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Innovative Food Science and Emerging Technologies 36 (2016) 120–127

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Antimicrobial and antioxidant activity of essential oil from pink pepper tree (Schinus terebinthifolius Raddi) in vitro and in cheese experimentally contaminated with Listeria monocytogenes Guilherme da Silva Dannenberg ⁎, Graciele Daiana Funck, Fábio José Mattei, Wladimir Padilha da Silva, Ângela Maria Fiorentini Department of Agroindustrial Science and Technology, Federal University of Pelotas, 96010-900, Pelotas, Brazil

a r t i c l e

i n f o

Article history: Received 10 March 2016 Received in revised form 8 June 2016 Accepted 13 June 2016 Available online 15 June 2016 Keywords: Antimicrobial activity Antioxidant activity Biopreservation Pink pepper tree Essential oil Cheese

a b s t r a c t Natural compounds with preservative activity have gained prominence in the field of food science as an alternative to traditional additives. To be effective, biopreservatives must have antioxidant and/or antimicrobial activities, characteristics often found in the essential oils (EO). This study aimed to verify the antimicrobial and antioxidant activity of EO from pink pepper tree fruit. Antimicrobial activity was evaluated in vitro on 18 bacteria, and in situ (Minas-type fresh cheese) against Listeria monocytogenes during storage (30 days/4 °C). The EO from ripe fruit showed the greatest activity in in vitro tests (MBC of 6.8 mg/mL for L. monocytogenes) and exhibited biopreservative activity in situ, having reduced the development of L. monocytogenes by 1.3 log CFU/g in 30 days. The values of peroxides and malonaldehydes were reduced by 3 Meq O2/Kg and 0.15 mg MDA/Kg, respectively, in 30 days. Results demonstrate that this EO has the potential to act as a preservative in food. Industrial relevance: The pink pepper tree (Schinus terebinthifolius Raddi) is a plant with favorable features for industrial use, but little exploited by the food industry so far. In this work, the essential oil (EO) of the pink pepper tree was presented as an alternative to us of preservatives traditionally applied in food. For this, antimicrobial and antioxidant activities of the EO were evaluated and discussed, analyzing its effects initially in vitro and after in situ, in order to infer the technological potential for application this extract may have use as a food biopreservative. © 2016 Elsevier Ltd. All rights reserved.

1. Introduction In all sectors of the food industry, preservation of food products is extremely important (Sokmen et al., 2004) and principally involves controlling the growth of microbes (Kulisic, Radonic, Katalinic, & Milos, 2004), which are responsible for generating risks to consumer health as well as food spoilage. Microbial contamination has a significant influence on the quality of food, and can compromise both safety, due to the presence of pathogenic bacteria, and preservation status, via the multiplication of spoilage bacteria that reduce product shelf-life. In industry, synthetic preservatives are typically used to control these undesirable microbiological and chemical alterations in food (Bajpai, Baek, & Kang, 2012). However, these additives are not pleasing to many consumers, who demand foods without what they perceive as artificial and harmful chemicals (Calo, Crandall, O'Bryan, & Ricke, 2015), generating a strong demand for products with fewer synthetic additives as well as natural substitutes (Alzoreky & Nakahara, 2003). This increased demand illustrates the relevance of this topic beyond the fields ⁎ Corresponding author. E-mail address: [email protected] (G. da Silva Dannenberg).

http://dx.doi.org/10.1016/j.ifset.2016.06.009 1466-8564/© 2016 Elsevier Ltd. All rights reserved.

of food technology and public health to the disciplines of economics and marketing. In this context, natural substances that demonstrate biopreservative activity with similar or even greater capacity than synthetic preservatives are gaining prominence (Al-fatimi, Wurster, Schr, & Lindequist, 2007). Essential oils (EO) are secondary metabolites produced by plants, which confer resistance to adverse conditions such as climatic variation and insect and microorganism attack, and they are among the class of natural extracts used as an alternative to synthetic preservatives. EO are composed of a large number of biologically active molecules (Kavoosi & Rowshan, 2013), which confer various properties including antimicrobial and antioxidant activity (Salgueiro, Martins, & Correia, 2010). Besides these properties of interest, the availability of raw material as well as the ease of cultivation of the plant are important factors that may restrict or encourage the industrial use of certain EO. The pink pepper tree (Schinus terebinthifolius Raddi), also known as pink pepper, native to Brazil, Paraguay, and Argentina, is widely distributed throughout South America, especially on the Brazilian coast (Agra, Silva, Basílio, Freitas, & Barbosa-Filho, 2008). Studies on the EO from this species are limited; however, previous work determined the chemical composition, identifying predominance of monoterpenes such as α-

