Universidade Estadual Paulista “Júlio de Mesquita Filho” Faculdade de Ciências Farmacêuticas Plant-based nanoemulsions with essential oils as edible coatings: A novel approach for strawberry preservation Josemar Gonçalves de Oliveira Filho Tese de Doutorado apresentada à Pós- Graduação em Alimentos e Nutrição para obtenção do título de Doutor em Alimentação e Nutrição. Área de concentração: Ciência de Alimentos Orientador: Prof. Dr. Marcos David Ferreira Araraquara 2022 Plant-based nanoemulsions with essential oils as edible coatings: A new approach for strawberry preservation Josemar Gonçalves de Oliveira Filho Tese de Doutorado apresentada à Pós- Graduação em Alimentos e Nutrição para obtenção do título de Doutor em Alimentação e Nutrição. Área de concentração: Ciência de Alimentos Orientador: Prof. Dr. Marcos David Ferreira Araraquara 2022 Diretoria do Serviço Técnico de Biblioteca e Documentação – Faculdade de C iênc ias Farmacêuticas UNESP C ampus de A raraquara Oliveira Filho, Josemar Gonçalves de. O486p Plant-based nanoemulsions with essential oils as edible coatings: A new approach for strawberry preservation / Josemar Gonçalves de Oliveira Filho. – Araraquara: [S.n.], 2022. 175 f. : il. Tese (Doutorado) – Universidade Estadual Paulista. “Júlio de Mesquita Filho”. Faculdade de Ciências Farmacêuticas. Programa de Pós Graduação em Alimentos e Nutrição. Área de Concentração em Ciência de Alimentos. Orientador: Marcos David Ferreira. 1. Óleo essencial. 2. Nanoemulsão. 3. Cera de carnaúba. 4. Amido de aratura. 5. Nanocristais de celulose. I. Ferreira, Marcos David, orient. II. Título. 33004153070P3 Esta f icha não pode ser modif icada UNIVERSIDADE ESTADUAL PAULISTA Câmpus de Araraquara CERTIFICADO DE APROVAÇÃO TÍTULO DA TESE: Plant-based nanoemulsions with essential oils as edible coatings: A novel approach for strawberry preservation AUTOR: JOSEMAR GONÇALVES DE OLIVEIRA FILHO ORIENTADOR: MARCOS DAVID FERREIRA Aprovado como parte das exigências para obtenção do Título de Doutor em ALIMENTOS E NUTRIÇÃO, área: Ciência dos Alimentos pela Comissão Examinadora: Prof. Dr. MARCOS DAVID FERREIRA (Participaçao Virtual) EMBRAPA Instrumentacao Agropecuaria Profa. Dra. MARIANA BURANELO EGEA (Participaçao Virtual) Departamento de Ciência e Tecnologia de Alimentos / Instituto Federal Goiano - Câmpus Rio Verde Profa. Dra. ELAINE CRISTINA PARIS (Participaçao Virtual) Embrapa Instrumentação / Empresa Brasileira de Pesquisa Agropecuária Profa. Dra. KATIA SIVIERI (Participaçao Virtual) Departamento de Alimentos e Nutrição / Faculdade de Ciências Farmacêuticas - UNESP- Araraquara Araraquara, 04 de março de 2022 Faculdade de Ciências Farmacêuticas - Câmpus de Araraquara - Rodovia Km 1 , 14800903 http://www2.fcfar.unesp.br/#!/pos-graduacao/alimentos- e-nutricao/CNPJ: 48.031.918/0025-00. Dedico-o à minha família, professores, amigos e a todos que cruzaram meu caminho durante o doutorado. Agradecimentos À Fundação Amparo à Pesquisa do Estado de São Paulo – FAPESP, pela bolsa no país, processo 2018/24612-9. À Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – CAPES, pela bolsa no país Código de Financiamento 001 Ao professor Dr. Marcos David Ferreira pela orientação ao longo de todo o doutorado. À Dra. Henriette Cordeiro Monteiro de Azeredo pela confiança e colaboração com essa pesquisa. Aos analistas, bolsistas e estagiários da Embrapa Instrumentação pelo apoio durante a realização dos experimentos. Em especial: Joana, Silviane, Adriana, Viviane, Milene, Ítalo, Beatriz. À Faculdade de Ciências Farmacêuticas - UNESP. À Embrapa Instrumentação pela estrutura laboratorial. Obrigado! iv Resumo Objetivo: O objetivo deste estudo foi avaliar o potencial de óleos essenciais (OEs) como fungicidas naturais e sua presença em revestimentos bio- nanocompositos baseados em amido de araruta, nanoemulsão de cera de carnaúba e nanocristais de celulose para aplicação na manutenção da qualidade e conservação pós-colheita de morangos durante o armazenamento. Metodologia: Os OEs das espécies Hortelã-verde (Mentha spicata), Hortelã- pimenta (Mentha x piperita), Palmarosa (Cymbopogon martinii) e Ho wood (Cinnamomum camphora) foram caracterizados por CG-SM e CG-FID, e a atividade antifúngica foi avaliada por diferentes métodos in vitro, e in vivo contra os principais fungos pós-colheita de morango. O efeito da incorporação da cera de carnaúba (0-15% em peso) utilizando a tecnologia de emulsão ─ micro- e nanoemulsão ─ nas características (físicas, tecnológicas e ópticas) de filmes/revestimentos de amido de araruta foi investigado. Em seguida, foram desenvolvidos filmes/revestimentos à base de amido de araruta com nanoemulsão de cera de carnaúba, nanocristais de celulose e óleos essenciais de Mentha spicata e Cymbopogon martinii foram produzidos usando a técnica de casting. Os filmes foram caracterizados quanto às propriedades de barreira à água, tração, térmica, ótica e microestrutura, bem como atividade antifúngica in vitro contra Rhizopus stolonifer e Botrytis cinerea. Morangos foram recobertos com os revestimentos desenvolvidos e foram avaliados durante o armazenamento (0, 3, 6, 9, e 12 dias) em relação ao teor de sólidos solúveis, pH, acidez titulável, perda de massa, firmeza, taxa de respiração, coloração, contagem de fungos totais, bactérias mesófilas aeróbias, deterioração fúngica visual, teor de antocianinas, ácido ascórbico, fenóis totais e atividade antioxidante. Resultados: Os principais componentes dos óleos essenciais foram mentol (45,37%), mentona (20,13%), isomentona (16,94%), acetato de mentila (3,81%), pulegona (1,89%), α-terpineno (1,88%), isopulegol (1,83%), neoisomentol (1,19%), e α-terpineol (1,08%) para óleo essencial de M. piperita, linalol (98,39%) para óleo essencial de C. camphora e geraniol (83,82%), acetato de geranila (7,49%), linalol (2,48%) e cariofileno (1,33%) para o óleo essencial de C. martini. A maior atividade antifúngica foi promovida por M. spicata e C. martinii nos métodos de contato direto e contato de vapor, diluição em micro-poços e ensaio de germinação de esporos, e in vivo na fase de vapor em morangos inoculados artificialmente com R. stolonifer e B. cinerea. A presença da cera de carnaúba nos filmes aumentou suas características hidrofóbicas, reduzindo a solubilidade em água, a umidade, a permeabilidade ao vapor de água e a estabilidade térmica, além de melhorar as propriedades de barreira à luz. Os filmes com nanoemulsão apresentaram menor permeabilidade ao vapor de água e propriedades de barreira à luz, bem como melhor resistência à tração e microestrutura mais lisa do que os filmes realizados com microemulsão. Enquanto a incorporação dos nanocristais de celulose diminuiu o teor de umidade e a permeabilidade ao vapor de água dos filmes, os nanocristais de celulose e o óleos essenciais diminuíram a transparência e afetaram a microestrutura dos filmes. A incorporação dos óleos essenciais de M. spicata e C. martinii melhorou a estabilidade térmica e conferiu excelente atividade contra fungos que v deterioram a fruta. Os filmes demonstraram uma excelente barreira contra o crescimento de fungos, permeabilidade ao vapor de água e luz UV/vis. Os revestimentos carregados com óleos essenciais de M. spicata e C. martinii foram capazes reduzir significativamente a severidade de B. cinerea e R. stolonifer em morangos artificialmente inoculados. Os revestimentos bio-nanocompósitos melhoraram a estabilidade dos morangos durante o armazenamento, minimizando a perda de massa, e alterações na cor, textura (exceto os revestimentos com óleo essencial de C. martinii), sólidos solúveis, pH, acidez titulável, teor compostos fenólicos, antocianinas, ácido ascórbico e atividade antioxidante em comparação com os morangos sem revestimentos (controle). Além disso, os revestimentos com óleos essenciais de M. spicata e C. martinii apresentaram atividade antimicrobiana, reduzindo a deterioração visual por fungos e as contagens de bactérias aeróbicas mesófilas e fungos e leveduras durante o armazenamento. Conclusão: Pode-se concluir que os revestimentos bio-nancompósitos, principalmente os carregados com óleo essencial de M. spicata, podem ser usados como materiais de revestimentos antimicrobianos para preservar morangos frescos durante a pós-colheita. Palavras-chave: óleo essencial; nanoemulsão; cera de carnaúba; amido de aratura, nanocristais de celulose. vi Abstract Objective: The aim of this study was to evaluate the potential of essential oils (EOs) as natural fungicides and their presence in bio-nanocomposite coatings based on arrowroot starch, carnauba wax nanoemulsion, and cellulose nanocrystals for application in quality maintenance and postharvest conservation of strawberries during storage. Methodology: The EOs of the species Green Mint (Mentha spicata), Peppermint (Mentha x piperita), Palmarosa (Cymbopogon martinii) and Howood (Cinnamomum camphora) were characterized by GC-MS and GC-FID, and the antifungal activity was evaluated by different methods in vitro, and in vivo against the main strawberry postharvest fungi. The effect of incorporating carnauba wax (0-15% by weight) using emulsion technology ─ micro- and nanoemulsion ─ on the characteristics (physical, technological