Contemporary Agriculture Vol. 67, No. 1, Pp , BIURET AND BRADFORD METHODS SUITABILITY FOR PROTEIN QUANTIFICATION IN RAPESEED MEAL*

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1 Contemporry Agriculture Vol. 67, No. 1, Pp , The Serin Journl of Agriculturl Sciences ISSN (Online) UDC: 63(497.1)(051) Originl scientific pper UDC: DOI: /contgri BIURET AND BRADFORD METHODS SUITABILITY FOR PROTEIN QUANTIFICATION IN RAPESEED MEAL* Hristo KALAYDZHIEV 1, Pety IVANOVA 1, Glin UZUNOVA 2, Ivn MANOLOV 3, Vesel CHALOVA 1 Summry: Being ttrctive for insects, non-geneticlly modified rpeseed is vlule for mintining environmentl iodiversity. Primrily, the rpeseed is n importnt industril crop which is used for production of vegetle oil. Oil extrction from rpeseeds results in the genertion of sustntil mounts of rpeseed mel which is used either s protein rich feed dditive or s source for preprtion of protein contining ingredients for food industry. Both pplictions require frequent evlution of protein content. Although Kjeldhl method is considered stndrd, it is not pproprite for routine evlution of protein content in protein extrcts. The im of the study ws to evlute suitility of iuret nd Brdford methods for protein quntifiction in rpeseed mel extrcts. After consecutive triple extrction of proteins with wter, 5% NCl, 70% ethyl lcohol nd 0.1 N NOH, protein evlution of ech lumin, gloulin, prolmin nd glutelin extrction liquot demonstrted overll lower protein content y Brdford method compred to iuret method. The most pronounced differences in protein content were oserved with prolmin frction where three fold higher protein concentrtions in ech extrction liquot ws oserved when iuret method ws pplied for the evlution. Comprtive quntifiction of the totl protein of ech of the four frctions followed similr trend of lower protein content evlution y Brdford method. Overll results indicted iuret method s more suitle for protein quntifiction in rpeseed mel extrcts which ws confirmed y comprison with dt otined y Kjeldhl method. Key words: rpeseed mel, protein quntifiction, spectrophotometric methods, Kjeldhl method. INTRODUCTION Rpeseed is mjor technicl plnt which cultivtion worldwide chieved stedy growth over the lst 20 yers. After surpssing cotton production t the eginning of 2000, it ecme the second most cultivted oilseed crop fter soyen (Crré nd Pouzet, 2014). The lrgest producers re Chin, Indi, Cnd nd the Europen Union. Rpeseed/cnol oil is used either for food purposes or iodiesel production s the lter ccounts for 57-70% of the totl iofuel genertion in the EU (Zentková nd Cvengrošová, 2013). Due to incresed interest to rpeseed oil, the worldwide production of rpeseeds hs een predicted to enhnce in ner future (Crré nd Pouzet, 2014). Beside economic impct, rpeseed is importnt for mintining nturl iodiversity. The right yellow color of the flower nd shllow lloction of nectr ttrct vrious ees, flies, nd utterflies (Kunin, 1997; Pudsini et l., 2015). After studying rpeseed insect diversity, Milovc et l. (2012) reported totl of 8,384 specimens from 11 insect orders with previling specimens elonging to Dipter nd Coleopter. Divers groups of cteri including Bcillus, Fhvoucterium, Micrococcus nd Rthyicter hve een oserved in ssocition with cnol plnts roots s well (Germid et l., 1998). The extrction of oil from rpeseeds results in genertion of rpeseed mel, y-product, which mount my rech up to 48% of the totl quntity of rpeseeds used (Ivnov, 2012). In the interntionl production of deftted 1 Hristo Klydzhiev, MS, Doctorl Student, Pety Ivnov, PhD, Assistnt Professor, Vesel Chlov, PhD, Associte Professor, University of Food Technologies, Deprtment of Biochemistry nd Moleculr Biology, 26 Mrits Blvd, Plovdiv 4002, Bulgri 2 Glin Uzunov, PhD, Associted Profesor, University of Food Technologies, Deprtment of Technology of Tocco, Sugr, nd Vegetle Essentil Oils, 26 Mrits Blvd, Plovdiv 4002, Bulgri 3 Ivn Mnolov, PhD, Full Professor, Agriculturl University, Deprtment of Agrochemistry nd Soil Science, 12 Mendeleev Str., Plovdiv 4000, Bulgri Corresponding uthor: e-mil: veselchlov@gmil.com, Tel.:

