Chiet Xuat Chitin 7199

i
Lun vn
Nghin cu ti u ha qu trnh
sn xut Chitin theo phng
php sinh hc kt hp ha hc
ii
LI CM N
Qua 2 thng n lc phn cui cng vi s gip tn tnh ca cc
thy c v bn b em hon tt ti ny. Qua y em xin by t lng bit
n su sc n Tin s: Nguyn Duy Nht v c gio: Phan Th Thng
ngi tn tnh truyn t nhng kin thc trong qu trnh th tp v trc
tip hng dn, ch bo nhng kinh nghim qu bu em hon thnh tt
ti.
Em xin by t lng bit n trn trng nht ti cc thy c trong khoa
cng ngh ho phn tch nhit tnh truyn t cho em nhng kin thc
trong nhng nm hc va qua. Em xin by t lng bit n n cc thy, c
ph trch phng th nghim ho phn tch, cng thy c b mn cng ngh
ho, b mn ho phn tch v cng ngh thc phm, cc anh ch v c ch
Vin nghin c v ng dng cng ngh Nha Trang to iu kin thun
li trong sut thi gian thc tp.
Chn thnh cm n cc bn sinh vin lp CH 29, cng cc bn sinh
vin thc tp ti phng th nghim Vin nhit tnh gip ng vin
em.
Cui cng con xin by t lng bit n su sc n b, m knh mn
cng anh ch em thn yu. Nhng ngi ng h nhit tnh c vt cht ln
tinh thn trong qu trnh hc tp v thc hin ti.
Sinh vin
Trn Th M Chu
iii
MC LC
LI CM N...........................................................................................................
MC LC...............................................................................................................
DANH MC CC BNG......................................................................................
DANH MC CC BNG......................................................................................
DANH MC CC HNH......................................................................................
LI M U..........................................................................................................
PHN 1....................................................................................................................
TNG QUAN V CNG NGH SN XUT V NG DNG CA
CHITIN-CHITOSAN................................................................................
1.1. TNG QUAN V CHITIN-CHITOSAN......................................................
1.1.1. S tn ti ca Chitin-Chitosan trong t nhin.................................................
1.1.2. Cu trc v tnh cht ca Chitin-Chitosan......................................................
1.1.2.1Cu trc ca Chitin-chitosan..........................................................................
1.1.2.2. Tnh cht ca Chitin-Chitosan.....................................................................
1.1.3. ng dng ca Chitin-Chitosan.......................................................................
1.1.3.1.Trong y hc v m phm...............................................................................
1.1.3.2.Trong cng nghip thc phm.......................................................................
1.1.3.3. ng dng trong nng nghip.......................................................................
1.1.3.4. ng dng trong sinh hc..............................................................................
1.1.3.5. ng dng trong cc ngnh cng nghip khc..............................................
1.2. GII THIU V PH LIU TM V CNG NGH SN XUT
CHITIN-CHITOSAN...................................................................................
1.2.1. Gii thiu v ph liu tm..............................................................................
1.2.2. Cng ngh sn xut Chitin-Chitosan............................................................
1.3. TNH HNH NGHIN CU SN XUT CHITIN-CHITOSAN TRN
TH GII V VIT NAM......................................................................
1.3.1. Sn xut chitin, chitosan theo phng php ha hc....................................
iv
1.3.2. Sn xut chitin, chitosan theo phng php ha hc ci tin:.......................

1.3.3. Sn xut chitin, chitosan theo phng php s dng enzyme:......................
PHN 2. NGUYN VT LIU V PHNG PHP NGHIN CU...........
2.1. NGUYN VT LIU NGHIN CU.........................................................
2.1.1. Nguyn liu u tm.....................................................................................
2.1.2. Enzyme Protease..........................................................................................
2.1.3. Acid benzoic (C6H5COOH)..........................................................................
2.1.4. Nguyn liu khc..........................................................................................
2.2. PHNG PHP NGHIN CU.................................................................
2.2.1. Phng php thu nhn mu..........................................................................
2.2.2. B tr th nghim xc nh cc thng s k thut..........................................
2.2.2.1. B tr th nghim xy dng ng chun ca phng php
Microbiuret v phng php Biuret..............................................................
2.2.2.2. B tr th nghim xc nh thnh phn ha hc ca ph liu u tm
th chn trng...............................................................................................
2.2.2.3. Th nghim xc nh ch kh protein ti u.........................................
a.
Kh ln 1: dng enzyme alcalase...........................................................
b.
Kh ln 2 dng NaOH:...........................................................................
2.2.2.4. Th nghim xc nh ch kh khong...................................................

a.
Kh ln 1: Dng acid benzoic.................................................................
b.
Kh ln 2: Dng acid chlohydric............................................................
2.3. CC PHNG PHP PHN TCH HA HC:[1].................................

2.3.1. Xc nh hm lng m bng phng php sy nhit 105oC theo
TCVN 3700-1990.........................................................................................
2.3.2. Xc nh hm lng khong bng phng php nung 600oC....................
2.3.3. Xc nh hm lng protein theo phng php Microbiuret:.......................
2.3.3.1. Dng c.....................................................................................................
2.3.3.2. Ha cht.....................................................................................................
2.3.3.3. Tin hnh...................................................................................................
2.3.4. Xc nh hm lng Protein theo phng php Biuret.................................

2.3.4.1. Dng c.....................................................................................................
2.3.4.2. Ha cht.....................................................................................................
2.3.4.3. Tin hnh...................................................................................................
2.3.5. Phng php x l s liu............................................................................
PHN 3. KT QU NGHIN CU V THO LUN...................................
3.1. Kt qu ng chun xy dng c theo phng php
Microbiuret v phng php Biuret.........................................................
3.1.1. Kt qu xy dng ng chun ca phng php Microbiuret :..................
3.1.2. Kt qu xy dng ng chun ca phng php Biuret:............................
3.2. Thnh phn ha hc ca ph liu tm th...................................................
3.3. Kt qu nghin cu cng on kh protein bng enzyme Alcalase..........
3.4. Kt qu nghin cu cng on kh protein bng NaOH...........................
3.5. Kt qu nghin cu cng on kh khong bng acid benzoic.................
3.6. Kt qu nghin cu cng on kh khong bng HCl...............................
PHN 4. KT LUN V XUT KIN...................................................
4.1. Kt lun:.........................................................................................................
4.2. Mt s xut cn tip tc nghin cu:......................................................
TI LIU THAM KHO.....................................................................................
PH LC.................................................................................................................
1) Phng php quy hoch thc nghim ti u ha cho cng on
kh protein t ph liu tm bng enzyme Alcalase..............................................
kh protein t ph liu tm bng NaOH:..............................................................
kh khong t ph liu tm bng C6H5COOH:...................................................
kh khong t ph liu tm bng HCl:...............................................................
5) Mt s thit b s dng trong ti:...................................................................
vi
vii
DANH MC CC BNG
Bng 1. Mt s ch tiu cht lng ca chitosan t v tm s theo phng
php x l kim mt giai on (Trn Th Luyn, 2003).............................
Bng 2. Mt s ch tiu cht lng ca chitosan sn xut theo quy trnh
Papain (Trn Th Luyn, 2003)...................................................................
Bng 3.B tr th nghim chy ng chun ca phng php Microbiuret.........
Bng 4. B tr th nghim chy ng chun ca phng php Biuret.................
Bng 5. B tr th nghim theo qui hoch thc nghim vi bin o ca cng
on kh protein bng enzyme Alcalase....................................................
Bng 6. B tr th nghim tm phng n ca cng on kh protein bng
enzyme Alcalase.........................................................................................
on kh protein bng NaOH.....................................................................
NaOH..........................................................................................................
on kh khong bng C6H5COOH...........................................................
Bng 10. B tr th nghim tm phng n ca cng on kh khong bng
C6H5COOH................................................................................................
on kh khong bng HCl........................................................................
HCl.............................................................................................................
Bng 13. Thnh phn ha hc ca ph liu tm th chn trng (%)......................
Bng 14. Kt qu hm lng protein cn li cc ch kh protein bng
enzyme Alcalase.........................................................................................
Bng 15. Th nghim tm phng n ca cng on kh protein bng
enzyme Alcalase......................................................................................
viii
Bng 16. Kt qu hm lng protein cc th nghim ti u theo ng dc nht..........

Bng 17. Kt qu hm lng protein cc ch kh protein bng NaOH.........
NaOH.........................................................................................................
Bng 19. Kt qu hm lng protein cc th nghim ti u theo ng dc nht..........
Bng 20. Thnh phn ha hc ca ph liu tm th Chn Trng sau khi kh l
kh protein bng enzyme Alcalase v NaOH iu kin ti u................
Bng 21. Kt qu hm lng khong cn li trong ph liu tm cc ch
kh khong bng C6H5COOH....................................................................
Bng 22. Th nghim tm phng n ca cng on kh khong bng
C6H5COOH.................................................................................................
Bng 23. Thnh phn ha hc ca ph liu tm th Chn Trng sau khi kh
protein bng enzyme Alcalase, NaOH v C6H5COOH iu kin ti u.
...................................................................................................................55
kh khong bng HCl................................................................................
Bng 25. Th nghim tm phng n ca cng on kh khong bng HCl
..............................................................................................................................
Bng 26. Kt qu o OD330nm..............................................................................
Bng 27. Kt qu o OD570nm..............................................................................
Bng 28. Bng b tr th nghim............................................................................
Bng 29.Cc th nghim tm phng n..............................................................
Bng 30. Cc th nghim tm phng n ca cng on kh protein bng
enzyme Alcalase...........................................................................................
Bng 31.Cc s liu dng tnh ton....................................................................
Bng 32. Kt qu thc nghim ti u ha cng on thy phn kh protein
bng enzyme ..........................................................................................................
Bng 33.Cc thng s ti u...................................................................................
Bng 34. Bng b tr th nghim.............................................................................
ix
Bng 35.Cc th nghim tm phng n..............................................................

Bng 36.Cc th nghim tm phng n ca cng on kh protein bng
NaOH...........................................................................................................
Bng 37. Cc s liu dng tnh ton...................................................................
Bng 38.Kt qu thc nghim ti u ha cng on kh protein bng NaOH
..............................................................................................................................
Bng 39.Cc thng s ti u.................................................................................
Bng 40. Bng b tr th nghim............................................................................
Bng 41. Cc th nghim tm phng n...........................................................
Bng 42. Cc th nghim tm phng n ca cng on kh khong bng
C6H5COOH................................................................................................
Bng 43.Cc s liu dng tnh ton..................................................................
Bng 44. Kt qu thc nghim ti u ha cng on kh khong bng C6H5COOH .
..............................................................................................................................
Bng 45. Cc thng s ti u................................................................................
Bng 46. Bng b tr th nghim...........................................................................
Bng 47. Cc th nghim tm phng n...........................................................
NaOH.........................................................................................................
Bng 49. Cc s liu dng tnh ton.................................................................
Bng 50. Kt qu thc nghim ti u ha cng on kh khong bng HCl.
..............................................................................................................................
Bng 51. Cc thng s ti u................................................................................
DANH MC CC HNH
Hnh 1.Cu to ca Chitin...............................................................................4
Hnh 2. Cu to ca Chitosan..........................................................................5
Hnh 3.Quy trnh ca Stevens (2002) Hc Vin Cng Ngh Chu ............15
Hnh 4.Quy trnh sn xut chitosan t v tm hm ca Hackman................17
Hnh 5.Quy trnh nhit ca Yamashaki v Nakamichi (Nht Bn)................17
Hnh 6.Quy trnh sn xut ca Php..............................................................18
Hnh 7.Quy trnh ca Minh Phng, trng i hc Thy Sn................20
Hnh 8. Quy trnh sn xut Chitosan Trung tm cao phn t thuc Vin
khoa hc Vit Nam.............................................................................21
Hnh 9. Quy trnh sn xut Chitosan t v tm S bng phng php ha hc
vi mt cng on x l kim (Trn Th Luyn, 2003).....................22
Hnh 10. Quy trnh ca Trung tm Ch bin i hc Thy sn....................24
Hnh 11. Quy trnh sn xut chitin ca Holanda v Netto (2006)..................25
Hnh 12. Quy trnh s dng Enzyme papain sn xut chitosan................26
Hnh 13.Quy trnh d kin sn xut..............................................................32
Hnh 14.Quy trnh sn xut Chitin khng dng acid benzoic.(i chng)....33
Hnh 15.Cng thc ca phc Biuret..............................................................42
Hnh 16. Phng trnh ng chun ca phng php Microbiuret.............45
Hnh 17. Phng trnh ng chun ca phng php Biuret......................46
Hnh 18. Kt qu nghin cu ti u ha ch thy phn bng enzyme theo
phng php ng ln dc ca BoxWillson...........................................49
Hnh 19. Kt qu nghin cu ti u ha ch ngm NaOH theo phng
php ng ln dc ca BoxWillson.............................................51
Hnh 20. Kt qu nghin cu ti u ha ch ngm C6H5COOH theo
phng php ng ln dc ca BoxWillson................................54
Hnh 21. Kt qu nghin cu ti u ha ch ngm HCl theo phng php
ng ln dc ca BoxWillson......................................................57
Hnh 22.Thit b n nhit Memmert..............................................................19
xi
Hnh 23. Thit b o Uvmini-1240................................................................19

