ASIAN JOURNAL OF CHEMISTRY

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1 Asin Journl of Chemistry; Vol. 26, No. 16 (2014), ASIAN JOURNAL OF CHEMISTRY Improvement of Antioxint Ativity of Whey Protein Hyrolyzte y Conjugtion with Glyosyltion CHANGYAN SUN 1,2, DEHAI LI 3, QIAN LIU 1 n BAOHUA KONG 1,4,* 1 College of Foo Siene, Northest Agriulturl University, Hrin , Heilongjing Provine, P.R. Chin 2 Heilongjing Diry Inustry Tehnil Development Center, Hrin , Heilongjing Provine, P.R. Chin 3 College of Forest, Northest Forestry University, Hrin , Heilongjing Provine, P.R. Chin 4 Synergeti Innovtion Center of Foo Sfety n Nutrition, Wuxi, Jingsu Provine, P.R. Chin *Corresponing uthor: Tel/Fx: ; E-mil: kongh63@hotmil.om Reeive: 10 Septemer 2013; Aepte: 16 Jnury 2014; Pulishe online: 28 July 2014; AJC This stuy ws onute to investigte the ntioxint tivity of whey protein hyrolyzte y onjugtion with gluosyltion. The retion onitions of whey protein hyrolyztes onjugte with gluose were optimize with the response surfe methoology using the entrl omposite rottle esign. The optimistion prmeters stuie were gluose onentrtion 8 %, temperture 94 C n retion time 3.2 h. Uner this optimum onition, the reuing power, hyroxyl ril svenging tivity, svenging of the ABTS ril, opper n iron helting, thiorituri i-retive sustnes of whey protein hyrolyztes y onjugtion with gluose through Millr retion re higher thn whey protein hyrolyztes. The result showe tht whey protein hyrolyztes y onjugtion with gluose h high ntioxint tivities. Keywors: Glyosyltion, Gluose, Whey protein hyrolyztes, Antioxint tivity, Response surfe methoology. INTRODUCTION Whey protein isolte (WPI), y-prout from heese inustry, owns high ontent of nutritionl onstituents n exellent funtionl properties, suh s emulsifying, foming n geltion. So milk whey proteins re n importnt soure of ingreients use in wie vriety of prouts 1. Whey proteins re gloulr proteins suh s β-ltogloulin (β-lg), α-ltlumin (α-l), ovine serum lumin (BSA) n immunogloulins, with β-ltogloulin eing the mjor omponent, lrgely responsile for the funtionlity of the totl whey protein system. Inspite of the enefiil properties of whey proteins, mny moifitions hve een use to further improve their funtionlity through physil, hemil or enzymti tretments to otin ingreients with new pplitions. However, some of these methos require noxious hemil regents, whih limit the use of suh moifie proteins s foo ingreients 2,3. An effetive metho to improve the funtionl properties of proteins, whih oes not require hemil tlysis, relies on the intertion of proteins with polyshries n smller rohyrtes vi Millr retion 4. Reports on the onjugtion of whey proteins with polyshries vi Millr retion re sre. There were some reports tht onjugtes of β-ltogloulin n extrn (43 kd) exhiite improve soluility n therml stility 5. Complexes of β-ltogloulin with extrn lso gve emulsions with exellent stility 6, whih inrese with the polyshrie size up to 150 kd 7. Similrly, the emulsifying tivity n soluility of BSA gretly inrese on onjugtion with gltomnn (22 kd). The finl funtionl properties of the protein-polyshrie onjugtes otine vi Millr retion epen on the protein onformtion, s well s on the prtiulr hrteristis of the polyshrie 8,9. As ompre to lrger moleules whey protein, the low retivity of the polyshries y the tthe moleules limits the extent of the Millr retion n iminishes susequent retions. Whey