Antimicrobial properties of starch films incorporated with chitosan nanoparticles: In vitro and in vivo evaluation

Journal Pre-proof Antimicrobial Properties of Starch Films Incorporated with Chitosan Nanoparticles: In Vitro and In Vivo Evaluation Ruzanna Ahmad Shapi’i, Siti Hajar Othman, Norhazirah Nordin, Roseliza Kadir Basha, Mohd Nazli Naim

PII:

S0144-8617(19)31270-6

DOI:

https://doi.org/10.1016/j.carbpol.2019.115602

Reference:

CARP 115602

To appear in:

Carbohydrate Polymers

Received Date:

5 August 2019

Revised Date:

5 November 2019

Accepted Date:

9 November 2019

Please cite this article as: Shapi’i RA, Othman SH, Nordin N, Basha RK, Naim MN, Antimicrobial Properties of Starch Films Incorporated with Chitosan Nanoparticles: In Vitro and In Vivo Evaluation, Carbohydrate Polymers (2019), doi: https://doi.org/10.1016/j.carbpol.2019.115602

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Antimicrobial Properties of Starch Films Incorporated with Chitosan Nanoparticles: In Vitro and In Vivo Evaluation Ruzanna Ahmad Shapi’i1,2, Siti Hajar Othman1, 2, *, Norhazirah Nordin1, Roseliza Kadir Basha1, and Mohd Nazli Naim1 1

Department of Process and Food Engineering, Faculty of Engineering, Universiti Putra

Malaysia, 43400 UPM Serdang, Selangor, Malaysia. 2

Materials Processing and Technology Laboratory, Institute of Advanced Technology,

* Corresponding author Siti Hajar Othman

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Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia.

Address: Department of Process and Food Engineering, Faculty of Engineering, Universiti

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Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia.

Fax: +603 8946 4440

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E-mail address: [email protected]

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Tel: +603 8946 6350

Abstract

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Highlights  Antimicrobial activity of starch/CNP films is dependent on the concentration of CNP.  15 to 20% w/w starch/CNP films can inhibit bacterial growth.  CNP inhibit gram-positive bacteria efficiently compared to gram-negative bacteria.  Shelf life of cherry tomatoes wrapped in starch/CNP films improved.

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Chitosan nanoparticles (CNP) were synthesized via ionic gelation and used for the preparation of starch-based nanocomposite films containing different concentration of CNP (0, 5, 10, 15, 20% w/w). Antimicrobial properties of starch/CNP films was evaluated via in vitro (disc diffusion analysis) and in vivo (microbial count in wrapped cherry tomatoes) study. It was found that inhibitory zone of the 15 and 20% of starch/CNP films were clearly observed for all the tested bacteria including Bacillus cereus, Staphylococcus aureus, Escherichia coli and Salmonella typhimurium. In vivo study revealed that the starch/CNP film (15% w/w) was more

efficient to inhibit the microbial growth in cherry tomatoes (7 x 102 CFU/g) compared to neat starch film (2.15 x 103 CFU/g) thus confirmed the potential application of the films as antimicrobial food packaging.

Keywords Active packaging, antimicrobial agent, biopolymer, cherry tomato, chitosan, starch film

1. Introduction

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Over the last decades, synthetic polymers such as polypropylene (PP), polyethylene terephthalate (PET) and polystyrene (PS) have been widely used as food packaging material. However, synthetic polymers are usually made up from petroleum which is non-degradable, thus end up an excessive amount of disposable on landfill which could lead to severe earth pollution (Othman et al., 2019). Alternatively, biopolymers such as cellulose, chitosan, Tara

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gum, fish gelatin and starch can be utilized to produce food packaging material due to their biodegradability properties. Among them, starch is one of the promising biopolymers to be

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used as food packaging material because of its abundant sources, renewable, and low price (Kowalczyk et al., 2015; Luchese et al., 2018). The structure of starch consists of branched and

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linear chains of glucose monomers, known as amylose and amylopectin which could affect the properties of the produced films such as mechanical, barrier and thermal properties. According to Mishra and Rai (2006), composition of amylose and amylopectin in starch is dependent on

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the origin of starch which can be categorized into cereal (maize, rice, and corn) and tuber (cassava, yam, potato, and sweet potato). Cassava and corn starches consist of 18% and 28% of amylose respectively which enough to produce films forming solution (Tavares et al., 2019).

