LOT MO DAU Ngay nay, véi sy phat trién manh mé cita khoa hoc ky thugt img dung, dc biét la cdc nganh céng nghé sinh hoc, y hoc va cong nghiép ché bién thye phim thi vige danh gia chat lugng thye pham dang rat duge quan tam va chi y. Trong s6 cac chi tiéu ding dé danh gia chat lugng thyc pham nhu ham lugng dudng, ham lugng lipit, cdc chat khodng, céc chat Vitamin ... thi ham lugng protein chita trong thye pham la chi tigu quan trong hon ca. Trong nhitng nim vira qua, & nuéc ta van dé vé sinh va an toan thye pham dang tro thanh m6t dé tai néng bong, thu hut duge sy cha y cia rat nhiéu nha khoa hoe. Vi vay, muc dich ciia khod lufn nay Li bude dau xde dinh him lugng cde axit amin va protein trong thyc pham. N6i dung nghién eitu cia ban khod lun nay bao gom: * Xay dymg mét phutong php cho phép x4c dinh nhanh va tuong déi chinh xde him lugng cdc axit amin va protein trong thye phim. * Ung dung phuong phap trén vao viée danh gia so b6 chat lugng ctia mot sé loai thyc phim.CHUONG 1: TONG QUAN 1. PROTEIN VA CAC CHUC NANG [1,2,3]. 1.1. Dinh nghia. Protein la polyme sinh hoc ctia L-6-aminoaxit két hop voi nhau bang lién két peptit. 1.2. Phin loai. Dya vao thanh phin hod hoe cdc protein duge phan thanh hai nhém Ién: * Protein don gian: - Cae L-6-aminoaxit - Polypeptit gdm hai hay vai chyc aminoaxit lién két voi nhau. - Protein gdm vai chye aminoaxit tré lén lién két voi nhau. * Protein phttc tap: Phan tir cia né bao gm phan protein va phan khong phai protein goi la" nhém ngoai ". Tuy theo ban chat hod hoc cla nhém ngoai, c6 thé phan thanh cde nhém nhé nhu : - Metaloprotein (nhém ngoai la ion kim loai nhu Fe™’, Zn’). - Lipoprotein (nhém ngogi Ia lipit). - Glycoprotein (nhém ngoai la gluxit). - Phosphoprotein (nhém ngoai li phosphat, vi dy casein stra). - Nucleoprotein (nhém ngoai la axit nucleic). - Cromoprotein(nhém ngoai la cdc chat mang mau). 1.3. Ham hong. G déng vat cd ham Iweng cao hon thyc vat, & ngudi protein chiém ti 45% trong Iugng khé. Ty Ié protein ciing khac nhau gitta cdc b6 phan trong co thé sng.Bang 1: Ham lurgng protein trong m6t sé nguyén ligu dong vat va thire va Nguyén ligu % Protein Nguyénligu | % Protein Gan 18-19 Moi 13-16 Tim 16-18 Oc 11-12 Thit gia sic 16-22 38 89 Trig gia cam 13-15 Hen aS Sita bo 35 Dau trong 34-40 Ca 17-21 Lac 23-27 Tom 19-23 Ngo 3-10 Mye 17-20 Gao 7S 1.4, Chire nang. Protein la thinh phan khéng thé thiéu duge cia tat cd cde co thé séng. Protein li nén ting vé cdu trac va chite nang cia co thé sinh vat. Duéi day la mot 86 chite nang quan trong cua protein: 1. Xtic tae. Ce protein cé chite ning xtic tac cde phan img sinh hoc goi Ia enzim. Hau hét cde phan tmg ctia co thé séng tir nhimg phan img don gidn nhét nhu phan img hydrat hod, phan img khtr nhém cacboxyl dén nhiing phan tmg phite tap nhu sao chép ma di truyén ... déu do enzim xuc tac. 2. Van tai. Protein déng vai tro nhu 1d cdc phutong tign vn tai chuyén ché cdc chit trong co thé séng. Vi dy, hemoglobin, mioglobin (6 dong vat cé xuong séng), hemoxiamin (6 d6ng vat khong xuong s6ng) két hgp véi O2 roi van chuyén O, dénkhap cdc m6 va co quan trong co thé. Hemoglobin con van tai cd CO) va H'. Glubolin lai van tai Fe. 3. Van dong. Nh protein tryc tiép tham gia trong qué trinh chuyén d6ng nhu : co co, chuyén vi tri cia nhiém sac thé trong qua trinh phan bao, di déng cia tinh tring, 6 dong vat cé xuong séng, sy co co duge thyc hign nho chuyén dong trugt trén nhau cia hai loai sgi protein: sgi to chia protein miozin va sgi manh chita cdc protein actin, troponiozin, troponin. 4, Bao vé. Dé la cdc khang thé trong mau dOng vat c6 xuong séng. Ching cé kha ning nhan biét , "bat" nhimg chat la x4m nhfp vao co thé nhu protein la, virut, vi khuan hodc té bao la va loai ching ra khdi co thé. Vi dy, cdc interferon 1a nhimg protein do té bao déng vat c6 xuong séng téng hgp va tiét ra dé chéng lai su nhiém virut. Tac dyng cia cac interferon rat manh, chi cin 6 néng 46 107'M da co higu qua khang virut 16 rét. Interferon két hgp vao mang nguyén sinh chat ctia cdc té bao khie trong co thé va cam img trang thai khang virut cua ching. Cc protein tham gia trong qué trinh déng mau cé vai tr bio vé cho co thé séng khoi bj mat mau. 6 mét sé thue vat c6 chira cdc protein c6 tac dung ddc déi voi dong it, ngay ca 6 liéu lugng rat thap ching cing cé tac dung bao vé thye vat khoi sy pha hoai cia dong vat. 5. Diéu hoa. M6t sé protein cé chire nang diéu hoa qua trinh thong tin di truyén, diéu hoa qué trinh trao déi chat. Protein diéu hoa qué trinh biéu hién gen, nhu cdc protein reprexo 6 vi khuan cé thé lam ngtmg qué trinh sinh tong hop enzim cua cdc gen tuong img. G co thé bic cao sy digu hoa hoat déng biéu hign gen theo mét co ché phite tap hon nhung ede protein cing déng vai trd quan trong.Ce protein cé hoat tinh hormon, cde protein te ché dic higu enzim déu cé chite nang dieu hoa nhiéu qua trinh trao d6i chat khac nhau, 6. Truyén xung than kinh. M6t s6 protein c vai trd trung gian cho phan img tra Idi cia té bao than kinh 46i véi cdc kich thich dc higu. Vi dy vai trd cia sic t6 thi gidc rodopxin 6 mang luéi mat, 7. Cau trie. Cac protein nay thuéng c6 dang sgi nhu sclerotin trong Iép v6 ngoai cla cén tring, fibroin ciia to tim, to nhén, colagen, elastin cia mé lién két, m6 xuong. 8. Dy trir dinh dung. Protein con 1a chat dinh dung quan trong cung cAp cdc axit amin cho phéi phat trién. Vi dy, albumin trong long trang trimg, casein trong sita, gliadin trong hat lua mi, zein cua ngé. 1.5. CAU TAO PHAN TU PROTEIN [1,2,3]. 