World Applied Sciences Journal 17 (Towards the Traceability of Halal and Thoyyiban Application): 51-56, 2012 ISSN IDOSI Publications, 2012

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1 World Applied Sciences Journl 17 (Towrds the Trceility of Hll nd Thoyyin Appliction): 51-56, 2012 ISSN IDOSI Pulictions, 2012 Detection of Cholesterol Oxidtion Products (COPs) in Rw nd Chilled Storge of Chicken Susges Formulted with Chicken Ft nd Red Plm mid Frction A.R. Alin, M.I. Fhmi, Z.H. Shzmwti, M.J. Them Juhn, J. Jurini nd A. Siti Mshitoh 1 Institute of Hll Reserch nd Mngement, Universiti Sins Islm Mlysi, Bndr Bru Nili, Nili, Negeri Semiln, Mlysi 2 Fculty of Science nd Technology, Universiti Sins Islm Mlysi, Bndr Bru Nili, Nili, Negeri Semiln, Mlysi Astrct: The purpose of this pper is to determine the effect of different lipid sources (niml nd plnt) during chilled storge on the formtion of cholesterol nd cholesterol oxidtion products in susges formulted with chicken ft nd red plm mid frction. The commercil smple, s represented y the chicken susge nd the mechnicl deoned met susge ws nlyzed s comprison. The susges were produced in plnt scle for two tches, vcuum pcked nd stored t -4 C or chilled condition. At time intervls of week 0, 1, 2 nd 3, the susges were nlyzed using gs chromtogrphy with flme ioniztion detector for determining cholesterol nd cholesterol oxidtions products, which were 25-hyroxycholesterol, -epoxycholesterol, - epoxycholesterol nd 7-ketocholesterol. This study showed the vrile mount of compounds nlyzed throughout the period of nlysis, 25-hydroxycholesterol detected in t week 3 (0.77 ppm). The significntly high mount of cholesterol ws detected in ( ppm) t week 3. The different type of susges formulted with chicken ft nd plm mid frction which were niml nd plnt fts, respectively did not show ny significnt chnges towrds the formtion of cholesterol nd COPs throughout the storge period in chilled condition. It is recommended for future works to prolong the period of storge to otin concrete result t the end of nlysis, nlyze the compounds using gs chromtogrphy with mss spectrometry to improve the detection limit nd to expnd the reference stndrd of cholesterol oxidtion products to e used s the compounds my vries. Key words: Cholesterol Cholesterol oxidized products Susges INTRODUCTION chin of the molecule. Oxidtion of lipids nd sterol (cholesterol) follows the sme oxidtion ptterns such s Lipids re orgnic compounds tht re found in utoxidtion, photoxidtion nd enzymic oxidtion tht living orgnisms nd these lipids re solule in nonpolr will produce hydroperoxides [3]. solvents [1]. Lipids include ll types of fts, oil, The hyroperoxides derived from oxidtion of phospholipids, sterols nd wxes [2] cn undergo unsturted ftty cids ply significnt role to fcilitte ltertions during the storge of food with consequent cholesterol oxidtion t -5 doule ond, which is more losses in nutritionl vlue. Bsiclly, cholesterol present sensitive to oxidtion [3]. In ddition, the foods with high in niml origin foods will undergo utoxidtion during cholesterol contents such s egg, whole milk, met, se processing s well s during storge which cn produce food cn hve COPs tht re present in our diet. However, toxic products. Cholesterol oxidtion products (COPs) the fresh foods contin low levels of COP nd the levels re similr to cholesterol, which contin n dditionl my go up during processing nd storge (time nd functionl group, for instnce hydroxyl, ketone or n condition) [4]. Generlly, het, ph, light, oxygen, wter epoxide group in the sterol nucleus nd/or on the side ctivity nd the presence of unsturted ftty cids re Corresponding Author: Shzmwti Zm Hshri, Fculty of Science nd Technology, Universiti Sins Islm Mlysi, Bndr Bru Nili, Nili, Negeri Semiln, Mlysi. Tel: , E-mil: shzzh@gmil.com. 51

