GVHD: PGS.

TS L Vn Vit Mn

S BIN TNH CA WHEY PROTEIN DI TC NG CA P SUT CAO

Ni dung I. II. III. IV. V. VI. I. Gii thiu Trong vng khong 20 nm tr li y, cc nh khoa hc ngy cng quan tm nhiu hn ti vic p dng phng php x l p sut cao (high pressure treatment; HPT) vo sn xut thc phm. p sut cao (100MPa 1000MPa) ngoi tc dng c ch vi sinh vt cn lm thay i cc tnh cht vt l v ha l ca thc phm. HPT l phng php duy nht kh d thay th cho phng php x l nhit v c p dng sn xut mt s loi thc phm nh mt tri cy, jelly, st, ham. Tuy nhin, n nay vn cha c sn phm ngun gc t sa no trn th trng c sn xut bng phng php ny. HPT c nhng u im vt tri nh: c th c ch vi sinh vt nhng khng nh hng n gi tr dinh dng ca thc phm; tha mn c nhu cu ca ngi tiu dng v thc phm sch, t cht bo qun. Ngoi ra phng php ny cn mang li cho thc phm nhng tnh cht ha l v cm quan khc bit. y l c s phng php ny khc phc c nhc im v chi ph u t v vn hnh, c th c tip tc t hy vng vo vic p dng thc tin trong tng lai. Hin nay, nhng nghin cu v p sut thy tnh trong ngnh sa c th chia thnh nhng nhm nh: nghin cu nh hng ca HPT ln sa; ln bn thnh phm (nh whey protein concentrate, whey protein isolate, sa tch bo...) v ln cc sn phm t sa (ph mai, sa chua). Nhm nghin cu v nh hng ca HPT ln sa li c th chia thnh nhng hng nh hn nh: nh hng ca HPT n micelle casein; nh hng n tnh cht cm quan ca sa; nh hng n whey protein trong sa v tng tc ca chng vi casein; nh hng ca HPT i vi nhng loi sa khc nhau (b, d, cu...). Trong bi bo co ca mnh, em xin trnh by li mt cch tng quan v nhng nh hng ca HPT n s bin tnh ca thnh phn whey protein trong sa, c ch cu s bin tnh whey protein trong sa, nhng yu t nh hng, nhng tnh cht ca sa b thay i c lin quan n bin tnh whey protein v kh nng ng dng trong thc phm. Cn ni thm rng whey protein c ni n trong bi ch chung thnh phn protein tan trong nc, ch khng phi l ch phm whey protein concentrate hay whey protiein isolate hay dch whey thu c sau khi ng t casein. Nhng tnh cht, biu hin ca whey protein c nghin cu khi chng nm trong mi trng dch sa c y cc thnh phn protein nh sa ti. Bi bo co ca em da ch yu trn 2 bi tng quan ca 2 tc gi Thom Huppertz [1] v Rosina LopezFandino[2]. Phn cc yu t nh hng n bin tnh ca protein c s dng mt s kt qu ca bi bo ca tc gi Skelte G. Anema [3]. Whey protein c nhiu loi, trong chim thnh phn ln nht l -Lactoglobulin(Gii thiu v phng php dng p sut cao Cch xc nh mc bin tnh whey protein Cc yu t nh hng C ch ca hin tng bin tnh nh hng ca bin tnh whey protein bi p sut cao ti nhng tnh cht ca sa Kt lun