1.5.1. Thanh phan cc nguyén té ciia protein. TAt ca cde protein déu chita cdc nguyén t6 C, H, O, N. Mt sé con c6 chita mét Iugng nho S. Ty 1é phan tram khdi lugng cdc nguyén t6 nay trong phan tir protein nhur sau: C: 50-55% O: 21-24% N: 15 - 18% H: 6.5-7.3% S: 0-0.24%Ngoai cdc nguyén t6 trén, mét sé protein con chita m6t lugng rat nho cdc nguyén 16 khac nhu: P, Fe,Zn, Cu, Mn, Ca 1.5.2. Don vj cdu tao co sé cia protein: L-c-aminoaxit. Tuy protein rat da dang vé cdu trac va dam nhan nhiéu chite nang nhu vay song hau nhu déu xay dung nén cdc dai phan tir ia minh cha yéu tt 20 L-o- aminoaxit bang lién két peptit. Do vay, ciing cé thé xem cdc aminoaxit nay 1a san pham cudi cig ctia sy thuy phan peptit va protein. Valin, mét dai dign cita c-aminoaxit 6 dang khong ion c-aminoaxit va ludng tinh 6 pH trung tinh FR oo tee -of nh ° Dang khéng ion Hinh 1: Cau trie cita c-aminoaxitBiéu dién trén kh6ng gian 3 chiéu Cae déng phan lap thé Hinh 2: CAu trac lap thé cia a-aminoaxit Nhu vay cau tao cla ching gidng nhau & ché cing co nhom cacboxyl - COOH va nhém amin - NH; déu gin vao nguyén ti C 6 vi tri a, con khée nhau béi cu tgo cita mach bén R. @ lam cho Khi thiéu tham chi chi mét trong cdc axit amin can thiét co tl protein duge ting hgp it hon protein bi phan giai, két qui din dén mat can bing di véi nito, Ham Iugng cde axit amin khéng thay thé A tile gitta chung trong phan tir protein 1 mét tiéu chuan quan trong dé danh gia chat lugng protein. Trong dung dich, cc aminoaxit thuémg 6 dang ion ludng tinh mang cing m6t luc dign tich duong (+) do nhém -NH) & dang -NH;" va dign tich am (-) do nhém -COOH ¢ dang -COO’. Ching hip thy anh sang, dae biét 1a dung dich cua 3 aminoaxit chita nhan thom: Tryptophan, tyrozin va phenylalanin hap thy kha manh 4nh sing UV o bude séng gan 280nm.‘Trong dung dich, tuy thuge vao pH cia méi trudng né cé thé lA ion bring tinh, hoge mang dign (+) hode (-). Gid tri pH ma & 46 amino axit t6n tai dang ion ludng cyc duge goi la diém dang dign va pH duge goi la pl. Vi cac mach bén R khac nhau gitta cdc aminoaxit nén méi aminoaxit cé mt diém dang dién riéng. Tai diém ding dign, ting dign tich cua aminoaxit bang 0. Con & bat ky diém pH nao thap hon pI hod cao hon pl cdc aminoaxit déu mang dign tich duong hod am, diéu nay tuy thude dign tich mach bén R ciia chinh n6. Vi vay, trong thye t8 dic tinh nay duge str dung dé phan téch va xdc dinh céc aminoaxit. 1.5.3. Peptit. Peptit li nhimg phan tir sinh hoc cu tao tir m6t vai cho téi hang chye amino axit. Peptit tao thanh ttr cdc aminoaxit thong qua phan img néi dau -NH2 cua phan tir nay vi dau -COOH cia phan tir kia tao ra lign két peptit.Hinh 3: Sy hinh thanh ciia m@t dipeptit nh 4: Sy hinh thanh cia m@t tetrapeptit. HiOcbital a-cacbon, Dic trumg lién két d6i ciia bO khung peptit Cae gée va d6 dai lin két Hinh 5: C4u tric cia bo khung peptit. Cu trac cia lién két peptit cho thay hau hét cdc lién két bat bién -C=O va - N-H la song song hoae gan song song va hoi bj xodn xung quanh lién két C-N. Chinh sy lai tgo cita ocbital 1m trong lién két d6i C=O véi d6i dign tit con lai ela N da lim cho mat phiing lign két tré nén vimg chic (xem hinh 7) va nhér cdc mat phing nay da tao ra lién két bac 2 (phién gap B) d6i véi cdu tric ctta protein. Ngoai lién két peptit, d6i khi gitra cc aminoaxit con cé lién két disunfua ndi 2 géc xystein voi nhau. Lién két nay cé vai tro quan trong gidp hinh thanh cau tric khéng gian cua rat nhiéu protein, dic biét protein ngoai bao cé chttc nang sinh hoc quan trong nhu hoemon, immunoglobulin va khang thé Peptit c6 tinh chat ly hod khéng khac nhiéu so véi aminoaxit vi cing chtra mot nhém -NH2 va -COOH ty do giéng nhu aminoaxit. Sy khac nhau & day chi la do cde mach bén R cia géc amino axit tham gia chudi peptit gay ra. Cac peptit bi thuy phan hoan toan béi dung dich HCI 6N & 110°C trong 24h, hoc béi dung dich kiém 8 cho céc amino axit ty do.Lién két peptit con bj thuy phan béi enzim thuy phan protein goi la proteazo. Ching c6 mat 6 tat ca cde té bao va mé té bao dé thyc hign nhiém vy chu yéu la phan gidi protein. 1.5.4, Cau trie khong gian cia protein, CAu trac khéng gian cua protein la tap hgp khong gian cua tat cd cdc nguyén tir trong phan tt, Ngudi ta phan biét 4 bac cau trac eta protein nhu sau a. Cu trac bac 1 1a trinh ty sip xép cdc aminoaxit va vi tri lién két disunfua trong chudi polypeptit. Cau trac bac 1 khéng cé cau hinh khong gian dac hiéu. b. Cau trie bac 2 phan anh sy sip xp c6 quy ludt trong khong gian ciia cdc aminoaxit trong chudi polypeptit, phé bién hon ca 1a cdu tric xodn o va cdu trac phién gép nép B c. Cu tric bac 3 phan anh tuong quan khéng gian cita cdc aminoaxit trong chudi polypeptit. Trong dé bao ham sy xoan hoge udn cia cdc cau tric bac 2 tao nén cdu hinh khéng gian dc thi rigng cho timg loai protein. 4d. Cu trie bac 4 phan anh tuong quan khéng gian gitta cdc sgi polypeptit (hay cde phn duéi don vi) trong phite hgp phan tir protein. 1.5.4.1, Cau tric bie 1 Cac géc aminoaxit trén chudi polypeptit duge sp xép theo mét trat ty nhat dinh va lién quan véi chitc nang sinh hgc ciia protein mét cach hét site chat ché. Vi sy t6 hop khac nhau ctia 20 aminoaxit da din dén cdc loai protein khac nhau, Cac protein cé chtre nang sinh hoc giéng nhau thudng cé cau tric bac 1 kha gidng nhau, Nhu vay co thé thay trong bat ky cdu trac bac 1 nao cia protein bao gid cing c6 nhimg doan quan trong bat bude phai cé trinh ty sip xép amino axit khéng thay déi, ngoai ra 6 mét sé vi tri ndo dé cé thé cé sy thay déi ma van khong anh huéng dén chite ning euia protein. 