Antimicrobials as an Innovative Tool for the Shelf-Life Enhancement of Fruits

RANJITHA K* and HARINDER SINGH OBEROI

Division of Post-Harvest Technology and Agricultural Engineering, ICAR-Indian Institute of Horticultural Research,

Hessaraghatta Lake (PO), Bengaluru 560089, Karnataka, India

'Corresponding author. E-mail: This email address is being protected from spam bots, you need Javascript enabled to view it

ABSTRACT

Microbiological spoilage is a primary reason for the perishable nature of horticultural produce. Even though conventional pesticides are highly effective in decay control, their postharvest use has been discouraged due to their ill effects on health and the environment. This has encouraged the scientists and industry to look for broad spectrum and safe alternatives.

This chapter details the updated information on the natural and synthetic alternatives for shelf life enhancement of fiuits. Plant-derived antimicrobial principles, like isothiocyanates, terpenoids, alkaloids, phenolics, aldehydes, and the essential oils, organic compounds of microbial origin, namely, aldehydes, ethanol, acetic acid, bacteriocins, and antibiotics as well as animal- derived compounds, namely, chitosan, lactoferrin, lysozyme, etc. Find their important role in postharvest spoilage management. Among the above- mentioned compounds, chitosan or its derivatives, as well as essential oils, are looked upon with great hope for future use. For a wider practical use, a multidisciplinary research is required to integrate them in packaging materials, encapsulation methods for their slow and sustained release, finding out the best combination for their synergistic action, chemical modifications to obtain their more potent derivatives, etc. in a variety of fruits.

INTRODUCTION

Fresh fruits are highly perishable due to physiological and microbiological changes resulting in tissue damage and loss in texture eventually leading to senescence. Physiological deterioration originates within the fruit tissues, but microbiological spoilage usually occurs due to external biological contaminants such as fungi and bacteria. A certain level of physiological changes is essential for ripening, however high-physiological activity beyond this stage leads to senescence, which may be checked by using agents for tissue firming, enzyme inhibition, phytoalexins, ethylene inhibition, etc. These agents alter the physiology and delay onset of microbial invasion, even though they do not possess direct antimicrobial activity. Information on the physiological aspects of spoilage and their control are well compiled in the available scientific literature (Wills, 2007; Siddiqui, 2015).

A spectrum of fungi and bacteria causes postharvest spoilage of fruits, but major postharvest diseases are caused by species of the fungi belonging to Alternaria, Aspergillus, Botrytis, Fusarium, Geotrichum, Colletotri- chum, Phoma, Monilima, Penicillium, Rhizopus, and Sclerotinia and of the bacteria Enviuia, Xanthomonas, etc. Initial tissue breakdown by primary pathogens is often rapidly followed by an invasion by a broad spectrum of weak pathogens and saprophytes, thus magnifying the damages caused by primary pathogens (Narayanasamy, 2008).

Conventionally, fungicides and antibiotics are used to control postharvest pathogens of fruits. Despite the efficacy of these molecules to achieve a convincingly good level of postharvest disease control, the use, however, has been curbed time-to-time by regulatory agencies citing the scientific evidences on their carcinogenicity, teratogenicity, residual toxicity, recalcitrance, environmental pollution, and resistance build-up in pathogen, and any other detrimental effect on health. Consequently, alternative molecules with antimicrobial action coupled with low mammalian toxicity for postharvest management of perishables are being continuously explored. It is more advantageous if such molecules possess “food additives” or “generally recommended as safe” characteristics and thus get exemption from “residue tolerances” in agricultural commodities (Bautista Banos, 2014).

Organic and inorganic salts, plant extracts, essential oils, glucosinolates, polyphenolics, antimicrobial peptides, chitosan, etc., are the low toxicity antimicrobial molecules of importance in shelf life extension of fruits (Fig. 9.1). Apart from these, antagonistic microbes against the pathogens are also useful. This chapter updates the progress in research on the use of antimicrobials as novel tools for shelf life enhancement of fresh fruits.

Types and source of antimicrobials with potential use for shelf life extension of fruits

FIGURE 9.1 Types and source of antimicrobials with potential use for shelf life extension of fruits.

