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Food Control 39 (2014) 214e221

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Food Control

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Control of bacteria growth on ready-to-eat beef loaves byantimicrobial plastic packaging incorporated with garlic oil

Suet-Yen Sung a, Lee Tin Sin a,*, Tiam-Ting Tee a, Soo-Tueen Bee a, A.R. Rahmat b,W.A.W.A. Rahman b

aDepartment of Chemical Engineering, Faculty of Engineering and Science, Universiti Tunku Abdul Rahman, Jalan Genting Kelang, 53300 Setapak, KualaLumpur, MalaysiabDepartment of Polymer Engineering, Faculty of Chemical Engineering, Universiti Teknologi Malaysia, 81310 UTM Skudai, Johor, Malaysia

a r t i c l e i n f o

Article history:Received 6 September 2013Received in revised form3 November 2013Accepted 9 November 2013

Keywords:Antimicrobial packagingGarlic oilBeef loaves

* Corresponding author. Tel.: þ60 3 4107 9802; faxE-mail address: direct.tinsin@gmail.com (L.T. Sin).

0956-7135/$ e see front matter � 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.foodcont.2013.11.020

a b s t r a c t

This study was designated to ascertain the effectiveness of low density polyethylene (LDPE) based filmincorporated with garlic oil for inhibition of food pathogen on ready-to-eat beef loaves. The blown filmextrusion method was employed to produce film samples added with garlic oil in 2, 4, 6 and 8% w/w aswell as sample with 0% w/w which served as control throughout the study. Besides, several analyseswere also conducted to determine the water vapour barrier properties, thermal stability and bondinginteraction of the plastic packaging as influenced by the incorporation of garlic oil. The outcomes ofchallenge test showed that regardless of the garlic oil amount (2e8% w/w), the antimicrobial plasticpackaging was able to reduce the number of Listeria monocytogenes on beef loaves after 3, 6, 9 and 15days of storage at 4 �C. However, there were insignificant effects on both Escherichia coli andBrochothrix thermosphacta. For water vapour barrier properties, films with higher amount of garlic oilproved to have weaker barrier properties. There was lack of significant difference in the thermal stabilityfor all samples when tested with thermogravimetry analyser. Also, the infrared analysis indicated garlicoil does not change the polymer structure.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Cooked meat for retail sale is often subjected to microbialcontamination. Among the microorganisms, L. monocytogenes andEscherichia coli were the most common bacteria that found in beefproducts that contributed to food-borne diseases. Consuming foodscontaminated by L. monocytogeneswould lead to fatal diseases suchas meningitis and pneumonia which often occurs in the new born,pregnant women and elder folks. L. monocytogenes has beeninvolved in food-borne illness outbreak at 1998 and 1999 whichcaused 21 deaths among 100 reported cases. In fact in year 2002, 10deaths occurred in United States related to consumption ofcontaminated meat (Ye, Neetoo, & Chen, 2008). As reported by thegovernment of United States in year 2000, the annual economic lossassociated with L. monocytogeneswas $2.3 billion. E. coli O157:H7 isalso a facultative anaerobe foodborne illness bacterial that partic-ularly problematic for the beef industry. Consuming foodscontaminated by E. coli O157:H7 could cause hemorrhagic colitis

: þ60 3 4107 9803.

All rights reserved.

disease resultant in bloody diarrhea, severe cramping and occa-sional vomiting that last for 2e9 days (Feng, 2000). For moreserious case, it could cause fatal as reported by Mead, Slutsker, andDietz (1999) where 52 people out of total 1843 were death.

In order to prevent the growth of pathogenic and spoilage mi-croorganisms on Ready-to-Eat (RTE) meat products, antimicrobial(AM) packaging has been developed. AM packaging is defined aspackaging containing AM agents and this packaging is moreeffective compared to direct adding of AM agents into foods. This isbecause direct adding of AM agent can cause rapid diffusion of theagent into foods; consequently, the active substances denaturizedby food constituents rapidly. Whereas, AM packaging offers slowand continuous migration of AM agent from packaging material tofood surfaces which enables AM agent to maintain at high con-centration over a long period (Quintavalla & Vicini, 2002). Exampleof the AM agents used commercially in packaging systems includedsilver substituted zeolite, chlorine dioxide, ethanol, sulfur dioxide,triclosan, and horseradish extract.

