Journl of Chromtogrphy A, 859 (1999) 77 86 www.elsevier.com/ locte/ chrom Anlysis of bovine whey proteins in soyben diry-like products by cpillry electrophoresis C. Grcı-Ruiz, M. Torre, M.L. Mrin b,b, * Centro de Tecnologı de los Alimentos y Servicios Biosnitrios, Universidd de Alcl, Ctr. Mdrid Brcelon Km. 33.600, 28871 Alcl de Henres (Mdrid), Spin b Deprtmento de Quımic Anlıtic, Fcultd de Ciencis, Universidd de Alcl, Ctr. Mdrid Brcelon Km. 33.600, 28871 Alcl de Henres (Mdrid), Spin Received 2 Mrch 1999; received in revised form 9 July 1999; ccepted 3 August 1999 Abstrct The simultneous seprtion of bovine whey proteins [-lctlbumin nd b-lctoglobulin (A1B)] nd soyben proteins ws performed, for the first time, by cpillry electrophoresis. Different experimentl conditions were tested. The most suitble consisted of 0.050 M phosphte buffer (ph 8) with 1 M ure nd 1.2 mg/ ml methylhydroxyethylcellulose, UV detection t 280 nm, 15 kv pplied voltge, nd 308C temperture. Quntittion of bovine whey proteins in commercil powdered soyben milk mnufctured by dding bovine whey to its formultion ws performed using the clibrtion method of the externl stndrd. Direct injection of solution of the powdered soyben milk only enbled quntittion of -lctlbumin in the commercil smple. Detection of b-lctoglobulin (A1B) required cid precipittion of the solution of the smple in order to concentrte bovine whey proteins in the superntnt prior to the nlysis of this protein in the whey obtined. Since -lctlbumin could lso be quntitted from the injection of the whey, the simultneous determintion of -lctlbumin nd b-lctoglobulin (A1B) ws possible upon cid precipittion of the powdered soyben milk solution. Detection limits obtined were 14 mg/ g sol. for -lctlbumin nd 52 mg/ g sol. for b-lctoglobulin (A1B) which represent protein concentrtions bout 60 mg/ 100 g smple for -lctlbumin nd 100 mg/ 100 g smple for b-lctoglobulin (A1B). 1999 Elsevier Science B.V. All rights reserved. Keywords: Whey; Soyben; Food nlysis; Proteins; Lctlbumin; Lctoglobulins 1. Introduction some individuls, nd this is why soyben diry-like products (soyben milks or soyben infnt formuls Bovine whey proteins, constituted minly by - mong others) re n interesting lterntive [1,2]. lctlbumin (-LA) nd b-lctoglobulin (A1B) [b- LG (A1B)], my cuse some llergic rections to *Corresponding uthor. Deprtmento de Quımic Anlıtic, Fcultd de Ciencis, Universidd de Alcl, Ctr. Mdrid Brcelon Km. 33.600, 28871 Alcl de Henres (Mdrid), Spin. Tel.: 134-91-8854-935; fx: 134-91-8854-971. E-mil ddress: mluis.mrin@lcl.es (M.L. Mrin) However, bovine whey proteins re sometimes dded to gret number of commercil soyben diry-like products to increse their nutritionl vlue. Since this ddition must be indicted on the lbel of these products in order to prevent the consumption of the niml proteins by llergic people, the development of nlyticl methods to detect bovine whey proteins in soyben diry-like products is therefore essentil 0021-9673/ 99/ $ see front mtter 1999 Elsevier Science B.V. All rights reserved. PII: S0021-9673(99)00838-9
78 C. Grcı-Ruiz et l. / J. Chromtogr. A 859 (1999) 77 86 to control the qulity of mny products commercil- Sigm. The soyben protein isolte (SPI), tken s ized s 100% vegetble. stndrd of soyben proteins, ws from ICN (Auror, Different techniques such s high-performnce OH, USA). The powdered soyben milk ws liquid chromtogrphy (HPLC) [3 11] or cpillry commercil product for humn consumption purelectrophoresis (CE) [12,13] hve been used to chsed from locl mrket in Alcl de Henres nlyze soyben or bovine whey proteins. However, (Mdrid, Spin). Its composition ccording to the the simultneous seprtion of soyben