GVHD: PGS.TS L Vn Vit Mn Lg), -Lactalbumin(-La), immunoglobulin v serum albumin. 3 bi bo ni trn ch yu nghin cu trn 2 i tng l -Lg v -La. V vy trong bi bo co ca mnh, em xin khng cp n 2 protein cn li. II. Xc nh mc bin tnh whey protein Di p sut cao, whey protein trong sa s b bin tnh. Theo Lopez-Fandino, o mc bin tnh ca whey protein, ta da vo tan ca chng pH 4,6. mu sa khng qua HPT, khi a v pH 4,6 th whey protein vn tan trong nc. Tuy nhin, khi b bin tnh, tan pH ny b gim i. Sau khi ly tm (tc phn khng tan), o hm lng whey protein mu qua v cha qua HPT ( pH 4,6) v so snh, ta s bit c mc bin tnh ca whey protein. o hm lng whey protein, Anema dng phng php sc k in di trn gel polyacrylamide (Polyacrylamide gel electrophoresis; PAGE). C th s dng thm cht kh sodium dodecyl sulfate (SDS-PAGE) hoc khng dng cht kh (native-PAGE). Mc bin tnh whey protein cn c trn thu hp cc peak ca -Lg v -La ca mu qua HPT so vi mu khng qua HPT. III. Cc yu t nh hng n mc bin tnh whey protein Cc yu t nh hng n mc bin tnh ca whey protein khi x l HP c th chia lm 3 nhm: bn cht ca whey protein, nhng yu t lin quan n k thut v cui cng l nhng yu t lin quan ti bn thn nguyn liu. III.1) Bn cht ca protein C hai bi bo ca Huppertz v Lopez-Fandino u cho rng -La bn vi p sut cao hn -Lg. p sut 100MPa th -Lg bt u b bin tnh, trong khi -La ch bt u bin tnh 400MPa (Hnh 1). Khi x l sa 400MPa trong 15pht nhit phng c n 80% -Lg b bin tnh.

Hnh 1: Mc bin tnh ca (a) -Lg v (b) -La di p sut cao 25oC (--), 40oC(--), 50oC(--) v 60oC (- -) trong 15 pht. Lopez-Fandino R.(2006) S nhy cm hn vi p sut cao ca -Lg so vi -La c gii thch bi nhiu nguyn nhn. C hai tc gi trn u c cch gii thch ging nhau. Nguyn nhn u tin l cu hnh khng gian ca -La cht ch (rigid) hn -Lg. Trong phn t ca -La c ti 4 lin kt disulfide (-S-S-) trong khi phn t -Lg ch c 2. Hn na, 2

GVHD: PGS.TS L Vn Vit Mn phn t -La c cu trc gp cun vi mt ion Ca2+ lin kt nm trung tm trong cu trc bc 4, trong khi phn t -Lg khng c lin kt vi Ca2+. Chnh v cu trc bn cht hn m -La kh b bin tnh hn -Lg.

Hnh 2: Minh ha c ch phn ng trao i thiol/disulfide Mt nguyn nhn khc quan trng hn l do trong phn t -Lg c mt nhm thiol t do (-SH), nhm ny d tham gia phn ng trao i thiol/disulfide vi lin kt disulfide khc. S c ch phn ng c v hnh 2. i vi nhm thiol t do ca -Lg th nhm disulfide gn nht (v khng gian) m chng c th phn ng l chnh cc lin kt disulfide trong phn t -Lg. Khi phn ng ny xy ra s lm thay i cu hnh khng gian ca phn t. III.2) Cc yu t k thut Theo c hai tc gi Huppertz v Lopez-Fandino th s bin tnh ca whey protein ph thuc vo cc yu t k thut nh p sut v nhit x l. Trn hnh 1 ta cng c th thy r rng vi cng mt iu kin nhit th p sut cng cao, mc bin tnh ca -Lg v -La cng nhiu. Tng t cng mt p sut, nhit cng cao ng vi mc bin tnh ca c 2 loi whey protein cng ln. Lopez-Fandino cn cho rn thi gian mu sa c duy tr p sut cao cng l mt yu t nh hng. D dng nhn thy rng thi gian x l cng di th mc bin tnh cng tng. Ngoi ra, Anema lm th nghim cho thy rng mc bin tnh ca whey protein cn ph thuc vo nng cht kh ca sa. III.3) Cc yu t lin quan n nguyn liu Thnh phn ha hc ca sa c nh hng n s bin tnh ca whey protein di p sut cao. C hai tc gi u khng nh pH tng c tc dng thc y s bin tnh ca whey protein; s c mt ca cc cht v hot nhm thiol (v d nh N-ethylmaleimide) lm gim mc bin tnh whey protein; v whey protein d b bin tnh trong sa hn so vi trong dung dch nc whey. Huppertz cn cho bit thm rng vic loi b Ca 2+ khi sa cng lm gim mc bin tnh ca whey protein; vic thm cht oxi ha (v d KIO3) vo sa lm tng s bin tnh ca -Lg nhng lm gim s bin tnh ca -La. a) pH Nh ta bit, s bin tnh ca protein c s tham gia ca phn ng trao i thiol/disulfide. pH cao c tc dng hot ha nhm thiol t do trong -Lg theo phn ng: Protein-SH Protein-S + H+
-

dng S-, nhm ny d dng phn ng vi cc lin kt disulfide (hnh 2). b) Cc cht v hot nhm thiol S c mt ca cc cht v hot nhm thiol c tc dng gim mc bin tnh ca protein. Cc cht ny lin kt vi nhm thiol ca -Lg lm chng khng th tham gia phn ng trao i thiol/disulfide. c) Hm lng Ca2+ pH ca sa th whey protein tch in m. S c mt ca ion Ca 2+ trong sa c tc dng trung ha mt phn in tch m ny, dn n vic lc y tnh in gia cc phn t whey protein gim. Nh hin tng trn 3

