do an glucosamin pp hoa hoc 9124

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Nghin cu iu ch glucosamin t v tm

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MC LC

T VN ..3 CHNG I: TNG QUAN ................................................................... 51.1. Ngun gc v s tn ti chitin - chitosan trong t nhin. ................................ 5 1.2. Cu trc ho hc, tnh cht l ho ca chitin........................................................... 6 1.2.1. Cu trc ho hc ca chitin. .....................................................................................6 1.2.2. Tnh cht l ho ca chitin. .......................................................................................8 1.3. Cu trc ho hc, tnh cht l ho sinh v c tnh ca chitosan............... 8 1.3.1. Cu trc ho hc ca chitosan. ...............................................................................9 1.3.2. Tnh cht l ho ca chitosan................................................................................ 10 1.3.3. Tnh cht sinh hc ca chitosan. ......................................................................... 11 1.3.4. c tnh ca chitosan. .............................................................................................. 11 1.4. Tnh hnh nghin cu sn xut v ng dng trong thc t ca chitin v chitosan Vit Nam v trn th gii. ................................................................................ 12 1.5. Cu trc ha hc, tnh cht l ha ca glucosamin. ......................................... 15 1.6. Cu trc ha hc, tnh cht vt l mt s mui ca glucosamin. ............ 17 1.6.1. Glucosamin hydroclorua.......................................................................................... 17 1.6.2. Glucosamin sulfat. ....................................................................................................... 17 1.6.3. Acetyl glucosamin. ........................................................................................................ 18 1.7. Dc l v dc ng hc ca glucosamin v mui ca n. ..................... 18 1.8. Mt s quy trnh sn xut chitin, chitosan trn th gii v ti Vit Nam........................................................................................................................................................... 21 1.8.1. Trn th gii. .................................................................................................................... 21 1.8.2. Vit Nam. ....................................................................................................................... 23 1.9. Quy trnh sn xut glucosamin hydroclorua (glu.HCl). ................................. 25 1.9.1. Quy trnh sn xut glu.HCl ca Trn Th Luyn. ..................................... 25 1.9.2. Quy trnh sn xut glu.HCl ca nh Rng. ....................................... 26

Chng II : I TNG V PHNG PHP NGHIN CU....................................................................................................................................................................... 27

2.1. i tng nghin cu.......................................................................................................... 27 2.2. Phng php nghin cu. .................................................................................................. 27

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CHNG III : THC NGHIM V KT QU ...................................... 29

3.1.Quy trnh iu ch chitin, chitosan v glucosamin hydroclorua. .............. 29 3.2. iu ch chitin t v tm. ................................................................................................ 30 3.3.iu ch chitosan bng cch deacetyl chitin. ........................................................ 31 3.4. iu ch glucosamin hydroclorua (glu.HCl). ...................................................... 32 3.4.1.iu ch glu.HCl t chitin. ..................................................................................... 32 3.4.2.iu ch glu.HCl t chitosan.................................................................................. 33 3.4.3. Tinh ch glu.HCl. .......................................................................................................... 34 3.5. Kho st nh hng ca thi gian n hiu sut iu ch glu.HCl t chitin. ........................................................................................................................................................ 39 3.6. Kho st nh hng ca nhit n hiu sut iu ch glu.HCl t chitin ......................................................................................................................................................... 39 3.7. Kho st nh hng ca nng axit HCl n hiu sut iu ch glu.HCl t chitin. .............................................................................................................................. 40

CHNG IV : KT QU V BN LUN .................................................. 414.1. iu ch chitin t v tm. ................................................................................................ 41 4.1.1.Qu trnh kh khong.................................................................................................. 41 4.1.2. Qu trnh loi b protein.........................................................................................41 4.1.3. Qu trnh ty mu (loi b Astaxanthin). ...................................................... 42 4.2. iu ch chitosan. .................................................................................................................. 43 4.3. iu ch glucosamin hydroclorua (glu.HCl)........................................................ 43 4.3.1. iu ch glu.HCl i t chitin. ............................................................................... 44 4.3.2. iu ch glu.HCl t chitosan. ............................................................................... 45

KT LUN ...................................................................................................................................... 48 TI LIU THAM KHO .................................................................................................. 49