In this study, garlic essential oil was used as the AM agent to beincorporated into plastic packaging film by using blown filmextruder. Garlic or Allium sativum in scientific name, has been usedwidely as medicine many years ago. It is used traditionally as food

Table 1Summary of film formulations.

Weight of LDPE(kg)

Weight of EVA(kg)

Weight of garlicoil (kg)

Weight percent of garlic oil(% w/w)

3.6 0.4 0.00 0 (Control)3.6 0.4 0.08 23.6 0.4 0.16 43.6 0.4 0.24 63.6 0.4 0.32 8

S.-Y. Sung et al. / Food Control 39 (2014) 214e221 215

preservative to inhibit the growth of pathogens and spoilage mi-croorganisms where a wide range of microorganisms includedbacteria, mold, fungi, parasites and viruses can be inhibited. Otherthan the AM benefit, garlic has been reported to reduce blood lipids,modulate cardiovascular, enhance immune functions, and havingantioxidant and anticancer properties. Up to date, there are only afew published papers related to garlic oil used as AM agent in foodpackaging system. Most of the studies were focus on edible film,and as per known, no research has studied garlic oil incorporated-plastic film produced by blown film extrusion technique. Thereason may be due to the AM agent in garlic, known as allicin, isvery unstable and easily decompose when process under hightemperature and pressure. However, the antimicrobial activitiesmay not loss since the breakdown products of allicin, i.e., diallylsulphide (DAS), diallyl disulphide (DAD), diallyl trisulphide (DAT)and ajoene have reported effective against certain type of micro-organisms (Corzo-Martinez, Corzo, & Villamiel, 2007; Tansey &Appleton, 1975). Therefore, this study was conducted to verify theAM effectiveness of blown film extruded AM film when used inactual beef packaging conditions. Besides, several analyses werealso carried out to determine the water barrier properties, thermalstability and bonding interaction of the plastic packaging asaffected by the incorporation of garlic oil.

2. Experimental

2.1. Materials

Deodorized garlic essential oil which contained more than 35%of allicin was purchased from Xiamen Forever Green Source Bio-chem Tech. Co. Ltd. For the packaging film, LDPE with 2.0 g/10 minof melt flow index was purchased from Titan Group wherebyethylene vinyl acetate copolymer (EVA) with the grade of UE629(10% vinyl acetate content) was supplied by USI Corporation,Taiwan. Tryptone Soya Agar (CM0131B) (TSA), Tryptone Soya Broth(CM0129B) (TSB), and saline peptone powder (CM0733B) werefrom Oxoid, United Kingdom which were supplied by Choice CareSdn. Bhd., Malaysia. Microbe strain used were typical beef con-taminants included gram-negative bacteria, E. coli (ATCC 10536)and gram-positive bacteria, L. monocytogenes (ATCC 13932) andB. thermosphacta (ATCC 11509). L. monocytogenes and E. coli arepathogenic bacteria and B. thermosphacta is beef spoilage bacteria.They were purchased from American Type Culture Collection(ATCC), United States.

2.2. Bacteria culture maintenance

The bacteria cultures were maintained according to the stepsdescribed in manual obtained from supplier. For ceasing bacteriaactivities, the bacteria strains were stored at�80 �C in Tryptone SoyBroth (TSB) that contained 20% glycerol. For experiment purpose,the bacteria were regularly subculture on Tryptone Soya Agar (TSA)and stored at 4 �C. In the preparation for challenge test, one colonyof bacteria was transferred from TSA into 50 ml Tryptone SoyaBroth (TSB) and incubated in incubator shaker at 37 �C, 200 rpm for18 h. Serial dilution was conducted to obtain required concentra-tion of bacteria.

2.3. Film preparation

LDPE film were added with various amount of garlic oil rangingfrom 0, 2, 4 6 and 8 weight percent (% w/w). Firstly, a masterbatchpreparation was prepared in such a way that the EVA was grindedto powder form using ball mill grinding machine and 2% w/w ofgarlic oil was added. The mixture was then mixed thoroughly by

using tumbler. LDPE pellets were added andmixed to get 5 batches.The process was repeated with 4, 6 and 8% w/w of garlic oilrespectively. The films were produced by blown film extruder. Thetemperature was set at 170 �C throughout all the zones from barrelto die, and the target film thickness was 40e50 mm. After extrusion,the films were wrapped with aluminium foil to prevent loss of AMagent by evaporation. Summary of film formulation is listed inTable 1.

