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1 As. J. Food Ag-Ind. 9, (4), Asin Journl of Food nd Agro-Industry ISSN Aville online t Reserch Article Liposome encpsultion of ntimicroil extrcts in pectin film for inhiition of food spoilge microorgnisms Orchorn Mekkerdchoo *, Psvdee Ptipsen nd Chleed Borompichichrtkul Deprtment of Food Technology, Fculty of Science, Chullongkorn University, Bngkok, Thilnd. *Author to whom correspondence should e ddressed, emil: orchorn_willim@hotmil.com This pper ws originlly presented t Food Innovtion Asi, Bngkok, Thilnd, August 9. Received 1 June 9, Accepted 4 Ferury 1 Astrct The purpose of this reserch ws to determine suitle method to encpsulte ntimicroil extrcts in liposome sed on pectin film to inhiit food spoilge microorgnisms. Clove oil, grlic oil nd pomegrnte extrcts were selected to e used in the experiments. Their ilities to inhiit food spoilge microorgnisms ginst Pseudomons sp. ATCC 5619, E. coli ATCC 59, E. coli ATCC 8739, Slmonell Typhimurium ATCC 3564, Slmonell Choleresuis ATCC 593, Lctocillus sp. TISTR 539 nd Lctocillus ske TISTR 89 ws found to e significntly positive. For liposome preprtion, criticl micelle concentrtion (CMC) of lecithin to form liposome ws 11 % w/w. Then emulsion of ntimicroil extrcts ws prepred using doule emulsion method. Oil phse contined lecithin solution in ethnol nd mixture of clove oil nd grlic oil (weight rtio of clove oil to grlic oil = 1:1) while wter phse contined pomegrnte extrct. The concentrtion of lecithin s vried to 1, 14 nd 16 % w/w nd rtio of lecithin solution to ntimicroil extrcts ws 1:3, 1:6 nd 1:9. Then stility of the emulsions ws exmined. The results showed tht the emulsion prepred with lecithin concentrtion of 1% nd rtio of lecithin solution to ntimicroil extrct of 1:6 gve the est stility nd good inhiition. All prepred emulsions showed positive result for cteril inhiition. Physicl properties of pectin film were studied t different pectin concentrtions (.5, 3, 3.5 nd 4% w/v) mixed with clcium chloride (3, 5, 7 nd 1% w/v) nd plsticizers (glycerol (GLY) nd soritol (SOR) t concentrtion of 4, 5 nd 6% of the weight of pectin into film mixture). The results showed tht t pectin concentrtion 4% w/v with clcium chloride 3% w/v nd using GLY 5% exhiited etter physicl properties (TS 19.9 MP, %E 7.85%, WVP 6.49 µg/msp) thn other tretments. Then liposome with ntimicroil extrcts ws dded into selected film condition t vried concentrtions (, 4 nd 6% w/w). Antimicroil effects of pectin film incorported with the liposome showed no significnt different (p.5) in cteril inhiition zone etween liposome concentrtions except those of 4 nd 6 % w/w hd highest inhiition zone ginst Lctocillus sp. TISTR 539 nd

2 As. J. Food Ag-Ind. 9, (4), Lctocillus ske TISTR 89 significntly. The physicl properties of pectin sed film with ntimicroil liposome hd following vlues; TS MP, %E %, WVP µg/msp, colour differentil ( E) nd film opcity Au x nm/µm. Keywords: liposome encpsultion, ntimicroil extrct, edile film, spoilge microorgnism, Thilnd Introduction Food is rich nutrient mtrix tht provides suitle environment for prolifertion of spoilge microorgnisms such s Pseudomons sp., Enterocterice nd lctic cid cteri in met nd met product [1] nd common food-orne pthogens such s Slmonell enteritidis, Escherichi coli []. Microil contmintion reduces the shelf-life of foods nd increses the risk of food orne illness, therefore dequte preservtion technologies must e pplied in order to preserve its sfety nd qulity, Although trditionl methods of preserving foods from the effect of microil growth such s refrigertion tht cn reduces the growth of microorgnisms ut cnnot kill microorgnisms [3, 4]. The consumer demnds high qulity, convenient, innovtive, regulr nd sfe food products with nturl flvors nd tste s well s n extended shelf-life. Moreover less chemicl preserved products re required [5-8] y consumers. It hs driven nturl ntimicroil compounds towrds the investigtion of replcing chemicl preservtives. Spices nd her extrcts re nturl compounds tht hve een widely studied on their nture ntimicroil ctivities nd found tht some exmple of they hve potentil for inhiition microorgnism in foods [, 4, 9-11]. Their importnt phytochemicl in those extrcts such s Eugenol from clove oil [1] or Allicin in grlic [13]. Moreover, those nturl extrcts re clssed s generlly recognized s sfe (GRAS) food dditives [4, 1, 13-14]. Unfortuntely, most nturl compounds re iologiclly instle ecuse they re sensitive to the environment (wter, oxygen, light) tht destroy their ntimicroil ctivities [15-16]. Currently, novel methods hve een introduced in order to improve their stilities nd their iovilility, y using encpsultion to prevent rectivity with the environment nd miniml compounds loss during storge. Therefore encpsultion provides prolonged ntimicroil ctivity in the finl product y grdutely diffusion out of ilyer memrne [17-1]. The enefit of using liposome encpsultion is oth of non-polr compound (oil phse such s essentil oil) nd polr compound (wter or ethnol phse) cn e encpsulted liposome-multilyer memrne []. Furthermore, liposome cn e constructed of nturl constituents such s lecithin from egg yolk or soy ens nd cholesterol so it is non-toxic. In ddition to hndle nd ppliction in food industry, liposome encpsultion of ntimicroil extrcts cn e incorported into pectin film for keeping structure nd stility of liposome during storge. Furthermore, the use of edile films in food protection nd preservtion hs recently incresed since they offer severl dvntges over synthetic mterils, such s eing iodegrdle nd environmentlly friendly [3]. When those film contct with food, moisture from food ws induced liposome memrne to slowly relese ntimicroil extrcts nd those extrcts will e trpped etween food surfce nd liposome memrne, thus helping mintin high concentrtions tht mke more efficient for inhiition food spoilge nd food pthogen microorgnisms [5, 4-5]. Moreover, edile film hs ilities to retrd moisture, oxygen, roms nd solute trnsporttion [6].

