COW MILK WHEY THROUGH EXPANDED BED ADSORPTION USING A HYDROPHOBIC RESIN

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1 Brazlan Journal of Chemcal Engneerng ISSN Prnted n Brazl Vol., No. 04, pp , October - December, 005 CONCENTRATION OF α-lactalbumin FROM COW MILK WHEY THROUGH EXPANDED BED ADSORPTION USING A HYDROPHOBIC RESIN L. S. Conrado, V. Veredas, E. S. Nóbrega and C. C. Santana * Department of Botechnologcal Process Engneerng; School of Chemcal Engneerng; State Unversty of Campnas, Phone (9) , Fax (9) , Rua Albert Ensten 500, Cdade Unverstára Zeferno Vaz, Barão Geraldo, Cx. P 6066, CEP , Campnas - SP, Brazl, E-mal: santana@feq.uncamp.br Department of Chemcal Engneerng, Federal Unversty of Campna Grande, Campna Grande - PB, Brazl. (Receved: October 0, 004 ; Accepted: Aprl, 005) Abstract - The desgn of novel applcatons for the use of new hgh-densty adsorbents n the drect recovery of protens requres analyss of the flud dynamcs and mass transfer characterstcs of the column used wth the sold-lqud system. In ths artcle we descrbe the operatng parameters related to the use of a hghdensty, hydrophobc resn (Streamlne Phenyl) for the recovery of α-lactalbumn from cow mlk whey n an expanded-bed adsorpton mode of operaton. The adsorpton sotherm, knetc curves and chromatographc curves for adsorpton and eluton were determned for α-lactalbumn recovered from whole whey. The experments were used to confrm the theoretcal model that nvolves the combnaton of resstance to the mass transfer n the lqud phase and dffusvty n the adsorbent resn pores. The model soluton was obtaned through the orthogonal collocaton method. A nonlnear multvarable optmzaton method was used to ft the model to expermental results. A purty of 79% was obtaned wth the adsorpton/eluton protocol pror to any polshng procedure. Keywords: α-lactalbumn; Mlk whey; Expanded bed adsorpton; Flud Dynamcs. INTRODUCTION Opportuntes for the commercal extracton of boproducts from food waste streams are ncreasng. Future ntatves to mprove the recovery of byproducts requre addtonal research to nvestgate possble uses for valuable waste stream components and to develop cost-effectve technques for ther recovery (Beszedts and Netzer, 98). One example of a potental feld for boproduct recovery s the extracton of protens from mlk whey usng chromatographc technques. A typcal composton of cheese whey s shown n Table. Most of the purfed protens derved from mlk products, such as whey, have mportant applcatons n clncal and veternary medcne, functonal food products and cell culture meda (Horton, 995). In ths work we examned the use of a hghdensty, hydrophobc resn (Streamlne Phenyl) for the recovery of α-lactalbumn (α-la) from cow mlk whey n an expanded-bed adsorpton mode of operaton. Noppe et al. (999) report a protocol for the fast and effcent purfcaton of α-la usng defatted cow mlk and the same resn as that used here, but wth EDTA and an eluton step nvolvng a Ca + -contanng buffer. The fundamental aspect of ths approach reles on the bndng capacty of α- lactalbumns for Ca + ons. These protens, whch wthout Ca + have hydrophobc characterstcs, undergo a sgnfcant conformatonal change wth *To whom correspondence should be addressed

