USE OF ESSENTIAL OILS FOR FOOD SAFETY: FORMULATIONS OF EASY-TO-USE FOOD-GRADE SPRAY SANITIZERS

: Motivated by combat against the spread of foodborne diseases, the formulations of simple and easy-to-use spray disinfectants containing Cinnamomum zeylanicum and Ocimum gratissimum essential oils were studied. Central composite experimental design was used in the development of stable products with optimal essential oil content for antimicrobial efficacy. It also allowed minimizing the alcohol content which was used to ensure miscibility between the essential oils and the aqueous phase. Trans -cinnamaldehyde was the major compound in the C. zeylanicum essential oil. Its low toxicity allowed the formulation of a risk-free product despite the great variability in the chemical composition of the essential oil. Cooked food coated with a spray containing 1% of this essential oil, in combination with Thymus vulgaris tincture, could be preserved for 3 days without refrigeration. Eugenol was the major component of O. gratissimum essential oil, followed by 1,8-cineole and methylchavicol. Sprays containing 1.5% of this essential oil were effective against Escherichia coli and Staphylococcus aureus . Xanthan gum proved to be a good essential oil-trapping agent under accelerated ageing conditions at 45 °C.


INTRODUCTION
The increasing resistance of microorganisms to antimicrobial chemicals is a global concern of the third millennium.The agri-food industry is one of the sectors facing this challenge.Every year 600 million foodborne illnesses are reported worldwide.Foodborne diseases are the cause of 7.69% of the world's annual deaths (Lee & Yoon, 2021).Street food has become a growing habit for people living in big cities.Foods sold on the streets are part of a country's cuisine culture (Sezgin & Sanlier, 2016).In low-and middle-income countries, street foods have gained socioeconomic interest in recent decades.On the one side, they contribute to attracting tourists by offering the possibility to promote and preserve cuisine culture.On the other side, they offer to local population, lowcost food with better nutritional quality in terms of protein and energy (Alfiero, Lo Giudice, & Bonadonna, 2017).The lack of good hygiene practices in street food also contributes to an outbreak of foodborne diseases (Gargiulo et al., 2022).
A multidisciplinary approach has been proposed by some scientists to combat the continuing spread and persistence of multidrug-resistant strains (Aslam et al., 2018).The use of essential oils for food safety represents a promising solution for controlling multidrug-resistant microorganisms along the food processing chain (Maurya, Prasad, Das & Dwivedy, 2021).Compared to conventional antimicrobials, essential oils are reported to be more natural, safer, environmentally friendly and better suited for use in food storage and preservation (Pandey, Kumar, Singh, Tripathi & Bajpai, 2017).
Spices and herbs improve the aroma and flavour of food while simultaneously providing some health benefits (Rafique, Baig, Mehboob, Zahid, & Irfan, 2022).Cinnamomum zeylanicum essential oil has demonstrated effective antimicrobial activities against strains responsible for food spoilage and infectious diseases.A recently conducted study revealed that its minimum inhibitory concentration ranged from 0.078 mg/mL to 1.25 mg/mL (Tomičić, Tomičić, Kocić-Tanackov & Raspor, 2022).Ocimum gratissimum essential oil has also been attributed to good activity against multidrugresistant isolates for foodborne and infectious diseases (Melo et al., 2019).
Thymus vulgaris ethanol extract has demonstrated a very good antioxidant capacity and an antibacterial effect (Akin, & Saki, 2019).These two qualities are required to preserve food from rancidity and microbiological degradation.For this reason, it was associated with C. zeylanicum essential oil in this study.
Therefore, the present study aimed to formulate two eco-friendly disinfectants based on essential oils intended for utilisation on hand and food contact surfaces.A spray formulation was chosen due to easier utilisation, mainly for fast-food restaurants and street food vendors.The third spray product formulation was intended for direct use on cooked food to extend its shelf life.

