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

Thesis (PhDFoodSc)--Stellenbosch University, 2017.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.AFRIKAANSE OPSOMMING: Vrugtesiektes in granate (Punica granatum L.) word dikwels deur ’n wye reeks swamme en bakterieë veroorsaak en lei tot groot finansiële en na-oes verliese in die waardeketting, asook verliese aan voedsaamheid. Die doel met hierdie studie was om na-oes siektes by granate in Suid-Afrika te identifiseer en om die raklewe van vars en minimaal geprosesseerde granate met die hulp van chitosan en essensiële olies (EOs) te verbeter. Om die identiteit van patogene op na-oes granate asook hulle bronne in die waardeketting vas te stel is monsters van blare en vrugte op verskillende stadiums van hulle ontwikkeling vanaf kommersiële vrugteboorde in die Weskaap, Suid-Afrika versamel. Die monsters is geneem van die kultivars ‘Herskawitz’ (mid-oes) en ‘Wonderful’ (laatoes). Swamme is vanaf gesonde en heel granaatblomme (oop en toe), onvolwasse blomme, onvolwasse groen vrugte en ryp granate, asook blare geneem. Die geïsoleerde swam patogene is deur die filogetiese ontleding van die interne transkribeerde spasieerder (ITS: ITS1 and ITS2) van die atoom ribosomale DNA en die 5.8S ribosomals RNA gene identifiseer. Aspergillus niger Tiegh., Cytospora spp., Clonostachys spp., Embellisia eureka E.G. Simmons, Pestalotiopsis spp., Nigrospora oryzae en Rhizopus stolonifer Ehrenb. is met nie-besmette plantmatriaal assosiëer. Die belangrikste bederfpatogene is vanaf oppervlakte gesteriliseerde monsters geneem en hulle is Alternaria spp., Aureobasidium pullulans, Botrytis spp., Penicillium spp., en Pilidiella granati Sacc. (syn. Coniella granati [Sacc.] Petr. & Syd.). Die oopblom stadium het die hoogste voorkoms van bederfspatogene en dieselfde patogene is op no-oes vrugte gevind. Toetse is gedoen op die hoof na-oes bederfpatogene naamlik Botrytis sp., Penicillium sp. en P. granati. Dit is die eerste keer wat P. granati op granate in Suid Afrika rapporteer is. ’n Instument (RFLP) is ontwikkel vir die opsporing van granaat na-oes patogene. Hierdie instrument kan help met die monitering van granaatpatogene in vrugteboorde en pakhuise, Die antiswam aktiwiteit van kropdop chitosan (0-10 g/L) and fludioxoniel (0-1.0 g/L) is op Botrytis sp., Penicillium sp. en P. granati wat vooraf van granate verkry is, getoets. Hoe sensitief die patogene is vir krapdop chitosan baseer op EC50 waardes (konsentrasies wat ’n 50% vermindering in swamvlok groei veroorsaak) toon dat P. granati (EC50 0.47 g/L) die mees senstief is, gevolg deur Botrytis sp. (EC50 1.19 g/L) en Penicillium sp. (EC50 2.21 g/L). Wat betref fludioxoniel was Penicillium sp. (EC50 0.02 mg/L) die sensitiefste gevolg deur P. granati (EC50 0.48 mg/L) en Botrytis sp. (EC50 0.09 mg/L). Die voorafbehandeling van vrugte (voorkomende behandeling) met chitosan lei tot beter siektebeheer (30-66% vermindering in bederf) invergeleke met die gebruik van chitosan na die inspuiting van patogene as ’n genesende strategie (18-38%). Die aanwending van chitosan (0, 2.5, 7.5 and 15 g/L) as n eetbare bedekking op minimaal geprosesseerde granate voor koelberging verminder die telling (P < 0.05) van mesofiliese bakterieë, swamme en skimmels. Verder hou die chitosan behandeling ook die fisiko-chemiese eienskappe van die granate (totale oplosbare vastestowwe (TSS), titreerbaarheid, klamheid, kleur, fermheid en askorbiensuur) in stand. Die bevindinge toon dat krapdop chitosan beskou kan word as ’n potensiële swamdoder vir die na-oes siektebeheer van beide vars en minimal geproseseerde granate ’n Opvolgstudie is gedoen om die antimikrobiale eienskappe van chitosan te verbeter. Chitosan is gebruik as ’n polimeriese draer van vlugtige essensiële olies EOs (kaneel, lemongrass and oregano). Die EOs is getoets vir antiswam aktiwiteite teen Botrytis sp., Penicillium sp., en P. granati. Lemon grass was die minste effektief en het nie die swam patogene heeltemal inhibeer nie, terwyl die oregano EO die sterkste was en P. granati deur waterdamp en direkte kontak heeltemal inhibeer. Die in vivo aanwending van chitosan-oregano as ’n eetbare bedekking het swamgroei effektief beheer (59-100%) maar het ’n negatiew effek op die skil gehad. As dit as flies gebruik is, het die chitosan-oregano flies nog steeds (P < 0.05) die bederf van die granate met 34-100% verminder sonder om die skil te benadeel. Die bevindinge bewys dus dat die aanwending van chitosan-EO gebaseerde fliese potensiaal het in die verpakking van vrugte. Om die gebruik van EOs in antimikrobiale verpakking verder te verbeter is kaneel en oregano EOs toegemaak in capsules met β-cyclodextrin (β-CD) en ’n nanovessel matrieks gebaseer op chitosan and poliviniel alkohol (PVA) om die termiese onstabiliteit van die EOs te verminder en om langer vrylating te bewerk. ’n GC-MS analise toon dat die β-CD heelwat (P < 0.05) meer doeltreffend is in die kapsule form van kaneel (4.86%), invergeleke oregano EO (1.75%). Dieselfde EO konsentrasies is egter verkry toe die EO inkorporeer is in chitosan-baseerde nanovesels. Die EO-baseerde β-CD mikrokapsules en die nanovesels het albei ’n antimikrobale effek op Botrytis sp. en kan dus in antimikrobale verpakking gebruik word. Tydens hierdie studie is die hoof na-oes bederfpatogene van granate in die Weskaap Streek van Suid-Afrika identifiseer en is daar verder vasgestel dat die vrugteboord die hoofbron van hierdie patogene is. Daar is bewys dat krapdop chitosan op sy eie of in kombinasie met EOs granaat na-oes patogene op vars en minimal geprosesseerde granate kan beheer. Die blootstelling van granate aan EO emulsies verminder die bederf maar het ook ’n negatiewe effek op die voorkoms van die skil. Maar die blootstelling van die vrug aan indirekte kontak met EOs in kapsules beheer die bederf sonder om die voorkoms van die skil te affektee