2 Klydzhiev et l. Contemporry Agriculture, 67 (1) 87-92, mels, the rpeseed mel rnks second fter the soyen mel. It cn e used directly s protein rich dditive in feed industry or s source for preprtion of protein isoltes nd concentrtes (Newkirk, 2009; Tn et l., 2011). The utiliztion of the rpeseed mel requires frequent evlution of protein content ecuse of vrious resons which include ut re not limited to estlishment of nutritive chrcteristics for proper diet formultion, conformity to regultory specifictions, uthentiction of protein-rich ingredients or sources (DuPont et l., 2005; Bonftti et l., 2008). Detiled description of current methods for totl protein mesurement in food nd feed is provided y Moore et l. (2010). Protein quntifiction y Kjeldhl method is considered stndrd nd relile. Mny interntionl lortories use it s reference procedure due to its ccurcy regrdless of the physicl stte of the smple (Owusu- Apenten, 2012). However, the technique is lor-intensive, long lsting nd expensive nd therefore, it is less pplicle for routine nd extensive evlution of protein content. It is especilly vlid for the production of proteinrich ingredients which include protein extrction nd optimiztion steps resulting in numerous smples for evlution. Spectrophotometric methods including iuret nd Brdford methods re simple, rpid, reltively inexpensive nd do not require elorte equipment which mke them suitle for routine nlyzes (Owusu-Apenten, 2012). However, their ccurcy cn e interfered y concomitnt compounds such s phenols present in mels (Hortin nd Meilinger, 2005; Ivnov et l., 2013). The purpose of this study ws to evlute suitility of iuret nd Brdford methods for protein quntifiction in rpeseed mel extrcts. MATERIAL AND METHODS Rpeseed mel smple preprtion. Rpeseed mel ws provided y locl compny (Oliv AD, Polski Trmesh). It ws produced from rpeseeds yield 2016 fter moisture reduction t C nd oil extrction with hexne t C. The mel ws grinded nd sifted to collect mm prticles. To reduce ntinutritionl compounds in the protein source, the rpeseed mel ws sujected to 4-step tretment with ethnol (96%)/wter mixture (65/35) t mel to solvent rtio of 25% (w/v) for 30 min t room temperture (23 C) s descried y Chnon et l. (2007). The residue ws collected, dried in ir nd stored in closed continer for further nlyzes. All regents used were of nlyticl grde. Rpeseed protein frctiontion. Proteins were sequentilly extrcted from the ethnol treted rpeseed mel with wter, 5% NCl, 70% ethyl lcohol nd 0.1 N NOH to otin lumin, gloulin, prolmin nd glutelin frctions respectively (Tn et l., 2012). Ech frctionl extrction ws repeted three times with decresing mel/residue to solvent rtion from the first to the third extrction s it follows: 1:10, 1:5 nd 1:2.5. The extrction liquots were collected seprtely for protein quntifiction. All extrctions were performed t room temperture (23 C) for 30 min under constnt gittion. Protein quntifiction. Kjeldhl method ws used to determine totl nitrogen content in the ethnol treted rpeseed mel. The result ws multiplied y 6.25 to convert to crude protein (AOAC, 1990). Protein content in extrcts ws evluted y iuret (AACC, 1983) nd Brdford methods (Brdford, 1976). Bovine serum lumin ws used to generte stndrd curves. Sttisticl nlysis. All extrctions nd nlyses were performed in triplicte. Presented results re verged mens ± stndrd devition (SD). Dt were nlyzed y one-wy nlysis of vrince (ANOVA) using Sttgrphics Centurion sttisticl progrm (version XVI, 2009) (Stt Point Technologies, Ins., Wrrenton, VA, USA). Men differences were estlished y Fisher s lest significnt difference test for pired comprison with significnce level α = RESULTS Protein content of ech rpeseed frction ws significntly higher (P< 0.05) when evluted y iuret method (Fig. 1). The highest difference (more thn 3-folds) ws estlished with prolmin frction followed y tht of the gloulins. Although still significnt, less thn 1.5-fold difference in protein contents mesured y the two methods ws oserved with glutelin frction. 88

3 Klydzhiev et l. Contemporry Agriculture, 67 (1) 87-92, Protein content, % Alumin Gloulin Prolmin Glutelin Biuret method Brdford method Figure 1. Protein content of ethnol treted rpeseed mel protein frctions evluted y iuret nd Brdford methods - Vlues with different superscripts differ significntly (P<0.5) Similr trend ws oserved fter evlution of the protein content of ech extrction liquot of frction (Tle 1). The most remrkle discrepncy in protein contents ws oserved with the second extrction of the prolmin frction where the protein level estimted y iuret method exceeded more thn 6-flods the protein level of the sme frction estimted y Brdford method. Tle 1. Protein content comprison of extrction liquots during protein frctiontion of ethnol treted rpeseed mel proteins Protein frction Extrction No: Protein content, %* Biuret method Brdford method Alumin Gloulin Prolmin Glutelin ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.03 *Protein content ws clculted on n ir-dry mtter sis Results for protein content otined y Kjeldhl, iuret nd Brdford methods differed significntly (P<0.05). Comprison of the two methods to Kjeldhl mesurement demonstrted closer protein estimtion of iuret method thn the Brdford one (Fig. 2) 89