Materials
Thymus vulgaris (thyme) was grown in a suburb of the capital city of Antananarivo.The aerial parts were carefully washed, dried at a temperature of 50 °C for 2 h and then ground.The hydroalcoholic extract of the dried powder was prepared as described by Olah et al. (2017) with slight modifications.Briefly, 30 g of the powder was macerated in 300 mL of a mixture of water and ethanol in the ratio 3:7 at ambient temperature.After 15 days, the mother tincture was recuperated through filtration.
Cinnamomum zeylanicum bark (cinnamon) was harvested on the east coast of Madagascar, near Ambalatenina village.Ocimum gratissimum leaves (African basil) were collected in the central highlands, near the capital Antananarivo.The essential oils were distilled using a Clevenger apparatus.The extraction time was 5 h for C. zeylanicum bark and 3h 30 min for O. gratissimum.
Pharmaceutical grade ethanol was purchased from ITW Reagents, PanReacAppliChem, Milano, Italy.Citric acid, white vinegar and baking soda were food-grade of brand Leader Price, France.Xanthan gum was imported from Shaanxi Rainwood Biotech Co. Ltd, China.

Essential oil analysis
The density of essential oil at 20 °C was measured with a pycnometer.The refractive index was measured with an Abbe refractometer according to the French Standard NFT 75 -112 (AFNOR, 1992).Rotary Index was determined as described by the French standard NFT 75 -113 (AFNOR, 1992).The apparatus was a Jobin Yvon Laurent (France) polarimeter with a tube of 100 mm length.
The volatiles were identified using a Girdel 300 series gas chromatography equipped with a flame ionization detector.The injection temperature was set at 250 °C.The detection temperature was also 250 °C.The capillary column was a DB-WAX with 0.32 mm internal diameter and 30 m length.Temperature programming was set from 60 °C to 220 °C with a gradient of 3 °C/min.Hydrogen was used as a carrier gas in constant flow mode (60 mL/min).

O N L I N E F I R S T
The identification of the components was done by calculating the retention index (RI) using a series of C8-C22 n-alkanes in comparison with local database and with the online NIST Database (NIST WebBook SRD 69).

Experimental design
In spray product formulations, the excipient was water.Most of the ingredients were water soluble.However, the main active ingredients i.e. the essential oils were not soluble.In order to obtain a miscible mixture alcohol was added.The main difficulty in the formulation was the correct ratio between the aqueous phase, the alcohol and the essential oil, taking into account the amount of essential oil required for optimal antimicrobial effectiveness.On the other hand, the alcohol concentration should be minimized to avoid side effects on the skin or through penetration (Himabindu, Tanish, Kumari & Nayab, 2020).The experimental design enabled the correct adjustment of these parameters.
The experimental design was based on a previous formulation study, with the necessary modifications (Llinares, Santos, Trujillo-Cayado, Ramírez, & Muñoz, 2018).A central composite model with three points was chosen.Each experiment was carried out in triplicate.The variables selected were the relative proportions of alcohol, aqueous phase and essential oil.The response variable was the stability of the formulation, evaluated through the miscibility of the mixtures.The design table for the formulation of the food spray, the hand-sani tizing spray, and the surface-cleaner spray are presented in Table 1.To formulate the foodpreservative spray, the required amount of citric acid was first diluted in distilled water to prepare Mixture 1. Thyme extract was mixed with alcohol and essential oil added before stirring for 3 minutes to form Mixture 2. The two mixtures were then added and homogenized for 3 minutes.The hand-sanitizing spray was only a mixture of water, alcohol and essential oil.For the surface cleaner spray, baking soda was added to a mixture of water and vinegar and stirred for 3 minutes.Xanthan gum was then added and the mixture was heated to 55 °C for 10 minutes.After cooling, the essential oil, previously diluted in alcohol was added, and the whole was stirred for another 10 minutes.

Preference test of the food-preservative spray
The ranking test was performed according to Sharif, Butt, Sharif and Nasir (2017).The home-use method was chosen.Sixty five untrained panellists, males and females, of various ages participated in the study.Sprays containing 0.5%, 1% and 1.5% essential oil were assigned a 3 digit code.
Participants were asked to rate these samples, on a scale from 1 to 5 with 1 being very bad and 5 very good.As these were untrained panellists, no description of odour, taste or appearance was particularly required.All they had to do was express their preferences based on all these characteristics combined.

Efficacy test of the food-preservative spray
The food preservative spray was applied three times to coat the surface of previously prepared pasta dishes.Good hygiene practice was respected for their preparation (Kamboj, Gupta, Bandral, Gandotra & Anjum, 2020).Thereafter, plates were stored under two different conditions: at room temperature and in a refrigerator.Microbiological testing was carried out after 3 days (72h).From each plate, 10 g of samples were taken and diluted with buffered peptone water for microbiological testing.