1.5.4.2. CAu trie bie 2: gdm xoin a, phié nép B va xoan colagena. CAu tric xoin @ : Doan mach polypeptit xoin chat lai Lim cho nhimg nhém peptit(-CO-NH-), C, tao thanh phan bén trong ctia Idi xoan, cdc mach bén R quay ra phia ngoai. CAu tric nay duge gitt ving chu yéu nhé lién két hidro duge tao thanh gitta nhém cacbonyl ctia mét lién két peptit voi nhém -NH ctia lién két tiép sau cach nhau 3 géc aminoaxit trén cimg m6t mach polypeptit. Tat ca cdc nhom -CO va -NH cé trong lién két peptit cla mach polypeptit déu tao thanh lién két hidro v6i nhau nhu vay, nghia la ctr mdi nhém -CO-NH- déu cé thé tao thanh 2 lin két hidro véi 2 nhom -CO-NH- khac. Céc lién két hidro duge tao thanh véi sé lugng téi da, bao dam 46 bén vig cau tric xodn a. Chiéu xoan c6 thé xoan phai (thuan chigu kim déng hé) hode trai, nhung xoan @ trong phan tir protein thuéng 1a dang xoan phai. Chiéu dai cac doan xoan o trong phan tir protein thuéng khong dai, ngin hon 40A, b, Cau tric phién gp nép B : Xuat hién trong fibroin cia to tim, né khéc v6i xoan o & mot sé diém cha yéu sau: - Doan mach polypeptit c6 cdu tric phién gap nép B thuong dudi dai ra chit khong cugn chat nhu xoan a. Khoang cach trén truc gita 2 géc aminoaxit ké nhau 143,5A, con é xodn a 1 1,5A. - Lién két hidro cén duge tao thanh gitta 2 mach polypeptit khac nhau, & ké nhau, c6 thé cimg huéng hay nguge huéng voi nhau. Trong phan tr cua nhieu protein hinh cdu cudn chit cdn gap 1u trie “quay B”. © dé mach polypeptit bi dio huéng d6t ng6t. Dé [a do tao thinh lign két hidro gitta nhém -CO ctia lién két peptit thir n véi nhém -NH cia lién két peptit thr n#2.Hinh 6a: Xo4n a Hinh 6b: Phién gap nép 6 ¢. Cau trie xoan colagen ; Kiéu cau tric nay xuat hign trong phan tir colagen, Thanh phan aminoaxit cla colagen rat dic biét so v6i cdc protein khac: glixin chiém khoang 35%, prolin chiém khoang 12% téng s6 aminoaxit trong phan tt. Ngoai ra colagen cdn chtta 2 aminoaxit it gap trong cdc protein khac [a hidroxiprolin va hidroxilizin. Don vi c4u trac cia colagen la tropocolagen bao gdm 3 mach polypeptit bén vao nhau thanh mot “day cap” siéu xodn,Hinh 7: Cau trie euia sgi colagen a. CAu tric bac! va 2 cia phan tir tropocolagen. b, Phan tir tropocolagen gdm 3 chudi polypeptit bén voi nhau. c. Sy sip xép ciia cdc phan ti tropocolagen trong sgi colagen. d, Vét vin ngang thé hién cdc phan ttr 6 cac day ké nhau xép Iéch nhau mot khoang cach 64 nm. ¢. Hinh anh thu duge qua kinh hién vi dign tu. 1.5.4.3, Cau trie bie 3: Cau trac nay phan anh tong quan trén pham vi toan bd két va tuong tac yéu cé vai tro quyét dinh git 6n dinh cdu trac bac 3. V6i lién két nay cdc phan tir cé xu thé cun chat lai, cae gdc ky nude quay vao trong, cdc géc ua nude 6 trén bé mat phan ti. Khi bién tinh phan tir protein bang cdc tac nhan hod hoe hode vat ly tire 1a pha vo lién két Van de Van, lién két hidro, khir cau sunfua, phan tir protein sé bi dudi ra dong théi thay d6i m6t sé tinh chat cua nd, dac biét 1atinh tan va hoat tinh sinh hoc. Sy bién tinh nay cé thé thuan nghich néu nhu ching cé thé tré lai trang thai cau trie ban dau va khéng thudn nghich néu nhu vinh vién khéng tré lai trang thai cdu trac ban dau. V6i nhiét d6 cao thuong pha vé tat cd cde lién két lam cho phan nt protein bj bién tinh hoan toan, vi dy nau riéu cua, canh trig. Cac dung méi hitu co, mudi amoni, chat tay rita thudng tac dng nhe nhang hon chi pha vo tuong téc ky nuéc. D6 pH ciing lam thay déi tong dién tich cua phan tir protein va pha vo mét phan lién két hidro nén citing duge xem la mét yéu 16 gay bién tinh. (a) (b) Hinh 8: Cau tric bic 3 cia phan tir protein ty nhién (a) va bién tinh (b) 1.5.4.4, CAu trite bac 4: Rat nhidu protein chiza 2 hoac nhiéu soi polypeptit cdn goi 14 phan dui don vi (subunit) giéng nhau hoc khac nhau. Do vay cau tric bac 4 phan anh tuong tac khéng gian gitta cdc phan nay trong phan tir protein,C&u tryc bae 4 Hinh 10: 4 bac edu tric cua protein 1.6. MOT SO TINH CHAT QUAN TRONG CUA PROTEIN.1.6.1. Khdi lung va hinh dang phan tir protein. Protein cé khéi Iugng phan tir tuong déi I6n, c6 dang hinh cau hoae dang sgi. Protein hinh cau tan trong nuée hoae dung dich mudi loang, rat hoat dong vé mt ho hoe. Cac protein hinh sgi tong déi tro vé mat hod hoc, chi yéu c6 chite nang co hoc. Vi du, colagen cia da, xuong, syn, gan, rang; keratin cia t6c, long, fibroin ctia to, miozin cita co... [1,2,3]. 1.6.2. Tinh chat lwéng tinh cia protein. Cling nhur aminoaxit, protein ciing 1a chit dign ly luGng tinh vi trong phan tir protein cdn nhiéu nhém phan cye cia mach bén, Cé thé digu chinh pH dé téch riéng cdc protein ra khoi hén hop ctia ching. Khi pH = pl cdc protein dé dang két tu lai v6i nhau lm cho protein két tia [1,3,6]. 