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pinene, β-Pinene, myrcene and limonene, followed by a lower concentration of sesquiterpenes such as D-germacrene (Cavalcanti et al., 2015; Gois et al., 2016). Other studies have verified the in vitro antimicrobial activity of the EO from pink pepper tree fruits (Aumeeruddyelalfi, Gurib-fakim, & Mahomoodally, 2015). The antioxidant activity has been evaluated in EO from pink pepper tree leaves (Uliana, Fronza, Vargas, Andrade, & Scherer, 2016). However, previous studies with EO from pink pepper tree were restricted to evaluation in vitro. To our knowledge, there are no studies that have evaluated pink pepper tree EO in food (in situ). As previously reported, the antimicrobial and antioxidant activity of pink pepper tree extracts coupled with the wide availability of plant material justify carrying out research for future industrial applications. The application of EO in foods may initially be considered in view of the fruit and pink pepper extracts having been used for human consumption without reports of health damage. The fruits have been used as condiments in cooking for many years. Extracts of this plant are also widely used in popular medicine for the treatment of various diseases. The pharmaceutical industry makes use of this EO in cosmetics for its aroma (Uliana et al., 2016), and EO are classified by the FDA as GRAS (generally recognized as safe) (Ghabraie, Vu, Tata, Salmieri, & Lacroix, 2016). These factors justify studies evaluating the pink pepper EO as a food preservative; however, for effective commercial application in the food industry, detailed toxicological studies need to be performed to ensure safety. The purpose of this study was to verify the antimicrobial and antioxidant activity of EO from pink pepper tree fruit at two ripening stages by qualifying and quantifying in vitro antimicrobial activity against 18 food bacterial strains; it also aimed to evaluate the preservative activity of EO in situ in Minas-type fresh cheese experimentally contaminated with Listeria monocytogenes.

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in a parallel work (unpublished data), identified β-myrcene (41%), βcuvebene (12%) and limonene (9%) as major constituents of these extracts. 2.3. Bacterial strains The microorganisms used in this study (Table 1) were selected based on their importance in food, both as undesirable bacteria (pathogenic and spoilage), or as technologically applicable bacteria (starter and probiotic cultures), and were obtained from the Food Microbiology Laboratory's bacteria collection in the Department of Agroindustrial Science and Technology (DCTA) at UFPel. 2.4. Antimicrobial activity in vitro 2.4.1. Disk-diffusion The antimicrobial activity of the EO was initially evaluated by the disk-diffusion technique (CLSI, 2012b) with adaptations. Bacteria were activated in Brain Heart Infusion broth (BHI-Oxoid®) for 12 h, and bacterial concentration was adjusted to 8.18 log CFU/mL (0.5 McFarland) in peptone water (Acumedia®). Inoculum were spread uniformly with a sterile swab (Absorve®) on the surface of petri plates (Cralplast®, 90 × 15 mm) containing 4 mm of Mueller-Hinton Agar (Oxoid®) with pH 7 ± 0.2. A sterile paper disk (Laborclin®) 6 mm in diameter was added to the center of each plate, upon which 10 μL of EO were placed. The petri plates were incubated at 37 °C, and the presence of inhibition zones around the paper disk were verified after 24 h with a digital pachymeter (King.tools®). The plates of starter cultures and probiotic bacteria were incubated in anaerobic jars (2.5 L - Permution®) with AnaeroGen sachets (Oxoid®), at 37 °C for 48 h. Antimicrobial activity of EO against each bacterial strain was tested in triplicate, and the evaluation was repeated a second time.

