213 Journal of Food Protection, Vol. 79, No. 2, 2016, Pages 213–219 doi:10.4315/0362-028X.JFP-15-245 Copyright Q, International Association for Food Protection
Inactivation of Escherichia coli, Listeria monocytogenes, and Salmonella Enteritidis by Cymbopogon citratus D.C. Stapf. Essential Oil in Pineapple Juice CAROLINE JUNQUEIRA BARCELLOS LEITE,1 JOSSANA PEREIRA DE SOUSA,1 ´ ALBERTO DA COSTA MEDEIROS,1 MARIA LUCIA ´ ˜ 1 JOSE DA CONCEIC ¸ AO, 2 ˜ VIVYANNE DOS SANTOS FALCAO-SILVA, AND EVANDRO LEITE DE SOUZA1* 1Laboratory
of Food Microbiology, Department of Nutrition, Health Sciences Center, and 2Laboratory of Genetics of Microorganisms, Department of Molecular Biology, Center of Exact and Natural Sciences, Federal University of Para´ıba, Joa˜o Pessoa, 58051900, Brazil MS 15-245: Received 5 June 2015/Accepted 13 August 2015
ABSTRACT In the present study, the efficacy of Cymbopogon citratus D.C. Stapf. essential oil (CCEO) to provoke a 5-log CFU/ml (5log) inactivation in a mixed composite of Escherichia coli, Listeria monocytogenes, and Salmonella enterica serovar Enteritidis in pineapple (Ananas comosus (L.) Merril) juice (48C) was assessed. Moreover, the effects of CCEO on the physicochemical and sensory quality parameters of pineapple juice were evaluated. The MIC of CCEO was 5 ll/ml against the composite mix examined. For L. monocytogenes and E. coli inoculated in juice containing CCEO (5, 2.5, and 1.25 ll/ml), a 5-log reduction was detected after 15 min of exposure. This same result was obtained for Salmonella Enteritidis incubated alone in pineapple juice containing CCEO at 5 and 2.5 ll/ml. Overall, Salmonella Enteritidis was the most tolerant and L. monocytogenes was the most sensitive to CCEO. The physicochemical properties (pH, titratable acidic [citric acid per 100 g], and soluble solids) of pineapple juice containing CCEO (2.5 and 1.25 ll/ml) were maintained. Juice containing CCEO (2.5 and 1.25 ll/ml) exhibited similar scores for odor, appearance, and viscosity compared with juice without CCEO. However, unsatisfactory changes in taste and aftertaste were observed in juices containing CCEO. These results suggest that CCEO could be used as an alternative antimicrobial compound to ensure the safety of pineapple juice, although CCEO at the tested concentrations negatively impacted its taste. Therefore, further studies are needed to determine the balance between microbial safety and taste acceptability of pineapple juice containing CCEO. Key words: Antibacterial effects; Essential oil; Pathogenic bacteria; Pineapple juice
Pineapple (Ananas comosus (L.) Merril) fruits and industrial by-products have a strong flavor and intense aroma and are used for fresh consumption and the production of a variety of derived industrial products, including juices (19). Pineapple fruit is a good source of fiber, vitamins, minerals, and phenolic compounds, with notable antioxidant capacities and potential health benefits (7). Because pineapple fruits are grown in the ground, it is virtually impossible to prevent microbial contamination of the rind of these fruits. Thus, when pineapple fruits with contaminated rinds are cut, different pathogenic and spoilage microorganisms can be transferred to the pulp (edible part) and, consequently, to the juice during or after extraction. Pineapple juice could be considered safe from pathogenic bacteria because of its intrinsic characteristics (low pH and high acidity) that create a hostile environment for bacterial survival. However, reports of the incidence, survival, and growth of pathogenic bacteria, such as Escherichia coli, Salmonella spp., and Listeria monocyto* Author for correspondence. Tel: þ 55 83 3216 7807; Fax: þ 55 83 3216 7094; E-mail: [email protected].
genes, in highly acidic fruits, such as apples, oranges, cranberries, and lemon juices, have changed the belief that there is small concern that fruit juices may harbor pathogenic bacteria (8, 25). Moreover, several outbreaks of E. coli and Salmonella spp. associated with the consumption of unpasteurized apple and orange juices have been reported (14, 18). Although L. monocytogenes has not often been implicated in outbreaks associated with unpasteurized fruit juices (10), this bacterium has previously been isolated from apple and raspberry juices (12, 36). Because of the acidity of pineapple juice (pH 3 to 4), similar to that of apple and orange juice, its potential to harbor pathogenic bacteria and cause foodborne illness remains a major concern. Thus, issues surrounding the safety of unpasteurized fruit juices have been of considerable interest to industry and to all those concerned with public health. The U.S. Food and Drug Administration (FDA) implemented a hazard analysis and critical control point (HACCP) regulation that includes a performance criterion for juice safety, commonly referred to as the juice HACCP regulation (38). The regulation requires juice processors to achieve a 5-log reduction of target microorganism(s) of public health significance when making
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juice, but this regulation does not require use of a specific method to achieve this inactivation level. Currently, pasteurization remains the dominant preservation procedure for eliminating pathogenic and spoilage organisms from juices. However, the heating used in pasteurization can cause a significant loss of nutrients (primarily vitamins and minerals) and undesirable changes in the fresh color and taste of juices (9). Although some fresh juices not formulated with chemical preservatives have been available in the marketplace, the juice industry still commonly adds chemical preservatives to control bacteria in their products (11). However, consumer concern about the potential risks of foods that contain chemicals and pathogenic microorganisms (32) has led to the demand for safer, fresher, and healthier foods with no or few chemical preservatives, thus creating a market demand for natural, nonthermal, and feasible technologies to ensure the microbial safety of juices (17). Innovative nonthermal technologies, such as pulsedelectric fields and high hydrostatic pressure, have been successfully applied to control pathogenic bacteria in juices (11). Essential oils (EOs) have also received increasing attention for their potential as inhibitors of microorganisms in fruit juices (30). The EOs are generally recognized as safe at the doses typically used in foods (1, 5) and have been approved by the FDA for use as flavoring agents in foods and beverages (37). The EO obtained from Cymbopogon citratus D.C. Stapf. (lemongrass) (CCEO) has shown widespectrum antimicrobial activity, including the inhibition of pathogenic and spoilage microorganisms (27, 31, 30). A variety of pineapple products (such as frozen pulp, unpasteurized juice, ready-to-eat mixed fruit salads, and minimally processed slices) containing ground C. citratus leaves are available in the market. These products are well accepted and are regarded as typical value-added products because of the distinct but pleasant ‘‘lemon-like’’ refreshing taste. Thus, pineapple juice could represent a potential food matrix to exploit the antimicrobial properties of CCEO. In the present study, we assessed the efficacy of CCEO to induce a 5-log reduction of a mixed composite of the potentially pathogenic bacteria E. coli, L. monocytogenes, and Salmonella enterica serovar Enteritidis in pineapple juice. The effects of CCEO on the physicochemical and sensory quality parameters of pineapple juice were assessed.