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Films produced from starch are generally odorless, tasteless, colorless, non-toxic and biologically degradable (Dang & Yoksan, 2016; Nordin et al., 2018). Unfortunately, starch

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films exhibit poor mechanical and barrier properties compared to synthetic plastics which limit the application of starch films for food packaging application (Yun et al., 2019). Incorporation of nanofiller into biopolymer may produce bio-nanocomposites which exhibit

good tensile strength, thermal stability, and barrier properties (Shapi’i et al., 2019). Nanoparticles such as zinc oxide and titanium dioxide can act as antimicrobial agents which are able to produce antimicrobial food packaging material when used as filler. Antimicrobial food packaging incorporated with antimicrobial agents will gradually release the antimicrobial agents to the food surface to inhibit the microbial growth and extend the shelf life of food (Dairi

et al., 2019). However, there are many speculations on the toxicity of nanoparticles due to its small dimension (<100 nm) which has high potential to penetrate human tissue thus harmful to human health (Hannon et al., 2015; He et al., 2019). Alternatively, nanoparticles that synthesized from natural biopolymer such as cellulose, chitin, and chitosan can be used as nanofiller (Othman, 2014; Shapi’i & Othman, 2016). They are generally edible and not harmful to human health. Among them, chitosan nanoparticles (CNP) exhibit good antimicrobial properties and have been used in food packaging (Antoniou et al., 2015), food coating (Pilon et al., 2015), textiles industry (Ali et al., 2011), water treatment (Sivakami et al., 2013) and others. A study on genotoxicity of CNP by De Lima et al. (2010) revealed that 180 mg/mL of

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60 nm CNP did not affect the mitotic index, thus indicating that CNP at mentioned concentration was not toxic.

CNP can be synthesized using various methods such as ionic gelation, reverse emulsion, precipitation and polyelectrolyte complexation (Hussain & Sahudin, 2016; Shapi’i et al., 2017). Ionic gelation method is the most promising method to produce CNP in food industry due to

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its non-toxicity (Al-Qadi et al., 2012; Rampino et al., 2013). During ionic gelation process, the cations in chitosan (CH) were cross-linked with the polyanions in sodium tripolyphosphate

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(TPP) thus forming a spherical shape of CNP spontaneously (Othman et al., 2019). A few studies have revealed that addition of CNP into the biopolymers could produce antimicrobial

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film. A study by Antoniou et al. (2015) found that incorporation of CNP into Tara gum film led to the formation of inhibitory zone on the gram-negative bacteria agar and gram-positive bacteria agar particularly E. coli and S. aureus respectively. CNP tends to form larger diameter

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of inhibitory zone on S. aureus compared to E. coli due to the different mechanisms of antimicrobial activity of CNP against gram-positive bacteria and gram-negative bacteria. They also found that increase in concentration of CNP from 5 to 10% resulted in an increase in

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diameter of the inhibitory zone on both types of bacteria. However, further increase in concentration of CNP slightly reduced the inhibitory zone due to the agglomeration of CNP in

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the film matrix. Therefore, concentration of CNP might be one of the most important parameters that affect the antimicrobial properties of the film. Furthermore, Pilon et al. (2015) has investigated the effect of CNP coating on the microbiological quality of fresh-cut apple slice. They found that coating apple slices with CNP could inhibit molds and yeasts, and mesophilic and psychotropics bacteria thus revealed the potential use of CNP in antimicrobial film. Increase in consumer demands for ready to eat food such as mixed salads, fresh-cut fruits, and sandwiches, forcing the researcher and industry to find new food preservation technique

that can control the microbial growth and lengthen the shelf life of food. Antimicrobial packaging is a promising preservation technique that can inhibit microbial growth in the food product and lengthen the shelf life of food. Thus, the aim of this work has been directed towards developing starch/CNP films as antimicrobial food packaging material. The optimum concentration of CNP that could inhibit microbial growth was investigated using disc diffusion method (in vitro). Then, the real application of starch/CNP film was demonstrated on the cherry tomatoes and the microbial growth in cherry tomatoes was evaluated for 10 days storage period (in vivo).