2 50 L. S. Conrado, V. Veredas, E. S. Nóbrega and C. C. Santana and other metal ons that makes them more hydrophlc. By usng Trs-EDTA buffer n the adsorpton step the Ca + ons are removed, makng the proten more hydrophobc and ncreasng ts bndng to hydrophobc adsorbents. The eluton step usng Ca + ons permts recovery of α-lactalbumn because of the reversble change n the hydrophlc character. Although expanded-bed chromatography Ca + offers the advantage of reducng the number of steps by savng recovery steps n ths applcaton, there s stll a need for process engneerng data to make the scale-up more relable. We therefore also examned some mportant process tools such as adsorpton sotherms, adsorpton knetc curves and chromatographc curves for α-la recovered from whole whey. Table : The man proten components of cheese whey from bovne mlk Proten Concentraton (g/l) Molecular weght (kda) Isoelectrc pont β-lactoglobuln α-lactalbumn BSA IgG, IgA, IgM Lactoperoxdase Lactoferrn Mlk Serum MATERIAL AND METHODS Casen partally precptated bovne mlk serum was used n all of the adsorpton experments. Adsorbent The expanded bed adsorbent used n these experments was a STREAMLINE Phenyl obtaned from Amersham Pharmaca Botech (Sweden). Ths resn has phenyl groups coupled to the STREAMLINE matrx va uncharged, chemcally stable ether lnkages, whch results n a hydrophobc medum wth mnmal onc propertes. The average densty of the adsorbent was. g.ml -. The partcle sze ranged from 00 to 300 µm wth an average sze of 8 µm measured by the laser lght-scatterng method usng a Masterszer S detector (Malvern Instruments Ltd.). The mean partcle porosty (ε p ) was 0.65, measured by the moment method (Arnold et al., 985). Column The column used was a STREAMLINE 5, wth an nternal dameter of 5 mm and a total heght of meter (Fgure ). Varous settled bed heghts (H 0 ) (0.5 cm, 5 cm, cm, 5 cm and 30 cm) were used to study the flud dynamcs and a bed heght of 0 cm was used to determne the breakthrough curves. Buffer System Equlbrum buffer: 50 mm Trs -.5 mm EDTA. Adsorpton buffer: 50 mm Trs - 35 mm EDTA. Wash buffer: 50 mm Trs -.5 mm EDTA. Eluton buffer: 50 mm Trs -.5 mm CaCl. The ph of all soluton buffers was 7.5. Fludzed Bed Studes Rchardson and Zak (954) showed that there was a correlaton between bed heght and superfcal velocty (u) for varous settled bed heghts. The Rchardson and Zak parameters of Equaton, n and U t (termnal settlng velocty), were determned from the regresson lne of a logarthmc plot of porosty versus superfcal velocty. All experments were done usng the equlbrum buffer. U n =ε () U t Resdence Tme Dstrbuton Resdence tme dstrbuton was measured to determne the axal dsperson n the lqud phase as a functon of bed expanson startng wth fxed bed heghts of 0 cm and 0 cm. Lnear velocty was vared from 3.4 to 4.86 cm/mn usng the equlbrum buffer. A pulse of the 5 ml tracer flud (0% aqueous acetone soluton) was appled to the column, and the UV absorbance (80 nm) was montored contnuously wth a UV spectrophotometer. Equlbrum and Adsorpton Knetcs Dlutons of casen partally precptated bovne mlk serum contanng dfferent concentratons of proten were ncubated n a shakng water bath at room temperature (Fgure ). The adsorbent used n these experments was STREAMLINE Phenyl. The total proten and α-lactalbumn concentratons n the supernatant were montored at varous tme ntervals based on absorbance at 80 nm and by HPLC analyss usng a Mono Q onc-exchange column calbrated wth α-lactalbumn (85% purty, purchased from SIGMA). Brazlan Journal of Chemcal Engneerng

3 Concentraton of α-lactalbumn from Cow Mlk Whey 503. Pumps P-5000 and P-6000,. Valve V7, 3. Loop 500 µl, 4. Loop de 700 ml, 5. Mxture, 6. Thermostatc bath, 7. Column of expanded bed, 8. Detector of UV (54-80 nm), 9. Regster, 0. Computer,. Fractons collector,. Ion-selectve electrode, 3. ph-meter/mv, 4. Buffer Fgure : Expermental set-up used for the expanded-bed column experments. Fgure : Expermental set-up for batch experments Chromatography The chromatographc experments were done n the same column (Fgure ) as that used for the expanded-bed experments (STREAMLINE SP 5 column), coupled to a P-5000 Amersham Pharmaca pump wth a UV-montorng absorbance cell mounted at the column ext. Mlk serum was ntroduced nto the column from the bottom usng a superfcal velocty of cm/mn, whch caused the 0 cm hgh fxed bed to expand.5-fold. The expanded-bed adsorpton step was done usng the frontal mode of operaton and was followed by the applcaton of the remanng soluton. The expanded bed was washed untl the absorbance at 80 nm had returned to the baselne. The eluton step was done after the volume n the bed had settled. Samples of the eluted peak were analyzed by SDS-PAGE electrophoress and HPLC. The HPLC column peaks were calbrated wth α- lactalbumn, β-lactoglobuln and casen (all from Sgma). Brazlan Journal of Chemcal Engneerng Vol., No. 04, pp , October - December, 005