3 As. J. Food Ag-Ind. 9, (4), The overll ojectives of the present reserch were to determine suitle method to encpsulte ntimicroil extrcts in liposome sed on pectin film to inhiit food spoilge microorgnisms which hve potentil for food ppliction nd e le to develop guideline to pply ntimicroil extrcts s nturl preservtive. Mterils nd Methods Orgnisms nd cultures Typicl food spoilge microorgnisms nd common food-orne pthogens used in this study were Escherichi coli ATCC 59, Escherichi coli ATCC 8739, Slmonell Typhimurium ATCC 3564, Slmonell Choleresuis ATCC 593, Pseudomons sp. ATCC 5619 (Deprtment of Medicl Science, Ministry of Pulic Helth (Nonthuri, Thilnd)) nd Lctocillus sp. TISTR 539, Lctocillus ske TISTR 89 (Thilnd Institute of Scientific nd Technologicl Reserch (Pthum Thni, Thilnd)). Stock cultures were stndrdized through 4 hours growth cycles in pproprite roth (nutrient roth (NB, Himedi, Indi) for E. coli ATCC 59, E.coli ATCC 8739, S. Typhimurium ATCC 3564, S. Choleresuis ATCC 593, Pseudomons sp. ATCC 5619 or lctocilli MRS roth (Himedi, Indi) for Lctocillus sp. nd L. ske t neroic condition. Determintion of ntimicroil ctivity of clove oil, grlic oil nd pomegrnte extrcts Antimicroil ctivity ws determined y disc diffusion method [7]. The cteri cultures from stndrdized culture were grown then inoculted in fresh medium (NB or MRS) t 37 O C. After 4 hours of growth, ech microorgnism ws inoculted y sterile cotton sw on the surfce of nutrient gr or lctocilli MRS gr plte. Susequently, sterile pper discs (Antiiotic- Testlttchen pper disc, Durn, USA) (6 mm in dimeter) sturted either with clove oil or grlic oil or pomegrnte pericrp extrcts (hexne extrct, ethyl cette extrct, chloroform extrct, ethnol extrct nd queous extrct) were plced on surfce of ech inoculted plte. Stndrd ntiiotic (Tetrcycline (Becton, Dickson nd Compny, USA), Chlormphenicl (Benex Limited, USA)) ws simultneously used s positive control. The plte ws incuted t 37 O C for 4 h under eroic conditions except Lctocillus sp. nd L. ske incuted in n tmosphere enriched in cron dioxide. After this period, it ws possile to oserve inhiition zone. The nticteril ctivity ws evluted y mesuring the inhiition zone dimeter oserved. The sensitivity to the different ntimicroil solutions ws clssified y the dimeter of the inhiition zone s: not sensitive, dimeters less thn.8 cm; sensitive, dimeters cm; very sensitive, dimeters cm; nd extremely sensitive, dimeters lrger thn. cm [8]. All nlyses were done in duplicte. Study of rtio of lecithin solution nd ntimicroil extrcts for liposome preprtion y using doule emulsion method nd screening for the ntimicroil ctivity of ntimicroil extrcts fter liposome encpsultion y disc diffusion method Determintion of the criticl micelle concentrtion (CMC) of lecithin solution Soyen lecithin (phosphtidylcholine >96.4, Merck, Germny) ws suspended in wter y homogeniztion t, rpm for 5 minutes, vried concentrtion from.1- % w/w. Surfce tension ws determined y pendent drop method employing goniometer. Surfce tension ws clculted y the computer progrm. After tht, grph of surfce tension of lecithin solution nd log of concentrtion of lecithin solution ws plotted to determine the CMC point.

4 As. J. Food Ag-Ind. 9, (4), Preprtion of liposome y doule emulsions method The W1/O/W doule emulsions were prepred y modified two-step emulsifiction form [9]. Oil phse contined lecithin solution in ethnol nd mixture of clove oil nd grlic oil (weight rtio of clove oil to grlic oil = 1:1). The concentrtions of lecithin used were 1, 14 nd 16 % w/w. The weight rtios of lecithin solution to oil mixture were 1:, 1:4 nd 1:6. The wter phse ws solution of pomegrnte extrct with ethnol. The mount of the pomegrnte extrct in the wter phse ws vried in order to otin weight rtio of lecithin solution to her extrcts (clove oil, grlic oil nd pomegrnte extrct of equl weight) of 1:3, 1:6 nd 1:9. Formtion of W1/O/W doule emulsions y lipid mixture solution (O) nd queous solution (W1) of weight rtio :1 ws emulsified with homogenizer (Ystrl X1/5, Bllrechten-Dottingen, Netherlnds) t, rpm for 1 minutes nd immeditely sonicted (1 Hz, 4 w, 3 minutes) with proe-type sonictor (Dr.hielscher Up4s, Germny) ll process were done in twice times until the mixture ecme uniform did not seprte for t lest 3 min fter soniction. The primry emulsions were then mixed with queous solution (W; In this study, W1 s the sme solution of W) of 3:4 weight rtio, nd emulsified t 18, rpm for 1 minutes to form W1/O/W doule emulsions. The emulsion ws extruded three times through 1 nm polycronte memrnes using n extruder TM (Northern Lipids, Cnd) for controlled liposome size. The temperture ws kept under 4 C using wter th during liposome preprtion. Stility of Liposome emulsion Emulsion smples were stored in well closed ottles with nitrogen gs nd stored in refrigertor t 4±1 C. Assessment of emulsion ws done during everydy storge for seven dys. The percentge of seprtion ws clculted nd compred with totl hight of emulsion [3]. All nlyses were done in triplicte. Antimicroil ctivity of liposome incorported of ntimicroil extrcts Antimicroil ctivities of liposomel incorportion of ntimicroil extrcts (clove oil, grlic oil nd pomegrnte extrct) were determined y disc diffusion method [7] ginst microorgnisms (listed in section 1) used in this study. Lecithin solutions (1, 14 nd 16 % w/w) were used s negtive control. The nticteril ctivity ws evluted y mesuring oserved the inhiition zone dimeter. All nlyses were done in duplicte. Study of suitle rtio of pectin nd clcium chloride, plsticizer nd liposome incorported of ntimicroil extrcts for preprtion of pectin film Preprtion of pectin film Pectin films preprtion ws modified from [31]. Pectin (.5, 3, 3.5 nd 4 % w/v, Himedi, Indi) were suspended in distilled wter (4 ml) t 9 O C fter stirring for 3 min. Then dding wrm CCl solution (3, 5, 7 nd 1 % w/v, Ajx Finechem, Austrli) t 1 to 3 prts of pectin forming solution. After tht the mixture ws stirred for 1 min. The solution ws degssed nd sonicted twice with th-type sonictor (Ultrsonik, Fisher scientific worldwide, Germny) for 5 min. The solution ws put into 45 O C wter th for control temperture. The solutions were csted onto crylic plte using film coter (Film coter PI-11, Jpn) followed y drying in n oven t 4 O C until film ws dried. All the films were plced on chmer t 5% ± 5 RH for 4 hours. Determintion of the mechnicl properties.