2. Material and methods 2.1. Pink pepper tree The fruit samples used in this study were collected from adult trees located on the campus of the Federal University of Pelotas (UFPel) in Capão do Leão, RS, 31°48′0459″ latitude and 52°24′5532″ longitude, and were botanically identified as Schinus terebinthifolius Raddi based on similarity to UFPel Department of Botany herbarium specimen 25.131. Both immature (green) and mature (red) fruit were harvested. Fruit color was determined by colorimeter (MINOLTA® CR 300). Green fruit was considered to be those having mean values of L = 37.90; a = − 2.28; b = 10.54, and mature fruit with color intensity equal to or higher than mean values of L = 36.42; a = 17.19; b = 6.75. The value “L” refers to luminosity varying from white to black, “a” to the coloration in the interval from red to green, and “b” to the coloration in the interval from yellow to blue (Lorenzo, Gómez, & Fonseca, 2014). 2.2. Extraction of essential oils Pink pepper tree fruit were frozen in liquid nitrogen and ground in a ball mill (MARCONI® MA 350), and EO was extracted by hydrodistillation using clevenger apparatus. The resulting EO was dehydrated by centrifugation at 6600g for 60 s in a refrigerated centrifuge (Eppendorf F® Centrifuge 6430R) and subsequently filtered with anhydrous sodium sulfate (Na2SO4 - SYNTH®). EO was stored in an amber glass bottle, and maintained at −80 °C until analysis. The yield (y) was calculated as the ratio between the mass of the obtained EO (m2) and fruit mass used in the process (m1): y = (m2/ m1) × 100. The green fruits and mature fruits presented means EO yield (w/w) of 1.4% and 3.6% respectively. Both EO were slightly yellowish translucent liquid, and presented refractive index appropriate to for pure EO as defined by the Brazilian pharmacopoeia (1.481). The EO chemical composition, performed by chromatographic analysis GC/MS

2.4.2. Minimum inhibitory concentration (MIC) The lowest concentration of EO required to inhibit the growth of the tested microorganisms was determined by the broth microdilution technique (CLSI, 2012a) with adaptations. Dilutions of the EO were performed in Tryptone Soy Broth supplemented with 0.6% yeast extract (TSB-YE-Oxoid®) for L. monocytogenes and Brain Heart Infusion Broth (BHI-Oxoid®) for all other bacteria. For both types of broth, pH was adjusted to 7.2 to 7.4, and 3% tween 80 (Vetec®) was added. Essential oils were initially diluted to 25% (m/v) in the cultivation broths, producing concentrations of 217.58 mg/mL and 218.23 mg/mL of EO from green fruits (GEO) and EO from ripe, mature fruits (MEO), respectively, due to the difference in density between them (0.870 and 0.873 g/mL). Serial dilutions were then performed in 96-well microdilution plates until the minimum concentrations of 0.11 mg/mL of GEO and 0.10 mg/mL of MEO were reached. Bacterial concentrations were adjusted to 1.5 × 108 CFU/mL (0.5 McFarland) and analyzed by spectrophotometer (Jenway® 6705) at a wavelength of 625 nm until absorbance read between 0.08 and 0.1 (CLSI, 2012b). Ten microliters of the inoculum were added to each well of the microdilution plates that contained 190 μL of the respective mixtures of broth and EO. Inoculum in broth without inhibitors was used as a positive control, and broth without inoculum was used as a negative control. The plates were incubated at 37 °C for 24 h, and readings were performed with a plate reader (Robonik® Readwel plate) at a wavelength of 620 nm (Martins, Arantes, Candeias, Tinoco, & Cruz-Morais, 2014; Ojeda-sana, Baren, Elechosa, Juárez, & Moreno, 2013). 2.4.3. Minimum bactericidal concentration (MBC) The lowest concentration of GEO and MEO capable of inducing microbial cell death was determined by inoculating Muller-Hinton (Oxoid®) agar plates with 10 μL aliquots from the wells that did not grow in the MIC test. Microdilution plates were incubated at 36 °C,

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Bacteria

Listeria monocytogenes Staphylococcus aureus Streptococcus mutans Bacillus cereus Corynebacterium fim Escherichia coli Salmonella typhimurium Shigella dysenteriae Enterobacter aerogenes Pseudomonas aeruginosa Aeromonas hydrophila Lactobacillus plantarum Leuconostoc mesenteroides Lactobacillus delbrueckii subsp. bulgaricus Lactococcus lactis subsp. lactis Bifidobacterium animalis subsp. lactis Bifidobacterium animalis subsp. lactis Lactobacillus brevis

Gram

Positive Positive Positive Positive Positive Negative Negative Negative Negative Negative Negative Positive Positive Positive Positive Positive Positive Positive