nd niml lbel of the product ws the following: soyben whey proteins hs only been recently chieved by protein isolte, bovine whey, clcium phosphte, reversed-phse (RP) HPLC [14,15]. Thus, RP- lecithin, vitmins A, C, D, B 12, thimine, riboflvin, HPLC method hs been developed to seprte soy- nicin, pntotenic cid, folic cid nd minerls. ben nd bovine whey proteins simultneously in pproximtely 20 min nd to quntify these proteins 2.2. Apprtus from the resulting whey upon cid precipittion of diry-like soyben product [14]. An ultrrpid de- The cpillry electrophoresis instrument ws tection of bovine whey proteins in powdered Model 279A-HT from Applied Biosystems (Norsoyben milk ws lso chieved by perfusion RP- wlk, CT, USA), equipped with n UV detector, HPLC in very short nlysis time (bout 5 min) temperture-controlled cpillry comprtment nd n [15]. utosmpler. Dt tretment ws performed with In spite of the interesting possibilities tht CE Turbochrom cquisition system (Perkin-Elmer, Nortechniques offer concerning protein seprtion wlk, CT, USA). Spectr nd derivtives for peks of [16,17], s fr s we know, there is no reference in 3D stndrds nd smple were obtined in HP CE the literture to the ppliction of this technique for system (Hewlett-Pckrd, Wldbronn, Germny) the simultneous seprtion of soyben nd bovine equipped with n on-column diode rry detection whey proteins. This is why in this work CE hs been 3D (DAD) system nd HP CE Chemsttion softdirected towrd ttining two objectives: (i) to wre. The fused-silic column employed [60 cm (40 seprte simultneously soyben proteins (globulins) cm to the detector)350 mm I.D.3360 mm O.D.] ws from bovine whey proteins [-LA nd b-lg (A1B)] obtined from Polymicro Technologies (Phoenix, nd (ii) to quntify bovine whey proteins in commer- AZ, USA). Temperture ws kept constnt nd equl cil powdered soyben milks in which these proteins to 308C. Injection ws performed by vcuum (67.73 were included in their formultion. kp during 1 s). Electrolytic solutions were degssed in Pent ultrsonic system (Mrtorell, Spin). A Model 93 ph 2. Experimentl meter (Rdiometer Copenhgen, Bgsverd, Denmrk) ws employed to djust the ph of the buffers. 2.1. Chemicls nd smples Centrifugtion ws performed t 2000g for 20 min in Sorvll RD-5B refrigerted superspeed centrifuge All regents were of nlyticl grde. Boric cid (DuPont, Newtown, CT, USA). nd disodium phosphte nhydrous were obtined from Schrlu (Brcelon, Spin); 2-(N-cyclohexyl- 2.3. Procedure mino)ethnesulfonic cid (CHES) ws purchsed from Sigm (St. Louis, MO, USA); sodium hy- Buffers were prepred s follows: (i) the cids or droxide, sodium sulfte nhydrous nd ure were slts (CHES, boric cid nd disodium phosphte) nd supplied from Pnrec (Brcelon, Spin); methyl- dditives (sodium sulfte, MHEC nd ure) were hydroxyethylcellulose (MHEC) ws purchsed from weighed nd dissolved in HPLC-grde wter; (ii) Aldrich (Milwukee, WI, USA). All solutions were solutions were sonicted during 5 min; (iii) ph of prepred with HPLC-grde wter (Milli-Q system; solutions ws djusted t vlue close to 8 with Millipore, Bedford, MA, USA). concentrted solution of sodium hydroxide (borte Stndrds of -LA nd b-lg (A1B) were from nd CHES buffers) or with the phosphoric cid in the