and optical) of arrowroot starch films/coatings was investigated. After that, films/coatings were developed based on arrowroot starch with carnauba wax nanoemulsion, cellulose nanocrystals and essential oils of M. spicata and C. martinii were produced using the casting technique. The films were characterized for water barrier, tensile, thermal, optical and microstructure properties, as well as in vitro antifungal activity against Rhizopus stolonifer and Botrytis cinerea. Strawberries were coated with the developed coatings and were evaluated during storage (0, 3, 6, 9, and 12 days) for soluble solids content, pH, titratable acidity, mass loss, firmness, respiration rate, color, total fungi count and aerobic mesophilic bacteria counts, visual fungal deterioration, anthocyanin, ascorbic acid, and total phenols contents, and antioxidant activity. Results: The main components of essential oils were menthol (45.37%), menthone (20.13%), isomentone (16.94%), menthyl acetate (3.81%), pulegone (1.89%), α-terpinene (1.88%), isopulegol (1.83%), neoisomenthol (1.19%), and α-terpineol (1.08%) for essential oil of M. piperita, linalool (98.39 %) for essential oil of C. camphora and geraniol (83.82%), geranyl acetate (7.49%), linalool (2.48%) and caryophyllene (1.33%) for the essential oil of C. Martini. The greatest antifungal activity was promoted by M. spicata and C. martinii in the direct contact and steam contact methods, micro-well dilution and spore germination assay, and in vivo in the vapor phase in strawberries inoculated artificially with R. stolonifer and B. cinerea. The presence of carnauba wax in the films increased their hydrophobic characteristics, reducing water solubility, moisture, water vapor permeability and thermal stability, in addition to improving light barrier properties. Films with nanoemulsion showed lower permeability to water vapor and light barrier properties, as well as better tensile strength and smoother microstructure than films made with microemulsion. While the incorporation of cellulose nanocrystals decreased the moisture content and water vapor permeability of the films, the cellulose nanocrystals and essential oils decreased the transparency and affected the microstructure of the films. The incorporation of essential oils from M. spicata and C. martinii improved thermal stability and provided excellent activity against fungi that spoil the fruit. The films demonstrated an excellent barrier against mold growth, permeability to water vapor and UV/vis light. Coatings loaded with essential oils of M. spicata and C. martinii were able to sis, nificantly reduce the severity of B. cinerea and R. stolonifer in artificially inoculated strawberries. Bio-nanocomposite coatings improved the stability of strawberries during storage, minimizing mass loss, and changes in color, texture (except coatings with C. martinii essential oil), soluble vii solids, pH, titratable acidity, phenolic, anthocyanins, and ascorbic acid contents, and antioxidant activity compared to uncoated strawberries (control). Furthermore, coatings with essential oils from M. spicata and C. martinii showed antimicrobial activity, reducing visual deterioration by fungi and the counts of aerobic mesophilic bacteria and fungi and yeasts during storage. Conclusion: It can be concluded that bio-nancomposite coatings, mainly those loaded with M. spicata essential oil, can be used as antimicrobial coating materials to preserve fresh strawberries during post-harvest. Key-words: essential oil; nanoemulsion; carnauba wax; aratura starch, cellulose nanocrystals. SUMÁRIO Pagina Resumo iv Abstract vi Introdução 9 Capítulo 1. Nanoemulsions as edible coatings: A potential strategy for fresh fruits and vegetables preservation 12 Introduction 14 Edible coatings – an overview 16 Methods to apply edible coatings 19 Nanomaterials in edible coatings 21 Fundamentals of nanoemulsions 22 Plant-based nanoemulsions as edible coatings on fruits and vegetables postharvest 25 Trends in materials based on nanoemulsions with potential for application in the preservation of fruits and vegetables 33 Potential toxicity, limitations, and regulatory aspects of nanoemulsions 35 Conclusion 36 Reference 37 Capítulo 2. Chemical composition and antifungal activity of essential oils and their combinations against Rhizopus stolonifer and Botrytis cinerea in strawberries 45 Introduction 47 Material and methods 48 Results and discussion 52 Conclusion 68 Reference 68 Capítulo 3. New approach in the development of edible films: The use of carnauba wax micro- or nanoemulsions in arrowroot starch-based films. 73 Introduction 75 Material and methods 76 Results and discussion 80 Conclusion 93 Reference 93 Capítulo 4. Arrowroot starch-based films incorporated with a carnauba wax nanoemulsion, cellulose nanocrystals, and essential oils: A new functional material for food packaging applications 103 Introduction 105 Material and methods 106 Results and discussion 110 Conclusion 122 Reference 123 Capítulo 5. Bio-nanocomposite edible coatings loaded with essential oils to preserve quality and improve shelf life of strawberry cv. ‘Oso Grande’ 130 Introduction 132 Material and methods 133 Results and discussion 134 Conclusion 139 Reference 155 Considerações finais 162 Referências 164 Apêndices 166 9 Introdução O morango é uma fruta não climatérica caracterizada por apresentar sabor único, ser altamente desejável e ser uma fonte relevante de compostos bioativos devido aos altos níveis de vitamina C, vitamina E e compostos fenólicos, assim como antocianinas, pigmentos que conferem cor vermelha ao morango e estão relacionadas a benefícios para a saúde (1). Devido à sua alta taxa de respiração, textura macia e sensibilidade à temperatura, choques mecânicos e vibrações, os morangos possuem vida pós-colheita curta. Isso pode resultar em um alto grau de deterioração por vários agentes patogênicos, o que, por sua vez, impacta em mudanças no pH, acidez titulável, teor de sólidos solúveis totais, perda de cor, firmeza e massa, resultando em deterioração e reduzindo a vida útil (2). Os morangos são tradicionalmente tratados com diferentes fungicidas para controlar a deterioração pós-colheita. Entretanto, estes deixam resíduos que apresentam riscos para os seres humanos e o meio ambiente, e os consumidores têm exigido cada vez mais uma produção de alimentos com alta qualidade e vida útil prolongada, com o mínimo de conservantes químicos sintéticos. Assim, medidas eficazes para o aumento da vida útil devem ser exploradas em relação aos mercados distantes, mantendo a qualidade nutricional da fruta (3). Existe, portanto, uma enorme demanda por tecnologias alternativas pós-colheita que devem oferecer proteção contra doenças pós-colheita e distúrbios fisiológicos, além de retardar a senescência, e, com isso, melhorar o manuseio e manutenção da qualidade de frutas, como o morango, durante a pós-colheita (4). Um dos métodos propostos é a aplicação de revestimentos comestíveis incorporados com agentes antimicrobianos naturais como os óleos essenciais (OEs). Resultados promissores para preservar a qualidade dos produtos hortícolas frescos, controlando a deterioração pós-colheita e ampliando a vida útil têm sido encontrados (5, 6, 7). Os revestimentos comestíveis são geralmente baseados em polissacarídeos, proteínas e lipídeos, isolados ou em combinação. Nas frutas e vegetais, esses revestimentos compostos baseados em polissacarídeos ou proteínas associados a lipídeos são usualmente utilizados para atingir boas características de barreira a gases e à umidade proporcionadas pelos componentes polimérico e lipídico, respectivamente (8). As propriedades desses revestimentos, como a adesão e propriedades mecânicas, ainda, podem ser melhoradas incorporando nanoestruturas de reforço, como os nanocristais de celulose (9,10). Os nanocristais de celulose têm atraído grande atenção por serem renováveis e ambientalmente benignos, naturalmente abundantes, biodegradáveis, biocompatíveis e com excelentes propriedades mecânicas (11). Os consumidores estão cada vez mais preocupados com a segurança e qualidade dos alimentos, impulsionando a demanda pelos chamados “revestimentos baseados em plantas”, ou seja, revestimentos à base de produtos naturais de origem vegetal que não apresentam nenhum dano à saúde do consumidor se consumidos. O uso de ceras e compostos de origem animal tem sido limitado por consumidores veganos e vegetarianos, 10 consumidores que são alérgicos a produtos de origem animal (como a quitosana) e crenças religiosas que não encorajam o consumo de animais (9). Os OEs são compostos aromáticos derivados do metabolismo secundário das plantas (12) que exercem fortes atividades antibacterianas, antivirais e antifúngicas, estimulando sua aplicação como antimicrobianos naturais (13). A preocupação crescente dos consumidores com a segurança dos conservantes químicos sintéticos levou ao aumento da tendência à utilização de OEs como agentes bioativos naturais na indústria alimentícia (14). Eles apresentarem baixa toxicidade em mamíferos, menor efeito ambiental e natureza volátil, o que facilita seu uso em baixas concentrações seguras para o consumo. Além disso, os consumidores aceitam os OEs mais prontamente porque são amplamente utilizados em práticas culinárias gerais. Eles também são ecologicamente corretos e são conhecidos como agroquímicos de “risco reduzido” (15). A utilização de nanoemulsões como revestimentos incorporadas com agentes bioativos, como os OEs, tem emergido como uma ferramenta potencial para aplicação em frutas (16). A formulação de nanoemulsões pode melhorar as propriedades de barreira do revestimento devido ao tamanho reduzido das gotículas dispersas e maior homogeneidade em comparação com emulsões convencionais (18). Neste contexto, o objetivo do presente trabalho foi avaliar o potencial de OEs como fungicidas naturais e sua presença em revestimentos nanoestruturados baseados em nanoemulsões para aplicação na manutenção da qualidade e conservação pós-colheita de morangos, como proposta de uso de agentes naturais de origem vegetal como materiais ativos para recobrimento de frutas. Na Figura 1 está apresentado o esquema geral da organização dos capítulos. 