ATCC 7644 ATCC 6538 ATCC 700610 ATCC 11778 NCTC 7547 ATCC 8739 ATCC 14028 ATCC 13313 ATCC 13048 ATCC 15442 IOC/FDA 11036 AJ2a ATCC 8293 INCQS 00383 DY13 LAFTI B94® Bb-12® ATCC 367

and the lowest concentration that showed no growth after 24 h was determined (Ait-ouazzou et al., 2011). 2.5. Antioxidant activity in vitro The in vitro antioxidant activity of the EO was determined by capture capacity of the free radical DPPH (2,2, difenil-2-picrilhidrazil), following the methodology proposed by QUASSINTI (2013). The calibration curve was plotted by correlating absorbance and the concentration of the free radical DPPH measured at 515 nm. Eight dilutions of the free radical (10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM e 80 μM) were utilized. Serial dilution of the EO was performed in acetone (SYNTH®). The analysis was conducted in microdilution plates, containing each dilution in triplicate, a blank with only solvent, and the eight dilutions of the free radical DPPH for the construction of the curve. Absorbance was measured after 30 min at a wavelength of 515 nm on a plate reader (Robonik® Readwel plate). Results were calculated according to HUSSEIN (2013) and expressed in EC50, referring to the concentration of EO required to reduce the initial concentration of the free radical DPPH by 50%. 2.6. Preparation of the food system (cheese) To test the antimicrobial activity of pink pepper tree EO in situ, Minas-type fresh cheese contaminated with L. monocytogenes (ATCC 7644) was treated with MEO, as MEO showed higher antibacterial activity in the in vitro test. The cheese was made with pasteurized milk (Santa Silvana®), which was heated and maintained at 37 °C, and then supplemented with 0.2% calcium chloride (CaCl2 - SYNTH®) to obtain a concentration of 200 mg.kg−1 ionic calcium. Protein coagulation was promoted enzymatically with the application of 0.1% (v/v) chymosin (Chr.Hansen®) with coagulation power 1:3000/75 IMCU. After 1 h of rest, the clot was cut into cubes of about 5 cm3 and gently agitated for 15 min to promote syneresis. The casein mass was processed in a laminar flow hood and divided into eight equal portions that were used to test antimicrobial activity of MEO, as shown in Table 2. The L. monocytogenes strain was activated in broth (TSB - Oxoid®) and cultured for 24 h on TSA agar (Oxoid®) with yeast extract. The concentration was adjusted to 107 CFU/mL starting from a microbial suspension equivalent to the standard of 0.5 McFarland (1.5 × 108 CFU/ g), with a final cell concentration of 2 × 104 CFU/g used in treatments CL, L1, L2, and L3. For treatments that included pathogen inoculation, equal aliquots of inoculum were added to each portion of the cheese. The same procedure was adopted for the implementation of MEO. After mixing, the casein masses were placed in polypropylene containers, enclosed with a plastic film (Polyvinyl chloride - PVC), and stored for 30 days at 4 °C (±2 °C) with a minimum of 80% humidity. Each treatment was tested in duplicate, and treatments were analyzed at 13 different time periods during storage (0, 1, 2, 3, 6, 9, 12, 15, 18, 21, 24, 27, and 30 days after inoculation) for a total of 416 pieces of cheese.

Isolated from artisanal sausage. a

Probiotic and starter cultures

Foodborn pathogen and spoilage bacteria

2.7. Antioxidant activity in situ

Category

Table 1 Bacteria used to evaluate antimicrobial activity of pink pepper tree EO.

Strain

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The antioxidant potential of MEO in situ was evaluated by two chemical oxidation parameters indicative of the degree of deterioration. Peroxides (primary) and malonaldehyde (secondary), oxidative compounds formed in the lipid oxidation process, were quantified in cheese using the peroxide value analysis (Association of Analytical Chemists, 2012) and Thiobarbituric acid reactive substances (TBARS) (Yildiz-Turp & Serdaroglu, 2010) respectively. 2.8. Antimicrobial activity in situ In situ antimicrobial activity of MEO was determined by quantification of L. monocytogenes in cheese using the method described by HEGDE (2007) with adaptations. Twenty-five grams of cheese

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(±0.1 g) from each treatment were homogenized in 225 ml of water with 0.1% bacteriological peptone (Oxoid®) and subjected to serial dilutions. The resulting solutions were then used to surface inoculate (0.1 ml) chromogen (Oxoid®) agar plates. Incubation was carried out at 37 °C for 48 h, and bacterial colonies were then visually identified and counted. – 0.2% – – – 0.2% 0.2% 0.2%

Pathogen inoculuma (v/m)

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2.9. Statistical analysis Data were submitted to an analysis of variance (ANOVA) in STATISTICA version 6.1 (StatSoft, France), adopting a 95% confidence interval. Mean separations were based on Tukey's test and were considered significant at the 0.05 level unless otherwise noted. 3. Results

– – 0.7% 1.4% 2.0% 0.7% 1.4% 2.0%

MEO (m/m)