5 As. J. Food Ag-Ind. 9, (4), Film chrcteriztion Film thickness Film thickness ws mesured y using testing film strips (3 x 15 cm) t nine different positions using micrometer (Dil Thickness Guge 731, Mitutoyo, Tokyo, Jpn;.1 mm limit). Then ll the vlues were verged. Tensile strength (TS) nd percentge of elongtion t rek (%E) Tensile strength (TS) nd percentge of elongtion t rek (%E) of the smple film (3 x 15 cm) were mesured using Intron Texture Anlyzer (Intron 5565, USA). It ws set t n initil distnce etween the grip of 5 cm nd cross-hed velocity of 3 mm/s. TS of the film ws clculted y dividing the mximum strength y the initil cross-sectionl re. The %E ws clculted y dividing the initil distnce etween the grip from the elongted distnce until the time tht the film reks. Wter vpor permeility (WVP) Wter vpor permeility (WVP) of the film ws mesured ccording to the ASTM E96-95 (1999). Colour differences ( E) nd opticl properties Film colour ws determined y Minolt colorimeter CR 4 Series(Osk, Jpn) clirted with stndrd (Y = 93., x =.3133, y =.319). The CIE L scle ws used, lightness (L) nd chromticity prmeters * (red green) nd * (yellow lue) were mesured. Test smples were performed y plcing the film smples over the stndrd nd the mesurement were tken s the verge of five points of ech smple. Smples were nlyzed in triplictes, recording five mesurements for ech smple. Colour differences ( E) were lso clculted y the following eqution: colour differences ; E = ( L*) + ( *) + ( *) Where L* = L* - L *; * = * - * ; * = * - *; L *, *, * the colour prmeter vlues of the stndrd nd L*, *, *, the colour prmeter vlues of the smple. Film opcity ws determined [3]. Film smple ws cut into rectngle nd plced on the internl side of spectrophotometer cell. The sornce (t 6 nm) ws recorded for ech smple using spectrophotometer (V-53PC, Jpn). The opcity ws expressed s sornce units per nnometers nd divided y film thickness (AU nm/lm). The mesurement ws repeted five times for ech tretments of film, nd the verge vlue ws used. Microstructure studies y scnning electron microscopy (SEM) Morphologicl investigtion of the surfce nd cross section of the films were investigted using scnning electron microscopy (JSM-541 LV, Jpn). The smples were sputter-coted with gold prior to exmine to llow for conductivity. Effects of the type nd plsticizer concentrtions on mechnicl properties of pectin film Suitle film conditions ws selected in section 4.1 y judging on mechnicl properties (cler, strength, flexile nd low wter vpor permeility (WVP) vlue). Selected pectin film conditions were prepred s descript y 4.1. The two plsticizer re soritol (SOR) nd glycerol (GLY) vried concentrtion t 4, 5 nd 6 %w/w of pectin were dded. All film mechnicl properties were determined of the s the method descried in section 4..