SALTS

Inorganic salts, like carbonates and bicarbonates, were used for posthar- vest systems during the early 1920s. Interest in such salts was revived in the 1980s due to the regulatory restrictions of postharvest fungicides. The antimicrobial effect is due to the reduction in fungal cell turgor pressure with consequent plasmolysis, leading to inability to sporulate. In addition to this, the induction of phytoalexins in the host tissue also is perceived as a mechanism for pathogen inhibition (Yousef et al., 2014). A 60-150 s dip in 40-50°C of 2-3% sodium carbonate (SC) or sodium bicarbonate (SBC) aqueous solutions was sufficient citrus green and blue molds caused by Penicillium digitatum and Penicillium italicum, reduced decay on longterm cold stored fruit. Sodium salts are more effective than other carbonate salts and their antifungal activity was higher on oranges than on mandarins (Palou et al., 2002). Since then, SC and SBC have been the most common food preservatives used for controlling decay in citrus pack houses worldwide. Organic salts like sodium benzoate (SB) and sodium parabens (Moscoso-Ranhrez et al., 2013; Montesinos-Herrero et al., 2016), etc., also are useful for postharvest uses, especially in citrus fruits (Duan et al., 2016).

VOLATILE ORGANIC COMPOUNDS

Volatile organic compounds (VOCs) typically constitute a complex mixture of low-molecular weight lipophilic compounds of plant and microbial origin. Their primary function for source organism is a defence against predators and pathogens, attraction of pollinators and seed dispersers, signaling, etc. Previously published reports have demonstrated that volatiles emitted by plants including among others ethanol (ETOH), acetaldehyde, and benzal- dehyde are promising alternatives for controlling commercially important postharvest pathogens on a great variety of fruits and some vegetables. Nevertheless, in the context of technology transfer, their performance should be also considered at pilot and commercial levels to evaluate the extension of their use at these stages when applied thr ough solution or vapor under different environments. Before successfully adopting these alternative methods in different packages, storage rooms, and packinghouses, it is necessary to correlate the effect obtained at laboratory within a commercial context, including quality and sensorial evaluations. Besides instead of focussing on advantages alone, studies should include their limitations because of the possibility of producing toxic effects, following regulatory issues. VOCs suffer from oxidation, volatilization, and prompt reaction with other fruit components. An adequate formulation favoring their slow release could improve their application, for example, using microcapsules with a sustained-release dosage as a pesticide; the active ingredients are dispersed into a few microns to several hundred microns by physical and chemical means. Frequently, the VOCs emitted by biological control agents provide only a limited contribution to the control of pathogens; therefore, the emission of VOCs forms a part of their mode of action (Di Francesco et al., 2015); when the microorganisms are not in direct contact with the pathogen, the VOC production can be considered to be a partial mechanism of the action. Although bacteria and yeasts showed great potential as biofumigants under airtight conditions, their practical application requires further investigation considering their potential to behave as a spoilage and safety concern organisms.

ALDEHYDES

Aldehydes, such as acetaldehyde, benzaldehyde, and (2E) hexanal are plant- derived VOCs with antifungal properties. The sources of these aldehydes are different. For example, acetaldehyde accumulates during ripening in several fruits, while benzaldehyde is the major component of almond bitter oil. 2E- hexenal, commonly called as “leaf aldehyde” is an organic volatile with fresh leaf green odor, naturally occurring in many plant species. At a cellular level, these lipophilic compounds exert their antimicrobial activity thr ough a mechanism of cell membrane damage, compromising its integrity and cell permeability. Such compounds deter conidia germination and mycelial development in pathogens.

Studies have shown that acetaldehyde reduces the growth of R. stolonifer, Penicillium digitatum, Colletotrichum musae, and Envinia carotovora with minimum inhibitory concentration values ranging from 0.88 to 0.91 mmol L'1 (Abd Alla et al., 2008). This antifungal volatile was also very effective in totally reducing the growth of Colletotrichum acutatum over a 7-day period at 23°C at concentrations of 0.56 mL L_1 (Almenar et al., 2007). In fresh “Midway” strawberry and “Sultanina” and “Perlette” grape, concentration of 10% completely inhibited both rots; while in grapes, reduction in soft rot incidence was approximately 90% at 0.25% concentration after 8 days of storage. Conidial survival on the Citrus reticulata fruit surface was 100% controlled after 12-h exposure to acetaldehyde (Mari et al., 2016).

In situ antifungal activity of 2E-hexenal is well proven against postharvest fungi, such as Alternaria alternata, Bacillus cinerea, Aspergillusflavus, Penicillium expansum, Colletotrichum acutatum, and Monilina laxa (Neri et al. 2006a, 2006b). About 93% reduction in blue mold infection on pears (Pyrus communis) was observed at 20°C storage after 7 days of application of 2E hexenal @ 12.5mL L'1. Anthracnose disease of “Camarosa” strawberry was also reported to be inhibited by this compound in a dose dependent manner. Brown rot on various cultivars of apricots, nectarines, peaches, and plums was effectively controlled by exposing fruit to 2E-hexenal@ 20mL L~‘ over a 20 h-period at 20°C. However, some fungal infections, like lenticel rot caused by Neofabraea alba in apple could not be controlled, even by applying a higher concentration of 25mL L'1 (Neri et al, 2009). Hexanal is an efficient fumigant in controlling mold on seedless table grapes, pears, strawberries, bananas, apples, pineapples, and melons (Mari et al, 2016).

