APPLICATION OF ZINC-OXIDE NANOPARTICLES BASED ANTIMICROBIAL FILM TO INHIBIT THE GROWTH OF ESCHERICHIA COLI AND SALMONELLA SPP. IN DEBONED CHICKEN MEAT - Unika Repository

APPLICATION OF ZINC-OXIDE NANOPARTICLES BASED ANTIMICROBIAL FILM TO INHIBIT THE GROWTH OF ESCHERICHIA COLI AND

PRACTICAL TRAINING REPORT

This practical training report is submitted for the partial requirement for

Bachelor Degree

By:

ANGELA NOVITA

15.I1.0063

DEPARTMENT OF FOOD TECHNOLOGY

FACULTY OF AGRICULTURAL TECHNOLOGY

SOEGIJAPRANATA CATHOLIC UNIVERSITY

SEMARANG

2018

APPLICATION OF ZINC-OXIDE NANOPARTICLES BASED

ANTIMICROBIAL FILM TO INHIBIT THE GROWTH OF

ESCHERICHIA COLI AND SALMONELLA SPP. IN DEBONED

CHICKEN MEAT

Practical Training at Fu Jen Catholic University, New Taipei, Taiwan

By:

ANGELA NOVITA

Student ID : 15.I1.0063

Faculty: Agricultural Technology

Semarang, May 14th 2018 Department of Food Technology

Faculty of Agricultural Technology Soegijapranata Catholic University

Practical Training Advisor I Practical Training Advisor II Dr. Shaun Chen Stefani Amanda Harmani, S.TP, M.Sc.

NPP: 0581 2017 318

Dean

Dr. R. Probo Y. Nugrahedi S.TP, M.Sc

NPP: 0581 2001 244 ii

PREFACE

Praise in the name of Jesus Christ, because only by His grace and blessing, the author would have the opportunity to undergo the practical training and finish the report smoothly. This report is the complete accountability from the practical training which was done in Taipei, Taiwan that took place from 4

of January until 4

th of March, 2018.

During the training the author did the research entitled: “Application of Zinc-Oxide

Nanoparticles Based Antimicrobial Film to Inhibit the Growth of Escherichia coli and Salmonella spp. in Deboned Chicken Meat”. This report was written as a requirement to acquire Bachelor Degree of Food Technology. The author would not be able to finish these tasks alone, and only by huge support and guidance given by the great and very helpful people around the author these report could be finished. Special thanks for:

1. Almighty God that always blessed, saved and guided author in every step of practical training in Taiwan.

2. R. Probo Y. Nugrahedi, M.Sc. as dean of faculty of agricultural, Soegijapranata Catholic University, for giving me the opportunity to join the internship program.

3. Dr. Shaun Chen as my advisor who advised and supported me all the time when I did this research.

4. Stefani Amanda S.TP, M.Sc. for helping me and advising me to make this practical training report better.

5. Huang zi yu (Joanne), a master student in Fu Jen University, who was always helping me done my research and whom without her I could not finish my research.

6. My family, Mom, Dad, and Brother who always supported me and cheered for me everyday.

7. My dearest friends Eileen Nathania, Fanny Margareta Phoa, Christopher Hendra, and Evan Fajar who always supported and cheered me for the thing I had gone through.

8. All of my dearest friends in Taiwan, Donna, Dave, Giant, Maurine, Theresia who made my times while in Taiwan were such an unforgettable moments. iii 9. Last but not least, I would like to thanks to all my beloved friends from EP 308,

Wen yu xian, Wu yi ru, Cheng ya wen, Zeng bo xuan, Lin jie yin and all others friends who I can’t say it one by one that always supported me and accompanied me. The author realized that this report is still far from perfect and there are still many shortcomings due to the limitation of the author. However, the author hope that this report can still be an inspiration and provide useful information for all the readers.

Semarang, May 14th 2018 Angela Novita

Author

TABLE OF CONTENT

ENDORSEMENT SHEET................................................................................................i PREFACE.........................................................................................................................ii TABLE OF CONTENTS................................................................................................ iv LIST OF TABLES...........................................................................................................vi LIST OF FIGURES.........................................................................................................vii

LIST OF TABLES

vii

LIST OF FIGURES

viii

1. INTRODUCTION

1.1 Background

Novel food technology is developing rapidly in this modern era and people currently expect something more than delicious and nutritious. However, there is also a rapid growing of consumer s’ interest in consumption of products with secured shelf life and controlled quality. Food safety also become one of the major concern all over the world, and consumers recognize the necessity to buy a safer food product. Due to those reasons, food manufacturers are looking for solutions to fulfill every progressive and rapidly changing demands of consumers. Therefore, many researches focusing on active food packaging have been accomplished and those developments effectively lead to longer shelf life and good quality. Nowadays, packaging is an essential process in modern trade goods, because it can guarantee preserving the quality of food products, protecting packed products against external conditions, providing safety of food products, and making storage, transportation, and distribution of products easier.

