Characterization and application of Lippia alba (Mill) and Cymbopogon citratus D.C. Stapf. essential oils as natural sanitizers in coriander

The food industry has been increasingly challenged by greater legal requirements to ensure food and consumer safety. On contrast to synthetic compounds, which are often not completely efficient or which can have toxic effects on consumers’ health (Gómez-López et al., 2008), natural antimicrobial agents have been gaining prominence as a market trend, mainly because they are seen as an alternative to using these sanitizers (Souza et al., 2009; Azerêdo et al., 2011; Sousa et al., 2012; Oliveira et al., 2015).


Introduction
The food industry has been increasingly challenged by greater legal requirements to ensure food and consumer safety.On contrast to synthetic compounds, which are often not completely efficient or which can have toxic effects on consumers' health (Gómez-López et al., 2008), natural antimicrobial agents have been gaining prominence as a market trend, mainly because they are seen as an alternative to using these sanitizers (Souza et al., 2009;Azerêdo et al., 2011;Sousa et al., 2012;Oliveira et al., 2015).
Essential oils (OEs) are complex, volatile, lipophilic natural compounds characterized by a strong odor.These compounds are composed of different classes, mainly monoterpenes, sesquiterpenes and oxygenated derivatives, which can vary in quantity, quality and composition according to the climate, soil composition, plant organ, age and stage of the vegetative cycle (Bakkali et al., 2008;Valeriano et al., 2012;Tohidi et al., 2017).They are produced by plants as secondary metabolites and known for their medicinal properties; their antimicrobial potentials on deteriorating and pathogenic microorganisms in foods stand out, as well as promoting benefits to various food matrices (Bakkali et al., 2008).
Studies of the antibacterial activity of EOs against foodborne pathogens have been carried out mainly in vitro, and more recently in food matrices such as the EOs of Mentha piperita order to understand their effects on bacteria, which appears to be related to their ability to cause damage to the cell wall, since typical lipophilic compounds can go through cell walls and the cytoplasmic membrane, affecting its structure and making it permeable (Burt, 2004;Bakkali et al., 2008).Prior to any substances being indicated for use in foods, it is important that they are all evaluated for risk in order to ensure the safety of the product to consumer's health (U. S. Food and Drug Administration, 2017).
Among the vegetables widely consumed in natura and susceptible of contamination, we can emphasize coriander (Coriandrum sativum L.) (Oliveira et al., 2015).Coriander is a little studied herb, although it is consumed almost all over the world due to its excellent nutritional characteristics in cooking, high vitamin content, medicinal properties, and its considerable value and socioeconomic importance (Silva et al., 2013).However, studies have shown undesirable percentages of thermotolerant coliforms indicating poor storage, inadequate hygienic conditions during cultivation and handling and fecal contamination (Silva et al., 2016), which requires its sanitization with antimicrobial agents in order to ensure and maintain microbiological quality at safe and efficient levels (Beuchat et al., 2001).Thus, the aim of this study was to evaluate the potential application of LAEO and CCEO as natural sanitizers in coriander.

Extraction and characterization of EOs
The aerial parts of Lippia alba (Mill) and Cymbopogon citratus D.C. Stapf.species (800 g of each) were collected in the city of Pombal, state of Paraíba, Brazil, located at 06º 46 '12" South latitude and 37º 48' 07" West longitude, between January and March 2016 in the evening, since this is the period of the highest active principle concentration of plants, as well as better yields in production (Manganotti et al., 2011).Next, they were transported to the Laboratory of Grain and Cereals Technology, UFCG -Pombal for EO extraction by hydrodistillation in a Clevenger extractor (Farmacopéia Brasileira, 1988) for 3 hours at an approximate temperature of 80 °C.The yield of each extracted EO was then calculated (Fonseca & Librand, 2008).
Total phenolic components of the EOs were determined using Folin-Ciocalteu and gallic acid as standards, with absorbance measured at 765 nm (Singleton et al., 1999).Analyzes were performed in triplicate and expressed as mg of GAE (Gallic Acid Equivalent) per g of analyzed EO.