ISOTHIOCYANATES

Isothiocyanates are an extensive group of p-thioglucoside- N-hydroxy sulphate anionic compounds predominantly occurring in their precursor form as glucosinolates in Brassicaceae plants (Delaquis and Mazza, 1995). Glucosinolates are hydrolyzed by the endogenous enzyme myrosinase

(thioglucoside glucohydrolase E. C. 3.2.1.147) producing not only ITCs, but also a combination of nitriles, thiocyanates, and oxazolidine thiones depending on the side chain in glucosinolates and hydrolysis conditions. Isothiocyanates inhibit microbes by different ways, namely, uncoupling oxidative phosphorylation in the mitochondria of fungi and thus hindering ATP synthesis, formation of reactive oxygen species (ROS) that leads to an intolerable level of oxidative stress in fungal cells, and irreversible binding with sulfhydryl groups, disulfide bonds and amino groups of proteins (Ribes et al., 2017).

Allyl isothiocyanates (AITC) present in mustard are veiy powerful antimicrobial isothiocyanates (Kosker et al., 1951). Even though it is highly fungicidal in vitro, the difficulty of the chemical in accessing the pathogen inside the fruit is the requirement of longer duration of the treatment that is approximately 3-6 h (Mari et al., 2008). A few studies reported the effectiveness of AITC treatment on small fruits such as mulbeny (Chen et al., 2015), blueberry (Wang et al., 2010), and strawberry (Ugolini et al., 2014). Preliminaiy in vitro (Manyes et al., 2015) and in vivo (Ugolini et al., 2014) data evidenced that this product can be considered safe since the estimated daily intakes were always lower than the AITC admissible daily intake. The AITC microcapsule encapsulation exhibited an efficacy of above 90% against tomato rot with a considerable extension of its shelf-life (Wu et al., 2015). The possibility to routinely apply VOCs emitted by ITCs is still under investigation. However, the results are promising, while the main issues that remain are a sustainable formulation, the correct exposure time and the adequate concentration, probably needing to be tailored for each fruit species in order to avoid phytotoxic effects.

ESSENTIAL OILS

Essential oils (EOs) are volatile secondaiy metabolites from plants with promising food preservation efficiency. Eveiy essential oil is typified by the diversity and level of different volatile organic molecules in them (Table 9.1). These lipophilic molecules act on microbial cells in multiples ways, likeenzyme binding, membrane destabilization, alteration of membrane permeability, granulation of cytoplasm, etc. Due to the lipophilic nature, essential oils result in cell membrane modification of the microbes, affecting the permeability of the cell membrane, subsequently causing leakage of cell components (da Cruz et al., 2013). Essential oils act synergistically in preventing fungal decay. Nguefack et al. (2012) reported that when oxygenated monoterpenes, such as citral, thymol, or carvacrol, were applied together with terpene hydrocarbon p-cymene, the p-cymene facilitated the entry of oxygenated monoteipenes into the cell by modifying the cell membranes. This resulted in a synergistic activity by combination treatment of the above terpenes. Furthermore, essential oils, such as thyme oil act as elicitor to stimulate the induced defense mechanism in avocado by upreg- ulating gene expression and the enzyme activities of pathogenesis-related proteins, such as (PRP) as chitinase and 7,3-7>-glucanase (Sellamuthu et al., 2013; Bill et al., 2014).

TABLE 9.1 Volatile Compounds Present in Essential Oils of Various Plants.

Plant (scientific name)

Major components

Allspice (Pimento dioica)

Eugenol, methyl ether cineol

Basil (Ocimum baslicum)

d-linalool, methyl chavicol

Black pepper (Piper nigrum)

Monoterepenes, sesquiterpenes

Bay (Laurus nobilis)

Cineol, 1-linalool, eugenol, geraniol

Caraway seed (Carum caivi)

C'arvone, limonene

Celery seed (Apium graveolens)

d-Limonene

Cinnamon (Cinnomum zeyktnicum)

Cinnamic aldehyde, 1-linalool, p-cymene, eugenol

Clove (Sygium aromaticum)

Eugenol, cariophylline

Coriander (Coriandrum sativum)

d-linalool, d-a-pinene, p- pinene

Cumin (Cuminum cyminum)

Cuminaldehyde, p-cymene

Fennel (Foeniculum vulgare)