2.4. Challenge test

The antimicrobial activity of the films was tested on Ready-to-Eat (RTE) beef loaves. Once purchased from local retailer, themeats were steamed to cooked and cut into loaf shape thatweighted 5 g per piece. In order to sterilize the beef loaves, everysingle side of the meat surfaces was exposed to UV light for15 min prior to test. The meat were then randomly divided into 3sets for different bacteria inoculations, and each set divided intofive lots for different packaging formulation, i.e., garlic oil in 0, 2, 4,6 and 8% w/w respectively. For inoculation of bacteria on beef, 0.1ml of each bacteria strain (E. coli, L. monocytogenes and B. ther-mosphacta) with concentration of 106e107 cfu/ml was transferredonto top and bottom surfaces of meat and spread evenly to obtainbacteria concentration of approximately 105 cfu/g. The sampleswere left for 5 min to allow the inoculums to soak and attached tothe meats before wrapped with plastic films containing 0, 2, 4, 6and 8% w/w of garlic oil respectively. The meats were tightly con-tacted with the films and the three open sides of films were sealedand stored immediately at 4 �C for 15 days to mimic the normalretail display temperature (Torstveit & Magnussen, 1998). Thecounts of bacteria were determined by using serial dilution methodimmediately. All these were done after inoculation and periodicallyat the end of 3, 6, 9 and 15 days of inoculation. For determinationthe number of bacteria growth on the sampling days, two packagesfor each formulation were opened. The bacteria on beef wereextracted by adding 50 ml saline peptone water into each sampleand homogenized with laboratory blender (Bagmixer) for 2 min.0.1 ml homogenatewas pipette transferred into centrifuge tube andserially diluted with 0.9 ml saline peptone water. 0.1 ml of eachdiluted homogenate were then transferred onto TSA plate andincubated at 37 �C (E. coli and L. monocytogenes) and 25 �C(B. thermosphacta) in incubator chamber. After 24 h, the number ofcolonies formed was calculated and expressed as cfu/g.

2.5. Water vapour barrier properties

The water vapour barrier properties of the film samples weredetermined in term of water vapour transmission rate (WVTR) at23 �C and 60% relative humidity (RH) according to the ASTM E96/E96M-10 (ASTM, 2010). The circular test cup was filled with silicagel (desiccant) activated at 200 �C (0%RH) and then sealed by thetest films. It was covered with a lid having 60 mm of opening (filmtested surface area) which allowed vapour to pass through. Thecups were placed inside a control chamber which was maintained

Fig. 1. Effect of garlic oil-incorporated plastic films on the growth of L. monocytogeneson RTE beef loaves stored at 4�C.

Fig. 2. Effect of garlic oil-incorporated plastic films on the growth of E. coli on RTE beefloaves stored at 4�C.

S.-Y. Sung et al. / Food Control 39 (2014) 214e221216

at 60 %RH and 23 �C. The test was carried out in triplicate for eachfilm formulation. The weight gain of the test cups was recordedperiodically until a constant rate of weight gain was attained. Theamount of water vapour transferred from surrounding air throughthe film into the desiccant can be calculated and expressed asWVTR in g/m2/day.

2.6. Fourier transform infrared (FTIR) spectroscopy

Fourier transform infrared spectroscopy (FTIR) analysis wascarried by using Thermo Scientific Nicolet iS50 FT-IR spectrom-eter. In this analysis, the type of chemical bonds (functional groups)and molecular structures of the polymer matrix and garlic com-ponents were identified. The wavelength of the analyzer was set inthe range of 400e4000 cm�1. The sample was placed on a plate inthe analyzer and FTIR spectra were recorded.

2.7. Thermogravimetric analysis (TGA)

TGA analysis was conducted using Mettler Toledo thermogra-vimetric analyser TGA/SDTA851e. Samples of 5e6 mg were cutfrom the plastic films. The sample was heated from 25 �C to 600 �Cat heating rate of 20 �C/min under nitrogen gas flow.