protein hyrolyztes with enzyme n proue smll moleulr proteins n n enhne the retivity of the polyshries. Severl proteins, suh s whey proteins, β-ltogloulin, ovlumin, lysozyme, rie proteins n soy proteins, hve een onjugte with vrious sugrs, in orer to improve their funtionlity n het stility properties β-ltogloulin moifie y shries showe stronger ril-svenging tivity n/or n influene miroorgnism growth, therefore hving the potentil to e use s foo preservtion itive. However, little informtion is ville out the ntioxint tivity y the intertion of whey protein hyrolyztes n

2 5088 Sun et l. Asin J. Chem. polyshries. The im of this stuy is to evlute the ntioxint tivity of intertion of whey protein hyrolyztes with polyshries n vi Millr retion. EXPERIMENTAL Whey protein isolte (WPI, 95 % protein) ws purhse from Dviso Foos Interntionl, In., (Minnesot, MN, USA). 2,2'-Azinois(3-ethylenzothizoline-6-sulfoni i mmonium slt) (ABTS), ferrozine, potssium ferriynie n L-leuine were purhse from Sigm Chemil Co. (St. Louis, MO, USA). All other hemils use in the present stuy regents were of nlytil gre. Preprtion of whey protein hyrolyztes (WPH): Whey protein isolte (WPI) solution (5% protein onentrtion) ws prehete wter th t 95 C for 5 min to unfol the protein struture 15. The hete-trete WPI ws then hyrolyze for 5 h with Allse, the rtio of enzyme to WPI is 2:100 n the enzyme hyrolyzte onition is ph 8.5, t 65 C. The protein hyrolyzte ws lyophilize n store t 4 C. Preprtion of WPI hyrolyzte polyshries onjugte: Aqueous mixtures of proteins n gluose, ltose n frutose (1:1, w/w) ws trnsferre to srew-sele tues, tightly ppe n hete in wter th (Tisite Instrument, Tinjin, Chin) t 50, 60, 70, 80 n 90 C. The smples were hete t ifferent temperture for 0, 1, 2, 3, 4, 5 h n then were oole immeitely in ie wter. Millr retion prouts (MRPs) were kept t 4 C until nlyse. Experimentl esign: Response surfe methoology ws use for investigting the influene of three inepenent vriles (temperture, onentrtion n retion time; these re the response vriles) on reuing power. In the present work, the experiments were performe oring to rottle entrl omposite esign (CCD). Bse on the impliit ssumption tht the errors re unorrelte with eh other n with the inepenent vriles n hve equl vrine, the metho of lest-squre regression ws use to fit the t to qurti moel 16. The entrl (0) n xil (± 1) levels of eh vrile were esignte s -1 (80 C; 7 %; 2 h), + 1 (100 C; 9 %; 4 h), 0 (90 C; 8 %; 3 h), respetively (Tle-2). The oe vlues of the experimentl ftors n ftor levels were use in the response surfe nlysis (Tle-2). The experiments were rrie out in triplite. Determintion of ntioxint tivity Reuing power: The reuing power of Millr retion prouts ws etermine using the metho of Yen n Duh with slight moifition 17. An liquot (0.5 ml) of Millr retion prouts (10-fol ilution) ws mixe with 2.5 ml of soium phosphte uffer (0.2 M, ph 6.6) n 2.5 ml of 1 % potssium ferriynie. After inution t 50 C for 20 min, 2.5 ml of 10 % trihloroeti i (w/v) were e. The mixture ws entrifuge t 3000 g for 10 min. An liquot (2.5 ml) of the superntnt ws mixe with 2.5 ml of istille wter n 0.5 ml of ferri hlorie (0.1 %) n the sorne t 700 nm ws mesure fter inute for 10 min. The sorne inrese is initive of n inrese in the reuing power. ABTS ril svenging tivity: The metho esrie y Re et l. 18 with slight moifition ws use to mesure the ABTS ril svenging tivity. Briefly, 100 µl of