C. Grcı-Ruiz et l. / J. Chromtogr. A 859 (1999) 77 86 79 cse of phosphte buffer; (iv) once prepred, these tion of peks ws mde by setting the bseline from seprtion medi were sonicted during 5 min nd vlley to vlley. As b-lg (A1B) protein showed filtered [through 0.22-mm disposble sterile poly- two peks in the electropherogrm of the stndrd sulfone filters (Alltech, Deerfield, IL, USA)] before nd the smple, the clibrtion in this cse ws mde injection. considering the ddition of both pek res. Smple solutions were esily prepred weighing The CE method developed ws vlidted by nd dissolving n pproprite mount of the pow- evluting the precision (repetbility nd reproducidered soyben milk in ech seprtion medium, bility) nd the ccurcy. sonicting nd filtering before injection. When the Detection limits were individully clculted from whey protein b-lg (A1B) could not be detected by the clibrtion curve s the concentrtion of bovine direct injection of the smple solution in the CE whey proteins giving signl equl to the intercept system, preconcentrtion step ws pplied in order plus three times the stndrd error of the stright line to concentrte this protein. This preconcentrtion [19]. ws mde following the method described by the Interntionl Diry Federtion (15 October 1993) [18] nd consisted of n cid precipittion (2 M HCl, 3. Results nd discussion ph 4.6) of freshly prepred solution corresponding to glss of milk (bout 12 g of powdered soyben 3.1. Simultneous seprtion of soyben nd milk in 250 ml of Milli-Q wter) nd subsequent bovine whey proteins by CE centrifugtion (2000 g during 20 min) to precipitte the mjor frction of soyben proteins nd, simul- In order to select the most suitble experimentl tneously, to concentrte the bovine whey proteins in conditions for the simultneous seprtion of soythe superntnt. ben nd bovine whey proteins, some preliminry At the beginning of the dy, the column ws experiments were performed. Mixtures of stndrds rinsed with HPLC-grde wter (5 min), followed by of these proteins were injected in different ex- 0.1 M sodium hydroxide (5 min), nd finlly HPLC- perimentl conditions in which the nture of the grde wter (5 min). Typiclly, nlyses were per- seprtion buffer, dditives, detection wvelength formed utomticlly by the equipment described nd the pplied voltge were modified. before using run sequence tht included the Two buffers of inorgnic nture (borte nd following steps: (i) 2-min rinse with HPLC-grde phosphte) nd nother of orgnic nture (CHES) wter; (ii) 2-min rinse with 0.1 M sodium hydroxide; were used with nd without some dditives s (iii) 2-min rinse with HPLC-grde wter; (iv) 4-min sodium sulfte, MHEC nd ure. Sodium sulfte ws rinse with the seprtion medium (buffer nd ddi- used s modifier of the electroosmotic flow (EOF) tives); (v) hydrodynmic smple injection from the becuse the increse of ionic strength of the buffer smple vil; nd (vi) smple seprtion run for 15 decreses the EOF nd improves resolution [20]. min with the seprtion medium in inlet nd outlet MHEC ws employed to prevent dsorption of vils. All nlyses were performed in triplicte. proteins to the inner wll of the cpillry [12,20]. Ure, denturing gent for proteins, ws used t 2.4. Quntittive nlysis nd method vlidtion low concentrtion (1 M) to increse protein solubiliztion [20,21]. Bsic ph vlues (ner 8) were Quntittive nlysis ws performed using the employed. This ph vlue ws considered pproprite clibrtion method of the externl stndrd (four to nlyze both bovine whey nd soyben proteins points were used to obtin ech clibrtion plot, ech becuse t this ph they re negtively chrged nd point corresponding to the nlysis by triplicte of the dsorption to the negtively chrged inner wll of ech stndrd solution). Thus, solutions contining the cpillry is minimum. known concentrtions of -LA nd b-lg (A1B) When CHES buffer modified with 1 M ure ws stndrds were prepred individully in the elec- used t bsic ph vlues, poor efficiency nd restrolytic solution to built the clibrtion lines. Integr- olution were obtined in the seprtion of the