4 504 L. S. Conrado, V. Veredas, E. S. Nóbrega and C. C. Santana Mathematcal Formulaton A mathematcal model that descrbes proten adsorpton on macroporous solds was presented by Horstmann and Chase (989) and Carrére (993) and adopted n the present work. It ncludes the mass transfer n the lqud flm, the dffuson n the partcle pores (adsorbent resn) and the surface adsorpton rate. The mathematcal formulaton s the followng: Mass Balance for a Sold Partcle Correspondng to the Dfferental Equaton that Descrbes the Solute (Proten) Dffuson n the Partcle Pores (Adsorbent Resn): e C C C q p = ed p ef + t r r r t () where ε p s the partcle porosty, C s the proten concentraton n the lqud phase nsde the partcle pores, q s the proten concentraton n the sold phase, D ef s the effectve dffuson coeffcent and t and r are the temporal and spatal (radal) varables, respectvely. The ntal and the boundary condtons are the followng: t = 0 C = 0 r (3) In the partcle center: C r = 0 r = 0 (4) On the surface: r = R C ε pd ef =k f (Cb-C = r= R) r (5) where R s the partcle radus and k f s the external mass transfer coeffcent. Accordng to Equaton 5, the rate of mass transfer through the lqud flm s related to the overall concentraton of proten n the lqud phase C b and to the concentraton of proten n the lqud phase of the partcle pores C wth a radus of R. Adsorpton Knetcs on the Pore Surface: where k and k are the adsorpton and desorpton knetcs constants, respectvely; q m s the maxmum bndng capacty of adsorbent and q s the quantty adsorbed n a tme nterval t. Assumng a very fast adsorpton rate on the surface, the Langmur equaton at equlbrum ( q / t.= 0) s: qmc q = k + C d where k d = (k / k ). Wth ths smplfcaton Eq.() becomes C C C ε p =ε pdef + t r r r C qmaxk t K + C d ( ) d (7) (8) Mass Balance n the Bulk Lqud Phase wth Overall Proten Concentraton dcb 3vk f (C b C) r= R dt RV = (9) where v s the adsorbent volume and V s the lqudphase volume. The ntal condtons of Equaton (9) are t 0 C = b 0 = C (0) where C o s the ntal lqud-phase proten (BSA) concentraton. Defnng the followng dmensonless varables: x t r R C = T = C0 td kr ef f = B m = R ed p ef T b C = C b 0 () The set of former equatons mples the followng results: Mass balance on a sold partcle: q t = KC(q q) Kq m (6) qmk d Θ Θ Θ + = + e p(kd + C 0Θ) τ x x x () Brazlan Journal of Chemcal Engneerng

5 Concentraton of α-lactalbumn from Cow Mlk Whey 505 τ = 0 Θ = 0 x (3) Θ x = 0 = 0 x Θ B = τ ε m x = ( Θ Θ ) b x= Mass balance n the bulk lqud phase: dθ b 3v = Bm Θb Θ x dτ V ( = ) (4) (5) (6) t = 0 Θ b = (7) When the varable transformaton u = x s appled to Equatons () and (5) and the spatal varable s dscretzed through the orthogonal collocaton method (Fnlayson, 980; Vlladsen and Mchelsen, 978; Yao C. and Ten, 99), we have qmk dθ d j + = ε p(kd + C 0Θj) dτ N+ N+ = 4u B Θ + 6 A Θ j jk k jk k k= k= (J =,,N) (8) t = 0 Q k = 0 k =,, N+ (9) B Nk + Θ k = Θb Θ N+ x= (0) N+ m x= A, ( ) k= where A and B are the matrces of orthogonal collocaton. There are N collocaton ponts n the 0 < u < range, and one boundary pont u N+ =. The pont u = 0 s not ncluded because the symmetry condton requres the frst dervatve to be zero at u = 0 and ths condton s bult nto the tral functon. The ordnary dfferental Equatons (6) and (8) together wth the respectve ntal condtons (Equatons (l7) and (9)) and the boundary condtons (Equatons (0) and (6)) form an ordnary dfferental equaton system that can be ntegrated by known methods, such as the fourthorder Runge-Kutta method (Fnlayson, 980; Yao and Ten, 99). RESULTS AND DISCUSSION Study of Bed Flud Dynamcs Fgure 3 shows that bed heght depended on superfcal velocty (U) and calculated bed porosty (ε). Rchardson and Zak s (954) correlaton parameters, n and U t (termnal settlng velocty), were determned by lnear regresson from a logarthmc plot of porosty versus superfcal velocty (Table ). The values for these parameters and the partcle termnal velocty agreed wth Rchardson and Zak s correlaton and wth the Stokes equaton for low Reynolds number partcleflud dynamcs. Bed heght (cm) cm 5 cm cm 5 cm 30 cm Lnear velocty (cm/mn) Fgure 3: Expanson characterstcs of the resn bed Brazlan Journal of Chemcal Engneerng Vol., No. 04, pp , October - December, 005