GVHD: PGS.TS L Vn Vit Mn m cc phn t protein d li gn v lin kt vi nhau (v d nh thng qua phn ng trao i thiol/dsulfide). V vy vic loi b calci phosphate ra khi sa c tc dng lm gim s bin tnh ca whey protein. d) Cc cht oxi ha Cc cht c tnh oxi ha (v d nh KIO3) oxi ha nhm thiol ca phn t -Lg theo phn ng 6Protein-SH + IO3- > 3Protein-SS-Protein + 3H2O + IPhn ng trn l phn ng oxi ha ch khng phi phn ng trao i thiol/disulfide (v khng lm xut hin ion S-). Phn ng ny ch lm bin tnh bn thn phn t -Lg. V vy m khi c thm KIO3 vo sa th bin tnh ca -Lg tng cn bin tnh ca -La gim. e) Bn cht mi trng Khi tn ti trong mi trng sa hay skim milk (cha ng t), whey protein d b bin tnh bi p sut hn khi tn ti trong mi trng nc whey. Nguyn nhn l v trong sa hay sa tch bo c -casein, l mt trong nhng cht tham gia phn ng vi whey protein khi chng bin tnh (iu ny s c gii thch r hn trong phn co ch phn ng). Ngoi ra, trong sa cng cha hm lng Ca2+ cao hn nc whey, mt yu t gp phn lm bin tnh whey protein ( gii thch trn). f) Nng cht kh Theo th nghim ca Anema th khi nng cht kh ban u trong sa trc khi qua HPT cng cao th mc bin tnh ca whey protein cng gim. Tuy nhin, mt quan st khc rt ra t th nghim ca ng l s ph thuc ca s bin tnh vo nng cht kh ch tn ti vng p sut <300MPa. p sut >300MPa hu nh khng c s khc bit v mc bin tnh whey protein gia cc mu sa c nng cht kh khc nhau. vng p sut <300MPa, do s bin tnh ch yu l thun nghch (tc cha c s lin kt ca b-Lg vi protein khc, vn ny s c ni k hn phn IV) v vy whey protein c th c lm bn bng nhng thnh phn khc, v d nh lactose. iu ny gii thch v sao p sut <300MPa th vic tng nng cht kh s lm gim bin tnh ca protein. IV. C ch ca hin tng bin tnh whey protein Trong bi bo ca mnh Huppertz trnh by c ch ca s bin tnh protein nh sau: Di p sut cao, -Lg b mt cu trc bc 3 v 4 (ch yu l cu trc cun ), lm l ra gc thiol t do vn b giu bn trong. Nhm thiol ny lin kt vi -casein, -La, -Lg thng qua phn ng trao i thiol/disulfide. Phn ng ny ch xy ra trong thi gian gi p sut ch khng xy ra khi nh p. Khi nh p, nhng phn t b dui mch nhng cha kp phn ng vi cc protein khc s ly li cu trc gp/cun v tr nn hu nh ging vi phn t protein cha bin tnh. iu ny c chng t bng th nghim o cng hng t (nuclear magnetic resonance; NMR) vi dng proton. Ph cng hng l nh nhau vi mu c v cha qua HPT. T th nghim ca mnh, Anema a ra gii thch cn k hn v ng hc ca qu trnh bin tnh ca whey protein. Theo ng, s bin tnh ca whey protein c 2 dng: thun nghch v khng thun nghch. Dng bin tnh thun nghch l s dui mch do s yu i ca lin kt k nc v lin kt hidro, nu phn t ch b bin tnh thun nghch th sau khi gim p, phn t s ti lp cu trc bc 3 hoc 4 nh ban u. Dng bin tnh khng thun nghch l s lin kt ca nhng protein dui mch (khi l ra nhm thiol trong phn t) vi cc protein khc. S bin tnh thun nghch chim u th vng p sut <300MPa. S bin tnh khng thun nghch chim u th vng p sut >300MPa. 4