Millr retion prouts (10-fol ilution) ws mixe with 3 ml ABTS + solution (sorne of 0.70 ± 0.01 t 734 nm). The mixture ws inute in rk for 6 min n the sorne t 734 nm ws re. The perentge of ABTS ril svenging tivity ws lulte s follows: ABTS + Svenging tivity = (A - A s) 100/A (1) where A is the sorne of the ontrol (ABTS + solution without Millr retion prouts) n A s is the sorne of the smple. Hyroxie ril svenging tivity: Hyroxie ril svenging tivity ws etermine oring to the metho of Lee et l. 19 with some moifitions. Briefly, retion mixtures ontining 1 ml of phosphte uffer (0.1 mm ferri hlorie, mm EDTA, 1.5 mm H 2O 2, 2.5 mm eoxyriose, 0.1 mm sorte, ph 7.4) n 0.1 ml of Millr retion prouts (10-fol ilution) were inute t 37 C for 1 h. Aing 1 ml of TCA (2.8 %) n 1 ml TBA (0.5%). The mixture ws inute t 80 C for 0.5 h. The sorne of the retion mixtures ws mesure t 532 nm. The perentge of hyroxie ril svenging tivity ws lulte s follows: 100 Hyroxie ril svenging tivity = (A A s ) (2) A where A is the sorne of the ontrol (solution without Millr retion prouts) n A s is the sorne of the smple. Metl heltion tivity: Determintion of helting tivity on Fe 2+ n Cu 2+ helting tivity were etermine y the metho of Dinis et l. 20 with moifitions. Cu 2+ : The smples were mixe with 1 ml of 2 mm CuSO 4, 1 ml 10 % pyriine n 20 µl 0.1 % pyrotehol violet inute for 10 min t room temperture. The mount of free opper in the solutions ws otine from the sorne rtio A632. Fe 2+ : One milliliter smple solution ws mixe with 1.85 ml oule istille H 2 O n 0.05 ml 2 mm FeCl 2, the mixture ws llowe to rest t room temperture for 30 s. The retion mixture thus otine ws lter e with 0.1 ml 5 mm ferrozine n mixe, sorne t 562 nm ws etermine with spetrophotometer fter 10 min resting time t room temperture n 5 min entrifugtion t 3000 rpm. For the lnk, the ssy ws onute in the sme mnner ut oule istille H 2 O ws e inste of smple solution. The perentge of helting tivity ws lulte s follows: Chelting tivity % = (1 - A562 nm smple/a562 nm lnk) 100 (3) where A562 nm smple is the sorne of smple n A562 nm lnk is the sorne of the lnk. Antioxint tivity ssy in liposome system: The ntioxint tivity of glyosylte whey protein hyrolyztes ws initilly ssesse y mens of thiorituri i-retive sustnes (TBARS) nlysis in n oxiizing liposome system. Liposomes were prepre from soyen phosphtiylholine oring to the metho esrie y Deker n Hultin 21. A series of mixe solutions of 5 ml of liposome solution with 1 ml of the glyosylte whey protein hyrolyztes (5-fol ilution) were prepre. The ontrol solution ws prepre

3 Vol. 26, No. 16 (2014) Improvement of Antioxint Ativity of Whey Protein 5089 y mixing 1 ml of wter inste of 1 ml of the glyosylte whey protein hyrolyztes with 5 ml of the liposome solution. Oxition ws initite y ing 0.1 ml of 50 mm FeCl 3 n 0.1 ml of 10 mm soium sorte into the liposome/ protein solution. Smples were inute in 37 C wter th for 1 h n lipi oxition ws immeitely etermine using the TBARS nlysis proeure. The onentrtions of TBARS (seonry prouts of lipi oxition), with or without the presene of glyosylte whey protein hyrolyztes, were etermine oring to the metho outline y Kong n Xiong 22. Sttistil nlysis: All the experiments were run in triplite. Dt were nlyze using the Generl Liner Moels proeure of Sttistix 8.1 softwre pkge (Anlytil Softwre, St. Pul, MN) for miroomputer. Anlysis of vrine (ANOVA) ws one to etermine the signifine of