3. Results and discussion

3.1. Challenge test

Owing to the increasing demand for RTE products due to thenew trends inmodern lifestyle, this research is focusing on RTE beefloaves. RTE foods are foods that will be served after heatinginstantly. Thus, it is important to stop harmful bacteria fromgrowing throughout the RTE foods storage period. The bacteriaselected in this study were typical beef pathogenic bacteria e

L. monocytogenes and E. coli; and meat spoilage bacteria e

B. thermosphacta. L. monocytogenes is an important foodbornepathogen which often contaminates foods during the post pro-cessing phase and its presence in RTE foods is not tolerated inmanycountries (Iseppi et al., 2008). E. coli is bacteria that most commonlyfound in beef products. It contributed to 44.2% total foodborneoutbreaks in beef products reported internationally between 1988and 2007 (Greig & Ravel, 2008). Consuming of foods contaminatedwith E. coli can cause food poisoning. Meanwhile, B. thermosphactaoften contributes to meat spoilage. By reducing the growth rate ofthis bacterium, the shelf-life of food can be extended.

Fig. 1 shows the growth behavior of L. monocytogenes on artifi-cially contaminated RTE beef that packaged in films with differentamount of garlic oil. Regardless on the amount of garlic oil addedinto plastic packaging, the beef samples having similar bacteriagrowth curve where the number of L. monocytogenes increasedrapidly within the 15 days of storage at 4 �C. The survival ofL. monocytogenes in meat products is mainly depends on the tem-perature and pH value during storage. According to Aldsworth,Dodd, and Waites (2009), although the optimum growth temper-ature for L. monocytogenes is 30e35 �C, it is capable to grow attemperature range of 0e42 �C. Barker and Park (2001) also sug-gested the optimum pH for L. monocytogenes growth is 7e8 butthey can still growing in the range of 5e10 too. In this study, the pHvalue of beef samples was 6.6 � 0.1 which was expected to supportthe growth of L. monocytogenes. Results indicated that RTE beefproducts that stored at refrigerator having high potential forcausing health problems when the products were contaminatedwith L. monocytogenes.

Throughout the storage period, L. monocytogenes grew mostrapidly on beef samples packaged in control film. The counts of

L. monocytogenes reached 7 log cfu/g within 4 days of storage andreached the initial stage of stationary phase on day 7. Whereas, theincorporation of garlic oil into plastic films can effectively reducethe bacteria growth rate. As in the case of film with 8% w/w garlicoil, the counts of L. monocytogenes in this treatment were 1.98 logcfu/g lower than those in control film on the 6th day of storage andonly reach the initial stage of stationary phase on day 10. Accordingto Quintavalla and Vicini (2002), the effectiveness of AM packagingwas attributed to the entrapment of AM agents within polymerchains that offered slow and continuous migration of the agentsfrom packaging material to food surfaces. Besides, Herath (2010)studied on the release kinetics of AM agents into food stimulantsalso suggested that the intermolecular interaction between AMagents and polymer was one of the factors to maintain the AMagents in high concentration over a long period. On the other hand,the AM effectiveness of filmwith 2% w/w garlic oil was comparablewith film containing higher amount of garlic oil. The further addi-tions of garlic oil only slightly increased the AM effectiveness ofpackaging at the end of storage day, thus, 2% w/w garlic oil wassufficient to suppress L. monocytogenes in this study. This was alsoin agreement with Du et al. (2009) reported 0.5% w/w garlic oil in

Fig. 3. Effect of garlic oil-incorporated plastic films on the growth of B. thermosphactaon RTE beef loaves stored at 4�C.

Fig. 4. Effect of different amount of garlic oil on water vapour transmission rate(WVTR) of film samples.

S.-Y. Sung et al. / Food Control 39 (2014) 214e221 217

tomato cast film can effectively suppressed the growth ofL. monocytogenes. While Kim, Choi, and Kang (2007) also reportedthat garlic oil was highly effective against different strains ofL. monocytogenes.