1.6.3. Khi hoa tan, protein tao thanh dung dich keo. Cac phan tir keo c6 kich thuée h chat dung dich keo protein, sy két tia protein. lon, khéng di qua mang ban tham, Ngudi ta si: dung tinh chat nay dé tinh sach protein khoi cdc chat phan tr thap bang phuong phap tham tach. Hai yéu 6 dim bao d6 bén dung dich keo protein la: - Su tich dign cimg dau cua cae phan tir protein 6 (pH # pl). - Lép vo hydrat bao quanh phan tir protein. Ngudi ta thuéng ding cde mudi v6 co nhu (NH,):SO, hay cae dung méi hitu co nhu axeton, etanol dé tao két tia protein nhung phai tién hanh 6 nhiét d6 thap. Cac yéu t6 gay bién tinh khéng thudn nghich thudng duge str dung dé loai bé protein ra khoi dung dich: ding nhigt, cde axit manh, cae kim loai nng...[1,3,5,6]. 1.6.4. Kha nang hap thy tia tir ngoai cia protein Dung djch protein cé kha nang hap thy anh séng tir ngoai & ving cé bude séng 180-220nm va 250-300nm [3,6]1.6.5. Kha nang thuy phfn cia protein C&c phan tir protein cé thé bj thiy phan bing axit, kiém hay enzim cho cae polypeptit va cudi cling la cdc aminoaxit [1,3,6] 2. CAC PHUONG PHAP XAC DINH HAM LUQNG AMINOAXIT VA PROTEIN. 2.1. Cae phan tng mau cia axit amin va protein. 2.1.1, Phan tg Biure. Day a phan img dic trumg ciia lién két peptit. Trong mi truéng kiém, cic hop chat co chita ttr hai lién két peptit tro nén c6 thé phan img voi CuSO, tao thanh phic chat mau xanh tim, tim, tim d6 tuy thugc vao sé ugng lién két peptit nhiéu hay it. Céc phan img xay ra_nhu sau: * Phan img tao biure. 2H)N- C- NH, ——» H)N- C— N= C- NH, === HN=(~ N= C=NH i I T I l ° oO NH, 0 OH OH Ure Biure (xeto) Biure (enol) * Phan img véi protein. Ciing tuong ty nhu truéng hgp cua biure, cdc lién két peptit 6 dang enol (trong mdi truéng kiém) ctia cde phan tir protein tao phite voi Cu”: H 2HN= €— N= C= NH + Cu(OH) + NaOH —> I OH OHDang phic hop nhu trén (v6i bén nguyén tir nito tham gia tao lién két phdi tri) thudng c6 mau dé (hép thy cye dai & bude séng 520-535nm). Trong trudng hop chi cé hai hay ba nguyén ti N tham gia tao lin két thi phtte sé cé mau tim hoe mau xanh (hap thy ctte dai & nhiing bude séng tuong ing la 540-580 va 615-670nm). Vi vay, thong thuéng san pham phan img cia cdc polypeptit khac nhau sé co mau thay di tir xanh tim dén dé tim. Phan tg biure thudng duge img dung dé dinh lugng protein [1,3,6,9]. 2.1.2. Phan tng voi ninhidrin. Céc aminoaxit khi phan tg véi ninhidrin 6 nhigt dO cao cho san phim cé mau xanh tim (hap thu cye dai é khoang bude séng 570nm). Day ld phin img chung cho tat cd cdc aminoaxit c6 chtra nhom NHp 6 vj tri Cs. Ngoai o-aminoaxit, céc peptit, protein, mudi amoni va amoniac cing cho phan tmg nay. Co ché phan img kha phite tap nhur sau: Du tién do téc dung eta cdc aminoaxit voi ninhidrin sé xudt hign phic chat dang bazo-schiff. Sau dé xay ra sy chuyén nhém, tiép theo la sy dé cacboxyl hod va su phan tach phite chit thanh andehit vi aminodixetohidrinden. -H,9 + HpN- Clr COOH = R N7 pe COOH I R oO Aminoaxit Bazo - Schiff Chuyén nhém va loai CO, CHRAminodixetohidrinden lai ngung ty véi mét phan tr ninhidrin khac va hinh thanh nén mt hop chat méi, déng théi bj enol hod va chuyén thinh dang cé mau, HH c=O + | ( Nib H oO Aminoxetohidrinden 90 LO I ! (lO oO Phttc mau xanh tim: Khi 6 cdc dung méi hitu co (axeton, etanol,piridin ... thudmg duge dimg 4é pha ninhidrin) thi sé xay ra phan (mg sau: 0 q t 29 ~ Op} - To cn R Phitc cé mauSan pham cua phan tmg nay cé chtra gdc R cla aminoaxit ban dau. Gée nay sé quyét dinh mau sac khac nhau (xanh da trdi, 4...) cla cdc hop chat khi phan img vi ninhidrin, Dic biét phite duge tao thinh gitta prolin (co chia nhém imin) va ninhidrin cé mau ving tuoi, khdc biét hin véi mau do cdc aminoaxit khée tgo nén trong phan tmg. Do vay, ninhidrin cdn duge sir dyng cho phan (mg mau dic trung dé phat f oO + we] — CuK + CO) + H,0 i oO Oo COOH prolin: Phire mau ving Phan tmg voi ninhidrin cé d6 nhay cao, do dé thuong duge ding dé dinh tinh va dinh long protein [1,3,6]. 2.1.3. Phan img véi axit HNO. Dusi tic dyng cua HNO, aminoaxit bj deamin hod tao thinh nito 6 dang khi. Phan img nay duge sit dung dé dinh lrgng céc o-aminoaxit (phuong phap Van Slyke) [6]. Phan tmg xay ra nhu sau: “OOH COOH I H.N— GH + O=N-OH —> HO- CH +N) +H,0 R R 2.1.4, Phan tg Pauli 4é phat hign histidin va tirozin.Khi tée dung véi axit diazobenzensunfonic (thudc thir diazo), histidin tao thanh phite chat mau man chin, cén tirozin tao thanh phire chat mau da cam [6]. Cie phan img xay ra nhu sau: * Su tao thanh axit diazobenzensunfonic: NaNO, + HCI — new f + NaCl + 2H,0 L=‘*3 * Phan tg ciia tirozin: HH N| + Na,CO; {9 +2 oy rr CH= CH- COOH NH) Zz 9 HL + o=p{_\nz N \=0 + H,CO; ONa ONa CH) HE COOH NH)Phire mau da cam. 2.1.5, Phan tng Millon dic trung cho tyrozin. ‘Thuéc thir Millon chinh la mudi thuy ngan nitrat trong HNO; dic. Khi_ cho thuéc thir Millon tac dyng voi nhan phenol cia tirozin sé tao nén hop chat nitrotirozin-thuy ngén cé mau dé. Phan tmg xay ra_nhur sau: HH g OHg Oo) ZX + HgNO; + HNO; —> + H,0 CH)- GIF COOH CHy- gi COOH NH), NH, Phan img Millon duge sit dung dé phat hign tyrozin va cac hyp chat phenol. Cha y khéng cho qué nhiéu thudc thir Millon vi HNO; cé trong thuéc thir sé tac dung véi protein cho mau vang [6]. 