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adjusted using 1 M NaOH), washed with sterile distilled water, and dried for 1 h in a safety cabinet. Next, the fruits were aseptically peeled, chopped, and mixed with distilled water (1:1 rate) using a domestic blender (for 3 min). The mixture was filtered using a triple-cheesecloth layer and was sterilized by autoclaving (1218C, 1.1 atm, for 15 min). The juice was stored in 50-ml aliquots at 208C, and when required, an aliquot was thawed under refrigeration (4 6 18C) and was used for subsequent assays. The juice samples were submitted to physicochemical analysis after autoclaving (previous experiments revealed that the assessed physicochemical parameters in pineapple juice were unchanged after autoclaving). The noninoculated juice samples used in sensory analysis were prepared 24 h before assessing acceptability, and microbiological analysis revealed the satisfactory microbiological quality of these juice samples according to current Brazilian legislation (3). Test strains and composite preparation. The L. monocytogenes (ATCC 7644), E. coli (UFPEDA 224), and Salmonella Enteritidis (UFPE 414) were obtained from the Collection of Microorganisms, Department of Antibiotics, Federal University of Pernambuco (Recife, Brazil). The stock cultures were maintained in BHI broth plus glycerol (15 g/100 ml) at 208C. Each strain was grown in BHI broth at 378C for 20 to 24 h (stationary growth phase), harvested through centrifugation (4,500 3 g, 15 min, 48C), washed twice in sterile saline solution (0.85 g/100 ml), and resuspended in sterile saline solution to obtain cell suspensions at which the optical density reading at 660 nm ranged from 0.08 to 0.13. This suspension provided viable cell counts of approximately 8 log CFU/ml of each strain when pour plated in BHI agar (21, 40). The mixed composite of the three strains tested was obtained by mixing the obtained bacterial suspensions at a ratio of 1:1:1 for a maximum of 15 min after the preparation of each pure suspension (final count of each strain approximately 8 log CFU/ml) (28). Identification of CCEO constituents. The constituents in CCEO were identified through gas chromatography coupled with mass spectrometry (GC-MS; CGMS-QP2010 Ultra, Shimadzu, Kyoto, Japan) performed under the following conditions: RTX5MS capillary column (30 m by 0.25 mm by 0.25 lm); program temperature, 60 to 2408C (38C/min); injector temperature, 2508C; detector temperature, 2208C; carrier gas, helium adjusted to 0.99 ml/min; ionizing energy, 70 eV; and mass range, 40 to 500. The spectral bank of GC-MS, NIST/EPA/NIH Mass Spectral Database (version 1.7, National Institute of Standards Technology, Norwalk, CT) was used to identify the individual essential oil constituents. The constituents were quantified after the areas of each detected constituent were normalized and expressed as a percentage area.
MATERIALS AND METHODS CCEO and pineapple fruits. The CCEO (batch 0061; pH 6.59) was obtained from Laszlo Aromatherapy Ltd. (Belo Horizonte, Brazil). The CCEO emulsions were prepared in brain heart infusion (BHI) broth (pH 7.40; Himedia, Mumbai, India) at concentrations of 80 (pH 7.25), 40 (pH 7.29), 20 (pH 7.31), 10 (pH 7.35), 5 (pH 7.36), 2.5 (pH 7.38), 1.25 (pH 7.39), 0.625 (pH 7.39), and 0.312 (pH 7.39) ll/ml using Tween 80 (1.5%, vol/vol; Himedia) as a stabilizing agent (28). The pineapple (A. comosus) fruits were purchased in the commercial maturation stage from a local wholesale distributor and were selected for uniformity in size, form, appearance, and the absence of mechanical injuries or visible infections. For juice preparation, the fruits were surface disinfected after a 15-min immersion in a sodium hypochlorite solution (150 ppm, pH 7.2
Determination of the MIC of CCEO. A modified microtiter plate assay was used to determine the MIC of CCEO according to a standard method (6), with minor modifications related to the use of a stain to detect bacterial growth and survival (40). Approximately 50 ll of CCEO solution (80 to 0.312 ll/ml) was dispensed into each well of a 96-well microplate containing 100 ll of BHI broth. Subsequently, 50 ll of bacterial suspension was added to each well (final viable cells count of approximately 7 log CFU/ml). The microplate was loosely wrapped with cling wrap to prevent bacterial dehydration and CCEO volatilization. Each plate included either a control (without CCEO), inoculated (positive control), or uninoculated (negative control) sample. The system was statically incubated at 378C for 24 h. Subsequently, a 30-ll aliquot of resazurin (0.01 g/100 liters, wt/vol) (Inlab, Sa˜o Paulo, Brazil), prepared in aqueous solution, was added to each well. The color
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TABLE 1. Gas chromatography–mass spectrometry analysis of the essential oil from C. citratus D.C. Stapf.a Constituents
% in essential oil
6-Methyl-5-hepten-2-ona Dipentyl-ketone Linalool Neral Geraniol Geranyl-acetate Caryophyllene
1.77 1.06 1.03 31.74 46.16 4.34 2.02
a
Constituents detected in concentration 1%.
changes were visually assessed after 20 min at 378C. Bacterial growth was indicated as color changes from purple to pink (or colorless). The MICs were confirmed as the lowest concentrations of CCEO capable of inhibiting bacterial growth. Effects of CCEO on survival of bacterial strains in pineapple juice. The effects of different CCEO concentrations on the survival of the bacterial mixed composite in pineapple juice under refrigerated storage were assessed using the viable cell count method. Initially, 150 ll of the tested bacterial mixed composite was inoculated into 5,850 ll of separated juice samples (final viable cell counts of approximately 7 log CFU/ml) containing CCEO at the desired final concentrations (5, 2.5, 1.25, and 0.6 ll/ ml). The different systems were gently hand shaken for 30 s and were subsequently incubated at 4 6 18C. At different exposure times (5, 10, 15, 30, and 45 min; 1, 2, 4, 8, 12, 24, 48, and 72 h), a 20-ll aliquot was obtained from each system to directly inoculate, using the microdrop technique (15), onto selective agar: eosin– methylene blue agar (Himedia) for E. coli, Listeria selective agar plus Listeria Selective Supplement II (Himedia) for L. monocytogenes, and Salmonella-Shigella agar (Himedia) for Salmonella Enteritidis (34). In parallel, 100-ll aliquots of each system were serially diluted in sterile saline solution; subsequently, 20 ll of each dilution was inoculated onto the same selective medium for each type of bacteria. Control juices (without CCEO) were similarly assayed. The plates were incubated at 378C for 24 to 48 h. Plates inoculated with aliquots collected from juice samples containing CCEO were incubated for 24 h longer, at adequate temperature, than the samples collected from control juice. The results are expressed as duration of exposure to each tested concentration of CCEO to establish a 5-log CFU/ml reduction in initial viable cell counts (CFU per milliliter at time zero) of each of the tested strains comprising the mixed composite (log N0 N, where N0 and N were the initial count and count after incubation, respectively, for indicated times at 48C). The detection limit of the viable cell count procedure was 2 log CFU/ml for all experiments and strains tested. Analysis of physicochemical and sensory parameters of pineapple juice. Samples of pineapple juice containing CCEO (2.5 and 1.25 ll/ml) were assessed for some physicochemical quality parameters at 0 (baseline, just after the addition of CCEO) and 72 h of storage at 4 6 18C. The titratable acidity was determined using phenolphthalein as an indicator with 0.1 N NaOH, and the results were expressed as grams per 100 g (equivalent of citric acid). pH values were determined using a digital potentiometer (model Q400AS, Quimis, Sa˜o Paulo, Brazil). The soluble solids content (8Brix) was determined using a digital
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refractometer (model HI 96801, Hanna Instruments, Sa˜o Paulo, Brazil) (2). Sensory evaluation was performed via acceptability tests. Sixty panelists (20 to 35 years old) were preselected according to interests and frequency of pineapple juice consumption. Panelists worked in individual booths with controlled temperature and lighting. Each panelist received three juice samples containing the different concentrations of CCEO tested (1.25 and 2.5 ll/ml). The CCEO was incorporated in juice samples 24 h prior to the sensory test and was kept at 4 6 18C. Samples of juice without CCEO were tested as controls. The different juice samples were served in 30-ml aliquots in white disposable cups coded with a randomized three-digit number. Immediately after removal from refrigeration, the samples were served simultaneously, using a blind method of random sequence. The panelists were asked to use low-salt crackers and water to cleanse their palates between the assessed samples. The acceptability of appearance, odor, viscosity, taste, aftertaste, and overall acceptability were evaluated on a 9-point hedonic scale, ranging from 1 (dislike very much) to 9 (like very much) (35). Reproducibility and statistics. All assays were performed in triplicate in three independent experiments (repetitions), and the results are expressed as the average of the tests. For assays to determine the MIC, the result was expressed as a modal value because the MIC was the same in all repetitions. For physicochemical and sensory parameters, statistical analyses were performed to determine significant differences (P 0.05) among treatments using analysis of variance, followed by Tukey’s post hoc test using the computational software GraphPad Prism 6.0 (GraphPad Software Inc., La Jolla, CA).
