Antimicrobial effects of chitosan and essential oils on postharvest diseases of pomegranate fruit

[摘要] ENGLISH ABSTRACT: Pomegranate (Punica granatum L.) fruit diseases often caused by a range of fungi and bacteria, pose significant financial, nutritional and postharvest losses along the value chain. This study aimed at identifying pomegranate postharvest diseases in South Africa and improving the shelf life of whole and minimally processed pomegranates using chitosan and essential oils (EOs). To identify pomegranate postharvest pathogens and their origin in the value chain, samples of leaves and fruit at different development stages were collected from commercial orchards of cultivars 'Herskawitz' (mid harvest) and 'Wonderful' (late harvest) located in the Western Cape region of South Africa. Fungi were isolated from healthy and intact pomegranate flowers (open, closed, diheased), immature fruitlets buds, immature green fruit and ripe pomegranate fruit as well as leaves. Isolated fungal pathogens were identified using phylogenetic analysis of the internal transcribed spacer (ITS: ITS1 and ITS2) of the nuclear ribosomal DNA and the 5.8S ribosomal RNA genes.Aspergillus niger Tiegh., Cytospora spp., Clonostachys spp., Embellisia eureka E.G. Simmons, Pestalotiopsis spp., Nigrospora oryzae and Rhizopus stolonifer Ehrenb., were associated with non-disinfected plant materials. The major pomegranate spoilage pathogens were isolated from surface sterilised samples and comprised of Alternaria spp., Aureobasidium pullulans, Botrytis spp., Penicillium spp., and Pilidiella granati Sacc. (syn. Coniella granati [Sacc.] Petr. & Syd.). The open flower stage had the highest incidence of spoilage pathogens and the same pathogens were isolated from fruit at postharvest. Pathogenicity tests were carried out on the major postharvest spoilage pathogens isolated namely Botrytis sp., Penicillium sp. and P. granati. This is the first report of P. granati on pomegranate fruit in South Africa. A restriction fragment length polymorphism (RFLP) tool was developed for the detection of pomegranate postharvest pathogens. This tool will be important in the monitoring of pomegranate pathogens in orchards and packhouses.The antifungal activity of crab shell chitosan (0-10 g/L) and fludioxonil (0-1.0 g/L) was tested against Botrytis sp., Penicillium sp. and P. granati previously isolated from pomegranate. Pathogen sensitivity to crab shell chitosan based on the EC50 values (concentration causing a 50% reduction in mycelial growth) showed that P. granati (EC50-0.47 g/L) was the most sensitive followed by Botrytis sp. (EC50-1.19 g/L) and Penicillium sp. (EC50 2.21 g/L). For fludioxonil Penicillium sp. (EC50 0.02 mg/L) was the most sensitive followed by P. granati (EC50 0.48 mg/L) and Botrytis sp. (EC50 0.09 mg/L). Pre-treating wounded fruit (preventive treatment) with chitosan prior to pathogen inoculation gave better disease control (30-66% decay reduction) compared to introducing the chitosan after pathogen inoculation as a curative strategy (18-38%). Applying chitosan (0, 2.5, 7.5 and 15 g/L) as an edible coating on minimally processed pomegranate arils prior to cold storage significantly (P<0.05) lowered counts for mesophilic aerobic bacteria, yeast and moulds. In addition, the chitosan treatment also maintained the physico-chemical attributes of the arils (total soluble solids (TSS), titratable acidity (TA), moisture, colour, firmness, total phenolics, anthocyanins and ascorbic acid). The findings demonstrate that crab shell chitosan can be considered as a potential green fungicide for postharvest disease management of both whole and minimally processed pomegranate fruit.A follow up study to enhance the antimicrobial properties of chitosan was conducted using chitosan as a polymeric carrier of volatile EOs (cinnamon, lemongrass and oregano). The EOs were assayed for antifungal activity against Botrytis sp., Penicillium sp., and P. granati. Lemon grass was the least effective EO as it failed to provide complete inhibition of any of the fungal pathogens while oregano EO was the most potent as it gave complete inhibition of P. granati by both vapour and direct contact methods. In vivo application of chitosan-oregano as an edible coating effectively controlled fungal growth by 59-100% but induced negative effects on the fruit skin. When applied as active film, the chitosan-oregano film still significantly (P < 0.05) reduced fruit decay by 34-100% without elucidating cosmetic damage to the fruit rind. The findings revealed the potential application of chitosan-EO based films in developing antimicrobial based active food packaging systems.To further improve use of EOs in antimicrobial packaging, cinnamon and oregano EOs were encapsulated in β-cyclodextrin (β-CD) and a nanofibrous matrix based on chitosan and polyvinyl alcohol (PVA) to reduce the thermal instability of the EOs and achieve prolonged release. A GC-MS analysis revealed that the β-CD was significantly (P < 0.05) more efficient in encapsulation of cinnamon EO (4.86%), compared to oregano EO (1.75%). However, similar EO concentrations were obtained when the essential oils were incorporated into electrospun chitosan based nanofibres. The EO based β-CD microcapsules and nanofibres both had antimicrobial effect on Botrytis sp. and thus could be used in antimicrobial packaging.This study identified the major postharvest spoilage pathogens of pomegranates in the Western Cape Region of South Africa and further determined that the orchard was the major source of these pathogens. Crab shell chitosan independently and in combination with EOs was shown to effectively control pomegranate postharvest pathogens on whole and minimally processed fruit. While exposing the pomegranate fruit to direct contact with EO emulsions reduced postharvest spoilage it also resulted in a negative cosmetic appearance of the fruit rind. However, exposing fruit to in-direct contact with encapsulated EOs controlled postharvest spoilage without affecting the fruit cosmetic appearance.

[发布机构] Stellenbosch University