Microencapsulation of Korean Mistletoe (Viscum album var. coloratum) Extract and Its Application into Milk

Transcription

1 299 Asin-Aust. J. Anim. Sci. Vol. 21, No. 2 : Februry 28 Microencpsultion of Koren Mistletoe (Viscum lbum vr. colortum) Extrct nd Its Appliction into Milk N. C. Kim, J. B. Kim 1 nd H. S. Kwk* Deprtment of Food Science nd Technology, Sejong University, 98 Kunj-dong, Kwngjin-ku, Seoul , Kore ABSTRACT : This study ws designed to develop microencpsulted Koren mistletoe extrct, to determine the stbility in vitro nd to exmine its ppliction in milk. Coting mterils used were polyglycerol monosterte (PGMS) nd medium-chin tricylglyderol (MCT). The highest efficiency of microencpsultion ws 78.3% with 15:1:4 (w/w/v) s PGMS : mistletoe extrct : distilled wter nd 66.1% with 15:1 (w/w) s MCT : mistletoe extrct. The size of microcpsule ws bout 3. nd 19.5 μm with PGMS nd MCT, respectively. When microcpsules of mistletoe extrct were incubted in simulted gstric fluid t ph 2 for 6 min, 14.8 nd 17.2% of lectin ws relesed from cpsules which were coted with PGMS nd MCT, respectively. Comprtively, 83.2 nd 87.3% of lectin ws relesed in simulted intestinl fluid (ph 8) fter 6 min incubtion of cpsules coted with PGMS nd MCT, respectively. The subsequent study determined the chnges of physicochemicl nd sensory chrcteristics of milk with fortifiction of the mistletoe extrct microcpsules during 12 dy storge. TBA vlue ws significntly lower in microcpsule-dded groups thn in the uncpsulted mistletoe extrct-dded group during the storge. When 1 ppm microencpsulted mistletoe extrct ws dded, the L-, - nd b- vlues nd viscosity were not significntly different from those of the control. In ddition, the relese of lectin from mistletoe extrct over 12 dys ws 8.3 nd 9.5 mg/1 ml in milk contining microcpsules mde by PGMS nd MCT, respectively. All sensory ttributes showed significnt difference in uncpsulted mistletoe extrct-dded milk compred with other groups. The present study indicted tht microcpsules of Koren mistletoe extrct could be pplied to milk nd microcpsules coted with PGMS were effectively relesed in simulted intestinl environment. (Key Words : Microencpsultion, Koren Mistletoe Extrct, Polyglycerol Monosterte, Medium-chin Tricylglycerol, Milk) INTRODUCTION Mistletoe is semi-obligte prsite plnt tht lives on mny different kinds of trees including ok trees. The mistletoe hs been utilized for long period of time s folk medicine for hypertension nd cncer in Fr Est Asin countries, s well s in Europe (Prk et l., 21). Extrcts from the Europen mistletoe (Viscum lbum L.) re widely used in cncer prevention nd in n djuvnt chemotherpy of humn cncer (Stoev et l., 21). The fvorble effects of extrcts from the Europen mistletoe hve been known for over 7 yers for the tretment of hypertoni, inflmmtory diseses nd lso cncer (Kwj et l., 1986; Hjto et l., 1989; Frnz, 1991; Kuttn, 1993; Gbius et l., * Corresponding Author: H. S. Kwk. Tel: , Fx: , E-mil: kwkhs@sejong.c.kr 1 School of Bioscienec nd Food Technology, Hndong Globl University, Nmsong-ri, Heunghe-up, Puk-ku, Pohng, Kyungpuk , Kore. Received June 28, 27; Accepted September 15, ). Although mistletoe extrcts re known for chemotherpy of vrious diseses, there is very little knowledge bout its stbility inside the body nd biologicl nd physiologicl functions. Also, regrdless of its roles, lectin, which is the min constituent, could be instble nd destroyed in the processing by temperture nd ph, etc. The Koren mistletoe extrct hs high viscosity, undesirble flvor nd color. In order to overcome these problems, the microencpsultion technique my be good ppliction for mistletoe fortifiction to food system. Microencpsultion, which shows potentil s crriers of enzymes in the food industry, could be good vehicle for the ddition of mistletoe to food (Bersen ev et l., 199; Jckson nd Lee, 1991). Currently, there is considerble interest in developing encpsulted minerls, flvors nd enzymes. Among severl fctors to be considered, choice of coting mteril is the most importnt nd depends on the chemicl nd physicl properties of the core mteril, the process used to form microcpsules, nd the ultimte