2 World Appl. Sci. J., 15 (Towrds the Trceility of Hll nd Thoyyin Appliction): 51-56, 2011 the mjor fctors tht influence COP formtion during Sigm Chemicl Compny. The purities of the stndrds food processing or storge [4, 5]. COPs hve een shown rnged from 95% to 99%. Chemicls (hexne nd to promote the formtion of ftty plques in the rteries, isopropnol) used for chromtogrphy nlysis were toxic to living cells, mutgenic nd possily crcinogenic chromtogrphy grde (Merck) nd ll other nlyticl ffects in oth in vivo nd in vitro studies. COPs lso grde solvents were from Sigm Chemicl. cuse cell memrne dmge nd inhiition of cholesterol iosynthesis. Severl investigtions hve demonstrted Susges Formultion nd Preprtion: Two types of tht 25-OH cholesterol nd triol re toxic gents cusing chicken susges formulted with different type of fts, therosclerosis. The presence of -epoxy cholesterol nd i.e. chicken ft (CF), red plm mid frction () which re -epoxy cholesterol hs een reported to e crcinogenic niml ft nd plnt lipid, respectively, were prepred in [6, 7]. plnt scle y using fcilities nd ingredients provided y The consumption of pre-cooked foods of niml locl met products mnufcturer, OmCorp Sdn. Bhd. origin is ecoming more populr in the world due to the (Kjng, Selngor, Mlysi). Two tches of susges low cooking time. However, the intke of cholesterol oxide were produced s replictions of the nlysis. contining foods hs d ffects on helth. Thus, it is susge ws purchsed from two different mrkets ut importnt to identify nd quntify these products in foods with similr rnds tht represent two tches of the in the mrket [8, 9]. From the nutritionl point of view, susge. susges re n importnt source of proteins of high Five kilogrm of ech type of susges (chicken iologicl vlue [10]. However, this trditionl processed susges formulted with chicken ft nd chicken met product show some negtive spects s susge formulted with red plm mid frction) were consequence of their high niml ft content, including prepred. The susge ws prepred y dding chicken cholesterol nd COPs. The reltively high cholesterol rest met into 150 litres owl cutter. All the level is one of the enhncing fctors for some diseses ingredients were homogenized etween 10 to 15 mins. The like coronry hert diseses [11]. susge tter mixtures were trnsferred to n utomted Although the people hve commonly consumed stuffer nd the cellulose csing with 10 mm in dimeter vrious process met products for mny yers, little ws used to stuff the susge. Then, it ws stemed for informtion is ville on the occurrence of cholesterol n hour t 80 C, rinsed with cold wter nd the csing ws nd COPs in commercilly mnufctured susges. Few peeled. Upon cooling, the tches were vcuum-pcked integrted studies cn e found in the literture on nd stored t 4 C. cholesterol nd, COPs, virtully none verifying the effect of storge on these products. Thus, the ojectives of this Tretment of Smples: The susges were treted y study re to investigte the chilled storge susges nd storing it chilled (4 C) nd nlyzed for COPs t time the type of fts, prticulrly chicken ft nd red plm mid intervls of 0, 1, 2 nd 3 weeks. Prior to tht, moisture frction on the formtion of cholesterol nd COPs, in content of susges ws determined y using moisture comprison with commercilized chicken susge in the nlyzer. mrket or chicken susge. Anlysis of Cholesterol Oxidtion Products (COPs): MATERIALS AND METHODS Sponifiction of COPs: Cholesterol nd cholesterol oxidtion products were sponified s descried y Smple: Red plm mid frction were otined from Sldnh et l. [12]. Approximtely 2 g of ground Crotino Sdn Bhd (Psir Gudng, Johor, Mlysi). susges were weighed respectively into 100 ml of Chicken, chicken ft nd ingredients of susges were conicl flsk. A totl of 10 ml of KOH (1 M) in 95% supplied y OmCorp Sdn. Bhd. (Kjng, Selngor, ethnol ws dded to the smple t room temperture for Mlysi). 22 hrs in the drk. For the extrction of the unsponifile mtter, 5 ml of distilled wter nd 10 ml of hexne were TM Chemicls: Ftty cids stndrd mixtures (Supelco 37), dded to the smples nd the mixture ws shken y ws purchsed from Sigm Aldrich (M) Sdn Bhd. using vortex. Then, the hexne frction tht ws locted Reference stndrd of cholesterol nd COPs; 5 - t the upper lyer ws seprted nd trnsferred into cholestne, 25-hydroxycholesterol (25-OH), 7- test tue. The extrction with 10 ml of hexne ws ketocholesterol (7-keto), -epoxycholesterol, - repeted for nother two times nd lter dried under epoxycholesterol, 5 -cholestne were purchsed from nitrogen flow. 52