Hnh 24.My li tm.......................................................................................20
Hnh 25.Thit b Vortex.................................................................................20
Hnh 26.S bt mu ca dung dch BSA sau khi cho thuc th Biuret.........20
Hnh 27. Cng thc cu to ca Sodium citrate............................................21
Hnh 28. Mono Sodium Citrate Anhydrous...................................................21
Hnh 29. Dung dch protein v thuc th Biuret...........................................21
Hnh 30. Thy phn u tm.........................................................................21
Hnh 31. u tm c v hot enzyme sau khi thy phn(dng nhit)..22
Hnh 32. Ph liu tm sau khi thy phn.......................................................22
Hnh 33. Ph liu tm sau khi thy phn bng enzyme Alcalase v x l
NaOH.................................................................................................22
Hnh 34. Ph liu tm kh protein v kh khong ln 1 bng
C6H5COOH........................................................................................
Hnh 35. Ph liu tm(PLT) kh protein bng enzyme(tri), PLT kh
protein bng enzyme v NaOH..........................................................23
Hnh 36. PLT kh protein bng enzyme v NaOH(tri), PLT kh
protein v kh khong bng C6H5COOH...........................................23
LI M U
1. Tnh cp thit ca ti:
Vit Nam hin nay ch yu sn xut chitin-chitosan theo phng php ho
hc c s dng cc loi ho cht vi nng cao, thi gian x l di gy nh hng
ti cht lng chitin-chitosan v cc ch phm khc c sn xut t ph liu gip
xc. V vy, vic nghin cu mt quy trnh kt hp phng php ho hc vi
phng php sinh hc ng thi gim ti a lng ho cht s dng l rt cn thit
gp phn gim chi ph sn xut, nng cao cht lng sn phm.
Ngoi ra, cn c rt nhiu hng nghin cu s dng ph liu gip xc sn xut cc
mt hng c gi tr khc nh: Astaxanthin, thc n chn nui, chit rt cht mi
nhng cng ngh gy nhim nn khng cho php tn dng cc cht c hot tnh
khc.
Do vic s dng ngun ph liu t v tm c trin khai ti rt nhiu
cc khu vc trong nc. Vic tn dng ph liu v tm tp trung ch yu vo sn
xut thc n cho gia sc, cc sn phm Chitin-Chitosan, glucosamine,
oligosaccharide Tuy nhin theo iu tra ban u th trong cng ngh sn xut
Chitin-Chitosan trong nc hin nay u thc hin ch yu theo phng php ha
hc dn n cht lng cha cao, hu ht cc c s sn xut Chitin-Chitosan cha
c mt h thng x l nc thi t tiu chun. Nguyn nhn ch yu l do hm
lng cht l lng, trong ch yu l cc cht c ngun gc t protein. Chng c
tnh cht l rt kh lng trong qu trnh x l. Nh vy, nu chng ta c th thu hi
protein sau qu trnh thy phn bng enzyme Alcalase th s tn dng c ngun
cht dinh dng trong ph liu u v tm lm bt dinh dng cho ngi, thc
n cho gia sc, gia cm, t nng cao nng cao c gi tr ca nguyn liu, gim
ti cho qu trnh x l nc thi, hn ch s nhim mi trng. ng thi vic s
dng thy phn bng enzyme s hn ch vic s dng ha cht, gy nhim mi
trng. Trong thc t hin nay ti cc c s sn xut phi thu nhn nguyn liu t
cc khu vc xa, khng tp trung v khi lng khng ln gy kh khn v chi ph
vn chuyn. Do vy vic nghin cu ng dng ch thy phn bng enzyme gp
phn ko di thi gian bo qun nguyn liu, gim chi ph do phi hoc sy kh
nguyn liu, gim s nhim mi trng do khng vn chuyn tm thi, ng thi
loi b mt phn cc cht khong, protein.
Xut pht t thc t trn, c s ng ca B mn Cng ngh Ch binKhoa Ch bin-Trng i hc Nha Trang, di s hng dn ca c Thc s Ng
Th Hoi Dng, em thc hin ti: Nghin cu ti u ha qu trnh sn
xut Chitin theo phng php sinh hc kt hp ha hc.
2.Mc ch ca ti:
Xc nh cc iu kin ti u kh protein t ph liu tm th chn trng
(Penaeus vannamei) bng enzyme Alcalase v NaOH, ng thi xc nh cc iu
kin ti u kh khong bng acid benzoic kt hp HCl nhm gim thiu ha cht
s dng, gim nhim mi trng, nng cao cht lng Chitin.
3. Tnh khoa hc v thc tin ca ti:
Thnh cng ca ti s c p dng ti cc c s sn xut chitin vi mc
ch tn dng ngun protein t u tm, hn ch s dng ha cht nhm gim
nhim mi trng v sn xut ra chitin-chitosan c cht lng cao v ng dng vo
cc lnh vc c bit nh ng dng trong y hc, m phm. ti cng l ngun
ti liu hu ch phc v cho cng tc nghin cu chuyn su v lnh vc ny.
4. Ni dung ca ti:
- Tng quan v cng ngh sn xut chitin-chitosan
- Nghin cu sn xut Chitin bng phng php sinh hc
Ti u ha qu trnh kh protein bng enzyme kt hp vi NaOH
Ti u ha qu trnh kh khong c s dng acid benzoic
- xut quy trnh.
PHN 1.
TNG QUAN V CNG NGH SN XUT V NG DNG CA

CHITIN-CHITOSAN.
1.1.
TNG QUAN V CHITIN-CHITOSAN
1.1.1. S tn ti ca Chitin-Chitosan trong t nhin

Chitin v dn xut ca n l Chitosan l nhng polysaccharide mch thng,
chng ph bin trong t nhin ch sau cellulose, Chitin tn ti c ng vt v thc
vt. ng vt thy sn, Chitin tn ti rt nhiu, c bit l v tm, cua, gh,
V vy, chng l ngun nguyn liu di do sn xut Chitin-Chitosan.
Trong ng vt: Chitin l thnh phn cu trc quan trng ca v bao ca mt
s ng vt khng xng sng nh cn trng, nhuyn th, gip xc, giun trn,
Chitin c coi l cht to xng hu c chnh ng vt khng xng sng.
Trong thc vt: Chitin c trong vch t bo ca nm v mt s loi to
chlorophyceae. Chitin tn ti trong t nhin dng tinh th. N c cu trc gm
nhiu phn t c ni vi nhau bng cu ni hydro v to thnh mt h thng
dng si t nhiu c t chc. Trong t nhin rt t gp dng tn ti t do ca Chitin,
n lin kt di dng phc hp Chitin-protein,Chitin vi cc hp cht hu c,
khi tn ti nh th Chitin c s khng i vi cc cht thy phn, ha hc v
enzyme. Do n gy kh khn cho vic tch chit v tinh ch. Ty thuc vo c
tnh ca c th v s thay i tng giai on sinh l m trong cng mt loi, ngi
ta c th thy s thay i v hm lng cng nh cht lng ca Chitin.
Trong t nhin, Chitosan rt him gp, ch c trong vch mt s lp vi nm
(c bit: zygomycetes, mucor,) v vi loi cn trng nh thnh bng ca mi
cha. S deacetyl bng kim, Chitin to thnh Chitosan v tan c trong dung dch
acid acetic long.
1.1.2. Cu trc v tnh cht ca Chitin-Chitosan
1.1.2.1 Cu trc ca Chitin-chitosan
Chitin-Chitosan l mt polysaccharide nn c cu trc dng chui.
Trong : Chitin : R : -NH-COCH3
Chitosan : R : -NH2
CHITIN: Chitin l mt polysaccharide mch thng, n c cu trc tuyn tnh

gm cc n v N-acetyl-glucosamine ni vi nhau nh cu -1,4glucoside.
Cng thc phn t: (C8H13O5N)n
Phn t lng : M = (203,19)n
Trong n ph thuc vo ngun gc nguyn liu:
i vi tm hm : n = 700800
i vi cua :
n = 500600
i vi tm th:
n = 400500
Cng thc cu to:
Hnh 1.Cu to ca Chitin.

CHITOSAN: Chitosan l mt polysaccharide mch thng, gm cc phn t D1,4glucosamine. Khi x l kim c t Chitin ta thu c Chitosan.
Cng thc phn t : (C6H11O4N)n
Phn t lng:
M= (161,07)n
Cng thc cu to :
Hnh 2. Cu to ca Chitosan.
1.1.2.2. Tnh cht ca Chitin-Chitosan
CHITIN:
Chitin c mu trng, khng tan trong nc, trong kim, trong acid long v
cc dung mi hu c khc nh ete, ru. Chitin ha tan c trong dung dch m
c, nng ca mui thyoxyanat liti (LiSCN) v mui thyoxyanat canxi (Ca(SCN) 2)
to thnh dung dch keo.
Chitin n nh vi cht oxy ha nh KMnO 4, nc javen, NaClO, ngi ta
li dng tnh cht ny kh mu cho Chitin.
Chitin c kh nng hp th tia hng ngoi bc sng 884890 cm.
Chitin l mt polysaccharide ngun gc t nhin, c hot tnh sinh hc cao, c tnh
ha hp sinh hc v t phn hy trn da. Chitin b men lysozyme, mt loi men ch
c c th ngi, phn gii thnh monome N-acetyl-D-glucosamine.
Chitin kt tinh dng v nh hnh, kh ha tan trong dung dch amoniac
(NH3), khng ha tan trong thuc th Schueizer-Sacrpamonia. iu ny c th l do
s thay i nhm hydroxy (-OH) ti v tr C 2 bng nhm acetamic (NHCOCH3)
ngn cn s to thnh cc phc hp cn thit.
Khi nung nng Chitin trong dung dch NaOH c th Chitin s b kh mt
gc acetyl to thnh Chitosan. Khi un nng Chitin trong acid HCl c th Chitin s
b thy phn to thnh Glucosamine 85,5%, acid acetic 14,5%.
CHITOSAN:
c tnh c bn ca Chitosan: Chitisan c ngun gc thin nhin, khng c,
dng an ton cho ngi trong thc n, thc phm, dc phm, c tnh ha hp sinh
hc cao vi c th, c kh nng t phn hy sinh hc, c nhiu tc dng sinh hc a
dng: c kh nng ht nc, gi m, khng nm, khng khun vi nhiu chng loi
khc nhau, kch thch tng sinh t bo ngi, ng vt, thc vt, c kh nng nui
dng t bo trong iu kin ngho dinh dng.
Tnh cht ha hc : Chitosan l cht rn, xp, nh, mu trng ng, khng
mi, khng v, ha tan d dng trong dung dch acid long. Loi Chitosan c khi
lng trung bnh thp t 100000400000 hay c dng nhiu nht trong y t v
trong thc phm.
Tnh cht sinh hc: Chitosan c nhiu tc dng sinh hc a dng nh : tnh
khng nm, tnh khng khun vi nhiu chng loi khc nhau, kch thch s pht
trin tng sinh ca t bo, c kh nng nui dng t bo trong iu kin ngho
dinh dng, tc dng cm mu, chng sng u.
Ngoi ra, Chitosan cn c tc dng lm gim cholesterol v lipid mu, lm to
vi ng mch v h huyt p, iu tr thn mn tnh, chng ri lon ni tit.
Vi kh nng thc y hot ng ca cc peptid- insulin, kch thch vic tit
ra insulin tuyn ty nn Chitosan c dng iu tr bnh tiu ng. Nhiu
cng trnh cng b kh nng khng t bin, kch thch lm tng cng h thng
min dch c th, khi phc bch cu, hn ch s pht trin ca cc t bo u, ung
th, HIV/AISD, chng tia t ngoi, chng nga, ca Chitosan.
1.1.3. ng dng ca Chitin-Chitosan:[2][5][10]
1.1.3.1.
Trong y hc v m phm
Dng lm ph gia trong k ngh bo ch dc phm:

T dc n, t dc chnh, t dc dn thuc, mng bao phim, vin nang
mm, nang cnglm cht mang sinh hc gn thuc, to ra thuc polymer tc
dng chm ko di, lm hot cht chnh cha bnh nh: Thuc iu tr lin vt
thng, vt phng, vt m v trng, thuc b dng c th: H lipid v cholesterol
mu, thuc cha bnh au d dy, tiu ng, xng khp, vim khp, vim xng,
long xng, chng ng t mu, khng nm, khng khun, iu tr suy gim min
dch, c kh nng hn ch s pht trin ca t bo u, t bo ung th v chng HIV.
Dng lm vt liu y sinh: Da nhn to, mng sinh hc, cht nn cho da nhn to, ch
khu phu thut, m cy ghp
Trong m phm chitosan c b sung vo kem chng kh da, kem lt mt tng
bm dnh, tng ha hp sinh hc vi da, chng tia cc tm
1.1.3.2. Trong cng nghip thc phm[3]
Chitosan c xem nh mt ph gia to cng, to keo, phn lp v kh
axit ca tri cy v ung, tng cng mi v t nhin. To mng bao gi thc
phm, hoa qu, rau ti. L mt polyme dng an ton cho ngi, li c hot tnh
sinh hc a dng, chitosan c coi l thnh phn b dng a vo thc phm,
bnh ko, nc gii kht, thc n vt nui v thy sn. Chitosan s dng chng
hin tng mt nc trong qu trnh lm lnh, lm ng thc phm.
1.1.3.3. ng dng trong nng nghip[2][5]
Dng bo qun ht ging, tng cng kh nng ny mm ca ht, tc nhn
chng nm, chng vi khun gy bnh cho mi trng xung quanh.
Ngoi ra, chitosan cn dng lm cht kch thch sinh trng cy trng, thuc chng
bnh o n, kh vn cho la.
1.1.3.4. ng dng trong sinh hc
Lm gi th hot ha cho cng ngh c nh enzyme v cc t bo vi sinh
vt, lm cht mang s dng trong sc k chn lc, mng lc sinh hc, tng hp
polymer sinh hc.
1.1.3.5. ng dng trong cc ngnh cng nghip khc.
Trong cng nghip dt:
Chitosan c dng h vi: c nh hnh in hoa, u im c th thay th
c h tinh bt bng chitosan lm cho vi hoa, ti, si bn chu c c xt, b mt
p, bn trong kim.
Lm vi chu nc, khng bt la: Ha tan chitosan trong dung dch acid
acetic long cng vi axetat nhm v axit stearic thu c hn hp. Hn hp ny
em sn ln vi, khi kh to thnh mng mng, chc, bn, chu nc v khng bt
la. Vi ny c s dng sn xut bo h lao ng.
Lm si Chitin: Ngm chitosan trong dung dch Na 2SO4 bo ha ri em ko
si, ra trong nc nhit cao thu c ging si gai. em si ny trn vi si
cellulose t l 30% thu c si Chitin-cellulose. Kh nng bt mu thuc nhum
cng tng khi ta tng h si chitin.
Trong cng nghip giy:
Chitosan c tc dng lm tng bn ca giy, ch cn thm trng lng bng 1%
trng lng ca giy th s lm tng gp i bn ca giy khi m t, tng nt
khi in. Cc loi giy ny dng lm giy v sinh, giy in, ti giy.
Trong ngnh phim nh:
Phim chitosan c nht rt cao, khng tan trong nc, acid. cng c ci
thin bng cch tng hp c chitosan, ri x l phim bng dung dch acid.
ng dng trong m phm:
Chitosan c s dng trong sn xut kem chng kh da, do bn cht
chitosan c nh d dng trn biu b da bi nhng nhm NH4 + thng c cc
nh khoa hc gn vi nhng cht gi nc hoc nhng cht lc tia cc tm. V vy
chitosan l gch ni gia hot cht ca kem v da.
1.2. GII THIU V PH LIU TM V CNG NGH SN XUT
CHITIN-CHITOSAN.
1.2.1. Gii thiu v ph liu tm
Vit Nam ngun nguyn liu tm l rt di do, c thu t 2 ngun chnh
l nh bt t nhin v nui trng. c bit, nui tm pht trin mnh trong
nhng nm gn y v tr thnh ngnh kinh t mi nhn. Din tch nui tm
tng t 250.000 ha nm 2000 ln n 478.000 ha nm 2001 v 540.000 ha nm
2003. Nm 2002, gi tr xut khu thu sn t hn 2 t USD, trong xut khu
tm ng lnh chim 47%, ng th 2 sau xut khu du kh. Nm 2004, xut khu
thu sn t gi tr 2,4 t USD, chim 8,9% tng gi tr xut khu c nc trong

tm ng lnh chim 53% tng gi tr xut khu thu sn. Nm 2006 kim ngch
xut khu thy sn qua mc 3 t t 3,31 t USD, tng gn 600 triu USD so vi
nm 2005, trong mt hng tm truyn thng chim v tr u bng xp x 1,5 t
USD, chim 44,3 % tng kim ngch xut khu. Nm 2007, tng kim ngch xut
khu thy sn t 3,75 t USD tng 12% so vi nm 2006
Mc tiu xut khu thu sn n nm 2010, Vit Nam phn u t gi tr 4 4,5 t USD. nh hng n nm 2020, ch bin xut khu thy sn tip tc l
ng lc thc y pht trin nui trng thu sn, khai thc thu sn v mang li
nhiu li ch kinh t ngnh, nng cao thu nhp v i sng lao ng ngh c. Tm
c d kin t khong 483 nghn tn nguyn liu phc v cho xut khu
khong 390.000 tn nm 2010. Theo thng k ca Trung tm Nghin cu Ch bin
Thy sn, i hc Thu sn th lng ph liu nm 2004 ti Vit Nam c tnh
khong 45.000 tn ph liu, nm 2005 c tnh khong 70.000 tn/nm. Trn Th
Luyn (2004) cho bit trong v tm ti chitosan chim khong 5% khi lng,
trong v tm kh khong 20-40% khi lng. Nh vy hng nm c th sn xut
gn 5000 tn chitosan phc v sn xut trong nc v xut khu, mang li hiu qu
kinh t cho ngnh Thu sn .
Ph liu tm (PLT) l nhng thnh phn ph thi t cc c s ch bin tm
bao gm u, v v ui tm. Ngoi ra, cn c tm gy thn, tm lt v sai quy
cch hoc tm b bin mu. Tu thuc vo loi v phng php x l m lng ph
liu c th vt qu 60% khi lng sn phm. C th ly tm cng xanh
Macrobrachium rosenbergii lm v d, u tm chim ti 60% trng lng tm.
u tm s Penaeus monodon cng chim ti 40% trng lng tm. Vi sn phm
tm lt v, rt ch lng, lng ui v v ui ca tm chim khong 25% trng
lng tm. i vi tm th, lng ph liu u tm chim 28% v v chim 9%,
nh vy tng lng ph liu v u tm th l 37%. Lng ph liu ny c th
gim t nhiu bng cch nng cao hiu qu lt v nh cc thit b v cng ngh ch
bin tt hn. Gim lng ph liu t khu ch bin hoc tm gii php ti s dng
10
chng ang tr nn ph bin nh mt phng cch gip lm tng li nhun cho