80 C. Grcı-Ruiz et l. / J. Chromtogr. A 859 (1999) 77 86 proteins s indicted in Tble 1. In contrst, when sponding to b-lg (A1B). Identifiction of ll these phosphte nd borte buffers were used t ph close peks ws done by injecting individully into the CE to 8 with the dditives indicted in Tble 1, five system ech bovine whey protein stndrd nd SPI. peks (or six peks when dditives such s N2SO4 On the other hnd, when 0.100 M borte buffer with or MHEC were dded to the buffers, mking pek 1 0.030 M N2SO4 nd 0.2 mg/ml of MHEC (ph 8) prtilly resolved into two peks) were seprted ws employed to nlyze stndrd mixture of when mixtures of stndrds of bovine whey proteins bovine whey proteins nd SPI, the electropherogrm nd SPI were injected. As n exmple, Fig. 1 shows obtined ws very similr to tht corresponding to the effect of some dditives on the seprtion of the phosphte buffer showing two peks t migrtion mixture of proteins (bovine whey proteins nd SPI) times of bout 5 nd 7 min for soyben proteins, one with phosphte buffer. It cn be observed tht pek of bout 6 min due to -LA, nd two prtilly efficiency nd resolution [especilly for the two resolved peks t bout 9 min corresponding to peks corresponding to b-lg (A1B)] improved b-lg (A1B). Despite the good resolution obtined when dditives (1 M ure nd 1.2 mg/ ml MHEC) for the stndrd proteins with both phosphte nd were dded to 0.05 M phosphte buffer (ph 8). borte buffers, minor mtrix effect ws observed Under these conditions, the electropherogrm ob- with phosphte buffer when injecting rel smples tined showed two peks t migrtion times bout 6 into the CE system. nd 10 min for soyben proteins (peks 1 nd 3 in Severl detection wvelengths were lso investi- Fig. 1), one pek bout 9 min corresponding to -LA gted s shown in Tble 1. In fct, wvelengths from (pek 2 in Fig. 1), nd two prtilly resolved peks 214 nm (corresponding to peptide bonds) to 280 nm t bout 11 min (peks 4 nd 5 in Fig. 1) corre- (corresponding to the mximum bsorption of ro- Tble 1 Results obtined in some preliminry experiments performed to simultneously seprte soyben nd bovine whey proteins Buffer Additives ph l V I Anlysis Observtions (nm) (kv) (ma) time (min) 0.050 M CHES 1 M Ure 8.9 230 20 4 7 Poor efficiency nd resolution 7.9 254 20 2 7 of stndrd proteins 0.050 M Phosphte None 8.0 280 15 52 13 Good resolution of stndrd proteins 0.030 M N2SO4 8.0 280 15 92 13 Good resolution of stndrd proteins 1 M Ure 8.0 225 20 85 9 Mximum signl-to-noise rtio 8.0 235 20 85 9 ws obtined for bovine whey proteins 8.0 245 20 85 9 t 280 nm 8.0 254 20 85 9 8.0 265 20 85 9 8.0 280 20 85 9 1 M Ure, 0.2 mg/ ml MHEC 8.0 245 20 85 14 Very good resolution of stndrd 1 M Ure, 0.6 mg/ ml MHEC 8.0 245 30.100 6 proteins employing 1.2 mg/ ml of 8.0 245 20 75 9 MHEC nd n pplied voltge of 15 kv 8.0 245 15 52 14 1 M Ure, 1.2 mg/ ml MHEC 8.0 245 15 52 12 8.0 280 15 52 12 0.100 M Borte 0.030 M N2SO4 8.0 214 20 66 10 Poor resolution of stndrd proteins 8.0 254 20 66 10 0.2 mg/ ml MHEC, 0.030 M N2SO4 8.2 254 20 78 10 Good resolution of stndrd proteins A mixture of SPI, -LA, nd b-lg (A1B) stndrds ws used. Concentrtions (mg/g sol., s is bsis) rnged from 2 to 13 for SPI, from 0.3 to 1.2 for b-lg (A1B) nd from 0.1 to 0.9 for -LA. b Injection by vcuum, 67.73 kp during 1 s.,b