LAEO and CCEO cytotoxicity
The bioassay with Artemia salina L. was based on the technique described by Meyer et al. (1982).Each test tube contained 5 mL of saline solution (1:1 seawater and distilled water) plus varying concentrations of EOs (0 to 500 μg/mL) and ten nauplii.The number count of dead larvae was performed after 24 hours and was used for calculating the LD50 by the linear regression method.The criteria for classifying EO cytotoxicity against Artemia salina L. based on the values found for LD50 was defined by Meyer et al. (1982) and Nguta et al. (2012), where values of LD50 > 1000 μL/mL are considered non-toxic, those between 500 and 1000 μL/mL are slightly toxic, those between 100 and 500 μL/mL are moderately toxic and those < 100 μL/mL as strongly toxic.The tests were carried out five times.

Determination of Minimum Inhibitory Concentration (MIC)
The broth macrodilution technique was used for determining LAEO and CCEO Minimum Onhibitory Concentration (MOC), in which the test strains were inoculated into brain and heart infusion broth (BHO) and peaked to the surface of test tubes containing Base Agar Blood (BAB), added to increasing doubled concentrations of EOs (0 to 10 μL/mL), and then the tubes were incubated in an oven at 37 °C for 24 hours, in duplicate.After the incubation period, the lowest EO concentration that did not show visible microbial growth was considered the minimum inhibitory concentration (Clinical and Laboratory Standards Onstitute, 2015).

Sanitary Potential of LAEO and CCEO against the naturally contaminant microbiota of coriander
The plant chosen for evaluating the sanitizing power of EOs was coriander (Coriandrum sativum L.), since it is an easily accessible herb with ample production in the Northeast region of Brazil.About 1000 grams of coriander cultivated with agro-ecological form without the use of pesticides were purchased from the open market of Pombal-PB in the month of April of 2016.
Sanitary potential of LAEO and CCEO was evaluated against the naturally contaminant microbiota of coriander, where 200 g portions were rinsed under running water and submitted to four treatments: 500 mL of distilled water -positive control; 500 mL of sodium hypochlorite with 100 ppm free chlorine -negative control; 500 mL of LAEO aqueous solution at the defined MOC with Tween 20 (0.1% w/v); or 500 mL of CCEO aqueous solution at the defined MOC with Tween 20 (0.1% w/v).The solutions were kept under light agitation for 5 min at 28 °C in order to ensure homogenization of the solutions and complete immersion of the herbs/leaves during the treatments.Next, the samples were then taken from the different solutions and stored in plastic trays, covered with PVC film and kept under refrigeration for 48 h (Souza et al., 2009).The sanitizing potential of the EOs on coriander during storage was determined at different time intervals (0, 24 and 48h) by evaluating the presence of coliforms at 45 °C, S. aureus and Salmonella spp., according to the methodology described by Silva et al. (2010) to evaluate the behavior of sanitizers during storage.

Statistical analysis
LD50 (the dose required to kill 50% of the A. salina L. nauplii population) determination was based on the PROBOT analysis (Finney, 1962) using the BioStat® software ( 2009) with 95% confidence.For the MOC determination assays, the results were expressed as modal values.Statistical analysis was performed to determine significant differences (p<0.05) using ANOVA with OriginPro8 ® .

EO characterization
The evaluated EOs presented higher extraction yield (Table 1) than that reported in the literature for the maximum values LAEO 0.54% (Nogueira et al., 2007) and similar to that reported by Koshima et al. (2006), 0.55% to CCEO, which was related to variables such as seasonality, plant development, and temperature, among other factors (Gobbo-Neto & Lopes, 2007).The EOs presented densities above 0.90 g/mL.
Total phenolic contents were 15.29 and 18.81 mg of GAE/g for LAEO and CCEO, respectively, which was related to the antimicrobial capacity of these components (Shan et al., 2007), as they have the ability to go through and disintegrate cell membranes, and their bioactivity is linked to this mechanism (Naidu, 2000).Port's et al. (2013) studying herbal infusions from the Amazonian region of Brazil met values of 27.37 and 13.61 mg GAE/g for infuse Lippia alba and Cymbopogon citratus, respectively, demonstrating value above the determined to LAEO and below to CCEO.

