Application of essential oils and polyphenols as natural antimicrobial agents in postharvest treatments: Advances and challenges

Abstract The use of natural antimicrobial agents is an attractive ecological alternative to the synthetic fungicides applied to control pathogens during postharvest. In order to improve industrial production systems, postharvest research has evolved toward integration with science and technology aspects. Thus, the present review aims to draw attention to the achieved advances and challenges must be overcome, to promote application of essential oils and polyphenols as antimicrobial agents, against phytopathogens and foodborne microorganisms during postharvest. Besides that, it attempts to highlight the use of coating and encapsulation techniques as emerging methods that improve their effectiveness. The integral knowledge about the vegetable systems, molecular mechanisms of pathogens and mechanisms of these substances would ensure more efficient in vitro and in vivo experiences. Finally, the cost‐benefit, toxicity, and ecotoxicity evaluation will be guaranteed the successful implementation and commercialization of these technologies, as a sustainable alternative to minimize production losses of vegetable commodities.

Taking into account, that postharvest process is focused on food safety and food properties conservation (nutritional, taste, aroma, and good appearance), the study about disinfection of foodborne bacteria, which threaten the health of consumers, is among the aspects that must be considered during postharvest research also (Gómez-López, 2012;Leyva et al., 2017;Meireles, Giaouris, & Simıes, 2016).The application of chemical microbicides continues to be the main form of pathogens control in different contexts. However, in the last decade, their intensive use has caused great concern about important issues related to environmental and human health (Arias & Toledo, 2018;García-Robles, Medina-Rodríguez, Mercado-Ruiz, & Báez-Sañudo, 2017;Mari, Bautista-Baños, & Sivakumar, 2016;Palou, Ali, Fallik, & Romanazzi, 2016). This situation is inducing to reinforce the regulations regarding the allowed maximum residue limit in the products ( Regulation (EU) No 528/2012).
Taking into account the importance of the research and techno-industrial strategies development, related with implementation of postharvest conservation technologies, the present work aims to update the state of the art about the study of EOs and PPhs as antimicrobial agents with potential use in postharvest field. Besides that, some challenges to be overcome for the industrial applications and commercialization of these antimicrobials in postharvest treatments should be evaluated.

| E SS ENTIAL OIL S AND P OLYPHENOL S A S NATUR AL ANTIMICROB IAL AG ENTS
Among the plant extracts that favor the development of efficient management strategies against microbial contamination, EOs have been the most investigated compounds among other antimicrobials (Kumar & Kudachikar, 2018;Mary et al., 2016;Rao, Chen, & McClement, 2019).

| E SS ENTIAL OIL S
The EOs are included among VOCs that are produced as secondary metabolites by plants and microorganisms. They have been extensively studied as aromas and flavoring substances, appreciated for food applications (Bosquez-Molina, Bautista-Baños, Morales-López et al., 2009). Furthermore, they have received a great attention for their antimicrobial action, useful during postharvest process (Kumar & Kudachikar, 2018;Mari et al., 2016). As GRAS (Generally Considered as Safe) substances, they have been applied in the agro-food market, and some of them such as thyme (Thymus vulgaris L.), clove (Syzygium aromaticum L. Merr. & L.M.Perry), and mint oil (Mentha rotundifolia L. Huds) have included in the pesticide database by the European Commission (http://ec.europa.eu/food/plant/ plagu icida s/eu-pesti cidas-datab ase-redir ect/index_en.htm) (Mari et al., 2016).
The extraction of EOs is possible from the plants that belong to several typical botanical orders, using different parts of the tree (bark and wood, flowers, fruit, peel, leaves, roots, and exudates) (Kumari et al., 2014;Mari et al., 2016). EOs mainly contain volatile terpenoids (monoterpenoids and sesquiterpenoids) with different characteristic functional groups (Rao, Chen, & McClement 2019).
Interestingly, all foodborne pathogens tested were sensitive to first oil and two of them, S. aureus and Listeria monocytogenes, to second one (MIC ≤ 1.25 μl/ml) while the beneficial food-related bacteria (Lactobacillus sp.) were not affected (MIC ± 10 μl/ml). Finally, Cinnamomum zeylanicum Blume oil showed a broader antimicrobial activity on all microorganisms analyzed.
Concerning the phytopathogenic bacteria, some reports have been founded about their sensitivity to EOs. Thus, the antimicrobial activity of 19 EOs was screened by Borboa-Flores et al.. (2010) in order to control Clavibacter michiganensis subspecies michiganensis, which causes serious losses to tomato crops. Six of them were selected by their bactericidal activity at dilutions 1:1, 1:5, and 1:10.
The best results were shown by Lippia palmeri S. Wats, T. vulgaris L., and C. zeylanicum J. Presl. The variance analysis revealed significant difference between these EOs, their concentrations as well as interaction between both two parameters in the bacterial growth inhibition.
Special attention has been dedicated to Colletotrichum gleosporioides because it is a cause of anthracnose, which provokes great losses in fruit production (Bosquez-Molina et al., 2010;Kumar & Kudachikar, 2018;Yilmaz et al., 2016). During in vitro and in vivo investigations with different EOs against anthracnose in apple was demonstrated that their fungicidal activity depends on the content of the metabolites carvacrol, thymol, cimeno, pinene, linalool, and ether acetate. However, their activity depends on the synergic action with the other minority components.
Recently, research has been focused on characterization of the specific major EOs extracts components present in different oils in relation to their antibacterial and antioxidant properties. Indeed, most of the constituents of common EOs that exhibit high antimicrobial efficacy are phenols, followed by oxygenated terpenoids.

