Schinus terebinthifolius (Anacardiaceae) essential oil for minas frescal cheese preservation Óleo essencial de Schinus terebinthifolius (Anacardiaceae) na conservação do queijo minas frescal Aceite esencial de Schinus terebinthifolius (Anacardiaceae) para la conservación del queso

Microorganism control in food has been a challenge due to the legislation restrictions of each country and the increase in the demand for food without chemical preservatives. Plant essential oils have antimicrobial action and are promising for the use in the food industry. Our study aimed to evaluate the chemical composition and the antimicrobial effect of Schinus terebinthifolius Raddi fruit essential oil on minas frescal cheese. The essential oil was extracted by hydrodistillation, and the chemical compounds were identified by gas chromatography coupled to mass spectrometry. The major identified compounds were hydrocarbon monoterpenes (57.0%), mainly: α-pinene (22.2%), limonene (17.0%), carvone (10.2%) and β-phellandrene (7.9%). The cheese was produced, the main microorganisms were isolated, and the minimum inhibitory concentration was determined through broth microdilution test. The cheese samples were soaked. The pink pepper fruit essential oil was added to the cheese samples at the concentration of 20000 μL/mL superficially or to the micelle. S. terebinthifolius fruit essential oil was efficient to control minas frescal cheese microorganisms mainly when applied by superficial addition. The essential oil is a potential source of studies to develop applications for the control of microorganisms in cheese such as Research, Society and Development, v. 9, n. 11, e1619119760, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i11.9760 3 minas frescal. Further studies may exploit the impact of the essential oil in minas frescal cheese acceptability.


Introduction
Minas frescal fresh cheese is obtained by enzymatic coagulation of the nonpolar fraction of milk casein which is separated but not matured (Brasil, 2004). This cheese is very appreciated in Brazil and has an estimated consume of 119,561.7 kg/year in 2019 (Veríssimo et al., 2019). Minas-type cheese has fat content ranging from 25.0% to 44.9% and moisture over 55.0% (Brasil, 2004). Due to its physical and chemical characteristics, it is an ideal means for the growth of pathogenic rotting microorganisms (Carvalho et al., 2007) and, therefore, with shelf life of up to 20 days (ABIQ, 2020).
Chemical preservatives are utilized to control microorganism growth in fresh cheese (Gonçalves et al., 2011). The preservatives have been associated with toxic reactions in the metabolism as well as with allergy triggering, behavioral changes and possible risks of cancer development (Polônio & Peres, 2009). Potassium sorbate (Ordoñez et al., 2020) and natamycin (Aparício et al., 2016) have usually been applied in the superficial treatment with fungicide and bactericide functions. Sodium nitrate has also been reported to be used to avoid swelling of minas frescal cheese (Mello, 2007), and to inhibit microorganism contamination, specially Clostridium bacteria (Perry, 2004). Research, Society and Development, v. 9, n. 11, e1619119760, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i11.9760 Koul et al. (2008) alerted that the use of synthetic fungicide could develop resistant fungal strains and recommended essential oil of biological base to be used as fungicides, boosting the search for plant antimicrobials (Anyanwu & Okoye, 2017). Sivakumar & Bautista-Baños (2014) recommended the utilization of essential oils to preserve food; Burt (2004) advise concentrations from 0.1 to 6% of essential oils to preserve food and as aromatizing agents which are also generally considered safe (GRAS) (Burt, 2004;Sivakumar & Bautista-Baños, 2014;Tedesco et al., 2020). Therefore, essential oils are considered promising for food preservation because they present antimicrobial and antioxidant activity (Bouhdid et al., 2010).
Schinus terebinthifolius Raddi, from the family Anacardiaceae, popularly known as pink pepper (Brazilian pepper tree) or aroeira in Brazil (Gomes et al., 2013) is a native plant of the Atlantic Forest (Cesário & Gaglianone, 2013). S. terebinthifolius fruit is very utilized in culinary and has approximately 70 g of essential oil/kg (Bortolucci et al., 2019). S. terebinthifolius essential oils present antimicrobial activity mainly against Gram-positive bacteria and fungi from the genus Candida (Pires et al., 2004). Thus, due to the antimicrobial activity of the oil and the high perishability of minas-type cheese, our study aimed to determine the chemical composition of S. terebinthifolius fruit essential oil, and to apply the oil on minas frescal cheese. Our results showed that the oil can be used to control microorganisms in cheese and expand the utilization of plant compounds to preserve food.  (Santos et al. 2013). After the distillation, the essential oil was stored in an amber flask at 4 °C (Brasil, 2010). Research, Society and Development, v. 9, n. 11, e1619119760, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i11.9760 6