ngnh thu sn.
Nhiu cng trnh nghin cu cho thy t l ca ph liu tm t 30-70%
(Watkin v cng s, 1982 ; Evers v Carroll [19], trung bnh khong 50% so vi
khi lng tm cha ch bin. Halanda v Netto (2006) cho rng ph liu tm c
th chim 50-70% so vi nguyn liu [20]. Phn ln tm c a vo ch bin
di dng bc v, b u. Phn u thng chim khi lng 34-45%, phn v,
ui v chn chim 10-15% trng lng ca tm nguyn liu. Tuy nhin, t l ny
tu thuc vo ging loi v giai on sinh trng ca chng [5] [6].
Cu to v thnh phn sinh ha ca v tm[5]
Lp ngoi cng ca v tm c cu trc chitin-protein bao ph, lp v ny
thng b ha cng khp b mt c th do s lng ng ca mui canxi v cc cht
hu c khc nm di dng phc tp do s tng tc gia protein v cc cht
khng ha tan.
V chia lm 4 lp chnh:
Lp biu b
Lp mu.
Lp canxi.
Lp khng b canxi ha.
- Lp biu b, lp mu, lp canxi ha cng do s lng ng ca canxi. Lp
mu, lp canxi ha, lp khng b canxi ha cha chitin nhng lp biu b th khng
- Lp mu: Tnh cht ca lp ny do s hin din ca nhng th hnh ht ca
vt cht mang mu ging dng melanin. Chng gm nhng ti kh hoc nhng
khng bo. Mt vi vng xut hin nhng h thng rnh thng ng c phn nhnh,
l con ng cho caxi thm thu vo.
- Lp biu b: nhng nghin cu cho thy lp mng nhanh chng b bin
bi Fucxin, c im pH =5,1 khng cha chitin. N khc vi cc lp v cn li, bt
mu xanh vi anilin xanh. Lp biu b c lipid v vy n cn tr tc ng ca acid
nhit thng hn cc lp bn trong. Mu ca lp ny thng vng rt nht.
11
- Lp canxi ha: Lp ny chim phn ln lp v, thng c mu xanh tri

u khp.
- Lp khng b canxi ha: Vng trong cng ca lp v c to bi mt
phn tng i nh so vi tng chiu dy bao gm cc phc chitin protein bn
vng khng c canxi v puinone.
Thnh phn sinh ho ca v tm
Protein: Thnh phn protein trong ph liu tm thng tn ti hai dng
Dng t do: Dng ny l phn tht tm t mt s tm b bin i c vt
ln vo ph liu hoc phn tht cn st li trong u v ni tng ca u tm. Nu
cng nhn vt u khng ng k thut th phn protein b tn tht vo ph liu
nhiu lm tng nh mc tiu hao nguyn vt liu, mt khc ph liu kh x l hn.
Dng phc tp: dng ny protein khng ha tan v thng lin kt vi
chitin, Canxi Carbonate, vi lipid to lipoprotein, vi sc t to proteincarotenoit
nh mt phn thng nht quyt nh tnh bn vng ca v tm.
Chitin: Tn ti di dng lin kt bi nhng lin kt ng ha tr vi cc
protein di dng phc hp chitin-protein; lin kt vi cc hp cht khong v cc
hp cht hu c khc gy kh khn cho vic tch v chit chng.
Canxi: Trong v, u tm, v gh c cha mt lng ln mui v c, ch
yu l mui CaCO3, hm lng Ca3(PO4)2 mc d khng nhiu nhng trong qu
trnh kh khong d hnh thnh hp cht CaHPO 4 khng tan trong HCl gy kh
khn cho qu trnh kh khong.
Sc t: Trong v tm thng c nhiu loi sc t nhng ch yu l
Astaxanthin.
Enzyme: Theo tp ch Thy sn (s 5/1993) hot enzyme protease ca
u tm khong 6,5 n v hot /g ti. Cc enzyme ch yu l enzyme ca ni
tng trong u tm v ca vi sinh vt thng tr trn tm nguyn liu.
Ngoi thnh phn ch yu k trn, trong v u tm cn c cc thnh phn
khc nh: nc, lipid, phospho,
1.2.2. Cng ngh sn xut Chitin-Chitosan
12
Mc d chitin phn b rng ri trong t nhin nhng n khng c tm thy

dng tinh khit. Chitin trng thi t nhin th lin kt vi protein, lipid, sc t v
canxi . V vy, n cn phi c lm sch trc khi s dng cho bt k mc ch
thng mi no. Phng php dng phn tch v tinh sch chitin phi m bo
ly i khong v tn dng c cc hp cht c gi tr khc. Do , nhiu phng
php c p dng cho vic thu hi chitin. Hn na, tnh hu ch ca cc ngun
chitin khc nhau ph thuc vo s sn c ca nguyn liu, phng php n gin,
hm lng chitin, v s ph hp tn thu cc sn phm c gi tr khc.
Hin nay vic lm sch chitin bao gm hai bc chnh:
- Kh khong: Loi b khong bng acid hoc l mt tc nhn to phc.
- Kh protein: Tch protein bng kim hoc mt enzyme protease.
Hai bc ny c th i v tr cho nhau ph thuc vo phng php thu hi
protein, carotenoid v hn na l ng dng chitin. Chitin s dng nh mt cht hp
th hay h tr enzyme nn kh khong trc, bi v bc ny ly i mui khong
v bo v cu trc chitin m bo s deacetyl polysaccharid khi x l kim nh
kh protein. Tng cng mc deacetyl gia tng cc nhm amino t do tham gia vo
qu trnh hp ph v gn kt protein. Tuy nhin, thu hi protein th nn thc hin
bc kh protein trc. Khi , sn lng protein v cht lng l ln nht . Sau
qu trnh kh khong v kh protein, sn phm c ty mu bng acetone hoc
hydrogen peroxide. Bc ny l khng cn thit v ph thuc vo yu cu ca sn
phm cui cng. C hai phng php chnh sn xut chitin: phng php ha
hc v phng php sinh hc.
-Phng php ha hc: Qu trnh kh khong c thc hin bng vic s
dng HCl hoc acid hu c khc hoc kt hp c hai nhit phng vi c ch
nh sau:
CaCO3 + 2HCl = CaCl2 + CO2 + H2O
Ca3(PO4)2 + 6HCl = 3CaCl2 + 2H3PO4
Cc yu t nh hng ti qu trnh ny l:
13
a. Nng acid: Nng acid qu thp s khng kh c ht khong dn n

sn phm cn nhiu tp cht. Nng qu cao s gy t mch chitin, gim
cht lng sn phm.
b. T l acid/nguyn liu: Nu qu nh th s khng kh ht khong, nu qu
ln s nh hng xu i vi mch, tn chi ph.
c. Nhit , thi gian x l: hai yu t ny cng rt quan trng, nh hng n
cht lng sn phm. Nu thi gian x l di v nhit cao th sn phm b
nt s rt kh x l sau ny, ng thi s nh hng ti nht ca sn phm
sau ny. Nu nhit thp v thi gian x l ngn th khong s khng b loi
trit .
Thng thng khi tin hnh nhit cao th thi gian phi ngn nu c
iu kin ta c th x l nhit thp thi gian di th cht lng s tt hn.
-Phng php sinh hc: Trong phng php sinh hc ch khc ti cng on
kh protein v deacetyl khng s dng ho cht m c th s dng h vi khun,
nm men hoc cc enzyme loi b protein mt cch trit . Vic deacetyl c
thc hin bi enzyme deacetylase. Sn phm chitosan thu c c cht lng cao
do khng b nh hng nhiu bi ho cht [6]
Vic s dng phng php sinh hc cng gp phi rt nhiu kh khn nh
gi thnh sn phm c th s cao tu thuc vo loi enzyme s dng, vic loi b
hon ton protein c th t c bng phng php ho hc nhng khng th t
c bng phng php sinh hc . V vy, ngi ta c th kt hp hai phng php
ny nhm khc phc nhng nhc im ca tng phng php. Hin nay, mt trong
nhng kh khn trong phng php ho hc sn xut chitin l th tch cht thi
c cha cc cht n mn, cc cht l lng kh x l qu ln. Nhng cht ny l do
trong cng on kh khong v kh protein sinh ra. Chnh v vy, cn thit phi c
cc bin php x l trc khi thi ra mi trng v iu ny lm cho gi thnh sn
phm tng ln. Qu trnh sn xut chitin bng phng php ho hc c th gy nn
s thu phn polymer (Simpson v cng s, 1994; Healy v cng s, 1994), bin
i tnh cht vt l (Gagne v Simpson, 1993) v gy nhim mi trng (Allan v
14
cng s, 1978) [20]. iu ny l do khng xc nh c bn cht hot ng ca

ho cht cng nh s khc nhau v hm lng chitin trong nguyn liu. Ngc li,
trong phng php sinh hc th th tch cht khng ln, protein sau qu trnh thy
phn bng enzyme c th c thu hi lm bt dinh dng, thc n cho gia sc, gia
cm, cc cht khc nh lipid, cc sc t cng c thu hi. Hn na s hn ch
c vic x l mi trng. V vy, mun sn phm chitin c c s ng nht
hn v cc c tnh l ho th chng ta phi p dng nhng phng php x l nh
hn nh vic s dng enzyme.
Legarraeta v cng s (1996) s dng enzyme protease v vi khun c
kh nng to protease tch protein nhm thay th cho phng php ho hc. Qu
trnh ny gip tn dng ti a gi tr ca ngun ph liu v hn ch nh hng n
mi trng. Hall & De Silva (1994) xut mt phng php kh khong n
gin bng vic s dng ln men lactic nh l mt phng php bo qun ph liu.
Phng php ny l dng chua ban u c pht trin cho bo qun ph liu tm
pandan trc qu trnh ch bin kh hu nhit i.
1.3. TNH HNH NGHIN CU SN XUT CHITIN-CHITOSAN TRN TH
GII V VIT NAM
Vic nghin cu v dng tn ti, cu trc, tnh cht l ha ng dng ca
Chitosan c cng b t nhng nm 30 ca th k XX. Nhng nc thnh
cng trong lnh vc nghin cu sn xut chitosan l: Nht, M, Trung Quc, n
, Php.
Cho n nay trn th gii c nhiu quy trnh sn xut chitin-chitosan, vi
nhiu ngun nguyn liu khc nhau nhng ch yu l v tm, cua, gh nh:[5]
1.3.1. Sn xut chitin, chitosan theo phng php ha hc
- Quy trnh ca Stevens
15
Ph liu tm ti
Kh protein
(NaOH 4%, t = 24 gi,
to=30oC)
Cao hn 1%
Kim tra hm lng protein
Kh khong
(HCl 4%, t=24 gi, to=30oC)
Cao hn 1%
Kim tra hm lng khong
Chitin
Hnh 3.Quy trnh ca Stevens (2002) Hc Vin Cng Ngh Chu
16
Quy trnh sn xut Chitosan t v tm hm ca Hackman:

V tm hm
Ngm HCl
Ra trung tnh,sy kh, nghin mn
Ngm HCl
Li tm
HCl 2M
to phng
t = 5h
w/v= 1/10
HCl 2M
to phng
t = 48h
w/v = 1/2.5
Ra trung tnh
Ngm NaOH
NaOH 1M
to = 100oC
t= 42h
w/v = 1/2,5
Li tm
Ra trung tnh
Ngm NaOH
Li tm
Ra trung tnh
Ra sch bng li tm
Lm kh
Chitin dng bt mu kem
NaOH 1M
To = 100oC
T= 12h
w/v = 1/2,5
17
Hnh 4.Quy trnh sn xut chitosan t v tm hm ca Hackman

Nhn xt: Quy trnh ny gm nhiu cng on, thi gian sn xut ko di 65
gi nn ch c ngha trong cng tc nghin cu th nghim v khi a ra sn xut
i tr th thit b cng knh, tn km, ha cht t tin, d hao ht khi sn xut.
-
Quy trnh thy nhit ca Yamashaki v Nakamichi (Nht Bn):

V cua kh
Kh cht v c
HCl 2M
to=120oC
t= 1h
Ra trung tnh
Sy kh
Kh protein v chitin
NaOH 15M
to= 150oC
t = 1h
Ra trung tnh
Sy kh
Chitosan
Hnh 5.Quy trnh nhit ca Yamashaki v Nakamichi (Nht Bn)
Nhn xt: Quy trnh n gin ha cng on, rt ngn ng k thi gian
sn xut so vi cc quy trnh khc. Ha cht s dng t (HCl v NaOH), chitosan
thu c c tinh khit cao. Tuy nhin sn phm chitosan thu c c nht
thp do nhit x l cc cng on kh cao.
1.3.2. Sn xut chitin, chitosan theo phng php ha hc ci tin:
18
Quy trnh sn xut ca Php:

V tm
Hp chn, phi kh
Xay nh
Tch protein
NaOH 3,5%
to = 65oC
t = 2h
w/v = 1/10
Ra trung tnh
Ngm HCl
Ngm aceton
Ngm NaOCl
HCl 1N
to phng
t = 2h
w/v = 1/10
To phng
T = 30 pht
w/v = 1/5
NaOCl 0,135%
to phng
t = 6 pht
w/v = 1/10
Ra trung tnh
Deacetyl chitin
Ra trung tnh
Chitosan
Hnh 6.Quy trnh sn xut ca Php
NaOH 40%
to = 85oC
t = 4h
w/v = 1/4
19
u im: Quy trnh sn xut ny rt ngn c thi gian sn xut rt nhiu.