| P OLYPHENOL S
The PPhs are secondary metabolites widely distributed in plants, where they fulfill various functions related to growth and reproduction, allelopathy, and protection against pathogens, predators, diseases, and UV radiation. Usually, they do not have a significant toxic effect depending on their concentration and gallic acid content (Bakowska- Barczak & Kolodziejczyk, 2011;Manchón, 2013). Usually, they are classified as flavonoids and nonflavonoids compounds.
Dozens of different flavonoids may be present in the same plant species. Some of them are conjugated with various sugars and further distinguished by the number and arrangement of the hydroxyl groups, and degree of alkylation and glycosylation (Manchón, 2013).  (Table 1).

Many investigations about the
Phenolic extracts of these plants have been shown a broad spectrum of antimicrobial activity mainly against foodborne microorganisms (Table 1) (Table 1). In general, a broad range of concentrations of different extracts has been used but in the majority of papers the authors report values of growth inhibition less than 50%, results that are must be improved in order to obtain a good efficient during in vivo tests.  Therefore, interesting antifungal effect was showed by phenolic and carotenoid extracts of "chiltepin" (Capsicum annum var. Glabriusculum) against F. oxysporum and A. alternata strains in relation to mycelial growth inhibition and conidial germination inhibition (Rodríguez-Maturino et al., 2015) (Table 2).