C. Grcı-Ruiz et l. / J. Chromtogr. A 859 (1999) 77 86 81 Fig. 1. Electropherogrms corresponding to the injection of mixture of SPI, -LA nd b-lg (A1B) stndrds employing different dditives in the seprtion medi. Conditions: detection wvelength, 280 nm; pplied voltge, 15 kv; injection by vcuum, 67.73 kp during 1 s.; temperture, 308C; concentrtion s is bsis: SPI, 13.04 mg/g sol.; -LA, 0.39 mg/g sol.; nd b-lg (A1B), 1.18 mg/g sol.; cpillry [60 cm (40 cm to the detector)350 mm I.D.3360 mm O.D.]. Seprtion medi: () 0.050 M phosphte buffer (ph 8); (b) 0.050 M phosphte buffer, 0.030 M sodium sulfte (ph 8); (c) 0.050 M phosphte buffer, 1 M ure, 1.2 mg/ml MHEC (ph 8). Peks: 1,35soyben proteins; 25-LA; 4 nd 55b-LG (A1B). mtic mino cids such s tyrosine nd tryptophn) presence of bovine whey proteins in these products, were investigted. At first, 245 nd 254 nm were quntittion of these proteins in commercil powchosen s better wvelengths to detect proteins in dered soyben milk contining bovine whey proteins stndrds mixtures. However, when commercil ws chieved. The experimentl conditions used for powdered soyben milk ws injected it ws observed these nlyses were those previously chosen: 0.05 M tht the most suitble wvelength to detect bovine phosphte buffer (ph 8) with 1 M ure nd 1.2 whey proteins corresponded to 280 nm, for which mg/ ml MHEC, detection wvelength, 280 nm, nd mtrix signls did not pper. In these conditions, the pplied voltge, 15 kv. detection of -LA ws possible by direct injection of Fig. 2 shows the electropherogrm obtined when the smple dissolved in the seprtion buffer. the powdered soyben milk, dissolved in the sep- Finlly, lthough different vlues of pplied volt- rtion medi, ws directly injected into the CE ges rnging from 15 to 30 kv were tested (see Tble system. In this figure, peks corresponding to soy- 1), vlue of 15 kv ws chosen s compromise ben proteins (1 nd 3) nd to -LA contined in the between current intensity nd nlysis time. powdered soyben milk (pek 2) cn be observed. Fig. 2b shows the electropherogrm corresponding to 3.2. Quntittive nlysis of bovine whey proteins the sme smple spiked with the stndrd of -LA. in commercil soyben diry-like product The increse observed in the re of pek 2 when spiking the rel smple with -LA stndrd enbled In order to show the potentil of the CE method to identify the pek 2 s the pek corresponding to developed concerning the qulity control of soyben -LA protein. This fct ws lso corroborted by diry-like products s regrds to the detection of the compring the spectr nd derivtives of pek 2
82 C. Grcı-Ruiz et l. / J. Chromtogr. A 859 (1999) 77 86 Fig. 2. Electropherogrms corresponding to the injection of commercil powdered soyben milk in which bovine whey proteins were included in its formultion. Conditions: 0.050 M phosphte buffer, 1 M ure, 1.2 mg/ ml MHEC (ph 8); other experimentl conditions s in Fig. 1. Concentrtion s is bsis: () powdered soyben milk, 29.39 mg/g sol.; (b) powdered soyben milk, 29.66 mg/g sol. spiked with 0.16 mg/g sol. -LA. Peks s in Fig. 1: 6 nd 75unknown peks. obtined in the smple with tht of the -LA superntnt supplemented with b-lg (A1B) stnstndrd. drd. The increse observed in the res of the two Fig. 2 nd b show tht peks corresponding to peks corresponding to b-lg (A1B) when spiking soyben proteins (1 nd 3) nd -LA (pek 2) cn the smple with b-lg (A1B) stndrd showed tht be observed by direct injection of solution of the peks 4 nd 5 corresponded to this bovine protein. smple; however, peks corresponding to b-lg (A1 This point ws lso corroborted compring the B) were not observed. In fct, peks 6 nd 7 were spectr nd derivtives for peks in the smple n in unknown peks which did not pper when SPI or the stndrd, when DAD ws used. -LA or b-lg (A1B) stndrds were injected into For quntittive nlysis of bovine whey proteins the CE system. A study of ll peks obtined using in the powdered soyben milk, the externl stndrd diode-rry detection indicted tht these peks did method ws used for clibrtion nd individully not correspond to protein mteril. performed for ech bovine whey protein. Liner To detect the b-lg (A1B) present in the pow- reltionships were obtined for the vrition of the dered