wrmness and cut-surface browning Post-cut dipping of apple slices in NatureSeal and the storage of treated slices at 4 °C inhibited softening and enzymatic browning as indicated by Xesh Wrmness and whiteness index of cut surfaces (Table 3). Incorporation of vanillin into the NatureSeal solution did not change these two major quality attributes of both ‘Empire’ and ‘Crispin’ apples slices.

Table 1 Inhibitory eVect of vanillin on the growth of eight selected pathogenic and spoilage microorganisms Microorganism

Parameter

Vanillin Concentration (mM) 0

Pathogenic/indicator E. coli cfu/ml % inhibition

1.5

3

6

12

18

8.72 £ 104a –

8.42 £ 104a 3

3.53 £ 104b 60

2.39 £ 104c 73

6.37 £ 102d 99

6.35 £ 102d 99

E. aerogenes

cfu/ml % inhibition

1.03 £ 105a –

1.03 £ 105a 0.3

8.96 £ 104b 13

8.43 £ 104c 18

2.79 £ 104d 73

1.53 £ 103e 99

P. aeruginosa

cfu/ml % inhibition

6.47 £ 104a –

6.65 £ 104a ¡3

6.46 £ 104a 0.1

3.58 £ 104b 45

6.06 £ 103c 91

1.00 £ 103d 98

S. Newport

cfu/ml % inhibition

1.15 £ 105a –

1.09 £ 105b 6

9.12 £ 104c 21

7.33 £ 104d 37

1.87 £ 104e 84

2.35 £ 103f 98

cfu/ml % inhibition

2.71 £ 104a –

1.60 £ 104b 41

8.39 £ 103c 69

5.87 £ 102d 98

1.0 £ 10d 100

1.0 £ 10d 100

L. casei

cfu/ml % inhibition

1.18 £ 105a –

1.17 £ 105a 0

1.16 £ 105c 0.5

1.16 £ 105c 0.8

3.03 £ 104d 74

1.8 £ 103e 98

P. expansum

cfu/ml % inhibition

1.50 £ 104a –

9.49 £ 103b 37

1.42 £ 103c 91

9.40 £ 102d 94

9.00 £ 102e 94

8.84 £ 102e 94

S. cerevisiae

cfu/ml % inhibition

5.49 £ 103c –

9.06 £ 103b ¡65

1.31 £ 104a ¡139

1.32 £ 103d 76

1.44 £ 102e 97

1.16 £ 102e 98

Spoilage C. albicans

DiVerent superscripts in each raw indicate signiWcant diVerences in the mean at D 0.05.

H.P.V. Rupasinghe et al. / Food Research International 39 (2006) 575–580 Table 3 Mean Xesh Wrmness and color of ‘Empire’ and ‘Crispin’ apple slices treated with or without vanillin Cultivar

Post-cut treatment

Mean quality attribute Firmness (N)

Empire

Water NatureSeal

3.51a 4.38b

61.9a 69.4b


4.31b

69.1b

T**, ENS, D* Crispin

Flesh color (Whiteness Index)

T**, ENS, D**

Water NatureSeal

3.42a 4.22b

56.6a 64.5b


4.30b

64.1b

T**, ENS, D**

T**, ENS, D**

DiVerent superscripts in each column indicate signiWcant diVerences in the mean at D 0.05. T, treatments; E, experiments; D, days at 4 °C storage; NS, not signiWcant; ¤,¤¤ , signiWcant at p < 0.01, 0.001, respectively.

4. Discussion The growing consumer demand and potential food safety concerns associated with fresh-cut produce has led to an interest in the investigation of the use of natural antimicrobial agents to suppress microbial growth on produce in order to extend shelf life and meet consumer expectation. Particularly, treating apple slices with commercial post-cut dipping solution NatureSeal™ allows up to 21 days retention of original color and Wrmness of refrigerated apple slices but the total microbial load could increase to unacceptable levels during the same period (Rupasinghe et al., 2005). We propose that incorporation of a GRAS antimicrobial agent that is compatible with the chemical properties of the post-cut dipping solution could improve the food safety and quality of fresh-cut apples and other produce. In nature, an enormous number of eVective antimicrobials exist, however, only a limited number have found application for use in foods (Brul & Coote, 1999; Gould, 1996). Vanillin, a low molecular weight plant phenolic molecule, is a unique food additive with multiple properties. Natural and synthetically produced (nature identical) vanillin has been widely used by the food industry as a GRAS Xavoring agent of variety of food products, and its organoleptic feature is well accepted by consumers (Hocking, 1997; Ramachandra Roa & Ravishankar, 2000). Vanillin is an eVective antioxidant in complex foods containing polyunsaturated fatty acids and its incorporation into dried foods (e.g. cereals) has shown a greater keeping quality than similar products left untreated (Burri, Graf, Lambelet, & Loliger, 1989). Previous studies have demonstrated the antimicrobial activity of vanillin against yeasts and moulds (Castañón et al., 1999; Cerrutti & Alzamora, 1996; Cerrutti et al., 1997; Fitzgerald et al., 2003; Fitzgerald et al., 2004b; López-Malo et al., 1995, López-Malo, Alzamora, & Argaiz, 1997, 1998; Matamoros-León et al., 1999) although the reports on antibacterial properties of vanillin are limited