2 3 Kim et l. (28) Asin-Aust. J. Anim. Sci. 21(2): properties desired in microcpsules. For microencpsultion, severl reserchers hve used coting mterils such s milk ft, gr, nd geltin, etc. responsible for enzyme, flvor nd iron microencpsultion in foods (Brun nd Olson, 1986; Mgee nd Olson, 1981, b). In the present study, ftty cid esters such s polyglycerol monosterte nd medium-chin triglyceride were used since those hve been proven s effective coting mterils in out previous studies (Kwk et l., 21; Kwk et l., 23). However, no study hs exmined the efficiency of mistletoe extrct microencpsultion using ftty cid esters, nd the stbility in vitro. Therefore, the objectives of this study were to develop the optimum conditions for microencpsulted Koren mistletoe extrct, to exmine the stbility of microcpsules in simulted gstrointestinl fluids in vitro, nd to pply the cpsules into milk for development of functionl milk. MATERIALS AND METHODS Mterils For the microencpsultion of Koren mistletoe (Viscum lbum colortum) extrct, polyglycerol monosterte (PGMS) nd medium-chin tricylglycerol (MCT) were used s coting mterils. Those were purchsed from Il- Shin Emulsifier Co., LTD. (Seoul, Kore). Preprtion of plnt mterils of the Koren mistletoe Mistletoes, living on ok trees, were collected on Jnury in the Andong re (Kore). Upon collecting, the mistletoe leves were immeditely stored t -8 C. Distilled wter through the ion-exchnge column ws dded into the freezed Koren mistletoe leves, cut by mixer for 2 min nd then stirred for 16 h t 4 C. The superntnt ws obtined by centrifugtion (1, rpm, 3 min, 4 C), filtered through membrne filter nd then freeze-dried for brown powdered form. Preprtion of microcpsule Microcpsules of Koren mistletoe extrct were mde by MCT or PGMS, which were selected s mjor coting mterils from our previous study (Kwk et l., 21). The rtios of coting mteril to core mteril were 5:1, 1:1, 15:1 nd 2:1 to mximize Koren mistletoe extrct content nd stbility of microcpsules, nd mixed t 1,2 rpm for 2 min with stirrer. An irless pint spryer (W-3, Wgner Spry tech. Co., Mrkdorf, Germny) nebulized coting mteril-koren mistletoe emulsion t 4 C into cylinder contining.5% polyethylene sorbitn monosterte (Tween-6) solution t 5 C. The dimeter of the nozzle orifice ws.4 mm. The chilled fluid ws centrifuged t 4,52 g for 12 min to seprte microcpsule suspension. Microcpsules were formed s lipid solidified in the chilled fluid. For PGMS microencpsultion, the distilled wter ws dditionlly dded becuse PGMS is highly viscous s described in the previous study (Kwk et l., 23). The PGMS nd distilled wter were mixed with the rtio of 15:4 (w/v), heted to 55 C for 2 min, cooled to 4 C. Mistletoe extrct ws then dded into the emulsion nd stirred with 1,2 rpm for 3 sec for sprying. Both MCT nd PGMS microencpsultion were done in triplicte. Microencpsultion efficiency of mistletoe extrct The dispersion fluid of microencpsultion ws ssyed for untrpped lectin in Koren mistletoe extrct (.5% of Koren mistletoe extrct is ssumed s lectin). One milliliter of the dispersion fluid ws tken nd filtered through Whtmn No. 54, followed by membrne filtrtion (di..4 μm, Whtmn Interntionl Limited, Mdistone, Englnd). The solute ws stored in the refrigertor until nlysis. Lectin ssy in Koren mistletoe extrct were mesured by enzyme-linked immunosorbent ssy (ELISA) by the method described by Yoon et l. (21). Flt-bottomed microtitrtion pltes were coted with 1 μl per well of 1st ntibody (rbbit nti-kml-c polyclonl ntibody) in 5 mm crbonte-bicrbonte buffer (ph 9.6) by overnight incubtion t 4 C or 2 h t 37 C. Excess ntibody ws wshed off with distilled wter contining.5% Tween-2 nd the coted wells were blocked with 2 μl per well of 2% bovine serum lbumin (BSA) in sline for 2 h t 37 C. Therefter, smple or corresponding stndrd required for the clibrtion curve (KML-C, top 2 μg/ml seril dilution) were dded to ech well in duplicte. The plte ws incubted t 37 C for 2 h. After wshing, 2nd ntibody tht mouse nti-kml-c monoclonl ntibody peroxidse conjugte t working