11 Figura 1. Esquema geral da composição dos capítulos. Capítulo 1: Nanoemulsions as edible coatings: A potential strategy for fresh fruits and vegetables preservation Capítulo 2: Chemical composition and antifungal activity of essential oils and their combinations against Rhizopus stolonifer and Botrytis cinerea in strawberries Capítulo 3: New approach in the development of edible films: The use of carnauba wax micro-or nanoemulsions in arrowroot starch-based films Capítulo 4: Arrowroot starch-based films incorporated with a carnauba wax nanoemulsion, cellulose nanocrystals, and essential oils: A new functional material for food packaging applications Capítulo 1: Nanoemulsions as edible coatings: A potential strategy for fresh fruits and vegetables preservation Capítulo 2: Chemical composition and antifungal activity of essential oils and their combinations against Rhizopus stolonifer and Botrytis cinerea in strawberries Capítulo 3: New approach in the development of edible films: The use of carnauba wax micro-or nanoemulsions in arrowroot starch-based films Capítulo 4: Arrowroot starch-based films incorporated with a carnauba wax nanoemulsion, cellulose nanocrystals, and essential oils: A new functional material for food packaging applications Capítulo 1 Capítulo 2 Capítulo 3 Capítulo 4 Capítulo 5 12 Capítulo 1. Nanoemulsions as edible coatings: A potential strategy for fresh fruits and vegetables preservation Review manuscript published in Foods. de Oliveira Filho, J.G.; Miranda, M.; Ferreira, M.D.; Plotto, A. Nanoemulsions as Edible Coatings: A Potential Strategy for Fresh Fruits and Vegetables Preservation. Foods 2021, 10, 2438. https://doi.org/10.3390/foods1010243 13 Nanoemulsions as edible coatings: A potential strategy for fresh fruits and vegetables preservation Abstract: Fresh fruits and vegetables are perishable commodities requiring technologies to extend their postharvest shelf life. Edible coatings have been used as a strategy to preserve fresh fruits and vegetables in addition to cold storage and/or controlled atmosphere. In recent years, nanotechnology has emerged as a new strategy for improving coating properties. Coatings based on plant-sources nanoemulsions in general have better water barrier, mechanical, optical and microstructural properties in comparison with coatings based on conventional emulsions. When antimicrobial and antioxidant compounds are incorporated into the coatings, nanocoatings enable the gradual and controlled release of those compounds over the food storage period better than conventional emulsions, hence increasing their bioactivity, extending shelf life and improving nutritional produce quality. The main goal of this review is to update the available in-formation on the use of nanoemulsions as coatings for preserving fresh fruits and vegetables, pointing to a prospective view and future applications. Keywords: nanotechnology; wax coating; natural antimicrobials; essential oils; nanocoatings; post-harvest; bioactive compounds; quality; preservation methods; nanomaterials 14 1. Introduction Fruits and vegetables are important sources of minerals, vitamins, and fibers, which are essential for human well-being, and their consump-tion has been associated with several beneficial effects on human health. The demand for those benefits has considerably increased over the years due to consumer preference for natural products and changes in lifestyle [1]. In this sense, fruits and vegetables are an important component of the human diet. After they are harvested, fruits and vegetables continue the respira-tion process, consume O2 and release CO2 and water. Consequently, li-pids, proteins, organic acids, and carbohydrates are metabolized, and en-ergy replacement is compromised, as the vegetable or fruit is separated from the mother plant [2]. Over time, quality characteristics such as color, flavor, weight, nutritional value, and bioactive compounds continue to deteriorate as a result of senescence [3]. The water released during the res-piration process plays an important role in the postharvest quality of fresh fruits and vegetables and can result in loss of nutritional value, soft tex-ture, sagging, wrinkling, and withering [4]. Although waxes have been used to preserve citrus fruit in ancient China, it was not until the twentieth century that edible coatings based on emulsions were developed to preserve the quality of fresh fruits and vege-tables [5]. These emulsions are typically formulated from oils (vegetable- or animal-derived), waxes (paraffin, carnauba wax, candellila, and bees-wax), and resins (shellac, wood rosin). Furthermore, polymer- based coat-ing solutions can have additional functionality when formulated with plant essential oils having antimicrobial activity [6]. Due to their hydro-phobic nature, oils and waxes have proven to be an efficient technology for fruits and vegetables preservation post-harvest, as they are able to minimize water loss, gas exchange and improve and/or preserve the physicochemical properties, such as color, firmness, fresh appearance, and microbial protection [7-10]. Recently, nanotechnology was introduced as a new tool for making coatings based on emulsions with improved properties and functionali-ties. Coatings are made of macro- or microemulsions (conventional) or nanoemulsions, for which the latter can be considered a conventional emulsion with very small particles. Droplets in nanoemulsions are on a nanoscale (particle radius less than 100 nm) dispersed in an 15 aqueous so-lution [11]. This changes the physical properties of the coating by further reducing moisture migration, gas exchange, oxidative reactions, and sup-pressing pathogenic growth (microorganisms), product deterioration and enhancing control of physiological disorders [12]. In addition, coatings based on nanoemulsions have shown to be promising vehicles for several active compounds, such as oil-soluble vitamins, antimicrobials, flavors, and nutraceuticals, which may further contribute to maintenance of food product quality attributes [13]. Figure 1 shows a survey of published scientific manuscripts on nanoemulsions as edible coatings for fruits and vegetables. The number of studies on the topic has increased considerably over the past few years, demonstrating the scientific community's increased interest in the topic. However, studies concerning in vivo biological efficiencies are limited [14] and applications on fruits and vegetables are even fewer. Thus, more re-search is essential to determine this technology's potential for future ap-plication on a commercial scale. In this context, the objective of this review is to update the available information on the use of nanoemulsions as coatings for preserving fresh fruits and vegetables. Figure 1. The distribution of publication related to ‘nanoemulsion as edible coating for fruits and vegetables’ (2005–2021): ScienceDirect databases). Data for 2021 is as of september. Year 2008 2010 2012 2014 2016 2018 2020 2022 N u m b e r o f p u b lic a ti o n 0 20 40 60 80 100 120 16 2. Edible coatings – an overview The first reports of the use of coatings on fruits appeared in the 12th century in China, where wax was applied to citrus (lemons and oranges) to reduce mass loss and preserve the fruit [15]. However, it was only in 1922 that the commercial scale application of waxes began in order to increase postharvest conservation of fruits and vegetables, thus reducing postharvest losses [16]. Currently, edible coatings are used as a strategy to increase the shelf life and postharvest quality of many fresh fruits and vegetables during storage [17,18]. Edible coatings are defined as thin layers applied on the fruit surface, forming clear films produced from food-grade materials and adding to, or as a substitute for, the waxes naturally present on the fruit surface. As these films become part of the food and are consumed as such (for fruits where the peel is consumed), the materials used in their composition must be GRAS (Generally Recognized as Safe), that is, be non-toxic and safe for food [19]. Edible coatings are formulated from various biopolymers such as polysaccharide, lipid, and protein compounds, or by combining materials resulting in improved properties (Table 1). They act as an obstacle to water vapor, gases, and solutes [20] as shown in Figure 2. 