The antimicrobial test of films against E. coli presented in Fig. 2shows that the bacteria count in all beef samples only increasedslightly throughout the 15 days storage at 4 �C. As in the case ofcontrol film, E. coli count only increased 0.342 log after 15 days ofstorage. This was reasonable since temperatures lower than 7 �Cusually could not support the growth of E. coli and the optimumtemperature for the growth is in the range of 35e40 �C. However,E. coli can still survive well in foods at refrigeration temperature inthe range of 3e7 �C within 5 weeks of storage period (ICMSF, 1996).Result shows that the counts of E. coli on beef packaged in garlic oil-containing films were consistently lower than those in the controlfilm sample throughout the storage period. However, the differencein counts between control film and AM films were low at�0.2 log cfu/g for all data points. Thus, it was concluded that garlicoil-containing films in this case having minimal AM effects onE. coli. Du et al. (2009) developed AM films also found that tomatofilms formulated with garlic oil at 0.5e3% w/w were not effectiveagainst E. coli (gram-negative bacteria). They were however effec-tive against L. monocytogenes (gram-positive bacteria) at all con-centration in agar disk diffusion test. This can be due to tworeasons, firstly, the high resistance of lipopolysaccharide layer ongram-negative bacteria towards garlic active compounds(Sivarooban, Hettiarachchy, & Johnson, 2008), secondly, the lack ofallicin in the AM films. There are many reports which claimed thatallicin in garlic is an effective bactericidal against E. coli (Ankri &Mirelman, 1999; Curtis, Noll, Stormann, & Slusarenko, 2004).However, allicin become unstable when exposed to high temper-ature and tends to decompose into sulfide compounds. The half-lifeof allicin is about a year at 4 �C, and 32 days at 15 �C, but only 1 dayat 37 �C (Fujisawa, Suma, Origuchi, Seki, & Ariga, 2008). Thus, it isexpected no allicin exist in garlic oil that has been exposed toelevated temperature in this study, whereby garlic oil has beenexposed to 170 �C during blown film extrusion process. Althoughthe breakdown products of allicin, sulfide compounds, having AMeffects against wide range of bacteria (Yin & Cheng, 2003), thesensitivity of E. coli to these compounds is relatively lowercompared to allicin (Sagdic & Tornuk, 2012). Thus, higher amount ofgarlic oil is needed to show inhibitory effects as reported by Seydimand Sarikus (2006) who found that whey protein isolated (WPI)film inhibit the growth of microbial on sprout only when highamount of garlic oil is incorporated.

B. thermosphacta is a competitive bacterium after Pseudomonasspp. that has been frequently reported as important spoilage bac-teria on beef products (Borch, Kant-Muermans, & Blixt, 1996;Doulgeraki, Ercolini, Villani, & Nychas, 2012; Nychas, Skandamis,Tassou, & Koutsoumanis, 2008). It is a facultative anaerobe that ableto grow under the presence and absence of oxygen. Under aerobiccondition, B. thermosphacta produces acetic, diacetyl, isovalericacids and 3-mthylbutanol as a result of metabolisms (Dainty,Edwards, & Hibbard, 1985). This is main cause of cheesy andslime odor of the food. B. thermosphacta has been detected in theaerobic flora of chilled meat (Nychas et al., 2008), therefore it is notsurprising that this bacteria could grow rapidly during retail displayprocess.

The growth of B. thermosphacta on beef loaves packaged in filmswith different amount of garlic oil is shown in Fig. 3. Under storagetemperature of 4 �C, the counts of B. thermosphacta increasedrapidly from 5.41 log cfu/g on day 0 to 9.01, 8.90, 8.92, 8.79 and8.86 log cfu/g for films with 0, 2, 4, 6 and 8% w/w garlic oil inrespective on day 6 of storage. This is similar with L. monocytogenes,where B. thermosphacta is also a gram-positive bacteria and it is

more resistance to AM agent compared to L. monocytogenes. Asobserved from the result curve, although filmwith garlic oil havinglower counts of B. thermosphacta, it is considered not effective sincethe differences in counts between control film and AM films werelow at <0.3 log cfu/g for all data points.

3.2. Water vapour barrier properties

Water vapour transmission rate (WVTR) represents the ease ofmoisture to penetrate and pass through a material (Li, Li, Kennedy,Peng, Yie, & Xie, 2006). It is important for a packaging to have goodwater vapour barrier properties not only to prevent excessivewaterloss from foods, but also resists moisture from atmosphere tomigrate into foods since water can accelerate microorganisms’growth and reduce shelf-life of foods. Fig. 4 shows the WVTR ofplastic filmwith different percentage of garlic oil incorporated. Theaddition of garlic oil on the film increased WVTR from 0.85 g/m2/day to 0.92, 1.27, 1.34 and 1.56 g/m2/day correspond to film with 2,4, 6 and 8% w/w of garlic oil. Although the hydrophobic compoundof garlic oil tends to inhibit the moisture contact, there was moreweight gain for film with higher garlic oil percentage. This wasprobably caused by the evaporation of volatile compound of garlicoil e sulphur components from the film and left behind the porousmatrix. The porous matrix of the film allowed the vapour

Fig. 5. FTIR spectra of (a) control film, (b) film with 2% w/w garlic oil, (c) film with 4% w/w garlic oil, (d) film with 6% w/w garlic oil, (e) film with 8% w/w garlic oil, ( ) ethylenegroups, and ( ) vinyl acetate groups.