strength (1:3,) in PBS ws dded into ech well nd incubted t 37 C for 1 h. After three wshings with.5% Tween-2 in distilled wter, the ntibody-ntigen rection ws reveled by dding 1 ml of substrte to ech well (.25 M 3,3',5,5' tetrmethylbenzidine, hydrogen peroxide in 5 mmol sodium cette buffer ph 5.2). The rection ws stopped fter 15 min by the ddition of 5 ml of 2 M sulfuric cid per well. The bsorbnce ws mesured t 45 nm. Microscopic observtion The micro-structurl imge of the cpsule ws mgnified by 1-folds with light microscope (Olympus Opticl Co., LTD., Jpn) nd photogrphed. In vitro study To determine the stbility of lectin in Koren mistletoe extrct in the stomch nd smll intestine, the simulted gstrointestinl solutions were prepred s follows: 1)

3 Kim et l. (28) Asin-Aust. J. Anim. Sci. 21(2): Tble 1. Microencpsultion efficiency of Koren mistletoe extrct with different rtios of coting to core mterils 1 Rtio (w/w) Microencpsultion efficiency (%) Coting mteril Core mteril PGMS 2 MCT d 43.1 b b c 39.6 b 1 Mens of triplicte. Mens in column without the sme letter re significntly different (p<.5). Other experimentl fctors: mixing temperture: 4 C, mixing speed: 1,2 rpm, centrifugl force: 2,49 g, nd centrifugl time: 15 min. 2 Polycylglycerol monosterte, 4 ml of distilled wter ws dded. 3 Medium-chin tricylglycerol. gstric fluid prepred in smple solution contining pepsin (ph 1.2) nd simulted into ph 2 using by 2 N HCl nd NOH, nd 2) intestinl fluid ws prepred in.1 M PBS buffer (1 ml, ph 7.4) contining 2 mg pncretin, 5 mg lipse, 1 mm cholic cid nd 1mM deoxycholic cid, nd simulted s ph 8. In gstric fluid, the microcpsules of mistletoe extrct in distilled wter were incubted t 37 C with 1 rpm shking, nd in intestinl fluid, they were incubted t 37 C with the smple collecting t 1 min intervl. The treted smples were centrifuged t 2,49 g nd the superntnt ws mesured for mistletoe extrct content relesed from the microcpsules. All tretments were triplicte. Stbility of microcpsules Thiobrbituric cid (TBA) test : The bsorbnce chnge by ddition of mistletoe into milk ws mesured using TBA test t 5 C for 12 dys. Oxidtion products were nlyzed spectrophotometriclly. The regent for the TBA test ws prepred immeditely before use by mixing equl volumes of freshly prepred.25 M TBA, which ws neutrlized with NOH nd 2 M H 3 PO 4 /2 M citric cid. Rections of the TBA test were strted by pipetting 5. ml of milk contining mistletoe extrct encpsulted or uncpsulted into glss centrifuge tube nd mixed thoroughly with 2.5 ml of TBA regent. The mixture ws heted immeditely in boiling wter bth for exctly 1 min nd cooled in ice. Ten millilters of cyclohexnone nd 1 ml of 4 M mmonium sulfte were dded nd centrifuged t 2,49 g for 5 min t room temperture. The ornge-red cyclohexnone superntnt ws decnted nd its bsorbnce t 532 nm mesured spectrometriclly in 1-cm light pth. All mesurements were run in triplicte. Lectin relese during storge : To mesure the stbility of lectin in Koren mistletoe extrct microcpsules, 1 mg of mistletoe extrct regrdless of encpsulted or uncpsulted, ws dded to mke 1 ppm, the mixture ws stored t 5 C for 12 dys, nd its stbility ws mesured t 2 dy intervls. The smples were centrifuged, nd the collected superntnt ws nlyzed for the determintion of lectin content relesed from microcpsules. All mesurements were mde in triplicte. Color nlysis Color vlue ws mesured using colorimeter (CR21, Tokyo, Jpn). Viscosity nlysis The viscosity of 5 ml milk smples ws mesured t 5 C using viscometer (VISCO STAR-L:J.P. Select S.A., Spin) with single spindle t 2 rpm. All smples were mesured in triplicte. Sensory nlysis Commercil whole milk (t every period) contining 1 ppm uncpsulted nd encpsulted Koren mistletoe extrct ws stored t 5 C for, 2, 4, 6, 8, 1 nd 12 dys. Seven semi-trined sensory pnelists were recruited from fculty nd grduted students in the Deprtment of Food Science nd Technology t Sejong University nd evluted in the milk smples throughout the study. The stringency, bitterness, yellowish, off-flvor nd herb flvor were scored on seven-point scle (1 = very slight, 3 = slight, 5 = strong