2.1.6. Phan tng ciia cdc axit amin chira lwu huynh (Phan tng Folia). Cée axit amin chita luu huynh nhu xistin, xistein, methionin duéi tae dyng cia kiém bj phan huy tao thanh natri sunfua (NaS) RSH + 2NaOH > NaS + ROH + 1,0 Thém chi axetat vio Na)S sé phan tmg tao thanh két tia nau den cia chi sunfiva (PbS) [6]. NazS + Pb(CH;COO); > 2CH;COONa + PbSY (két tia nau den)2.1.7. Phan tg Sakaguchi dic trung cho acginin. Day [i phan tg ding dé phat hign acginin. Khi cho acginin tac dyng voi hipobromua (NaBrO) va 6-naphtol sé tao thanh san phim c6 mau d6 cam [6] Phan tmg cé thé duge trinh bay nhu sau: ! | fan wok) oH NI + NaBrO + — NH + /2N) + NaBr + H,O (GHa)s C \ >? (GH)s “HNH gun coon COOH 2.1.8, Phan tg Adamkievic dc trung cho tryptophan. Trong méi trudng axit, tryptophan phan tmg véi nhiéu loai andehit tgo thinh nhimg san phim ngung két cé mau dé tim dac trung [6]. Cc phan img xay ra nhu sau: wk + CO, H Axit glioxilic Focmandéhit o HOOC— & + H)SO,—> ‘ HGoon Goo HN-CH GHENH, OOH “OOH — +10 Bis-2-triptophanylmetal “OOH “OOH qooH ‘OOH HN-CH GH-NH, HyN-H nee Gh vn CH Oxi hos NH fn Bis-2-triptophanylmetal biscrpophoyteachine 2.2.Dinh hrgng axit amin va protein, 2.2.1, Xde dinh N-amin bing phwong php chudn 46 focmol. Cae aminoaxit c6 thé phan (mg voi focmol trung tinh dé tao thanh metyl - aminoaxit. Focmol da khod nhém amin (NH) mang tinh kiém nén cé thé chuan 46 nhém cacboxyl trong phan ttr aminoaxit bing kiém. Dua vao long kiém da ding 48 chuan d6 sé tinh duge Iugng Nito cua aminoaxit [6,9]. Phan tmg duge biéuNH H N= CH) RCH + (=O —~> R-CH + HO COOH oH COOH Aminoaxit — Focmol Metyl-aminoaxit h rong protein bing phwong php Kjeldahl. Luong nito tong sé trong cac pham vat c6 ngudn géc sinh vat thudng duge xde dinh bang phuong phap Kjeldahl, Dé 1a ham lugng tong ctia céc dang nito hitu co va v6 co cé trong phim vat nghién ctu. Dé dinh lugng nito protein, ngudi ta thuong xéc dinh nito ting sé va nite phi protein theo phuong phap Kjeldahl, sau dé ly higu sé gitta hai dang nito nay rdi nhan voi hé sé tuong img [6,9]. Luu §: Cac protein thye vit oé ty 1¢ nite protein khoang 16.8%, do vay trong truéng hop nay hé sé nhan sé 14 5.95 (100 : 16.8). Cac protein d6ng vat c6 ty If nite protein khoang 16%, do vay trong truéng hop nay hé s6 nhan sé 1a 6.25 (100 : 16) (6). 2.2.2.1. Xac dinh nito tong sé theo phuong phap Kjeldahl. Dusi téc dung cita H,SO, dic 6 nhigt 9 cao, cdc hyp chat hiu co cé chira nito (N) bi phan huy va bi oxi hod dén CO; va H,O, con nito chuyén thinh amoniac va tiép tuc két hyp voi H2SO, tao thanh mudi amoni sunfat. Qué trinh duge tién hanh qua cdc bude sau: * V6 co hod nguyén ligu. R-CHNH)-COOH + H)SO, > CO, + H2O + (NH,),SO4 * Cat dam. (NH4):SOq + 2NaOH —» NaySO, + 2H:0 + 2NH37 Phan img xay ra trong binh himg: 2NH; + H)SOs —» (NH,):SO, Chuan 46 HySO, du 6 binh himg bing NaOH 0.1N.2.2.2.2. X4e dinh nito' phi protein. Dé xdc dinh N-phi protein can ding cdc dung mdi thich hop dé chiét rat tat ca cdc dang N-phi protein (vi du dung mdi li etanol 70%). Tuy nhién, trong dich chiét van con lin m6t vai loai protein, vi vay can ding chat két tua dé tach phan protein hoa tan trong qua trinh chiét rat, Két tua protein c6 thé tién hanh theo nhiéu céch nhu: bang etanol, axeton, cdc mudi kim loai nang, axit ho&c dun 6 nhiét 46 cao ... nhung thong dyng nhat 1a ding cdc chat két tia sau day: TCA 10-20%, poly nhém clorua (PAC) véi chat try keo ty dang 4m A101, polyme C310H (keo tu duong) Pb(CH;COO), 10-15%, CuSO, 10% trong hén hgp voi NaOH 10%, ty 16 1: 1 [5,6]. Sau khi di logi bd két tia, dich loc chi cdn chita cée dang N-phi protein. Dem v6 co hod dich nay va tiép tue phap Kjeldahl. 2.2. hanh xde dinh ham lugng nito theo phuong Dinh lvgng protein trong nguyén liéu. 1g sé bao gdm N-protein va N-phi protein. Muén xdc dinh ham luong protein trong nguyén ligu phai xac dinh N-téng s6 va N-phi protein, Ham lugng protein trong mau duge tinh theo céng thie sau: Protein (img) = (Neing sé ~ Nphi protin)-6,25 Hoac Protein (mg) = (Neing sé - Nphi protein)-5,95 2.2.3. Dinh lrgng protein theo phong phap Bradford. Ce protein khi phan img vi xanh Coomassie (Coomassie Brilliant Blue - CBB) sé hinh thinh hop chat mau cé kha ning hap thy anh sing & bude séng 595 nm, cudng 49 mau ty Ig véi néng d9 protein trong dung dich. Phuong phap cé 46 nhay cao, cho phép phat hign téi vai ug protein/ml, dé thye hign va tiét kigm thoi gian [6,9]h lugng protein theo phuong phap Lowry. Protein tac dung v6i thuéc thir Folin-Ciocalteux tao thanh sin pham mau xanh, Day 1a san phim khir cia photphomolipdat-photphovolframat bai phire chat Déng-Protein va cé d6 hap thy cuc dai & bude séng 750 nm. Cudng 46 mau cia hén hgp phan img ty 1§ thufn véi ndng d6 Protein trong mt pham vi cho phép Ding may so mau quang dign dé xdc dinh cuong d6 mau trong cc mau va so sinh vi dich protein tiéu chuan. Phuong phap Lowry sit dung két hop phan img Biure (tac dung én lién két peptit, muc 2.1.1.) va phan img véi thude tht Folin (tac dung lén cdc géc Tyrozin, Tryptophan, Histidin) dé tao phite cé mau dc trung. Phuong phap c6 46 nhay tuong déi cao, cho phép xac dinh duge dén néng d6 vai chuc pg protein, do vay duge sit dung rong rai dé xdc dinh nhiéu loai protein 6 dang tuong déi tinh khiét, Tuy nhién, nhuge diém ctia phuong phap li mau cia phite chat bj anh huong néu trong dung dich c6 chia mét sé chat nhu EDTA, sacarozo, dém Tris hay mét sé chat khtr nhu xistein, ditiotreitol ... [4,6,9] 2.2.5, Dinh tinh va dinh lgng aminoaxit bang phuong phap sic ky gidy. Phuong phap sac ky phan bé trén gidy dya vao su khdc nhau vé hé sé phan bé cdc chat tan gitta hai pha long khéng tron lin, Ngudi ta mang lén m6t diém 6 dau ban gidy sic ky mét giot dung dich nghién cttu réi nhing du gidy co mang dung dich nghién cttu dé vao chau dung méi hitu co linh dng bao hoa nude (nhu rrgu butilic). Trong qué trinh di chuyén chim cua dung méi qua éng mao dan cua gidy, cdc cau