6 506 L. S. Conrado, V. Veredas, E. S. Nóbrega and C. C. Santana Bed heght (cm) Table : Numercal values for the bed expanson parameters Expermental Rchardson & Zak coeffcent (n) (cm/mn) Expermental settlng velocty ( U t ) (cm/mn) Settlng velocty calculated wth the Stokes equaton (cm/mn) Adsorpton Equlbrum and Knetcs The equlbrum sotherm for the total protens and α-lactalbumn n mlk serum are shown n Fgures 4 and 5, respectvely. A Langmur-type sotherm was obtaned for the total protens and for the α-lactalbumn, whch was adsorbed wth other protens. The data ftted Equaton (7) and the parameters k d and q m (dssocaton constant and maxmum capacty of adsorpton) are shown n Table 3. The adsorpton knetcs data were used together wth the Horstmann and Chase mathematcal model to assess the mass transfer parameters n order to scale up the adsorpton process. The values of mass transfer resstance parameters D p and k f are m /s and m/s (Fgure 6), whch descrbe the adsorpton phenomena n the resn partcles relatvely well. Equlbrum capacty (mg of proten/mg of resn) ,0 0, 0, 0,3 0,4 0,5 0,6 Whey concentraton (mg/ml) Fgure 4: Equlbrum sotherm for total protens n the whole mlk whey Equlbrum capacty (mg of proten/mg of resn) 3,0,5,0,5,0 0,5 0,0 0,0 0, 0, 0,3 0,4 0,5 0,6 0,7 α-lactalbumn concentraton (mg/ml) Fgure 5: Equlbrum sotherm for α-lactalbumn Brazlan Journal of Chemcal Engneerng

7 Concentraton of α-lactalbumn from Cow Mlk Whey 507 Table 3: Equlbrum parameters for the adsorpton of a-lactalbumn and whole whey onto Streamlne Phenyl resn Qm (mg of proten/g of resn) k d (mg of proten/g de resn) α-lactalbumn Whole Bovne whey ,45 α-lactalbumn Concentraton (mg/ml),40,35,30,5,0,5,0,05 a-lactalbumn (.4 mg/ml) Model D p = 4.87x0-9 m /s K f = 6.9x0-3 m/s Tme (s) Fgure 6: Adsorpton knetcs for α-lactalbumn and the adjusted model Chromatography The breakthrough curves for fxed and expanded beds showed a smlar behavor (Fgure 7). SDS/PAGE 5% polyacrylamde gels were used to dentfy the protens n several steps of the process (Fgure 8). Lane n Fgure 6 shows the molecular mass calbraton protens and lane 3 shows the protens that were eluted from the resn wth the eluton buffer. Some α-la (4 kda), β-lactoglobuln (8 kda) and casen (30 kda) were found n ths pool. Lane 4, whch s representatve of the eluted fracton, contaned manly α-la together wth some traces of β- lactoglobuln and casen. The eluted fracton was analyzed by HPLC n order to determne the α- lactalbumn concentraton. The purty of α- lactalbumn after one cycle of adsorpton/eluton protocol was 79 % wth no prevous polshng procedure. Absorbance (80 nm) ADSORPTION WASH Expanded bed Fxed bed ELUTION Eluton Volume (ml) Fgure 7: Breakthrough and eluton curves for protens n mlk whey Brazlan Journal of Chemcal Engneerng Vol., No. 04, pp , October - December, 005