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T VN

Gip xc l ngun nguyn liu thy sn di do chim 1/3 tng sn lng nguyn liu thy sn Vit Nam. Trong cng nghip ch bin thy sn xut khu, t l c cu cc mt hng ng lnh gip xc chim t 70 - 80% cng sut ch bin. Hng nm cc nh my ch bin thi b mt lng ph liu gip xc kh ln khong 70.000 tn/nm [3]. Ngun ph liu ny l nguyn liu quan trng cho cng nghip sn xut chitin 1, chitosan 2, glucosamin 3 v cc sn phm c gi tr khc. Do vy vic nghin cu v pht trin sn xut cc sn phm t v tm l rt quan trng, nng cao gi tr s dng ph liu ny v lm sch mi trng. Sn phm chitin - chitosan c nhiu cng trnh nghin cu v ng dng trong thc t. Chitin c ng dng lm da nhn to v l nguyn liu trung gian cho cc cht quan trng nh chitosan, glucosamin v cc cht c gi tr khc. Chitosan c nhiu ng dng trong cc ngnh cng nghip, nng nghip, y dc v bo v mi trng nh: sn xut glucosamin, ch khu phu thut, thuc kem, vi, sn, cht bo v hoa qu, bo v mi trngVi kh nng ng dng rng ri ca chitin chitosan m nhiu nc trn th gii v c Vit Nam nghin cu sn xut cc sn phm ny. Glucosamin l mt hot cht qu c sn xut t v tm thng qua nguyn liu trung gian l chitin hoc chitosan. Glucosamin ch yu c s dng trong y hc cha bnh thoi ho khp. ngi gi, chc nng cng nh cu to ca khp c nhiu thay i, cc t bo ca khp thoi ho, tr nn km linh ng. Gn v dy chng phn on, ng vi, kh cn, tr nn km bn b, km co dn, khng chu c cng lc v d b tn thng. Sn tr nn c mu, x ha, gai xng, kh nc, rn nt vi nhiu tinh th canxi lm khp au. Khp co dui kh khn v mng hot dch mng v kh dn [1]. Trc y, trong iu tr cc bnh thoi ha khp ngi ta thng dng cc thuc thuc nhm corticoid hoc nhm khng vim gim au khng steroid NSAID (Non - Steroidal Anti - Inflammatory Drugs), nhng cc thuc thucNguyn c Duy Lp CN Ho Dc v Ho Cht BVTV K47 3

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http://www.ebook.edu.vn nhm ny ch iu tr triu chng v c nhiu tc dng ph. Vi cc thuc thuc

nhm corticoid thng gy nn cc tc dng ph: long xng, vim lot d dy, gim min dch. i vi cc NSAID th gy vim lot d dy[12]. T thp k 90 ngi ta pht hin ra glucosamin phc hi c cc sn khp, tc l cha c cn nguyn ca bnh vim, thoi ho khp [2]. c nhiu cng trnh nghin cu vi nhiu th nghim lm sng chng minh tc dng iu tr tn gc bnh thoi ho khp ca glucosamin, nht l dng phi hp vi dc liu thin nhin. Trn thc t glucosamin thng c s dng cc dng: glucosamin hydroclorua 4, glucosamin sulfat 5, N - Acetyl glucosamin 6. Hin nay thuc cha glucosamin c lu hnh trn 70 quc gia. Ti Vit Nam, glucosamin v cc thnh phm i t glucosamin vn ch yu l nhp khu v xu th s dng ngy cng nhiu. Trong khi ngun nguyn liu (v gip xc) ch yu iu ch ra glucosamin th di do. tn dng ngun nguyn liu ph thi, to ngun nguyn liu cho vic sn xut thuc cha bnh thoi ho khp v l nguyn liu cho vic nghin cu cc ng dng khc ca glucosamin chng ti t vn tin hnh ti Nghin cu iu ch glucosamin t v tm. Mc tiu ca ti nhm gii quyt cc vn sau : 1. Phn lp chitin chitosan t v tm ph thi. 2. iu ch glucosamin hydroclorua t chitin v chitosan..