Sn phm thu c rt sch c mu trng p do kh c sc t trit .
Nhc im: Do NaOCl l mt cht oxy ha mnh nn nh hng n mch
polymer lm cho nht ca sn phm gim mt cch r rt. Mt khc, aceton rt
t tin, tn tht nhiu, gi thnh sn phm cao. Cha k n cc yu t an ton sn
xut cng ngh ny kh p dng trong iu kin sn xut ca nc ta hin nay.
Vic nghin cu sn xut Chitosan v cc ng dng ca chng trong sn xut
phc v i sng l mt vn tng i mi nc ta. Nm 1978 trng i hc
Thy sn bt u nghin cu tch chit Chitin-Chitosan do Minh Phng thc hin.
- Quy trnh ca Minh Phng, Trng i hc Thy sn:
Nhn xt: Sn xut chitosan theo quy trnh ny sn phm to thnh c cht
lng kh tt, chitin c mu sc p. Song thi gian cn di, s dng nhiu cht
oxy ha do d lm nh hng n nht ca sn phm.
Gn y, khi chitosan tr thnh nhu cu trong nhiu ngnh cng nghip v c
gi tr th rt nhiu c quan nghin cu nh: trng i hc Thy sn, Trung tm
nghin cu polymer Vin khoa hc Vit Nam, X nghip Thy sn H Ni, Trung
tm Cng ngh v sinh hc Thy sn Vin nghin cu mi trng Thy sn 2,
tp trung vo nghin cu v ng dng cng ngh ny. Trong , cc kt qu cng
b gn y ca cc nh khoa hc thuc trng i hc Thy sn Nha Trang i
su nghin cu hon thin quy trnh sn xut bc cao hn theo hng gim thiu
s dng ha cht trong x l, ng dng cng ngh enzyme. Nhng kt qu
gp phn p ng yu cu cp bch x l ph liu ca tm ng lnh v trc
nhng yu cu khc khe hn v cht lng ca chitin, chitosan trn th trng y
tim nng hin nay.
20
V tm kh
Ngm HCl
HCl 6N
to phng
t = 48h
w/v = 1/2,5
Ra trung tnh
Ngm NaOH
NaOH 8%
to = 100oC
t = 2h
w/v = 1/2,5
Ra trung tnh
Ty mu
Chitin
Nu trong NaOH
NaOH 40%
to = 80oC
t = 24h
w/v = 1/1
Ra trung tnh
Chitosan
Hnh 7.Quy trnh ca Minh Phng, trng i hc Thy Sn.
Nhn xt : Chitin thu c c trng cao mc d khng c cng on ty
mu. Tuy nhin, li c nhc im l thi gian sn xut ko di, tiu tn nhn cng,
nng ha cht s dng cao kt hp vi thi gian s l di (cng on kh
khong) lm ct mch polymer trong mi trng acid dn n nht gim.
21
Quy trnh sn xut chitosan Trung tm cao phn t thuc Vin khoa
hc Vit Nam.
Nguyn liu
Ngm HCl
HCl 4%
to phng
t = 24h
Ra trung tnh
Nu trong NaOH
NaOH 3%
to = 9095oC
t = 3h
Ra trung tnh
Ngm HCl
HCl 4%
to phng
t = 24h
Ra trung tnh
Nu trong NaOH
NaOH 3%
to = 9095oC
t = 3h
Ra trung tnh
Nu trong NaOH c
NaOH 40%
to = 9095oC
t = 3h
Chitosan
Hnh 8. Quy trnh sn xut Chitosan Trung tm cao phn t thuc Vin khoa
hc Vit Nam
Nhn xt: Sn phm chitosan sn xut theo quy trnh ny c mu sc khng
p bng sn phm theo quy trnh ca Minh Phng, thi gian thc hin li ko
di, nhiu cng on.
22
Quy trnh sn xut Chitosan t v tm S bng phng php ha hc

vi mt cng on x l kim (Trn Th Luyn)
V tm ti
V tm kh
Ngm HCl
HCl 10%
to phng
t =5h
w/v = 1/10
Ngm HCl
HCl 10%
to phng
t= 5h
w/v = 1/5
Ra trung tnh
Kh protein, lipid
NaOH 40%
to = 802oC
t = 5h
w/v = 1/10
Ra trung tnh
Chitosan
Hnh 9. Quy trnh sn xut Chitosan t v tm S bng phng php ha hc
vi mt cng on x l kim (Trn Th Luyn, 2003)
Nhn xt: phng php x l kim mt giai on cho sn phm chitosan c
cht lng khng thua km so vi quy trnh thng thng vi hai giai on x l
kim. tng thi gian cn thit gim rt nhiu v nh vy nu so v mt kinh t th
y l phng php tt hn hn. Tuy nhin phng php ny vn cn nhc im
l dung dch NaOH c sau khi deacetyl c mu sm gy kh khn cho vic ti s
dng. Theo quy trnh cng ngh ny sn phm chitosan dt c c cc ch tiu cht
lng nh bng 1
23
Bng 1. Mt s ch tiu cht lng ca chitosan t v tm s theo phng php

x l kim mt giai on (Trn Th Luyn, 2003)
Ch tiu
Mu sc
Trng thi
m
Hm lng tro
Hm lng cc cht
Kt qu
Trng p
Mm mi
10%
0,023%
1,6%
Ch tiu
Deacetyl
Tng thi gian thc hin
Nts
Hiu sut
tan
Kt qu
76,25%
9,5 gi
8,07%
40,25
98,32%
14,48oE
Phn ng biure
m tnh
khng tan
nht
-Quy trnh ca Trung tm Ch bin i hc Thy sn.

Quy trnh ca trung tm ch bin c u im l n gin, khng i hi my
mc thit b phc tp. Do rt d dng sn xut ln. Tuy nhin thi gian sn
xut ko di v nng ha cht s dng cn kh cao, vn cha c hng x l
nc thi t qu trnh sn xut.
24
Ph liu tm
Ra s b
Kh khong
HCL 7%
v/w=5/1
To phng
T=24h
Ngm ra trung tnh

Kh protein
NaOH 6%
v/w=5/1
To phng
T=24h
Ra trung tnh
Deacetyl ha
Ngm ra trung tnh
Phi, sy
Chitosan
NaOH 40%
v/w10/1
To=80oC
T=6,5h
Hnh 10. Quy trnh ca Trung tm Ch bin i hc Thy sn
25
1.3.3. Sn xut chitin, chitosan theo phng php s dng enzyme:

-Quy trnh sn xut chitin ca Holanda v Netto[27]
Ph liu tm
pH = 8,5
to = 55oC
t l E/NL 3%
w/v = 1/1
Kh protein bng enzyme

Alcalase
Ly tm (4oC, 15 pht)
Phn b
Phn dch ni pha trn
Chit Astaxanthin bng du

nnh v hn hp dung mi
Sy lnh
Kh khong HCl 2,5%, 2h, to

phng
Bt protein
Ra trung tnh
Sy kh (60oC, 16h)
Chitin
Hnh 11. Quy trnh sn xut chitin ca Holanda v Netto (2006)
u im: Quy trnh ny rt ngn c thi gian sn xut rt nhiu. Sn phm
Chitin thu c c cht lng kh tt, mu trng p do kh c sc t trong cng
on chit astaxanthin. Ngoi ra cn tn thu c protein v astaxanthin mang li hiu qu
kinh t cao, ng thi gim thiu ng k lng ha cht s dng.
26
Nhc im: Enzyme t tin dn n chi ph gi thnh sn phm cao.

-
Quy trnh s dng Enzyme papain sn xut chitosan (Trn Th

Luyn, 2003)[6]
V tm kh
Ngm HCl
V tm ti
HCl 10%
To phng
T = 5h
w/v = 1/10
Ngm HCl
Ra trung tnh
Kh protein
Ra sch
Lm kh
Chitin
Deacetyl
Ra trung tnh
Ra trung tnh
Chitosan
Hnh 12. Quy trnh s dng Enzyme papain sn xut chitosan

(Trn Th Luyn, 2003)
HCl 10%
To phng
T = 5h
w/v = 1/5
27
Bng 2. Mt s ch tiu cht lng ca chitosan sn xut theo quy trnh Papain
(Trn Th Luyn, 2003)
Ch tiu
Kt qu
Ch tiu
Kt qu
Mu sc
Trng, trong
nht
15,25oE
Trng thi
Mm mi
Deacetyl
78,25%
m
10,10%
Nts
8,25%
Hm lng tro
0,68%
Hiu sut
41,25%
Hm lng cc
0,92%
tan
98,37%
cht khng tan
Nhn xt : Quy trnh papain cho sn phm c nht cao hn cc quy trnh
khc. c bit deacetyl, tan v hiu sut ca quy trnh c u th hn hn.
Nhng nng cao cht lng ca chitosan c th s dng enzyme papain thay th
cho NaOH kh protein trong v tm. c bit dch thy phn thu c s dng
cho cc mc ch thu hi protein v tn dng, iu chc chn mang li hiu qu
cao. Tuy nhin, cn nghin cu quy trnh x l tn dng dch thy phn ny. Cn
tip tc nghin cu v sn xut enzyme deacetylase thay th hon ton cho
NaOH c trong cng on deacetyl.
Gn y ti Nghin cu tch chit Chitin t u-v tm bng cc
phng php sinh hc (S dng Bromelanin trong dch p v da) do tc gi
Nguyn Vn Thit v Ngc T thc hin nhm thu nhn chitin t ph liu u
v tm bng phng php cng ngh enzyme.[12][13]
Nguyn liu phc v th nghim bao gm: ph phm u v v tm kh (Nam
nh), ph phm sau khi mua v c sy li cho kh gin nhit 40 oC ri nghin nh
thnh bt thu nhn chitin. V da c thu mua ch. Nghin cu th nghim tch
chit chitin bng phng php ha hc. Kt qu cho thy, lng chitin trung bnh
thu c t 100g nguyn liu bt u v tm kh l 7,4-7,5 g. Kt qu ny thp
hn nhiu so vi cc s liu cng b trong cc ti liu khc. Nguyn nhn l do
nhm nghin cu s dng u v tm cha nhiu tht. Hiu sut thu hi chitin
ph thuc vo mu nguyn liu cha nhiu hay t protein.
Tip tc tch chit chitin nh s dng enzyme bromelanin, tc gi cho rng,
vic x l bt v tm vi dch p b da khng ch loi c phn ln lng
28
protein ca v tm m cn loi c ht cc cht khong trong v tm. Dch p b

da c nhiu axit hu c c phn ng vi cc cht khong trong v tm. Phng
php ny t ra c nhiu u im nh: khng cn axit loi khong, tiu tn t xt
cho loi protein, hiu qu thu hi chitin cao hn, cht lng ph phm chitin nhn
tt hn v t gy nhim mi trng hn. Nhc im duy nht ca phng php
l mt nhiu thi gian thc hin quy trnh.
29
PHN 2. NGUYN VT LIU V PHNG PHP

NGHIN CU
2.1. NGUYN VT LIU NGHIN CU
2.1.1. Nguyn liu u tm
u tm th chn trng (Penaeus vannamei) c chn l i tng nghin
cu. u tm c ly t nguyn liu tm Th chn trng ch bin ti Cng ty C
phn Nha Trang Seafoods (F17), Khnh Ha. Nguyn liu sau khi ly c vn
chuyn ngay bng thng xp cch nhit c nhit < 5 oC v phng th nghim.
Nguyn liu trc khi s dng c ra sch, ro trong thi gian 5 pht. Trong
trng hp cha lm ngay th ra sch bao gi bo qun ng iu kin
nhit -20oC ti phng th nghim Trung tm Cng ngh sinh hc v Mi trng.
2.1.2. Enzyme Protease[15]
ti s dng enzyme Alcalase c mua ti cng ty Novozyme, Tp. H
Ch Minh. Enzyme Alcalase 2.4 FG thu c t Bacillus licheniformis. y l
enzyme c phn tch v tinh sch t ngun vi sinh vt. S dng enzyme Alcalase
cho php iu chnh d dng thy phn, tnh ton c lng base yu cu
duy tr pH khng i trong sut qu trnh thy phn. Chn enzyme ny cng da
trn c trng ca n cho kh nng khng ht nc ca cc amino acid vo giai
on cui, dn n sn phm thy phn khng c v ng (Adler-Nissen, 1986),
ng thi sn phm c s cn bng tt cc amino acid thit yu (Kristinsson v
Rasco, 2000).
Hot tnh 2,4 AU-A/g.
Nhit bo qun tt nht l 010oC.
iu kin hot ng ti u cho enzyme Alcalase AF 2.4 L: pH = 8
Nhit : 50 60 oC (122 1400F)
DH (%) ti a 15 25.
kim sot c tnh chc nng ca sn phm thy phn th nn dng phn
ng enzyme gn vi DH % xc nh. Tt c protease c th b bt hot bng cch
30
x l nhit 850C, thi gian 10 pht hoc protease Alcalase b bt hot ti pH = 4

hay thp hn trong khong 30 pht. Phn ng c th dng tc thi bng cch thm
vo cc acid thch hp nh: acid hydrochloric, phosphoric, malic, lactic, acetic .
Tng nhit tc thi kh t c di cc iu kin cng nghip v n c th
kh kim sot DH % do s thy phn vn tip tc trong sut giai on bt hot.
2.1.3. Acid benzoic (C6H5COOH)
Axit benzoic, C7H6O2 (or C6H5COOH), l mt cht rn tinh th khng mu
v l dng carboxylic acid aromatic n gin nht. Tn ca n c ly theo gum
benzoin, l mt ngun iu ch axid benzoic. Acid yu ny v cc mui ca n
c s dng lm cht bo qun thc phm. y l mt cht ban u quan trng
tng hp nhiu cht hu c khc. Acid benzoic c pht hin vo th k 16.
Vic chng ct kh gum benzoin c m t ln u tin bi Nostradamus
(1556), v sau l bi Alexius Pedemontanus (1560) v Blaise de Vigenre
(1596). Justus von Liebig v Friedrich Whler xc nh cu trc ca acid benzoic
vo nm 1832. H cng nghin cu quan h gia acid hippuric v acid benzoic.
Nm 1875, Salkowski pht hin ra kh nng khng nm ca acid benzoic, do
n c s dng bo qun mt s loi tri cy ...
CTPT: C6H5COOH
Phn t gam : 122,12 g/mol
B ngoi : Cht tinh th rn, khng mu
T trng : 1,32g/cm2, solid
im nng chy: 122,4oC (395oK)
si : 249oC (522oK)
ha tan trong nc: Tan c ( nc nng )(3,4g/l 25oC)
ha tan trong methalnol, diethylether : tan c
acid (pKa): 4,21
2.1.4. Nguyn liu khc
NaOH tinh th dng phn tch.
HCl dng phn tch
31
2.2. PHNG PHP NGHIN CU

2.2.1. Phng php thu nhn mu
Nguyn liu u tm c thu ti phn xng ch bin, Cng ty C phn
Nha Trang Seafoods (F17). Yu cu NL phi ti, khng c mi l, khng b bin
, khng ln tp cht. NL sau khi ly cho ngay vo thng xp cch nhit c cha
nc v vn chuyn ngay v phng th nghim. NL c ra sch v xay nhuyn
(trong c 100% l u tm , tt c cc mu th nghim u c xay bi cng
mt thit b v kch thc mu sau khi xay l 0,5 0,8 cm) v tin hnh lm th
nghim ngay. Trong trng hp cha lm ngay th ra sch, cn cho vo ti polyme
( mi ti 1 kg), bo qun ng iu kin nhit -20oC.
2.2.2.
B tr th nghim xc nh cc thng s k thut
Phng php b tr th nghim: dng phng php qui hoch thc nghim
S b tr th nghim tng qut
M t quy trnh
Nguyn liu v u tm c xay nh v kh protein bng enzyme protease
(Alcalase) trong mi trng c pH v t l E/S theo t l nghin cu. Tin hnh
thu phn protein chn ra thi gian, t l E/S, nhit thch hp. Sau khi tm
c ch ti u ta tin hnh thu phn ri lc tch ring phn dch v phn b.
Phn dch tn dng thu protein v asthaxanthin dng sn xut thc n chn nui.
Phn b c ra sch, phi kh, tip tc kh protein bng NaOH, kh khong
bng C6H5COOH v HCl, mi cng on cng tin hnh thc hin chn c
ch ti u (nng , thi gian x l) sau ra sch v thu c Chitin.
Quy trnh d kin sn xut chitin t ph liu tm:
32
u tm
Xay nh
Tch protein bng enzyme Alcalase
T l E/NL:[0,1-0,5%]
Nhit : [40-60oC]
Thi gian:[4-10h]
Dch protein
Kh protein bng NaOH
Kh khong bng acid benzoic