8 508 L. S. Conrado, V. Veredas, E. S. Nóbrega and C. C. Santana Fgure 8: SDS/PAGE of protens fractons CONCLUSIONS A procedure for the concentraton of α- lactalbumn from cow mlk whey was developed usng a hgh-densty, hydrophobc resn and an expanded-bed column. The expanson characterstcs as well as the equlbrum and adsorpton knetc data were determned for future scale-up of the process. The mathematcal formulaton and the numercal method descrbe farly well the adsorpton knetcs for α-lactalbumn, but need to be mproved. The expermental breakthrough and eluton curves and HPLC analyss of the eluate showed that a purty of 79% was obtaned for α-lactalbumn after one cycle of the adsorpton/eluton protocol, pror to any polshng procedure. ACKNOWLEDGMENTS The authors would lke to thank Prof. Valdemro C. Sgarber, Department of Nutrton, School of Food Engneerng, State Unversty of Campnas, for the whole whey samples and for the many dscussons on the of ths subject. NOMENCLATURE A matrx of orthogonal collocaton B matrx of orthogonal collocaton B m Bot number C* equlbrum concentraton, C b lqud-phase proten concentraton, C partcle pore proten concentraton, C o ntal bulk lqud-phase proten concentraton, D ef effectve dffuson coeffcent, m mn - H o ntal heght of the bed k adsorpton knetc constant, cm -3 g -3 mn - k desorpton knetc constant, mn - k d dssocaton constant, k f external mass-transfer m mn - convectve coeffcent, N number of orthogonal collocaton ponts n Rchardson and Zak s parameter q* equlbrum concentraton, q sold-phase proten concentraton, q m maxmal capacty of the sold phase, R partcle radus, m r spatal(radal) varable, m R determnaton coeffcent t temporal(tme) varable, mn U superfcal velocty u dmensonless spatal varable Brazlan Journal of Chemcal Engneerng

9 Concentraton of α-lactalbumn from Cow Mlk Whey 509 U t termnal settng velocty v sold-phase volume, cm 3 V lqud-phase volume, cm 3 x dmensonless spatal varable Greek Letters Γ * equlbrum concentraton,. 0-3 g g - Γ m maxmal capacty of the sold. 0-3 g g - phase, ε bed porosty ε p partcle porosty τ dmensonless temporal varable Θ dmensonless concentraton Indexes B j k ndex, refers to bulk lqud phase ndex, refers to pore lqud phase ndex, refers to orthogonal collocaton pont ndex, refers to orthogonal collocaton pont REFERENCES (j =,,N) (k =,,N+) Arnold, F.H., Blanch, H.W. and Wlke, C.R., Lqud- Chromatography Plate Heght Equatons, Journal of Chromatography, 330 (), pp (985). Beszedts, S. and Netzer, A., Proten Recovery from food processng wastewaters, B&L Informatons Servces, Toronto, Ontaro, pp (98). Carrére, H., Mlk Whey Protens Extracton through Ion Exchange Chromatography n Fludzed Beds: Modelng and Optmzaton, Ph.D. dss., Insttut Natonal Polytechnque de Toulouse, Toulouse, (993) (n French). Fnlayson, B.A., Nonlnear Analyss n Chemcal Engneerng, McGraw-Hll, New York (980). Horstmann, B.J. and Chase, H.A., Modelng the Affnty Adsorpton of Immunoglobuln-G to Proten-A Immoblzed to Agarose Matrces, Chem. Eng. Res. Des., vol. 67, pp (989). Horton, B.S., Commercal Utlzaton of Mnor Mlk Components n the Health and Food Industres, Journal of Dary Scence, 78, pp (995). Karau, A., Benken, C., Thömmes, J. and Kula, M.R., The Influence of Partcle Sze Dstrbuton and Operatng Condtons on the Adsorpton Performance n Fludzed Beds, Botechnology and Boengneerng, vol. 55, n o, pp (997). McKenzey, H.A., Mlk Protens and Molecular Botechnology, vol., Academc Press (970). Noppe, W., Haezebrouck, P., Hanssens, I. and De Cuyper, M., A Smplfed Purfcaton Procedure of Alpha-Lactalbumn from Mlk usng Ca+dependent Adsorpton n Hydrophobc Expanded Bed Chromatography, Boseparaton, vol. 8, nos. -5, pp (999). Rchardson, J.F. and Zak, W.N., Sedmentaton and Fludzaton: Part I, Trans. Inst. Chem. Eng. 3, pp (954). Vlladsen, J.E. and Mchelsen, M.L., Soluton of Dfferental Equaton Models by Polynomal Approxmaton, Prentce Hall Inc., New Jersey, pp. 445 (978). Yao C.C. and Ten C., Approxmaton of Intrapartcle Mass-Transfer n Adsorpton Processes Nonlnear-Systems, Chemcal Engneerng Scence, 47(), pp (99). Brazlan Journal of Chemcal Engneerng Vol., No. 04, pp , October - December, 005