GVHD: PGS.TS L Vn Vit Mn Lin quan n s bin tnh bt thun nghch ca whey protein di p sut cao, Huppertz v LopezFandino u c cng quan im khi cho rng sau khi dui mch lm l ra cc nhm thiol th -Lg tham gia lin kt vi cc micelle casein v -La. S lin kt ca -Lg vi -La, thng qua phn ng trao i thiol/disulfide, l d hiu v chng d gp nhau trong dch sa, do cng l protein tan trong nc. Tuy nhin c hai tc gi u cho rng mt phn -Lg cn tham gia phn ng tao i thiol/disulfide vi -casein, v thc t l -casein cng c lin kt disulfide trong phn t. gii thch cho gi thit trn, Huppertz dn li th nghim ca chnh mnh. Th nghim nh sau: u tin, ta bit rng khi ly tm sa s thu c phn cn (sediment) v phn lng (supernatant). Vi mi mu sa bt k, -Lg chia thnh 2 phn l phn to cn (l thnh phn s nm trong phn cn sau khi ly tm; sedimentable) v phn khng to cn (phn nm trong dung dch sau khi ly tm; non-sedimentable). -Lg to cn l cc -Lg lin kt vi micelle casein hoc kt hp vi nhau thnh nhng khi gm nhiu phn t -Lg. -Lg khng to cn l cc phn t -Lg n l, phn t -Lg kt hp vi mt phn t casein hoc vi nhng mnh micelle khng to cn. Khi x l HP, phn -Lg to cn s tng ln,v phn -Lg khng to cn s gim xung. Khi x l HPT vi mu sa khng cha micelle casein, Huppertz thu c kt qu sau: Khi ly tm mu sa ny th kt qu l phn b-Lg khng to cn khng thay i. Tuy nhin, nu a pH v 4.6 trc khi ly tm th phn -Lg to cn c tng. iu ny chng t trong mu sa trn, -Lg c b bin tnh (do -Lg gim ha tan khi a v pH 4,6) nhng cc t hp -Lg khng ln hoc qu t to cn. V vy ta kt lun c rng khi b bin tnh th a s -Lg s kt hp vi micelle casein v lng -Lg kt hp vi nhau l khng ng k. Ngoi ra, Huppertz cho rng mt phn whey protein bin tnh cng lin kt vi protein trong mng bao ca ht cu bo i vi mu sa nguyn c qua HPT, cng thng qua lin kt trao i thiol/disulfide. V. nh hng ca bin tnh whey protein bi HPT n tnh cht ca sa V.1 nh hng n kh nng to cream Trong 3 bi bo, ch c bi bo ca Huppertz cp n nh hng ca HPT n tnh cht ca sa nguyn kem. Khi sa nguyn kem c qua qu trnh HPT, whey protein bin tnh lin kt vi protein ca mng bao ht cu bo. iu ny khin cc ht cu bo kh lin kt vi nhau thnh tng khi ln v tch pha. Chnh v vy, kh nng to cream (to vng) ca sa nhn chung s gim. Tnh cht ny th hin qua s gim vn tc to cream v lng cream to ra cng gim. V.2 nh hng n kh nng ng t bng rennin C hai bi bo ca Huppertz v Lopez-Fandino u c nhc n nh hng ca s bin tnh whey protein di p sut cao ti kh nng ng t bng rennin ca sa tch bo. Qu trnh ng t bng rennin gm 2 giai on: giai on 1 l giai on chnh, din ra s thy phn c hiu phn t -casein bi enzyme rennin, gii phng 2 phn on caseinmacropeptide v para--casein; giai on 2 din ra s lin kt cc ht casein li vi nhau ch yu nh vo lin kt k nc do lp o bo v -casein mt i vai tr bo v. HPT c nhng nh hng tch cc ti qu trnh ng t bng rennin. Mc d nhng nh hng tch cc ny khng lin quan ti whey protein nhng cn thit nhc li hiu c nh hng ca whey protein, s c ni sau. HPT nh hng tch cc ti qu trnh ng t bng rennin bi nhiu nguyn nhn. HP lm ph v mt phn cc ht cu bo v lm mt phn -casein ha tan vo pha lin tc. iu ny lm gim in tch m 5