nd 7 = very strong), nd overll cceptbility were lso scored on seven-point scle (1 = dislike extremely, 3 = dislike modertely, 5 = like modertely nd 7 = like extremely). Sttisticl nlysis Dt from ech experiment were nlyzed by nlysis of vrince (ANOVA) using SAS progrm (1985) nd differences mong tretments were determined by Duncn s multiple test t p<.5, unless otherwise stted. RESULTS Microencpsultion PGMS : When the rtio of PGMS to distilled wter ws 15g:4 ml, the optimum rtio of PGMS to mistletoe extrct, (5:1, 1:1, 15:1 nd 2:1) ws exmined s shown in Tble 1. Efficiency of the microencpsultion incresed up to 15:1 (w/w) (coting to core rtio) with 4 ml distilled wter in spry solution nd decresed with 2:1 rtio. When PGMS ws 15 g nd mistletoe extrct ws 1 g, the efficiency of microencpsultion ws 74.5% s the highest vlue. This result ws in ccordnce to our previous study (Kwk et l., 21), indicting tht too much ddition of distilled wter for micorencpsultion reulted in wek-microcpsule cot, therefore, efficiency ws decresed. Severl studies (Kwk et l., 23; Lee et l., 23; Choi et l., 26) hve reported the possibility of PGMS s coting mteril for efficient formtion of microcpsules.

4 32 Kim et l. (28) Asin-Aust. J. Anim. Sci. 21(2): () 5 Lectin relese (%) PGMS microcpsule MCT microcpsule (b) Time (min) Figure 2. Koren mistletoe extrct relese from microcpsules with PGMS or MCT incubted in simulted gstric fluid in vitro. Simulted gstric fluid contining pepsin nd djusted to different phs with HCl nd NOH t incubted t 37 C for 6 min. Ech br represents n verge of three trils. Ech br indictes stndrd devition nd br with different letters re significntly different (p<.5). Figure 1. Photomicrogrph of microencpsulted Koren mistletoe extrct with polyglycerol monosterte () nd mediumchin tricylglyerol (b). The photogrph ws tken t 4 mgnifiction. When iron ws microencpsulted by PGMS, the efficiency ws 75% (Kwk et l., 23). Other studies using scorbic cid (Lee et l., 23) nd chitooligoscchride (Choi et l., 26) s core mteril indicted tht 94.2% nd 92.1% of microencpsultion efficiency ws found, respectively. MCT : The efficiency of microencpsultion by MCT is lso shown in Tble 1. The efficiency ws the highest (54.4%) when the cot to core rtio ws 1:1. Significnt differences were not found between those of 1:1 (54.4%) nd 15:1 (52.9%) (p>.5). In the cse of 2:1, MCT ws left over in the upper lyer of dispersion fluid fter centrifugtion. Therefore, the optimum rtio of MCT to mistletoe ws found to be 15:1, even though left over MCT ws still found in the upper lyer. Our previous study indicted tht the efficiency of isoflvone microencpsultion incresed stedily up to 15:1 of cot-to-core rtio nd ws the gretest (7.2%) when isoflvone ws microencpsulted with MCT (Kim et l., 26). Since the microencpsultion efficiency with MCT hs been lower thn tht with PGMS in our studies, it is indicted tht MCT my not be the dequte coting mteril for microencpsultion. Microscopic observtion Photomicrogrph of microencpsulted mistletoe with PGMS or MCT is shown in Figure 1. In the cse of both PGMS nd MCT microcpsules, the sizes were vrious nd the verge sizes were bout 3 nd 19.5 μm, respectively (Figure 1). Microscopic exmintion of microcpsules reveled sphericl prticles nd microcpsules contining mistletoe extrct hd smooth surfces nd evenly distributed pockets of mistletoe solution in microcpsules mde by both PGMS nd MCT. In vitro study This study ws conducted to determine whether the microcpsules relesed Koren mistletoe extrct during simulted gstric intestinl conditions (Figure 2). When incubted t ph 2, less thn 3% lectin ws relesed from the microcpsules t 1 min incubtion nd it ws incresed up to 13-15% t 3 min nd plteued therefter. From 1-3 min incubtion, there ws drmtic increse from to 15%. To determine how effectively mistletoe extrct ws relesed in the intestine, simulted intestinl fluid (ph 8) ws prepred with the presence of pncretin nd bile slts, nd incubted during t 37 C for 6 min (Figure 3). When both ph nd the durtion of incubtion incresed, lectin relese from mistletoe microcpsules incresed drmticlly.