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Chng I: TNG QUAN

1.1. Ngun gc v s tn ti chitin - chitosan trong t nhinChitin - chitosan l mt polysacharit tn ti trong t nhin vi sn lng rt ln (ng th hai sau xellulose). Trong t nhin chitin tn ti trong c ng vt v thc vt . Trong ng vt, chitin l mt thnh phn cu trc quan trng ca cc v mt s ng vt khng xng sng nh: cn trng, nhuyn th, gip xc v giun trn. Trong ng vt bc cao monome ca chitin l mt thnh phn ch yu trong m da n gip cho s ti to v gn lin cc vt thng da. Trong thc vt chitin c thnh t bo nm h zygenmyctes, cc sinh khi nm mc, mt s loi to... [13]. Chitin - chitosan l polysacharit c m khng c, c khi lng phn t ln. Cu trc ca chitin l tp hp cc monosacharit (N-acetyl--D-glucosamine) lin kt vi nhau bi cc cu ni glucozit v hnh thnh mt mng cc si c t chc. Hn na chitin tn ti rt him trng thi t do v hu nh lun lun ni bi cc cu ni ng tr (coralente) vi cc protein, CaCO3 v cc hp cht hu c khc.

Hnh 1 : Chitin v v tm

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http://www.ebook.edu.vn Trong cc loi thy sn c bit l trong v tm, cua, gh, hm lng

chitin - chitosan chim kh cao ao ng t 14 - 35% so vi trng lng kh [5]. V vy v tm, cua, gh l ngun nguyn liu chnh sn xut chitin - chitosan. V mt lch s, chitin c Braconnot pht hin u tin vo nm 1821, trong cn dch chit t mt loi nm. ng t tn cho cht ny l Fungine ghi nh ngun gc ca n. Nm 1823 Odier phn lp c mt cht t b cnh cng m ng gi l chitin hay chiton, ting Hy lp c ngha l v gip, nhng ng khng pht hin ra s c mt ca nit trong . Cui cng c Odier v Braconnot u i n kt lun chitin c dng cng thc ging vi xellulose.

CH2OH O

CH2OH O O OH O NHCOCH3 CH2OH O O OH NH2 O

1)

O

OH

NHCOCH3 CH2OH O

2)

O

OH NH2

CH2OH O

CH2OH O O OH OH O

3)

O

OH OH

Hnh 2 :1) Chitin; 2) Chitosan; 3) Xellulose

1.2. Cu trc ho hc, tnh cht l ho ca chitin 1.2.1. Cu trc ho hc ca chitinChitin 1 c cu trc tinh th rt cht ch v u n. Bng phng php nhiu x tia X. Ngi ta chng minh c chitin tn ti ba dng cu hnh :, , - chitin [15]. Cc dng ny ca chitin ch do s sp xp khc nhau v hng ca mi mt xch (N-acetyl-D-glucosamin) trong mch.Nguyn c Duy Lp CN Ho Dc v Ho Cht BVTV K47 6

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http://www.ebook.edu.vn C th biu din mi mt xch ny bng mi tn sao cho phn u ca mi

tn ch nhm CH2OH, phn ui ch nhm NHCOCH3, th cc cu trc , , chitin c m t nh sau:

- Chitin

- Chitin

- Chitin

- chitin c cu trc cc mch c sp xp ngc chiu nhau u n, nn ngoi lin kt hydro trong mt lp v h chui, n cn c lin kt hydro gia cc lp do cc chui thuc lp k nhau nn rt bn vng. Do cc mt xch sp xp o chiu, xen k thun li v mt khng gian v nng lng. y cng l dng ph bin trong t nhin. , - chitin do mt xch ghp vi nhau theo kiu song song ( - chitin) v hai song song mt ngc chiu ( - chitin), gia cc lp khng c loi lin kt hydro. Dng - chitin cng c th chuyn sang dng - chitin nh qu trnh axetyl ha cho cu trc tinh th bn vng hn. Qua nghin cu v s thy phn chitin bng enzym hay axit HCl m c th ngi ta thy rng chitin c cu trc l mt polyme c to thnh t cc n v N-Acetyl--D-glucosamine lin kt vi nhau bi lin kt -(1-4) glucozit. Cng thc cu to ca chitin:O OH O O HO NHCCH3 O OH HO O NHCH2CH3 O HO O NHCH2CH3 O OH O

1

Tn gi : Poly(1-4)-2-acetamido-2-deoxy--D-glucose; Poly(1-4)-2acetamido-2-deoxy--D-glucopyranose.