Efficacy test of the surface-cleaner and the hand sprays
Before formulating the final spray products, the antimicrobial activity of the essential oil of O. gratissimum was evaluated.After formulation, the sprays were divided into two samples.The first sample was kept at ambient temperature.
The second sample was subjected to an accelerated ageing test at 45 °C according to the Guidelines on stability testing by CTFA & COLIPA (2004).The efficacy tests of these samples were carried out 2 and 6 weeks after formulation.
All For the spray efficacy test, the same quantity of MHB, inoculum, and resazurin was filled as previously described for the essential oil.
Columns 1 to 3 contained 50 µL of the hand spray, which was stored at room temperature with 1.5%, 2.5%, and 3.5% of essential oil in triplicate.Columns 4 to 6 contained 50µL of the corresponding products stored at 45 °C.The same operations were performed with the surface cleaner in columns 7 to 12.After 18 h of incubation at 37 °C, subculturing in a petri dish containing a Plate Count Agar medium (Himedia, Dinhori, Nashik India) was carried out.The presence or absence of a colony was observed after incubation at 37 °C for 24 h.
O N L I N E F I R S T

Statistical analysis
The experimental design was carried out using Chemoface 1.6, free software.
Statistical analysis for the ranking test of the sprays containing 0.5%, 1% and 1.5% essential oil was performed using JASP 0.15.0.0 software (Love et al., 2019).Descriptive analysis was conducted to compare the scores attributed to each sample by the 65 participants.Dot plots were used to analyse the variation in the results.

Physico-chemical quality of essential oils
The physico-chemical characteristics of C. zeylanicum bark and O. gratissimum leaves essential oils are summarized in Table 2.
C. zeylanicum bark essential oil characteristics are consistent with commercially available oils (NHR Organic oils, 2017; Eden Botanicals, 2017).The results for O. gratissimum essential oil are comparable to that of Vietnamese essential oil which has a specific gravity of 0.945 and a refractive index of 1.526 (Huong et al., 2020).However, for the latter, the rotary power is +15.6.For the essential oil from Tanzania, the values are very different with a specific gravity of 0.87 and an optical rotation of -110.0,only the refractive index was similar: 1.516 (Malima, Massaga, Malecela & Andrew, 2013).

Chemical composition of the essential oils
The chemical composition of C. zeylanicum bark essential oil and its comparison with literature data are presented in Table 3.To highlight the variability of C. zeylanicum essential oil composition, our samples were compared to essential oils of samples from two other locations on the Eastern coast of Madagascar, reported elsewhere.The tested sample originated from Ambalatenina.The two other locations were East Fenerive and East Ilaka (Randriamampionona, 2010).Literature data on composition of cinnamon essential oil from Iran (Alizadeh Behbahani, Falah, Lavi Arab, Vasiee & Tabatabaee Yazdi, 2020), Turkey (Unlu, Ergene, Unlu, Zeytinoglu & Vural, 2010) and Sri Lanka (Paranagama et al., 2001) were also included in Table 3.
The essential oil studied contained 79.22% oxygenated terpenes and 13.85% terpene hydrocarbons.Trans-cinnamaldehyde is the major constituent of all the essential oils (Table 3) with relative proportions ranging from 50% to 80%.There was a significant difference in the relative proportions of the compounds of C. zeylanicum essential oils from the same Eastern Madagascan region.Eugenol content varied from 0.3% to 7.6%.Essential oil from Sri Lanka contained compounds that are non-existent in oils from other locations, like coumarin and alpha ylangene.Trans-cinnamaldehyde is endowed with antibacterial (Firmino et al., 2018), antifungal (Shreaz et al., 2016), acaricidal (Nwanade et al., 2021) and anti-inflammatory activities.WHO suggests an acceptable daily intake of 0.7 mg/kg body for cinnamaldehyde.
A study reported that an intake of 400 mg daily during a clinical trial resulted in no major side effects (Gunawardena et al., 2015).The very low toxicity of this essential oil allows an easy formulation of the food spray despite the high variability of cinnamaldehyde, without a major risk of side effects.
The chemical composition of O. gratissimum leaves essential oil originated from Madagascar revealed that it consisted of 92.82% oxygenated terpenes and 3.34% hydrocarbon terpenes (Table 4).This sample was compared with essential oils reported from Brazil (Mohr, Lermen, Gazim, Gonçalves & Alberton 2017;Melo et al., 2019) and Kenya (Matasyoh et al., 2007).Brazilian samples presented two chemotypes: linalool / 1,8-cineole chemotype and eugenol chemotype.This latter is comparable to the Madagascar and Kenya samples.Eugenol is a broad-spectrum antimicrobial agent which is active for both gram-positive and gram-negative strains.It can be used for pharmaceuticals, food preservation and cosmetics (Marchese et al., 2017).It is well known as an anaesthetic,antioxidant and anti-inflammatory agent.
In a formulation of hand-sanitizing spray, it should be noted that eugenol is a skin penetration enhancer.Randriamampionona, 2010; d Alizadeh Behbahani et al., 2020; e Unlu, et al., 2010; f Paranagama et al. 2001   O N L I N E F I R S T  Mohr et al., 2017; d Melo et al., 2019; e Matasyoh et al., 2007  ND-Not documented, t-compounds