the min effets. Signifint ifferenes (P < 0.05) etween mens were ientifie using Tukey proeures. RESULTS AND DISCUSSION With the im to hieve the mximum level of whey protein glyosyltion, ifferent kins n onentrtion of reuing sugr were selete for the glyosyltion retion. Totl reuing power is one of the importnt methos whih re utilize to investigte ertin mterils own in vitro ntioxint tivity. Tle-1 show tht the glyosylte whey protein hyrolyztes prepre with gluose h higher reuing power thn those prepre with ltose n frutose. Fig. 1 show tht the hnges in reuing power of whey protein hyrolyztes y onjugtion with ifferent gluose onentrtion through Millr retion. The ntioxint tivity of whey protein glyosyltions inrese with the inrese of the hyrolyzte time. This is onsistent with the results of Kim n Lee 23. They foun tht the reuing power of glyosyltion prout inreses with the inrese of heting time in the retion system of gluose n three kins of mino is (glyine, glyylglyine, triglyine) (Fig. 1). The ntioxint tivity of whey protein glyosyltions lso inrese with the inrese of gluose onentrtion from 3 to 9 %. The ntioxint tivity of whey protein hyrolyztes onjugtion with gluose onentrtion of 9 n 8 % is higher (p < 0.05) thn the onentrtion from7 to 3 %. When the retion time is over 3 h, the ntioxint tivity of whey protein glyosyltions inrese slowly. Similr reltion of ril-svenging tivity with nture of the sugr ws oserve y Chevlier et l. 24. TABLE-1 CHANGES IN REDUCING POWER OF VARIOUS GLYCOSYLATED WHEY PROTEIN HYDROLYZATES DURING DIFFERENT HEATING TIMES Heting times Totl reuing power (OD 700nm ) (h) Gluose Ltose Frutose ±0.003 ea 0.043±0.009 ea 0.032±0.025 A ±0.027 A 0.136±0.011 A 0.076±0.008 B ±0.003 A 0.274±0.014 A 0.201±0.012 B ±0.021 A 0.346±0.012 B 0.300±0.022 C ±0.014 A 0.367±0.035 B 0.331±0.014 B ±0.051 A 0.455±0.022 B 0.334±0.054 C Note: -e : mens with the ifferent letter within row re signifint ifferene (P < 0.05); A-C : mens with the ifferent letter within olumn re signifint ifferene (P < 0.05). Reuing power (OD 700 nm ) Fig % 5 % 6 % 7 % 8 % 9 % Time (h) Chnges in reuing power of vrious gluose onentrtion glyosylte whey protein hyrolyztes uring ifferent heting times Note: -e : mens with the ifferent letter within the sme time re signifint ifferene (P < 0.05) Effet of temperture on reuing power: Fig. 2 shows tht the hnges in reuing power of vrious glyosylte whey protein hyrolyztes uring ifferent heting temperture. The ntioxint tivity of whey protein glyosyltions inrese with the inrese of the temperture n retion time. The ntioxint tivity of whey protein hyrolyztes onjugtion with gluose t temperture of 90 C is higher (p < 0.05) thn the temperture from 50 to 80 C. This result is mye ue to the sening temperture ws signifintly moifie in the onjugte solution system, whih shoul e srie to the hnges in whey protein enturtion n ggregtion ehviour upon the tthment of gluose 25. The ntioxint tivity of whey protein hyrolyztes onjugtion with gluose is lso inrese with the inrese of retion time. Reuing power (OD 700nm ) Fig C 60 C 70 C 80 C 90 C Time (h) Chnges in reuing power of vrious glyosylte whey protein hyrolyztes uring ifferent heting temperture n times [Note: - : mens with the ifferent letter within the sme time re signifint ifferene (P < 0.05)] Optimum retion onitions: Gluose onentrtion, retion time n retion temperture were onsiere s the three ftors; three-ftor to three-level response surfe e e e