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2. Materials and methods 2.1. Materials

Chitosan (low molecular weight) and sodium tripolyphosphate (TPP) were purchased from Sigma-Aldrich, USA. Tapioca starch (amylose: 19%, amylopectin: 81%) was obtained from

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Thye Huat Chan Sdn Bhd (Brand Kapal ABC, Thailand). Acetic acid and glycerol were

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purchased from R&M Marketing, UK.

2.2. Preparation of CNP

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All parameters in this work were fixed based on the optimum parameters that produced the most stable and smallest size of CNP as reported by Gokce et al. (2014) with some modification of initial concentration of CH. Firstly, CH solution (5, 10, 15, 20% w/w of solid starch) were

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prepared by dispersing chitosan flakes (0.15, 0.3, 0.45, 0.6 g) into 50 mL aqueous acetic acid solution (1% v/v) for 30 minutes using magnetic stirrer (FAVORIT HS0707V2, Indonesia). Then, the pH of the solution was adjusted to optimum pH 4.6 using NaOH. Different

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concentrations of TPP solution were prepared according to the optimum ratio of chitosan to TPP (5:1) by dissolving TPP powder (0.03, 0.06, 0.09, 0.12 g) in 50 mL distilled water.

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CNP was spontaneously obtained upon the addition of 50 mL of TPP solution drop by drop

to the 50 mL CH solution under vigorous magnetic stirring at room temperature (25ºC) for 30 minutes. Then, ultrasonic probe (QSonica Q500, USA) was used to disperse the CNP in the suspension for 15 minutes with a sequence of 1 minute sonication and 10 seconds of rest at an amplitude of 50%. The beaker containing CNP was placed in the ice bath during ultrasonication to ensure that the temperature of CNP suspension was in the favorable range (30 to 35ºC). The

particle size distribution of CNP suspensions was then characterized using particle size analyzer (Malvern Instruments, UK).

2.3. Preparation of antimicrobial film An amount of 3 g tapioca starch was dispersed in 100 mL distilled water-glycerol solutions in order to obtain 3% w/w suspensions. The composition of glycerol added into the starch solution was 25% w/w of the dry starch solid weight. Then, the solution was heated with continuous stirring until gelatinized completely at 75 ºC. The starch solution was cooled down to 50 ± 2 ºC before mixed with CNP suspension.

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The starch/CNP film solution was prepared by mixing 100 mL CNP suspension with 100 mL gelatinized starch solution and stirred for 30 minutes. Then, the solution underwent sonication for 5 minutes to produce a homogeneous solution. An amount of 50 mL of the solution was poured into an acrylic petri dish (diameter: 14 cm) and left in the air-conditioned room (20˚C) for 48 hours on the flat table. Then, the petri dish containing starch/CNP film was

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dried at 40 ˚C for 5 hours in a ventilated oven to constant weight. A neat starch film without the addition of CNP was also prepared as the control.

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After drying, the film was peeled off from the petri dish and conditioned in a desiccator

30ºC) (Xu et al., 2005).

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containing saturated magnesium nitrate (R&M Marketing, UK) solution (RH: 51%, Temp:

2.4. Transmission electron microscopy (TEM)

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Morphological properties of the CNP and starch/ CNP films were observed using

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transmission electron microscope (TEM) at x50K magnification.