Ra
Kh khong bng HCl

Ra
Chitin
Hnh 13.Quy trnh d kin sn xut.
Ra
CM = 13%
Thi gian: 810h
Nhit phng
CM= 0,0120,018M
Thi gian: 410h
Nhit phng
CM=0,60,8M
Thi gian: 410h
Nhit phng
33
Sau khi tm c cc thng s ti u ca cc cng on, ta tin hnh sn

xut Chitin theo cc thng s ti u ca cng on kh protein bng enzyme v
NaOH, cn cng on kh khong ta ch dng HCl, khng dng C6H5COOH.
Theo Nguyn Vn Ton v cng s nghin cu [22], cng on kh
khong ch dng HCl 1,1M; ngm trong 12 gi 30 oC c p dng i vi ph
liu u v tm, kt qu hm lng khong cn li trong Chitin l 1,110,01%.
y i tng nguyn liu c nghin cu l ph liu u tm, u tm thng th
hm lng khong cao hn cho nn em d kin quy trnh i chng vi nng
HCl l 1,2M; v ngm trong 16 gi nhit phng. Quy trnh d kin sn xut
i chng nh sau:
u tm
Xay nh
Tch protein bng enzyme

Alcalase
B tch protein
Kh protein bng NaOH
Kh khong bng HCl
T l E/NL:0,42%
Nhit : 56oC
Thi gian:8,8 gi
Dch protein
Nng :2,4%
Thi gian:12,2 gi.
Nhit phng
Nng :1,2M
Thi gian:16 gi
Nhit phng
Chitin
Hnh 14.Quy trnh sn xut Chitin khng dng acid benzoic.(i chng)
34
2.2.2.1. B tr th nghim xy dng ng chun ca phng php

Microbiuret v phng php Biuret
Xy dng ng chun cho phng php Microbiuret:
Ha tan 0.1 gam huyt thanh b (BSA) trong NaOH 3% to thnh cc nng
khc nhau sao cho nng protein trong dch thuc khong t 0.05-0.5g/l.
Chun b 7 ng nghim sch, nh s th t t 1 n 7, sau cho cc dung
dch ha cht vo cc ng nghim vi th tch v th t nh sau:
Bng 3.B tr th nghim chy ng chun ca phng php Microbiuret
ng nghim
Nng
S ml dch BSA(ml)
S ml NaOH 3%(ml)
0,05
0,2
3,8
0,1
0,4
3,6
0,2
0,8
3,2
0,3
1,2
2,8
0,4
1,6
2,4
0,5
Sau ly mi mu 4ml, thm vo 200l thuc th Microbiuret,vortex

cho u v sau 15 pht mang i o bc sng 330nm. (S dng ng 0 lm mu
blank).
Xy dng ng chun cho phng php Biuret:
Pha dung dch chun BSA: Ha tan 0,5 gam BSA vo trong 50ml nc ct,
khuy dn dn khi BSA tan ht ri nh mc ln 100ml bng nc ct (Hm lng
BSA: 10mg/ml).
Xy dng ng chun: Chun b 6 ng nghim sch, nh s th t t 0-5 sau
cho cc dung dch ha cht vo cc ng nghim vi th tch v th t nh sau:
Bng 4. B tr th nghim chy ng chun ca phng php Biuret
35
Dung dch ha cht
ng nghim
0
BSA chun (ml)
0,2
0,4
0,6
0,8
Nc ct (ml)
0,8
0,6
0,4
0,2
Thuc th Biuret (ml)
Hm lng BSA(mg/ml)
10
Sau khi cho y cc ha cht, cc ng nghim trn nhit phng

trong thi gian 30 pht.
Sau khi , dung dch trong cc ng nghim trn c o mt quang hc
bc sng 570nm c gi tr OD570 (S dng ng 0 lm mu blank)
2.2.2.2. B tr th nghim xc nh thnh phn ha hc ca ph liu u tm
th chn trng
Xc nh hm lng protein theo phng php Biuret.
Xc nh hm lng khong bng phng php nung 600oC
2.2.2.3. Th nghim xc nh ch kh protein ti u
a. Kh ln 1: dng enzyme alcalase
Th nghim c b tr theo phng php quy hoch thc nghim [8][9]
Mc ch ca qu trnh ti u l chn mt ch cng ngh thch hp cho
qu trnh loi b protein t u tm bng enzyme Alcalase sao cho hm lng
protein kh c l tt nht.
Phn tch nh hng ca cc yu t n qu trnh thy phn, qua cc th
nghim thm d v k tha kt qu nghin cu trc y, ti chn cc yu t c
nh nh sau:
- pH = 8
- T l nguyn liu / nc l 1:1
- Cn li 3 thng s: t l enzyme b sung, nhit v thi gian thy phn nh
hng n hiu sut kh protein. ti nghin cu quy hoch thc nghim phn tch hi
36
quy 3 yu t, sau ti u ha qu trnh bng phng php ng dc nht tm

gi tr ti u.
Cc thng s cn ti u l:
- Nng enzyme trong khong: [0,1%-0,5%]
- Nhit thy phn trong khong: [40oC-60oC]
- Thi gian thy phn trong khong: [4h-10h]
Hm mc tiu l: Hm lng protein cn li trong mu sau khi thy phn
(Y) (% theo vt cht kh) Min.
on kh protein bng enzyme Alcalase.
N U1(nng %)
U2(nhit
)
Y(%)
U3(thi gian)
X0
X1
X2
X3
0,1
40
-1
-1
-1
0,5
40
-1
-1
0,1
60
-1
-1
0,5
60
-1
0,1
40
10
-1
-1
0,5
40
10
-1
0,1
60
10
-1
0,5
60
10

enzyme Alcalase
STT
U1
U2
U3
0,3
50
10
0,3
50
0,3
50
U1: nng enzyme (%)
11
U2: nhit thy phn (oC)
Y(%)
37
U3: thi gian thy phn (gi)

b. Kh ln 2 dng NaOH:
Cc th nghim ca mu kh protein bng NaOH theo phng php quy
hoch thc nghim:
Kh protein bng xt long l phng php s dng xt nng thp thy
phn cc lin kt peptit ca protein khng ha tan to thnh cc dng peptit, pepton, acid
amin ha tan trong nc v d dng loi b ra khi nguyn liu. Nh trn ni protein
trong ph liu tm tn ti di hai dng l dng t do thng l phn tht trong u,
chn, ui tm v phn protein lin kt vi chitin v cc thnh phn khc v vy mun
loi b protein ra khi nguyn liu phi s dng cc tc nhn ha hc hoc sinh hc
thy phn protein. Tuy nhin, phng php sinh hc khng th loi b protein mt cch
trit c, do ta phi kt hp c 2 phng php sinh hc v ha hc m bo
hm lng protein cn li trong Chitin l < 1%.
Phng trnh phn ng biu din qu trnh kh protein nh sau:
NaOH
H2N-CH-CO-NH-CH-CO-
H2N-CH-CO-NH-CH- + H2N-CH-COOH
to cao
R1
R2
R1
Polypeptid
Peptid
R2
R3
Acid amin
Qu trnh kh protein thng km theo qu trnh kh lipid do lipid c th phn ng

vi kim to thnh x phng theo phng trnh phn ng sau:
CH2OCOR1
CH2OH
CHOCOR2 + 3NaOH
CHOH + R1COONa + R2COONa + R3COONa
CH2OCOR3
CH2OH
38
Triaxylglycerol
Glycerol X phng
Thng thng hm lng ca lipid trong nguyn liu khng cao do vy

khng cn nghin cu loi b lipid trong cng ngh sn xut chitin-chitosan v phn
nh lipid b thy phn theo phn ng trn.
Cc yu t c nh:
- Nhit : 40oC
- T l mu em i x l NaOH/dung dch NaOH l: 1/10(w/v)
- Nng NaOH trong khong: [0,1%-0,3%]
- Thi gian x l NaOH trong khong: [8h-10h]
Hm mc tiu l: Hm lng protein cn li trong mu sau khi thy phn
on kh protein bng NaOH
N
U1(Nng
NaOH(%))
U2 (Thi
gian (h))
X0
X1
X2
0,1
-1
-1
0,3
-1
0,1
10
-1
0,3
10
Y(%)
Bng 8. B tr th nghim tm phng n ca cng on kh protein bng NaOH.

STT
U1
U2
0,2
11
0,2
11
0,2
11
U1 : Nng NaOH(%)
U2: Thi gian x l NaOH(gi)
Y(%)
39
2.3.4.3.
Th nghim xc nh ch kh khong
a. Kh ln 1: Dng acid benzoic

Cc th nghim ca mu kh khong bng acid benzoic theo phng
php quy hoch thc nghim
Mc ch ca cng on ny l nghin cu kh nng kh khong ca axt
benzoic trn nguyn liu l u tm c kh protein bng enzym Alcalaza v
NaOH iu kin ti u.
Cng on kh khong c thc hin bng cch ngm ph liu tm trong
dung dch acid benzoic. Cc thng s ti u c xc nh bng phng php quy
hoch thc nghim v cc tnh ton c thc hin trn phn mm MS.Excel 2003.
Theo phng php quy hoch thc nghim (c trnh by phn ph lc)
xc nh c cc thng s ti u cho cng on kh khong nh sau:
Cc yu t c nh:
- Nhit : nhit phng
- T l mu em i kh khong/dung dch acid benzoic =1/15(w/v)
- Nng C6H5COOH trong khong: [0,012M- 0,018M]
- Thi gian x l C6H5COOH trong khong: [4h-10h]
Hm mc tiu l: Hm lng khong cn li trong mu sau khi kh khong
on kh khong bng C6H5COOH
N
U1 (nng
U2 (thi gian (h))
C6H5COOH (M))
X0
X1
X2
0,012
-1
-1
0,018
-1
0,012
10
-1
0,018
10
Y(%)
40

C6H5COOH
STT
U1
U2
0,015
0,015
0,015
Y(%)
U1 : Nng C6H5COOH(M)
U2: Thi gian x l ngm C6H5COOH (gi)
b. Kh ln 2: Dng acid chlohydric
Cc th nghim ca mu kh khong bng HCl theo phng php quy
hoch thc nghim:
Sau khi kh protein bng enzyme v NaOH th % hm lng khong cn li rt
ln, dng acid benzoic khng th loi b khong mt cch trit c, do ta phi
kt hp s dng c acid h c kt hp acid v c m bo hm lng khong cn li
trong Chitin l < 1%.
Mc ch ca cng on ny l nghin cu kh nng kh khong ca axt
chlohydric trn nguyn liu l u tm c kh protein bng enzym Alcalaza
v NaOH v kh khong mt phn bng acid benzoic iu kin ti u.
Cng on kh khong c thc hin bng cch ngm ph liu tm trong
dung dch HCl. Cc thng s ti u c xc nh bng phng php quy hoch
thc nghim v cc tnh ton c thc hin trn phn mm MS.Excel 2003.
Theo phng php quy hoch thc nghim (c trnh by phn ph lc) xc
nh c cc thng s ti u cho cng on kh khong nh sau:
Cc yu t c nh:
- Nhit : nhit phng
- T l mu em i kh khong/dung dch HCl =1/10(w/v)
41
- Nng HCl trong khong: [0,6M- 0,8M]

- Thi gian x l HCl trong khong: [4h-10h]
Hm mc tiu l: Hm lng khong cn li trong mu sau khi kh khong (Y) (%
theo vt cht kh) Min.
on kh khong bng HCl.
N
U1(Nng
HCl(M))
U2 (Thi
gian (h))
X0
X1
X2
0,6
-1
-1
0,8
-1
0,6
10
-1
0,8
10
Y(%)
Bng 12. B tr th nghim tm phng n ca cng on kh khong bng HCl

STT
U1
U2
0,7
0,7
7
0,7
U1 : Nng HCl (M)
Y(%)
U2: Thi gian x l ngm HCl (gi)

2.3.
CC PHNG PHP PHN TCH HA HC:[1]
2.3.1. Xc nh hm lng m bng phng php sy nhit 105 oC theo

TCVN 3700-1990.
2.3.2. Xc nh hm lng khong bng phng php nung 600oC.
2.3.3. Xc nh hm lng protein theo phng php Microbiuret:
Nguyn l: Trong mi trng kim, protein kt hp vi Cu++ thnh mt phc
cht mu tm (phn ng biuret). Mu sc ca phc cht t l vi s liu peptid (CO-NH) ca protein v gn nh khng ph thuc vo nng tng i gia
albumin v globulin.
42
Hnh 15.Cng thc ca phc Biuret.

2.3.3.1. Dng c
-Dng c thy tinh(ng nghim, cc thy tinh, ng ong, bnh nh mc,
bnh tam gic, pipette), vt liu thng thng ca phng th nghim
- Micropipet (100-1000l)
-Thit b o UV-Vis mini1240
2.3.3.2. Ha cht
- NaOH
- Na2CO3 (Sodium Cabonate)
- CuSO4.5H2O
- Na3C6H5O7.2H2O (Sodium citrate)
2.3.3.3. Tin hnh
Pha dung dch thuc th Microbiuret:
Ly 173g Sodium citrate v 100g Sodium carbonate, em ha tan trong
nc nng nhng khng nc si.
Ly 17,3g CuSO4 ha tan trong 100ml nc ct v thm vo hn hp trn.
Sau thm nc ct vo cho 1 lt.
Xy dng ng chun: c b tr nh ni phn b tr th nghim
X l mu:
43
Ly 1g mu + 20ml NaOH 3% 80oC,trong 6 gi. Sau , hn hp ny

c lm lnh nhit phng, ri sau mang i lc. Khi lc, dng t 10-15ml
NaOH 3% ra b.
Sau em dch i li tm 5000 vng/15 pht Ly dch Pha long sao
cho hm lng protein nm trong di bc sng.
Ly 4ml + 200l Microbiuret o bc sng 330nm.
Kt qu o c tng ng vi gi tr y (OD330nm), da vo phng trnh
ng chun tnh ra c hm lng protein c trong 1 ml dch. T tnh ra c
phn trm protein c trong mu theo cng thc tnh sau:
% protein
V .C.100
m.1000.(100 Mc) / 100
V: Th tch mu sau khi pha long(ml)

C: Hm lng protein trong 1 ml(mg/ml)
m: Khi lng mu em i (gam)
Mc: m ca mu em i phn tch (%)
2.3.4. Xc nh hm lng Protein theo phng php Biuret:
2.3.4.1. Dng c: ging nh phng php Microbiuret.
2.3.4.2. Ha cht:
NaOH
CuSO4.5H2O
C4H4NaKO6.4H2O (Kali Natri tartrat)
BSA (Bovine serum albumin)
2.3.4.3.
Tin hnh:
Pha thuc th Biuret: Ha tan 2,35375 gam CuSO4.5H2O v 8,0571 gam
C4H4NaKO6.4H2O trong 500ml nc ct, khuy u v cho thm 300ml NaOH

10%. Dung dch trn c khuy trn u ri lm y n 1000ml bng nc ct .
Xy dng ng chun: c b tr nh ni phn b tr th nghim.
X l mu:
Cch 1: Ngm 3-5gam nguyn liu kh hoc 3-10 gam nguyn liu t trong
NaOH 4% vi t l 1:10 ti 1:15w/v. Sau em 95oC trong 6 gi.
44
Cch 2: X l mu ging nh trn nhng khng 95 oC trong 6 gi m

nhit phng 12 gi, sau nng nhit ln 95oC trong 1 gi.
Sau khi : i vi dch thy phn thu c, nu khng 100ml th thm
nc ct vo cho 100ml. Ly khong 10ml dch thy phn em i lc chn
khng bng giy lc Whatman Filter paper.
Phn dch lc c em i phn tch hm lng protein bng thit b o UVVis mini1240.
2.3.5. Phng php x l s liu: Cc s liu c x l theo phn mm Excel
2003. Mi th nghim b tr song song 3 th nghim, mi th nghim lp li 3 ln,
ly kt qu trung bnh cng 3 ln th nghim.
PHN 3. KT QU NGHIN CU V THO LUN
45
3.1.
Kt qu ng chun xy dng c theo phng php Microbiuret v

phng php Biuret:
3.1.1. Kt qu xy dng ng chun ca phng php Microbiuret :

Kt qu o c ( ph lc)
Phng trnh ng chun.
Hnh 16. Phng trnh ng chun ca phng php Microbiuret

3.1.2. Kt qu xy dng ng chun ca phng php Biuret:
Kt qu o mt quang hc ca cc mu ng chun ( ph lc)
46
Hnh 17. Phng trnh ng chun ca phng php Biuret.