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http://www.ebook.edu.vn Cng thc phn t: [C8H13O5N]n

Phn t lng : Mchitin = (203,09)n

1.2.2. Tnh cht l ho ca chitin [3]Chitin c mu trng hay mu trng pht hng, dng vy hoc dng bt, khng mi, khng v, khng tan trong nc, trong mi trng kim, axit long v cc dung mi hu c nh ete, ru Nhng tan trong dung dch c nng ca mui thioxianat liti (LiSCN) v thioxianat canxi (Ca(SCN)2) to thnh dung dch keo, tan c trong h dimetylacetamid - LiCl 8% [4], tan trong hexafluoroisopropyl alcohol (CF3CHOHCF3) v hexafluoracetone sesquihydrate (CF3COCF3.H2O) [16]. Chitin c kh nng hp thu tia hng ngoi c bc sng 884 - 890cm-1. Chitin n nh vi cc cht oxy ho mnh nh thuc tm (KMnO4); oxy gi (H2O2); nc javen (NaOCl - NaCl), li dng tnh cht ny m ngi ta s dng cc cht oxy ho trn kh mu cho chitin. Khi un nng trong dung dch NaOH m c (40 - 50%), nhit cao th chitin s b mt gc acetyl to thnh chitosan:Chitn CH2OH NaOH 40-50% OH T0cao NHCOCH3 Chitosan CH2OH OH NH2

Khi un nng trong axit HCl m c, nhit cao th chitin s b ct mch thu c glucosamin:CH2OH Chitn OH NHCOCH3 HCl 36% T0cao CH2OH Glucosamin OH NH2

Phn ng este ha : - Chitin tc dng vi HNO3 m c cho sn phm chitin nitrat. - Chitin tc dng vi anhydrit sunfuric trong pyridin, dioxan v N,N-dimetylanilin cho sn phm chitin sunfonat.

1.3. Cu trc ho hc, tnh cht l ho sinh v c tnh ca chitosan

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1.3.1. Cu trc ho hc ca chitosan [3]Trong s cc dn xut ca chitin th chitosan 2 l mt trong nhng dn xut quan trng v n c hot tnh sinh hc cao v c nhiu ng dng trong thc t. Vic sn xut chitosan tng i n gin, khng cn dung mi, ha cht c hi, t tin. Chitosan thu c bng phn ng deacetyl ha chitin, bin i nhm N-acetyl thnh nhm amin v tr C2. Do qu trnh kh acetyl xy ra khng hon ton nn ngi ta qui c nu deacetyl ha (degree of deacetylation) DD > 50% th gi l chitosan, nu DD < 50% gi l chitin [13]. Chitosan c cu trc tuyn tnh t cc n v 2-amino-2-deoxy--Dglucosamine lin kt vi nhau bng lin kt -(1-4) glucozit. Cng thc cu to ca chitosan:

OH O O HO NH2 OH HO O

NH2 O HO

OH O O NH2

2

Tn gi khoa hc: Poly(1-4)-2-amino-2-deoxy--D-glucose; poly(1-4)-2amino-2-deoxy--D-glucopyranose. Cng thc phn t: [C6H11O4N]n Phn t lng: Mchitosan = (161,07)n Qua cu trc ca chitin - chitosan ta thy chitin ch c mt nhm chc hot ng l -OH (H nhm hydroxyl bc 1 linh ng hn H nhm hydroxyl bc 2 trong vng 6 cnh) cn chitosan c 2 nhm chc hot ng l -OH, -NH2, do chitosan d dng tham gia phn ng ha hc hn chitin. Trong thc t cc mch chitin - chitosan an xen nhau, v vy to ra nhiu sn phm ng thi, vic tch v phn tch chng rt phc tp.

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1.3.2. Tnh cht l ho ca chitosan [3]Chitosan c mu trng ng hoc mu vng nht, tn ti dng bt hoc dng vy, khng mi, khng v, nhit nng chy 309 - 3110C. Chitosan c tnh kim nh, khng tan trong nc, trong kim nhng ho

tan c trong dung dch axit hu c long nh: axit acetic, axit fomic, axit lactic, to thnh dung dch keo nht trong sut. Chitosan ho tan trong dung dch axit acetic 1 - 1.5%. nht ca chitosan trong dung dch axit long lin quan n kch thc v khi lng phn t trung bnh ca chitosan (y cng l tnh cht chung ca tt c cc dung dch polyme) [6]. Chitosan kt hp vi aldehit trong iu kin thch hp hnh thnh gel, y l c s by t bo, enzym. Chitosan phn ng vi axit m c, to mui kh tan. Chitosan tc dng vi iod trong mi trng H2SO4 cho phn ng ln mu tm. Mt s dn xut chitosan [26]:OH O O HO + Chitosan salts NH3 O O HO OH O HN O O HO OH O NH O O HO OH O N=CHR Schiff base OH OH O O O HO NH2 Chitosan NHR N-alkylchitosan OH O HO O NHCH2COOH O O HO O O O