5 Kim et l. (28) Asin-Aust. J. Anim. Sci. 21(2): Control(no ddition) Uncpsulted Lectin relese (%) PGMS microcpsule MCT microcpsule Absorbnce (53 nm) PGMS microcpsule MCT microcpsule Time (min) Figure 3. Koren mistletoe extrct relese from microcpsules with PGMS or MCT incubted in simulted intestinl fluid in vitro. Simulted intestinl condition included enzymes such s lipse (5 mg) nd pncretin (2 mg) nd incubted t 37 C for 6 min. Ech br represents n verge of three trils. Ech br indictes stndrd devition nd br with different letters re significntly different (p<.5). Less thn 3% of the entrpped mistletoe extrct ws relesed t ph 8 from to 1 min nd drmtic increse (bout 3 times) ws observed t 3 min incubtion nd mintined therefter. When incubted t ph 8, 8-82% lectin ws relesed from microcpsules mde by MCT or PGMS t 3 min incubtion nd therefter, respectively. Stbility of microcpsules during storge TBA test : The effect of mistletoe extrct fortifiction in milk on chemicl oxidtion (s mesured by the TBA test) during 12 dys of storge is shown in Figure 4. The tretment ws divided into 4 different groups s follows: (1) control, no ddition of mistletoe extrct microcpsules; (2) Uncpsulted, 1 ppm of uncpsulted Koren mistletoe extrct, 1 mg/1 ml mistletoe extrct-dded milk; (3) PGMS microcpsule, 1 ppm of PGMS encpsulted mistletoe extrct; (4) MCT microcpsule, 1 ppm of MCT encpsulted mistletoe extrct. In ll groups, TBA bsorbnce incresed proportionlly to storge period. In the 1 ppm of uncpsulted mistletoe extrct-dded group, TBA bsorbnce incresed drmticlly from.11 to.34 from to 12 dys. TBA bsorbnce ws significntly lower in the encpsulted group thn in uncpsulted group t 12 dys of storge. When 1 ppm of mistletoe extrct ws dded, the TBA bsorbnce difference between uncpsulted (.34) nd encpsulted groups (.24 nd.27 for PGMS nd MCT, Storge period (dy) Figure 4. Effect of microencpsulted Koren mistletoe extrct on bsorbnce chnge (TBA vlue) in milk stored t 5 C for 12 dys. Relese of lectin (mg/1 ml) PGMS microcpsule MCT microcpsule Figure 5. Relese of lectin from Koren mistletoe extrct microcpsules in milk stored t 5 C for 12 dys. b Storge period (dy) respectively) ws significnt t 6 dys of storge nd therefter. This result indicted tht chemicl lipid oxidtion proceeded more rpidly in milk with uncpsulted mistletoe extrct dded thn in milk with encpsulted mistletoe extrct dded regrdless of coting mterils. Lectin relese : To exmine the stbility of microencpsules during the storge, 1 ml of microencpsulted solution (1 mg/ml) ws mixed with 1 ml of commercil milk, nd the mixture stored t 5 C for 12 b b

6 34 Kim et l. (28) Asin-Aust. J. Anim. Sci. 21(2): Tble 2. Color vlue of microencpsulted Koren mistletoe extrct in milk stored t 5 C for 12 dys 1 Tretment L-vlue -vlue b-vlue Control b 5.32 Uncpsulted b b PGMS microcpsule b 5.5 MCT microcpsule b Mens of triplictes. Mens in column with sme letter re not significntly different (p<.5). 2 Mrket milk stored t 5 C for 1 dy (no microcpsule dded). dys. The relese of lectin from the mistletoe extrct microcpsules ws then determined fter, 2, 4, 6, 8, 1 nd 12 dy s shown in Figure 5. The relese of lectin (%) from mistletoe extrct microcpsules mintined bout 4 mg/1 ml up to 4 dys nd incresed therefter. When PGMS ws used s coting mteril, 6.1% of lectin ws relesed t 6 dys nd incresed up to 8.3% t 12 dys. Comprtively, when MCT ws used, 6.6 nd 9.5% lectin were relesed t 6 nd 12 dys of storge, respectively. In ddition, significnt difference in lectin relese between the mistletoe extrct microcpsules ws found. Color The color vlues of milk smples with the ddition of encpsulted nd uncpsulted mistletoe extrct were shown in Tble 2. The color chnge ws significnt with uncpsulted mistletoe extrct, while