3.2. Thnh phn ha hc ca ph liu tm th:
Kt qu phn tch thnh phn ha hc ca ph liu tm th chn trng c
trnh by ti bng 3.1.
Bng 13. Thnh phn ha hc ca ph liu tm th chn trng (%)
Ch tiu
Protein
Lipid
Tro
Chitin
Thnh phn
ha hc (%)
49
4,7
25,2
18,3
(Tnh theo vt cht kh)

Kt qu phn tch cho thy tm th chn trng (Penaeus vannamei) c hm
lng protein trong ph liu tm kh cao. Kt qu ny cho thy cn s dng
phng php sinh hc thy phn protein tn dng lng protein c cht
lng vo vic ch bin thc n gia sc, ng thi nghin cu hon thin quy
trnh sn xut chitin va c hiu qu kinh t, va mang ngha mi trng.Hm
lng khong trong u tm cng kh cao, hm lng lipid v hm lng Chitin
c tham kho.[13]
47
3.3.
Kt qu nghin cu cng on kh protein bng enzyme Alcalase:

Cng on kh protein ny c thc hin bng cch b sung enzyme
Alcalase thy phn ph liu tm. Cc thng s ti u c xc nh bng

phng php quy hoch thc nghim v cc tnh ton c thc hin trn phn
mm MS.Excel 2003.
xc nh c cc thng s ti u cho cng on kh protein nh sau:
Bng 14. Kt qu hm lng protein cn li cc ch kh protein bng
enzyme Alcalase
N U1(nng %)
U2(nhit
)
U3(thi gian) X0 X1 X2 X3
Y(%) lng
protein cn li
0,1
40
-1 -1 -1
19,6183
0,5
40
1 -1 -1
11,8941
0,1
60
-1 1 -1
13,023
0,5
60
1 -1
14,233
0,1
40
10
-1 -1 1
11,5383
0,5
40
10
1 -1 1
10,5916
0,1
60
10
-1 1
9,9975
0,5
60
10
9,3423

enzyme Alcalase
N0
U1
U2
U3
Y(%)
0,3
50
12,67
10
0,3
50
12,7970
11
0,3
50
13,8167
Phng trnh hi quy ca hm lng protein xc nh c nh sau:

Y= 12,5298 1,0145*X1 0,8808*X2 2,1623*X3
(1)
48
Tho lun: Phng trnh (1) tng thch vi thc nghim :qa trnh thy
phn kh protein ca ph liu tm bng enzyme ph thuc vo nhiu yu t nh:
nng enzyme, nhit , thi gian Qua phng trnh trn ta thy quy lut bin
i ca hm lng protein l khi tng nng enzyme (X1), nhit thy phn
(X2) v thi gian kh protein (X3) th hm lng protein cn li trong ph liu tm
(Y) gim xung, nhng thi gian thy phn v nng enzyme nh hng ti hm
lng protein nhiu hn so vi nhit thy phn. T phng trnh (1) nu mun
kh protein mt cch trit ta phi tng nng enzyme, thi gian kh protein v
nhit thy phn; song vic tng cao nng enzyme, nhit thy phn v ko
di thi gian kh protein nh th no cho hp l tit kim c thi gian v chi
ph sn xut m vn m bo c lng protein cn li trong ph liu tm l t
nht. Nh vy xut pht t mc c s, tin hnh lm th nghim ti u theo ng
dc nht v thu c kt qu cc th nghim tip theo bng 16
Bng 16. Kt qu hm lng protein cc th nghim ti u theo ng dc nht
Bng kt qu th nghim ti u ha
Tn
U1(nng %)
U2(nhit )
U3(thi gian)
Y(%)
Mc c s
0,3
50
Bc lm trn
0,04
0,6
Th nghim 12
0,34
52
7,6
9,451
Th nghim 13
0,38
54
8,2
9,2528
Th nghim 14
0,42
56
8,8
8,0102
Th nghim 15
0,46
58
9,4
8,655
T bng th nghim trn ta nhn thy th nghim th 14 hm lng protein

cn li trong ph liu tm l thp nht.
49
Hnh 18. Kt qu nghin cu ti u ha ch thy phn bng enzyme theo

phng php ng ln dc ca BoxWillson.
Nhn xt v tho lun: B tr th nghim theo phng php quy hoch thc
nghim ton hc cho php dn ti im ti u nht. Kt qu nghin cu c ngha
ton hc cao vi tin cy l 95%. im ti u t c th nghim th 14 vi nng
enzyme l 0,42%, nhit 56oC v ng vi thi gian tng ng l 8,8 gi.
3.4.
Kt qu nghin cu cng on kh protein bng NaOH:

Cng on kh protein c thc hin bng cch ngm ph liu tm trong dung
dch acid NaOH long. Cc thng s ti u c xc nh bng phng php quy hoch
thc nghim v cc tnh ton c thc hin trn phn mm MS.Excel 2003.
xc nh c cc thng s ti u cho cng on kh protein nh sau.
Bng 17. Kt qu hm lng protein cc ch kh protein bng NaOH
U1 (nng %) U2 (thi gian)
X0
X1
X2
Y (%) lng
protein cn li
-1
-1
1,78
-1
1,04
14
-1
1,34
14
0,82
50
Bng 18. Th nghim tm phng n ca cng on kh protein bng NaOH

N0
U1
U2
Y(%)
11
1,1214
10
11
1,0764
11
11
1,1319
Phng trnh hi quy ca hm lng protein xc nh c nh sau :

Y= 1,245 0,315*X1 0,165*X2 (2)
Tho lun: Phng trnh (2) tng thch vi thc nghim v qua phng
trnh trn ta thy quy lut bin i ca hm lng protein l khi tng nng dung
dch NaOH (X1) v thi gian kh protein (X2) th hm lng protein trong ph liu
tm (Y) gim xung, nhng nng NaOH nh hng ti hm lng protein nhiu
hn. T phng trnh (2) nu mun kh protein mt cch trit ta phi tng nng
NaOH v thi gian kh protein; song vic tng cao nng NaOH v ko di
thi gian kh protein s gy nh hng ln cu trc ca chitin-chitosan lm ct
mch polymer t lm gim nht dung dch chitosan. Nh vy xut pht t
mc c s, tin hnh lm th nghim ti u theo ng dc nht v thu c kt
qu cc th nghim tip theo bng 19
Bng 19. Kt qu hm lng protein cc th nghim ti u theo ng dc nht
Tn
U1 (nng %)
U2 (thi gian)
0,2
11
Bc lm trn
0,2
0,6
Th nghim 9
2,2
11,6
1,08
Th nghim 10
2,4
12,2
0,96
Th nghim 11
2,6
12,8
0,91
Th nghim 12
2,8
13,4
0,86
Mc c s
Bc chuyn ng
Y(%)
51
T bng th nghim trn ta nhn thy th nghim th 10 hm lng protein

trong chitin tng i thp tng ng vi nng NaOH l 2,4%, 12,2 gi. Nu
nng cao nng protein v thi gian x l kh protein th hm lng gim khng
ng k. Mt khc, tng nng NaOH lm tng chi ph sn xut v gim nht
chitosan. Hm lng protein trong chitin nh hng ln n cht lng chitin v
protein lm gim tan ca chitin trong acid HCl c v nh hng ti cht lng
ca cc ch phm c sn xut t chitin nh: Glucosamin, chitosan
Khi hm lng protein trong chitin cao th trong cng on deacetyl ta phi
tng hm lng NaOH v ko di thi gian deacetyl kh ht protein v lm tng
deacetyl. Nhng nng NaOH cng cao v thi gian x l kh protein cng di
th d gy ct mch glucoside t lm gim nht dung dch chitosan. V vy
em quyt nh chn cc thng s ti u cho cng on x l kh protein nh trn.
T cc th nhim phn ti u ho cng on kh protein theo quy hoch
thc nghim ta v c th sau:
Hnh 19. Kt qu nghin cu ti u ha ch ngm NaOH theo phng php

ng ln dc ca BoxWillson
52
Nhn xt:
Qua th trn ta thy khi nng NaOH thp th d thi gian c ko di th
hm lng protein trong chitin cng rt cao (1,34%). Khi nng NaOH tng dn
v thi gian kh protein tng dn th hm lng protein gim dn. nng NaOH
2,4% protein trong mu <1% l hm lng protein t tiu chun ca chitinchitosan hin nay. nng NaOH 2,4% cho hm lng protein tng i thp
(0,96%), nng NaOH 2,6% v 2,8% hm lng protein cn li trong ph liu
tm thp hn 0,96% nhng nng NaOH cao c nh hng n cht lng ca
Chitin-Chitosan sau ny, mc khc cn tn thm chi ph. V vy c th chn nng
NaOH=2,4% trong thi gian 12,2 gi lm thng s ti u cho cng on kh
protein.
Vy thng s ti u cho cng on kh protein l: Nng NaOH=2,4%,
thi gian x l 12,2 gi, t l w/v=1/10, nhit (401 oC).
3.5.
Kt qu nghin cu cng on kh khong bng acid benzoic:

Ph liu tm qua khu x l enzyme v NaOH c thnh phn ho hc
c trnh by bng 20.

Bng 20. Thnh phn ha hc ca ph liu tm th Chn Trng sau khi kh l
kh protein bng enzyme Alcalase v NaOH iu kin ti u
STT
1
2
3
Thnh phn
Hm lng (%) trong ph liu tm
Protein
0,96
Khong
62,4
m
13,86
T bng 20 ta thy sau khi x l protein bng enzyme Alcalase v NaOH
hm lng protein gim ng k (hiu qu kh protein 98%). Nhng hm lng

khong cn rt nhiu v mi trng kh protein bng enzyme l mi trng kim
(pH=7,2). Mt phn khong c tch ra do phn protein lin kt vi khong c
tch ra. Nhng thnh phn phn trn theo khi lng ca hm lng khong tng
do hm lng protein trong ph liu gim
53
Cng on kh khong c thc hin bng cch ngm ph liu tm trong dung
dch acid benzoic. Cc thng s ti u c xc nh bng phng php quy hoch thc
nghim v cc tnh ton c thc hin trn phn mm MS.Excel 2003.
xc nh c cc thng s ti u cho cng on kh khong nh sau.
kh khong bng C6H5COOH
U2
U1 (nng
N
X0
X1
X2
Y(%)
C6H5COOH (M)) (thi gian (h))
1
0,012
-1
-1
35,21
0,018
-1
34,38
0,012
10
-1
25,09
0,018
10
18,57
Bng 22. Th nghim tm phng n ca cng on kh khong bng C6H5COOH

N0
U1
U2
Y(%)
0,015
26,05
10
0,015
25,14
11
0,015
26,23
Phng trnh hi quy ca hm lng khong xc nh c nh sau:

Y= 26.3125 - 1.8375*X1 - 6.4825*X2 (3)
Quy lut bin i ca hm lng khong l khi tng nng dung dch acid
benzoic (X1), tng thi gian kh khong (X2) th hm lng khong trong ph liu
tm (Y) gim xung, nhng thi gian ngm acid benzoic nh hng ti hm lng
khong nhiu hn . T phng trnh (3) nu mun kh khong mt cch trit ta
phi tng nng acid benzoic, thi gian kh khong; song vic tng cao nng
acid benzoic, ko di thi gian kh khong s gy nh hng ln cu trc ca
chitin-chitosan lm ct mch polymer t lm gim nht dung dch chitosan.
54
Nh vy xut pht t mc c s em tin hnh cc th nghim ti u theo ng dc

nht.
Tn
U1(nng M)
U2(thi gian)
Mc c s
0,015
7
H s bj
-1,8375
-6,4825
Bc chuyn ng
3
3
Bc lm trn
0,0006
0,6
Th nghim 9
0,0156
7,60
Th nghim 10
0,0162
8,20
Th nghim 11
0,0168
8,80
Th nghim 12
0,0174
9,4
Y(%)
25,01
22,29
20,83
19,02
Hnh 20. Kt qu nghin cu ti u ha ch ngm C6H5COOH theo phng

php ng ln dc ca BoxWillson.
Nhn xt: T cc th nghim v nhn vo th, nhn thy khi tng nng
acid benzoic, thi gian kh khong th hm lng khong gim dn, khi nng
C6H5COOH 0,0168M, thi gian ngm 8,8 gi th hm lng khong cn li l
20,83%. Khi tip tc tng nng benzoic v thi gian kh khong th hm lng
khong gim khng ng k. Do vy nu chitin-chitosan yu cu hm lng khong
thp v nht cao th ta c th chn cc thng s ti u cho qu trnh kh khong
55
gim c nng C6H5COOH v thi gian x l C6H5COOH sau ny nhm

m bo c cht lng ca Chitin-Chitosan mt cch tt nht. Ta c th chn cc
thng s ti u cho qu trnh kh khong nh sau: nng C 6H5COOH l
0,0168M, thi gian ngm 8,8 gi.
3.6.
Kt qu nghin cu cng on kh khong bng HCl:

Ph liu tm qua khu x l acid benzoic c thnh phn ho hc c
trnh by bng 3.1.