Of the 18 bacteria initially submitted to the disk diffusion test, 15 (83%) were sensitive to essential oils from fruit at both ripening stages (Table 3), presenting inhibition zones ranging from 29.75 mm (L. plantarum) to 53.14 mm (C. fim). However, the inhibition zones of MEO were significantly larger than those of GEO in 10 cases (67% of inhibition), while the inhibition zones of GEO were not larger than MEO in any bacteria tested and were similar to those of MEO in five bacteria tested (33% of inhibition). All gram-positive bacteria tested were sensitive to both essential oils, with MEO showing larger inhibition zones than GEO in 10 (83%) gram-positive bacteria, and both essential oils showing similar activity in two (17%) gram-positive bacteria tested. Three of the gram-negative bacteria tested in this study (50%) were resistant to both essential oils evaluated. Among the bacteria that were inhibited, MEO had a larger inhibition zone than GEO for S. dysenteriae, and no difference in inhibition zone was observed between the two EO in P. aeruginosa and A. hydrophila. Differences in bioactivity (evaluated by MIC and MBC) against undesirable bacteria were seen between MEO and GEO. MEO was more effective against S. aureus, C. fim, and S. dysenteriae, and GEO was more effective against S. mutans and A. hydrophila. No difference in activity against B. cereus was seen between the essential oils. No difference in the minimum inhibitory concentration between MEO and GEO was observed for L. monocytogenes and P. aeruginosa. However, a smaller concentration of MEO was needed to promote death (MIC) of both bacteria than GEO. GEO had higher activity than MEO in all bacteria with technological applications with the exception of L. mesenteroides.

% = Percentage of the total mass of casein (100%). a L. monocytogenes bacteria at a concentration of 1.107 CFU/g.

CC CL O1 O2 O3 L1 L2 L3

3.2. Antioxidant activity in vitro

Treatment

Table 2 Treatments used to tests the antimicrobial activity of mature pink pepper tree essential oil in Minas-type fresh cheese inoculated with L. monocytogenes.

3.1. Antimicrobial activity in vitro

Both essential oils showed antioxidant activity, as the concentration of the free radical DPPH was reduced. The EC50 (concentration of EO required to reduce the initial concentration of the free radical DPPH by 50%) was 21.7 ± 0.8 for GEO and 23.5 ± 1.2 μg/mL for MEO; however, the statistical analysis showed no significant difference between the antioxidant activity of these two EO. 3.3. Antimicrobial activity in situ The in situ test showed a similar trend for L. monocytogenes development in all treatments during the 30 days of refrigerated storage, with a gradual increase in cell counts during this time (Fig. 1). However, the addition of MEO at different concentrations resulted in different bacterial growth kinetics. In the control treatment without addition of MEO, the pathogen increased from 4.5 log CFU/g at the starting point to 7.1 log CFU/g on the 30th day of storage. Treatment L3, which consisted of addition of 2% MEO, had the lowest bacterial counts among all treatments, and ranged from 4.3 log CFU/g at time zero to 5.8 log CFU/g at

GEO

13.598 13.598 0.850 3.400 13.598 27.197 – – – 13.598 3.400 3.400 1.700 3.400 1.700 1.700 1.700 3.400

MEO

1.704 6.820 0.852 27.278 6.820 6.820 – – – 6.820 6.820 0.426 13.639 13.639 6.820 13.639 13.639 6.820

1.704 6.820 0.852 27.278 6.820 6.820 – – – 6.820 13.639 0.852 13.639 13.639 6.820 13.639 13.639 13.639

Fig. 1. Effect of different concentrations of mature pink pepper tree fruit essential oil (MEO) on the development of L. monocytogenes over 30 days of refrigerated storage (4 °C ± 2) on Minas-type fresh cheese.

a

Probiotic and starter cultures

Results are expressed as mean ± standard deviation; in each line, different letters correspond to the significant difference (p b 0.05).

6.799 6.799 0.850 3.400 13.598 27.197 – – – 6.799 1.700 0.850 0.850 1.700 1.700 1.700 0.850 0.850 42.70 ± 0.19 b 40.86 ± 0.31 b 39.97 ± 0.81 b 42.62 ± 0.06 b 53.14 ± 0.78 b 38.80 ± 0.03 b 00.00 ± 0.00 a 00.00 ± 0.00 a 00.00 ± 0.00 a 41.33 ± 1.03 a 41.36 ± 0.12 a 42.49 ± 0.05 b 38.93 ± 0.17 b 37.03 ± 0.03 b 32.70 ± 1.11 a 35.81 ± 0.83 a 41.12 ± 0.44 b 38.19 ± 0.98 a