On awareness of consumer demands, Department of Food Technology at Soegijapranata Catholic University sets up a training program for students to improve their knowledge and research skill. Experiences in food technology could be gained not only by curricular process in university, but also with a working experience in research laboratory. In this program, students are given an opportunity to either join a food industry and work in that company or to take a part of in-house training as part of research group in other universities. While the students do a field work, either in a company or in other universities, they will experience the real practice of food industry and gain more knowledge about how food researchs actually conduct in the industry. This experience will be one thing that students need once they graduate and have to work in the food industrial field.

For this project, the in-house training are chosen. Students who choose an in-house training have to go to the selected universities which will be their facilitator of the research. The department of food science of Fu Jen Catholic University in Taiwan is one of the selected universities as the facilitator for research. Through this program, students will be offered an opportunity to conduct their research abroad, learn different analytical instruments and also to experience the cultural diversity. This program can be held because there is a student

2 exchange mutual agreement between Soegijapranata Catholic University and Fu Jen Catholic University.

The title of the present project is “Application of Zinc-Oxide Nanoparticles Based

Antimicrobial Film to Inhibit the Growth of Escherichia coli and Salmonella spp. in Deboned Chicken Meat”. The advisor of this research is Dr. Shaun Chen, an Associate Professor of Food Science Department, Fu Jen Catholic University, Taiwan. The supervisor of this study is Joanne Huang, a graduate student of Departement of Food Science, Fu Jen Catholic University, Taiwan.

1.2 Purpose of Practical Training a.

To give an experience on how a food science research are conducted abroad with new environment.

To improve and broaden knowledge and experience that could be usefull in the real industrial or scientific field in the future.

To give an opportunity to learn how to adapt in new circumstances and society in other country with different culture.

To give an opportunity to meet new friends and build an international network.

1.3 Time and Place of Practical Training

The practical training was conducted in the Faculty of Food Science, Fu Jen Catholic

th th University, New Taipei City, Taiwan, and took place between 4 January to 4 March 2018.

Figure 1. Map of Fu Jen University, New Taipei City, Taiwan

3 The location of Fu Jen Catholic University is located in 242, New Taipei City, Xinzhuang District, 中正路510號, Taiwan (Telephone : +886 2905 2000 ).

2. INSTITUTION PROFILE

2.1 Fu Jen Catholic University

Fu Jen Catholic University is the first Catholic university in China established by the Catholic Church and operates under the auspices of the Holy See and the Ministry of Education Republic of China, Taiwan. Fu Jen Catholic University is a comprehensive, pontifical university built in Beijing in 1925 and was rebuilt in Taiwan in 1961. In 1925, Catholic University was founded impeaching by the Catholic church by the Benedictines of St. Vincent Archabbey in the USA. In 1927, the Beijing government approved the trial run and the name officially changed to Fu Jen Catholic University. Moved by the Christian understanding of love and inspired by the high ideals of Confucian education, it adopted the name "Fu Jen", meaning assistance and benevolence, to give expression to its universal vision and mission realized through holistic education in the Chinese cultural context. Fu Jen Catholic University has a history of more than 92 years and has provide the country with well-educated students characterized by integrated physical, social, intellectual, aesthetic, moral and spiritual development. The University also hopes to serve society through various additional academic programs and community services. Aided by extensive scientific research, Fu Jen is committed to the pursuit of truth and the integration of Western and Chinese cultural values so as to promote the well-being of the human family and strengthen world solidarity.

For the formation of students, the University supports a well-balanced division between general education and professional training with a special emphasis on humanistic discipline, which helps students foster lofty sentiments and enrich their lives when they start their careers after graduation. Moreover, to stimulate international academic exchange and collaboration, and to promote cultural dialogue, the University has established sister-school relationships with renowned universities worldwide. Fu Jen comprises 11 colleges (Liberal Arts, Education, Communication, Arts, Medicine, Science and Engineering, Foreign Languages, Human Ecology, Law, Management, Social Sciences), offering 48 undergraduate program s, 47 master’s programs and 11 doctoral programs. Fu Jen has 26,000 students and over 160 student associations and clubs. The university has about 120 sister schools worldwide and strives to provide students with a diversified, holistic, interdisciplinary, and international learning environment.