Gas chromatography-mass spectrometry of Schinus terebinthifolius fruit essential oil
The chemical identification of the essential oil was done by a gas chromatographer (Agilent 7890 B) coupled to a mass spectrometer (Agilent 5977A) (GC-MS), equipped with a capillary column fused with silica HP-5MS UI Agilent (30 m x 0.250 mm x 0.25 μm). The analysis conditions were as following: injector temperature at 220 o C, injection volume of µL and injection rate in split mode (1:30). The initial column temperature was 60 °C (2 min), with heating ramp of 2°C/min up to 180 °C (4 min). From 180 °C to 260 °C, a heating ramp of 10 °C/min was determined. From 260 ° C to 300 °C, a heating ramp of 40 ° C/min was utilized (Cavalcanti et al., 2015). The transfer line was kept at 285 ºC, and the ionization source and quadrupole at 230 °C and 150 °C, respectively. The utilized carrier gas was helium with a flow of 1 mL/min. The detection system was EM in "scan" mode, in the mass/charge (m/z) rate of 40-550 m/z with 3-min "solvent delay". The oil samples were diluted in the ratio of 1:10 with dichloromethane. The chemical compounds were identified comparing their mass spectra to the mass spectra from WILEY 275 as well as comparing their retention indices (RI) which were obtained by utilizing a homologous n-alkane standard series (C7-C26) (Adams, 2017).

Isolation of minas frescal cheese natural microbiota
In order to produce minas frescal cheese, in natura milk was kept at 70 ºC for 30 minutes and cooled in water bath to 34 ºC. Next, a commercial chymosin coagulating agent, 75 International Milk Coagulating Unit -IMCU, liquid Estrella ® curd (4 mL for each 8 L of milk) was used. The mixture was kept at rest for 50 min until coagulation. The resulting curd was cut in 1-2 cm cubes with a sterile cutter, slowly agitated (5 min) and then 10 g of cooking salt (NaCl) was added. After that, the precipitate was separated in perforated plastic molds (5 cm x 5 cm x 5 cm) to allow liquid draining for 12 hours under refrigeration (7 ± 0.5 ºC).
Later, they were removed from the molds and stored in closed polypropylene recipients at 8º C (Ribeiro, 2015).
After the cheese preparation, the microorganisms, naturally found in cheese, were isolated to determine the minimum inhibitory concentration (MIC) of the essential oil.
Samples of 1g from different parts of the cheese were collected, ground and homogenized in an Erlenmeyer flask containing peptone water at 0.1%. Serial dilutions were carried out in peptone water at 0.1% next, 1 mL of each dilution was sown in 15 mL of standard agar Research, Society and Development, v. 9, n. 11, e1619119760, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i11.9760 7 medium count pattern (PCA) (Himedia ® ) in Petri dishes by Pour Plate technique, and incubated for 24 hours in an oven at 35 °C. The microorganism growth was evaluated and quantified by total count method. The microorganism isolation was done in nutrient agar medium by streak method and the identification was carried out by Gram coloration. Only plates with growth lower than or equal to 300 colonies per plate were considered for the identification and isolation of microorganisms.

Antimicrobial activity of the essential oil against isolated microorganisms by broth microdilution
Broth microdilution method using 96-well microplate was utilized for the assays according to the Clinical and Laboratory Standard Institute (CLSI) and the protocols M27-A2 and M7-A5, modified for natural products (CLSI, 2015). A replication in the culture medium of the isolated strains was done for 8 hours before determining the minimum inhibitory concentration (MIC). Next, for each isolated microorganism, a standard suspension in saline solution was obtained and adjusted to 0.15 of McFarland scale in order to obtain 1.5 × 10 4 CFU/mL. The essential oil solution was prepared with distilled water using Tween 80 (2%, volume/volume; Sigma-Aldrich, USA) as emulsifier, from an initial concentration of 40,000 µg/mL. Then, the serial dilution of 1:2 wells in the microplate, containing 100 µL of culture medium, was carried out. After the essential oil dilution, 5 µL of the inoculum was added to all column wells. Each microplate includes positive controls (culture medium without the essential oil) and negative controls (non-inoculated culture medium). The microplates were incubated at 35 °C for 24 h. The antibiotics streptomycin (Sigma P7794) and ketoconazole (Sigma-Aldrich®) were used as positive control (1 mg/mL in sterile saline solution), and tween aqueous solution as negative control. The reading was done with the addition of 10 μL of developer, 2, 3,5-cloreto of triphenyl tetrazolium (Reatec®) at 1.0% in each well, followed by microplate incubation at 37º C for 10 min. The minimum inhibitory concentration (MIC) of the essential oil was defined as the lowest concentration capable of inhibiting bacterial growth.