4 Klydzhiev et l. Contemporry Agriculture, 67 (1) 87-92, Protein content*, % c Kjeldhl method Biuret method Brdford method Method used for evlution Figure 2. Totl protein content of ethnol treted rpeseed mel evluted y Kjeldhl, iuret nd Brdford methods *Protein contents y iuret nd Brdford methods were clculted s sum of the protein level of ech frction, lumin, gloulin, prolmin nd glutelin. -c Vlues with different superscripts differ significntly (P<0.5) DISCUSSION Totl content of phenolic cids in cnol/rpeseed mel vry from 1.54% to 1.84% clculted on dry mtter sis (Nczk et l., 1986). To reduce the level of the phenolic compounds nd nti-nutrients such s glucosinoltes, which would worsen the qulity of susequently produced rpeseed mel protein- derivtives, the rpeseed mel ws sujected to 4-step ethnol tretment. Previously, y using the sme procedure Ivnov et l. (2017) chieved more thn four-fold reduction of phenols lowering their level to 0.25%. Most proly, the pretretment step of the rpeseed mel is the cuse for no formtion of interfering color t 540 nm which ws noticed when ethnol nontreted rpeseed mel extrcts were quntified for proteins y iuret method (dt not pulished). The protein contents of lumin, gloulin, prolmin nd glutelin frctions of the industril rpeseed mel evluted y iuret method (Fig. 1) were close to dt reported y Tn et l. (2012) for industril tosted mel. Similrly, prolmins were found to e the minor frction of the smple nlyzed. The ethnol pre-tretment step used in this study should lso e ccounted for the reltively low prolmin content reported herein. Brdford method, when used for protein quntifiction of either rpeseed mel protein frction or n extrction liquot, led to 2 to 3 folds lower results compred to iuret method. This could e prtilly explined y the mechnism of interction of Coomssie Brillint Blue G250 dye with the nlyte. The dye inds to polypeptides with moleculr weight greter thn D s the numer of the molecules strongly correltes to the totl numer of rginine, histidine nd lysine (Owusu-Apenten, 2012). According to Klockemn et l. (1997), lysine is the first limiting mino cid in rpeseed protein. In ddition, the industril procedure for oil extrction nd rpeseed mel production include enhnced therml tretment steps which increse the rectivity of ε-mino group of the lysine rdicl (Kwok et l., 1998). It esily goes into rection with sugrs which reduces lysine rdicl vilility for susequent interction with Coomssie Brillint Blue G250 dye. According to Brimer et l. (1995), non-enzymtic glyction of proteins interferes with their quntittive evlution nd my result in up to 20% underestimtion when Brdford method is used. Phenolics nd other compound from the rpeseed mel my complex with proteins (Aider nd Brn, 2011) which dditionlly reduces their ccessiility to the Coomssie Brillint Blue G250 dye. Since the interction etween the dye nd proteins is electrosttic, differences in the mino cid composition of the frctionl proteins s well s those contined in the extrction liquots is the most prole explntion for the different extent of reduction of the results otined with Brdford ssy (Tle 1). Kjeldhl method for protein quntifiction is sed on smple minerliztion followed y determintion of totl nitrogen content (Owusu-Apenten, 2012). In contrst, iuret nd Brdford procedures involve protein extrction steps. However, the choice of extrction procedure highly influences the extent of protein recovery (Pedroche et l., 2004; Tn et l., 2011). In ddition, type of the smples should lso e ccounted for the differences oserved mong protein yields chieved. For exmple, Tn et l. (2012) reported lowest protein recovery for industril tosted mel 90