4 5090 Sun et l. Asin J. Chem. experiment is esigne to ientify the optimum retion onitions, the optimum retion onitions re shown in Tle-2. TABLE-2 CODED VALUES OF CORRESPONDING ACTUAL VALUES OF INDEPENDENT VARIABLES IN RESPONSE SURFACE DESIGN Ftor Level Temperture ( C) Gluose onentrtion Time (h) Aoring to Box Benhnken's entrl omposite experiment esign priniples, on the sis of the single-ftor experimentl stuy, temperture, gluose onentrtion n retion time were selete s the inepenent vrile, totl reuing power s the evluting initor, to esign 17 groups of threeftor to three-level response surfe experiments, mong whih 13 re ftoril experiments n 4 re entrl experiments whih re use to estimte the experimentl error. Experimentl sheme n results re shown in Tle-3. Tle-4 shows tht the results of regression nlysis n vrine nlysis. Sequene numer TABLE-3 DESIGN AND RESULTS OF THE RESPONSE SURFACE METHODOLOGY C (gluose onentrtion) B (temp.) A (time) Y (totl reuing power) (OD 700 nm ) Through the nlysis of the sttistil nlysis softwre Sttistix 8.1, the qurti regression moel ws otine: Y = A B C A B C AB AC BC Tle-4 shows tht the effet of B, A 2, B 2, AC terms were signifint (P < 0.05). This inites tht in the liner terms, retion time hs signifint effet on the experiment results (P < 0.05); in the intertion terms, gluose onentrtion n retion time hve signifint mutul effet on the experiment results (P < 0.05); n in the qurti terms, oth gluose TABLE-4 VARIANCE ANALYSIS FOR THE FITTED REGRESSION EQUATION Ftor DF Coeffiient Sum of Men estimte Squres Squre F Vlue P Vlue Moel A E E B C A B C AB E E AC BC Resiul Lk of fit Pure error Cor totl R onentrtion n retion temperture hve signifint effet on the experiment results (P < 0.05). The onrete mutul funtions re shown from Figs. 3 to 5. Totl reuing power Fig. 3. Totl reuing power B: temperture A: gluose onentrtion Response surfe stereogrm of temperture (B) n gluose onentrtion (A) C: time A: gluose onentrtion Fig. 4. Response surfe stereogrm of time (C) n gluose onentrtion (A)

5 Vol. 26, No. 16 (2014) Improvement of Antioxint Ativity of Whey Protein Totl reuing power C: time B: temperture Fig. 5. Response surfe stereogrm of time (C) n temperture (B) Tle-4 shows the results of vrine nlysis, euse the liner reltions re ovious etween the vriles n the inepenent vriles, the moel regression is evient (P < 0.05). The moel R 2 equls 0.828, initing tht the moel is well fit for the experiment. The p test lso ws use to evlute the signifine of the prmeters for the moel n the lk of fit, there ws no signifine in the lk of fit (p = ) in the moel. This inite tht the moels oul e use to preit responses 26. The liner reltions re ovious etween the inepenent vriles n the response vlues n so it n e pplie in the theoretil preition of the retion. Getting ifferentition for regression eqution n mking it equls zero n get the mximum point of the surfe, the optimum level vlues of the three mjor ftors is following: gluose onentrtion is 8.08, temperture is C n time is 3.18 h. The optimum reuing power of the response surfe is In orer to fit the prtil proutive onitions, the ove onitions were orrete: gluose onentrtion is 8 %, temperture is 94 C n retion time is 3.2 h. Uner suh onitions we i the ientifition experiment n the reuing power is s result, whose reprouiility is etter thn the theoretil preition (Tle-5). Smple Whey protein hyrolyztes (10 