17 Figure 2. Main functions of edible coatings on fruit and vegetables. The mechanism of action for coatings on fruit is similar to packaging with a modified atmosphere; the coating produces a physical barrier that modifies gas exchange between the interior of the fruit and the surrounding atmosphere, increasing the concentration of CO2 and decreasing O2 [30]. This environment can effectively decrease respiration rate, conserve stored energy, delay microbial growth, and therefore, extend the useful life of the fruit [31]. The coating efficiency depends on the coating thickness formed on the fruit surface, since there is a negative correlation between thickness and coating permeability [32]. Another important point is related to low permeability coatings, based on resins such as shellac, for example, which can restrict gas exchange almost entirely, leading to the accumulation of CO2 within the fruit, and the production of compounds resulting from the fermentation process that can cause off-flavor, such as acetaldehyde and ethanol, thus affecting fruit quality [18,33]. Edible coating CO2 CO2H2O H2O 18 Table 1. Summary of diverse structural materials frequently used for edible coating. 1 Material Main matrices Positive points Negative points References Polysaccharide Starch, chitosan, alginate, cellulose, and its derivatives, and pectin Good gas and mechanical barrier properties Poor moisture barrier due to hydrophilic nature [21,22] Lipid Animal, vegetable waxes and resins, vegetable oil, and fatty acids Good moisture barrier properties with a shiny appearance Poor mechanical and gas barrier properties [18,23,24] Protein Gelatin, casein, whey protein, zein, soy protein, myofibrillar protein, and quinoa protein Good gas barrier properties without anaerobic conditions Brittle and susceptible to cracking [25] Composite Combination of polysaccharide and/or protein with lipids Good moisture and gas barrier properties Formation of non- homogeneous emulsion [26-29] 2 19 In addition to maintaining quality and postharvest conservation of fruits and vegetables, the coating materials can also act as carriers of compounds such as food coloring, flavoring, antimicrobials, antioxidants, antagonistic microorganisms, among others [34,35]. In this sense, several natural bioactive compounds have been incorporated into edible coating materials such as essential oils [36-38], plant extracts [39,40], vitamins [34], antagonistic microorganisms [41,42], antibrowning or firming agents in fresh cut fruit. [43,44]. 3. Methods to apply edible coatings The effectiveness of coatings in preserving fresh fruits and vegetables is influenced by the application method, which will be chosen according to the nature of the food to be coated, the surface attributes, the rheological properties of the solution, and the main purpose of the coating [45]. The adhesion of coatings to food surfaces is essential for performance of their intended function [16,46]. Wettability is used to quantify the interfacial interaction that occurs between the food surface and the coating. This variable must be taken into account when assessing the performance of the coating solution on the food surface [31]. Dipping (Figure 3a), spraying (Figure 3b), and hand coating (Figure 3c) techniques are the most common methods for applying edible coatings to fresh fruits and vegetables. Other techniques such as fluidized bed and foaming are also available; however, these techniques are rarely used on commercial and laboratory scales [45]. 20 Figure 3. Dipping (a), spraying (b), and (c) hand coating techniques to apply edible coatings. On a laboratory scale, immersion is one of the main methods used for coating fruits due to its simplicity, without dependence on equipment, and uniformity of film obtained. In this method, the entire surface of the food is submerged in the film-forming solution at a constant speed, allowing full surface coverage, ensuring complete surface wetting [47]. After application, the excess solution is drained to eliminate the overload of film-forming solution on the fruit surface [48]. Finally, the food is dried with the excess solvent and liquid being evaporated to leave the film in contact with the food surface. Drying can take place at room temperature or using a heated air tunnel after draining the solution. This technique allows the application of coating solutions with a wide viscosity range [46]. A negative point of this technique is the possibility of cross- contamination from fruit to fruit during the immersion process due to the accumulation of residues and microbial organisms [45]. To avoid this problem, products that will be coated must be properly cleaned and sanitized, and the coating solution replaced frequently [15]. According to Raghav et al. [16], in general, fruits and vegetables are immersed for 5-30 seconds in the coating solution. (a) (b) (c) Advantages (A) / Disadvantage (D) (A) Entire surface of the food comes into contact with the film-forming solution: complete wetting; Used to apply filmogenic solutions that have a wide viscosity range. (D) Possibility of contamination of the coating solution; Use of large volumes of solution. (A) Homogenic coating; Use of smaller volumes of solution; Avoids the possibility of contamination of the coating solution. (D) Viscous solutions cannot be sprayed; tends to clog equipment. (A) Use of smaller volumes of solution; It avoids the possibility of contamination of the coating solution. (D) Non-uniform coating thickness; non-continuous coating formation. 21 In turn, the spraying technique, most popular in packing houses, provides a homogeneous and attractive coating. In addition, it avoids the possibility of contaminating the coating solution [49]. This process increases the liquid surface through the formation of drops and distributes them over the food surface [45]. During spray application, the fruit or vegetable is placed on a plate or rotating rollers at a coordinated speed, under dispersing nozzles activated manually or automatically. This procedure is repeated until the desirable coating thickness is achieved. A drawback of this technique is that viscous solutions cannot be sprayed as they clog the equipment [50]. Another method to apply a filmogenic solution is by gloved hands to the fruit surface. Fruits can be coated by spreading a uniform amount of coating solution by hand while wearing latex gloves. It is appropriate on a laboratory scale to avoid solution contaminations and to minimize waste of experimental coating solutions during screenings. However, a negative aspect consists of the non- homogeneous film thickness formed on the entire fruit surface [18], [35]. 4. Nanomaterials in edible coatings In recent years, nanotechnology has been used as an important tool to increase the storage period for food products. The application of nanoscale particles provides different and improved properties compared to particles with larger size. Related to foods, nanotechnology has a wide spectrum of uses in films and coatings due to the improved features they impart [51]. Figure 4 shows the advances in the development of nanosystems incorporated with food-grade ingredients, which makes it feasible to explore functional modifications in food coating materials that include nanoemulsions, polymeric nanoparticles, nanostructured lipid transporters, nanotubes, nanocrystals, nanofibers, and others [52]. Nanosystems, when incorporated into matrices based on hydrocolloids (proteins or carbohydrates), give rise to nanocomposites, which are the combination of two or more materials, one of which is on a nanoscale, in order to improve coating properties [52,53]. 22 Figure 4. Nanomaterials in edible coatings The main changes due to use of nanosystems in nanocomposite coatings refer to the water barrier, optical and microstructural mechanical properties, and the antimicrobial and antioxidant effects. Nanoparticles in coatings potentiate these activities when antimicrobial or antioxidant compounds are incorporated in the coating, by enabling their gradual and controlled release over the period of fruit storage, sometimes under different storage conditions, hence improving bioavailability of these compounds over time [52,54]. The improvements in these properties are important to guarantee food quality maintenance as well as to reduce the development of decay microorganisms (bacteria, filamentous fungi, and yeasts) and action of free radicals that deteriorate food and reduce shelf life [55]. Another advantage of adding active agents to nanosystems is that a smaller proportion of these substances is necessary to obtain good activity; therefore, the use of these compounds in low concentrations does not negatively affect food sensory properties [12]. 