| MECHANIS MS OF AC TION
Regarding the action mechanisms of EOs and PPhs as antimicrobial agents, it is important to consider that their effectiveness will depend not only on the concentration and chemical nature, but also on the susceptibility and concentration of the pathogen, and even of the microbial strain characteristics. This capacity will vary depending on the composition and nature of the microbial cell surface (Melgarejo & Postilla, 2011;Rao et al., 2019). Furthermore, it is important to know and understand the mechanisms of action of pathogens, depending on the characteristics of the fruit and environmental con- Four possible mechanisms of action have been defined for antimicrobial substances: interference in the cellular structure; interference in cellular biosynthesis; inhibition of the energy mechanism and multisite activity (Calvo & Martínez-Martínez, 2009;Melgarejo & Postilla, 2011;Rodríguez-Maturino et al., 2015). Besides that, the environmental factors such as temperature, pH, medium composition, etc. and contact time will play an important role, as well (Barkai-Golan, 2001;Sandoval-Contreras et al., 2017). In the case of EOs, due to their hydrophobic nature, they could penetrate through bacterial cell outer membranes and cytoplasmic membranes into the interior of cell and thus disintegrate its structures. It renders them more permeable, causing the leakage of cellular components or inactivating the enzymes responsible for the cell wall synthesis and provoking molecular changes in their structure. In addition, the EOs have antioxidant properties effective in retarding the process of lipid peroxidation and scavenging free radicals (Rodríguez-García et al., 2016). In the same manner, these structural modifications take place in fungi and would cause morphological variations of the hyphae and inhibition of conidial germination (Cushnie & Lamb, 2005;Rao et al., 2019;Rodríguez-García et al., 2016).
In general, EOs and PPhs may provoke a physical, chemical, or biochemical change in the microorganisms, and different constituents may operate by different mechanisms and may target different kinds of microbes, such as Gram-positive and Gram-negative bacteria, yeasts, or molds, because they differ in the composition of their cell membranes. Therefore, it is difficult to predict how susceptible pathogens are and why the susceptibility varies from strain to strain after applying these compounds.
It has been proved that some tree EOs inhibit respiration and also causing potassium ion leakage in both Gram-positive bacteria and Gram-negative bacteria, and the most of their constituents are phenols (thymol, eugenol, and carvacrol), followed by oxygenated terpenoids (Rao et al., 2019).
Regarding PPhs some mechanisms are reported: formation of complexes with soluble and extracellular proteins, generating a disruption of the fungal cell wall (Rodríguez-Maturino et al., 2015); reduction of the decomposition of H 2 O 2 , which affect many pathogens since it becomes highly reactive oxygen species promoting oxidative damage; action as a proton exchanger, and the resulting collapse of proton motive force and depletion of adenosine triphosphate (ATP) eventually lead to cell death (Rao et al., 2019).
PPhs could inactivate the essential amino acid synthesis caused by interference in the reactions of phosphoenolpyruvate, erythrose-4-phosphate, and shikimic acid. This favors the production of tryptophan and decreases the production of phenylalanine or tyrosine, modifying the structure of some proteins essential for the formation of fungal appressorium structure (Pagnussatt et al., 2013).
It has been shown that PPhs act at the membrane level by modifying the polar heads of the lipid molecules. Some results demonstrated that their antimicrobial activity is associated with the presence and position of a free hydroxyl group bonded directly to a C6 aromatic ring as a system for electron delocalization, that favor their ability to modify the microbial cell membrane integrity.
Furthermore, the hydroxyl group has a key role in the inactivation of microbial enzymes such as ATPase, histidine decarboxylase, amylase, and protease. Inhibition of ATPase may be important for cell death due to disturbed cellular respiration (Rao et al., 2019). For instance, tannins are able to block the activity of catalase, provoking a lethal effect on some pathogens (Zhao & Drlica, 2014).
Also, they could inhibit enzymes involved in ergosterol synthesis, the main component of the fungal cell membrane, reducing its intracellular content (Campoy & Adrio, 2017;Rao et al., 2019). Besides that, it is important to take into consideration that EOs and PPhs compounds also could induce fruit resistance processes to pathogenic microorganisms, as other natural compounds do (chitosan, fructooligosaccharides, and vegetable hormones), provoking a sequential reaction by activation of particular genes and biosynthesis of antimicrobial substances .

| US E OF COATING S AND FILMS FOR NATUR AL ANTIMICROB IAL S AG ENTS' APPLI C ATI ON
In order to prevent postharvest diseases, the natural antimicrobial agents can be used in vapor or liquid systems, mixed with surfactants in immersion tanks during the packaging process or in wax formulations. In the practice, these compounds have been incorporated   In the complex host/antimicrobial compound/pathogen system,  (Fernández et al., 2017;Mari et al., 2016). Besides that, a greater emphasis has been placed on the currently films development from new natural biopolymers, due to the growing demand for sustainable food production and opportunities to open new markets from nontraditional agricultural products and wastes. Recently, the use of conventional raw materials (tubers, rice, amaranth, and quinoa) has allowed to obtain films and coatings with good mechanical and barrier properties (Mari et al., 2016).