OCH2CH2SO3O O O HO NH3+ Sulfoethylchitosan OCH2COOH O O O HO NHCH2COOH N,O-carboxymethylchitosan OCH2CH2CN O O O HO NHAc Cyanoethylchitin OCS2-Na+ O O O HO NHAc Chitin xanthogenate O Na+ -O

Crosslinked Chitosan

N-carboxymethylchitosan OH O HO O NHAc Chitin O OCH2CHOHR O O O HO NHAc Hydroxyalkylchitin OCH2COOH O O O HO NHAc Carboxmethylchitin

O-Na+ O

O NHAc

O RH2CO

OCH2R O

O NHAc

Alkalichitin

Alkylchitin

Hnh 3 : S cc dn xut i t chitin, chitosan.

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1.3.3. Tnh cht sinh hc ca chitosan [5]Chitosan khng c, dng an ton cho ngi [18]. Chng c tnh ho hp

sinh hc cao vi c th [19], c kh nng t phn hu sinh hc [20]. 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 [21]. Ngoi ra, chitosan cn c tc dng lm gim cholesterol v lipid mu, h huyt p [22], iu tr thn mn tnh, chng ri lon ni tit [23]. Vi kh nng thc y hot ng ca cc peptit - 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 cc t bo u, ung th, HIV/AIDS, chng tia t ngoi, chng nga ca chitosan [24].

1.3.4. c tnh ca chitosan [5]Vo nm 1968, K. Arai v cng s xc nh chitosan hu nh khng c, ch s LD50 = 16g/kg cn nng c th, khng gy c trn sc vt thc nghim v ngi, khng gy c tnh trng din [25]. Nghin cu tim chitosan theo ng tnh mch trn th, cc tc gi kt lun: chitosan l vt liu ho hp sinh hc cao, n l cht mang l tng trong h thng vn ti thuc, khng nhng s dng cho ng ung, tim tnh mch, tim bp, tim di da, m cn s dng an ton trong ghp m [25]. Dng chitosan loi trng lng phn t trung bnh thp tim tnh mch, khng thy c tch lu gan. Loi chitosan c DD 50%, c kh nng phn hu sinh hc cao, sau khi tim vo bng chut, n c thi tr d dng, nhanh chng qua thn v nc tiu, chitosan khng phn b ti gan v l lch. Nhiu tc gi ch r nhng li im ca chitosan: tnh cht c hc tt, khng c, d to mng, c th t phn hy sinh hc, ha hp sinh hc khng nhng i vi ng vt m cn i vi cc m thc vt, l vt liu y sinh tt lm mau lin vt thng [27].

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1.4. Tnh hnh nghin cu sn xut v ng dng trong thc t ca chitin v chitosan Vit Nam v trn Th gii [3]Trc y ngi ta th chit tch chitin t thc vt bin nhng ngun nguyn liu khng p ng nhu cu sn xut. Tr lng chitin phn ln c ngun gc t v tm, cua. Trong mt thi gian, cc cht ph thi ny khng c thu hi m li thi ra ngoi gy nhim mi trng. Nm 1977 Vin k thut Masachusetts (M), khi tin hnh xc nh gi tr ca chitin v protein trong v tm, cua cho thy vic thu hi cc cht ny c li nu s dng trong cng nghip. Phn protein thu c s dng ch bin thc n gia sc, cn phn chitin s c dng nh mt cht khi u iu ch cc dn xut c nhiu ng dng trong lnh vc cng nghip [16]. Vic nghin cu sn xut chitin - chitosan v cc ng dng ca chng trong sn xut phuc v i sng l mt hng nghin cu tng i mi m nc ta. Vo nhng nm 1978 n 1980 Trng i hc Thu sn Nha Trang cng b quy trnh sn xut chitin - chitosan ca k s Minh Phng, nhng cha c ng dng c th trong sn xut. Gn y trc yu cu x l ph liu thu sn ng lnh ang ngy cng cp bch, trc nhng thng tin k thut mi v chitin - chitosan cng nh tim nng th trng ca chng thc y cc nh khoa hc ca chng ta bt tay vo nghin cu hon thin quy trnh sn xut chitin - chitosan bc cao hn, ng thi nghin cu cc ng dng ca chng trong cc lnh vc sn xut cng nghip. Hin nay Vit Nam c nhiu c s khoa hc ang nghin cu sn xut chitin - chitosan nh: Trng i Hc Nng Lm - thnh ph H Ch Minh; Trung tm nghin cu polyme - Vin Khoa Hc Vit Nam; Vin Ho thuc phn Vin Khoa Hc Vit Nam ti thnh ph H Ch Minh; Trung tm cng ngh v sinh hc Thu sn - Vin nghin cu nui trng Thu sn 2. min Bc, Vin Khoa Hc Vit Nam kt hp vi X nghip thu sn H Ni sn xut chitosan v ng dng trong lnh vc nng nghip ng la