present in trace quantities
Genotoxicity and immunotoxicity of eugenol have been reported, but extensive research on its chronic toxicity is very limited (Nejad, Özgüneş & Başaran, 2017).

Microbiological activity of O. gratissimum essential oil
Essential oil of O. gratissimum exhibited antimicrobial activity against both tested micro-organisms.The minimum inhibitory concentration (MIC) was 11.36 µg/mL and the minimum bactericidal concentration (MBC) was 14.20 µg/ml against E. coli strain.In case of S. aureus strain, MIC/MBC was 12.12/15.15µg/mL, respectively.O. gratissimum essential oil from Brazil has shown MIC and MBC of 1000µg/mL for these two strains (Melo et al., 2019).Interestingly, both the Madagascan and Brazilian essential oils had around 74% eugenol as the main active principle.The main dif-ference resided in the content of 1,8cineole which was 15.16% for the Brazilian essential oil, and 9.34% for the Madagascan essential oil.The Madagascan essential oil contained, in addition to this, 8.47% of methylchavicol.It could be then suggested that the synergy between eugenol, 1,8-cineole and methylcha-vicol resulted in a better activity than the synergy between eugenol and 1,8cineole alone.Methylchaviol has good antimicrobial activity and it has been studied for integration in bio-film polymers for food preservation (Suppakul, Sonneveld, Bigger, & Miltz, 2011).

Spray formulations
Taking into account the previous results, and the toxicity of eugenol (Nejad et al., 2017) the relative percentage of O. gratissimum essential oil was limited between 1.5% and 3.5%.For the C. zeylanicum essential oil, the percentage was between 0.5 and 1.5%.3D-surface response graphs in the function of essential oil percentages are reported in Fig. 1.

O N L I N E F I R S T
The hand-sanitizing spray presented the largest optimal area.For different concentrations of essential oil, the aqueous phase amount could be varied between 78-94 g with 12-15 g of alcohol.The food-preservative spray was the most sensitive to the variation of essential oil quantity.For a small amount of essential oil, the optimal area was quite large.As the amount of essential oil increased, the optimum area for relative quantities of alcohol and aqueous phase decreased very quickly.A formulation with 1.5% essential oil showed poor stability and 15% alcohol is needed with 83.5% of the aqueous phase.For the surface cleaner spray, the optimum area also decreased with increasing amount of essential oil but it was less sensitive than in the case of the handsanitizing spray.The hand-sanitizing spray composition allowed the most stable formulation and the food-preservative spray with the thyme extract was the most restrictive.

Characterization of the food-preservative spray A) Ranking test
The results for the ranking test of the food preservative spray are presented in Table 5, and the corresponding distributions for the preference scores are shown in the dot plots (Fig. 2).Doutoum, Tidjani., Markhous, Kimassoum, & Nadlaou, 2019 O N L I N E F I R S T The maximum score was attributed to sample A with 0.5% of essential oil, for which the mean is 4.338.None of the scores for the 3 samples had a normal distribution.For sample A, the tendency was ascending from score 2 to score 5.It means that the spray was appreciated by the majority of the panellists who gave good scores of 4 and 5.
The dominant score for the spray with 1% of essential oil was 3 which means that most of the panellists find it neither good nor bad.There was no significant difference between the average scores for the sample B and C. However the high standard deviation for sample C showed large spread range of data indicating that the opinions are divergent for the spray with 1.5% essential oil.
Considering the above results on the consumer preference for low essential oil content and the stability of the formulation described previously, only sprays with 0.5% and 1% essential oil were chosen for efficacy testing.Given the fact that thyme mother tincture already contained alcohol, using an additional 15% to stabilize the formula at 1.5% essential oil would result in high alcohol content.