Fig. 6. A section of FTIR spectra of (a) control film, (b) filmwith 2% w/w garlic oil, (c) filmwith 4% w/w garlic oil, (d) filmwith 6% w/w garlic oil, (e) filmwith 8% w/w garlic oil. Arrowrepresented S¼O group at 1050 cm-1.

S.-Y. Sung et al. / Food Control 39 (2014) 214e221218

Fig. 7. TGA weight lost (%) curves of LDPE/EVA film with (a) 0% w/w garlic oil, (b) 2% w/w garlic oil, (c) 4% w/w garlic oil, (d) 6% w/w garlic oil, and (e) 8% w/w garlic oil.

S.-Y. Sung et al. / Food Control 39 (2014) 214e221 219

transmission and contributed to the weight gain. These results canbe verified by the study from Pranoto, Rakshit, and Salokhe (2005)where the weight gained was greater with the higher amount ofgarlic oil added into alginate films. The respective authors sug-gested that it might be due the structure of alginate film waschanged by addition of garlic oil which resulted in intermolecularextension and furthermore, loosening the compactness of thestructure. Hence, it encouraged the water vapour to pass throughthe film.

3.3. Fourier transform infrared (FTIR) spectroscopy

FTIR is used to monitor the functional group and structuralchanges of film samples at molecular level through a detail spectral

analysis (Ahmad, Benjakul, Prodpran, & Agustini, 2012). In thisstudy, FTIR was performed to identify the interaction betweenpolymer matrixes with garlic oil. Fig. 5 represents the spectra ofLDPE/EVA films incorporated with different amount of garlic oilranging from 0 to 8% w/w. In overall, there was lack of significantdifferences between the spectra. The spectra with garlic oil incor-porated were similar to the control film. All characteristic peaks areidentical such as at the bands of 2800e3100 cm�1, 1740 cm�1,1464 cm�1, 1375 cm�1, 1241 cm�1, 1021 cm�1, 729 cm�1 and720 cm�1. These bands can be related to absorbance of ethylenegroups as 2920 cm�1, 2850 cm�1, 1470 cm�1, 1375 cm�1, 729 cm�1

and 720 cm�1, and vinyl acetate groups as 1740 cm�1, 1240 cm�1,1020 cm�1. (Jamroz, 2003). These shows that the absence of majorstructural changes in the polymer matrixes. Similar results were

Table 2Decomposition temperature of LDPE/EVA film incorporated with different amountof garlic oil.

Garlic oil (% w/w) Ti (�C) Tp (�C)

0 318 4642 317 4634 318 4636 320 4628 319 462

S.-Y. Sung et al. / Food Control 39 (2014) 214e221220

observed in chitosan film incorporated with A. sativum extract(Pranoto et al., 2005).

Nevertheless, there were someminor distortions of bands at theabsorbance range of 1030e1200 cm�1. A very weak peak can beobserved at 1050 cm�1 for films added with garlic oil (Fig. 2). Thispeak represented S<ce:glyph name¼"dbnd"/>O bond (Ili�c et al.,2012) that existed in garlic oil active component, namely ajoene.Ajoene was expected to be responsible for the antibacterial activityby inhibiting sulfhydryl group (eSH)-containing enzymes in mi-croorganisms. Since only small quantity of garlic oil extract hasbeen added into plastic film, the amount of ajoene existed was verymuch lower than the polymer chains, and thus the peak repre-sented S<ce:glyph name¼"dbnd"/>O bond was relatively weaker.Besides, the absorbance at 1131 cm�1 and 1177 cm�1 in control filmwere integrated into one at 1153 cm�1 in filmwith 2%w/w garlic oiland 1161 cm�1 in films with 4e8% w/w garlic oil. As the amount ofgarlic oil increased, this peak behaved sharper significantly (Fig. 6).Peak at 1177 cm�1 in control film is probably representing carbonyl,C<ce:glyph name¼"dbnd"/>O stretching of ketone structurewhich formed by main chain scission of EVA copolymer (Jin, Chen,& Zhang, 2010) during the film forming process. In the addition ofgarlic oil, this peakwas overwritten by peak at 1153 cm�1 (filmwith2% w/w garlic oil) and 1161 cm�1 (film with 4e8% w/w garlic oil)which was most likely due to the vibration of CeOH groups ofamino acids (Wong, Wong, Caputo, Godwin, & Rigas, 1991) fromgarlic oil. Other important vibration represented the existence ofgarlic oil active components such as SeC bond (700e800 cm�1)was hardly to be detected since it was probably obscured by thevibration of ethylene units.