5 Currently, Fu Jen provides 11 colleges (Liberal Arts, Education, Communication, Arts, Medicine, Science and Engineering, Foreign Languages, Human Ecology, Law, Management, Social Sciences) with 48 departments, 47 master programs, 23 in-service master programs, 11 doctoral programs, and also 16 departments in School of Continuing Education. There are seven goals of Fu Jen Catholic University, such as human dignity, the meaning of life, academic research, community awareness, dialogue with cultures, religious cooperation, and spirit of service.

Figure 2. Logo of Fu Jen University

2.2 Faculty of Food Science

The Department of Family Studies and Nutrition Sciences was established in 1963. The department was grouped into two sections, Family Studies section and Nutrition Sciences section. Nutrition Sciences was merged with the Food Sciences section as the Department of Nutrition and Food Sciences in 1971. The Graduate Institute of Nutrition and Food Sciences was established and started to offer a master’s degree program in 1983. The doctoral program was joined to the Institute in 1995. Food Sciences section became an individual department in 2006. The De partment of Food Science offers Bachelor’s degree program and Master’s degree program.

2.3 Mission of Faculty

Uphold the spirit of pursuing truth, goodness, beauty and holiness, the Department of Food Science at the Fu Jen Catholic University integrates basic sciences with latest technology for excellence in education, research, and service. The Department of Food Science at the Fu Jen Catholic University are committed to promote the healthier, tastier and safer foods for improving eating quality, human health and wellness.

2.4 Faculty Member

Figure 3. was the description of the member of Food Science Faculty in Fu Jen University, Taiwan.

Director of Human Ecology

Professor Bing-Hui Chen

Director of Food Science

Assistant Professor Tsung-Yu Tsai Professor Chiwei P. Chiu Professor John Tung Chien Food Enzymology Lab. Food Physicochemistry Lab.

Associate Professor Jung-Feng Hsieh Associate Professor Meng I-Marie Kuo Associate Professor Rey-May Huang Associate Professor Shaun-Chen Food Microbiology Lab. Nutraceuticals & Food Processing Lab.

Assistant Professor Bang-Yuan Chen Assistant Professor Tsai-Hua Kao Food Biochemistry Lab.

Assistant Professor Chun-Ping Lu Figure 3. Faculty Member of Food Science, Fu Jen University, Taiwan

3. RESEARCH PROJECT

3.1 Research Overview

The topic of this research is “Application of Zinc-Oxide Nanoparticles Based Antimicrobial Film to Inhibit the Growth of Escherichia coli and Salmonella spp. in Deboned Chicken Meat”. The objective of this project was to develop an antimicrobial film immobilizing zinc- oxide nanoparticles, and then applied it to inhibit the growth of Escherichia coli and

Salmonella spp. on deboned chicken meat. Antimicrobial Films were prepared using 4

different concentrations of ZnO nanoparticles, including 0 ; 0.3 ; 0.5 ; 0.7 g in 100 g alginate gel. The next step was the analysis for the antimicrobial films, which were divided into 2 analysis, first was to determine the physical properties and the second was to determine the antimicrobial activities. Tensile strength and elongation at break tests were achieved to measure the physical properties of antimicrobial film. Agar diffusion method was used to evaluate the antimicrobial effects against the food pathogen. Bacterial strains used in this study were Escherichia coli and Salmonella spp. After the antimicrobial effects were determined, the antimicrobial films were applied to deboned chicken product which were

inoculated with activated culture with an approximately concentration of 10 - 10 CFU/mL, which is a high enough concentration to show a noticeable decline in the case of inhibitory action, but low enough to increase noticeably in the absence of inhibitory activity. After 3 days of incubation, the colonies of Escherichia coli and Salmonella spp. were counted manually and all the results were subjected to statistic analysis.

3.2 Background of Research

Nowadays, people demand food products which are minimally processed, easy to prepare, and ready to eat. Those demands lead to pose major challenges in terms of safety and quality. One of essential technology to maintain food quality and safety is food packaging, thus, food manufacturers look forward to providing novel and active packaging to extend shelf life and provide better quality, due to the growing of consumer interest in consumption of safer and tastier food products.