5 Klydzhiev et l. Contemporry Agriculture, 67 (1) 87-92, compred to the other smples tested (Austrlin cnol (Brssic npus) mel, mustrd (Sinpis l) mel nd pretosted industril mel) suggesting high influence of therml tretment on protein soluility. Therefore, equl protein content mesured y Kjeldhl nd iuret nd Brdford methods pplied fter protein extrction, cnnot e expected. Considering the 40% protein recovery from industril tosted mel when Osorne extrction method is used, reported y Tn et l. (2012), totl protein content evluted y iuret method ws closer to tht of the Kjeldhl method (Fig. 2). Possile inccurcy of Kjeldhl results my lso e ssumed due to reltively high non-protein nitrogen compounds in plnts (Izhki, 1993; Fujihr et l., 2001). By using dt of totl mino cid content of some plnt smples from Nigeri, Mgomy et l. (2014) clculted specific conversion fctors vrying from 3.24 to 5.39 which implied considerle overestimtion of the protein content if conversion coefficient of 6.25 is used. CONCLUSION Biuret nd Brdford methods re widely used for totl protein quntifiction. Although oth spectrophotometric ones, the methods differ in their principles nd potentil drwcks. The use of Brdford procedure for the evlution of protein content of either protein frctions or extrction liquots resulted in 2 to 3 fold lower results thn iuret method. Comprison to Kjeldhl method s well s dt reported in literture indicted etter suitility of iuret method for determintion of the protein content of extrcts otined from industril rpeseed mel fter 4-step queous ethnol tretment. ACKNOWLEDGEMENT This reserch is prt of doctorl study supported y University of Food Technologies-Plovdiv, Bulgri REFERENCES AACC: Method 46-15: Crude protein 5-minute Biuret method for whet nd other grins. Approved methods of the Americn Assocition of Cerel Chemists, 8th edition, Americn Assocition of Cerel Chemists, St. Pul, MN, USA, AIDER, M., BARBANA, C.: Cnol proteins: composition, extrction, functionl properties, ioctivity, pplictions s food ingredient nd llergenicity A prcticl nd criticl review. Trends in Food Science & Technology, 22:21-39, AOAC. Officil Methods of Anlysis, Assocition of Officil Anlyticl Chemists, Wshington, DC, USA, BONFATTI, V., GRIGOLETTO, L., CECCHINATO, A., GALLO, L., CARNIER, P.: Vlidtion of new reversed-phse highperformnce liquid chromtogrphy method for seprtion nd quntifiction of ovine milk protein genetic vrints. Journl of Chromtogrphy A, 1195: , BRADFORD, M.: A rpid nd sensitive for the quntittion of microgrm quntities of protein utilizing the principle of proteindye inding. Anlyticl Biochemistry, 72: , CARRÉ, P., POUZET, A.: Rpeseed mrket, worldwide nd in Europe. OCL-Oilseeds nd fts, Crop nd Lipids, 21:D102-D114, CHABANON, G., CHEVALOT, I., FRAMBOISIER, X., CHENU, S., MARC, I.: Hydrolysis of rpeseed protein isoltes: Kinetics, chrcteriztion nd functionl properties of hydrolystes. Process Biochemistry, 42: , DUPONT, F.M., CHAN, R., LOPEZ, R., VENSEL, W.H.: Sequentil extrction nd quntittive recovery of glidins, glutenins, nd other proteins from smll smples of whet flour. Journl of Agriculturl nd Food Chemistry, 53: , FUJIHARA, S., KASUGA, A., AOYAGI, Y.: Nitrogen-to-protein conversion fctors for common vegetles in Jpn. Journl of Food Science, 66: , GERMIDA, J.J., SICILIANO, S.D., RENATO DE FREITAS, J., SEIB, A.M.: Diversity of root-ssocited cteri ssocited with field-grown cnol (Brssic npus L.) nd whet (Triticum estivum L.). FEMS Microiology Ecology, 26:43-50, HORTIN, G.L., MEILINGER, B.: Cross-rectivity of mino cids nd other compounds in the iuret rection: Interference with urinry peptide mesurements. Clinicl Chemistry, 51: , IVANOVA, P., KALAYDZHIEV, H., RUSTAD, T., SILVA, C.L.M. nd CHALOVA, V.I.: Comprtive iochemicl profile of protein-rich products otined from industril rpeseed mel. Emirtes Journl of Food nd Agriculture, 29: , IVANOVA, P., KOLEVA, L., CHALOVA, V.: Appliction of iuret nd Brdford methods for quntittive determintion of proteins in sunflower products. Scientific Works of University of Food Technologies-Plovdiv, LX: , IVANOVA, R.: Rpeseed - The Culture of Present nd Future. Videnov & Son, Sofi, Bulgri, IZHAKI, I.: Influence of nonprotein nitrogen on estimtion of protein from totl nitrogen in fleshy fruits. Journl of Chemicl Ecology, 19: , KLOCKEMAN, D.M., TOLEDO, R., SIMS, K.A.: Isoltion nd chrcteriztion of deftted cnol mel protein. Journl of Agriculturl nd Food Chemistry, 45: , KUNIN,W.E.: Popultion size nd density effects in pollintion: pollintor forging nd plnt reproductive uccess in experimentl rrys of Brssic ker. Journl of Ecology, 85: , KWOK, K., SHIU, Y., YEUNG, C., NIRANJAN, K.: Effect of therml processing on ville lysine, thimine nd rioflvin content in soymilk. Journl of the Science of Food nd Agriculture, 77: ,

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