3.4. Thermogravimetric analysis (TGA)

The study on thermal stability of plastic films with differentpercentage of garlic oil was conducted through TGA under nitrogenatmosphere. Fig. 7 shows the TGA weight loss curves as thefunction of temperature obtained for LDPE/EVA films containing 0,2, 4, 6 and 8% w/w of garlic oil. For all the formulations, only singlepeak of weight loss corresponds to the polymer blends wereobserved. This may be due to the percentage of garlic oil addedwas insignificant for the detection. Besides, it was expected thatsome amount of garlic oil components may be lost during theblown film extrusion process which exposed the additives toelevated temperature.

Table 2 summarizes the initial decomposition temperature (Ti)and peak decomposition temperature (Tp) obtained from TGA data.As it can be seen, no significant distinction was observed for Ti andTp values in all the samples. LDPE/EVAmatrix started to decomposeat around 318 �C with peak decomposition temperature atapproximately 463 �C. Results indicated that the incorporation ofgarlic oil has not affected the thermal stability of the plastic pack-aging. Similar result was obtained by Suppakul, Miltz, Sonneveld,and Bigger (2006) who incorporated linalool and methylchavicolinto LDPE-based film. The authors found that linalool only slightlyreduced the Tp of LDPE from 466 �C to 461 �C.

4. Conclusions

The result of challenge test showed that L. monocytogenes andB. thermosphacta grow rapidly on the RTE beef loaves with pH6.6 � 0.1 and storage temperature of 4 �C (i.e. retail temperature).These bacteria grew rapidly onpackaged beef samples especially forcontrol sample. This indicated RTE beef products that were stored atrefrigerator during retail display possessed high potential to causehealth problems when the products were contaminated by bacteriainitially. Nevertheless, plastic film incorporated with 2% w/w garlicoil has sufficiently reduced the growth rate of L. monocytogenes onRTEbeef after 3, 6, 9 and15days of storageat 4 �C.However, garlic oilAM was lack effective on both E. coli and B. thermosphacta since thedifference in bacteria counts between control film and AM filmswere low at �0.3 log cfu/g for all data points.

For water vapour barrier properties, the incorporation of garlicoil has slightly reduced the film water vapour barrier properties.The addition of garlic oil on the film increased WVTR from 0.85 g/m2/day to 0.92, 1.27, 1.34 and 1.56 g/m2/day correspond to filmwith2, 4, 6 and 8% w/w of garlic oil, respectively. Although the hydro-phobic compound of garlic oil tends to inhibit the moisture contact,however the evaporation of volatile compound of garlic oil from thefilm had left behind the porousmatrix that allowedwater vapour topass through.

The study of film thermal stability conducted by TGA testshowed that the incorporation of garlic oil has not affected thethermal stability of the plastic packaging. The films started todecompose at about 318 �C with peak decomposition temperatureat approximately 463 �C. The existent of garlic oil was not traceablethroughout the test because some amount of garlic oil componentsmay be lost during the blown film extrusion process which exposedthe additives to elevated temperature. Whereby, S<ce:glyphname¼"dbnd"/>O bond of ajoene is observable by using FTIRtechnique at absorbance wavelength of 1050 cm�1. Ajoene wasexpected to be responsible for the antibacterial activity.

Overall, this research indicated that the plastic film containedgarlic oil has good potential to be used as food packaging especiallyto inhibit L. monocytogenes.

Acknowledgements

This research was financially supported by Ministry of Science,Technology and Innovations (MOSTI) of The Federal Government ofMalaysia-Putrajaya under Science Fund 03-02-11-SF0128. Specialthanks to Nigel Foong from Universiti Tunku Abdul Rahman,Kampar Campus on assisting thermogravimetry analysis.

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