| EN C APSUL ATION OF NATUR AL ANTIMICROB IAL COMP OUNDS
In postharvest processes as in food practice, applications of encapsulation methods have been linked with ensuring controlled releasing and avoiding undesirable structural and bioactivity changes of HBVC. Micro-and nano-encapsulation are two major ways of this technology, able to improve product functionality. Recently, there have been found remarkable interest in development of these nano-scale delivery systems for this compounds due to their advantages-high encapsulation efficiency and loading capacity, enhanced bioavailability, improved stability, sustained release profile, and masking undesirable flavors (Esfanjani & Jafari, 2016;Fathi, Martín, & McClements, 2014;Kamil et al., 2015;Liang et al., 2017;Maes, Bouquillon, & Fauconnier, 2019;Shishir et al., 2018;Yu et al., 2018 et al., 2015). It has shown that the technique, nature of the coating material and HBVC also greatly influenced the encapsulation capacity (Ballesteros et al., 2017).
Concerning the antimicrobial effectiveness of micro-and nanoparticles, some attractive results have been obtained (Table 3).
An improvement in the antimicrobial action of monoterpenoid phenols, carvacrol, and thymol was observed when they were added as microcapsules in flexible plastic film coating (Guarda et al., 2011). In  (Table 3). In this sense, nanoencapsulation and delivery of phytochemicals of tropical fruit by-products was studied by Silva, Hill, Figueredo, and Gomes (2014). They demonstrated the advantages of the use of this matrix for antioxidant and antimicrobial applications. The same manner, nanoencapsulation of hydrophobic phytochemicals and PPhs of guabiroba fruit (Campomanesia xanthocarpa O. Berg) was achieved by Pereira, Hill, and Zambiazi (2015) and Pereira et al. (2018).
Evaluation of capsules with these compounds against the high-resistant Gram-positive bacterium, L. innocua, showed a significantly greater growth inhibition in comparison with nonencapsulated compounds. Furthermore, capsules of polyphenolic extracts of passion fruit (Passiflora edulis Sims) in PLGA were obtained using the coprecipitation method (Oliveira, Ferreira, & Gomes, 2017). These authors observed 23.8%-79% efficiency and a greater antimicrobial action in comparison with the whole extracts (Table 3).
Therefore, β-cyclodextrin (CD) and their derivative hydroxypropyl-β-cyclodextrin have been used successfully also for encapsulation of PPhs as eugenol and (+) catechin and (−)-epicatechin, improving their stability (Table 3). In vitro assays showed inhibitory effect of complex CD-eugenol on of Peronophythora litchii colony growth in a concentration-and time-dependent manner (MIC = 0.2 g), while in vivo assays showed its impact on reduction of the decay index of treated fresh litchi fruit. After exposure to CD-EG, the surface of fungal hyphae and/or sporangiophores became wrinkled, with folds and breakage. Besides that, damage to cell walls and membrane structures was confirmed (Gong, Li, Chen et al., 2016). On the other hand, investigation about complexation of two isomers, (+)-catechin and (−)-epicatechin, with hydroxypropyl-b-cyclodextrin in Tris-HCl buffer solutions at pH 6.8-8.0 using isothermal titration calorimetry, was carried out.
Stability study indicated that a complex with CD showed a stronger but different protection effect on isomers, depending on their molecular structure (Liu et al., 2016).