5. Fundamentals of nanoemulsions Emulsions are generally made of two immiscible liquids, commonly oil and water, forming a relatively stable mixture. Generally, emulsions are systems that contain a dispersed and continuous phase and can be classified according to the three-dimensional organization of the oil and water phases. Oil-droplets dispersed within an aqueous phase is named oil-in-water (O/W) emulsion, whereas water-droplets dispersed in the oil phase is classified as water-in-oil Nanosystems in edible coatings Size range: 100 nm or smaller Nanoemulsions Polymeric nanoparticles Solid lipid nanoparticles Nanotubes and nanofibers Others Improved edible coating Improve the technological properties Gradual release of bioactive compounds Minimal effect on the sensory properties of food Low impact on the sensory properties of the food, masking the taste or smell of the coating material Increase the bioactivity of compounds Increase the solubility of the bioactive lipids which enhances antimicrobial activity Enables the gradual and controlled release of bioactive compounds into the food over the storage period 23 (W/O) emulsion, and they are the most common emulsions [14,56,57]. Figure 5 shows the schematically structures of O/W (Figure 5 A) and W/O (Figure 5 B) emulsions, emphasizing the micelles structure dispersed in the continuous phase. Emulsions are classified into three main classes according to thermodynamic stability, stable mechanisms, and physical properties: macroemulsion or conventional emulsion, nanoemulsion, and microemulsion. Conventional and nanoemulsions are thermodynamically unstable, while the microemulsion is stable. The droplet mean radius for conventional emulsions are bigger, which distinguishes them from nanoemulsions with a radius of less than 100 nm [11,57,58]. The droplet size in nanoemulsions is a key-point that influences their capability to improve the bioavailability of added hydrophobic substances, such as carotenoids [58], and increase antimicrobial essential oil properties [59] or oil compounds [60]. The nanoemulsion classes will be further discussed in this article, with the focus of nanoemulsions as edible nanocoatings. Figure 5. Schematic representation of (A) oil-in-water (O/W) and (B) water-in-oil (W/O) emulsions, representing micelle structure dispersed in continuous phase for each system. 24 5.1. Nanoemulsions and production methods The small size of particles in nanoemulsions allows potential ad-vantages over conventional emulsions, such as greater stability concern-ing particle aggregation and gravitational separation, in addition to high optical transparency, modification of the physical properties of the coating and increased bioavailability of bioactive-loaded lipid droplets [57]. Free nanoemulsion-based delivery systems increased the bioaccessibility of vitamins (D) and carotenoids ( - carotene and curcumin) [58,61]; however, studies have demonstrated that bioactive-loaded nanoemulsions prepared with a biopolymer mixture can be trapped in the matrices and decrease bioaccessibility. Nanoemulsions need energy for their formation which is provided by mechanical equipment or physical and chemical properties of the sys-tem. Procedures using mechanical energy are called high energy methods and use microfluidizers, high-pressure homogenizers, and ultrasonic homogenizers. The methods that employ the system's physical and chemical properties are categorized as low energy, such as spontaneous emulsification, phase inversion temperature, and emulsion inversion methods [54,57]. When high-energy methods are employed, the surfactants help break oil- droplets inside the homogenizer by decreasing interfacial tension, thus promoting smaller droplets and preventing droplet aggregation. A high shear rate is necessary to break the droplet to form nano-droplets, and is generally achieved by high-pressure homogenizers, as the use of high energy generates forces that can break the droplets in the dispersed phase [56,57]. Those methods are well established in the food industry and can be adapted for nanoemulsion production. On the other hand, for low en-ergy methods, surfactants promote small droplet spontaneous formation due to their ability to generate extremely low interfacial tensions under specific conditions. Therefore, the surfactants utilized are extremely important because the emulsion pH stability, ionic strength, heating, cooling and storage are mainly determined by the amphiphilic molecule chosen [56,57]. The amphiphilic material, such as surfactants, phospholipids, proteins and polysaccharides, reduces the interfacial tension and maintains droplet stability. Emulsions (O/W or W/O) (Figure 6 A and B) are the most stable systems, 25 however in unusual regimes, multiple emulsions such as W/O/W and O/W/O (Figure 6 C and D) may be formed and are usually ex-tremely unstable to coalescence [14,54,56]. Most fruits and vegetables con-tain a high-water volume; therefore, among emulsions, the O/W type (Fig-ure 6A) is the most explored for food systems due to the possibility of loading the oil-droplets with lipophilic key- compounds surrounded by water [14], [54]. Figure 6. Representation of most common emulsion (A) oil-in-water (O/W) and (B) water-in-oil (W/O), and multiple emulsions (C) water-in-oil-in-water (W/O/W) and (D) oil-in-water-in-oil (O/W/O). 5.2. Surfactants Surfactants can be classified according to their electrical characteristics as ionic, non-ionic, and zwitterionic. Most foods surfactants are ionic, such as esterified monoglycerides, which are mainly negatively charged and can form nanoemulsions using low or high energy. Non-ionic surfactants also can be used for both methods and have low toxicity and irritability, including compounds such as Tween® (condensate of sorbitol fatty acid esters and ethylene oxide) and Span ® (a family of fatty acids sorbitan). On the other hand, zwitterionic surfactants contain two or more ionizable groups with opposite charges, and consequently, they can have a negative, positive or neutral charge depending on the pH solution. For example, this group includes lecithin, a phospholipid widely used in foods [57,62]. One of the main aspects of an emulsion formulation is the choice of surfactant. The Hydrophilic-Lipophilic Balance (HLB) system was developed, which represents the balance of the size and strength of the polar and non-polar groups [62]. It demonstrates molecule properties as amphiphilic compounds using a numerical scale, assigning higher HLB values as the substance is more 26 hydrophilic [62]. However, the HLB system only considers the properties of the surfactant itself. For this reason, the hydrophilic-lipophilic deviation (HLD) system is another approach to the behavior exhibited by surfactant-oil-water and usually more suitable in formulations [57,63]. In addition, proteins, polymers with amphiphilic properties, and combinations of polymers and surfactants can act as emulsifiers [64]. Studies have demonstrated the importance of modulating nanoemulsions composition and structure to achieve higher digestion and absorption in the gastrointestinal tract and to efficiently deliver compounds such as vitamins and nutraceuticals [54,58,65,66]. Therefore, the choice of emulsifier is of extreme importance since it can improve carotenoids bio-accessibility, for example. In a study performed by Yao et al. (2019) [65], the authors demonstrated the relationship between carotenoids bio-accessibility from spinach and co-ingesting with excipient nanoemulsions: nanoemulsions containing different ratios of medium or long-chain triglycerides in the oil phase composition decreased β- carotene bioaccessibility when the ratio of medium-chain triglycerides was increased. The findings were credited to the formed micelles ability to hold the carotenoids in their hydrophobic domains. 6. Plant-based nanoemulsions as edible coatings on fruits and vege- tables postharvest 6.1. Coatings based on essential oil nanoemulsions One of the main features can be in the form of antimicrobial nanoemulsions, for example, nanoemulsions based on plant essential oils, which are associated with biopolymers such as alginate, chitosan, and starch, among others. It has been shown that when essential oils are encapsulated in nanoemulsions, they have less impact on the sensory properties of the food, masking the taste or smell of the core material (coating), yet providing better biological activity of essential oils due to the increase in the surface area [67]. In this way, it is possible to use low doses of bioactive material, increasing the transport of active ingredients through biological membranes, thus intensifying the bioavailability of bi-oactive compounds, in addition to less interaction with other components of the food matrix. Other advantages are the low mass 27 transport of com-pounds into and out of the coating, less impact on optical, barrier and microstructural properties and greater coating stability [68,69]. Essential oils have received special attention as active ingredients applicable in food coatings, due to their potent antimicrobial and antioxi-dant activities [70]. Essential oils are volatile aromatic substances of low molecular weight (for example, phenolic compounds, such as monoter-penes, flavonoids and phenolic acids) produced by plants (for example, cinnamon, thyme, lavender, ginger, palmarosa, lemongrass, mint, citrus