2.5. In-vitro antimicrobial test In-vitro antimicrobial test was done to investigate the effect of varying CNP concentration

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on the ability of starch/CNP film to act as an antimicrobial film. The test was done by disc diffusion assay according to Pelissari et al. (2009). Two types of gram-positive (B. cereus and S. aureus) and two types of gram-negative (E. coli and S. typhi) bacteria were obtained from Microbiology Laboratory, Faculty of Food Science and Technology, Universiti Putra Malaysia, then were cultured in nutrient agar (Merck, Germany) for 24 hours at 37 °C. After 24 hours, the cultured bacteria were inoculated into saline water until the concentration of bacteria were 108 CFU/mL which was standardized using the McFarland scale. Then, 0.1 mL of bacteria in

the saline water was spread onto the petri dish containing Mueller-Hinton agar (Merck, Germany). Starch/CNP films were cut aseptically into 1.5 cm discs using a puncher and sterilized under UV light before conducting the antimicrobial tests. Then, all discs were placed carefully into the petri dish that contained bacteria culture and incubated at 37 °C for 24 hours in the incubator. After 24 hours, the inhibition area of the film discs was examined to determine the ability of the film to inhibit bacterial growth. The test was carried out in triplicate for each sample.

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2.6. In-vivo antimicrobial test Both starch/CNP film and neat starch film were used to wrap cherry tomatoes in order to demonstrate their real application of the films on the food product. Then, microbial count in the tomato cherries was determined to investigate the ability of the films to inhibit microbial growth during the storage time.

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Cherry tomatoes were purchased from Kea Farm, Cameron Highlands, Pahang, Malaysia and subjected to a selection regarding physical integrity, diameter, and color. The tomatoes

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were washed under running tap water and then immersed in hypochlorite solution (200 mg L−1) for 10 minutes. The tomatoes were then dried using a hair dryer (Philips, Malaysia) for 40 s.

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The tomatoes were manually wrapped in the films (neat starch film and starch/CNP film) and then, stored in a chiller at 10 °C (±2 °C) and RH 95 % up to 10 days according to the storage condition of tomatoes suggested by USDA (2016). Microbiological test was performed on day

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0, 3 and 10.

The samples of 10 g of the tomatoes were mixed with 90 ml of peptone water (0.1% w/v, Friedemann Schmidt, Australia), and homogenized in a blender. Then, serial dilutions of

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tomato extracts were prepared in peptone water. The total plate count of bacteria was determined using Plate Count Agar (Fluka, USA) after incubation at 37 ˚C for 48 hours. The

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test was performed in triplicate and means were reported. The colonies of bacteria were counted and calculated using the following equation:

Colony

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number of colonies x dilution  CFU  unit   volume of culture plate  mL 

2.7. Statistical analysis

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Analysis of variance (ANOVA) was applied on the in-vivo test results using Minitab Software (Minitab Inc., State College, Pa., USA) to evaluate the P-value.

3. Results and discussion 3.1. CNP and starch/CNP films

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CNP was first synthesized using ionic gelation process whereby the initial concentration of CH was varied from 0 to 20% w/w. Figure 1 (a) shows the TEM image of CNP synthesized using 15% w/w of CH. The shape of CNP was almost round which was consistent with the study by Dudhani and Kosaraju (2010) whom produced the CNP via ionic gelation between CH and TPP for encapsulation of catechin as drug delivery agent. The nominal particle size of

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CNP was found to be in the range of 60 nm to 110 nm which confirmed the formation of CNP in nanometer size dimension. Meanwhile, Figure 1 (b) shows the TEM image of 15% w/w

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starch/CNP films whereby the black and round particles represent the CNP dispersed in the starch matrix film. It was found that the CNP were well dispersed in the starch matrix and that

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no agglomeration was formed thus produced homogenous starch/CNP film. It is worth noted that well dispersion of CNP in the matrix may result to the good antimicrobial properties.

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Figure 1:

500 nm

200 nm

TEM images of (a) CNP, (b) starch/CNP films (magnification at x50K).