Microbial contamination on food products is a serious issue that can increase the risk of foodborne illness and reduce the shelf life of food products (Quintavalla & Vicini, 2002). Outbreaks cause by the foodborne pathogens such as Escherichia coli and Salmonella spp.

8 continue to draw public attention related to food safety issues. Animal products, including raw meat, poultry, fish are more risky for consumers because those are most likely to contain pathogen. There has been a significant increase in the consumption of poultry and its processed products since the 1960s (Capita et al., 2002). Because of the increase of poultry consumption, the microbial safety of those food products becomes more important for producers, public health officials, especially consumers. There are several different microorganisms in living poultry animals such as Escherichia coli and Salmonella spp. Typically, those microorganisms grow on poultry skin, feathers, and in alimentary tract (Kozacinski et al., 2006). Therefore, contamination of poultry carcasses during slaughtering and processing occurs easily during the change of live animals to meat for human consumption.

Escherichia coli is an organism that is part of the normal microflora in the intestinal tract of

human and warm-blooded animals (NSW Food Authority, 2009). The risk of the food contaminated by Escherichia coli can cause many problems to human health, such as enteritis and several extraintestinal diseases (e.g wound infections), mastitis, urogenital infections, septicaemia and meningitis (Johnsen et al., 2001). Salmonella spp. are the most common causes of human foodborne diseases linked to poultry (Hafez, 2005). Gastrointestinal symptoms of salmonellosis can develop from few as 15 to 20 cells after ingesting, and the symptomps include nausea, vomiting, abdominal cramps, diarrhea, and headaches (U.S. FDA, 1992) Therefore, development of new antimicrobial is needed to ensure food safety and extend shelf life. Antimicrobial packaging is one of the effective methods in maintaining the safety and quality of food products during storage. The antimicrobial agent in this packaging will migrate slowly to the surface of the food product from the packaging system, that leads to maintain desirable safety and quality of food products. Based on the antimicrobial capacity, development of antimicrobial films to protect fresh and processed foods against pathogens and extend the shelf life of foods is becoming the new trend in food safety research and one of the potential antimicrobial agent is Zinc-Oxide (Du et al., 2009).

The objectives of this research were to develop an antimicrobial film immobilizing zinc- oxide nanoparticles and to investigate the effect of different concentrations of zinc oxide

9 nanoparticles on antimicrobial activity and mechanical properties of zinc-oxide antimicrobial film. The zinc-oxide antimicrobial film then applied on deboned chicken meat to evaluate the effectiveness on inhibiting the growth of Escherichia coli and Salmonella spp.

3.3 Literature Review

3.3.1 Alginic Acid for Development of Antimicrobial Film

Alginic acid is a polysaccharide which naturally contains carboxyl groups in constituent residue, therefore this material has various abilities for preparation of functional materials. Since alginic acid has high reproducibility and availability as a natural resource, this material can be used as a source of biodegradable or edible films (Lazarus, West, Oblinger, and Palmer, 1976). Furthermore, edible films prepared from alginate form a strong films, but exhibit poor water resistance due to their hydrophilic nature (Guilbert, 1986; Kester & Fennema, 1986). Alginate is able to react with polyvalent metal cations, especially calcium ions to produce strong gels or insoluble polymers (King, 1983 in Rhim, 2004).

Edible film or edible coating is able to carry some food additives, such as antimicrobials, antioxidants, colorants, flavors, and spices (Han, 2001 in Pranoto et al., 2005). The incorporation of antimicrobial agents into edible film or edible coating localizes the functional effect at the food surface. The antimicrobial agents are not directly released to the food surface, thus, they remain at high concentrations for extended periods of time (Ouattara

et al., 2000 in Pranoto et al., 2005). Antimicrobial biodegradable films demonstrate an

effective way to inhibit the growth of food-borne pathogens and spoilage microorganisms, therefore it is beneficial to preserve food safety and prevent product spoilage (Du et al., 2009). In addition, the use of biodegradable packaging also contributes to reduce the municipal solid waste problem.

3.3.2 Zinc Oxide (ZnO) Nanoparticles

Zinc has a wide array of vital physiological functions. In human body, the majority of dietary zinc is absorbed in the upper small intestine. The contents of luminal of the duodenum and jejunum, especially phytate, have a major effect on the percentage of absorption of available zinc. Albumin is the major transporter of zinc in both portal and systemic circulation. A Maximal Tolerable Daily Intake (MTDI) of zinc is 30 mg zinc/day or 0.5 mg/kg/day

10 established by Health Council of the Netherlands (1998) in agreement with the recommendation of the Commission of the European Communities (1993).