Application of essential oil to minas frescal cheese
The cheese samples were added with essential oil at the concentration of 20000 μL/mL by superficial application or by addition to the mass after coagulation at a concentration based Research, Society and Development, v. 9, n. 11, e1619119760, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i11.9760 8 on the results of MIC for isolated microorganisms. For the superficial application, a pipette was used for the dripping of the essential oil diluted in distilled water with tween at the concentration of 20 mg/mL, using 500 µL (20 µL/cm 2 ). In the treatment of sample by mixture, the same amount of essential oil was added in the cheese mass when preparing it.
The cheese without essential oil addition was utilized as control. Later, the samples were stored in closed plastic recipients under refrigeration at 8º C for 15 days. Each treatment was carried out in triplicate and analyzed at 0, 5 and 10 days of storing. The antimicrobial activity was determined by quantifying the total aerobic mesophilic microorganisms (APHA, 2001). After the preparation, samples of 1 g were collected from the analyzed cheese. The portions were separately diluted in peptone water at 0.1% and diluted in series. After the dilutions, 1 mL of each dilution in 15 ml of PCA medium in Petri dish was sown and incubated for 24 hours at 35°C. Next, the total microorganism count was carried out.

Statistical analysis
All assays were done in triplicate. The results were expressed in arithmetic mean ± standard deviation and analyzed by analysis of unidirectional variance (ANOVA) followed by Tukey HSD test (honestly significant difference) with α = 0.05 to determine the statistical significance of the results. The analysis was done using Statistica® 8.0 software.
In the evaluation and isolation of cheese microbiota, the presence of Gram-positive bacteria and septate filamentous fungus was observed. The average values of MIC for the isolated bacterium 1 was 6.67 ± 2. 88 mg/mL and for the isolated bacterium 2 was 13.34 ± 5.77 mg/mL, and for the fungus 1.9 mg/mL. For the positive control, the average MIC value was 6.25 ± < 0.01 mg/mL for streptomycin and 0.35 ± 0.12 mg/mL for ketoconazole, respectively (Table 2). There was no significant difference (p ≤ 0.05) when comparing the average MIC values of the control streptomycin to isolate 1; however, when compared to isolated 2, the essential oil presented MIC 2-fold higher than the positive control (p ≤ 0.05). Research, Society andDevelopment, v. 9, n. 11, e1619119760, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i11.9760 These results show the potential of pink pepper fruit essential oil as a development inhibitor of isolated contaminating microorganism of cheese.   The average count results of total aerobic mesophilic microorganisms (CFU/g) of the cheese samples submitted to treatments with essential oil by superficial application or addition to the mass are shown in Table 3. The in situ test presented similar tendency for the development of microorganisms in all treatments during 15 days of refrigerated storage, with a gradual increase in the cell count during this period. However, the addition of essential oil resulted in a kinetic difference of the bacterial growth. In the control treatment (without addition of essential oil), the microbial count increase from 6.78 log CFU/g up to 8.49 log Research, Society andDevelopment, v. 9, n. 11, e1619119760, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i11.9760 11 CFU/g in the 15-day storage period. The treatment of essential oil superficial application presented a lower bacterial count between the treatments, from 5.73 log CFU/g up to 8.34 log CFU/g. In the 10-to-15-day period, the cheese samples added with essential oil had log CFU/g from 10 to 12% lower (p ≤ 0.05) than the control.

Discussion
Our results showed that Schinus terebinthifolius Raddi essential oil has inhibitory activity in minas frescal cheese microbiota.  (Silva et al. 2012). Silva et al. (2012) reported that (+)-α-pinene inhibited the secretion of phospholipase and esterase enzymes by microorganisms. The antimicrobial activity of S. terebinthifolius Raddi essential oil can be explained by the minimum inhibitory concentrations obtained by monoterpene compounds. Therefore, the compounds of pink pepper fruit essential oil may have acted on the control of microorganisms of minas frescal cheese. Dannenberg et al. (2016) showed, for ripe fruit essential oil, high antimicrobial activity with MIC values of 1.7 mg/mL, 6.8 mg/mL and 0.85 mg/mL against S. aureus, L. monocytogenes and B. cereus. Also, the essential oil exhibited biopreservative activity of frescal queijo. For these authors, S. terebinthifolius essential oil is a potential biopreservative, but not yet much used by the food industry.
terebinthifolius fruit essential oil was efficient to control microorganisms of minas frescal cheese, mainly by superficial application. The essential oil is a source of studies for the development of applications to control microorganisms in minas frescal cheese. Further studies may explore the impact of the essential oil on the acceptability of minas frescal cheese. Research, Society andDevelopment, v. 9, n. 11, e1619119760, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i11.9760