GVHD: PGS.TS L Vn Vit Mn b mt cc micelle v lm gim lc y tnh in gia chng. Vic gim mt che ph ca -casein bn ngoi micelle cng lm gim tnh ho nc, tng tnh k nc ca lp mng micelle, khin chng d kt hp vi nhau hn ( giai on 2). Kch thc ca cc ht micelle gim khin cho khi ng cng chc hn. Nu nh sau khi x l HPT ta bo qun sa trong mt khong thi gian nht nh 20 oC th nhng nh hng tch cc ti kh nng ng t (tc ng t tng, thi gian ng t gim, cng chc khi ng...) s b mt i v trong khong thi gian cc micelle dn hi phc li trng thi nh trc khi qua HPT. Tuy nhin, HPT cng c nhng tc ng cn tr s ng t casein bng rennin. C ch ca nhng tc ng c lin quan n s bin tnh whey protein. Nh ni, khi -Lg b bin tnh, chng lin kt vi micelle casein, c th l vi -casein thng qua phn ng trao i thiol/disulfide. Lin kt ny lm gim kh nng tip xc ca rennin vi -casein, dn n vic lm gim tc ca giai on 1 trong qu trnh ng t. Tng t, lin kt ny cng gy kh khn trong giai on hai v n ngn cn cc ht micelle lin kt li vi nhau. iu ny gii thch v sao khi x l sa p sut <= 250MPa (s lng lin kt gia -Lg v -casein cha nhiu), thi gian ng t c rt ngn nhng 250MPa th thi gian ng t ca sa khng i. V khi tng p sut tc dng ln trn 250MPa th thi gian ng t thm ch cn tng ln. Khi thm KIO3 vo dung dch (cht ny ngn cn phn ng trao i thiol/disulfide) th khng c hin tng tng thi gian ng t sa qua HPT 600MPa. Nh vy, mc gim kch thc micelle v mc bin tnh whey protein s quyt nh n mc nh hng ca HPT n kh nng ng t ca sa. Kch thc micelle cng nh, sa cng d ng t. Whey protein cng bin tnh nhiu, sa cng kh ng t. Nu ta gia nhit sa ln 65 oC trong 30 pht hoc 90oC trong 10 pht ri em x l HP (>400MPa) th khng xy ra hin tng tng thi gian ng t m ngc li, thi gian ng t gim dn khi p sut tng. iu ny cho thy r rng khi whey protein b bin tnh trc (bi nhit) th HP ch tc ng n kch thc ca micelle. VI. Kt lun Qua 2 bi tng quan v mt bi nghin cu, ta thy nhng nghin cu v nh hng ca HPT n tnh cht ca whey protein t c nhiu bc tin ng k. Mc bin tnh ca tng loi whey protien, cc yu t nh hng v c ch ca hin tng ny c lm sng t. S bin tnh ca whey protein c th l tch cc trong trng hp bo qun sa khi n ngn chn s to vng sa. Tuy nhin torng cng nghip sn xut b hay ph mai th s bin tnh whey protein li c tc ng xu n cc qu trnh sn xut. Nhng hiu bit ny, cng vi s pht trin ca k thut phn tch, thit b chu p sut cao v thit b o c; cng vi s hiu bit v nh hng ca HPT n micelle casein, gip con ngi tng bc nm vng c c ch ca nhng tc ng m HPT mang li cho sa. Tuy nhin, cn nhiu nghin cu hn na v nh hng ca HPT ti gi tr dinh dng cng nh cm quan ca sa. Ch khi c c nhng hiu bit nn tng y th nhng nh sn xut thc phm mi lm ch c qu trnh HPT trong tng lai.

GVHD: PGS.TS L Vn Vit Mn Ti liu tham kho [1] Huppertz T., Fox P. F., de Kruif K. G., Kelly A. L. (2006). High pressure-induced changes in bovine milk proteins: A review. Biochimica et Biophysica Acta, 1764(3), 593-598. [2] Lopez-Fandino R. (2005). High pressure-induced changes in milk proteins and possible applications in dairy technology. International Dairy Journal, 16(10), 11191131. [3] Anema S. G. (2008). Effect of milk solids concentration on whey protein denaturation, particle size changes and solubilization of casein in high-pressure-treated skim milk. International Dairy Journal, 18(3), 228235.