slight but not significnt chnge ws found with both encpsulted mistletoe extrct smples. The L-vlue in uncpsulted mistletoe extrct-dded group ws significntly lower thn ll other encpsulted-dded nd control groups (p<.5). No difference ws found between encpsulted microcpsule-dded groups with both PGMS nd MCT microcpsule groups nd control group. The -vlue ws significntly lower in uncpsulted mistletoe extrct-dded group thn those in other groups (p<.5), which indicted less brown color with mistletoe extrct. Koren mistletoe extrct microcpsule-dded groups with PGMS nd MCT were not significntly different from the control group, which ws the commercil milk without ny ddition. The b-vlue chnge ws similr to those of L- nd -vlues. These results indicted tht the chnges of color vlue were remrkbly msked by the microencpsultion. Viscosity The viscosity of uncpsulted mistletoe extrct-dded group ws significntly higher thn those in encpsulteddded nd the control groups t every storge periods (p<.5) (Figure 6). When 1 ppm of uncpsulted mistletoe extrct ws dded into milk smples, viscosity ws 1.23 cps t the initil period nd kept incresing Viscosity (cp) Storge period (dy) Control (no ddition) Uncpsultd PGMS microcpsule MCT microcpsule Figure 6. Effect of microencpsulted Koren mistletoe extrct on viscosity in milk stored t 5 C for 12 dys. throughout the storge period. However, viscosity vlues in milk smples dded by encpsulted mistletoe extrct with PGMS nd MCT were bout.83 nd.79 cps t the initil period, respectively, which ws similr to tht of the control (.86 cps). Viscosity vlue incresed stedily throughout the storge period nd reched up to.92 cps (PGMS) nd.99 cps (MCT) t 11 dy storge nd those were not significntly different from tht of the control (.84 cps). These results my indicte tht microencpsultion of mistletoe extrct could prevent significnt chnge of viscosity in milk. Sensory nlysis The sensory properties of mistletoe extrct-fortified milk stored t 5 C for 12 dys were evluted (Tble 3). When 1 ppm of microencpsulted mistletoe extrct hd been dded into milk, stringency score ws significntly different from tht of the control nd Koren mistletoe extrct-dded groups regrdless of microencpsultion throughout the storge (p<.5). When 1 ppm of uncpsulted mistletoe extrct hd been dded, stringency score ws drmticlly higher thn those of microencpsulted groups with PGMS nd MCT (p<.5). The scores of bitterness nd yellowish, no difference ws found between the control nd microencpsulted groups, however, those were significntly higher in milk-dded uncpsulted Koren mistletoe extrct compred with other groups (p<.5). Not much difference ws found in offflvor nd herb flvor mong groups. In respect of the overll cceptbility, the control nd 1 ppm microencpsulted mistletoe extrct-dded groups showed high consumer preference throughout the storge, while uncpsulted mistletoe extrct-dded milk showed less thn

7 Kim et l. (28) Asin-Aust. J. Anim. Sci. 21(2): Tble 3. Sensory scores of Koren mistletoe extrct microcpsule-dded milk stored t 4 C for 12 dys 1 Sensory description 3 Storge period Tretment 2 (dy) Astringency Bitterness Off-flvor Herb flvor Yellowish 4 Overll cceptbility Control 1. c 1. b b 5.14 Uncpsulted c PGMS microcpsule 1.36 b 1.14 b b 4.16 b MCT microcpsule 1.47 b 1.34 b b 4. b 2 Control 1. c 1.2 b c 4.89 Uncpsulted c PGMS microcpsule 1.5 b 1.6 b b 4.3 b MCT microcpsule 1.51 b 1.5 b b 3.78 b 4 Control 1.25 c 1.2 c 1.67 b b 4.89 Uncpsulted c PGMS microcpsule 1.58 b 1.74 b 1.83 b b 3.89 b MCT microcpsule 1.66 b 2.18 b 1.79 b b 3.96 b 6 Control 1.41 c 1.22 c 1.63 c 1. b 1.35 c 4.38 Uncpsulted c PGMS microcpsule 1.65 b 1.76 b 1.93 bc 1.24 b 1.85 b 3.66 b MCT microcpsule 1.69 b 2.15 b 2.28 b 1.2 b 1.96 b 3.29 b 8 Control 