3 World Appl. Sci. J., 15 (Towrds the Trceility of Hll nd Thoyyin Appliction): 51-56, 2011 Tle 1: Susge Formultion Percentge of Ingredients (%) Ingredients Chicken Ft Plm Mid Frction Chicken Brest Met Chicken Skin (CSF) 5 - Crotino Plm Mid Frction () - 5 Wter Emulsion Textured Vegetle Protein 3 3 Slt Phosphte Potto Strch Milk Powder Tndori Powder Onion Powder Chili Powder Wonderhot Powder Munich Powder Derivtiztion of COPs: After drying the solvent under nitrogen gs, 100 µl of Tri-Sil regent ws dded nd the test tue ws kept t 80 C for 60 mins. The solvent ws evported under strem of nitrogen gs nd the TMS-ether derivtives were dissolved in 1 ml of hexne (chromtogrphy grde). The test tue of smples nd reference stndrds were then sonicted in n ultrsonic th for 1 min nd were centrifuged for 3 mins t 13,000 rpm. The smples nd reference stndrds were stored t -20 C for susequent nlysis y GC within one week fter derivtiztion [13]. Determintion of COPs y Gs Chromtogrphy with Flme Ioniztion Detector (GC-FID): Anlysis of COPs s their TMS-ether derivtives ws crried out y GC fitted with flme ioniztion detector (GC-FID). A cpillry column of DB-5 (20 mm x 0.1 mm x 0.1 µm) ws used to seprte the COPs. The method ccording to Uhyseker et l. [13] ws pplied in this study. The column temperture ws set t 150 C nd held for 5 min, incresed to 280 C t the rte 50 C/min, mintined t 280 C for 7.6 min. The temperture then ws incresed to 300 C t 4 C/min nd held for 5.4 mins. The detector ws set t 320 C. The heter ws put t 290 C with hydrogen flow for 300 ml/min nd ir flow t 40 ml/min. Identifiction nd purity percentges were performed using TMS ether derivtives of COPs stndrds. Sttisticl Anlysis: The dt were sujected to one wy nlysis of vrince (ANOVA), Duncn Multiple Rnge Rest, the vlue of men nd stndrd devition y using SAS version 9.2 nd Enterprise Guide version 4. RESULT AND DISCUSSION Tle 2 showed tht ech compound of COPs s well s cholesterol did not hve similr nd consistent pttern in term of mount t time intervl of weeks nlyzed. They were vritions in etween smples of susges nlyzed; CF, nd. Cholesterol tht ws determined in or commercilized susge t week 3 showed the highest vlue ( ± ) when compred to others; CF ( ±0.3229) nd (0.8899±0.1827). The pttern ws similr for the mount of 25-hydroxycholesterol ( ± ) in t week 3, despite of the minute mount of it in susge ( ±0.1058) nd not detected t ll in CF susge. It is lso noted tht there ws no 25-hyroxycholesterol detected throughout the weeks of nlysis for CF susge. Longer storge period my e needed for the compound to e present t detectle mount. This ws in prllel with smple of rw pork chops tht the initil COPs were non-detectle limit (limit of detection ws 5-10 ng of COPs) nd even fter 8 dys of refrigerted storge, detectle COPs were only present in some smple [14]. This my explin the undetected mount of -epoxycholesterol t the erlier week of nlysis. In nother COP compound nlyzed, - epoxycholesterol ws detected in significntly higher mount t week 0 of nlysis for CF susge ( ± ) nd susge ( ± ) when compred to week 1 (6.8968±3.9801) nd week 2 (1.9939±0.5780). In contrry, ( ±4.8293) ws significntly higher s nlyzed t week 3, in comprison to week 2. This compound, however, incresed significntly t week 3 in susge. It cn e due to the ntioxidnt constituents tht present in, minly the tocopherols, tocotrienols nd crotene eing used throughout the storge period to scvenge the free rdicls formed t the erlier process of susge production, the mnufcturer of red plm mid frction stted tht the ft contined more thn 400 ppm of tocopherols nd tocotrienols, s well s 406 ppm of crotene. Menwhile, the presence of -epoxycholesterol in ll susges ws not detected t week 0. In CF susge, it hs een detected t week 1 (2.1240±2.9140), while in nd susges it ws detected t week 3 of nlysis, with the mount of ± nd ±0.9196, respectively. The compound however ws not detected t the susequent week of nlysis in nd susges. The lter presence of this COP 53