Anethole

Garlic (Allium sativum)

Diallyl disulphide, diallyl trisulphide, allicin, diethyl sulphide

Lemon grass (Cymbopogon citratus)

Citral, geraniol

Marjoram (Origanum marjorana)

Linalool, methyl chavicol, cineol, eugenol, terpininiol

Mustard (Brassica juncea)

Allyl isothiocyanate

Onion (Allium сера)

Propyl disulfide

Oregano (Origanum vulgare)

Thymol, carvacrol, a-pinene, p-cymene

Parsley (Petroselinium crispum)

a-pinene, fenol-eter-apiol

Rosemary (Rosemarinus officinalis)

Borneol, cineol, camphor, a-pinene, bornyl acetate

Sage (Salvia officinalis)

Thujone, cineol, borneol, thymol, eugenol

Thyme (Thymus vulgaris)

Thymol, carvacrol, 1-linalool, geraniol, p-cymene

Indian basil (Ocimum tenuiflorum)

Eugenol, estragole, ocimene, caryophyllene- p , bergamotene

Vanilla (Vanilla planifolia)

Vanillin, vanillic acid, p-hydroxy benzoic acid coumaric acid

Source: Davidson et al. (2005).

Mycelial growth and spore production are the two most important stages of fungi development affected by EOs (Farzaneh et al., 2015; Phillips et al., 2012; Pekmezovic et ah, 2015). Overall, various important postharvest fungi including A. altemata, B. cinerea, C. gloeosporioides, R. stolonifer, Aspergillus spp, and Penicillium spp, can be effectively controlled by EO extracts from botanical plants including Dill (Anethum graveolens), Basil (Ocinium selloi), summer savory (Satureja hortensis), laurel (Lauris nobilis), and oregano (Origanum vtdgare), among others. Among these, the uses of thyme and oregano oils are studied in depth owing to their high efficiency against several fungi. Thyme oil is highly effective at concentrations as low as 0.10 mL L'1. For example, anthracnose in avocado (Persea americana) is reduced by about 80-90% with thyme oil (66.7 mL L'1), mentha (Mentha piperita) and lemongrass (Cymbopogen nardus) (106 mL L'1) (Sellamuthu et ah, 2013). Barrera-Necha et ah (2008) evaluated nine EOs in controlling anthracnose on papaya fruit and found that cimiamon and clove (Syzygium aromaticum) EOs at 50 mg mL'1 concentration provided the best control. The disease reduction obtained in this case was more than 87% compared to 65% in the untreated fruit. De Corato et ah (2010) and Sliao et ah (2013), found that gray mold of kiwifruit, strawbeny and blueberry can be effectively controlled by laurel, tea tree (Melaleuca alternifolia), cinnamon, and peppermint EOs.

METHYL CYCLOPROPANE

1-methyl cyclopropane (1-MCP) is a gaseous agent widely known for its efficiency in delaying ripening and yellowing. A recent report gave an additional potential use for this compound as an antimicrobial for extending postharvest life of fruits. The 1-MCP application was able to suppress anthracnose of postharvest mango fruit by directly inhibiting spore germination and mycelial growth of Colletotrichum gloeosporioides. (Xu et ah, 2017). The mechanism of suppression is thought to be due to the induction of ROS generation, damage of the mitochondria and destruction of the integrity of plasma membrane of spores, thus significantly suppressing spore germination and mycelial growth.

ACETIC ACID

Acetic acid (AAC) has shown promising results in controlling P. expansum and B. cinerea on apple at a concentration (2.7 and 4.0 mg L'1) and storage temperature (0°C) (Sholberg et al, 2001). In further experiments, it was also shown that AAC fumigation could effectively control stem end infections in pears and green mold in citrus (Sholberg et al., 2004, Smilanick et al., 2014)

ETHANOL

ETOH, a widely used sanitizing agent, can be used for control of postharvest diseases. Control of grey mold disease on table grapes using ETOH during low storage temperatures is well established. This is done by direct immersion or by fumigation by placing the compound in containers with ETOH pads. Even though antimicrobial, the phytotoxicity is a commonly reported problem with ETOH use in fresh produce (Candir et al., 2011). Mango pathogens, like Cun>ularia lunata and Pestalotia mangiferae, etc., are inhibited by a combination treatment of ETOH at 300 mL L_1, followed by heating at 50°C for 60 s (Gutierrez-Martinez et al., 2012). Other fruits, such as grapes, sweet cherries, Chinese bayberry, and banana have also benefited from ETOH applications (Romanazzi et al., 2007, Zhang et al., 2007; Bai et al., 2011; Salazar and Serrano, 2013).

 
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