GEO MEO

41.23 ± 0.09 a 35.22 ± 0.79 a 31.39 ± 0.77 a 31.20 ± 1.67 a 40.58 ± 1.00 a 35.44 ± 0.76 a 00.00 ± 0.00 a 00.00 ± 0.00 a 00.00 ± 0.00 a 44.31 ± 0.45 a 40.16 ± 0.29 a 37.75 ± 1.00 a 29.75 ± 0.60 a 33.52 ± 0.37 a 34.34 ± 0.08 a 37.04 ± 0.01 a 34.47 ± 0.08 a 36.70 ± 0.12 a S. aureus L. monocytogenes B. cereus S. mutans C. fim S. dysenteriae E. aerogenes E. coli S. typhimurium P. aeruginosa A. hydrophila L. mesenteroides L. plantarum L. delbrueckii subsp. bulgaricus L. brevis L. lactis subsp. lactis B. lactis B. bifidum Foodborne pathogen and spoilage bacteria

GEO

Bacteria Category

Inhibition zone (mm)a

MIC (mg/mL)

MBC (mg/mL)

MEO

G. da Silva Dannenberg et al. / Innovative Food Science and Emerging Technologies 36 (2016) 120–127 Table 3 Results of antimicrobial activity tests (agar disk diffusion test, minimum inhibitory concentration, and minimum bactericidal concentration) of essential oil of green (GEO) and mature pink pepper tree fruit (MEO) against important food bacteria.

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the end of the assay. Treatment L3 had significantly fewer bacteria than the control from day 1 through day 30, and than all other treatments beginning at day 6 and continuing for the duration of the test. The application of 1.4% MEO in treatment L2 and 0.7% MEO in treatment L1 showed similar results, with statistically lower counts than the control treatment except on day 15, and higher counts than treatment L3 from day 6 on. Differences in bacterial count between treatments L1 and L2 were seen on days 2, 9, 12, and 30, where treatment L2 had lower counts. Over the 30 days of the in situ test, bacterial counts increased from 4.4 to 6.5 log CFU/g in treatment L1 and from 4.3 to 6.3 log CFU/ g in treatment L2. 3.4. Antioxidant activity in situ Over the 30 days of storage, cheese from all treatments had lower peroxide values when compared to the control treatment (without MEO application), demonstrating the inhibitory activity of this extract in the initial stage of the lipid oxidation process (Fig. 2) Among the three treatments analyzed, treatment O3, with the highest essential oil concentration (2%), showed the lowest peroxide values and differed significantly from treatments O1 and O2 from day 9 through day 30. Treatments O1 and O2 with 0.7% and 1.4% MEO, respectively, had lower

Fig. 2. Effect of different concentrations of mature Brazilian pepper tree fruit essential oil (MEO) on peroxide values during 30 days of refrigerated storage (4 °C ± 2) of Minastype fresh cheese.

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peroxide values than the control starting from day 6, and higher peroxide values than the O3 treatment values starting from day 9. Treatment O2 had lower peroxide values than O1 on days 3, 12, 18, and 30. All treatments showed similar behavior, with an initial increase in malonaldehyde levels (MDA), which are secondary oxidation compounds, followed by a reduction. However, from day 6 through day 30, treatment O3 showed significantly lower TBARS than the control (Fig. 3). Additionally, treatments O1 and O2 showed significant differences in TBARS value compared to the control from the 12th day of storage. An inverse relationship between the essential oil concentration and antioxidant activity as measured by TBARS was seen among the three MEO treatments tested, where the higher the EO concentration, the lower the activity.

4. Discussion Essential oils extracted from green and mature (red) pink pepper tree fruit showed antimicrobial activity against 15 (83%) of the 18 bacteria tested in this study, demonstrating that molecules with antimicrobial activity such as terpenes, terpenoids and phenylpropenes exist among the secondary metabolites tested (Hyldgaard, Mygind, Meyer, & Debabov, 2012), which are produced by plant with the purpose of protecting against opportunistic bacteria (Tajkarimi, Ibrahim, & Cliver, 2010). Similar results were found in the literature for essential oil of Schinus molle (Martins, Arantes, Candeias, Tinoco, & Cruz-Morais, 2014), another representative of the Anacardiaceae family. In addition to conferring activity against bacteria, the inhibition zones observed in the agar disk diffusion test, which ranged from 29.75 and 53.14 mm, demonstrate significant antimicrobial activity, as inhibition zones larger than 18 mm in a study of pink pepper tree bark extracts were considered very active (Santos et al., 2007). The fact that both GEO and MEO showed activity against several strains of both Gram-positive and Gram-negative bacteria indicates that the tested EO demonstrate broad spectrum antibacterial activity independent of cell wall type. Essential oils typically contain several bioactive molecules, and may be composed of as many as 45 different compounds (Djenane, Yangüela, Montañés, Djerbal, & Roncalés, 2011). This diversity of structures allows for the presence of different modes of action (Carson, Mee, & Riley, 2002), which are responsible for acting on different cellular targets (S. Burt, 2004). Essential oils have several different modes of action, including disturbances of bacterial cell walls by forming pores that result in permeability and allow for the liberation of cellular components, reduction of intracellular pH, and changes in

