ANTIMICROBIAL EFFECT OF EDIBLE COATING CONJUGATED WITH SILVER NANOPARTICLE ON PAPAYA (Carica papaya L. cv.

Eksotika)

ABSTRACT Edible coatings are used on many products to control moisture transfer, gas exchange and oxidation process. In addition, these films and coatings can be incorporated with active ingredients that can enhance the nutritional and safety aspects of the products. Antimicrobial agents are one of the common active ingredients incorporated into edible coatings. Silver nanoparticles are proposed as antimicrobial agent for this study. Many tropical fruits for example papaya eksotika are prone to spoilage due to microbial infection and also wounding during handling. There are several microorganisms of concern including Colletotrichum gloesporioides, Erwinia, Botryodiplodia, Fusarium, Escherichia coli and also Staphylococcus aureus. Applying edible coating onto the fruit may reduce the spoilage and contribute to the extension of shelf life. Thus, the main objective of this study is to determine the antimicrobial effect of edible coating incorporated with silver nanoparticles on papaya eksotika. The optimum concentration of silver nanoparticles as antimicrobial agent will be determined using the minimum inhibitory concentration (MIC) and also disk diffusion methods before being incorporated into the edible coating. In this study, chitosan and carrageenan will be used as coatings. The edible coating incorporated with silver nanoparticles will then be applied onto the fruit by either spraying or brushing. Coated fruit will be stored at two different temperatures, (ambient temperature (262C;7010%RH) and 151C; 6010%) for 2-8 weeks. The fruit samples during storage will be analysed for the changes in their

physicochemical properties including texture (texture analyzer), colour (colourimeter), total soluble solids (refractometer), titratable acidity (titration), pH (pH meter) and also vitamin C content (titration). Effectiveness of silver nanoparticles against the mentioned bacteria and fungi during storage will also be evaluated. The total plate count and also presence of yeast and mould during storage period will also be determined. Statistical analysis of experimental data will be done by using Minitab 14.

Keywords: Edible coating; chitosan; carrageenan; silver nanoparticles; antimicrobial effect; papaya eksotika

1.0 INTRODUCTION Fruits and vegetables can be classified as climacteric or non-climacteric. Climacteric fruits continue to ripen after harvest and show more dramatic changes in their physiological activity (Maftoonazad & Ramaswamy, 2005). The quality of fruits is usually judge based on their appearance and freshness. However, fruits are easily injured especially during handling. This alters the integrity of fruit and induces wounding stress and spoilage (Rojas-Grau et al., 2007). Controlled atmosphere storage and modified atmosphere storage have been used for preserving fruits by reducing their quality changes and quantity losses during storage (Park, 1999). Edible coatings are alternative storage methods for fresh products and have attracted increasing attention because of environmental considerations and the trends toward the use of convenience food (zden & Bayindirli, 2002). In addition, edible coatings on fresh fruit can provide an alternative to modified atmosphere storage by

reducing quality changes and quantity losses through modification and control of the internal atmosphere of the individual fruits (Park,1999). Application of edible films and coatings on whole fruit are quite popular. Studies have shown that semipermeable coatings are capable to improve the storage life of perishable fruits (Worrell et al., 2002). For an example, shelf life of mango was extended by coating treatments due to delayed ripening and softening, reduced decay and weight loss (Baldwin et al., 1999). Edible coatings have been used in the fresh cut industry to reduce the deleterious effects that minimal processing imposes on intact vegetable tissues (Rojas-Grau et al., 2009). Edible coatings may contribute to extend the shelf life of fresh cut fruits by reducing moisture and solute migration, gas exchange, respiration and oxidative reaction rates (Baldwin et al., 1996). The application of edible coating have been expanded due to its high potential to carry active ingredients such as antibrowning agents, colourants, flavours, nutrients, spices and antimicrobial compound that can extend product shelf life and reduce the risk of pathogen growth on food surfaces (Pranoto et al., 2005). Nowadays, nanotechnologies are being used to enhance the nutritional aspects of food by means of nanoscale additives and nutrients and nanosized delivery systems for bioactive compound (Bouwmeester et al., 2007). This also includes their application in edible coating. However, studies on the application of nanoparticles incorporated into edible coating on specific fruits are rather limited. One fruit of concern are the Eksotika papaya. Brazil, Mexico and Malaysia are the main exporting countries of papaya mainly to the European market (Cia et al., 2007) with Malaysia being the second most important exporter of Eksotika papaya. Thus, the

main objective of this study is to determine the antimicrobial effect of edible coating incorporated with nanosilver particle on eksotika papaya. In addition, suitable coatings will be screened and optimized before being applied on the fruit. The amount of nanosilver particle that will give the desired antimicrobial effect will also need to be determined.