6 As. J. Food Ag-Ind. 9, (4), Effect of concentrtion of ntimicroil extrcts in liposome on mechnicl properties nd ntimicroil ctivity of pectin film Selected pectin film conditions from section 4.3 were prepred s descried y section 4.1. Liposome ws dded y vried concentrtion t, 4 nd 6 %w/w of film solution nd stirring 1 minutes [33]. All film mechnicl properties were determined s method listed in section 4. nd ntimicroil ctivity of those films ws determined y diffusion technique (disc method). Film smples were cut into circle (6 mm in dimeter) from different position of film nd plced on culture plte. The nticteril ctivity ws evluted y mesuring the oserved inhiition zone dimeter. Results nd Discussion Antimicroil ctivity of clove oil, grlic oil nd pomegrnte extrcts Antimicroil ctivities of ntimicroil extrcts re presented in Tle 1. Clove oil hd significntly lrgest inhiition zone of every tested microorgnisms especilly Lctocillus ske (4.75 cm). Moreover, it lso hd ctivity ginst tested microorgnisms eqully or more thn stndrd ntiiotic especilly ginst to Slmonell Typhimurium, followed y pomegrnte extrcted y ethnol nd grlic oil. The ctive compound of clove oil is eugenol which is round 93-95% [1]. Its mechnism is distortion of the lipid-protein interction of cteril cell memrne nd rendering them more permele or ct on cteril enzyme such s ATPse [1]. Active compounds screening of ethnolic pomegrnte extrcts re sterol, flvonoid, triterpene, phenol nd tnnin, they hve ntimicroil ctivity to inhiit E.coli O157:H7 [34]. The ctive compound of grlic oil is llicin (dillyl thiosulfinte), the min ntimicroil effect of llicin is its chemicl rection with thiol groups of vrious enzymes such s lcohol dehydrogense, thioredoxin reductse nd RNA polymerse in cteri [35]. Tle 1. Inhiition zone of clove oil, grlic oil nd pomegrnte extrcts from ethnol y disc diffusion method. (6 mm in disc dimeter). Microil strin E. coli ATCC 59 E. coli ATCC 8739 Slmonell Typhimurium Dimeter of Inhiition zone 1, Clove oil Grlic oil Pomegrnte extrcts from ethnol Tetrcycline Chlormphenicl.5±.7.95±.7 d 1.48±.39 c.±.1.5±.6.5±.7 1.1±.5 d 1.48±.18 c.±.7.5±.11 3.±.1 1.5±.1 d 1.33±.4 e.±.7 c.6±.5 Slmonell.75±.35 1.±.14 e 1.73±.6 d.4±.13 c.9±.9 Choleresuis Pseudomons.33±.1 1.1±.14 c 1.8±.8 N/A N/A sp. Lctocillus 3.38± ±.7 c 1.56±.5 N/A N/A sp. Lctocillus ske 4.75±.1 1.5±.15 c.13±.8 N/A N/A - : no inhiition; N/A: Not detected 1 The venge of dimeter of Inhiition zone ± stndrd error Different letters within the sme row nd for the sme microil strin significnt different (p.5)

7 As. J. Food Ag-Ind. 9, (4), Study of rtio of lecithin solution nd ntimicroil extrcts for liposome preprtion y using doule emulsion method nd screening for the ntimicroil ctivity of ntimicroil extrcts fter liposome encpsultion y disc diffusion method. Criticl micelle concentrtion (CMC) of lecithin solution Criticl micelle concentrtion (CMC) is mininum concentrtion of n emulsifier (mphiphilic component) in solution t which micelles in the solution re formed. CMC is key prmeter for the optimiztion of emulsifier in liposome formultions. For liposome preprtion, emulsifier concentrtion for liposome preprtion must e more thn minimum concentrtion of emulsifier tht cn encpsult ntimicroil extrcts. Figure 1 shows plot of lecithin solution nd log of concentrtion of lecithin solution. In the plot, CMC ws determined round 11 % w/w. Therefore, selected concentrtion of lecithin solution for preprtion of liposome incorported with ntimicroil extrcts were 1, 14 nd 16 % w/w for further study. Surfce Tension (dyne/cm) CMC Concentrtion of lecithin solutions (% w/w) Figure 1. Surfce tension nd log of concentrtion of lecithin solution etween.1- % w/w. Storge stility nd ntimicroil ctivity of liposome incorported of ntimicroil extrcts of liposome emulsion After extrusion through memrne, liposome emulsion ws stored t 4 C to study the storge stility. It ws noticed tht the seprtion of emulsion could e divided into (1) queousemulsion () oil-emulsion (3) queous-oil-emulsion. The seprtion ws much evidence when storge time ws incresed. On other hnds when incresing concentrtion of lecithin solution, the seprtion ws slow. However, when concentrtion of ntimicroil extrcts ws incresed, tht induced the seprtion. A. B. C. D. Figure. Type seprtion of emulsion; emulsion crem (A), queous seprte nd emulsion crem (B), oil seprte nd emulsion crem (C) nd queous seprte, oil seprte nd emulsion crem (D).

8 As. J. Food Ag-Ind. 9, (4), the queous seprtion of totl high of emulsion (%) 7 6 cd 5 4 c Time of storge (dys) 1% 1:3 1% 1:6 1% 1:9 14% 1:3 14% 1:6 14% 1:9 16% 1:3 16% 1:6 16% 1:9 Figure 3. Percentge of queous seprtion which different concentrtion of lecithin solutions nd different rtio of lecithin solutions to ntimicroil extrcts, when stored t 4 C for seven dys. Different letters within different line nd for the sme dy, only 7 th dys significnt different (p.5) The oil seprtion of totl high of emulsion (%) e d c c Time of storge (dys) 1% 1:3 1% 1:6 1% 1:9 14% 1:3 14% 1:6 14% 1:9 16% 1:3 16% 1:6 16% 1:9 Figure 4. Percentge of oil seprtion which different concentrtion of lecithin solutions nd different rtio of lecithin solutions to ntimicroil extrcts, when stored t 4 C for seven dys. Different letters within different line nd for the sme dy, only 7 th dys significnt different (p.5) During extrusion, het nd pressure occurred t the memrne surfce tht prcticlly mde liposome roken nd relese ntimicroil extrcts therefore emulsions will e seprte fter extrusion [36]. The result showed the seprtion of emulsion most induced (fst nd most seprtion) when concentrtion of ntimicroil extrcts were highest while concentrtion of lecithin solution ws lowest. During the storge, liposome will ecome more heterogeneous fter tht liposome my fuse or ggregte, thus liposome ecme igger. Moreover, the lipids used in liposome formtion cn ecome oxidized or hydrolyzed, resulting in ilyer permeility chnges tht induced to lekge of liposome. From Fig. 3-4, rtio of lecithin solution to ntimicroil extrcts t 1:3 ws good form of emulsion.