The innovation of nanotechnology, which refers to Roco (1999) is the manufacture and use of materials with size of up to about 100 nm in one or more dimensions, has created great chances for the development of new materials as antimicrobial agents. In the study of Rai et

al. (2009), results demonstrated that inorganic compounds in nanosize impart strong

antibacterial activity at low concentrations because of their high surface area to volume ratio and different chemical and physical properties. Additionally, they are more stable in extreme conditions such as high pressures and temperature, considered non-toxic and some even contain mineral elements which are essential to the human body (Roselli et al., 2003). According to Bradley et al., (2011), most antibacterial inorganic materials are metallic nanoparticles and metal oxide nanoparticles, such as zinc oxide (ZnO). Currently, ZnO is one of the five zinc compounds that are listed as a generally recognized as safe (GRAS) material by the U.S. Food and Drug Administration (21CFR182.8991) (FDA, 2011). Furthermore, nanosized ZnO particles now are widely used as a functional inorganic material for coating in many applications.

Refers to Applerot et al. (2009) Escherichia coli has shown higher susceptibility toward ZnO nanoparticles compared to Staphylococcus aureus. The higher resistance shown by

Staphylococcus aureus can be explained because the differences of intracellular antioxidant

content between these two bacteria, such as carotenoid pigments in the interior of S. Aureus, which results in a greater oxidant resistance as well as the presence of potent detoxification agent such as antioxidant enzymes, particularly catalase. The functional activity of nanoparticles is most likely influenced by their size, therefore the antimicrobial activity of ZnO has been improved with a diminution of particle size (Zhang et al., 2007). This can be explained due to an increase in the surface area/volume ratio, which results in the increased reactivity of ZnO surface in nanometer size, since H

2 O 2 generation depends strongly on ZnO

surface area (Ohira et al., 2008 in Espitia et al., 2012). Therefore, a larger surface area will

result in more ROS (Reactive Oxygen Species), especially H O compounds on the surface of ZnO, thus smaller nanoparticles results in a greater antibacterial activity.

11 According to Wang et al., (2009) in Espitia et al., (2012), the toxicity of ZnO nanoparticles is

due to the solubility of Zn ions in the medium containing the microorganisms, however, the solubility of ZnO depends of their concentration and time. Thus, low concentrations of

solubilized Zn can trigger a relatively high tolerance by the microorganism. Intrinsic factor

of each microorganism could affect the metabolic processes of Zn ions, therefore differences in toxicity thresholds of ZnO nanoparticles in various microorganisms could be observed. When studying the effect of ZnO against E. coli at low concentrations, ZnO nanoparticles may actually increase bacterial growth. According to results reported by Padmavathy and Vijayaraghavan (2008) in Espitia et al., (2012), ZnO nanoparticle suspensions in lower concentration (0,01

– 1 mM) seem to have less antimicrobial activity

against E. coli and the presence of soluble Zn ions may act as nutrients for this microorganism.

3.3.3 Escherichia coli

Enterohemorrhagic bacteria, Eschericia coli (EHEC) 0157:H7 is one of the most important foodborne pathogens in food industry and has resulted in a large number of highly publicized and expensive recalls (Al-Qadiri et al., 2006 in Liu et al., 2009). EHEC 0157:H7 can survive in acidic foods and its infective dose is as low as 10-100 cells. Outbreaks owing to this foodborne pathogen have increased in recent years. Foods of various origins, including spinach, lettuce, mayonnaise, raw milk, undercooked ground beef and roast beef were implicated in illnesses and outbreaks caused by E. coli O157:H7 (Smith and Fratamico, 2005 in Liu et al., 2009). The illness caused by E. coli O157:H7 can lead to inflammation of the colon and gives rise to diarrhea and abdominal pain with bloody stools (Al-Holy et al.

2006 in Liu et al., 2009).