4 World Appl. Sci. J., 15 (Towrds the Trceility of Hll nd Thoyyin Appliction): 51-56, 2011 Tle 2: Cholesterol nd cholesterol oxidtion products for different types of susges treted in chilled storge s nlyzed t different time intervl Week Compounds Cholesterol CF ± ± ± ± ± ± ± ± ± ± ± epoxycholesterol CF ± ± ± ± ± ± ± ± ± ± ± ± epoxycholesterol CF ± ± ± ± ± OHcholesterol CF ± ± ± ketocholesterol CF ± ± Vlues re mens ± stndrd devition of the five nlysis (two tches nlyzed in triplicte); vlues t different superscript letters re significntly differences (P<0.05) etween the storge times. Mens followed y the sme letter do not differ significntly from ech other might e due to the formtion of it fter storing the composition tles, the rtio etween unsturted nd smples, which resulted in the trnsformtion of - sturted ftty cids in chicken is higher thn in other epoxycholesterol to its isomer, -epoxycholesterol. mets such s pork, eef nd mutton [19]. According to Ro et l. [15], -epoxycholesterol or The inconsistent pttern of cholesterol nd COPs cholesterol- -epoxide ws resistnt to chnge nd content in this study re in contrst with reserch Conchillo et l. [16] reported tht -epoxycholesterol ws findings y Bggio nd Brggnolo [20] tht showed no the most undnt COPs in rw smples nd in vcuum- COPs were significntly formed during the storge time in stored grilled chicken, which were in contrry to the result ny of smples of met products studied. They elieved otined in this study. tht the presence of spices nd nturl condiments in the The lest compound of COPs tht cn e detected met products which inhiit the formtion of COPs. ws 7-ketocholesterol which ws nlyzed t zero time Moreover, mny spices nd hers hve een shown to nlysis in CF (3.6602±8.1844) nd (2.0921±4.6781) imprt n ntioxidnt effect in food systems [21]. The susges. The undetected mount my e due to ntioxidnt properties of spices re relted to their limittion of the instrument or the unstle structure of phenolic contents. It ws found tht the ddition of sge the compound wherey it hs undergone trnsformtion in chicken met ws effective in controlling lipid nd process, or the compounds were not present in the smple cholesterol oxidtion [22]. On the other hnd, study y t ll. Lercker nd Rodriguez-Estrd [17] suggested tht Ro et l. [15] on wter ufflo met found tht COPs no significnt differences in the 7-ketocholesterol content increse on cooking nd storge. It ws lso supported were found mong the different cooking tretments, ut y Kowle et l. [23] on their study on mutton when the rw met studied were lredy presented t cooked nd stored t 4 C nd -0 C. considerly high initil 7-ketocholesterol level (3.5 ppm). The instrument nd detection method of cholesterol It ws reported tht the predominnt species ws nd COPs conducted y Bggio nd Brggnolo [20] reported to e 7-ketocholesterol, ccounting for lmost ws y using high performnce liquid chromtogrphy hlf of the totl COPs, followed y 7 - nd 7 - (HPLC) with diode rry detector which my ginst hydroxycholesterol nd oth epoxides in order of nlysis done y using gs chromtogrphy give decresing concentrtion [18]. Oxidtion rections re different result, s the cpility nd tretment of smples initited in the highly susceptile memrne ound were different when compred. The smples nlyzed y phospholipids, which contin reltive lrge mounts of HPLC do not need to e derivtized prior to nlysis s to polyunsturted ftty cids. This process prticulrly mke the cholesterol nd COPs voltile. Uhyseker ffects unsturted lipids, with poultry met eing one of nd collegue, [13] proved tht evlution of COPs would the most susceptile, ecuse, ccording to food e different etween GC-FID nd GC-MS. This ws gin 54