RESULTS Identification of CCEO constituents. The GC-MS analysis of CCEO showed the presence of seven different constituents in amounts higher than 1% of the EO total mass (Table 1). The monoterpenes geranial (b-citral isomer) (46.16%) and neral (a-citral isomer) (31.74%) were identified as the major constituents of CCEO. Other compounds, such as geranyl acetate (4.34%) and caryophyllene (2.02%), were detected in lower amounts. Antimicrobial assays. The MIC of CCEO was 5 ll/ml against the composite mix containing E. coli UFEPEDA 224, L. monocytogenes ATCC 7644, and Salmonella Enteritidis UFEPEDA 414 (data not shown). The incorporation of CCEO in pineapple juice at all tested concentrations (5, 2.5, 1.25, and 0.6 ll/ml) caused a decrease in viable counts of E. coli, L. monocytogenes, and Salmonella Enteritidis. For L. monocytogenes and E. coli inoculated in juice containing CCEO at 5, 2.5, or 1.25 ll/ml, a 5-log reduction in viable counts was detected after 15 min of exposure (Table 2). This same reduction (after 15 min) was observed for Salmonella Enteritidis alone when inoculated in juice containing CCEO at 5 or 2.5 ll/ml. A fourfold longer exposure time (1 h) was needed for a 5-log reduction of Salmonella Enteritidis in juice containing CCEO at 1.25 ll/ml compared with the other tested bacteria. In juice samples containing CCEO at 0.6 ll/ml, 5-log reductions in E. coli and L. monocytogenes counts were observed after 1 h and 45 min, respectively, whereas 12 h of
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TABLE 2. Exposure time to achieve 5-log CFU/ml reduction in a mixed population of E. coli, L. monocytogenes, and Salmonella Enteritidis in pineapple fruit juice containing different concentrations of CCEO and stored at 4 6 18 Ca Duration of exposure for a 5-log reduction in initial counts Concn of CCEO in pineapple juice (ll/ml)
E. coli
5 2.5 1.25 0.6 Control (0)
15 min 15 min 15 min 1h ND
a
L. monocytogenes
15 15 15 45 72
Salmonella Enteritidis
min min min min h
15 min 15 min 1h 12 h ND
CCEO, essential oil from C. citratus D.C. Stapf. ND, 5-log reduction was not detected.
incubation was needed to achieve a similar reduction of Salmonella Enteritidis. Because a 5-log reduction was observed, no viable cells were detected at longer assessed time intervals (up to 72 h). In juice samples without CCEO, only L. monocytogenes presented a 5-log reduction as detected after 72 h of cultivation. In the same samples, 3.3- and 1.29-log reductions were observed for E. coli and Salmonella Enteritidis, respectively, after 72 h of cultivation.
tested concentrations did not differ (P . 0.05) in scores for all assessed sensory attributes. Appearance, odor, and viscosity had a mean score of ‘‘like slightly’’ on the hedonic scale for samples of pineapple juice containing CCEO, whereas taste, aftertaste, and overall acceptability always had a mean score of ‘‘neither like nor dislike.’’ The mean scores for all assessed sensory parameters were ‘‘slightly like’’ for control juice samples.
DISCUSSION Physicochemical and sensory analysis of pineapple juice. Pineapple juice samples with or without CCEO (2.5 and 1.25 ll/ml) were evaluated for pH, titratable acidity, and 8Brix immediately after the addition of CCEO and after refrigeration (4 6 18C) for 72 h. The fruit juice samples with or without CCEO presented no difference (P . 0.05) in the evaluated physicochemical parameters at the assessed time intervals. Overall, the addition of CCEO preserved the assessed physicochemical properties (quality) of pineapple juice samples (Table 3). The addition of CCEO at 2.5 and 1.25 ll/ml did not affect (P . 0.05) the appearance, odor, and viscosity of pineapple juice after refrigeration for 24 h (Table 4). However, both tested CCEO concentrations negatively affected the taste and aftertaste of pineapple juice. Samples of pineapple juice without CCEO received the highest scores (P 0.05) for taste, aftertaste, and overall acceptability. Juice containing CCEO, at both tested concentrations, exhibited similar scores (P . 0.05) for odor, appearance, and viscosity compared with control samples (without CCEO). In addition, samples containing CCEO at both
The CCEO tested in this study inhibited the growth of a mixed composite of E. coli, L. monocytogenes, and Salmonella Enteritidis in synthetic broth. However, based on the detected MIC (5 ll/ml), the CCEO tested could be classified as a less effective bacterial inhibitor (1) of the target mixed bacterial composite. The detected MIC of CCEO could be intrinsically related with the individual constituents forming this EO, where neral (b-citral cisisomer [citral B]) and geraniol (a-citral trans-isomer [citral A]) were detected as major constituents. Based on the results of previous studies, the following approximate general rank (in decreasing order of antibacterial activity) of individual constituents is proposed: eugenol . carvacrol–thymol– cinnamic acid . basil methyl chavicol . cinnamaldehyde . citral-geraniol (5), where citral is not classified as possessing the strongest antimicrobial effects. The presence of high amounts of oxygenated monoterpenes in CCEO (77.90%), particularly the citral compounds neral and geraniol (terpene aldehydes), could be responsible for its inhibitory effects against the tested bacterial strains. Researchers have proposed that the antimicrobial activity of
TABLE 3. Physicochemical parameters of pineapple juice supplemented with or without CCEO and stored at 4 6 18 Ca Scores for physicochemical parameters after 0 and 72 h of storage, avg (6SD) Titratable acidityb
pH
8Brix
Concn of CCEO in pineapple juice (ll/ml)
0h
72 h
0h
72 h
0h
72 h
2.5 1.25 Control (0)
3.82 (60.31) 3.88 (60.45) 3.92 (60.24)
3.91 (60.21) 3.93 (60.33) 3.96 (60.36)
0.19 (60.03) 0.18 (60.02) 0.17 (60.03)
0.18 (60.02) 0.17 (60.04) 0.17 (60.02)
10.18 (60.81) 10.15 (60.76) 10.17 (60.55)
10.87 (60.68) 10.42 (60.55) 10.40 (60.43)
a
b
CCEO, essential oil from C. citratus D.C. Stapf. No significant differences (P . 0.05) were found among the mean values, according to Tukey’s test. Values are given as grams of citric acid per 100 g of juice.
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TABLE 4. Scores for sensory attributes of pineapple juice with or without CCEO and stored at 4 6 18 C for 24 ha Scores for sensory attributes, avg (6SD) Concn of CCEO in juice (ll/ml)
2.5 1.25 Control (0) a
Appearance
6.39 (61.84) 6.19 (62.27) 6.62 (61.81)
Odor A A A
6.43 (61.98) 6.44 (62.13) 7.03 (61.80)
Viscosity A A A
6.11 (61.58) 6.21 (61.66) 7.21 (61.45)
Taste A A A
5.25 (61.85) 5.67 (62.05) 7.97 (60.84)
Aftertaste A A B
5.38 (61.72) 5.75 (61.95) 7.25 (61.15)
Overall acceptance A A B
5.49 (61.75) 5.57 (61.94) 7.52 (61.07)
A A B
CCEO, essential oil from C. citratus D.C. Stapf. Acceptability of each attribute was evaluated using a 9-point hedonic scale, ranging from 1 (dislike very much) to 9 (like very much). Values followed by different letters, in the same column, are significantly different (P 0.05), according to Tukey’s test.