soyben milk nlyzed, previous step, pre- pek re s function of the concentrtion of -LA concentrtion by the protocol described in the Ex- (working concentrtion rnge from 0.036 to 0.356 perimentl section, ws necessry. Fig. 3 shows the mg/g sol. s is bsis) nd b-lg (A1B) (working electropherogrm obtined by direct injection of the concentrtion rnge from 0.122 to 0.927 mg/ g sol. superntnt (whey) obtined upon cid precipittion s is bsis) stndrds. Good liner correltions were of solution of the powdered soyben milk, nd Fig. found in both cses (r.0.999) (four points consid- 3b shows the electropherogrm corresponding to the ered for the clibrtion). The chrcteristics of these
C. Grcı-Ruiz et l. / J. Chromtogr. A 859 (1999) 77 86 83 Fig. 3. Electropherogrms corresponding to the whey obtined by cidic precipittion of solution (43.93 mg/ g sol. s is bsis) of commercil powdered soyben milk. Experimentl conditions s in Fig. 2. () Direct injection of the whey; (b) direct injection of the whey spiked with b-lg (A1B) (0.61 mg/g sol.). Peks s in Fig. 1: 6 nd 75unknown peks. clibrtion plots enbled the evlution of the CE in migrtion time nd 1.9% in pek re. On the method sensitivity s slopes of the stright lines other hnd, reproducibility for -LA content in the 24 obtined [2.74?10 g sol./ mg protein for -LA nd commercil soyben product nlyzed ws mesured 24 1.34?10 g sol./ mg protein for b-lg (A1B)] nd s the RSD obtined for the six vlues of condetermining detection limits for both proteins from centrtion determined by the CE method when six the intercept vlues [290 for -LA nd 545 for b-lg individul nlyses were performed. Results obtined (A1B)], not being significntly different from zero, re shown in Tble 2. It cn be observed tht the t-test, P,0.05 [19]. Detection limits obtined were RSD obtined ws equl to 3%. Likewise, repro- 14 mg/g sol. for -LA nd 52 mg/g sol. for b-lg ducibility of the b-lg (A1B) content in the whey (A1B) [bout 60 mg/100 g smple for -LA nd obtined fter cid precipittion of the sme product 100 mg/ 100 g smple for b-lg (A1B)]. ws evluted s the RSD obtined for the five To evlute the precision of the optimized CE vlues of concentrtion determined by the CE methmethod, repetbility nd reproducibility were lso od in five individul nlyses of the whey. In this studied. The repetbility in migrtion time nd pek cse, the RSD obtined ws less thn 3% (Tble 2). re ws determined (s reltive stndrd devition, Accurcy of the method ws evluted by cl- RSD) for six consecutive injections of solutions of culting the recovery (%) of ech bovine whey -LA or b-lg (A1B) stndrds. The RSDs were protein when known quntity of stndrd of the bout 2.4% for migrtion time nd 3.4% for pek corresponding protein ws dded to three solutions re in the cse of -LA (0.130 mg/ g sol. s is prepred with the powdered soyben milk nlyzed bsis) nd even minor for b-lg (A1B) (0.730 mg/g (in the cse of the -LA) or to three whey solutions sol. s is bsis) for which the RSD ws bout 0.6% obtined fter cid precipittion of the nlyzed
84 C. Grcı-Ruiz et l. / J. Chromtogr. A 859 (1999) 77 86 Tble 2 Reproducibility in the -LA (six individul determintions) nd b-lg (A1B) (five individul determintions) contents in commercil powdered soyben milk elborted with bovine whey proteins (clibrtion by the externl stndrd method) -LA b-lg (A1B) b c b c Cs n Concentrtion of Cs n Concentrtion of -LA determined (mg/ 100 g smple) 27.511 3 0.502 43.486 4 0.567 27.485 3 0.495 55.096 2 0.554 26.148 3 0.462 55.713 3 0.539 28.753 3 0.495 58.844 3 0.536 24.778 3 0.479 56.603 3 0.529 26.586 3 0.486 b-lg (A1B) determined (mg/100 g smple) Men vlue (RSD, %) 0.486 (2.97) 0.545 (2.81) Experimentl conditions s in Fig. 2. b