9 As. J. Food Ag-Ind. 9, (4), Tle. Inhiition zone of prepred emulsions incorported of ntimicroil extrcts y disc diffusion method. Concentr Dimeter of Inhiition zone (cm) tion of lecithin solutions (% w/w) Weight rtio of lecithin solutions to ntimicro il extrcts E.coli ATCC 59 E.Coli ATCC 8739 Slmonell Typhimurium 1 1 : 3.1±.7 c 1.8±.15 ns.±.5.3±.11 c 1.9±.5 1.9±.5 c 1 : 6.3±.8 cd 1.8±.7 ns.5±.1 c.9±.8 d.3±.15 c.4±.5 d 1: 9 1.9±. 1.9±. ns.5±.9 c 1.5±.7.5± ±.1 c 14 1 : 3.3±.13 cd 1.9±.11 ns 1.7±.7.±.5.± ±.9 c 1 : 6.±.7 cd 1.6±. ns.±. 3.±.13 d.±.14 c.1±.17 c 1: 9.±. cd 1.7±.1 ns.± ±. 1.8±.1 1.5± : 3.7±.1 e 1.9±.3 ns.8±.14 d.8±.15 cd.±.8 c 1.17±.8 1 : 6.7±.18 e 1.8±.18 ns.8±.16 d 3±.17 d.7±.1 d 1.5±.6 1: 9.4±. cd 1.6±.5 ns.6±.13 cd.7±.14 cd.7±.1 d 1.33±.11 Different letters within the sme column nd for the sme microil strin significnt different (p.5) The ntimicroil properties of liposome emulsion re shown in Tle. All emulsions showed positive result for cteril inhiition s the inhiition zones more thn.8 cm tht mens microorgnisms were sensitive to ntimicroil sustrtes. The highest ntimicroil ctivity of emulsions ws rtio of lecithin solution to ntimicroil extrcts 1:6 nd inhiition power of cteri will e incresing when the rtio of ntimicroil extrcts were incresing. Except rtio 1:9 s this rtio experience more seprtion thn other emulsions therefore less of ntimicroil extrcts were encpsulted. However, when concentrtion of lecithin solution ws incresed, inhiition power will e incresing ecuse emulsions hd more. Pseudomons sp. Lctocillus sp. Lctocillus ske inhiition zone (cm) e d cd d e c d c e f c d d c c d c E. coli ATCC 8739 E. coli ATCC 59 Slmonell Pseudomons Typhimurium sp. Lctocillus sp. Lctocillus ske Microorgnism 16% 1:6 clove oil grlic oil ethnolic pomegrnte extrct tetrcyline chlormphenicl Figure 5. Inhiition zone of prepred emulsions t concentrtion of lecithin solution 16 %w/w nd rtio of lecithin solution to ntimicroil extrcts 1:6 nd nturl ntimicroil extrcts without seprtion. Mens within the sme column with sme letter re not significntly different (p.5) When compred inhiition zone etween prepred emulsion (concentrtion of lecithin solution 16 %w/w nd rtio of lecithin solution to ntimicroil extrcts 1:6) nd nturl ntimicroil extrcts without encpsulted (Figure 5). Result shows tht emulsions enhnced ntimicroil

10 As. J. Food Ag-Ind. 9, (4), ctivities fter encpsultion more thn using single ntimicroil extrct without encpsultion. Moreover, when compred with ntiiotic, emulsions hd ctivity to ginst tested microorgnisms over stndrd ntiiotic especilly E.coli ATCC 59, Slmonell Typhimurium significntly. Therefore, the comintion of three ntimicroil extrcts together ws more effective to inhiit cteri. Liolios et l. (9) studied liposoml incorportion of crvcrol nd thymol isolted from the essentil oil of Orignum dictmnus L. nd ntimicroil ctivity ws founded to increse fter encpsultion. Liposome cn interct with cteri cells in mny wys for exmple inter-memrne trnsfer, contct relese, sorption, fusion nd phgocytosis. The mechnism of interction depends on cell type (cell wll/memrne composition), s well s the liposome memrne physicochemicl chrcteristics. The use of liposome improves the cellulr trnsport nd releses the ctive component in side cell tht the drmticlly incresed ntimicroil ctivity, fter liposome encpsultion [37]. From the experimentl, the results showed tht the emulsion prepred with lecithin concentrtion of 1 %w/w nd rtio of lecithin solution to ntimicroil extrct of 1:6 gve the est stility, good inhiition nd less using of lecithin. Study of suitle rtio of pectin nd clcium chloride, plsticizer nd liposome incorported of ntimicroil extrcts for preprtion of pectin film. Physicl properties of pectin film Pectin film hs film thickness vried from.-.45 mm. The incresing of thickness due concentrtion of pectin nd clcium chloride were incresed s film thickness depend on solute solid in film forming solution [38]. TS is mesure of film strength [39], TS of films vried from 4.93 to MP. When pectin concentrtion incresed enhnced TS while CCl concentrtion incresed reduced TS significntly ecuse pectin is gelling gent [4] so when pectin incresed, interction etween intermoleculr chins will e incresed nd film is stronger. SEM (Figure 7) showed more crcks while CCl incresed ecuse it creted mny junction zones nd pinholes tht mde film strength reduction. In ddition, excess clcium slt left in the films would interfere with the mesurement of cross-linked clcium nd my lso lter the mechnicl properties of the film [41]. %E is mesure of film stretch ility prior to rekge tht showed how much film flexiility. %E ws vried from 3.74% to 1.99 %, when concentrtion of pectin decresed while concentrtion of CCl incresed tht incresed %E significntly ecuse CCl is elsticlly ctive chin [4]. Moreover, when considering film s morphology, it ws found tht film structure ws less dense when CCl incresed. Generlly, the strength nd flexiility of films re negtively correlted [31]. WVP is mesure of ese of moisture to penetrte nd pss through mteril. WVP vried from 1.56 to µg/m s P. WVP vlues significnt incresed when Pectin nd CCl incresed ecuse pectin film is hydrophilic film, wht could e relted with higher numer of free hydroxyl group tht enhnced interctions with wter fvoring wter vpor trnsmission through the films [43]. Color of the film my influence the consumer cceptility of product [44] tht ws oserved y reding E. Pectin films ppered cler nd trnsprent. Experiments showed tht E vried from.17 to.45 did not different significntly when pectin nd CCl incresed, pectin films hd the gretest L*-vlue ut lowest *- nd *-vlues. It is generlly know tht E vlues less thn 3. cnnot e detected y humn eyes [45]. When compred E vlues with nother film tht ws founded pectin film hd E lower thn those of egg lumin films (1.7-.3) [46], HPMC mixture films (chitosn/ hydroxypropyl methylcellulose; ) [47]. film opcity vried from.91 to.99 Au nm/µm, film opcity vlues significnt decesed when CCl incresed. The otined results indicted tht films sed on pectin showed closed to vlues to