3.3.4 Salmonella spp.

Salmonella spp. is one of enteric pathogen in related with animal and slaughter hygiene. Meat

is one of implicated sources of human salmonellosis (EFSA, 2008 in Montserrat & Yosep, 2010). The two most common Salmonella serotypes are Thyphimurium and Enteriditis. It has been observed that Salmonella spp. usually survived during chilling. Human salmonellosis infections can lead to uncomplicated enterocolitis and thypoid fever, then lead to a serious disease involve diarrhea, headache, fever, and abdominal pain. Salmonella spp. can also cause systemic infections, lead to chronic reactive athritis (Echeita et al., 1999 in Montserrat

12 & Yosep, 2010). Salmonella serotypes have been identified to be multidrug resistant, with the spectrum of antibiotic resistance still increasing, and this high level of resistance could explain their rapid growth among poultry.

4. RESEARCH METHODOLOGY

4.1 Experimental Design

7 CFU/ml

–10
–10

Microbial Growth Analysis after 72 Hours Incubation at 7

2 CFU/ml

Inoculation of Salmonella spp. culture onto a Sterile Deboned Chicken Meat with Initial Number of Bacteria was

Application of Zinc Oxide Based Antimicrobial Film on Deboned Chicken Meat Inoculation of Escherichia coli culture onto a Sterile Deboned Chicken Meat with Initial Number of Bacteria was

Figure 4. Flowchart of Research Design Antimicrobial Film with 0 g ; 0.3 g ; 0.5 g ; and 0.7 g Zinc-Oxide (ZnO)

7 CFU/ml

Inoculation of Salmonella spp. culture onto a Nutrient Agar Plate with Initial Number of Bacteria was in the Range of 10

Inoculation of Escherichia coli culture onto a Nutrient Agar Plate with Initial Number of Bacteria was in the Range of 10

Determination of Antimicrobial Activity by Inhibitory Zone Analysis After 24 Hour Incubation

Determination of Elongation at Break (E)

Determination of Tensile Strength (TS)

Nanoparticles Determination of Mechanical Properties

2 CFU/ml

4.2 Preparation of Zinc-Oxide (ZnO) Based Antimicrobial Film

Dry ZnO nanoparticles from Sigma-Aldrich with primary sizes < 100 nm were prepared. A preset amounts of dry ZnO nanoparticles, including 0.3 ; 0.5 ; 0.7 g were mixed with 45 mL distilled water in a glass beaker. The glass beaker was placed in an ultrasonicator (Delta Ultrasonic Cleaner D150 H) for 60 minutes. After that, 5 mL of polyethylene glycol (PEG 400) as dispersant was used to improve the stability of the suspension (Xihong Li et al., 2009). The glass beaker was placed again in an ultrasonicator for 180 minutes. A solution of alginic acid sodium salt was then prepared, by mixing 1 g of alginic acid sodium salt powder with 50 mL distilled water in a glass beaker and stirred using a magnetic stirrer in a hotplate at 240 rpm until all the chemicals were dissolved. The ZnO nanofluid was then mixed with the alginic acid sodium salt solution, and placed in an ultrasonicator for 60 minutes. The mixed solution of ZnO nanofluid and alginic acid sodium salt was taken 40 mL and poured into a 15 cm diameter glass petridisc. The petridiscs were placed on a hotplate and heated at

o o

100 C for 1 hour and placed in an oven at 65 C until the solution dried and turned into a sheet of film. Dried film was soaked into 2% of CaCl

2 solution for 2 minutes to prevent curling of

the films during drying (Rhim, 2004) and then dried again at room temperature. Film made by mixing 2 g of alginic acid sodium salt alone with 100 mL distilled water was used as a control.

4.3 Determination of Mechanical Properties of Zinc Oxide Based Antimicrobial Film

Tensile Strength (TS) and elongation at break (E) were evaluated using a Texture Analyzer (Lotun Science Co. Ltd) according to the ASTM standard methods (ASTM D882 and ASTM D6287). Pre-conditioned films cut into 2.5 cm (W) x 7.2 cm (L) strips and mounted between the grips of the machine. Sample was pulled until break at a cross head speed of 1 mm/second and trigger force of 5 kg. TS and E were calculated using the following relationship:

ℎ ( ) = ( ℎ ℎ)

(%) = 100

The results of TS were expressed by MPa and the results of E were expressed by percentage (%). TS and E measurements for each type of film were replicated three times.

4.4 Determination of Antimicrobial Properties of Zinc Oxide Based Antimicrobial Film

4.4.1 Preparation of Mueller Hinton II Agar (MHA) for Agar Diffusion Method

Nutrient agar plates were prepared by adding 38 g MHA powder with 1 L distilled water in a glass beaker and shaked until completely dissolved. After that, MHA solution were sterilized

(121 C, 15 min), and then poured into petridishes asseptically.