fruits and fennel) or their isolated components (for example, eugenol, geraniol, menthol, limonene, carvacrol and linalool) that can reduce micro-bial growth in food, and have been studied as natural antimicrobials in food for decades [71]. However, their volatile nature, low water solubility and strong aroma limit their applications in foods. In this sense, using nanotechnological approaches is a promising strategy to enable the ap- plication of essential oils as natural antimicrobials in foods, overcoming their limitations and increasing their antimicrobial activity [52]. Table 2 presents the main types of nanoemulsions as edible coatings classified by matrix type and their impact on fruit and vegetable shelf life. Edible coatings based on nanoemulsions of essential oils have been studied as an alternative to prolong fresh fruit and vegetable shelf life. For example, a coating based on the nanoemulsion of lemon essential oil and chitosan increased the shelf life of arugula leaves by 7 days compared to a coating of chitosan or lemon oil alone [72]. Likewise, coatings based on modified chitosan and carvacrol nanoemulsions completely inhibited the growth of Escherichia coli on fresh green beans during the 11-day period under refrigeration [73]. Gundewadi et al. [74] also reported that the nanoemulsification of basil essential oil in an alginate coating was more effective than its respective microemulsion and presented better coating stability. In addition, when applied to okra fruits, nanoemulsion was more efficient in preserving texture, color and sensory characteristics compared to control fruits. The essential oil of nanoemulsified basil showed greater antifungal activity against fungal pathogens than micro-emulsions. Chu et al. [75] developed a pullulan coating with a cinnamon essential oil nanoemulsion for strawberry storage. The nanoemul-sion- based coating was more effective than other coatings in reducing loss of mass, 28 firmness, total soluble solids, acidity and controlling the growth of fungi and bacteria during fruit storage. In another study, Prakash, Baskaran & Vadivel [60], evaluated the ef-fect of a coating based on sodium alginate and citral nanoemulsion on the quality of fresh cut pineapples. Coatings based on nanoemulsions were effective at reducing microbial growth during storage. In addition, at a concentration of 0.2% of citral nanoemulsion, the coating reduced the presence of Salmonella enterica and Listeria monocytogenes after artificial in-oculation [60]. The coating based on nanoemulsions of lemongrass essen-tial oil, tween® 80 and alginate was more effective at preserving the char-acteristics of minimally processed Fuji apples than their respective con-ventional emulsions. The nanoemulsion coating inhibited the growth of artificially inoculated E. coli on fruits faster than conventional emulsions [59]. 29 Table 2. Main types nanoemulsions as edible coatings and their impact on fruit and vegetable shelf life Matrix Bioactive substance or lipid compound Production technique Functionality Fruit or vegetable Reference Carnauba wax Lemongrass essential oil Dynamic high pressure Increase the antimicrobial activity of the essential oil and improve the homogeneity and stability of the emulsion Plums (Prunus salicina) [76] Carnauba wax Lemongrass essential oil High shear probe and high pressure dynamic processing (DHP) Increase the antimicrobial activity of essential oil Grape berry (Vitis labruscana Bailey) [77] Chitosan Carvacrol, bergamot, mandarin, and lemon essential oils High- pressure homogenization Increase the antimicrobial activity of essential oils Green beans (Phaseolus vulgaris) [73] Sodium alginate Lemongrass essential oil Microfluidization Improve the stability of the emulsion and increase the antimicrobial activity of the essential oil Fresh-cut Fuji apples (Malus domestica 'Fuji) [59] Modified chitosan Lemon, mandarin, oregano or clove essential oils High pressure homogenization (HPH) Increase the antimicrobial activity of the essential oil and improve the homogeneity and stability of the emulsion Arugula leaf (Eruca sativa) [72] Candelilla wax Extract of tarbush High speed stirrer Improved the wettability of the nanocoating on the Fuji apple surface Fuji apple (Malus domestica 'Fuji) [78] Sodium alginate Basil essential oil Ultrasound Increase the antimicrobial activity of essential oil Okra (Abelmoschus esculentus) [74] Quinoa protein/chitosan Thymol 1200 rpm agitation Increase the antimicrobial activity of the active compound and improve dispersion in the matrix Strawberry (Fragaria × ananassa) [79] Hydroxypropyl methylcellulose Carnauba wax nano- emulsion High pressure homogenization (HPH) and mechanical stirring Reduce gas permeability and moisture loss ‘Redtainung’ Papaya (Carica papaya) [28] Pullulan Cinnamon essential oil Ultrasound Improve the distribution of oil in the matrix and increase its antimicrobial activity Strawberry (Fragaria × ananassa) [75] 30 Sodium alginate Citral Ultrasound Improve the dispersion of the active compound in the matrix and increase its antimicrobial activity Fresh cut pineapples (Ananas comosus) [60] Carnauba wax Oleic acid, Carnauba wax High pressure homogenization (HPH) Improve optical properties, and emulsion stability 'Nova' mandarins (Citrus reticulata) and ‘Unique’ tangors (C. reticulata · C. sinensis) [18] Chitosan Cellulose nanocrystal and oleic acid Ultra turrax homogenizer Increase coating stability at high humidity, adhesion on fruit surface and delayed ripening of pears Bartlett pears (Pyrus communis) [80] 31 6.2. Coatings based on plant-based wax nanoemulsions Commercial coatings based on approved waxes must meet state/national fruit and vegetable additive regulations and be considered safe for consumption. However, to improve the characteristics of wax-based coatings, they are combined with synthetic chemicals to pre-vent microbiological deterioration and to ensure homogeneous stability of the coating during product storage. Commercial coatings are typically formulated using oxidized polyethylene wax (a by-product of the petroleum industry), carnauba wax (from the leaves of the carnauba palm, Copernicia cerifera), candelilla wax (from the candelilla shrub, Euphorbia cerifera), and shellac (from the insect bug Kerria lacca) as matrices, combined with water and other agents such as oleic acid, morpholine, ammonia, polydimethylsiloxane antifoam, and others [81]. The compounds combined with waxes used as emulsifying, mois-turizing, and antimicrobial agents in commercial coatings, are mostly synthetic chemical products and could be a concern for human health [82]. As an example, morpholine is a base acting as a counterion to facili-tate fatty acids emulsification in waxes. In the presence of nitrite/nitrate, morpholine can form N- nitrosomorpholine, a potent mutagen and carcinogen [83,84]. N- nitrosomorpholine was not found on coated fruit surface, but the possibility of its formation in the gut from reaction of mor-pholine with dietary nitrates was considered; it was found at concentra-tions less than the safe dose of 4.3 ng/kg body weight/day, not enough to raise concerns [85]. Ammonia could be used as a replacement for morpholine [86], but its highly volatile and irritant nature makes it less easy to use than morpholine. Consumers are increasingly concerned about the safety and quality of food, driving the demand for so-called "environmentally friendly coat-ings”, that is, coatings based on natural products of plant origin that do not present any harm to the consumer's health if consumed. The use of waxes and compounds of animal origin has been limited by vegan and vegetarian consumers, consumers who are allergic to animal products (such as chitosan) and religious beliefs that do not encourage the consumption of animals [87]. Therefore, the demand for plant-based wax-based coatings is an important market for fresh fruits and vegetables, and nanotechnology is a promising tool to meet this demand by 32 improv-ing the properties of these coatings, especially wax-based ones, reducing the need of synthetic additives. Nanotechnology has been successfully used to produce plant-based waxes nanoemulsions, such as carnauba wax [18] and candelilla wax [78] without the addition of morpholine. Wax-based nanoemulsions can have improved barrier properties due to the small size of the droplets, promot-ing greater homogeneity compared to conventional emulsions, greater transparency, improved physico- chemical properties (optical, mechanical and barrier) and greater stability in comparison with conventional emul-sions [18,54,78]. In addition, these nanoemulsions can be used for the de-velopment of nanocomposite coatings, in combination with hydrocolloid components (polysaccharides and proteins) in order to improve the water barrier properties of these compounds and minimize the impact of the in-corporation of lipid compounds in the matrix hydrocolloids [29]. Lipid nanoemulsions made from plant-based waxes have shown greater effectiveness as edible coatings than conventional emulsions on fresh fruits and vegetables preservation (Table 2). The carnauba