579

(Cerrutti et al., 1997; Fitzgerald et al., 2004a; Jay & Rivers, 1984). In this study, we investigated the dose-dependent eVect of vanillin on selected pathogenic, indicator, and spoilage microorganisms: E. coli, E. aerogenes, P. aeruginosa, S. Newport, C. albicans, L. casei, P. expansum, and S. cerevisiae. The selection of these organisms was based on the broad categories of microorganisms that can be associated with refrigerated fresh-cut apples (Wiley, 1994). As far as we are aware, this is the Wrst report to illustrate inhibitory action of vanillin on E. aerogenes, L. casei, S. Newport, and P. expansum. Interestingly, all microorganisms tested were inhibited by vanillin at concentrations between 6 and 18 mM, with the exception of P. expansum (MIC > 18 mM). The MIC value range (6–18 mM) observed in the present study is in agreement with the previously reported MIC values for diVerent microorganisms (Fitzgerald et al., 2004b; Matamoros-León et al., 1999). For example, MIC value for S. cerevisiae reported by Fitzgerald et al. (2004b) was 17 mM. Moreover, MIC of vanillin for P. digitatum and P. italicum was 6.6 mM and the same for P. glabrum was 9.6 mM (Matamoros-León et al., 1999). It is interesting to note that the growth of S. cerevisiae was enhanced at 1.5 and 3 mM (sub-MIC) concentrations of vanillin. Fitzgerald et al. (2003) found that S. cerevisiae was able to convert vanillin to vanillyl alcohol and vanillic acid when incubated with sub-MIC levels of vanillin (1 mM). Therefore, it appeared that bioconversion of vanillin at sub-MIC levels was advantageous to S. cerevisiae while higher vanillin concentrations (>6 mM) were found to be inhibitory for the growth (Fitzgerald et al., 2003). For the Wrst time, we have demonstrated that incorporation of vanillin (12 mM) in the post-cut dipping solution of apple slices could inhibit the microbial growth during the 19-day post-cut storage by 37 and 66% in ‘Empire’ and ‘Crispin’ apple slices, respectively. Several cultivar speciWc factors could have contributed to the diVerent intensities of responses observed between ‘Empire’ and ‘Crispin’ apple slices, including: diVerences in surface pH, levels of essential nutrients (vitamins, minerals, and nitrogen-containing compounds) or natural phenolic compounds present on the Xesh of apples. The selection of 12 mM concentration for the above study was based upon the in vitro observations of MIC values and several other factors. Previous studies have shown that the activity of vanillin against microorganisms could be increased by lowering the incubation or storage temperature (Fitzgerald et al., 2004b; López-Malo et al., 1997). The incubation temperatures of microorganism cultures that were used in this study for the generation of MIC values were 28 and 30 °C. Therefore, we speculated that antimicrobial action of vanillin would be greater at the lower temperature (4 °C) used for post-cut storage of vanillin treated fresh-cut apples and therefore suYcient for control of aerobic microbial growth of fresh-cut apples. Furthermore, the bakery and beverage industry use vanillin as a Xavouring component at levels between 1.3 and 12.5 mM (Hocking, 1997). Our preliminary observations

580


also indicated that vanillin concentration beyond 12 mM could produce unacceptable Xavor and aroma for fresh-cut apples. Vanillin was also identiWed as a compatible additive for NatureSeal as its presence did not aVect the NatureSeal’s ability to act as an anti-browning and softening control agent. In conclusion, vanillin that is commonly used as a Xavoring agent in foods was found to be eVective in reducing growth of eight selected microorganisms in vitro and total microbial load on fresh-cut apple slices. Vanillin retained its eVectiveness as well as that of NatureSeal when the two chemicals were combined. Therefore, this study provides a new insight into the possible use of vanillin as a natural antimicrobial agent in processing of sliced apples and others. However, further research is required in order to obtain information about the organoleptic quality and consumer acceptance of fresh-cut apples treated with vanillin, before making a recommendation for its use as a preservative in post-cut dipping solution. Acknowledgements This research was funded jointly by the Healthy Futures for Ontario Agriculture program of the Ontario Ministry of Agriculture and Food (OMAF), Pride Pak Canada Ltd., Mississauga, Ont., and Ontario Apple Sales Group. References Alzamora, S. M., & Guerrero, S. (2003). Plant antimicrobials combined with conventional preservatives for fruit products. In S. Roller (Ed.), Natural antimicrobials for the minimal processing of foods (pp. 235– 249). Boca Raton, FL: CRC Press LLC. Beuchat, L. R., & Golden, D. A. (1989). Antimicrobials occurring naturally in foods. Food Technology, 43, 134–142. Bolin, H. R., & Huxsoll, C. C. (1991). Control of minimally processed carrot (Daucus carotova) surface discoloration caused by abrasion peeling. Journal of Food Science, 56, 416–421. Brul, S., & Coote, P. (1999). Preservative agents in foods. Mode of action and microbial resistance mechanisms, 50, 1–17. Burri, J., Graf, M., Lambelet, P., & Loliger, J. (1989). Vanillin: more than a Xavouring agent – a potential antioxidant. Journal of the Science of Food and Agriculture, 48, 49–56. Castañón, X., Argaiz, A., & López-Malo, A. (1999). EVect of storage temperature on the microbial and color stability of banana puree with addition of vanillin or potassium sorbate. Food Science and Technology International, 5, 51–58. Cerrutti, P., & Alzamora, S. M. (1996). Inhibitory eVects of vanillin on some food spoilage yeasts in laboratory media and fruit purees. International Journal of Food Microbiology, 29, 379–386.

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