LAEO and CCEO cytotoxicity
LAEO had biological activity against Artemia salina L. with LD50 of 41.56 (μg/mL), and with limits varying from 29.32 to 62.38 (μg/mL).CCEO presented LD50 of 18.85 (μg/mL), with limits ranging from 13.71 to 26.00 (μg/mL).The results showed that the EOs present high toxicity to Artemia salina and may consider bioassay as a preliminary means to determine the bioavailability of the EOs and not the real toxicity of the product.Some authors have reported LD 50 values below 250 μg/mL with anticancer, antifungal, antiviral, antimicrobial, insecticide and trypanosomicide (Nguta et al., 2012;Meyer et al., 1982;Macrae et al., 1988;Siqueira et al., 1998).

Sanitary potential of LAEO and CCEO
LAEO and CCEO had activity on microorganisms assayed with MOC ranging from 1.25 to 5.00 μL/ mL and 0.62 to 1.25 μL/ mL, respectively (Table 2).According to Aligiannis et al. (2001), based on the MOC of plant extracts we can classify their inhibition profiles as: strong inhibitors, with MOC up to 500 μg/ mL; moderate inhibitor -MOC between 600 and 1500 μg/ mL; and weak inhibitor, with MOC greater than 1500 μg/ mL.Thus, LAEO and CCEO may be regarded as strong microbial and potentially bioactive inhibitors.The MOC values for CCEO are in agreement with those found by Leite et al. (2016) in evaluating CCEO antimicrobial activity on E. coli, L. monocytogenes and Salmonella Enteritidis in pineapple juice.
The CCEO presented lower MOC values than those of LAEO, corroborating with the bioactivity observed in the cytotoxicity for Artemia salina L, where it also presented lower values of LD50.We can verify that the highest MOC values were observed for E. coli, followed by MOC values related to the two Salmonella serovars, especially in LAEO, which may be related to the structural characteristic of the cell wall presented by the gram-negative bacteria.According to Hyldgaard et al. (2012), gram-negative bacteria are generally less sensitive to the antimicrobial action than gram-positive bacteria.This occurs due to the action of the essential oils mainly occurring in the cell wall of the strains, which would require a higher concentration of the antimicrobial agent for inhibiting a gram-negative bacterium.
By determining cytotoxicity and minimum inhibitory concentration, EO application in safe and effective doses can be recommended.The MOC used for the application of LAEO and CCEO in sanitizing coriander was the lowest concentration of inhibition among all strains, corresponding to 1.25 and 0.62 μL/mL (Table 2), respectively, with the purpose of verifying its real efficiency when tested against naturally contaminant coriander microflora (Figure 1).
The microbiological analyzes performed on the coriander found the presence of coliforms at 45 °C, and S. aureus, as well as the absence of Salmonella spp.RDC Number 12 (Brasil, 2001) only requires the evaluation of the presence or absence of Salmonella spp./25 g for in natura vegetables/greens, considering that the food is fit for consumption as it meets the absence pattern of Salmonella spp.established by the legislation in force, although the presence of S. aureus pose potential health risks to consumers.
On Figure 1 we have the results of counts for S. aureus in coriander with different sanitizers during storage.The counts  were made based on reduction of initial count (zero storage time) cycles and 24 and 48 hours storage times of coriander under refrigeration.After 24 hours storage it is observed that treatment with CCEO was superior to treatment with sodium hypochlorite, whereas treatment with LAEO was statistically similar.This shows that even at low concentrations the EOs were efficient and that the sanitizing potential of the two increases with the passage of storage time.