EOs without phenolic compounds as major individual constituents, such as CCEO, could be associated with membrane disruption of target cells through lipophilic compounds, primarily terpene hydrocarbons (e.g., caryophyllene, a-pinene, p-cymene, and c-terpinene) (24). These terpene hydrocarbons induce swelling of the bacterial cell membrane to facilitate the transportation of citral into the cell (32), where this compound promotes dramatic effects on the cell envelope, proton motive force, and intracellular ATP content (33). Given that EOs could negatively impact the sensory acceptability of juices (26), the assays of bacterial inactivation were performed using pineapple juice samples containing the CCEO at concentrations similar to the MIC (5 ll/ml) and at concentrations as low as the 1/2 MIC (2.5 ll/ ml), 1/4 MIC (1.25 ll/ml), and 1/8 MIC (0.6 ll/ml). This approach was used to detect the lowest assayed CCEO concentrations capable of achieving the regulatory 5-log reduction of target pathogenic bacteria. Notably, CCEO was effective to cause 5-log decreases in E. coli, L. monocytogenes, and Salmonella Enteritidis at all tested concentrations in pineapple juice. These effects against target bacteria occurred at a maximum exposure time of 15 min when the EO was incorporated in juices at 5, 2.5, and 1.25 ll/ml, with the exception of Salmonella Enteritidis challenged with 1.25 ll/ml of the EO. Although some juices have previously been characterized as being hostile toward E. coli, L. monocytogenes, and Salmonella and as causing at least 5-log reductions in refrigerated storage (20, 29), only L. monocytogenes showed this reduction in pineapple juice samples without CCEO after incubation for 72 h. The rapid and steady inactivation effects of CCEO in pineapple juice are surprising considering the high MIC attributed to this EO in synthetic media. However, the intrinsic low pH and high acidity of pineapple juice probably contributed to these inhibitory effects, due to the increased sensitivity of bacteria to EOs at low pH values (mostly below pH 5.5, as found in pineapple juice) (13). The increase in inhibitory effects of EOs at low pH values has previously been cited as a consequence of the increase in the hydrophobicity of EOs, which provides easier dissolution of individual constituents in the lipids of target bacteria cell membrane (16). However, a previous study confirmed that high 8Brix levels and some intrinsic compounds (such as concentrated acids) might also play an important role in the bactericidal effects of juices (10). Thus, the intrinsic characteristics of pineapple juice might also induce the increased sensitization of tested bacteria to CCEO.
In view of the data obtained for the inactivation of test pathogenic bacteria, the ranking of bacterial sensitivity to CCEO in pineapple juice was L. monocytogenes . E. coli . Salmonella Enteritidis. This result is not surprising because gram-negative bacteria (e.g., E. coli and Salmonella Enteritidis) are generally less sensitive to EOs than gram-positive bacteria (e.g., L. monocytogenes) (5, 23). In gram-negative bacteria, the increased tolerance reflected the presence of the outer membrane, which limits the diffusion of hydrophobic compounds, such as the individual constituents of EOs, through lipopolysaccharide coverage (39). Considering that CCEO at 5, 2.5, and 1.25 ll/ml provoked the rapid reduction of pathogenic bacteria in pineapple juice, and considering that EOs impact the sensory aspects of juices (5), assays for assessing the impact on the quality parameters of pineapple juice were performed using CCEO at 2.5 and 1.25 ll/ml. Notably, CCEO did not alter the pH, titratable acidity, and 8Brix in pineapple juice samples. Moreover, samples with or without CCEO supplementation met the current Brazilian standard for unsweetened pineapple fruit juice, which requires titratable acidity (grams of citric acid per 100 g) 0.16 and 8Brix 6.0 (3, 4), and were consistent with data from previous studies on the quality of pineapple juices (19, 22). Although pineapple juice samples containing CCEO (at the tested concentrations) were acceptable for some sensory properties (appearance, odor, viscosity), noticeable unsatisfactory changes were found in taste, aftertaste, and overall acceptability. Thus, the overall acceptability of juice samples containing CCEO was likely affected by the taste and aftertaste perceptions of the panelists. Similar findings were reported for melon and watermelon juices containing citric acid (20 ll/ml) or cinnamon bark essential oil (2 ll/ml) and submitted to treatment with a high-intensity pulsed electric field (25). Hence, further studies about the reduction of the negative impacts of CCEO on some sensory aspects of pineapple juice are needed and should focus on the combination of CCEO at lower doses with other antimicrobial substances or procedures for potential use in juice preservation. This study demonstrates that CCEO effectively induces a 5-log reduction of a mixed composite of E. coli, L. monocytogenes, and Salmonella Enteritidis in pineapple juice in a short exposure time. Moreover, CCEO did not affect the physicochemical properties, particularly acidity, pH, and 8Brix, of pineapple juice. In contrast, CCEO negatively impacted the taste and aftertaste. Overall, these results suggest CCEO as an alternative antimicrobial substance to ensure the safety of pineapple juice; however,
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further studies regarding the combined use of CCEO (at lower doses than those tested in this study) with other nonthermal technologies are needed to establish a balance between safety and sensory aspects in this product.
ACKNOWLEDGMENT The authors thank the Coordination for Higher Personnel Improvements (CAPES, Brazil) for a scholarship awarded to the first author (C. J. B. Leite).
REFERENCES 1. Aligiannis, N. E., S. M. Kalpoutzakis, and J. B. Chinou. 2001. Composition and antimicrobial activity of the essential oils of two Origanum species. J. Agric. Food Chem. 49:4168–4170. 2. AOAC International. 2006. Official methods of analysis of AOAC International. AOAC International, Arlington, VA. 3. Brazil. 2003. Ministry of Agriculture, Livestock and Supply. Approve the technical regulation for establishment of identity and general quality parameters of tropical juice, the identity and general quality parameters of pineapple, acerola, spondia, cashew, guava, soursop, papaya, mango, mangaba, passion fruit and cherry; and the identity and general quality parameters pineapple, acerola, spondia, cashew, guava, soursop, papaya, mango, passion fruit, peach and cherry nectar. Normative Instruction number 12, 4 September 2003. (In Portuguese.) Available at: http://sistemasweb.agricultura.gov.br/sislegis/action/detalhaAto.do? method=consultarLegislacaoFederal. Accessed 4 April 2015. 4. Brazil. 2005. National Agency for Sanitary Surveillance. Approve the technical regulation for vegetable products, fruits and edible mushrooms. Resolution number 272, 22 September 2005. (In Portuguese.) Available at: http://portal.anvisa.gov.br/wps/wcm/ connect/ac09380047457ea18a84de3fbc4c6735/RDC_272_2005.pdf? MOD=AJPERES. Accessed 6 April 2015. 5. Burt, S. 2004. Essential oils: their antibacterial properties and potential applications in foods—a review. Int. J. Food Microbiol. 94:223–253. 6. Clinical and Laboratory Standards Institute. 2006. Methods for dilution and antimicrobial susceptibility tests for bacteria that grow aerobically. Approved standard M07-A7. Clinical and Laboratory Standards Institute, Wayne, PA. 7. Couto, D. S., L. M. C. Cabral, V. M. da Matta, R. Deliz, and D. G. C. Freitas. 2011. Concentration of pineapple juice by reverse osmosis: physicochemical characteristics and consumer acceptance. Food Sci. Technol. 31:905–910. 8. Duan, J., and Y. Zahao. 2009. Antimicrobial efficiency of essential oil and freeze-thaw treatments against Escherichia coli O157:H7 and Salmonella enterica ser. Enteritidis in strawberry juice. Food Microbiol. Saf. 74:131–137. 9. Elez-Mart´ınez, P., R. Soliva-Fortuny, and O. Mart´ın-Belloso. 2006. Comparative study on shelf life of orange juice processed by highintensity pulsed electric fields or heat treatment. Eur. Food Res. Technol. 222:321–329. 10. Enanche, E., and Y. Chen. 2007. Survival of Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes in cranberry concentrates at different 8Brix levels. J. Food Prot. 70:2072–2077. 11. Espina, L., M. Somolinos, A. Ait-Ouazzou, S. Cond´on, D. Garc´ıaGonzalo, and R. Paga´n. 2012. Inactivation of Escherichia coli O157:H7 in fruit juices by combined treatments of citrus fruit essential oils and heat. Int. J. Food Microbiol. 159:9–16. 12. Flessa, S., D. M. Lusk, and L. J. Harris. 2005. Survival of Listeria monocytogenes on fresh and frozen strawberries. Int. J. Food Microbiol. 101:255–262. 13. Gutierrez, J., C. Barry-Ryan, and P. Bourke. 2009. Antimicrobial activity of plant essential oils using food model media: efficacy, synergistic potential and interactions with food components. Food Microbiol. 26:142–150. 14. Han, Y., and R. H. Linton. 2004. Fate of Escherichia coli O157:H7 and Listeria monocytogenes in strawberry juice and acidified media at different pH values and temperatures. J. Food Prot. 67:2443–2452.