wax nanoemulsion coating showed less water loss, conferred gloss, and caused less ethanol production than shellac in coated 'Nova' mandarins (Citrus reticulata) and ‘Unique’ tangors (C. sinensis) [18]. In addition, the coating based on carnauba wax nanoemulsion exhibited greater gloss measurements, less changes in the fruit internal atmosphere and volatile profile, and consequently, better flavor compared to the conventional car-nauba wax emulsion [18]. Lipid nanoemulsions produced from waxes, such as carnauba or candelilla, have been shown to be suitable vehicles for carrying bioactive compounds, such as plant extracts and essential oils [78,88]. They can improve the physical stability of the active substances, and improve the bioactivity of these compounds, and due to the prolonged and slow diffusion, they reduce the impact of these substances on the sensory properties of fruits and vegetables [88]. De Léon-Zapata et al. [78], developed cande-lilla wax nanoemulsions added with tarbush extract and evaluated its ef-fect on the preservation of Fuji apples. The combination of extract and nanocoating reduced the size of the droplets and improved the zeta poten-tial and optical properties of the coating. When applied to Fuji apples, the nanocoating effectively reduced physico-chemical and 33 microbiological changes and delayed fruit senescence in comparison with the control treatment. In another study, a nanoemulsion of carnauba wax combined with lemongrass essential oil nanoemulsion was applied to plums [76]. The coatings were able to inhibit the growth of Salmonella typhimurium (Salmonella enterica) and E. coli O157: H7 inoculated plums during storage, and did not significantly affect their taste and appearance (brightness). In addition, nanoemulsion coatings were effective at reducing weight loss, ethylene production and respiration rate. Fruit coated with nanoemul-sions showed greater firmness and increase in phenolic compounds con-tent during storage in comparison with uncoated fruits [76]. A similar re-sult was observed in another study carried out by these authors with grape berries. The coating based on carnauba wax and lemongrass essen-tial oil nanoemulsion inhibited the growth of S. typhimurium and E. coli O157: H7 inoculated fruit. Lemongrass in nanoemulsions did not affect berry taste and improved their brightness. Coatings based on nanoemul-sions were also able to reduce weight loss and maintain firmness, phe-nolic compounds, and antioxidant activity in berries. The coatings demonstrated the potential to reduce microbiological contamination of grape berries by foodborne pathogens and prolong their shelf life. [77]. 7. Trends in materials based on nanoemulsions with potential for appli- cation in the preservation of fruits and vegetables New coating materials based on nanoemulsions with potential for application in fruits and vegetables have been developed in the last two years with the aim of contributing even more to the preservation of these products. One way to develop these functionalized materials is to combine composites with different properties to develop a functionalized coating material. For example, de Oliveira Filho, et al. [89] developed a function-alized coating combining arrowroot starch (biopolymeric matrix), carnauba wax nanoemulsion (to improve the water barrier properties of the coating), cellulose nanocrystals (to improve mechanical properties and stabilize the emulsion), and essential oils (to confer antimicrobial activity). The combination of compounds resulted in a coating material with excel- 34 lent water barrier, mechanical, thermal, optical, microstructural and an-timicrobial properties against fungi that attack fruits during post-harvest. Another increasingly explored trend in the development of new coatings based on nanoemulsions with better stabilities is the use of solid particles to form Pikering nanoemulsions, that is, nanoemulsions stabilized with solid particles such as cellulose nanocrystals [90], starch nano-crystals [91], γ-Al2O3 nanoparticles [92], cyclodextrin [93] among others. Pickering nanoemulsions have excellent stability due to irreversible adsorption that occurs between solid particles at the oil-water interface due to the high adsorption energy [94]. Another characteristic of these nanoemulsions is the ability to release active ingredients encapsulated under specific conditions, such as pH and temperature [93]. Almasi, Azizi & Amjadi [95] compared two coating materials based on pectin, one with marjoram essential oil encapsulated in a whey protein/inulin stabilized Pickering nanoemulsion, and the other with marjoram essential oil nanoemulsified with Tween 80. Coatings based on pectin with Pickering nanoemulsions presented mechanical and water barrier properties supe-rior to those based on standard nanoemulsion. In another study, López- Monterrubio et al. [96] developed highly efficient β-carotene nanoemulsions stabilized by a complex formed by hydrolyzed whey protein and pectin. The nanoemulsions showed good stability during the 30-day storage period with low formation of clumps. Deng et al. [80] developed coatings based on chitosan and Pickering nanoemulsion of oleic acid stabilized with cellulose nanocrystals and evaluated their effects on the postharvest conservation of green D'Anjou and Bartlett pears (Pyrus communis L.). The coating formulated with 5% cellulose nanocrystals showed strong adhesion to the fruit surface, show-ing greater gas barrier property compared to the commercial Semperfresh™ product, and presented a more homogeneous matrix, being effective in delaying ripening and increased the shelf life of pears during storage. Although the above new materials have been little studied in food systems, the results described in the literature are very encouraging. 8. Potential toxicity, limitations, and regulatory aspects of nanoemulsions 35 Nanoemulsions, due to the nanometric size of the droplets, may par-tially remain intact during digestion, representing potential safety risks related to the compounds used for their production (such as surfactants). They can be of concern in metabolic or hormonal dysregulation due to their rapid absorption compared to conventional emulsions, their ability to increase the bioavailability of bioactive agents to a toxic level, and the possibility of increased absorption by epithelial cells which can cause changes in the functionality of the gastrointestinal tract [97]. However, as they have a high surface area, nanoemulsions can also be quickly digested by enzymes from the gastrointestinal tract, reducing the possible toxic ef-fect that can occur due to their accumulation in organ cells [98]. In vitro studies were performed using cell cultures, usually models of normal cells such as fibroblasts, to investigate potential toxicity of nanoemulsions. Kaur et al. [99] reported that nanoemulsions based on to- copheryl polyethylene glycol succinate (TPGS), lemon oil, Tween-80 and water did not show toxicity in Hep G2 cells. In another study, Marchese et al. [100] observed that bergamot essential oil nanoemulsions showed cy-totoxic activity against Caco 2 cells at high concentrations. A limitation of these studies is the fact that authors have not previously exposed the nanoemulsions in simulated conditions of the gastrointestinal tract before contact with the cells. Knowledge about the potential toxicity of nanoemulsions in vivo is still limited and should be investigated [97]. The effect of nanoemulsions based on antimicrobial compounds, such as essential oils, on the gastro-intestinal tract is also poorly reported in the literature. This effect must be carefully studied, as antimicrobial compounds can influence the intestinal microbiota or epithelial cells of the gastrointestinal tract. In a recent study, Hort et al. [101] evaluated the toxicity of Miglyol and egg lecithin nanoemulsions using an in vivo model (male Wistar rats). The nanoemulsions were orally administered to rats for 21 days at lipid concentrations of 200, 400 or 800 mg/kg of body weight. The results of biochemical, hematological, oxidative stress and genotoxicity parame-ters showed that nanoemulsions could be considered safe for oral admin-istration, but high doses by the parenteral route could cause toxic effects. The few studies suggest that nanoemulsions formulated with GRAS ingredients do not exhibit strong cytotoxic effects. The nanometer size of the 36 droplets suggests that they are rapidly transformed into monoglycer-ides and free fatty acids in the small intestine, which are normal digestion products and should not have toxic effects [57]. As for regulatory aspects, essential oils and other antimicrobial agents are mainly regulated by the European Food Safety Authority (EFSA) in Europe and the Food and Drug Administration (FDA) in the United States [102]. However, for nanoemulsions there is no international authority that makes this regulation. The FDA addresses the regulation of nanotechnol-ogy products as guidance for industries. The European Council and Par-liament have regulated food nanotechnology as new food products or food ingredients [103]. 