3.2. In-vitro antimicrobial activity: Effect of CNP concentration on the antimicrobial activity of starch/CNP films For in vitro analysis, growth inhibition zones around the starch and starch/CNP films (inhibition halos) and growth inhibition under the films (area of contact with the agar surface) were visually examined. Figure 2 (a) and (b) shows the inhibitory zone of gram-positive bacteria that were B. cereus and S. aureus for neat starch and starch/CNP films. It can be seen from Figure 2 that no inhibition zone can be observed for control neat starch films (without CNP) for both B. cereus and S. aureus. This indicated that the bacteria colonies were able to

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grow under the neat starch films due to the absence of CNP as the antimicrobial agent.

Figure 2:

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5%

Inhibitory zones of neat starch films and starch/CNP films for gram-positive

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bacteria, (a) B. cereus, and (b) S. aureus. Addition of CNP into starch films resulted in the inhibitory zones of both gram-positive

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bacteria under the films as can be seen from Figure 2 (a) and (b). This was due to the binding of CNP to the cell wall of gram-positive bacteria which formed a polymer membrane on the surface of the bacteria cell. According to Antoniou et al. (2015), the formation of polymer membrane around the bacteria could act as a barrier that prevented the essential nutrients and oxygen to diffuse through the membrane of the bacteria cell. Lack of both nutrient and oxygen in bacteria cell slowed down the microbial activities, thus inhibited the microbial growth. It is worth noted that the inhibition zones of starch/CNP films were only observed under the films. No inhibition zone around the films was observed for all the film samples shown in Figure 2 8

which indicates that starch/CNP films only able to inhibit gram-positive bacteria that in direct contact with the film. This was due to the natural character of CNP that is non-volatile thus reduced the efficiency of CNP to diffuse through adjacent agar media and limited the antimicrobial activity of CNP on the agar that was not in direct contact with CNP (Dutta et al., 2009; Rodriguez-Nunez et al., 2012). These findings were consistent with the work of Tripathi et al. (2010) and Li et al. (2006). They reported that the inhibition zone of chitosan/PVA/pectin films and chitosan films against gram-positive bacteria can only be observed under the films. Figure 2 (a) and (b) also demonstrate that increase in the concentration of CNP (0 to 20% w/w) in starch films resulted in the increase of inhibition zone area under the films

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for both gram-positive bacteria. This was due to the high concentration of CNP that could inhibit more bacteria growth compared to the low concentration of CNP, thus led to efficient antimicrobial activity of the CNP. The similar trend of result was reported by Ali et al. (2011) who studied the effect of concentration of CNP on the antimicrobial activity of CNP against gram-positive bacteria for medical textile application. They found that increase in the

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concentration of CNP from 0.01 to 0.04% resulted in the increase in the antimicrobial activity of CNP against S. aureus from 92 to 100%. Study reported by Kowalczyk et al. (2015) revealed

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that blending of CH with other polymers such as starch and gelatin reduced the efficiency of antimicrobial activity of the films compared to the neat CH film. This was due to the decrease

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in the ratio of CH to the other polymers which led to the decrease in the amount of antimicrobial agent. It can also be clearly seen from Figure 2 (a) and (b) that 15% and 20% w/w of CNP were sufficient to inhibit both gram-positive bacteria as clear and full inhibitory zone area were

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observed under both 15% and 20% w/w starch/CNP films. Therefore, 15% w/w of CNP is the minimum concentration of CNP that is required to inhibit gram-positive bacteria. Furthermore, Figure 3 (a) and (b) shows the inhibitory zone of gram-negative bacteria that

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were E. coli and S. typhi of neat starch and starch/CNP films. It can be clearly seen that the control neat starch films show no inhibition zone for both gram-negative bacteria. Surprisingly,

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there was no inhibition zone under the starch/CNP films observed in Figure 3 which indicated that CNP was not able to inhibit the gram-negative bacteria. This result was consistent with the study reported by Hosseini et al. (2016) who investigated the antimicrobial properties of fish gelatin/CNP films. They found that fish gelatin/CNP films did not show inhibitory zone on both S. enteritidis and E. coli due to the formation of strong hydrogen bond between functional groups of fish gelatin (-OH, -NH2 and -COOH) and CNP (-OH and -NH2). Thus, for this study, it can be speculated that the formation of hydrogen bonds between functional groups of the starch film (-OH) and CNP (-NH2) that resulted in slow diffusion of CNP from polymer 9

matrix into the agar. It is worth noted that the natural chaaracteristic of CNP that is non-volatile

Inhibitory zones of neat starch film and starch/CNP films with or without CNP for

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gram-negative bacteria, (a) E. coli, and (b) S. typhi.