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37. U.S. Food and Drug Administration. 2009. 21 CFR 173. Title 21— Food and drugs, part 173—Secondary direct food additives permitted in food for human consumption. Available at: http://www.accessdata. fda.gov/scripts/cdrh/cfdocs/cfCFR/CFRSearch.cfm?CFRPart¼173. Accessed 22 May 2015. 38. U.S. Food and Drug Administration. 2001. Hazard analysis and critical control point (HACCP): procedures for the safe and sanitary processing and importing of juice. Fed. Regist. 66:6138– 6202. 39. Vaara, M. 1992. Agents that increase the permeability of the outer membrane. Microbiol. Rev. 56:395–411. 40. Wiegand, L., K. Hilpert, and R. E. Hancock. 2008. Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nat. Protoc. 3:163–175.
Inactivation of Escherichia coli, Listeria monocytogenes, and Salmonella Enteritidis by Cymbopogon citratus D.C. Stapf. Essential Oil in Pineapple Juice CAROLINE JUNQUEIRA BARCELLOS LEITE,1 JOSSANA PEREIRA DE SOUSA,1 ´ ALBERTO DA COSTA MEDEIROS,1 MARIA LUCIA ´ ˜ 1 JOSE DA CONCEIC ¸ AO, 2 ˜ VIVYANNE DOS SANTOS FALCAO-SILVA, AND EVANDRO LEITE DE SOUZA1* 1Laboratory
of Food Microbiology, Department of Nutrition, Health Sciences Center, and 2Laboratory of Genetics of Microorganisms, Department of Molecular Biology, Center of Exact and Natural Sciences, Federal University of Para´ıba, Joa˜o Pessoa, 58051900, Brazil MS 15-245: Received 5 June 2015/Accepted 13 August 2015
ABSTRACT In the present study, the efficacy of Cymbopogon citratus D.C. Stapf. essential oil (CCEO) to provoke a 5-log CFU/ml (5log) inactivation in a mixed composite of Escherichia coli, Listeria monocytogenes, and Salmonella enterica serovar Enteritidis in pineapple (Ananas comosus (L.) Merril) juice (48C) was assessed. Moreover, the effects of CCEO on the physicochemical and sensory quality parameters of pineapple juice were evaluated. The MIC of CCEO was 5 ll/ml against the composite mix examined. For L. monocytogenes and E. coli inoculated in juice containing CCEO (5, 2.5, and 1.25 ll/ml), a 5-log reduction was detected after 15 min of exposure. This same result was obtained for Salmonella Enteritidis incubated alone in pineapple juice containing CCEO at 5 and 2.5 ll/ml. Overall, Salmonella Enteritidis was the most tolerant and L. monocytogenes was the most sensitive to CCEO. The physicochemical properties (pH, titratable acidic [citric acid per 100 g], and soluble solids) of pineapple juice containing CCEO (2.5 and 1.25 ll/ml) were maintained. Juice containing CCEO (2.5 and 1.25 ll/ml) exhibited similar scores for odor, appearance, and viscosity compared with juice without CCEO. However, unsatisfactory changes in taste and aftertaste were observed in juices containing CCEO. These results suggest that CCEO could be used as an alternative antimicrobial compound to ensure the safety of pineapple juice, although CCEO at the tested concentrations negatively impacted its taste. Therefore, further studies are needed to determine the balance between microbial safety and taste acceptability of pineapple juice containing CCEO. Key words: Antibacterial effects; Essential oil; Pathogenic bacteria; Pineapple juice
Pineapple (Ananas comosus (L.) Merril) fruits and industrial by-products have a strong flavor and intense aroma and are used for fresh consumption and the production of a variety of derived industrial products, including juices (19). Pineapple fruit is a good source of fiber, vitamins, minerals, and phenolic compounds, with notable antioxidant capacities and potential health benefits (7). Because pineapple fruits are grown in the ground, it is virtually impossible to prevent microbial contamination of the rind of these fruits. Thus, when pineapple fruits with contaminated rinds are cut, different pathogenic and spoilage microorganisms can be transferred to the pulp (edible part) and, consequently, to the juice during or after extraction. Pineapple juice could be considered safe from pathogenic bacteria because of its intrinsic characteristics (low pH and high acidity) that create a hostile environment for bacterial survival. However, reports of the incidence, survival, and growth of pathogenic bacteria, such as Escherichia coli, Salmonella spp., and Listeria monocyto* Author for correspondence. Tel: þ 55 83 3216 7807; Fax: þ 55 83 3216 7094; E-mail: [email protected].
genes, in highly acidic fruits, such as apples, oranges, cranberries, and lemon juices, have changed the belief that there is small concern that fruit juices may harbor pathogenic bacteria (8, 25). Moreover, several outbreaks of E. coli and Salmonella spp. associated with the consumption of unpasteurized apple and orange juices have been reported (14, 18). Although L. monocytogenes has not often been implicated in outbreaks associated with unpasteurized fruit juices (10), this bacterium has previously been isolated from apple and raspberry juices (12, 36). Because of the acidity of pineapple juice (pH 3 to 4), similar to that of apple and orange juice, its potential to harbor pathogenic bacteria and cause foodborne illness remains a major concern. Thus, issues surrounding the safety of unpasteurized fruit juices have been of considerable interest to industry and to all those concerned with public health. The U.S. Food and Drug Administration (FDA) implemented a hazard analysis and critical control point (HACCP) regulation that includes a performance criterion for juice safety, commonly referred to as the juice HACCP regulation (38). The regulation requires juice processors to achieve a 5-log reduction of target microorganism(s) of public health significance when making
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juice, but this regulation does not require use of a specific method to achieve this inactivation level. Currently, pasteurization remains the dominant preservation procedure for eliminating pathogenic and spoilage organisms from juices. However, the heating used in pasteurization can cause a significant loss of nutrients (primarily vitamins and minerals) and undesirable changes in the fresh color and taste of juices (9). Although some fresh juices not formulated with chemical preservatives have been available in the marketplace, the juice industry still commonly adds chemical preservatives to control bacteria in their products (11). However, consumer concern about the potential risks of foods that contain chemicals and pathogenic microorganisms (32) has led to the demand for safer, fresher, and healthier foods with no or few chemical preservatives, thus creating a market demand for natural, nonthermal, and feasible technologies to ensure the microbial safety of juices (17). Innovative nonthermal technologies, such as pulsedelectric fields and high hydrostatic pressure, have been successfully applied to control pathogenic bacteria in juices (11). Essential oils (EOs) have also received increasing attention for their potential as inhibitors of microorganisms in fruit juices (30). The EOs are generally recognized as safe at the doses typically used in foods (1, 5) and have been approved by the FDA for use as flavoring agents in foods and beverages (37). The EO obtained from Cymbopogon citratus D.C. Stapf. (lemongrass) (CCEO) has shown widespectrum antimicrobial activity, including the inhibition of pathogenic and spoilage microorganisms (27, 31, 30). A variety of pineapple products (such as frozen pulp, unpasteurized juice, ready-to-eat mixed fruit salads, and minimally processed slices) containing ground C. citratus leaves are available in the market. These products are well accepted and are regarded as typical value-added products because of the distinct but pleasant ‘‘lemon-like’’ refreshing taste. Thus, pineapple juice could represent a potential food matrix to exploit the antimicrobial properties of CCEO. In the present study, we assessed the efficacy of CCEO to induce a 5-log reduction of a mixed composite of the potentially pathogenic bacteria E. coli, L. monocytogenes, and Salmonella enterica serovar Enteritidis in pineapple juice. The effects of CCEO on the physicochemical and sensory quality parameters of pineapple juice were assessed.