ti riéng biét cua hén hgp sé chuyén dich theo voi van toc khdc nhau, nho vay ma hén hop duge phan chia ra thinh cae cdu ti thanh phan. Su chuyén dich cia céc chat trong khi tién hanh sac ky cé thé gidi thich nhu sau: Cac soi xenlulozo cia gidy cé ai lye Ién déi véi nude va hap phy nuéc trong dung mdi hiru co bao hoa nude ( dong vai tro 1a chat mang ). O day pha tinh la nude, c6 chita trong nuée khoang 20%, cdn dung mdi hitu co 1a chat Jong linh dng khidung méi chay qua phan gidy c6 chtra dung dich nghién ctru thi m6t phan cdc chat chuyén vao dung méi ( pha d6ng ). Khi dung méi téi phan giay khong 6 chita cdc chat phan tich thi d6 lai xay ra qué trinh phan bé cac chat gitta pha tinh 1a nue va pha dng la dung méi hitu co. Lan nay, céc chat lai chuyén tir ( dung méi ) pha hirw co sang pha nuée. Nhu vay, cho dung méi chuyén dich lign tue qua gidy, cde chat phan tich sé duge chuyén tir digém mang ban dau dén m@t diém khac trén giay theo huwéng chay cla dung mdi do két qua cia qua trinh tach phan bé lién tyc gitra hai pha tinh va dng. Qué trinh phan tich nay duge tiém hanh trong thiét bj kin bao hoa ciia dung méi va nuéc. Khi tuyén dung méi da di duge doan dung 30 — 40 cm, ngudi ta gidy ra sdy kh6 va phun thud nhuém mau, nhé dé xac dinh duge vi tri cua cdc tir trén dai gidy. Dai gidy sau khi hién mau goi la sic ky dd. Trong thyc tién, hé sé van téc chuyén dich (ky higu la Rf) c6 y nghia rat lon. H6 sé van téc chuyén dich Rf la ti sé gitta quang duéng di duge cla chat so voi quing duéng chuyén dich ctia dung mdi. = Quing duéng dich chuyén cia chat / Quang duéng dich chuyén cla dung méi. Rf: La dai lugng dic trung va khéng di 4éi véi mdi chat trong nhimg diéu kién xe dinh. Tuy thugc vao huéng hode chiéu chuyén dich cua dung méi, ngudi ta phan biét mot sé dang sic ky nhu sau: * Sic ky nghich: Khi dung méi chay dich tir duéi lén trén. * Sac ky thuan: Khi dung moi chay tir trén xudng, * Shc ky vong tron: Khi dung mdi chuyén dich tir tam ra xung quanh. Ngoai ra, nguéi ta con phan biét: Sac ky m6t chiéu khi cho dung méi chuyén dich theo mt huéng nhat dinh. Sac ky hai chiéu khi cho dung méi chuyén dich theo mét chiéu nao dé, sau day sdy kh6 va cho dung méi chay theo mt chiéu khac vudng gée voi chiéu ban dau.Phuong phap sic ky gidy mt chiéu ( c6 thé thuan hodc nghich ) la phuong phap don gian nhat, chi ding mot hé dung mdi nao dé. Kha nang phan tich kém hon sic ky hai chidu, Trong sic ky thudn, hé s6 van téc chuyén dich Rf In, nhung cc vét mau thu duge khéng gon, con trong sic ky nghich dai lvgng Rf nhé hon, nhung vét mau Iai gon hon Dé dinh Iugng cdc axit amin ngudi ta thudng sir dung cée phutong phap sau: * So sinh bang mat thuong cuong 46 mau va dién tich cdc vét mau cia hop chat da cho véi cong 46 mau va dién tich cdc vét mau cia hon hgp déi chtmg da biét rd néng dd. * Do chiet suat cla dich chiét chat mau tir sac ky 46 bang m6t dung mdi thich hop. Sai s6 cia phuong phap nay khoang 3 — 6 %. * Ding densitomet do mat 46 mau cia céc vat trong khi cho anh sang xuyén qua, D6 chinh xéc ctia phép do tang len dén 10 ~ 15% [8,9] 2.2.6. Xe dinh aminoaxit va peptit bing sic ky long pha ngwge va sic ky long khéi pho ( thay xem va sira gitp em mue nay) [10]. Phuong phap méi cho phép xdc dinh nhay cdc aminoaxit va peptit 1A sir dung thude thir 2-(9-carbazole)-ethyl chloroformate (CEOC) véi my dd huynh quang (FL) da duge phat trién, Sy phat hign ra cdc din xudt da duge tién hanh bing phuong phap sic ky léng khdi phd. Nhom mang mau trong thudc thir 2-(9- fluorenyl)-ethyl chloroformate (FMOC) da duge thay thé bang carbazole,din dén mét téc nhan din xuat c6 tinh nhay quang 1a CEOC. CEOC cé thé phan logi cde peptit va aminoaxit dé dang va nhanh chong. Chat dan xudt du bén dé c6 kha nang phn tich boi phuong phap sic ky long higu ning cao (HPLC). Higu suat tao din xuat cue dai (gin 100%) duge quan sat véi sé mol thudc thir vugt qua 3-4 lan va nim trong khoang pH = 8.8 + 10. Cac dan xudt cé d6 nhay quang manh cho phép bom thang vao hén hgp phan img ma khong lam xo tron dang ké béi san pham phy. trong qué trinh phén huy tac nhan nhay quang nhu li 2-(9-carbazole)-ethanol va 2-(9-carbazole)-ethyl carbonate. Thém vao 46, qué trinh phat hign sw phan (mg véi din xudt CEOC duge so sénh véi qué trinh phat hign sy phan img thu duge bing FMOC. Ty 1é ACcroc /ACemoc = 1.00 + 1.82 cho sy cdm img phat huynh quang (FL) va AC'ceoc/AC'rmoc = 1.00 + 1.21 cho sy cdm tg tir ngoai - eye tim (UV). 6 day AC va AC’ la sy cdm tg tuong tmg gitta FL va UV. Sy phan tach cdc chit din xuat ca peptit va aminoaxit di duge t6i uu hod trén c6t Hypersil BDS C18. Phuong phap nay duge sir dung thinh céng dé phan tich protein thuy phan tir len va tir din xuat truc tip cia bia. CHUONG 2: THUC NGHIEM1. HOA CHAT VA DUNG CU. 1.1. Chuan bj hoa chat. 1.1.1. Pha thude thir. * Thuéc thir Biure (con goi la tac nhan Gornal). Cfn Sg CuSO,.5H,O hoa tan trong 495ml HO ta duge dung dich A (dung dich Sg CuSO;.5H,0 1%). Can 10g KNaCsH,0¢.4H20 (Kali Natri tactrat) hoa tan trong 490ml H,0 ta duge dung dich B (dung dich KNaCyH,O¢.4H;0 2%). Cfn 20.5g NayCO va 4g NaOH hod tan trong 1000ml H;0 ta duge dung dich C (dung dich NaCO; 2% trong NaOH 0.1). Pha th Lay Lml dung dich A + Iml dung dich B + 98ml dung dich C. thir Biure (chi pha truée khi ding): Thuéc thir Biure cé mau xanh nhat. Thudc thir Biure khéng ding duge trong méi truéng cé NH,’ vi