11 As. J. Food Ag-Ind. 9, (4), the other polyol-plsticized films nd some commonly used synthetic films such s low-density polyethylene (3.5 A6/mm) nd oriented polypropylene (1.67 A6/mm) [48]. Thickness (mm) Concentrtion of pectin (%w/v) Tensile strength (MP) Concentrtion of pectin (%w/v) CCl % CCl 3 % CCl 5 % CCl 7 % CCl 1 % A. CCl % CCl 3 % CCl 5 % CCl 7 % CCl 1 % B. % Elongtion t rek Concentrtion of pectin (%w/v) Wter vpor permeility (g/m/s/p)x Concentrtion of pectin (%w/w) CCl % CCl 3 % CCl 5 % CCl 7 % CCl 1 % C. CCl % CCl 3% CCl 5 % CCl 7 % CCl 1 % D. 4.5 color differentl Concentrtion of pectin (%w/v) film pocity (Auxnm/mm Concentrtion of pectin (%w/v) CCl % CCl 3% CCl 5 % CCl 7 % CCl 1 % E. CCl % CCl 3% CCl 5 % CCl 7 % CCl 1 % F. Figure 6. Physicl properties of pectin films: thickness (A), TS (B), %E (C), WVP (D), E (E) nd film opcity (F). Figure 7. The surfce (A,B,C, D) nd cross-section (E,F,G,H) of pectin films t concentrtion of pectin 3.5%(w/v) with CCl 3,5,7,1%(w/v).

12 As. J. Food Ag-Ind. 9, (4), The results showed tht pectin concentrtion 3% w/v with clcium chloride 3% w/v nd pectin concentrtion 4% w/v without or 3% w/v clcium chloride exhiited etter physicl compring to other film mixtures. Effects of the type nd plsticizer concentrtions on mechnicl properties of pectin film Selected pectin films from section 3.1 were incorported with soritol (SOR) nd gercerol (GLY) t vried concentrtion t 4, 5 nd 6 %w/w of pectin. The mechnicl properties of films re showed in Figure 8. The results indicted tht these properties vried with the nturl of plsticizers used. The thickness of films vried from mm. Glycerol-plsticized films ws thinner thn soritol-plsticized film significntly ecuse glycerol hs lower moleculr weight (9.9) thn soritol (18.). Therefore, the mount of glycerol is lwys less thn soritol t sme level of rtio to pectin [49]. Moreover, film thickness depend on solute solid in film forming solution so ll solid in film were pectin, CCl nd plsticizer [38] so plsticized film were thicker thn non- plsticized film significntly, glycerol-plsticized films more thin thn soritol due to their lowest solid content [49]. Both of glycerol nd soritol hd wter sorption properties tht mde film thicker [5]. TS vried from 14.9 to 6.44 MP nd decresed significnt s concentrtion of plsticizer incresed, TS in films plsticized y soritol ws significntly lrger thn in plsticized y glycerol t the sme concentrtion due to plsticizer molecules ind with wter molecules nd shield ctive centres long polymer chins, therey decresing intermoleculr interction [5] nd plsticizers in this studied is polyol tht hve hydroxyl group which is open chin tht mke competition etween plsticizer nd pectin for clcium. Since polyol plsticizers nd clcium from complex, plsticizer interfered with the formtion of junction zones. Therefore, junction zones re not so dense nd reduced polymerpolymer interction tht effected to TS decresing [51] nd films plsticized with glycerol, which s moleculr weight (MW) of 9, hd out twice higher mol content thn tht of soritol (MW 18) plsticized films [5] nd plsticizer with lower MW more film plsticiztion thn higher MW [49]. %E vried from 5.6 to1.69% nd decresed significnt s concentrtion of plsticizer incresed, glycerol-plsticized films hd significntly lrger %E thn in plsticized y glycerol t the sme concentrtion ecuse plsticizer wekened intermoleculr forces etween djcent polymer chins nd incresed free volume nd chin moility tht mde %E ws incresed [5]. The reson for glycerol-plsticized films hd lrger %E is glycerol hydroscopic chrcter tht tends to provide dditionl wter into the film mtrix nd the mtrix of the film ecome less dense nd under stress movement of polymer chins were fcilitted so incresed free volume nd chin moility[53]. WVP vried from 4.58 to 1.3 µg/m s P. The result showed tht WVP incresed s plsticizer concentrtion incresed, the films plsticized y glycerol hd significntly lger WVP vlues thn film plsticized y soritol t sme concentrtion. The ddition of plsticizer reduced the cohesive forces etween intermoleculr chin nd incresed free volume nd segmentl motions enhnce wter molecules diffused more esily nd higher WVP resulted [46]. glycerol hd significntly lger WVP vlues thn film plsticized y soritol due to glycerol ttriuted higher hygroscopicity reltive therefore possess higher cpcity to sor wter from environment thn soritol [53]. Moreover, film morphology (SEM, Figure 9) of soritol-plsticized films showed more compct nd denser structure thn glycerol-plsticized films so soritol significntly decresed film WVP compred with glycerol [5]. Experiments showed tht E vried from 1.59 to.76 nd did not different significntly etween film plsticized y soritol or glycerol ut E were reduced when compred with un-plsticizer films ecuse dded plsticizer hd cler, glossy nd white tht cn e reduced E vlues. Film opcity vried from 1. to 1.73 Au nm/µm nd did not different significntly etween film plsticized y soritol or glycerol ut film opcity vlues significnt decesed when compred with non-plsticizer films.