Fig. 3. Effect of different concentrations of mature Brazilian pepper tree fruit essential oil (MEO) on malonaldehyde production during 30 days of refrigerated storage (4 °C ± 2) of Minas-type fresh cheese.

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intracellular concentration of adenosine triphosphate (ATP) (S. Burt, 2004). Like most modes of action reported for EO involving the microbial cell wall, Gram-positive bacteria, which have a higher proportion of peptidoglycan in their structure (Nazzaro, Fratianni, & Martino, 2013), are more sensitive than Gram-negative bacteria. This is due to the hydrophobic characteristic of the constituents of EO. The hydrophobicity of the EO facilitates the passage of these molecules into the cell wall of Gram-positive bacteria, resulting in activity in the wall as well as the cytoplasm (S. A. Burt & Reinders, 2003). Results of this study, whereby all Gram-positive bacteria tested were sensitive to both EOs, support this. Lipopolysaccharide (LPS) present on the surface of the outer membrane of Gram-negative bacteria has a repelling effect on the hydrophobic components of EO, hindering their entrance into the cell wall (Trombetta et al., 2005). The presence of divalent ions such as Mg2 + in the middle of LPS will increase the cross-linking between the molecules, thus reducing the pore size and further limiting the passage of bioactive compounds (Dussault, Vu, & Lacroix, 2014). Bacteria with the ability to form biofilms are capable of producing exopolysaccharides, which serve as protection mechanisms, hindering the contact of antimicrobial agents with the cellular target (Kavanaugh & Ribbeck, 2012). This ability, along with the type of cell wall, explains the resistance of E. aerogenes, E. coli and S. Typhimurium to the EO seen in this work. However, three other Gram-negative bacteria that are able to form biofilms (S. dysinteriae, P. aeruginosa, and A. hydrophila) were susceptible to the extracts tested in this study. These results may be justified due to the existence of a wide variety of compounds in the EO with various modes of action specific to different cellular targets, which enable even Gram-negative bacteria to be more sensitive than Gram-positive bacteria (Klein, Rüben, & Upmann, 2013). Previous studies reported that the outer membrane of Gramnegative bacteria is not entirely impermeable to hydrophobic compounds such as those found in EO, allowing for their slow passage through the pores (Plésiat & Nikaido, 1992). Gustafson et al., (1998) verified the death of E. coli by tea tree EO without cell lysis, demonstrating that the active molecule(s) in this essential oil permeates the outer membrane of the pathogen. In most of the available literature, EO activity is tested only against bacterial pathogens and/or spoilage. However, this study examined the effects of EO on bacteria with technological applications such as starter cultures, widely used in industry for the production of milk and fermented meat (Lorenzo, Gómez, & Fonseca, 2014), and probiotic bacteria, commonly added to food to promote consumer health benefits (Davis, 2014). All bacteria with technological applications tested were sensitive to EO; however, in both the MIC and MBC tests, higher concentrations of MEO than GEO were necessary. Additionally, these concentrations were even higher than those found for the majority of undesirable bacteria (pathogenic and spoilage). This can be explained by the predominance of molecules which act specifically on pathogenic bacteria, such as proteins responsible for virulence factors or enzymes that are not found in lactic acid bacteria (LAB) (Nazzaro, Fratianni, & Martino, 2013). The higher resistance seen in LAB in the in vitro tests performed in this study is consistent with results obtained by other authors (Mathur & Singh, 2005). These results may be of interest technologically, since the MIC and MBC of MEO for most LAB were significantly greater than those in pathogenic and spoilage bacteria. Intermediate concentrations between the MIC for the LAB and undesirable bacteria could be utilized in a food system containing both types of microorganisms, whereby the EO would provide activity against the undesirable bacteria but not have deleterious effects on the LAB. Therefore, it can be suggested that MEO has the potential to be used as a biopreservative in fermented products without affecting the fermentation process. MEO was more effective against most pathogenic and spoilage bacteria tested than GEO, showing that changes in the concentration of active compounds and/or production of new bioactive molecules occur