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adjusted using 1 M NaOH), washed with sterile distilled water, and dried for 1 h in a safety cabinet. Next, the fruits were aseptically peeled, chopped, and mixed with distilled water (1:1 rate) using a domestic blender (for 3 min). The mixture was filtered using a triple-cheesecloth layer and was sterilized by autoclaving (1218C, 1.1 atm, for 15 min). The juice was stored in 50-ml aliquots at 208C, and when required, an aliquot was thawed under refrigeration (4 6 18C) and was used for subsequent assays. The juice samples were submitted to physicochemical analysis after autoclaving (previous experiments revealed that the assessed physicochemical parameters in pineapple juice were unchanged after autoclaving). The noninoculated juice samples used in sensory analysis were prepared 24 h before assessing acceptability, and microbiological analysis revealed the satisfactory microbiological quality of these juice samples according to current Brazilian legislation (3). Test strains and composite preparation. The L. monocytogenes (ATCC 7644), E. coli (UFPEDA 224), and Salmonella Enteritidis (UFPE 414) were obtained from the Collection of Microorganisms, Department of Antibiotics, Federal University of Pernambuco (Recife, Brazil). The stock cultures were maintained in BHI broth plus glycerol (15 g/100 ml) at 208C. Each strain was grown in BHI broth at 378C for 20 to 24 h (stationary growth phase), harvested through centrifugation (4,500 3 g, 15 min, 48C), washed twice in sterile saline solution (0.85 g/100 ml), and resuspended in sterile saline solution to obtain cell suspensions at which the optical density reading at 660 nm ranged from 0.08 to 0.13. This suspension provided viable cell counts of approximately 8 log CFU/ml of each strain when pour plated in BHI agar (21, 40). The mixed composite of the three strains tested was obtained by mixing the obtained bacterial suspensions at a ratio of 1:1:1 for a maximum of 15 min after the preparation of each pure suspension (final count of each strain approximately 8 log CFU/ml) (28). Identification of CCEO constituents. The constituents in CCEO were identified through gas chromatography coupled with mass spectrometry (GC-MS; CGMS-QP2010 Ultra, Shimadzu, Kyoto, Japan) performed under the following conditions: RTX5MS capillary column (30 m by 0.25 mm by 0.25 lm); program temperature, 60 to 2408C (38C/min); injector temperature, 2508C; detector temperature, 2208C; carrier gas, helium adjusted to 0.99 ml/min; ionizing energy, 70 eV; and mass range, 40 to 500. The spectral bank of GC-MS, NIST/EPA/NIH Mass Spectral Database (version 1.7, National Institute of Standards Technology, Norwalk, CT) was used to identify the individual essential oil constituents. The constituents were quantified after the areas of each detected constituent were normalized and expressed as a percentage area.
MATERIALS AND METHODS CCEO and pineapple fruits. The CCEO (batch 0061; pH 6.59) was obtained from Laszlo Aromatherapy Ltd. (Belo Horizonte, Brazil). The CCEO emulsions were prepared in brain heart infusion (BHI) broth (pH 7.40; Himedia, Mumbai, India) at concentrations of 80 (pH 7.25), 40 (pH 7.29), 20 (pH 7.31), 10 (pH 7.35), 5 (pH 7.36), 2.5 (pH 7.38), 1.25 (pH 7.39), 0.625 (pH 7.39), and 0.312 (pH 7.39) ll/ml using Tween 80 (1.5%, vol/vol; Himedia) as a stabilizing agent (28). The pineapple (A. comosus) fruits were purchased in the commercial maturation stage from a local wholesale distributor and were selected for uniformity in size, form, appearance, and the absence of mechanical injuries or visible infections. For juice preparation, the fruits were surface disinfected after a 15-min immersion in a sodium hypochlorite solution (150 ppm, pH 7.2
Determination of the MIC of CCEO. A modified microtiter plate assay was used to determine the MIC of CCEO according to a standard method (6), with minor modifications related to the use of a stain to detect bacterial growth and survival (40). Approximately 50 ll of CCEO solution (80 to 0.312 ll/ml) was dispensed into each well of a 96-well microplate containing 100 ll of BHI broth. Subsequently, 50 ll of bacterial suspension was added to each well (final viable cells count of approximately 7 log CFU/ml). The microplate was loosely wrapped with cling wrap to prevent bacterial dehydration and CCEO volatilization. Each plate included either a control (without CCEO), inoculated (positive control), or uninoculated (negative control) sample. The system was statically incubated at 378C for 24 h. Subsequently, a 30-ll aliquot of resazurin (0.01 g/100 liters, wt/vol) (Inlab, Sa˜o Paulo, Brazil), prepared in aqueous solution, was added to each well. The color
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TABLE 1. Gas chromatography–mass spectrometry analysis of the essential oil from C. citratus D.C. Stapf.a Constituents
% in essential oil
6-Methyl-5-hepten-2-ona Dipentyl-ketone Linalool Neral Geraniol Geranyl-acetate Caryophyllene
1.77 1.06 1.03 31.74 46.16 4.34 2.02
a
Constituents detected in concentration 1%.