mg/ml) Glyosylte whey protein hyrolyztes (10 mg/ml) TABLE-5 COMPARISON OF ANTIOXIDATIVE ACTIVITIES AMONG WHEY PROTEIN HYDROLYZATES, GLYCOSYLATED WHEY PROTEIN HYDROLYZATES AND COMMON ANTIOXIDANTS V Reuing Power (OD 700nm ) 0.165± ± Hyroxyl ril svenging tivity 9.22± ± 2.01 ABTS + svenging tivity 33.14± ± 1.33 Cu 2+ helting tivity 11.79± ± 2.15 Fe 2+ helting tivity 3.83± ± 0.36 V(0.01 %) 1.766± ± ± ± ± 0.57 Note: - :mens with the ifferent letter within row re signifint ifferene (P < 0.05) TBARS (mg/kg) 3.21± ± ± 0.79 Comprison of ntioxint tivities of whey protein hyrolyztes y onjugtion with gluose through Millr retion uner the optimum onition: Antioxint tivity ws nlyse y etermintion of the reuing power, hyroxyl ril svenging tivity, svenging of the ABTS ril, Copper n iron helting tivities of whey protein hyrolyztes y onjugtion with gluose through Millr retion n whey protein hyrolyztes (Tle-5). The omprison results of the ntioxint tivities of whey protein hyrolyztes y onjugtion with gluose through Millr retion n ntioxint V re shown in Tle-5. Reuing power ws etermine y mesuring reution of the Fe 3+ /ferriynie omplex to the ferrous form, whih ws monitore y mesuring the formtion of Perl's Prussin lue t 700 nm. The reuing power of whey protein hyrolyztes y onjugtion with gluose (0.939) is higher (P < 0.05) thn tht of whey protein hyrolyztes (0.165). The hyroxyl ril svenging tivity of whey protein hyrolyztes y onjugtion with gluose (55.37 %) is higher (P < 0.05) thn tht of whey protein hyrolyztes (9.22 %). The ABTS eoloriztion ssy n e use to etermine ntioxint tivity of oth lipophili n hyrophili moleules n is se on the retion of hyrogen onting ntioxints with the ABTS + ril, whih is intensely oloure n is etermine y mesuring sorne t 734 nm. The ABTS + of whey protein hyrolyztes y onjugtion with gluose (84.92 %) is higher (P < 0.05) thn tht of whey protein hyrolyztes (33.14 %). Similr results were oserve y Beermnn et l. 27. These t re onsistent with reports tht the ril svenging mehnism epens on the presene of hyrophoi mino is 28. Cheltion of metl ions hs n ntioxint effet euse the trnsition metls iron n opper tlyze the genertion of retive oxygen speies, inluing hyroxyl ril ( OH) n superoxie ril (O 2 - ), leing to oxition of unsturte lipis n promoting oxitive mge t ifferent levels 29. The Cu 2+ helting tivity n Fe 2+ helting tivity of whey protein hyrolyztes y onjugtion with gluose is n %, respetively. The Cu 2+ helting tivity n Fe 2+ helting tivity of whey protein hyrolyztes y onjugtion with gluose is higher (P < 0.05) thn tht of whey protein hyrolyztes. This result my e ue to the whey protein ws hyrolyze n proue some smll pepties; smll pepties n provie the highest opper helting tivity 30. The TBARS of whey protein hyrolyztes y onjugtion with gluose is lso higher (P < 0.05) thn tht of whey protein hyrolyztes. Tle-5 showe tht whey protein hyrolyztes y onjugtion with gluose emonstrte high ntioxint tivities. Conlusion A ftoril esign ws pplie to optimize the ntioxint tivities of whey protein hyrolyztes y onjugtion with gluose. The opertionl onitions selete to otin signifint ntioxint properties were gluose onentrtion is 8 %, temperture is 94 C n retion time is 3.2 h. Uner this optimum onition, whey protein hyrolyztes y onjugtion with gluose through Millr retion showe high ntioxint tivities thn whey protein hyrolyztes.

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