NH,’ tao phite voi Cu’ khong mau, bén nén khéng tao phite sinh mau duge véi polypeptit. * Thudc thir Folin-Ciocalteux. Hoa tan 100g NaVOy.2H,0 va 25g Na»MoO,.2H,0 vio 800ml nude cat. ‘Thém 50 ml dung dich HsPO; 80% va 100 ml HCI dam dc. Dun séi hn hop trong binh cau day tron dung tich 2000 ml vi éng sinh han nguge trong vong 10 gid. Co thé dun ngat quang, khong can phai dun lién tuc, nhung phai dun s6i du 10 gid. Sau khi dun xong thém vao hén hp 150g LiySO,, 50 ml nude edt va 5 gigt nude Br). Dun s6i 15 phut khéng cé éng sinh han ngugc dé loai trir lrgng Br; du. Dung dich thudc thir phai cé mau vang, khong duge cé mau xanh. Néu thudc thir cé mau xanh thi phai xir Ii v6i Brom lan thir hai. Sau khi lam ngudi dung dich, thém nude ct cho téi vach mite trong binh dinh mite dung tich 1000 ml. X4c dinh néng 46 cia thude thir bing cach chudn d6 dung dich Folin - Ciocalteux pha loang 10 lan véi dung dich NaOH 0.1N (ding chat chi th 1a phenolphtalein).Bao quan thuéc thir Folin trong binh thuy tinh mau, dé trong ti lanh, Sau 2-3, thing dung dich nga mau xanh thi thém vai gigt Brom va lai dun s6i 15 phut, dung dich c6 mau vang tré Trude khi ding, pha loang thudc thir bing nude cat sao cho néng 46 thude thir bang IN. Protein higén mau tim - xanh trong méi trang kiém do su oxy hoa ctia volframat va molipdat cdc géc tirozin va triptophan trong protein. Cae chat ure (0.5%), guanidin (0.5%), vonphramat natri (0.5%), sunfat natri (1%), nitrat natri (1%), clorat (0.5%), tricloaxetat (0.5%), rugu etylic (5%), ete (5%), axeton (0.5%), sunfat kém (0.1%), hydroxit bari (0.5%), sunfatamon (0.15%) - khéng lim gidm mau cia phan img. Glixin (0.5%) lam gidm dén 50% mau cla phan tg. Bém phosphat tir 0.1M tro Ién gay két tia phan mg mau. * Pha dung dich dgm phosphat e6 pH=8. Cén 11,8660g NasHPO; hoa tan trong 1000ml HO ta duge dung dich E. Can 9.0780g KH:PO, hoa tan trong 1000ml H,0 ta duge dung di F. Tiép theo, ding pipet lay chinh xac 5.5ml dung dich F cho vao binh dinh mite 100ml, thm dung dich E vao binh cho téi vach mite ta duge dung dich dém phosphat co pH=8. 1.1.2, Pha dung dich casein chuan. Cén 0.6250g casein (chita 80% la protein) chuyén vo binh dinh mie 500ml, thém dung dich dém phosphat co pH=8 vao binh, Dat binh trén may khudy tir va gitt nhiét d6 trong khoang 50-60°C. Sau khi casein da tan hét, ding nuéc cat dinh mic téi vach ta duge dung dich casein chudn cé néng 46 1 mgprotein/ml. Pha dung dich casein chuan cé néng d6 protein 1a SOug/ml tir dung dich casein chun 6 trén bang cach ding pipet hat chinh xéc Sml dung dich casein chuan 1 mg/ml cho vao binh dinh mite 100ml, thém nude ct vao binh, Mic déu, sau d6 dinh mite dén vach bang nude cat. Bang cach tuong ty ta sé pha duge cde dung dich casein chun cé néng 46 protein la 100, 150, 200, 300, 400, 500 agprotein/ml.C4e dung dich casein chudn trén duge ding dé lap duéng chuan xde dinh ham lugng cac aminoaxit va protein trong thye phim, 1.1.3. Cae hod chat kha. Ngoai cdc hoa cha trén, trong qua trinh lam thye nghigm con ding céc loai hoa chat khac nhu: HCI dam diac, HsPO; 85%, NazVO4.2H,0, Na2MoO,.2H,0, LipSO,, NaOH 0.1N, nuée Brom 1.2. Dung cy va thiét bi. * 10 dng nghigm sach, khé trong | gid go. * | pipet 10 ml c6 chia vach. * 1 pipet 5 ml cé chia vach. * 2 pipet 2 ml c6 chia vach. * 4 pipet | ml c6 chia vach. * 2 binh dinh mate 100 ml. * 1 binh dinh mate 500 ml. * 1 binh dinh mite 1000 ml. * 2 dng dong 100 ml. * 2 binh tam gide 250 ml. * 2 cdc dung tich 1000 ml. * | buret 25 ml. * | binh cau 3 cd + 1 6 gid do. * 1 sinh han hoi hu. * 1 bép dién. * May khuay tir c6 diéu chinh nhiét 46. * 2 cuvet day 1 em. * May do quang MC - 752. 2. Xae dinh ham Iwgng axit amin va protein trong m@t so thye pham. Nhu da trinh bay & phan téng quan, cdc aminoaxit va protein phan tmg véi hén hop thudc thir Biure + Folin-Ciocalteux tao hop chat mau xanh va duge doquang 6 bude séng 750 nm. Phuong phap nay di duge hoan thign tir lau, cdc digu kign t6i wu da duge xéc lap, vi vay ching tdi chon phuong phap nay dé xée dinh ham lugng cia céc aminoaxit va protein trong m6t sé loai thyc pham. 2.1. Quy trinh phan tich chung. Miu that: Lay chinh xac vao éng nghiém sach, khé 0.5 ml dung dich nghién ctru, thém vao 0.5 ml H;O va 2.5 ml thuéc thir Biure, lic déu va gitr & nhiét 46 phong 10 phiit. Sau dé thém chinh x4c 0.25 ml dung dich Folin-Ciocalteux néng 46 IN, lac déu, sau 30 phut dem so mau trén may 6 bude song 750 nm. Tw sé doc cia mau thi nghiém, d6i chiéu vi 46 thi chuan dé tinh ra ham lugng protein trong | ml dich nghién cu, tir dé tinh ra ham lugng % protein trong nguyén ligu. 1u thi nghiém, lam dng déi ching trong 46 thay Miu tring: Song song vi dung dich protein bang nude cat va tiép tue lam nhu d6i voi mau that.. 2.2. Dung dwéng chudn xac dinh ham lgng aminoaxit vA protein. Dé xdc dinh duge ham lugng cia cdc aminoaxit va protein trong thye phim ta phai xay dung dudng chuan. Dung pipet lay chinh xdc 1 ml cdc dung dich casein chudn cé néng dé protein lan lugt 14 50, 100, 150, 200, 300, 400, 500 j1g/ml cho vao 7 éng nghiém sach khé. Thém vao 2.5 ml dung dich thudéc thir Biure, lic déu va git 6 nhiét do phing 10 phuit. Sau dé thém chinh xéc 0.25 ml dung dich Folin-Ciocalteux néng d6 IN, lic déu, sau 30 phat dem so mau trén may & bude séng 750 nm duge két qua 6 bang duge két qua & bang 2. Bang 2: Mt d@ quang ciia cae dung dich protein chudn.Protein 