5 World Appl. Sci. J., 15 (Towrds the Trceility of Hll nd Thoyyin Appliction): 51-56, 2011 supported y Hur et l. [24] whom stted gret 8. Svge, G.P., P.C. Dutt nd M.T. Rodriguez-Estrd, discrepncies hve een oserved in the COPs dt otined from different lortories for the vrious mtrices nlyzed, which my e due to differences in the extrction procedures or the chromtogrphic methods. CONCLUSION The different type of susges formulted with chicken ft nd plm mid frction which were niml nd plnt fts respectively did not show ny significnt contriution towrds the formtion of cholesterol nd cholesterol nd COPs throughout the storge period in chilling condition. Some of the compounds were either undetectle or did not present t ll. It is recommended for future works to longer the period of storge to otin concrete result t the end of nlysis, nlyzing the compounds using gs chromtogrphy with mss spectrometry s to increse the detection limit nd expnding the reference stndrd of cholesterol oxidtion products to e used s the compounds my vries nd trnsformed. REFERENCES 1. Bruice, P.Y., Lipid. Essentil Orgnic Chemistry. Person Eduction Inc., pp: Murno, P.S., Humn Nutrition nd Food (Lipid- Those Essentil Ftty Acids). Understnding Food science nd Technology. Thomson Lerning Inc., pp: Lercker, G. nd M.T. Rodriguez-Estrd, Cholesterol oxidtion: presence of 7-ketocholesterol in different food products. J. Food Compost. Anl., 13: Pningvit, A.J., A.D. King, Jones nd B.G. Germn, Cholesterol oxides in foods of niml origin. J. Food Science, 60(6): Lrkeson, B., P.C. Dutt nd I. Hnsson, Effects of frying nd storge on cholesterol oxidtion in minced met products. J. Am. Oil Chem. Soc., 77: Osd, K., Cholesterol oxidtion products: other iologicl effects. In cholesterol nd phytosterol oxidtion products: Anlysis, occurrence nd iologicl effects, (Eds F. Gurdiol, P.C. Dutt, R. Codony nd G.P. Svge). Chmpign, Illinois: AOCS Press, pp: Dorgnes, C. nd G. Mrquez-Ruiz, Oxidize ft in foods. Curr. Opin. Clin. Nutr. Met. Cre., 6: Cholesterol oxides: their occurrence nd methods to prevent their genertion in foods. Asi Pc. J. Clin. Nutr., 11(1): Dutt, P.C. nd L.A. Appelqvist, Studies on phytosterol oxides I: Effect of storge on the content in potto chips prepred in different vegetle oils. J. Am. Oil Chem. Soc., 74: Beriin, M.J., J. Chsco nd G. Lizso, Reltionship etween iochemicl nd sensory qulity chrcteristics of different commercil rnds of slchichón. Food Control, 11(3): Muguerz, E., O. Gimeno, D. Ansoren, J.G. Blouks nd I. Astisrán, Effect of replcing pork ckft with pre-emulsified olive oil on lipid frction nd sensory qulity of Chorizo de Pmplon - A trditionl Spnish fermented susge. Met Science, 59(3): Sldnh, T., A.C.F. Swy, M.N. Eerlin nd N. Brggnolo, HPLC seprtion nd determintion of 12 cholesterol oxidtion products in fish: comprtive study of RI, UV nd APCI-MS detectors. J. Agric. Food Chem., 54(12): Uhyseker, S.J.K.A., T. Verleyen nd P.C. Dutt, Evlution of GC nd GC-MS methods for the nlysis of cholesterol oxidtion products. Food Chemistry, 84(1): Monhn, F.J., I. Gry, A.M. Booren, E.R. Miller, D.J. Buckley nd P.A. Morrissey, Influence of dietry tretment on lipid nd cholesterol oxidtion in pork. J. Agric. Food Chem., 40: Ro, V.K., B.N. Kowle, N.P. Bu nd G.S. Bisht, Effect of cooking nd storge on lipid oxidtion nd development of cholesterol oxidtion products in wter Bufflo met. Met Sci., 43: Conchillo, A., D. Ansoren nd I. Astisrn, Intensity of lipid oxidtion nd formtion of cholesterol oxidtion products during frozen storge of rw nd cooked chicken. J. Sci. Food Agric., 84: Lercker, G. nd M.T. Rodriguez-Estrd, Cholesterol oxidtion mechnisms. In Cholesterol nd phytosterol oxidtion products: Anlysis, occurrence nd iologicl effects. (Eds F Gurdiol, PC Dutt, R Codony nd GP Svge). Chmpign. Illinois: AOCS Press, pp: Zuillg, M.P. nd G. Merker, Quntifiction of three cholesterol oxidtion products in rw met nd chicken. J. Food Sci., 56(5):

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