13 As. J. Food Ag-Ind. 9, (4), Thickness (mm) hij j ghij ij ij efgh defg efgh fgh fghi de cd c Pectin 3%/CCl 3% Pectin 4%/CCl % Pectin 4%/CCl 3% Film conditions Tensile stregth (MP) j i h g cdef fg cdef cde def efg def cdef cdef cdef cd cde c Pectin 3%/CCl 3% Pectin 4%/CCl % Pectin 4%/CCl 3% Film conditions Plsticizer % Sor 4% Sor 5% Sor 6% Gly 4 % Gly 5% Gly 6% A. Plsticizer % Sor 4% Sor 5% Sor 6% Gly 4 % Gly 5% Gly 6% B. %Elongtion t rek ef c c def gh Pectin 3%/CCl 3% Pectin 4%/CCl % Pectin 4%/CCl 3% c Film conditions f h cd f cd g cdef W ter vpor permeility x1-6 (g /m /s /P ) efg cd cd cd def def c cd c i i Pectin 3%/CCl 3% Pectin 4%/CCl % Pectin 4%/CCl 3% Film conditions cde gh i fg cd Plsticizer % Sor 4% Sor 5% Sor 6% Gly 4 % Gly 5% Gly 6% C. Plsticizer % Sor 4% Sor 5% Sor 6% Gly 4 % Gly 5% Gly 6% D color differentl ghij cdefg cd defghij ij ghij cdefgh Pectin 3%/CCl 3% Pectin 4%/CCl % Pectin 4%/CCl 3% Film conditions cdefgh Plsticizer % Sor 4% Sor 5% Sor 6% Gly 4 % Gly 5% Gly 6% k E. Film opcity (Au x nm/mm) e cd c c c c Pectin 3%/CCl 3% Pectin 4%/CCl % Pectin 4%/CCl 3% Film conditions cde cde Plsticizer % Sor 4% Sor 5% Sor 6% Gly 4 % Gly 5% Gly 6% de F. Figure 8. Physicl properties of pectin plsticizer films: thickness (A), TS (B), %E (C), WVP (D), E (E) nd film opcity (F). Mens within the sme column with sme letter re not significntly different (p.5) Pectin 3 / C 3% Sor 4 Pectin 4 / C % Sor 6 Pectin 3 / C 3% Gly 4 Pectin 4 / C % Gly 6 Pectin 3 / C 3% Sor 4 Pectin 4 / C % Sor 6 Pectin 3 / C 3% Gly 4 Pectin 4 / C % Gly 6 Figure 9. The surfce nd cross-section of pectin films plsticized y soritol nd glycerol.

14 As. J. Food Ag-Ind. 9, (4), From experiment tht showed soritol-plsticized films hd thickness nd TS vlues more thn glycerol-plsticized while film plsticized y glycerol hd %E nd WVP vlues more thn film plsticized y soritol t sme concentrtion. The results showed tht t pectin concentrtion 4% w/v with clcium chloride 3% w/v nd using GLY 5% exhiited etter physicl properties thn other tretments. Effect of concentrtion of ntimicroil extrcts in liposome on mechnicl properties nd ntimicroil ctivity of pectin film Mechnicl properties Liposome ws dded into selected film condition t vried concentrtion (, 4 nd 6% w/w). It ws showed the effect of different concentrtions of liposome incorported into pectin film nd the resultnt chnge in the properties. The mechnicl properties of films re showed in Figure 1. The thicknesses of films vried from.5 to.53 mm. nd did not different significntly etween liposome concentrtions ut thickness vlues significnt incresed when compred with control films. This increse might e due to hydrophilic group of pectin interction fvoured y liposome (hydrophilic group outside) in film-forming solution nd tent to form lrger clusters in the film mtrix tht might e exhiited higher thickness [53]. TS vried from to MP. Incorportion of liposome tht encpsulted ntimicroil extrcts hd effect to TS of film, s seen in the reduced TS vlues when compred with control film ut concentrtion of liposome did not different significntly for TS vlues. It is the reson of reduced TS vlues ecuse of the presence oils in liposome proly interferes with ionic interctions fcilitted y C ions tht cused greter reduction of TS [54] or liposome my weken the strong interctions etween pectin molecules [53]. Generlly, incorportion of dditives other tht mke lower TS vlues [55]. On the other hnd, incorportion of liposome ws cresed %E when concentrtion of liposome incresed lmost two times tht showed liposome hd plsticizer properties. However, ddition of liposome higher thn 4%w/w reduced %E vlue. This is due to non-homogenous distriution of liposome tht mde ggregtes within the finl film structure [56]. WVP vried from 8.55 to 11 µg/m s P. Incorportion of liposome ffected the WVP of pectin films. WVP vlue tended to increse s higher concentrtion of liposome ws incorported. It is proly occurred due to ntimicroil extrcts might contriute to extend intermoleculr interctions of the structurl mtrix in pectin film. Therefore, it enhnced moisture pssing through the film [54]. In ddition, WVP depend on hydrophilic prt nd rtion of hydrophilic: hydrophoic in film [57] therefore in film tht corporte liposome which hd continuous phse ws wter (hydrophilic) so tht incresed hydrophilic prt of film tht result to WVP vlues were incresed. When considered film morphology (SEM, Figure 11) of liposome incorported in pectin film tht ws found film structure hd less dense nd porous structure so WVP vlues were incresed. However, pectin film with liposome still hd lower WVP vlues when compred with other films such s whey protein isolted film (116 g/m s P) [58], geltin film (16.9 g/m s P) [59], chitosn film (117 g/m s P) [6] nd pomce extrct in low methoxyl film (73. g/m s P) [5]. Addition of liposome ffected the ppernce of edile film in oth colour nd film opcity. E vried from 8.57 to nd incresed significntly s liposome dds more higher. The colour tended to yellowish s indicted y the increse of vlue nd L vlues decresed. The vlue produced y the incorportion of liposome tht hd grlic oil nd pomegrnte extrcts encpsulted nd L vlues decresed so it indictes tht the colour of the edile film tends to drken. Film opcity vried from.55 to 6.89 Au nm/µm. Film opcity vlues significnt incresed when liposome concentrtion incresed. Mostly, film trnsprency ws reduced s the ntimicroil gents were incorported [54, 61].