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Figure 3:

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also limit the efficiency of antimicrobial activity of CNP against gram-negative bacteria.

Comparing between Figure 2 and Figure 3, CNP was found to be more efficient in inhibiting gram-positive bacteria compared to gram-negative bacteria. According to Antoniou et al.

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(2015), gram-negative bacteria was more resistant to the CNP compared with gram-positive bacteria due to the different modes of antimicrobial activity of CNP against both types of bacteria. CNP (NH3+) tend to form a polymer membrane around the cell wall of gram-positive

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bacteria that is in direct contact with CNP thus preventing nutrient and oxygen supply for metabolic activity of the bacteria. For gram-negative bacteria, CNP will diffuse through the

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cell wall of bacteria and disrupted the cytoplasmic membrane which led to the leakage of bacteria. This leakage damaged the bacteria intracellular components and killed the bacteria

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cells. According to Qi et al. (2004), CNP is more efficient to penetrate through the gramnegative bacteria cell wall compared to bulk CH due to the smaller particle size of CNP. However, strong hydrogen bond of CNP and starch molecules resulted in slow diffusion of CNP from the starch matrix into the agar medium thus limit the efficiency of antimicrobial activity of CNP against gram-negative bacteria (Hosseini et al., 2016). The result of the present study was consistent to Antoniou et al. (2015) who found that Tara gum/CNP films had higher antimicrobial activity against gram-positive bacteria (S. aureus) compared to gram-negative bacteria (E.coli). Another study by Kowalczyk et al. (2015) also 10

reported that the gelatin/chitosan film was more efficient to inhibit gram-positive bacteria (B. cereus) compared to gram-negative bacteria (E. coli). Although the present work shows no inhibition zone for gram-negative bacteria, the colony of the bacteria was seen to decrease with the addition of CNP as can be observed from the color intensity of the bacteria colony under the films. This result revealed that the antimicrobial activity of CNP against gram-negative bacteria still occurr but not as efficient as the gram-positive bacteria.

3.3. In vivo activity: Application of the films as antimicrobial packaging Based on in vitro evaluation result, inhibition zone under 15% w/w starch/CNP film was comparable with that of 20% w/w starch/CNP film whereby both films were able to inhibit gram-positive bacteria.

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Thus, 15% w/w starch/CNP film was chosen to be used in the in vivo evaluation to minimize the usage of the CNP, thus reducing the production cost. In vivo antimicrobial activity of the films was done

on the cherry tomatoes to demonstrate the real application of the films as antimicrobial packaging film. Cherry tomatoes were chosen for in vivo analysis because it is one of the

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favorable vegetables owing to its high nutritional values and great taste. In this work, unwrapped cherry tomatoes (UW), cherry tomatoes wrapped using neat starch film (WST) and

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cherry tomatoes wrapped using 15% w/w starch/CNP film (WCN), were stored in a chiller (temperature: 10 ºC) for 10 days. Total plate count (TPC) of all cherry tomatoes were evaluated

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on day 0, 3, and 10 and the results were tabulated in Figure 4.

100000

ST

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1000

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acceptable contamination limit b

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cfu/g

10000

UW

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g d

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100

10 1

0

3 Days

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Figure 4: TPC of cherry tomatoes on day 0, 3, and 10. Different letters in the same graph indicate a statistical significant difference (P<0.05).