4.4.2 Culturing Escherichia coli and Salmonella sp.

Stock cell cultures were activated by taking one colony of each bacteria into a 9 mL buffered peptone water and vortexed to homogenized the bacterial culture. The cell cultures were then inoculated onto a nutrient agar plate and incubated for 24 hours in an agitating incubator.

4.4.3 Analysis of Inhibitory Zone

The agar diffusion method was used for determination of antibacterial effects of ZnO nanoparticles based films on bacterial strains. The films with different nano ZnO loaded were cut into 15 mm diameter disks and then placed on agar plates which had been seeded with 0.1

mL of activated cell culture. Initial number of bacteria was in the range of 10 CFU/ml,

–10 and the diameters of inhibitory zone on agar plates after 24 hour incubation were analyzed.

4.5 Application of Zinc Oxide Based Antimicrobial Film to Deboned Chicken Meat

4.5.1 Preparation of Tryptic Soy Agar (TSA) for Bacteria Growth

The TSA powder (38 g) were initially mixed with 1 L distilled water in a glass beaker and

shaked until completely dissolved. After that, TSA solution were sterilized (121

C, 15 min) then poured into petridishes asseptically.

4.5.2 Culturing Escherichia coli and Salmonella spp.

4.5.3 Application of Antimicrobial Film to Deboned Chicken Meat

Deboned chicken meat was purchased from local supermarket and stored at refrigeration temperature prior to antimicrobial tests. The deboned chicken meat was cut into small pieces (4 x 4 x 1 cm) and sterilized with 90% (v/v) ethanol irradiated with UV for 30 minutes. After

16 that, the sterilized meats were inoculated with 0,1 ml bacterial culture with initial number of

the bacteria was 10 CFU/ml and wrapped with films (ZnO incorporated and/or control), then

o stored at temperature 7 C for 72 hours.

4.5.4 Microbial Growth Analysis

After 72 hours incubation, the wrapping films from on meat samples were removed and the meat samples were mixed and washed with 20 mL peptone water for about 1 minute. After that, 1 mL peptone water used for washing the meat samples were taken using 1000 µL

micropipet (Socorex Acura 825) following placed in 9 mL peptone water to reach a 10

-3

dilution. The dilution was continued until 10 dilution solution was reached. An aliquot 0,1 mL of each dilution was taken, and placed onto a TSA plates, and spreaded on the agar surface with a sterile hockey stick. The dillution samples were taken duplicate. The samples

were incubated in the incubator at 37 C for 24 hours. Finally, Escherichia coli and

Salmonella spp. colonies were counted manually and expressed as colony-forming units per

mililiter. For computation, total colony per plate was divided by dillution factor and it is expressed as CFU/mL.

1 =

4.6 Statistical Analysis

Results of physical properties and microbial growth analysis were analyzed using one-way analysis of variance (ANOVA). The significant differences between treatment means were determined using Duncan Test New Multiple Range Test (DNMRT) at 95% confidence level. The statistical software used was IBM SPSS Statistics 20.

5. RESULTS AND DISCUSSION

5.1 Zinc Oxide Based Antimicrobial Film

Figure 5. shows the results of control antimicrobial film and ZnO based antimicrobial film with 3 different concentrations, including 0.3 ; 0.5 ; and 0.7 gs of ZnO. As expected, alginate films without ZnO (control) were transparent and pliable, conversely, the antimicrobial films in the present of ZnO were not translucent, that a milk white tinted color was observed (Rhim, 2004).

Control 0.3 g ZnO 0.5 g ZnO 0.7 g ZnO Figure 5. Zinc Oxide Based Antimicrobial Film

5.2 Mechanical Properties

Tensile strength (TS) and elongation at break (E) are the important mechanical properties in almost every packaging applications. Tensile strength is measured for film strength during stretching and elongation at break is the stretch ability prior to breakage (Krochta & Johnson 1997 in Pranoto, Salokhe, & Rakshit, 2005). Different TS and E values were observed with respect to ZnO contents, although all the films have the same duration and same concentration of CaCl