changes were visually assessed after 20 min at 378C. Bacterial growth was indicated as color changes from purple to pink (or colorless). The MICs were confirmed as the lowest concentrations of CCEO capable of inhibiting bacterial growth. Effects of CCEO on survival of bacterial strains in pineapple juice. The effects of different CCEO concentrations on the survival of the bacterial mixed composite in pineapple juice under refrigerated storage were assessed using the viable cell count method. Initially, 150 ll of the tested bacterial mixed composite was inoculated into 5,850 ll of separated juice samples (final viable cell counts of approximately 7 log CFU/ml) containing CCEO at the desired final concentrations (5, 2.5, 1.25, and 0.6 ll/ ml). The different systems were gently hand shaken for 30 s and were subsequently incubated at 4 6 18C. At different exposure times (5, 10, 15, 30, and 45 min; 1, 2, 4, 8, 12, 24, 48, and 72 h), a 20-ll aliquot was obtained from each system to directly inoculate, using the microdrop technique (15), onto selective agar: eosin– methylene blue agar (Himedia) for E. coli, Listeria selective agar plus Listeria Selective Supplement II (Himedia) for L. monocytogenes, and Salmonella-Shigella agar (Himedia) for Salmonella Enteritidis (34). In parallel, 100-ll aliquots of each system were serially diluted in sterile saline solution; subsequently, 20 ll of each dilution was inoculated onto the same selective medium for each type of bacteria. Control juices (without CCEO) were similarly assayed. The plates were incubated at 378C for 24 to 48 h. Plates inoculated with aliquots collected from juice samples containing CCEO were incubated for 24 h longer, at adequate temperature, than the samples collected from control juice. The results are expressed as duration of exposure to each tested concentration of CCEO to establish a 5-log CFU/ml reduction in initial viable cell counts (CFU per milliliter at time zero) of each of the tested strains comprising the mixed composite (log N0 N, where N0 and N were the initial count and count after incubation, respectively, for indicated times at 48C). The detection limit of the viable cell count procedure was 2 log CFU/ml for all experiments and strains tested. Analysis of physicochemical and sensory parameters of pineapple juice. Samples of pineapple juice containing CCEO (2.5 and 1.25 ll/ml) were assessed for some physicochemical quality parameters at 0 (baseline, just after the addition of CCEO) and 72 h of storage at 4 6 18C. The titratable acidity was determined using phenolphthalein as an indicator with 0.1 N NaOH, and the results were expressed as grams per 100 g (equivalent of citric acid). pH values were determined using a digital potentiometer (model Q400AS, Quimis, Sa˜o Paulo, Brazil). The soluble solids content (8Brix) was determined using a digital
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refractometer (model HI 96801, Hanna Instruments, Sa˜o Paulo, Brazil) (2). Sensory evaluation was performed via acceptability tests. Sixty panelists (20 to 35 years old) were preselected according to interests and frequency of pineapple juice consumption. Panelists worked in individual booths with controlled temperature and lighting. Each panelist received three juice samples containing the different concentrations of CCEO tested (1.25 and 2.5 ll/ml). The CCEO was incorporated in juice samples 24 h prior to the sensory test and was kept at 4 6 18C. Samples of juice without CCEO were tested as controls. The different juice samples were served in 30-ml aliquots in white disposable cups coded with a randomized three-digit number. Immediately after removal from refrigeration, the samples were served simultaneously, using a blind method of random sequence. The panelists were asked to use low-salt crackers and water to cleanse their palates between the assessed samples. The acceptability of appearance, odor, viscosity, taste, aftertaste, and overall acceptability were evaluated on a 9-point hedonic scale, ranging from 1 (dislike very much) to 9 (like very much) (35). Reproducibility and statistics. All assays were performed in triplicate in three independent experiments (repetitions), and the results are expressed as the average of the tests. For assays to determine the MIC, the result was expressed as a modal value because the MIC was the same in all repetitions. For physicochemical and sensory parameters, statistical analyses were performed to determine significant differences (P 0.05) among treatments using analysis of variance, followed by Tukey’s post hoc test using the computational software GraphPad Prism 6.0 (GraphPad Software Inc., La Jolla, CA).
RESULTS Identification of CCEO constituents. The GC-MS analysis of CCEO showed the presence of seven different constituents in amounts higher than 1% of the EO total mass (Table 1). The monoterpenes geranial (b-citral isomer) (46.16%) and neral (a-citral isomer) (31.74%) were identified as the major constituents of CCEO. Other compounds, such as geranyl acetate (4.34%) and caryophyllene (2.02%), were detected in lower amounts. Antimicrobial assays. The MIC of CCEO was 5 ll/ml against the composite mix containing E. coli UFEPEDA 224, L. monocytogenes ATCC 7644, and Salmonella Enteritidis UFEPEDA 414 (data not shown). The incorporation of CCEO in pineapple juice at all tested concentrations (5, 2.5, 1.25, and 0.6 ll/ml) caused a decrease in viable counts of E. coli, L. monocytogenes, and Salmonella Enteritidis. For L. monocytogenes and E. coli inoculated in juice containing CCEO at 5, 2.5, or 1.25 ll/ml, a 5-log reduction in viable counts was detected after 15 min of exposure (Table 2). This same reduction (after 15 min) was observed for Salmonella Enteritidis alone when inoculated in juice containing CCEO at 5 or 2.5 ll/ml. A fourfold longer exposure time (1 h) was needed for a 5-log reduction of Salmonella Enteritidis in juice containing CCEO at 1.25 ll/ml compared with the other tested bacteria. In juice samples containing CCEO at 0.6 ll/ml, 5-log reductions in E. coli and L. monocytogenes counts were observed after 1 h and 45 min, respectively, whereas 12 h of
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TABLE 2. Exposure time to achieve 5-log CFU/ml reduction in a mixed population of E. coli, L. monocytogenes, and Salmonella Enteritidis in pineapple fruit juice containing different concentrations of CCEO and stored at 4 6 18 Ca Duration of exposure for a 5-log reduction in initial counts Concn of CCEO in pineapple juice (ll/ml)
E. coli
5 2.5 1.25 0.6 Control (0)
15 min 15 min 15 min 1h ND
a
L. monocytogenes
15 15 15 45 72
Salmonella Enteritidis
min min min min h
15 min 15 min 1h 12 h ND
CCEO, essential oil from C. citratus D.C. Stapf. ND, 5-log reduction was not detected.
incubation was needed to achieve a similar reduction of Salmonella Enteritidis. Because a 5-log reduction was observed, no viable cells were detected at longer assessed time intervals (up to 72 h). In juice samples without CCEO, only L. monocytogenes presented a 5-log reduction as detected after 72 h of cultivation. In the same samples, 3.3- and 1.29-log reductions were observed for E. coli and Salmonella Enteritidis, respectively, after 72 h of cultivation.
tested concentrations did not differ (P . 0.05) in scores for all assessed sensory attributes. Appearance, odor, and viscosity had a mean score of ‘‘like slightly’’ on the hedonic scale for samples of pineapple juice containing CCEO, whereas taste, aftertaste, and overall acceptability always had a mean score of ‘‘neither like nor dislike.’’ The mean scores for all assessed sensory parameters were ‘‘slightly like’’ for control juice samples.
DISCUSSION Physicochemical and sensory analysis of pineapple juice. Pineapple juice samples with or without CCEO (2.5 and 1.25 ll/ml) were evaluated for pH, titratable acidity, and 8Brix immediately after the addition of CCEO and after refrigeration (4 6 18C) for 72 h. The fruit juice samples with or without CCEO presented no difference (P . 0.05) in the evaluated physicochemical parameters at the assessed time intervals. Overall, the addition of CCEO preserved the assessed physicochemical properties (quality) of pineapple juice samples (Table 3). The addition of CCEO at 2.5 and 1.25 ll/ml did not affect (P . 0.05) the appearance, odor, and viscosity of pineapple juice after refrigeration for 24 h (Table 4). However, both tested CCEO concentrations negatively affected the taste and aftertaste of pineapple juice. Samples of pineapple juice without CCEO received the highest scores (P 0.05) for taste, aftertaste, and overall acceptability. Juice containing CCEO, at both tested concentrations, exhibited similar scores (P . 0.05) for odor, appearance, and viscosity compared with control samples (without CCEO). In addition, samples containing CCEO at both
The CCEO tested in this study inhibited the growth of a mixed composite of E. coli, L. monocytogenes, and Salmonella Enteritidis in synthetic broth. However, based on the detected MIC (5 ll/ml), the CCEO tested could be classified as a less effective bacterial inhibitor (1) of the target mixed bacterial composite. The detected MIC of CCEO could be intrinsically related with the individual constituents forming this EO, where neral (b-citral cisisomer [citral B]) and geraniol (a-citral trans-isomer [citral A]) were detected as major constituents. Based on the results of previous studies, the following approximate general rank (in decreasing order of antibacterial activity) of individual constituents is proposed: eugenol . carvacrol–thymol– cinnamic acid . basil methyl chavicol . cinnamaldehyde . citral-geraniol (5), where citral is not classified as possessing the strongest antimicrobial effects. The presence of high amounts of oxygenated monoterpenes in CCEO (77.90%), particularly the citral compounds neral and geraniol (terpene aldehydes), could be responsible for its inhibitory effects against the tested bacterial strains. Researchers have proposed that the antimicrobial activity of
TABLE 3. Physicochemical parameters of pineapple juice supplemented with or without CCEO and stored at 4 6 18 Ca Scores for physicochemical parameters after 0 and 72 h of storage, avg (6SD) Titratable acidityb
pH
8Brix
Concn of CCEO in pineapple juice (ll/ml)
0h
72 h
0h
72 h
0h
72 h
2.5 1.25 Control (0)
3.82 (60.31) 3.88 (60.45) 3.92 (60.24)
3.91 (60.21) 3.93 (60.33) 3.96 (60.36)
0.19 (60.03) 0.18 (60.02) 0.17 (60.03)
0.18 (60.02) 0.17 (60.04) 0.17 (60.02)
10.18 (60.81) 10.15 (60.76) 10.17 (60.55)
10.87 (60.68) 10.42 (60.55) 10.40 (60.43)
a
b
CCEO, essential oil from C. citratus D.C. Stapf. No significant differences (P . 0.05) were found among the mean values, according to Tukey’s test. Values are given as grams of citric acid per 100 g of juice.