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with ripening, as has been previously demonstrated (Quassinti et al., 2013). In addition to microbial activity, both GEO and MEO demonstrated the ability to sequester the free radical DPPH at concentrations of 21.7 and 23.5 μg/mL, respectively. Previously, ALVES-SILVA (2013) showed that EO concentrations of 0.31, 88.2 and 89.6 mg/mL for basil, celery and coriander, respectively, were needed to obtain results similar to those seen in this study. No statistical difference between the two EO tested was observed, indicating no effect of fruit maturity on EO antioxidant capacity. The Minas-type fresh cheese food matrix used in this study is suitable for the multiplication of L. monocytogenes, given that the population of this pathogen in the control treatment (CL) increased from 4.5 to 7.1 log CFU/g after 30 days of refrigeration (4 °C ± 2). In the cheese, the concentration of MEO that corresponded to the MIC observed in the in vitro test for L. monocytogenes (L1) was not as effective in the control of this pathogen. This reduction in antimicrobial activity may be due to interactions of hydrophobic molecules with EO components of the food matrix, such as lipids, carbohydrates and proteins (Gutierrez, BarryRyan, & Bourke, 2008). These compounds can react with the EO extract, neutralizing and reducing the concentration of the active molecule(s), or hampering its contact with the target (Rattanachaikunsopon & Phumkhachorn, 2010). In addition, the food components may surround the microbial cells, protecting them from antimicrobial activity (Mejlholm & Dalgaard, 2002). The results obtained in this study emphasize the importance of performing antimicrobial activity tests in situ, and demonstrate that in order for this EO to be used as a food biopreservative, concentrations greater than the MIC determined in in vitro tests are necessary, considering that the antimicrobial compounds may be negatively impacted by the food. The use of MEO at concentrations greater than the MIC (treatments L2 and L3) resulted in a significant reduction of L. monocytogenes compared to the control and in proportion to the increase in concentration, which is in agreement with previous literature. Abdollahzadeh, Rezaei, and Hosseini (2014) applied 0.4, 0.8 and 1.2% thyme EO to minced fish meat contaminated with L. monocytogenes, and also found the reduction in pathogen count was proportional to the concentration of EO used during refrigerated storage (4 °C). Similarly, Alboofetileh, Rezaei, Hosseini, and Abdollahi (2014) evaluated films containing EO of marjoram, cloves, and cinnamon that were shown to be effective against S. aureus, E. coli, and L. monocytogenes, where activity increased sequentially with an increase in EO concentration. With respect to in situ antioxidant activity, MEO demonstrated activity against formation of primary oxidative compounds (peroxides) of the lipid peroxidation process, which prolongs the shelf life of the food by preserving sensory characteristics for a longer period of time (R. H. Olmedo, Asensio, Nepote, Mestrallet, & Grosso, 2009; R. Olmedo, Nepote, Mestrallet, & Grosso, 2008). Similar results were seen in a study investigating antioxidant activity of oregano and rosemary EO, where the peroxide value in cream cheese at 35 days of storage was also reduced (R. H. Olmedo, Nepote, & Grosso, 2013). In addition to lowering peroxide levels in the cheese, MEO decreased the concentration of secondary oxidative compounds (TBARS). The instability of these substances may explain why the increase was followed by a decrease over the 30 days of the test. Over time, both malonaldehyde and other carbonated products produced from peroxides degrade to form organic acids and alcohols, which, though not detected by the TBARS method, result in undesirable sensory characteristics (Ulu, 2004). 5. Conclusion The essential oils extracted from green (GEO) and mature (MEO) fruit of pink pepper tree demonstrated antimicrobial and antioxidant activity in vitro tests, with antimicrobial activity against six pathogenic bacteria, seven spoilage bacteria, and three bacteria with technological

applications, as well as antioxidant activity by reducing the free radical DPPH. The MEO presented particularly interesting behavior, by acting against undesirable bacteria at a low concentration and affecting bacteria with technological applications at higher concentrations. The application of MEO to Minas-type fresh cheese demonstrated its biopreservative capacity in situ, as the population of L. monocytogenes and the concentration of primary and secondary oxidative compounds decreased over the 30 days of cold storage. These results confirm the potential technological application of MEO as a biopreservative, making it a candidate for partial or total replacement of undesired synthetic preservatives currently used in food systems. 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