9. Conclusions and future perspectives The use of substances obtained from plant-based natural sources has emerged as a trend in the fresh fruit and vegetable market for coating ap- plications. The application of these compounds on a nanoscale has ad-vantages allowing a wider use in relation to particles on larger scales. Re-cent studies indicate that nanoemulsions play an important role in the development of a new generation of coatings with improved properties for the preservation of fresh fruits and vegetables. This emerging technology makes it possible to improve the physical stability and performance of ac-tive substances within an edible coating, bringing the possibility of in-creasing the quality and/or nutritional value of fruits and vegetables. Alt-hough the evidence published to date suggests that nanoemulsions ap-plied as edible coatings can extend the life of different fruits and vegeta-bles, there are other important aspects to explore before considering them on a commercial scale in future trends, such as the bioavailability of bio- active compounds incorporated in the nanoemulsions, potential toxicity and digestibility, for example. Most of the tested nanoemulsion coatings have antimicrobial properties; however, it can also be possible to produce and apply edible coatings with health-promoting substances. Author Contributions: writing—original draft preparation, J.G.O.F. and M.M.; writing—review and editing, M.D.F. and A.P.; visualization, J.G.O.F. and M.M.; supervision, M.D.F.; project administration, M.D.F.; funding acquisition, M.D.F. All authors have read and agreed to the published version of the manuscript. 37 Funding: This research was funded by FAPESP (process 2016/23419- 5,2018/10657-0, and 2018/24612-9), CAPES (001), CNPq (process 407956/2016-6; fellowship 310728/2019-3), Empresa Brasileira de Pesquisa Ag- ropecuária (Embrapa), Rede Agronano, and MCTI-SisNano from Brazil. Acknowledgments: Authors than Dr. Elizabeth Baldwin for reviewing the manuscript and constructive suggestions. Conflicts of Interest: The authors declare no conflict of interest. References 1. Dhandevi, P.; Jeewon, R. Fruit and vegetable intake: Benefits and progress of nutrition education interventions-narrative review article. Iranian journal of public health 2015, 44, 1309. 2. 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Food Measure 15, 1815–1825 (2021). https://doi.org/10.1007/s11694-020-00765-x Oliveira Filho, J. G., Silva, G., Egea, M. B., de Azeredo, H. M. C., & Ferreira, M. D. (2021). Essential oils as natural fungicides to control Rhizopus stolonifer- induced spoiled of strawberries. Biointerface Research in Applied Chemistry 11(5):13244 – 13251. 46 Chemical composition and antifungal activity of essential oils and their combinations against Rhizopus stolonifer and Botrytis cinerea in strawberries Abstract The chemical composition and antifungal activity of OEs Cymbopogon martinii, Cinnamomum camphora, Mentha spicata and Mentha piperita and their binary mixtures M1 (M. piperita and C. martinii), M2 (M. piperita and C. camphora), M3 (M. piperita and M. spicata), M4 (C. martinii and C. camphora), M5 (C. martinii and M. spicata), M6 (C. camphora and M. spicata) were evaluated against Rizhopus stolonifer and Botrytis cinerea by direct contact and contact methodology of steam. The major components were menthol (45.37%), menthone (20.13%), isomenthone (16.94%), menthyl acetate (3.81%), pulegone (1.89%), α-terpinene (1.88%), isopulegol (1.83%), neoisomenthol (1.19%), and α-terpineol (1.08%) for M. piperita essential oil, linalool (98.39%) for C. camphora essential oil, and geraniol (83.82%), geranyl acetate (7.49%), linalool (2.48%), and caryophyllene (1.33%) for C. martini essential oil. The greatest antifungal activity was promoted by M. spicata and C. martinii in the methods of direct contact and vapor contact. The best treatments were also verified for antifungal activity in vitro, by the methods of germination of spores and dilution in micro- wells, and in vivo in the vapor phase in strawberries artificially inoculated with R. stolonifer and B. cinerea. In the spore germination test, M. spicata and C. martinii EOs were the most active inhibitors of spore germination and, in the microwell dilution test, both EOs showed the lowest values of minimum inhibitory concentration (MIC) and fungicidal concentration (MFC). In vivo, the steamed strawberries from the EOs of M. spicata and C. martinii showed a significant reduction in the incidence and severity of infection by R. stolonifer and B. cinerea. Therefore, EOs M. spicata and C. martinii can be a potentially efficient and safe alternative for use as a natural vapor phase fungicide to control R. stolonifer and B. cinerea in fresh stored strawberries. Keywords: natural fungicides, essential oils, Cymbopogon martini, Mentha spicata. 47 INTRODUÇÃO Strawberry (Fragaria x ananassa Duch) is a very valued fruit for its flavor, and a relevant source of bioactive compounds like vitamins (such as vitamin C and E), phenolic acids, flavonoids and anthocyanins that are reported for promote health benefits (Ariza et al. 2016). It has a short postharvest life due to their high respiration rate, sensitivity to temperature and soft texture, which results in a high degree of deterioration by various pathogens like Botrytis cinerea during postharvest (Dhital et al. 2017). Rhizopus stolonifer is one of the most common fungi and is considered as one of the most devastating (Bautista-Banos et al., 2014). Rhizopus rot appears particularly on mature fruits, when temperatures are above 5°C, and spread rapidly infecting healthy fruits (Ogawa et al., 1995). Due to their wide array of hosts, rapid penetration and colonization, R. stolonifer has become an important control target for synthetic fungicides. The necrotrophic fungus B. cinerea is considered the most important postharvest pathogen of strawberries in the world, causing impactful losses to the fresh strawberry industry (Petrasch et al. 2019). That fungus was chosen as the second most important plant pathogen fungus in the world based on its effects in many plant species, causing gray rot (Dean et al. 2012). Traditionally, synthetic fungicides have been used to control postharvest fruit pathogens. However, its possible negative effects on human health and the environment, increased production costs. Besides that, the selection and appearance of strains resistant to a notable number of fungicides have led to attempts to find biological resources to replace those synthetic products (Leroch, Kretschmer, and Hahn 2011; Palou et al. 2016). In this context, essential oils have received prominence as natural fungicides for presenting strong antifungal activity against postharvest fruit pathogens, being considered natural, safe and biodegradable alternatives (Oliveira et al. 2019; Pedrotti, Silva Ribeiro, and Schwambach 2019;Tomazoni et al. 2017). Essential oils are metabolites derived from plant secondary metabolism and are made up of complex mixtures of volatile organic substances, such as sesquiterpenes and monoterpenes (Fisher and Phillips 2008; Salvia-Trujillo et al. 2015). The antifungal activity of essential oils is related to the presence of these compounds and their mechanism of action occurs through their hydrophobic nature, that allows it to interact with microbial membranes, causing cell lysis, 48 interrupting the proton's motor force, electron flow, transport active and inhibiting protein synthesis (Burt 2004; Nerio, Olivero-Verbel, and Stashenko 2010) In some cases, for essential oils to have effective antimicrobial activity in vivo, high concentrations of these compounds are necessary, which can result in a negative impact on foods sensory properties, since essential oils are characterized by strong odors. To avoid this adverse effect, the possible synergistic effect produced by the combination of different essential oils from plants has been reported as an efficient strategy to inhibit microbial development, reducing individual concentrations of essential oil and adverse sensory effects for in vivo application(Aguilar-González, Palou, and López-Malo 2015; Hossain et al. 2016), such as combinations of mustard (Brassica nigra) and clove (Syzygium aromaticum) essential oils on inhibition of B. cinerea in strawberries (Aguilar- González, Palou, and López-Malo 2015). However, only a few studies on the effects of combinations of essential oil have been reported in the literature. Thus, the objective of this study was to evaluate and compare the effects of essential oils and their binary mixtures against the growth of R. Stolonifer and B. cinerea in vitro and in vivo on postharvest strawberries. MATERIAIS E MÉTODOS Materiais Pippermint (Mentha piperita), palmarosa (Cymbopogon martinii) and ho wood (Cinnamomum camphora) essential oils were purchased from Laszlo Aromaterapia (Belo Horizonte, Minas Gerais, Brazil). Mint (Mentha spicata) essential oil was purchased from Ferquima Ind. e Com. Ltda. (Vargem Grande Paulista, São Paulo, Brazil). Fungal strains R. stolonifer CCT 0276 and B. cinerea CCT 1252 were obtained from Andre Tosello Foundation (Campinas, São Paulo, Brazil). Chemical composition of the es