1.39 c 1.54 b 1.76 c 1. b 1.47 c 4.36 Uncpsulted c PGMS microcpsule 1.56 c 1.81 b 2.15 bc bc 3.69 b MCT microcpsule 1.96 b 1.91 b 2.46 b b 3.34 b 1 Control 1.42 c 1.51 b 2.5 b 1. c 1.41 c 4.7 Uncpsulted c PGMS microcpsule 1.83 b 2.14 b 2.63 b 1.75 b 1.87 b 3.72 b MCT microcpsule 1.92 b 2.18 b 2.79 b b 3.58 b 12 Control 1.67 c 1.7 c 2.1 c 1. c 1.68 b 3.95 Uncpsulted c PGMS microcpsule 1.8 bc 2.23 bc 3.13 b 2.3 b 1.87 b 3.4 b MCT microcpsule 2.1 b 2.49 b 3.36 b 2.39 b 2.18 b 3.36 b 1 Mens within column by the sme letter t ech storge period re not significntly different (p<.5). 2 Control: mrket milk, Uncpsulted: uncpsulted mistletoe extrct-dded milk, PGMS microcpsule: microencpsulted mistletoe extrct by polyglycerol monosterte-dded milk, MCT microcpsule: microencpsulted mistletoe extrct by medium-chin triglyceride-dded milk. 3 The scle of strigency, bitterness, off-flvor, herbs flvor, color scores: 1 = very slight, 2 = slight, 3 = slight-moderte, 4 = moderte, 5 = modertestrong, 6 = strong, 7 = very strong. The scle of overll cceptbility score: 1 = dislike extremely, 4 = neither like nor dislike, 7 = like extremely. 4 Yellow color. score 2 throughout the storge. These resulted indicted tht Koren mistletoe extrct-dded milk ws shown to be well mintined by the encpsultion of mistletoe extrct during 12 dy storge. DISCUSSION It is well known tht the crude extrct of the Koren mistletoe (Viscum lbum colortum) contins immunomodultory effects. The extrct induces mouse mcrophges to produce cytokines, which leds to nticncer ctivity in mouse metstsis model system (Yoon et l., 1995). The lectin tht ws purified from the extrct proved to be responsible for the production of the sytokines nd the nti-tumor ctivity. Additionlly, the lectin lso induced poptotic cell deth of severl cncer cell lines (Yoon et l., 1999). On the bsis of the bove informtion, we decided to pply the Koren mistletoe extrct for milk fortifiction using microencpsultion process to overcome the undesirble flvor, color nd stbility. When microencpsultion efficiency of PGMS or MCT ws determined, the efficiency ws the highest with 15:1 or 1:1 (w/w) of cot-to-core rtio, respectively. In the cse of 2:1 (w/w), both PGMS nd MCT were leftover in the upper lyer of the dispersion fluid fter centrifugtion. Our previous study (Kwk et l., 23) showed the highest efficiency (75%) with 5:1:3 rtio (w/w/v) s coting to core mterils to distilled wter. Tht study showed tht the conditions for microencpsultion depended on the rtio of coting to core mterils, the viscosity of the spry solution, kinds of coting nd core mterils, nd food system to which it ws to be pplied. Lectin in Koren mistletoe extrct hs been recognized tht my confer significnt long-term benefits if incorported into the diet either nturlly s n integrl prt of the food or s food supplement. It is the concept tht hs resulted in the development of the reltively new field of functionl foods. Therefore, n experiment should be

8 36 Kim et l. (28) Asin-Aust. J. Anim. Sci. 21(2): performed to determine how stble the microcpsules were in the stomch nd how effectively they were relesed in the intestine. It is generlly ccepted tht for n effective uptke if nutritionl effect from microcpsules, severl problems need to be solved such s the cpsules hve to contin s much ctivity s possible, hve to resist the gstric nd intestinl fluids, nd hve to be cptured by the enterocytes before being relesed into the blood circultion. As expected, the present study indicted tht little mount of lectin in Koren mistletoe extrct ws relesed t low ph. Comprtively, those releses incresed drmticlly in neutrl ph, which ws similr condition to tht of the intestine. These results lso indicted tht microcpsules would be convenient