2.0 LITERATURE REVIEW Edible films and coatings are artificial barriers to gas diffusion. A barrier to gas diffusion on fruit and vegetable surfaces can reduce moisture loss and internal levels of oxygen, while increasing internal levels of CO2 and ethylene (Avena-Bustillos et al., 1997). The main film forming materials are biopolymers, such as proteins, polysaccharides, lipids and resins (Han & Gennadios, 2005). Proteins are commonly used film forming materials which include collagen, gelatin, casein and many others. Another group of film forming material, polysaccharide, includes starch, modified starch, carrageenan and chitosan. Besides that, beeswax, paraffin and shellac are example of lipids and resins that are also used as film forming materials (Han & Gennadios, 2005). It has mentioned in the literature that chitosan coating delayed the change in eating quality, reduced respiration rate and weight loss and partially inhibited the increase of polyphenol oxidase activity of longan fruit which is associated with peel discoloration (Jiang & Li, 2001). Similarly, applications of methyl cellulose coating to avocado fruits were shown to be beneficial in retarding the ripening behaviour (Maftoonazad & Ramaswamy, 2005).

Generally, applications of film forming biopolymers were combined with plasticizers and other additives (Han & Gennadios, 2005). Plasticizers are defined based on two purposes, which are to aid processing and modify the properties of the final product (Sothornvit & Krochta, 2005). Some edible films and coatings are supported with active ingredients in order to improve the quality and extending the shelf life of fruits. Antimicrobial agents are one of the active ingredients commonly used. Studies have been conducted to evaluate this application. The effect of lemongrass, oregano oil and vanillin incorporated in apple pureealginate edible coatings on the shelf life of fresh-cut Fuji apples was investigated (Rojas-Grau et al., 2007). They found that, the calcium chloride and N-acetylcysteine containing apple puree-alginate coatings helped to maintain firmness and color. Durango et al. (2006) found that the use of an antimicrobial coating consisting of chitosan added yam starch is a viable alternative in controlling the microbiota present in minimally processed carrot, since the growth of lactic acid bacteria, total coliforms, psychrotrophs, yeast and molds and mesophilic aerobes was substantially inhibited by the application of 1.5% chitosan coating. Antibacterial alginate based edible film has been studied by incorporation of garlic oil as a natural antibacterial agent (Pranoto et al., 2005). The results showed that garlic oil had antibacterial activity on four bacteria used in the study. Application of nanotechnology in food has become a new trend especially to develop new food products and new food packaging (Siegrist et al., 2008). Food scientists are creating nanocoatings for foods and food packages that serve as barriers to bacteria (Lagaron et al., 2005).

Eksotika papaya has the potential to be applied with the nanosilver particle incorporated edible coating. This is because the fruit are being largely exported and an efficient packaging is required that can help in extending their shelf life and maintain it freshness by the time they reach consumer. In addition, after harvesting papaya fruits become very perishable and can easily rot. Anthracnose is the main postharvest disease in Brazil and other countries (Cia et al., 2007). Thus, the application of edible coating with the addition of nanosilver particles are hoped to help overcome this problem.

3.0 MATERIALS & METHODS 3.1 Project flow Sourcing for papaya supply

Purchase of raw materials and chemicals

Stage 1 Screening for edible coating - based on literature

Stage 2 Antimicrobial effect of nanosilver particle - best concentration (analysis and standards)

Optimization of coating formulation - physiological analysis (colour, texture, weight loss, TA, TSS & pH)

Stage 3 Incorporation of nanosilver particles into edible coating - choose the best concentration - antimicrobial effect determination

Fruit coating - dipping

Stage 4 Storage study - Duration: 2-8 weeks - Condition: room temperature & 15C - physicochemical and microbiological analysis

Data analysis & write up

3.2 Identification of common fungi/bacteria that causes rottening in papaya The identification will be done by referring to literature for common disease causing fungi/bacteria in papaya. Cia et al. (2006) found that anthracrose is the main postharvest disease in papaya fruit with Colletotrichum gloesporioides as the causal agent. Thus the antimicrobial activity will focus on this microorganism. In addition, the antimicrobial activities of other major postharvest pathogens, Erwinia, Botryodiplodia and Fusarium will also be determined along with Escherichia coli and also Staphylococcus aureus.