Evaluating coliform counts at 45 °C during storage, it was observed that samples that were immersed in water had values of 3.6 NMP g -1 for time zero and 9.1 NMP g -1 for the 48 hours storage time.Already samples with sodium hypochlorite treatment showed similar values to LAEO treatment with values of 3.6 NMP g -1 for time zero and <3.0 NMP g -1 with 48 hours of storage.While samples with CCEO treatment showed counts of <3.0 NMP g -1 already at time zero, this count remained in the samples for the times of 24 and 48 hours.
The MOC values used to evaluate the EOs' sanitizing potential were effective in reducing coliform levels at 45 °C present in coriander at higher (CCEO) or equivalent (LAEO) levels to those achieved by the commercial sanitizer sodium hypochlorite for the same evaluated microorganisms.This data is relevant, since sodium hypochlorite is the commercial sanitizer of choice for sanitizing fruits and vegetables, and the studied EOs behaved with superior or equivalent efficacy to this commercially available sanitizing agent, as opposed to that found by Hyldgaard et al. (2012), where it is emphasized that the use of EOs as preservatives in foods has been limited because in practice higher concentrations of EOs are required to achieve the observed antimicrobial activity in vitro.However, the data presented in this study corroborate those reported by Leite et al. (2016), where CCEO was efficient in inactivating E. coli, L. monocytogenes and Salmonella Enteritidis in pineapple juice.Some authors have reported the use of Thymus vulgaris L. (time), Origanum vulgare L. (oregano) and Rosmarinus officinalis L. (rosemary) for antiseptic and sanitizing purposes in vegetables, unveiling the potential use of these substances in foods (Azerêdo et al., 2011;Souza et al., 2013;Oliveira et al., 2015;Siroli et al., 2015;Medeiros Barbosa et al., 2016).Azerêdo et al. (2011) and Souza et al. (2013) used Origanum vulgare L. and Rosmarinus officinalis L. EOs against microorganisms from minimally processed vegetables and in vegetable broth, they concluded that these EOs combined at sub-inhibitory concentrations were efficient in inhibiting the growth and survival of pathogenic and deteriorating microorganisms associated with minimally processed vegetables, being still sensorially acceptable and a potential alternative to replace synthetic sanitizers applied classically in vegetables, respectively.Oliveira et al. (2015) studied the effect of synergistic use of carvacrol and 1-8 cineol (major components of some EOs) against bacteria related to minimally processed vegetables, they concluded that in sub-inhibitory concentrations EOs were effective in inhibiting the growth and survival of L. monocytogenes, A. hydrophila and P. fluorescens in mixed culture.They also concluded that the EOs were responsible for decreasing cell viability, as it altered membrane permeability causing ultrastructural changes in the cells tested.
We found that the microbial counts decreased throughout the exposure period to LAEO and CCEO, demonstrating an increase in the efficiency of EOs as a function of time.The CCEO achieved greater microbial inhibition efficiency for all evaluated treatments.The food exposure time to the EOs, effective in reducing the microbial load of spoilage and pathogenic microorganisms, has also been observed by Sousa et al. (2012) and Medeiros Barbosa et al. (2016).The same was observed by Oliveira et al. (2015) when studying the synergistic inhibition of plant-associated bacteria in mixed culture by the major components of EOs, carvacrol and 1,8-cineol.
Although the EO concentrations applied to coriander were lower than the MOCs observed in the in vitro tests for E. coli (Table 2), the inhibition of this microorganism can be observed by direct contact with the CCEO at time 0, or within 24 hours for LAEO, demonstrating high potential for the practical application of these EOs in foods.

Conclusion
LAEO and CCEO reduced the microbial load present in coriander, demonstrating their potential as natural sanitizers and demonstrating superior or equivalent efficacy to 100 ppm sodium hypochlorite, a common commercially used sanitizer.By correlating the cytotoxic and antimicrobial results obtained, the EOs presented potential to be applied to food.