15 As. J. Food Ag-Ind. 9, (4), Thickness (mm) Tensile stregth (MP) liposome % liposome % liposome 4% liposome 6% liposome % liposome % liposome 4% liposome 6% Concentrtion of liposome (%w/w) A. Concentrtion of liposome (%w/w) B. %Elongtion t rek d c liposome % liposome % liposome 4% liposome 6% Wter vpor permeility (g/m/s/p)x c d liposome % liposome % liposome 4% liposome 6% Concentrtion of liposome (%w/w) C. Concentrtion of liposome (%w/w) D. color differentl d c liposome % liposome % liposome 4% liposome 6% Concentrtion of liposome (%w/w) E. Film opcity (Au x nm/mm) d c liposome % liposome % liposome 4% liposome 6% Concentrtion of liposome (%w/w) F. Figure 1. Physicl properties of pectin films incorported of liposome: thickness (A), TS (B), %E (C), WVP (D), E (E) nd film opcity (F) Mens within the sme column with sme letter re not significntly different (p.5). A. B. C. D. E. F. Figure 11. The surfce (A,B.C) nd cross-section (D,E,F) of pectin films incorported liposome t vried concentrtion, 4 nd 6% w/w respectively. The result showed tht TS of the film decresed while the %E, WVP, E nd film opcity incresed when compred to the control, the pectin film incorported with 4% w/w liposome hd highest %E nd the other of physicl properties were cceptle.

16 As. J. Food Ag-Ind. 9, (4), Antimicroil ctivity The results of the ntimicroil ssessment of pectin film incorported with ntimicroil liposome cteri re presented in Figure 1. The cteri selected here re commonly ssocited with food spoilge nd food orne pthogen. The result showed ntimicroil effects of pectin film incorported with the liposome hd no significnt difference. Except those of 4 nd 6 % w/w hd highest inhiition zone ginst Lctocillus sp. nd Lctocillus ske significntly. Inhiition zone (cm) ns ns ns ns ns E.coli 8739 E.coli 59 Slmonell choleresuis Slmonell Typhimurium Pseudomons sp. Lctocillus sp. Lctocillus ske Microorgnism % w/w 4% w/w 6% w/w Figure 1. Inhiition zone of pectin film incorported with the liposome y disc diffusion method Mens within the sme column with sme letter re not significntly different (p.5) Inhiition zone of pectin film incorported with the liposome hd lrger thn.8 cm tht men microorgnism is sensitive to ntimicroil extrcts [8]. This films showed most inhiition in E. coli nd secondly in Slmonell sp. significntly. However, incresing level of ntimicroil liposome t higher concentrtion did not revel significntly n inhiitory of Escherichi coli ATCC 59, Escherichi coli ATCC 8739, Slmonell Typhimurium ATCC 3564, Slmonell Choleresuis ATCC 593, Pseudomons sp. ATCC It ws generlly cused y the mximum cpility of polymer to crry ctive gents eside the occurrence of functionl groups interction phenomenon tht mde ctive compound cn not e relesed out of polymer [6. Generlly, ntimicroil ctivity lwys is higher when ntimicroil concentrtion incresed [63]. These results prove tht liposome could e immoilized in the pectin film nd susequently relesed, therey inhiiting trget microorgnisms.

17 As. J. Food Ag-Ind. 9, (4), Dimeter of inhiition zone (cm) E.coli 8739 ns E.coli 59 ns Slmonell choleresuis ns Slmonell Typhimurium Pseudomons sp. Microorgnisms Lctocillus sp. Lctocillus ske film with liposome %w/w film with liposome 6%w/w film with ethnolic pomegrnte extrct 4% w/w film with liposome 4% w/w film with ethnolic pomegrnte extrct % w/w film with ethnolic pomegrnte extrct 6% w/w Figure 13. Inhiition zone of prepred of pectin film incorported with the liposome nd film incorported pomegrnte extrcted from ethnol t sme concentrtion. Mens within the sme column with sme letter re not significntly different (p.5). When compred of pectin film incorported with the liposome nd film incorported with clove oil or grlic oil or pomegrnte extrcts t sme concentrtion showed in Figure 13. It ws found tht film incorported with clove oil or grlic oil, the inhiition zone of ws not oserved with tested cteri. However, incorportion of pomegrnte extrcts reveled wek inhiitory effect, indicted y miniml growth underneth film discs ws.7 cm tht less thn.8 cm. It mens microorgnisms were not sensitive to ntimicroil film [8]. Therefore, film incorported with ntimicroil extrcts without encpsultion hd ntimicroil ctivity less thn ntimicroil extrcts encpsulted efore incorporte in film. This due to lost of ntimicroil extrcts etween film preprtion or drying [64] especilly in edile films re enriched with essentil oils, the drying tempertures usully were employed to form the edile coting re high enough to voltilize high percentge of the ctive romtic components in essentil oil [65]. Conclusion From the experiment it ws found tht pectin concentrtion 4% w/v with clcium chloride 3% w/v nd using GLY 5% ws good film to use for incorporting liposome. The incorportion of ntimicroil liposome into pectin film leded to moderte chnges in pectin film physicl properties. When testing on ntimicroil ctivity, it ws founded tht microorgnism is sensitive to extremely sensitive to pectin film incorported with the liposome. Pectin film incorported with 4% w/w liposome ws selected due to it high ntimicroil ctivity tht gve lrge inhiition zone significntly to lctic cid cteri nd the other of physicl properties were cceptle. Therefore, n nticteril pectin film incorported with the liposome is promising nd hs good potentil in mny food pplictions.

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