Bng 23. Thnh phn ha hc ca ph liu tm th Chn Trng sau khi kh
protein bng enzyme Alcalase, NaOH v C6H5COOH iu kin ti u.
STT
Thnh phn
Hm lng (%) trong ph liu tm
1
2
3
Protein
Khong
m
0,96
20,83
12,23

kh khong bng HCl
N
1
2
3
4
U1(Nng
HCl(M))
0,6
0,8
0,6
0,8
U2
(Thi gian (h))
4
4
10
10
X0
X1
X2
Y(%)
1
1
1
1
-1
1
-1
1
-1
-1
1
1
4,98
2,24
2,86
0,73
Bng 25. Th nghim tm phng n ca cng on kh khong bng HCl

N0
U1
U2
Y(%)
0,7
1,94
10
0,7
1,81
11
0,7
1,79
56
Phng trnh hi quy ca hm lng khong xc nh c nh sau:

Y= 2.7025 - 1.2425 * X1 - 0.9325 * X2 (4)
Quy lut bin i ca hm lng khong l khi tng nng dung dch acid
chlohydric (X1), tng thi gian kh khong (X2) th hm lng khong trong ph
liu tm (Y) gim xung, nhng nng HCl nh hng ti hm lng khong
nhiu hn . T phng trnh (4) nu mun kh khong mt cch trit ta phi
tng nng HCl, thi gian kh khong; song vic tng cao nng HCl, ko di
thi gian kh khong s gy nh hng ln cu trc ca chitin-chitosan lm ct
mch polymer t lm gim nht dung dch chitosan. Nh vy xut pht t
mc c s em tin hnh cc th nghim ti u theo ng dc nht.
Tn
U1(nng %)
U2(thi gian)
Mc c s
0.7
7
H s bj
-1.2425
-0.9325
Khong bin thin
0.1
3
bj*khong bin thin
-0.12425
-2.7975
Bc chuyn ng
4
4
Bc lm trn
0.02
0.6
Th nghim 9
0.72
7.6
Th nghim 10
0.74
8.2
Th nghim 11
0.76
8.8
Th nghim 12
0.78
9.4
1.53
0.98
0.83
0.77
57
Hnh 21. Kt qu nghin cu ti u ha ch ngm HCl theo phng php

ng ln dc ca BoxWillson.
Nhn xt: T cc th nghim v nhn vo th, nhn thy khi tng nng
acid chlohydric v thi gian kh khong th hm lng khong gim dn, khi nng
HCl 0,74M, thi gian ngm 8,2 gi th hm lng khong cn li l 0,98%. Khi
tip tc tng nng HCl v thi gian kh khong th hm lng khong gim
khng ng k, mt khc, hm lng khong cn li cng < 1%. Do vy nu
chitin-chitosan yu cu hm lng khong thp v nht cao th ta c th chn
cc thng s ti u cho qu trnh kh khong gim c nng HCl v thi
gian x l HCl sau ny nhm m bo c cht lng ca Chitin-Chitosan mt
cch tt nht. Ta c th chn cc thng s ti u cho qu trnh kh khong nh sau:
nng HCl l 0,74M, thi gian ngm 8,2 gi.
Tho lun: T qui trnh i chng kh khong bng HCl nng 1,4M v
thi gian ngm l 16 gi nhit phng, khng dng C6H5COOH, ta thu c
Chitin, trong hm lng khong cn li l 0.78%, cn quy trnh d kin sn xut
th hm lng khong cn li 0,98%. Ta thy, Chitin thu c t 2 qui trnh trn u
c hm lng khong cn li l <1%, nhng qui trnh kh khong c s dng kt
58
hp vi C6H5COOH th cht lng Chitin s tt hn v thi gian x l HCl ngn hn

do gim tc ng khng c li n mch polysaccharid ca Chitin, acid hu c
c kh nng bo ton mch Chitin. Mc khc, khi x l acid benzoic n cn lm
yu lin kt gia protein-chitin-khong, do khi x l HCl s d dng tch khong
ra khi ph liu tm nng thp (0,74M) v thi gian ngn hn (8,2 gi). Khi
ch dng HCl thi th phi dng nng cao hn (1,2M), thi gian x l di hn
(16 gi), v nng v thi gian di nh vy s lm nh hng xu n mch
polysaccharid ca Chitin. Do , vic kt hp kh khong bng acid hu c v v
c l vn quan trng cn c nghin cu thm.
59
PHN 4. KT LUN V XUT KIN

4.1.
Kt lun:
1. nghin cu c iu kin kh protein bng enzyme v NaOH thch hp

sn xut chitin nh sau:
- iu kin kh protein bng enzyme Alcalase: Nng enzyme 0,42%,
nhit 56oC,thi gian l 8.8 gi,t l nguyn liu/nc l 1:1.
- iu kin kh protein bng NaOH: Nng NaOH 0,24%, nhit
40oC,thi gian l 12,2 gi, t l 1w/5v.
Kh protein t ph liu tm th chn trng (Penaeus vannamei) bng
enzyme Alcalase gim thiu ha cht s dng, gim nhim mi trng, ng
thi tn dng ngun protein t u tm lm thc n chn nui.
2. nghin cu c iu kin kh khong bng acic benzoic thch hp: nng
acid benzoic l 0,0168M, thi gian x l l 8,8 gi, nhit phng.
Tip tc kh khong bng acid chlohydric thch hp l: 0,74M,thi gian
ngm l 8,2 gi, nhit phng.
4.2. Mt s xut cn tip tc nghin cu:
-
Cn tip tc nghin cu cng on deacetyl hon thin qui trnh sn xut

Chitin-Chitosan theo phng php sinh hc
Cn phn tch cht lng Chitosan t chitin sn xut theo qui trnh xut
nh gi mc nh hng ca cc thng s cng ngh.
60
TI LIU THAM KHO

A. Ti liu ting Vit
1. ng Vn Hp, Minh Phng, Nguyn Thun Anh, V Ngc Bi
Phn tch thc phm kim nghim thy sn Nh xut bn Nng
nghip, 2006.
2. Nguyn Hu To, L Vn Lin, Bi Vn Chnh,V Ch Cng Vin
chn nui K thut ch bin, bo qun, v s dng ngun ph phm
nng nghip v hi sn lm thc n chn nui.
3. Nguyn Th Ngc T , 2003,76tr. Nghin cu dng vt liu Chitosan
lm ph gia thc phm m bo v sinh an ton thc phm. [Tn tp
ch]
4. Trn Th Luyn, 2004, Bo co tng kt d n sn xut th nghim cp
b sn xut chitin, Chitosan t ph liu ch bin thy sn, M s
B2002-33-01-DA,8-15
5. Trn Th Luyn, Minh Phng, Nguyn Anh Tun, 03/2005, Sn xut
cc ch phm k thut v y dc t ph liu Thy sn- NXB Nng
nghip.
6. Trn Th Luyn v cng s (2003), Nghin cu sn xut Chitosan t v
tm s bng phng php ha hc vi mt cng on x l kim, Tp
ch KHCN Thy sn, i hc Thy Sn, s 5, 18-20
7. Trn Th Luyn, (2006), Cc phn ng c bn v bin i ca thc
phm trong qu trnh cng ngh - NXB Nng nghip, 2-17
8. Nguyn Cnh (1993), Quy hoch thc nghim, Trng i hc Bch
Khoa Tp.H Ch Minh, 10-15
9. Phan Hiu Hin (2001), Phng php b tr th nghim v x l s liu
NXB Nng nghip,22-24.
10. M.T.DenSiKov, Nguyn Vn t, Bi Huy Thanh (1997), Tn dng
ph liu ca cng nghip thc phm, NXB Khoa hoc v K thut, H
Ni, 60-64
61
11. Roelof Schoemaker, Chuyn gia t vn (2005), Tn dng ph liu tm,

D n ci thin cht lng v xut khu Thy sn (Seaquip), NXB
Nng nghip, 10-15.
12. http://www.casep.com.vn/default.asp?ngng=&dk=22&id=2938
13. http://khcn.ntu.edu.vn/vn/tai_nguyen/danh_muc_tap_chi/20060403/200
702260902082.pdf.
14. Mt s n tt nghip trn th vin.
B. Ti liu ting Anh
15. Adler Niesen. J.,(1986), Enzyme Hydrosis of Food Proteins, Elsevier
Applied Science Publishers, New York.
16. Asbjorn Gildberg, Even Stenberg., (2000), A new process for advanced
utilisention of shrimp waste, pp.2-7.
17. Birch. G.G., Blakerough. N., and Parker. K.J., (1981), Enzymes and
Food Processing Applied, Science Publishers Ltd, London.
18. Bustos. R.O.,and Michael. H., (1994), Microbial deproteinisation of
waste prawn shell, Institution of Chemical Engineers Symposium
Series, Institution of Chemical Engineers, Rugby, England, pp. 13-15.
19. Dalev. P. G., Simeonova. L.S., (1992), An enzyme biotechnology for
thetotal utilization of leather wastes, Biotechnol Lett Vol. 14, pp.531534.
20. Gagne. N. and Simpson. B.K.,(1993), Use of proteolytic enzymes to
facilitate recovery from shrimp wastes, Food Biotechnol,pp. 253-263.
21. Holanda, H. D. D, Netto, F. M., 2006. Recovery of components from
shirmp (Xiphopenaeus kroyeri) processing waste by enzymatic
hydrolysis. Journal of Food Science, 71, 298-303.
22. Nguyen Van Toan, Chuen-How Ng, Kyaw Nyein Aye, Trung Si Trang
and Willem F.Stevens, Production of high-quality chitin and chitosan
from preconditioned shrimp shells, F. Chem Technol Biotechnol
81:1113-1118(1006)
62
ng
PH LC
Bng 26. Kt qu o OD330nm
ng nghim
Hm lng
BSA(mg/ml)
0,05
0,1
0,2
0,3
0,4
0,5
0,075
0,148
0,296
0,448
0,594
0,732
OD330nm
Bng 27. Kt qu o OD570nm

ng nghim
Hm lng BSA(mg/ml)
OD570
0,101
0,201
0,305
0,409
10
0,506
1) Phng php quy hoch thc nghim ti u ha cho cng on kh

protein t ph liu tm bng enzyme Alcalase
Phng php quy hoch thc nghim th s th nghim cn tin hnh nh gi
vai tr ca tng yu t l: N = nk
Trong :
N : S th nghim;
n: S mc, y n = 3
k: S yu t nh hng; k = 3
vy N = 23 = 8 th nghim.
Tnh cc h s ca phng trnh hi quy:
N
bj
X
i 1
ij
.Yi
y: j = 0, 1,2, k vi k l s yu t c lp.
N l s th nghim trong ma trn quy hoch thc nghim N=8
Bng 28. Bng b tr th nghim
N U1(nng %)
U2(nhit
)
U3(thi gian) X0 X1 X2 X3
Y(%)lng
protein cn li
0,1
40
-1 -1 -1
19,6183
0,5
40
1 -1 -1
11,8941
0,1
60
-1 1 -1
13,023
0,5
60
1 -1
14,233
0,1
40
10
-1 -1 1
11,5383
0,5
40
10
1 -1 1
10,5916
0,1
60
10
-1 1
9,9975
0,5
60
10
9,3423
Thay s ta tnh c cc h s:
b0 = 12,5298 ; b1 = -1,0145;
b2 = - 0,8808 ; b3= -2,1623
Phng trnh hi quy tuyn tnh dng rt gn l:

Y
12,5298 1,0145 * X1 0,8808 * X2 2,1623 * X3(1)
Kim nh tnh ngha ca cc h s trong phng trnh hi quy.

B tr 3 th nghim song song thy phn ph liu tm bng enzyme Alcalase theo
cc iu kin tm phng n. Cc th nghim c b tr nh sau:
Bng 29.Cc th nghim tm phng n

N
1
U1
03
U2
50
U3
7
Y
12,67
2
3
0,3
0,3
50
50
7
7
12,797
13,8167

enzyme Alcalase.
0
0
0
0 2
Y1u
Y10u Y 1
N0
A
Y10u Y 1
Y1
1
12,67
- 0,3912
0,1531
2
2 12,797
- 0,2642
0,0698
3
3
0
13,8167
Y
u 1
0
1u
0,6555
13,0618
0,4296
Y
u 1
0
1u
Y1
1,3081
Ta s c phng sai ti hin l:
Y
3
S
2
th
N0 3 :
u 1
0
1u
0 2
Y1
N0 1
A
0,3263
N0 1
s th nghim tm phng n
Kim nh ngha cc h s hi quy:

Cu trc:
H O : i 0
Ha : j 0
tj
Trong
s bj
bj
S bj
vi Sbj
Sth2
0,2019
N
l lch qun phng ca h s th j
Tnh ton ta c:
to= 62,0449
t1= 5,0237
t2 = 4,3616
t3 = 10,7075
Tra bng phn v phn b Student vi p= 0,05; f = N 0 - 1 = 2
Suy ra :
t 0.05 2 4,3
Nh vy cc h s t1 ; t 2 ; t 3 u c ngha. Khi phng trnh hi quy c dng:

Y1 =12,5298 1,0145*X1 0,8808*X2 2,1623*X3 (1)
Kim nh s tng thch ca m hnh thc nghim c thc nghim theo

tiu chun Fisher.
Cc s liu dng tnh ton phng sai d ( S th2 ) c cho bng sau:
Bng 31.Cc s liu dng tnh ton
Yi Yi
Yi Yi ) 2
19,6183 15,7066
3,9117
15,3013
11,8941 13,6776
-1,7835
3,1808
13,023
15,7066
-2,6836
7,2018
14,233
13,6776
0,5554
0,3085
11,5383
11,3819
0,1564
0,0244
10,5916
9,3529
1,2387
1,5344
9,9975
11,3819
-1,3844
1,9167
9,3423
9,3529
0,0106
0,0001
STT
1
2
Yi
Yi
Y Y
N
Phng sai d
S d2
i 1
(Y Y )
i 1
29,47
N 1
29,47
5,8936
83
Trong : N: S th nghim
1: S h s c ngha
S d2
12,5307
18,064
Tiu chun Fisher: F 2
0,6540
S th
Tra bng phn v phn b Fisher F (f1,f2) vi:
=0,05
f1=N-L=3: Bc t do ca t.
f2=N0-1=2 : Bc t do ca mu.
Tra bng ta c: F0,05(3,2) = 19,3
V F = 18,0641 < F0,05(3,2) = 19,3; nn phng trnh tng thch vi thc
nghim.
Nhn vo phng trnh hi quy (1), ta c nhn xt:
C 3 yu t nhit thy phn, thi gian thy phn v t l E/S u c nh

hng n mc thy phn nhng thi gian c nh hng mnh nht.
Cc h s b1, b2, b3 u cng du m, c ngha l Y(hm lng protein
cn li) ng bin vi X1 (t l E/S), X2 (nhit thy phn), X3 (thi gian thy
phn)
T phng trnh (1) ta tin hnh ti u ha theo phng php ng dc
nht. Chn bc chuyn ng ca nhit thy phn X1 l 2 oC. Cc bc chuyn
ng ca yu t X2, X3 c tnh theo cng thc di y:
1 2
b11
0,036
b2 2
3 1
b11
0,58
b3 3
Chn bc nhy thch hp vi X1 l 0,04.
Bng 32. Kt qu thc nghim ti u ha cng on thy phn kh protein

bng enzyme .
Tn
X1
X2
X3
Y
Mc c s
0.3
50
H s bj
-1.0145
-0.8808
-2.1623
Khong bin thin
0.2
10
bj j
-0.2029
-8.808
-6.4869
Bc j
0.036
0.58
Bc lm trn
0.04
0.6
Th nghim 9
0.34
52
7.6
9.451
Th nghim 10
0.38
54
8.2
9.2528
Th nghim 11
0.42
56
8.8
8.0102
Th nghim 12
0.46
58
9.4
8.655
Bng 33.Cc thng s ti u.