Concentrtion of the smple solution s is bsis (mg/g sol.) c Number of replictes for ech smple. commercil product [in the cse of the b-lg (A1 concentrtion of ech bovine protein in ech solution B)]. Tble 3 groups ll dt from which recovery ws clculted (mg/ g sol.). After dding different (%) for these proteins could be obtined. The concentrtions of -LA to the solutions of the powdered soyben milk employed contined 0.486 smple nd different concentrtions of b-lg (A1B) mg/ 100 g smple of -LA nd 0.545 mg/ 100 g to the whey obtined fter cid precipittion of ech smple of b-lg (A1B) (content determined by the solution of the smple, the totl expected (theoret- CE method, see Tble 2). Solutions were prepred icl) content of ech protein in ech smple solution by weighing nd dissolving n dequte quntity of or whey cn be obtined. The comprison between the powdered soyben milk (three solutions to study this theoreticl content nd tht determined by the the ccurcy of ech protein) nd the corresponding CE method enbled to obtin the recovery for ech Tble 3 Accurcy estimted s recovery (%) of the -LA nd b-lg (A1B) proteins dded to commercil powdered soyben milk elborted with bovine whey proteins b c d e f Protein Smple Cs Cp Cx C t (Cp1C x) Ct, exp Recovery (%) -LA Powdered soyben milk contining 27.818 0.136 0.046 0.182 0.188 103 0.486 mg -LA/ 100 g smple 27.716 0.136 0.106 0.242 0.251 104 (determined by the CE method) 27.701 0.136 0.168 0.304 0.318 105 Men vlue (RSD, %) 104 (0.54) b-lg (A1B) Powdered soyben milk contining 43.481 0.235 0.103 0.338 0.344 102 0.545 mg b-lg (A1B)/ 100 g smple 43.477 0.235 0.197 0.432 0.436 101 (determined by the CE method) 43.473 0.235 0.283 0.518 0.551 106 Men vlue (RSD, %) 103 (2.92) Experimentl conditions s in Fig. 2. b Concentrtion of the smple solution s is bsis (mg/g sol.). c Concentrtion of protein in the smple solution (mg/g sol.). d Concentrtion of protein stndrd dded to the solution of the smple (mg/g sol.). e Totl concentrtion of protein in the smple solution (mg/g sol.) clculted s the ddition of Cp nd C x. f Totl concentrtion of protein in the smple solution (mg/g sol.) determined by the CE method.
C. Grcı-Ruiz et l. / J. Chromtogr. A 859 (1999) 77 86 85 bovine protein. Tble 3 shows tht men recoveries these products re used to substitute diry products of 104% for -LA nd close to 103% for b-lg in cse of llergies to niml whey proteins, the (A1B) were obtined. possibility of detecting bovine whey proteins in The concentrtion of -LA found in the powdered soyben diry-like products hs gret interest from soyben milk by direct injection of solution of the the qulity control perspective. Quntittion of powdered soyben milk ws 0.48660.014 (mg/ 100 bovine whey proteins in powdered soyben milk g smple). The concentrtion determined for b-lg elborted with bovine whey in its formultion cn (A1B) in this soyben milk ws 0.54560.015 (mg/ be performed using the clibrtion method of the 100 g smple) nd ws obtined by direct injection externl stndrd. Direct injection of smple soluof the whey obtined fter cid precipittion of tion enbled determintion of -LA while the quntisolution of the powdered soyben milk. The injection ttion of b-lg (A1B) required cid precipittion of of the whey lso enbled determintion of -LA the solution of the powdered soyben milk to concentrtion in the commercil smple nlyzed by concentrte this protein. From the injection of the mesuring the re of the pek obtined in the whey obtined fter cid precipittion of the smple injection of the whey nd by interpolting this vlue solution not only b-lg (A1B) ws determined but of re in the corresponding clibrtion plot. The lso -LA cn be quntitted in the whey. Therefore, result