Thi Bnh v thu c mt s kt qu ng khch l. min Nam, Trung tm cng ngh v sinh hc thu sn phi hp vi mt s c quan khc: i Hc Y Dc thnh ph H Ch Minh, phn Vin Khoa HcNguyn c Duy Lp CN Ho Dc v Ho Cht BVTV K47 12

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http://www.ebook.edu.vn Vit Nam, Vin Khoa Hc nng nghip min nam, ang nghin cu sn xut

v ng dng chitin - chitosan trong lnh vc: nng nghip, y dc v m phm. Trong nng nghip, chitosan c s dng bo v cc ht ging nhm mc ch ngn nga s tn cng ca nm trong t, ng thi n cn c tc dng c nh phn bn, thuc tr su, tng cng kh nng ny mm ca ht [7]. Qua nghin cu nh hng ca chitosan v cc nguyn t vi lng ln mt s ch tiu sinh ho ca m la nhit thp th kt qu nghin cu cho thy chitosan vi lng lm tng hm lng dip lc v hm lng nit; ng thi hm lng cc enzym nh amylaza, catalaza hay peroxidaza cng tng ln. Ngy nay chitosan cn c dng lm nguyn liu b xung vo thc n cho tm, c, cua kch thch sinh trng. Nhng ng dng ca chitin - chitosan v nhng dn xut ca chng ngy cng pht trin. Mt s a vo ng dng nh l: ch khu t hu, da nhn to [4], thu knh chit xut, v mt s ng dng khc cn ang nghin cu nh: tc ng kch thch min dch, chng s pht trin ca khi u, c tnh lm gim cholesterol mu [10], tr bng nhit [11] Da nhn to c ngun gc t chitin, n ging nh mt tm vi v c bc p ln vt thng ch mt ln n khi khi. Da nhn to b phn hu sinh hc t t cho n lc hnh thnh lp biu b mi. N c tc dng gim au, gip cho cc vt so bng phc hi biu b nhanh chng. Trng i Hc Dc H Ni, i Hc Y H Ni, Trung tm khoa hc t nhin v cng ngh Quc gia cng ch to thnh cng loi da nhn to ny v bc u ng dng c hiu qu. Chitin - chitosan v cc oligome ca n c c tnh min dch do n kch thch cc t bo gi nhim v bo v min dch vi cc t bo khi u v cc tc nhn gy bnh. Nhng nghin cu gn y hng vo cc oligome, N-acetyl-glucosamin v glucosamin, cc cht ny c mt s tnh cht ca cc polyme tng ng nhng li c u th l tan tt trong nc do d dng c hp th. Hin nay trn th gii thnh cng vic s dng chitosan lm cht mang c nh enzym v t bo. Enzym c nh cho php m ra vic s dng rng ri enzym trong cng nghip, y hc v khoa hc phn tch. Enzym c nh c