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TABLE 4. Scores for sensory attributes of pineapple juice with or without CCEO and stored at 4 6 18 C for 24 ha Scores for sensory attributes, avg (6SD) Concn of CCEO in juice (ll/ml)
2.5 1.25 Control (0) a
Appearance
6.39 (61.84) 6.19 (62.27) 6.62 (61.81)
Odor A A A
6.43 (61.98) 6.44 (62.13) 7.03 (61.80)
Viscosity A A A
6.11 (61.58) 6.21 (61.66) 7.21 (61.45)
Taste A A A
5.25 (61.85) 5.67 (62.05) 7.97 (60.84)
Aftertaste A A B
5.38 (61.72) 5.75 (61.95) 7.25 (61.15)
Overall acceptance A A B
5.49 (61.75) 5.57 (61.94) 7.52 (61.07)
A A B
CCEO, essential oil from C. citratus D.C. Stapf. Acceptability of each attribute was evaluated using a 9-point hedonic scale, ranging from 1 (dislike very much) to 9 (like very much). Values followed by different letters, in the same column, are significantly different (P 0.05), according to Tukey’s test.
EOs without phenolic compounds as major individual constituents, such as CCEO, could be associated with membrane disruption of target cells through lipophilic compounds, primarily terpene hydrocarbons (e.g., caryophyllene, a-pinene, p-cymene, and c-terpinene) (24). These terpene hydrocarbons induce swelling of the bacterial cell membrane to facilitate the transportation of citral into the cell (32), where this compound promotes dramatic effects on the cell envelope, proton motive force, and intracellular ATP content (33). Given that EOs could negatively impact the sensory acceptability of juices (26), the assays of bacterial inactivation were performed using pineapple juice samples containing the CCEO at concentrations similar to the MIC (5 ll/ml) and at concentrations as low as the 1/2 MIC (2.5 ll/ ml), 1/4 MIC (1.25 ll/ml), and 1/8 MIC (0.6 ll/ml). This approach was used to detect the lowest assayed CCEO concentrations capable of achieving the regulatory 5-log reduction of target pathogenic bacteria. Notably, CCEO was effective to cause 5-log decreases in E. coli, L. monocytogenes, and Salmonella Enteritidis at all tested concentrations in pineapple juice. These effects against target bacteria occurred at a maximum exposure time of 15 min when the EO was incorporated in juices at 5, 2.5, and 1.25 ll/ml, with the exception of Salmonella Enteritidis challenged with 1.25 ll/ml of the EO. Although some juices have previously been characterized as being hostile toward E. coli, L. monocytogenes, and Salmonella and as causing at least 5-log reductions in refrigerated storage (20, 29), only L. monocytogenes showed this reduction in pineapple juice samples without CCEO after incubation for 72 h. The rapid and steady inactivation effects of CCEO in pineapple juice are surprising considering the high MIC attributed to this EO in synthetic media. However, the intrinsic low pH and high acidity of pineapple juice probably contributed to these inhibitory effects, due to the increased sensitivity of bacteria to EOs at low pH values (mostly below pH 5.5, as found in pineapple juice) (13). The increase in inhibitory effects of EOs at low pH values has previously been cited as a consequence of the increase in the hydrophobicity of EOs, which provides easier dissolution of individual constituents in the lipids of target bacteria cell membrane (16). However, a previous study confirmed that high 8Brix levels and some intrinsic compounds (such as concentrated acids) might also play an important role in the bactericidal effects of juices (10). Thus, the intrinsic characteristics of pineapple juice might also induce the increased sensitization of tested bacteria to CCEO.
In view of the data obtained for the inactivation of test pathogenic bacteria, the ranking of bacterial sensitivity to CCEO in pineapple juice was L. monocytogenes . E. coli . Salmonella Enteritidis. This result is not surprising because gram-negative bacteria (e.g., E. coli and Salmonella Enteritidis) are generally less sensitive to EOs than gram-positive bacteria (e.g., L. monocytogenes) (5, 23). In gram-negative bacteria, the increased tolerance reflected the presence of the outer membrane, which limits the diffusion of hydrophobic compounds, such as the individual constituents of EOs, through lipopolysaccharide coverage (39). Considering that CCEO at 5, 2.5, and 1.25 ll/ml provoked the rapid reduction of pathogenic bacteria in pineapple juice, and considering that EOs impact the sensory aspects of juices (5), assays for assessing the impact on the quality parameters of pineapple juice were performed using CCEO at 2.5 and 1.25 ll/ml. Notably, CCEO did not alter the pH, titratable acidity, and 8Brix in pineapple juice samples. Moreover, samples with or without CCEO supplementation met the current Brazilian standard for unsweetened pineapple fruit juice, which requires titratable acidity (grams of citric acid per 100 g) 0.16 and 8Brix 6.0 (3, 4), and were consistent with data from previous studies on the quality of pineapple juices (19, 22). Although pineapple juice samples containing CCEO (at the tested concentrations) were acceptable for some sensory properties (appearance, odor, viscosity), noticeable unsatisfactory changes were found in taste, aftertaste, and overall acceptability. Thus, the overall acceptability of juice samples containing CCEO was likely affected by the taste and aftertaste perceptions of the panelists. Similar findings were reported for melon and watermelon juices containing citric acid (20 ll/ml) or cinnamon bark essential oil (2 ll/ml) and submitted to treatment with a high-intensity pulsed electric field (25). Hence, further studies about the reduction of the negative impacts of CCEO on some sensory aspects of pineapple juice are needed and should focus on the combination of CCEO at lower doses with other antimicrobial substances or procedures for potential use in juice preservation. This study demonstrates that CCEO effectively induces a 5-log reduction of a mixed composite of E. coli, L. monocytogenes, and Salmonella Enteritidis in pineapple juice in a short exposure time. Moreover, CCEO did not affect the physicochemical properties, particularly acidity, pH, and 8Brix, of pineapple juice. In contrast, CCEO negatively impacted the taste and aftertaste. Overall, these results suggest CCEO as an alternative antimicrobial substance to ensure the safety of pineapple juice; however,
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further studies regarding the combined use of CCEO (at lower doses than those tested in this study) with other nonthermal technologies are needed to establish a balance between safety and sensory aspects in this product.
ACKNOWLEDGMENT The authors thank the Coordination for Higher Personnel Improvements (CAPES, Brazil) for a scholarship awarded to the first author (C. J. B. Leite).
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