obtined in this cse for the content of -LA in the CE method developed enbles the simultneous the powdered soyben milk studied ws quntittion of two bovine whey proteins in the whey 0.50460.019 mg/ 100 g smple. There were no obtined upon cid precipittion of the powdered significnt differences, in sttisticl terms, between soyben milk solutions (nlysis time close to 12 this result nd tht obtined by direct injection of min). Detection limits obtined were 14 mg/g sol. solution of the powdered soyben milk (t-test, P, for -LA nd 52 mg/g sol. for b-lg (A1B) which 0.05) showing tht the CE method optimized enbles represent protein concentrtions bout 60 mg/ 100 g to determine simultneously the bovine whey pro- smple for -LA nd 100 mg/ 100 g smple for teins present in soyben diry-like product by b-lg (A1B). injecting directly the whey obtined fter cid precipittion of solution of this product. The ppliction of sttisticl tests (t-test, P,0.05) [19] indicted tht b-lg (A1B) content in the Acknowledgements soyben milk obtined by the CE method ws The uthors thnk the University of Alcl de sttisticlly similr to those obtined by other HPLC Henres (project E002/ 98) nd the Comunidd methods such s conventionl RP-HPLC [14] Autonom de Mdrid (projects COR 0035/94 nd (0.2860.30) (mg/100 g smple) nd perfusion RP- 06G/047/96) for finncil support. They lso thnk HPLC [15] (0.45360.114) (mg/ 100 g smple) when C. Mrin for linguistic ssistnce. the sme milk ws nlyzed. Nevertheless, in determining the -LA content, differences sttisticlly significnt were found (t-test, P,0.05) between the results obtined by CE nd by HPLC, lthough the References result by the CE method ws close to those determined by conventionl RP-HPLC (0.3860.02) [1] Soy Protein Products Chrcteristics, Nutritionl Aspects nd Utiliztion, Soy Protein Council, Wshington, DC, 1987. (mg/ 100 g smple) or perfusion RP-HPLC [2] M.C. Grcı, M. Torre, M.L. Mrin, F. Lbord, CRC Crit. (0.35560.015) (mg/ 100 g smple). Rev. Food Sci. Nutr. 37 (1997) 361. [3] M.C. Grcı, M. Torre, F. Lbord, M.L. Mrin, J. Chromtogr. A 758 (1997) 75. [4] M.C. Grcı, 4. Conclusions M. Torre, M.L. Mrin, J. Chromtogr. Sci. 36 (1998) 527. CE hs proved to be useful in detecting bovine [5] B.D. Oomh, H. Voldeng, J.A. Fregeu-Reid, Plnt Foods Hum. Nutr. 45 (1994) 251. whey proteins in soyben diry-like products. Since [6] R.E. Peterson, W.J. Wolf, Cerel Chem. 69 (1992) 101.
86 C. Grcı-Ruiz et l. / J. Chromtogr. A 859 (1999) 77 86 [7] R.E. Buehler, T.T. VnToi, M.B. McDonld Jr., Seed Sci. [15] M.C. Grcı, M.L. Mrin, M. Torre, J. Chromtogr. A 822 Technol. 17 (1989) 193. (1998) 225. [8] R.J. Perce, Aust. J. Diry Technol. 38 (1983) 114. [16] Introduction to Cpillry Electrophoresis of Proteins nd [9] P. Resmini, L. Pellegrino, R. Andreini, F. Prti, Sci. Tec. Peptides, Beckmn Instruments, Fullerton, CA, 1991. Ltt.-Cs. 40 (1989) 7. [17] E. Szoko, Electrophoresis 18 (1997) 74. [10] M. de Frutos, A. Cifuentes, L. Amigo, M. Rmos, J.C. [18] Interntionl Diry Federtion, Questionnire 3293/ E, Oc- Diez-Ms, Z. Lebensm. Unters. Forsch. 195 (1992) 326. tober 1993. [11] M. Torre, M.E. Cohen, N. Corzo, M.A. Rodrıguez, J.C. [19] J.C. Miller, J.N. Miller, in: Estdıstic pr Quımic Anlı- Diez-Ms, J. Chromtogr. A 729 (1996) 99. tic, 2nd ed., Addison-Wesley, 1993, pp. 40 63. [12] M. Knning, M. Csell, C. Oliemn, LC?GC Int. 6 (1993) [20] R.P. Od, J.P. Lnders, in: J.P. Lnders (Ed.), Hndbook of 701. Cpillry Electrophoresis, CRC Press, Boc Rton, FL, [13] C. Grcı-Ruiz, M.C. Grcı, M. Torre, M.L. Mrin, 1993, p. 10. Electrophoresis 20 (1999) 2003. [21] H. Nishi, M. Mtsuo, J. Liq. Chromtogr. 14 (1991) 973. [14] M.C. Grcı, M.L. Mrin, M. Torre, Anl. Chem. 69 (1997) 2217.