Cc thng s ti u
Hm lng protein (%)
Nng enzyme Alcalase:4,2 %

Thi gian: 8,8 gi
Nhit : 56oC
8,0102

protein t ph liu tm bng NaOH:
Phng php quy hoch thc nghim th s th nghim cn tin hnh nh
gi vai tr ca tng yu t l:
N=nk
Trong : N : S th nghim;
n: S mc, y n=2
k: S yu t nh hng; k=2
vy N= 22 =4 th nghim.
bj
X
i 1
ij
.Yi
y: j = 0, 1,2, k vi k l s yu t c lp.
N
U1(nng %) U2(thi gian)
X0
X1
X2 Y(%)lng protein cn li
0,1
-1
-1
1,78
0,3
-1
1,04
0,1
14
-1
1,34
0,3
14
0,82
Thay s ta tnh c cc h s: b0 = 1,245 ; b1= -0,315;

b2= -0,165
Y
1.245 - 0.315*X1 - 0.165*X2

B tr 3 th nghim song song kh protein trong ph liu tm bng NaOH
theo cc iu kin tm phng n. Cc th nghim c b tr nh sau:
Bng 35.Cc th nghim tm phng n
N0
U1
U2
Y(%)
0,2
11
1,1214
10
0,2
11
1,0764
11
0,2
11
1,1319
Bng 36.Cc th nghim tm phng n ca cng on kh protein bng

NaOH
0
0
0 2
N0
Y1u0
A
Y10u Y 1
Y1
Y10u Y 1
1,1214
1,0764
1,1319
Y10u
u 1
1,1099
0,0115
0,0001
- 0,0335
0,0011
0,0220
0,0005
Y 0 Y 10
1u
u 1
0,001
Y
3
S
2
th
0
1u
u 1
N0 1
N0 3 :
0 2
Y1
A
0,0009
N0 1

Cu trc:
H O : i 0
Ha : j 0
tj
bj
S bj
vi Sbj
Trong
s bj
Sth2
0,0147
N
Tnh ton ta c:
to= 84,4553
t1= 21,3682
t2 = 11,1929
Suy ra : t0.05 2 4,3
Nh vy cc h s t 1,12 u c ngha. Khi phng trnh hi quy c
dng:
Y1 = 1,245 0,315*X1 0,165*X2 (2)

tiu chun Fisher. Cc s liu dng tnh ton phng sai d ( S th2 ) c cho
bng sau:
Bng 37. Cc s liu dng tnh ton

STT
Yi
Yi
Yi Yi
Yi Yi ) 2
1,78
1,7250
0,0550
0,003
1,04
1,0950
-0,0550
0,003
1,34
1,3950
-0,0550
0,003
0,82
0,7650
0,0550
0,003
Y Y
N
Phng sai d
S d2
i 1
N 1
(Y
i 1
Yi ) 2 0,001
0,0061
1: S h s c ngha
S d2
0.0061
6,96
Tiu chun Fisher: F 2
S th 0.0009
=0,05
Tra bng ta c: F0,05(2,2) =18,5
V F =6,96 < F0,05(2,2) =18,5; nn phng trnh tng thch vi thc nghim.
C 2 yu t nng NaOH, thi gian ngm u c nh hng n mc
kh protein nhng nng c nh hng mnh nht.
Cc h s b1, b2 u cng du m, c ngha l Y(hm lng protein cn li)
ng bin vi X1 (nng NaOH), X2 (thi gian ngm).
nht. Chn bc chuyn ng ca nng NaOH l 0,2%. Cc bc chuyn ng
ca yu t X2, X3 c tnh theo cng thc sau hay c chn theo kinh nghim.
2 1
b11
0,607
b2 2
10
Kt qu thc nghim ti u ha cng on thy phn kh protein bng

NaOH c ghi trong bng :
Bng 38.Kt qu thc nghim ti u ha cng on kh protein bng NaOH
Tn
X1
X2
Mc c s
0,2
11
H s bj
-2,3238
-2,6763
Khong bin thin
0,1
bj j
-0,3238
-8,0289
Bc j
0,2
0,607
Bc lm trn
0,2
0,6
Th nghim 9
2,2
11,6
1,08
Th nghim 10
2,4
12,2
0,96
Th nghim 11
2,6
12,8
0,91
Th nghim 12
2,8
13,4
0,86
Bng 39.Cc thng s ti u

Cc thng s ti u
Hm lng protein (%)
Nng NaOH:2,4 %
Thi gian: 12,2 gi
T l w/v: 1/15
0,96

khong t ph liu tm bng C6H5COOH:
Phng php quy hoch thc nghim th s th nghim cn tin hnh nhgi vai
tr ca tng yu t l:
N=nk
n: S mc, y n=2
11

N
bj
X
i 1
ij
.Yi
y: j = 0, 1,2, k vi k l s yu t c lp.
N
U1(nng M) U2(thi gian)
X0
X1
X2 Y(%)lng khong cn li
0,012
-1
-1
35,21
0,018
-1
34,38
0,012
10
-1
25,09
0,018
10
18,57
Thay s ta tnh c cc h s: b0 = 26,3125 ; b1= -1,8375;

b2= -6,4825
26,3125 1,8375*X1 6,4825*X2 (3)
Y
B tr 3 th nghim song song kh khong trong ph liu tm bng C 6H5COOH

Bng 41. Cc th nghim tm phng n
N0
U1
U2
Y(%)
0,015
26,05
10
0,015
25,14
11
0,015
26,23
Bng 42. Cc th nghim tm phng n ca cng on kh khong bng

C6H5COOH
0
0
0 2
N0
Y1u0
A
Y10u Y 1
Y1
Y10u Y 1
26,05
0,2433
0,0592
12
25,14
26,23
Y10u
u 1
25,8067
- 0,6667
0,4444
0,4233
0,1792
Y
3
u 1
0
1u
Y1
0,6829
Y
3
S
2
th
u 1
N0 1
N0 3 :
0 2
Y1
0
1u
A
0.3414
N0 1

Cu trc:
H O : i 0
Ha : j 0
tj
bj
S bj
vi S bj
Trong
s bj
S th2
0,2922
N
Tnh ton ta c:
to= 96,907
t1= 6,2893
t2 =22,1881
Suy ra : t 0.05 2 4,3
Nh vy cc h s t1,12 u c ngha. Khi phng trnh hi quy c dng:
Y1 = 26,3125 1,8375*X1 6,4825*X2 (3)

tiu chun Fisher. Cc s liu dng tnh ton phng sai d ( S th2 ) c cho
bng sau:
Bng 43.Cc s liu dng tnh ton
STT
Yi
Yi
Yi Yi
Yi Yi ) 2
13
35,21
36,6325
-1,4225
2,0235
34,38
32,9575
1,4225
2,0235
25,09
23,6675
1,4225
2,0235
18,57
19,9925
-1,4225
2,0235
Y Y
N
Phng sai d
S d2
i 1
N 1
(Y
i 1
Yi ) 2 8,09
4,047
1: S h s c ngha
Tiu chun Fisher : F
S d2
4,0470
11,853
2
S th 0,3414
=0,05
Tra bng ta c: F0,05(2,2) =18,5
V F =11,853 < F0,05(2,2)=18,5; nn phng trnh tng thch vi thc nghim.
C 2 yu t nng C6H5COOH, thi gian ngm u c nh hng n mc
kh khong nhng thi gian kh khong c nh hng mnh nht.
Cc h s b1, b2 u cng du m, c ngha l Y(hm lng khong cn li)
ng bin vi X1 (nng C6H5COOH), X2 (thi gian ngm).
nht. Chn bc chuyn ng ca nng C 6H5COOH l 0.0006M. Cc bc
chuyn ng ca yu t X1, X2 c tnh theo cng thc sau hay c chn theo
kinh nghim.
2 1
b11
0,59
b2 2
14
Kt qu thc nghim ti u ha cng on thy phn kh khong bng acid

benzoic c ghi trong bng :
Bng 44. Kt qu thc nghim ti u ha cng on kh khong bng C6H5COOH .
Tn
X1
X2
Mc c s
0,015
H s bj
- 1,8375
-6,4825
Khong bin thin
0,003
bj j
-0,000945
-1,845
Bc j
0,0006
0,59
Bc lm trn
0,0006
0,6
Th nghim 9
0,0156
7,6
25,01
Th nghim 10
0,0162
8,2
22,29
Th nghim 11
0,0168
8,8
20,83
Th nghim 12
0,0174
9,2
19,02
Bng 45. Cc thng s ti u

Cc thng s ti u
Hm lng khong (%)
Nng C6H5COOH : 0,0168M

Thi gian: 8,8 gi
T l w/v: 1/10
20,83

khong t ph liu tm bng HCl:
Phng php quy hoch thc nghim th s th nghim cn tin hnh nhgi vai
tr ca tng yu t l:
N=nk
n: S mc, y n=2
15
N
bj
X
i 1
ij
.Yi
y: j = 0, 1,2, k vi k l s yu t c lp.
N
U1(nng M) U2(thi gian)
X0
X1
X2 Y(%)lng khong cn li
0,6
-1
-1
4,98
0,8
-1
2,24
0,6
10
-1
2,86
0,8
10
0,63
Thay s ta tnh c cc h s: b0 = 2,7025 ; b1= -1,2425; b2= -0,9325

Y
2,7025 1,2425*X1 0,9325*X2 (4)

B tr 3 th nghim song song kh khong trong ph liu tm bng C 6H5COOH
Bng 47. Cc th nghim tm phng n
N0
U1
U2
Y(%)
0,7
1,94
10
0,7
1,81
11
0,7
1,79

NaOH
16
N0
Y1u0
1,94
1,81
1,79
Y10u Y 1
Y1
3
Y10u
u 1
1,8467
0
1u
0 2
Y1
0,0933
0,0087
-0,0367
0,0013
-0.0567
0,0032
Y
3
u 1
0
1u
Y1
0.0133
Y
3
S
2
th
u 1
N0 1
N0 3 :
0 2
Y1
0
1u
A
0,0066
N0 1

Cu trc:
H O : i 0
Ha : j 0
tj
bj
S bj
vi S bj
Trong
S th2
0,0407
s bj
Tnh ton ta c:
to= 65,7479 ; t1= 30,5113; t2 =22,89
Suy ra : t 0.05 2 4,3
Nh vy cc h s t1,12 u c ngha. Khi phng trnh hi quy c dng:
Y1 = 2,7025 1,2425*X1 0,9325*X2 (4)
Kim nh s tng thch ca m hnh thc nghim c thc nghim theo tiu chun
Fisher. Cc s liu dng tnh ton phng sai d ( S th2 ) c cho bng sau:
Bng 49. Cc s liu dng tnh ton
STT
Yi
Yi
Yi Yi
Yi Yi ) 2
4,98
4,8525
0,1275
0,0163
17
2,24
2,3675
-0,1275
0,0163
2,86
2,9875
-0,1275
0,0163
0,63
0,5025
0,1275
0,0163
Y Y
N
Phng sai d
S d2
i 1
N 1
(Y
i 1
Yi ) 2 0,07
0,0325
1: S h s c ngha
S d2
4.0470
4,9014
Tiu chun Fisher : F 2
S th 0.3414
=0,05
Tra bng ta c: F0,05(2,2) =18,5
V F =4,9014 < F0,05(2,2) =18,5; nn phng trnh tng thch vi thc nghim.
C 2 yu t nng HCl, thi gian ngm u c nh hng n mc kh
khong nhng nng c nh hng mnh nht.
Cc h s b1, b2 u cng du m, c ngha l Y(hm lng khong cn li)
ng bin vi X1 (nng HCl), X2 (thi gian ngm).
nht. Chn bc chuyn ng ca nng HCl l 0,02M. Cc bc chuyn ng
ca yu t X1, X2 c tnh theo cng thc sau hay c chn theo kinh nghim.
2 1
b11
0,579
b2 2
Kt qu thc nghim ti u ha cng on thy phn kh khong bng acid

chlohydric c ghi trong bng :
Bng 50. Kt qu thc nghim ti u ha cng on kh khong bng HCl.
18
Tn
X1
X2
Mc c s
0,7
H s bj
- 1,2425
-0,9325
Khong bin thin
0,1
bj j
-0,12425
-2,7975
Bc j
0,02
0,579
Bc lm trn
0,02
0,6
Th nghim 9
0,72
7,6
1,53
Th nghim 10
0,74
8,2
0,98
Th nghim 11
0,76
8,8
0,83
Th nghim 12
0,78
9,2
0,77
Bng 51. Cc thng s ti u

Cc thng s ti u
Hm lng khong (%)
Nng HCl : 0,74M

Thi gian: 8,2 gi
T l w/v: 1/10
0,98
5) Mt s thit b s dng trong ti:
Hnh 22.Thit b n nhit Memmert.
19
Hnh 23. Thit b o Uvmini-1240.
Hnh 24.My li tm.
Hnh 25.Thit b Vortex.
20
Hnh 26.S bt mu ca dung dch BSA sau khi cho thuc th Biuret.
Hnh 27. Cng thc cu to ca Sodium citrate.
Hnh 28. Mono Sodium Citrate Anhydrous.
21
Hnh 29. Dung dch protein v thuc th Biuret
Hnh 30. Thy phn u tm
Hnh 31. u tm c v hot enzyme sau khi thy phn(dng nhit)
22
Hnh 32. Ph liu tm sau khi thy phn
Hnh 33. Ph liu tm sau khi thy phn bng enzyme Alcalase v x l NaOH
Hnh 34. Ph liu tm kh protein v kh khong ln 1 bng C6H5COOH
23
Hnh 35. Ph liu tm(PLT) kh protein bng enzyme(tri), PLT kh

protein bng enzyme v NaOH
Hnh 36. PLT kh protein bng enzyme v NaOH(tri), PLT kh protein

v kh khong bng C6H5COOH

Chiet Xuat Chitin 7199

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i

1.3.2. Sn xut chitin, chitosan theo phng php ha hc ci tin:.......................

Kh ln 1: dng enzyme alcalase...........................................................

2.2.2.4. Th nghim xc nh ch kh khong...................................................

Kh ln 1: Dng acid benzoic.................................................................

Kh ln 2: Dng acid chlohydric............................................................

2.3. CC PHNG PHP PHN TCH HA HC:[1].................................

2.3.4. Xc nh hm lng Protein theo phng php Biuret.................................

Bng 16. Kt qu hm lng protein cc th nghim ti u theo ng dc nht..........

Bng 35.Cc th nghim tm phng n..............................................................

Hnh 23. Thit b o Uvmini-1240................................................................19

TNG QUAN V CNG NGH SN XUT V NG DNG CA

TNG QUAN V CHITIN-CHITOSAN

1.1.1. S tn ti ca Chitin-Chitosan trong t nhin

CHITIN: Chitin l mt polysaccharide mch thng, n c cu trc tuyn tnh

Cng thc cu to:

Hnh 1.Cu to ca Chitin.

Dng lm ph gia trong k ngh bo ch dc phm:

thu sn t gi tr 2,4 t USD, chim 8,9% tng gi tr xut khu c nc trong

chng ang tr nn ph bin nh mt phng cch gip lm tng li nhun cho

- Lp canxi ha: Lp ny chim phn ln lp v, thng c mu xanh tri

Mc d chitin phn b rng ri trong t nhin nhng n khng c tm thy

a. Nng acid: Nng acid qu thp s khng kh c ht khong dn n

cng s, 1978) [20]. iu ny l do khng xc nh c bn cht hot ng ca

Kim tra hm lng protein

Hnh 3.Quy trnh ca Stevens (2002) Hc Vin Cng Ngh Chu

Quy trnh sn xut Chitosan t v tm hm ca Hackman:

Hnh 4.Quy trnh sn xut chitosan t v tm hm ca Hackman

Quy trnh thy nhit ca Yamashaki v Nakamichi (Nht Bn):

Quy trnh sn xut ca Php:

u im: Quy trnh sn xut ny rt ngn c thi gian sn xut rt nhiu.

Quy trnh sn xut Chitosan t v tm S bng phng php ha hc

Bng 1. Mt s ch tiu cht lng ca chitosan t v tm s theo phng php

-Quy trnh ca Trung tm Ch bin i hc Thy sn.

Ngm ra trung tnh

Hnh 10. Quy trnh ca Trung tm Ch bin i hc Thy sn

1.3.3. Sn xut chitin, chitosan theo phng php s dng enzyme:

Kh protein bng enzyme

Phn dch ni pha trn

Chit Astaxanthin bng du

Kh khong HCl 2,5%, 2h, to

Nhc im: Enzyme t tin dn n chi ph gi thnh sn phm cao.

Quy trnh s dng Enzyme papain sn xut chitosan (Trn Th

Hnh 12. Quy trnh s dng Enzyme papain sn xut chitosan

protein ca v tm m cn loi c ht cc cht khong trong v tm. Dch p b

PHN 2. NGUYN VT LIU V PHNG PHP

x l nhit 850C, thi gian 10 pht hoc protease Alcalase b bt hot ti pH = 4

2.2. PHNG PHP NGHIN CU

B tr th nghim xc nh cc thng s k thut

Tch protein bng enzyme Alcalase

Kh protein bng NaOH

Kh khong bng acid benzoic

Kh khong bng HCl

Hnh 13.Quy trnh d kin sn xut.

Sau khi tm c cc thng s ti u ca cc cng on, ta tin hnh sn

Tch protein bng enzyme

Kh protein bng NaOH

Kh khong bng HCl

2.2.2.1. B tr th nghim xy dng ng chun ca phng php

Sau ly mi mu 4ml, thm vo 200l thuc th Microbiuret,vortex

Dung dch ha cht

BSA chun (ml)

Thuc th Biuret (ml)

Sau khi cho y cc ha cht, cc ng nghim trn nhit phng