| CHALLENG E S IN RE S E ARCH AND APPLIC ATION OF NATUR AL ANTIMICROB IAL S IN P OS THARVE S T SYS TEMS
According includes. More knowledge about the antimicrobial mechanisms of action of EOs and PPhs at the molecular level, rather than just at the cellular level is required (Basak & Guha, 2018;Gómez-López, 2012;Rao et al., 2019;Romanazzi et al., 2016;Wenchao et al., 2019).
Natural alternatives would have a better chance of success if they have been applied at optimum timing, and thus, the early and rapid detection of pathogens is important. Consequently, smart innovative tools and technologies are needed to improve the accuracy and promptness in diagnosing plant pathogens and foodborne microorganisms and their toxic metabolites (Albonico, Schutz, Caloni, Cortinovis, & Spicer, 2017;Cortés-Higareda et al., 2019). They will facilitate high-throughput analysis and efficient monitoring .
Finally, it is necessary to consider the role of genomic knowledge which gives information about gene functions, molecular receptors, and metabolic reactions that allow a better understanding of the physiological changes in fruit and vegetables and favors the optimization of treatments to control postharvest decay (Droby et al., 2016;Fernandez-Blanco, Frizzell, Shanon, Ruíz, & Connolly, 2016). On the other hand, recent progress in metagenomic technologies should be used to characterize the composition of microbiota on products and its dynamics (Droby et al., 2016).
Thus, this information may be useful to develop a new paradigm in postharvest biocontrol and in strategies of use of natural antimicrobials also. Therefore, more knowledge about the antimicrobial mechanisms of action of EOs and PPhs at the molecular level, rather than just at the cellular level is required (Rao et al., 2019).
Besides that, considering the value of nanotechnology-related combined preservation strategies and nanotechnology-related intel- to understand the decomposition mechanisms of these substances in organism (Mari et al., 2016). Among other relevant aspects, the organoleptic properties of the fruit are very important since the taste should not be affected by the treatment or by other quality attributes, related to the appearance, the nutrient concentration, etc. (Mari et al., 2016;Palou et al., 2016).
Finally, besides the satisfactory results at laboratory or semi-industrial level, a techno-economic feasibility study it is indispensable, in order to glimpse the cost-benefit relationship of the proposed production process. It will depend on infrastructure, raw material availability, local needs and technical facilities, among other factors. The integral approach, must be consider for costeffectiveness control programs of preharvest, harvest, and post-harvest processes.
For successfully development of this technology will be indispensable to attend the cost and energy sustainability, applying strategies related with circular economy. Mass and energy integration are necessary for reducing the costs and environmental impact.
The use of the biorefinery-based platform concept will enhance the efficiency of the plant residues utilization and to develop technological and marketing approaches for production of other HBVC and bioenergy (Moncada, Tamayo, & Cardona, 2014). Recently, a biorefinery model for PPhs, ethanol, and xylitol coproduction from spent blackberry pulp was developed by Davila, Rosenberg, and Cardona (2017). According to the sale-to-total-production-cost ratio, they demonstrated the importance of the mass and heat integration. The productivity values of 193.4, 6,912, and 452.2 kg/day of polyphenols, ethanol, and xylitol and yields of 3.88, 352.9, and 18.37 kg/ton of spent blackberry pulp were obtained, respectively.
On the other hand, it is necessary to develop cheaper technological alternatives, on the basis of efficient emergent extraction methods, and mainly drying techniques used during the raw material pretreatment, extracts concentration, and encapsulates formation.
In this sense, some studies about spray drying conditions were carried out in order to avoid high energy consumption. For instance, the best conditions for the energy required, production costs, and physicochemical characteristics of the cheese whey were determined by Domínguez-Niño, Cantú-Lozano, Ragazzo-Sánchez, Andrade-González, and Luna-Solano (2018). A dried product of 0.2165 kg/hr was obtained, with a moisture content of 2.08%, cost of 17.06 $/kg, and energy consumption of 2.0490 kW·hr/kg of dry product. Besides that, great interest to use and optimization of the renewable energies for production processes has been observed (Costales, 2010).

| CON CLUS IONS
The use of natural antimicrobial agents as EOs and PPhs is a more ecological alternative technology to the synthetic and chemical fungicides currently used to control pathogens in postharvest processes. Among emerging technologies, edible coatings, films, and encapsulation techniques are showing advances and ensure improvement of the treatment effectiveness.
However, there are still many limitations that make difficult the implementation of this technology as a control strategy. Among some, challenges must be overcome for the industrial application, is necessary to consider the variability in quantity and quality of natural products, depending on the source, extraction conditions, and characterization methods. It is needed to show their true effectiveness against the disease in order to evidence the individual and synergistic effects.
Concerning methods, smart innovative tools are needed to improve the accuracy and promptness in diagnosing plant pathogens, foodborne microorganisms, and their toxic metabolites. On the other hand, it is indispensable to increase experimental tests in vivo, in order to confirm the laboratory in vitro results, and to apply a holistic method of analysis of postharvest process for improving the efficiency of treatments. Finally, genomic knowledge will give information about gene functions, molecular receptors, and metabolic reactions, allowing a better understanding of the physiological changes in fruit and vegetables and favoring the optimization of treatments also. Besides that, more studies about the microbiome diversity and composition on harvested products and its variability after harvest and during storage are necessary.
Therefore, attention must be paid to their toxicological properties, mechanisms of action against the pathogens and the effect on the organoleptic properties of the products. All these aspects will favor the successful implementation and commercialization of natural antimicrobial postharvest technology, but a cost-benefit relationship, toxicity and ecotoxicity analysis will be indispensable in order to comply the sustainability of this technology.

ACK N OWLED G M ENT
This study was supported by the Consejo Nacional de Ciencia y Tecnología (CONACyT, México) for the scholarship granted to Laura Maryoris Aguilar Veloz (number 895760) and Yuliana Vázquez González (number 862248), CYTED thematic network code 319RT0576 ("Desarrollo Sostenible en Agroalimentación y Aprovechamiento de Residuales Industriales") and Spanish Ministry of Science.

CO N FLI C T O F I NTE R E S T
The authors declare that they do not have any conflict of interest and written informed consent was obtained from all of them.

E TH I C A L S TATEM ENTS
This study does not involve any human or animal testing.