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From Figure 4, it can be seen that TPC of cherry tomatoes on day 0 was 1 x 102 cfu/g and this value served as the reference point for other samples. The TPC of UW, WST, and WCN increased with the increase in the period of storage due to the bacteria growth over time. It was found that the microbial count of UW increased rapidly from 1 x 102 to 1.15 x 104 cfu/g (114.5fold increment) after 10 days period of storage. This was due to the cold and moist condition in the chiller with the availability of nutrients from tomatoes that provide good conditions for bacteria in cherry tomatoes to grow. Furthermore, existence of bacteria in the chiller that could survive in the cold condition such as psychrotrophic bacteria might also contaminated the UW tomatoes, thus increased the microbial count of UW (Djioua et al., 2010).

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Figure 4 also shows that the microbial growth of WST was lower than UW due to the role of the neat starch film as a barrier that could protect the tomatoes from the surrounding contamination.The microbial count of WST increased from 1 x 102 to 2.15 x 103 cfu/g (20.5fold increment) after 10 days period of storage which was still in the range of of ready to eat food that is below than 1 x 104 cfu/g (FSAI, 2016; NSW Food Authority, 2009). Addition of

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15% w/w CNP into starch film resulted in the significant improvement of the film against microbial growth. It was found that the microbial count for WCN was only increased from 1 x

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102 to 7 x 102 cfu/g (0.86-fold increment) after 10 days period of storage. Starch/CNP films were found to be more effective to inhibit the bacteria growth compared to the neat starch film

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due to the presence of CNP in the starch. Low microbial count of tomato that wrapped in starch/CNP film was due to the presence of NH3+ ion that formed a polymer membrane on the surface of the bacteria cell which led to the death of bacteria This result is consistent with the

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work by Vasconez et al. (2009) which reported that edible coating solution which consists of tapioca starch and CH was able to reduce the yeast population (Z. Bailii) on the salmon fillet throughout the 6 days storage period.

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It is worth noted that low microbial growth in tomato that packaged in starch/CNP film was also attributed to the tiny size of CNP that was incorporated in the starch film which was more

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efficient to inhibit microbial growth compared to bulk CH. Pilon et al. (2015) have demonstrated the application of CNP as edible coating solution on the fresh-cut apple slice. They investigated the effect of particle size of CNP (110 to 300 nm) and bulk CH on the antimicrobial activity of coated apple slices. They found that apple slices coated with 110 nm CNP solution resulted in lower microbial count compared to apple slices coated with 300 nm CNP solution and bulk CH solution. They reported that tiny size of CNP increased the surface interaction to microbial cells, thus increased the efficiency of antimicrobial activity againts bacteria growth (Pilon et al., 2015). Large surface area of CNP that in direct contact with the 12

apple slices increased the efficiency of NH3+ ion from CNP to form a polymer membrane on the surface of the bacteria cell which led to the death of microbes. 4. Conclusion The starch/CNP films were successfully produced and applied as antimicrobial food packaging on the cherry tomatoes. CNP was synthesized at different concentration of CH (5 to 20% w/w) via ionic gelation process and incorporated into the starch films to produce starch/CNP films. Morphological properties of both CNP and starch/CNP films were observed using TEM which revealed that the nominal particle size of CNP was in the range between 60 to 110 nm and was dispersed well in the starch film. In-vitro evaluation of antimicrobial activity

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of starch/CNP films found that CNP was more efficient to inhibit gram-positive bacteria compared to gram-negative bacteria. It was found that addition of 15% w/w of CNP into the starch film was sufficient to inhibit the gram-positive bacteria, thus it was chosen for the invivo evaluation. Starch/CNP film was found to exhibit the highest efficiency of antimicrobial

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activity due to the presence of CNP. In conclusion, starch/CNP films produced in this study is

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promising to be used as antimicrobial packaging material.

Acknowledgement We acknowledge the financial support provided by Putra Grant-Putra Graduate Initiative

of interests in this work.

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(IPS), Universiti Putra Malaysia, Malaysia (Vote no. 9573800). The authors declare no conflict

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