tool for lectin fortifiction becuse of n increse of bsorption by flvoring the uptke nd effective relese in the intestine. The present study showed possible ppliction in encpsulted lectin in Koren mistletoe extrct fortifiction using MCT or PGMS, which my be used effectively in the food system. ACKNOWLEDGMENT This reserch ws supported by grnd of locl industry new technology fusion development project, the Smll & Medium Business Administrtion (SMBA) in Degu, Kore. REFERENCES Bersen ev, E. A., A. A. Innov, T. P. Snsonov, E. M. Chernov, nd N. I. Orgvelidze Microencpsulted romtizers for te. Pishchevy Pomyshlennost, USSR 1: Brun, S. D. nd N. F. Olson Encpsultion of proteins nd peptides in milk ft. Encpsultion efficiency nd temperture nd freezing stbility. J. Microencpsultion 3(2): Choi, H. J., J. Ahn, N. C. Kim nd H. S. Kwk. 26. The effects of microencpsulted chitooligoscchride on physicl nd sensory properties of the milk. Asin-Aust. J. Anim. Sci. 19: Frnz, H In: Advnces in lectin reserch (Ed. H. Frnz, Kichi Ksi, J. Kochurek, S. Olsnes, L. M. Shnnon). pp. 4:33-5. Verlg Gesundheit nd Springer Verlg, Berlin. Gbius, H. J., S. Gbius nd S. S. Joshi Form ill-defined to the immunomodultory lectin: will there be reson for oncologicl of mistletoe: Plnt Med. 6:2-7. Hjto, T., K. Hostnsk, nd H. J. Gbius Modultory potency of the β-glctosidse-specific lectin from mistletoe (lscdor) on the host defense system in vivo in rbbits nd ptients. Cncer Res. 49: Jckson, L. S. nd K. Lee Microencpsulted iron food fortifiction. J. Food Sci. 56: Kim, N. C., B. J. Jeon, J. Ahn nd H. S. Kwk. 26. In vitro study of microencpsulted isoflvone nd β-glctosidse. J. Agric. Food Chem. 54: Kuttn, G Tumoricidl ctivity of mouse peripherl mcrophges treted with Viscum lbum extrct. Immunol. Investig. 22: Kwj, T. A., C. B. Dis nd S. Pentecost Recent studies on the nticncer ctivities of mistletoe (Viscum lbum) nd its lkloids. Oncology 43:42-5. Kwk, H. S., M. R. Ihm nd J. Ahn. 21. Microencpsultion of β-glctosidse with ftty cid esters. J. Diry Sci. 84: Kwk, H. S., K. M. Yng nd J. Ahn. 23. Microencpsulted iron for milk fortifiction. J. Agric. Food Chem. 51: Mgee, E. L. Jr. nd N. F. Olson Microencpsultion of cheese ripening systems: Formtion of microcpsules. J. Diry Sci. 64:6-61. Mgee, E. L. Jr. nd N. F. Olson. 1981b. Microencpsultion of cheese ripening systems: Stbility of microcpsules. J. Diry Sci. 64: Lee, J. B., J. Ahn, J. H. Lee nd H. S. Kwk. 23. The microencpsulted scorbic cid relesed in vitro nd its effect on iron biovilbility. Arch. Phrm. Res. 26: Prk, C. H., D. W. Lee, T. B. Kng, K. H. Lee, T. J. Yoon, J. B. Kim, M. S. Do nd S. K. Song. 21. cdna cloning nd sequence nlysis of the lectin genes of the Koren mistletoe (Viscum lbum colortum). Mol. Cells 12: SAS User s Guide: Sttistics, Version 5 Edition. SAS Institute, Inc., Cry, NC. Stoev, S., M. Frnz, R. Wcker, R. Kruspenhr, E. Guthohrlein, A. Mikhilov, C. Betzel nd W. Voelter. 21. Promry structure, isoforms, nd moleculr modeling of chitinbinding mistletoe lectin. Arch. Biochem. Biophys. 392: Yoon, T. J., Y. C. Yoo, O. B. Choi, M. S. Do, T. B. Kng, S. W. Lee, I. Azum nd J. B. Kim Inhibitory effect of Koren mistletoe (Viscum lbum colortum) extrct on tumor ngiogenesis nd metstsis of hemtogenous nd nonhemtogenous tumor cells in mice. Cncer Lett. 97: Yoon, T. J., Y. C. Yoo, T. B. Kng, E. Her, S. H. Kim, K. Kim, I. Azum nd J. B. Kim. 21. Cellulr nd humorl djuvnt ctivity of lectins isolted from Koren mistletoe (Viscum lbum colortum). Int. Immunophrmcol. 1: Yoon, T. J., Y. C. Yoo, T. B. Kng, K. Shimzki, S. K. Song, K. H. Lee, S. H. Kim, C. H. Prk, I. Azum nd J. B. Kim Lectins isolted from Koren mistletoe (Viscum lbum colortum) induce poptosis in tumor cells. Cncer Lett. 136:33-4.