2 soaking treatment. CaCl 2 is a salts with multivalent cations which

increase the gel strength due to the development of cross-linking between carboxyl group of

alginate and Ca . Therefore, the difference of TS and E values between the film were caused by the different composition of alginic acid and ZnO nanoparticles. Control film showed the greatest tensile strength (29,48 ± 2,61 MPa) among the test films, as reported previously (Rhim, 2004). The greatest TS in control film was regarded as 2 g of alginic acid used, compared to 1 g of alginic acid in the antimicrobial films. As the alginic acids contents were reduced, the strength of films were also decreased. This is due to the fact that alginic acid is a biopolymer and has colloidal properties, including stabilizing, thickening, film forming,

18 (Rhim, 2004). As the concentration of ZnO nanoparticles increased, the TS value decreased because of the presence of ZnO nanoparticles. The presence of ZnO nanoparticles in the films probably interferes with ionic interactions between Ca ions and alginate, which were supposed to help in forming a network, thus, cause a loss of TS values (Pranoto et al., 2005).

Generally, as TS value increases, the elongation at break (E) value decreases as shown in the Table 1. The E values between control, 0.3 g and 0.5 g ZnO containing films were not significantly different, while film with 0.7 g ZnO showed the highest percentage of E value and significantly greater than others. From those results, TS and E inversely proportional to each other, whereas the TS value increases, the E value decreases, and the interaction are shown in Figure 5-6. The present results agree with previous study by Lee, Shim, & Lee (2004). As explained previously, tensile strength (TS) is measured for film strength during stretching and elongation at break (E) is the stretch ability prior to breakage (Krochta & Johnson 1997 in Pranoto, Salokhe, & Rakshit, 2005). Therefore, if the TS value increases, thus the film strength during stretching is increasing. As a result of the increases of film strength, the stretch ability of the film prior to breakage is decreasing. Thus, it can be explained why TS value and E value inversely proportional. Decrease of TS value and increase of E value, resulting in more flexible and less brittle films. Films with such properties are beneficial as a biodegradable films to provide food protection and preservation during storage, furthermore to provide alleviation of enviromental pollution (Lee, Shim, & Lee, 2004).

Table 1. Effects of ZnO Concentration on Physical Properties of Antimicrobial Films Elongation at Break (%)

Concentration of ZnO (g) Tensile Strength (Mpa)

a a

1.46 ± 0.004 Control 29.48 ± 2.61

a c

2.19 ± 0.004 0.3 5.90 ± 2.02

a b

1.52 ± 0.004 0.5 10.09 ± 2.32

b c

3.22 ± 0.008 _a,b,c 0.7 2.64 ± 0.23 Within the same column, values not followed by the same superscript are significantly different (P < 0.05).

Control is film without zinc oxide.

19 Figure 6. Results of Tensile Strength

Figure 7.Results of Elongation at Break

5.3 Inhibitory Zone

5.4 Results of Escherichia coli

After 24 hours incubation, the effects of different ZnO concentrations on growth inhibition of

Escherichia coli were accomplished and the control film did not show effective antibacterial

properties. As seen on the figures, the control film did not have an effective antibacterial property against Escherichia coli as illustrated in Figure 7-10. The results of antimicrobial films containing nano ZnO were not as expected, where clear zones could not be determined. The results could be due to the insufficient diffuse of antimicrobial agents through agar gel.

0,00 5,00 10,00 15,00 20,00 25,00 30,00 35,00

0,00 0,20 0,40 0,60 0,80 Ten si le S tr e n g th ZnO Concentration (g)

Tensile Strength

Tensile Strength

0,00% 0,50% 1,00% 1,50% 2,00% 2,50% 3,00% 3,50%

0,2 0,4 0,6 0,8 E lo n g ation at B re ak ZnO Concentration (g)

Elongation at Break (%)

Elongation

20 Furthermore, as the amount of ZnO nanoparticles increased, the bacteriostatic action of the antimicrobial films were expected to increase too, thus increasing the diameter of the inhibitory zone (Hosseini, Razavi, & Mousavi, 2009). The ZnO based antimicrobial films did not show any inhibitory zone despite the antimicrobial activity of ZnO, this might be due to the ZnO agents did not diffused through the adjacent agar media during the agar diffusion test method, thus the organisms did not go through any direct contact with the active sites of ZnO and the antimicrobial agents could not inhibit the microorganism surrounding the film strips (Hosseini et al., 2009).

10 CFU/mL

APPLICATION OF ZINC-OXIDE NANOPARTICLES BASED ANTIMICROBIAL FILM TO INHIBIT THE GROWTH OF ESCHERICHIA COLI AND SALMONELLA SPP. IN DEBONED CHICKEN MEAT - Unika Repository