B) Efficacy test
Results of the microbiological testing on the third day of storage period are displayed in Table 6.
Sprays with 0.5% and with 1% of essential oil allowed the microbiological preservation of the food until day 3.The spray had a marked antifungal action and 0.5% essential oil was sufficient for preserving the food even without refrigeration.(Bylaite, Adler-Nissen, & Meyer, 2005).Thus, a slower evaporation of essential oil contrary to the hand sanitizing spray could be explained.Hence, the surface cleaner spray with 2.5% essential oil was efficient even after accelerated aging condition at 45 °C.
Many scientific reviews have demonstrated the effectiveness of the essential oils in fighting multiresistant strains.Essential oils are incorporated in nanofilms and nanoparticules for preservation purpose.Many researchers focus on the development of active food packagings containing essential oils (Guidotti-Takeuchi et al., 2022).These technologies are still expensive for less developed countries, and here we offer an inexpensive solution to help with food security.In addition, the presentation in the form of a spray is a very practical and easyto-use.The lack of facilities within street food vendors is among the reasons of the outbreak of foodborne diseases (Verma & Mishra, 2020).The availability of low cost and convenient disinfectant sprays may help in the application of the food safety regulation.

CONCLUSION
Results from this study indicated effectiveness of essential oils of C. zeylanicum and O. gratissimum incorporated in simple spray products against food-borne pathogens.The surface cleaner-spray supplemented with O. gratissimum essential oil (1.5%) and white vinegar showed effectiveness against E. coli and S. aureus.Moreover, the addition of xanthan gum enables trapping the essential oil within the product, which allowed retention of its effectiveness after treatment in an accelerated aging condition at 45 °C at a concentration of 2.5%.The formulation of a hand spray without xanthan gum required a higher content of essential oil (3.5%) after accelerated aging treatment.However, both sprays stored at room temperature were active at a concentration of 1.5% essential oil.The combination of C. zeylanicum essential oil with 7% T. vulgaris tincture allowed the preservation of cooked pasta for 3 days without refrigeration.These results provide scientific evidence and contribute to the development of potent, ecological, and safer food contact disinfectants as well as food preservative agents that can be good alternatives to synthetic chemicals.

Figure 1 .
Figure 1.Relative quantities of alcohol and aqueous phase in function of essential oil mass percentage

Figure 2 .
Figure 2. Dot plots showing the scores for each formulation of food preservative-spray

Table 1. Experiment designs of spray formulations. Run Food-preservative spray Hand-sanitizing spray Surface-cleaner spray Aqueous phase (g) Alcohol (g) EO (mass %) Aqueous phase (g) Alcohol (g) EO (mass %) Aqueous phase (g) Alcohol (g) EO (mass %)
tests in this study were performed using clinical strains of Escherichia coli and

Table 2 .
Physico-chemical characteristics of the selected essential oils

Table 3 .
Chemical composition of C. zeylanicum bark essential oil a Experimental retention indices ; b Reference values according to online NIST database (NIST WebBook SRD 69); c

Table 4 .
Chemical composition of the O. gratissimum leaves essential oil a Experimental retention indices; b Reference values according to online NIST database (NIST WebBook SRD 69); c

Table 5 .
Ranking test of the tested formulations of food preservative-sprays

Table 6 .
Efficacy test for the food-preservative spray

Table 7 .
Hand-sanitizing spray efficacy test

Table 8 .
Efficacy test for the surface cleaner spray

Table 8 .
Wandera (2022)dicated that even at accelerated aging conditions, the spray with 2.5% essential oil was efficient.Wandera (2022)has demonstrated that white vinegar is a good disinfectant in food preparation settings.It is active against the two strains E.coli and S. aureus.Our study demonstrated a good syner-O N L I N E F I R S T gistic effect of vinegar with O. gratissimum essential oil.Previous research showed that xanthan gum has good aroma trapping property