3.3 Screening and optimization of most suitable edible coating Most suitable edible coating for papaya will be screened based on literature. Best formulations for the coatings will be developed based on their effect on the physiological changes of papaya; colour, texture and weight loss, TA, TSS and also pH. The process parameters (homogenizing pressure, cycle, duration and temperature) will also need to be optimized.

3.4 Antimicrobial effect of silver nanoparticle The effectiveness of silver nanoparticle against Colletotrichum gloesporioides, Erwinia, Botryodiplodia, Fusarium, Escherichia coli and also Staphylococcus aureus will be tested at different concentration using minimum inhibitory concentration (MIC) and also disc diffusion method. This is to determine which concentration that will give the best antimicrobial effect. In addition to the antimicrobial test, other factors including permitted concentration according to standards and also application in previous studies

will also be taken into consideration in choosing the best concentration of silver nanoparticles.

3.5 Incorporation of silver nanoparticles into edible coating The chosen concentration of silver nanoparticles will be incorporated into the edible coating. The antimicrobial effect of the edible coating incorporated with silver nanoparticles Colletotrichum gloesporioides, Erwinia, Botryodiplodia, Fusarium, Escherichia coli and also Staphylococcus aureus will be determined using two different methods: i) qualitative evaluation with modified agar diffusion assay and ii) quantitative evaluation in liquid medium.

3.6 Fruit coating To choose same green & mature papaya Weighing of edible compounds

Washing

Mixing edible compound with distilled water Homogenization

Sanitizing in 200ppm sodium hypochlorite solution for 2 minutes

Edible coating emulsion Dry the papaya at ambient temperature for 15 minutes

Applying edible coating emulsion by dipping Dry coated papaya at ambient temperature

Papaya eksotika will be purchased from the local market or at an identified farm. Papayas will be washed, sanitized by immersion in 200ppm sodium hypochlorite solution for 2 minutes, rinsed and dried (Rojas-Grau et al., 2007). Coatings will be applied by dipping before being air flow dried at 20C for 3 hours (Durango et al., 2006).

3.7 Storage study Papaya eksotika will be coated with silver nanoparticle incorporated edible coating. The storage study will be conducted under two different temperatures (ambient temperature (262C; 1570%RH) and 151C; 6010%) for a period of 2-8 weeks. The effectiveness of the edible coating incorporated with silver nanoparticles will be tested based on physicochemical and microbial parameters. The physicochemical tests will include texture (texture analyzer), colour (colourimeter), total soluble solid

(refractometer), titratable acidity (titration), pH (pH meter) and also vitamin C content (titration). Microbiological analysis will include total plate count (TPC) and also yeast and mould. Effectiveness of silver nanoparticles against the mentioned bacteria and fungi during storage will also be evaluated.

4.0 EQUIPMENTS

Instruments that will be used for physicochemical analysis (texture analyzer, pH meter, colourimeter and refractometer) are available in research lab and also teaching lab. As for the microbiological tests, materials are currently available. Special Equipment Homogenizer Chromameter Texture analyzer Gas chromatography (GC) Auto titrator Particle size analyzer Facility Edible coatings and chemicals Glass ware Infrastructure Computer with Minitab 14.1 Printer and digital camera Description Available Available Available Available Available Available Description To be purchased Some need to be purchased Description Necessary for experimental design (RSM designs) Available Available

PROJECT SCHEDULE
2010 F M A M J J A S O N D 2011 J F M A M J J A S O N D

Project Activities Literature search/review and design of experiment X X X X X X X X X X X X X X X X X X X X X X X X X

2009 D J X X

Purchasing of solvents and glass wares

Sourcing for papaya supply

Preliminary studies for sample preparation Sample preparation according to the established experimental design Analysis of prepared samples

Storage study (antimicrobial test)

Data analysis and publication of scientific paper/reports Final report write up

X X X X X X X X

Milestone Experimental design developed and independent and response variables determined Optimum formulations for different edible coating emulsions identified Optimum process parameters for different edible coating emulsions identified Antimicrobial activity of edible coating emulsion conjugated with silver nanoparticles evaluated Submission of final report

X : Activities;

: Planned milestone

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