0.05 0.10 0.15 0.20 0.30 0.40 0.50 mg/ml Agso 0.124 0.136 0.147 0.159 0.178 0.209 0.231 So véi mau tring Aqso = 0.106, ta due két qua higu chinh nhu 6 bang 3. Bang 3: Mit d6 quang cita cde dung dich protein chudn (44 higu chinh). Protein 0.05 0.10 0.15 0.20 0.30 0.40 0.50 mg/ml Agso 0.018 0.030 0.041 0.053 0.072 0.103 0.125 Cée két qua trén duge biéu dién 6 hinh 11y= 0.2381x + 0.0053 R? = 0.9967 o 02 oe 06 [Protein] Hinh 11: Duong chudn xde dinh ham lugng aminoaxit va protein. Khi xr ly trén may tinh theo phuong trinh héi quy tuyén tinh ta tim duge phuong trinh lién quan Aqso = 0.2381 Cprocin + 0.0053 (1) Dudng chudn thye té khé thing nén phuong trinh (1) cé thé str dung nhanh két qua khi phan tich cdc mau 2.3. Phan tich nuée mam, 2.3.1. Quy trinh phan tich née mim, Ding pipet lay chinh x4c 2 ml dung dich nude mam can phan tich cho vao binh djnh mite 100 mi, thém nude cat vao binh, lac déu, sau dé dinh mite dén vach bang nude cat (dung dich mau). Tiép theo lay 0.5 ml dung dich mau cho vao mét vao éng nghiém 0.5 ml H,0 +2.5 ml Biure + 0.25 ng nghiém sach, khé. Thém ml Folin-Ciocalteux ndng 46 1N, lac déu, sau 30 phut dem so mau trén may & buéeséng 750 nm, Ham lugng aminoaxit va protein trong nuée mam duge tinh theo cOng thie sau: Ham Iugng Protein trong mau = Ham Ivong Protein tinh theo PTDC . 100 Ghi cha: Ham lugng protein tinh theo phuong trinh duéng chuan 1a sé mg protein/ml va duge tinh nhu sau: (Gi 4 tri A do duge - 0.106) - 0.0053 Ham lwong protein tinh thee PTC = © $$$ (mg/ml) 0.2381 2.3.2. Két qua phan tich, Két qua phan tich ham Iugng axit amin va protein trong nuée mam duge thé hign trong bang 4: Bang 4: Ham lugng cac axit amin va protein trong nuée mam, SIT Mau Mat d quang | Ham lvgng Protein A (mg/ml) 1 | Nuée mim Knorr 0.201 37.67 2__ | Nude mim Nhi cao cp Nha Trang 0.186 31.37 3 Nuée mam Nhi bigt Nha Trang 0.184 30.53 4 [Nude mim cao dam Cat Hai 0.180 28.85 3__ | Xidau Trung Quéc 0.176 27.17 (nuée cham dau nim) 6 | Nude trong Bibom Viet Tien o.171 25.07 7 | Nuéc mam thuéng 0.159 20.03Binh lugn: Qua bang sé ligu phan tich va két qua tinh duge ta thay ham lugng aminoaxit va protein trong nuée mim Ia rat cao (khoang 20-40 mg/ml). Hon nita, cdc loai nuéc mam déu cé mau khé dam, do d6 khi tién hanh phan tich ta cin pha long mau bang nude cat dé han ché sai s6 cua phép do. Ciing qua bang sé ligu phan tich ta thay cdc loai nuée mim khdc nhau thi ham Iugng aminoaxit va protein chia trong né la khde nhau va gid thanh cing khée nhau. 2.4, Phan tich bia. 2.4.1, Quy trinh phan tich bia. Dung pipet lay chinh xdc 0.1 ml mau bia can phan tich cho vao | éng nghiém sach, khd. Tiép theo, thém vao éng nghiém 0.9 ml H,O va 2.5 ml dung dich thuéc thir Biure, lic déu va gitt 6 nhigt 4 phong 10 phat, Sau d6 thém chinh xc 0.25 ml dung dich Folin-Ciocalteux néng d@ IN, lic déu, sau 30 phit dem so mau trén may & bude séng 750 nm. Ham lugng aminoaxit va protein trong bia duge tinh theo céng thite sau: Ham lvong Protein trong mau = Ham hugng Protein tinh theo PTBC . 10 Ghi cha: Him lvong protein tinh theo phuong trinh duéng chuan la sé mg protein/ml va duge tinh nhw sau: (Gi 4 tri A do duge - 0.106) - 0.0053 Ham lvong protein tinh theo PTBC =_§ —— (mg/ml) 0.2381 2.2.4.2. Két qua phan tich,Két qua phan tich him lugng cdc axit amin va protein trong bia duge thé hign trong bang 5: Bang 5: Ham lurgng céc axit amin vA protein trong bia. STT Mau Mat do quang A Ham long Protein (mg/ml) 1 Bia lon Heliken 0.208, 4.06 2 Bia lon Carberg, 0.203 3.85 3 | Bia lon Tiger 0.190 4 Bia lon Halida 0.180 5 Bia lon Ha Noi 0.174 6 Bia chai Ha Noi 0.171 7 Bia hoi Ha Noi 0.169 2.42 8 Bia thi céng Trung Hoa 0.151 1.67 Binh luan: Qua bang sé ligu phan tich va két qua tinh duge ta thay him lugng aminoaxit cdc logi bia vin da tr va protein trong bia 1d khéng cao (duéi 5 mg/ml). Tuy vay, thanh thuong higu ndi tiéng nhu Heliken, Carberg ...thi lrgng dam hoa tan chira trong né 1a cao hon han so v6i cdc loai bia khac. Dé cing 1a mét co sé dé gidi thich tai sao céc loai bia trén cé huong vi dim da, Idi cuén va gid thinh cao nhu vay. 2.5. Phan tich sira udng. 2.5.1. Quy trinh phan tich sia. Ding pipet lay chinh xdc 2 ml dung dich sita can phan tich cho vao binh dinh mic 100 ml, thém nuéc cat vao binh, lic déu, sau dé dinh mite dén vach bing nuée cat (dung dich mau). Tiép theo lay 0.5 ml dung dich mau cho vao mét éng nghiém sach, khé. Thém tiép vao dng nghiém 0.5 ml H;O +2.5 ml Biure + 0.25 ml Folin-Ciocalteux néng 46 IN, lic déu, sau 30 phut dem so mau trén may bude séng 750 nm. Ham lugng aminoaxit va protein trong nude mim duge tinh theo céng thie sau: Ham lIvong Protein trong mau = Ham lvong Protein tinh theo PTDC . 100 Ghi cha: Ham lugng protein tinh theo phuong trinh duéng chuan la sé mg protein/ml va duge tinh nhu sau: (Gia tri A do duge - 0.106) - 0.0053 Ham lvong protein tinh theo PTBC =| ——_______- (mg/ml) 0.2381 2.5.2. Két qua phan tich, Két qua phan tich ham Iugng cdc axit amin va protein trong sita uéng duge thé hign trong bang 6: Bang 6: Ham lugng cac axit amin va protein trong sita uéng. STT Mau Mat d6 quang A | Ham lvong Protein (mg/ml) 1 Sita b6 Méc Chau 0.190 33.05 2 Sita tuoi tigt tring Vinamilk 0.189 32.63 3 | Sita tuoi nguyén kem Izzi 0.185 30.95 4 | Sita Cé Gai Ha Lan 0.184 30.53 5 |Stta Nuvi 0.182 29.69 6 Sita chua Yomost 0.142 12.89 7 Sita Elovi (stra pha nudée cam tuoi) 0.136 10.37