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http://www.ebook.edu.vn s dng lu di, khng cn thay i cht xc tc. Nht l trong cng ngh lm

sch nc, lm trong nc hoa qu, s dng enzym c nh rt thun li v t hiu qu cao. Chitosan tho mn yu cu i vi mt cht mang c phn t lng ln, bn vng khng tan v n nh vi cc yu t ho hc. Do c cu trc tng t nh xellulose nn chitosan c nghin cu b sung vo lm nguyn liu sn xut giy. Chitosan lm tng bn dai ca giy, ng thi vic in trn giy cng tt hn. Trong sn xut giy qua nghin cu ngi ta thy nu b sung 1% chitosan th bn ca giy tng ln khi b t hay tng nt khi in [5]. C th thay h tinh bt bng chitosan h vi, n c tc dng lm t si bn, mn, bng p, c nh hnh in, chu c axit v kim nh. Chitosan kt hp vi mt s thnh phn khc sn xut vi chu nhit, vi chng thm, sn xut vi col Chitosan c s dng sn xut kem chng kh da do tnh cht ca chitosan l c th c nh d dng trn biu b ca da nh cc nhm NH+4. Cc nhm ny lin kt vi cc t bo sng ha ca da, nh vy m cc nh khoa hc nghin cu s dng chitosan lm cc loi kem dng da chng nng. Nh kh nng lm ng t cc th rn l lng giu protein v nh kh nng kt dnh tt cc ion kim loi nh Pb, Hg do chitin c s dng ty lc ngun nc thi cng nghip t cc nh my ch bin thc phm. Chitosan s dng chng hin tng mt nc trong qu trnh lm lnh, lm ng thc phm. Do chitosan c tnh cht dit khun, do n c to thnh mng mng bao gi thc phm chng m mc, chng mt nc. c tnh dit khun ca chitosan c th hin trn cc mt sau : Khi tip xc vi thc phm chitin - chitosan s ly i t cc vi sinh vt ny cc ion thit yu, v d nh ion Cu2+. Nh vy vi sinh vt s b cht do s mt cn bng lin quan n cc ion thit yu. Ngn chn ph hoi chc nng mng t bo. Gy ra s r r cc phn bn trong t bo.

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http://www.ebook.edu.vn Nh vy vic dng cht chitosan bao bc quanh b mt thc phm c th

ko di thi gian bo qun, gim s h hng do kh nng khng nm, khng khun ca n.

1.5. Cu trc ha hc, tnh cht l ha ca glucosaminKhi thy phn chitin trong mi trng axit HCl m c, cc mi ni amid v osid u b ph hy do thu c glucosamin 3 (l monome ca chitosan). Yu t nng axit v nhit thy phn rt quan trng, nu nng ca axit khng thch hp th qu trnh deacetyl ha v deosid ch dng li gii hn nht nh, nu nhit khng thch hp th sn phm cui cng l glucosamin c th b ging ha thnh nhng phn t n gin hn. Cng thc cu to ca glucosamin:NH2 OH OHC OH H C OH H C NH2 HO C H H C OH H C CH2OH O HO HO NH2 OH OH O OH CH2OH O OH OH

3OH

OH NH2

Tn UIPAC: (3R,4R,5S,6R)-3-amino-6-(hydroxymethyl)oxane-2,4,5-triol Tn gi khc: 2-Amino-2-deoxy-D-glucose; 2-amino-2-deoxy--Dglucopyranose; chitosamine; D-glucosamine; D-(+)- glucosamine. Cng thc phn t: C6H13O5N Phn t lng: Mglucosamin = 179,17

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Hnh 4 : Cu trc khng gian ca glucosamin Glucosamin l cht rn dng tinh th, khng mu, khng mi, im nng chy 880C, im phn hy 1100C, tan c trong nc v trong methanol si, hi tan trong methanol hoc ethanol, khng tan trong ether v chloroform. Mt s phn ng ca glucosamin [14]: Phn ng trng bc: Ging nh glucose, glucosamin cho phn ng trng bc kh r rng:C5H12O4NCHO + 2[Ag(NH3)]OH 500C + 3NH3 + H2O

C5H12O4NCOONH4 + 2Ag

Phn ng vi Cu(OH)2. Sn phm c mu xanh nht.CHO CHNH2 2 CHOH + Cu(OH)2 CHOH CHOH CH2OH CHO CHNH2 CHO CHO CHOH CH2OH Cu H CHO CHNH2 H OHC OHC CHOH CH2OH

+ 2 H2O

Phn ng vi C6H5CH=O. Phn ng xc nhn c mt ca NH2. Baz Shiff to ra di dng keo snh mu nu:

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Nghin cu iu ch glucosamin t v tmCHO CHO + H2SO4d 500C CHN CHOH CHOH CHOH CH2OH

http://www.ebook.edu.vnCHO CHNH2 CHOH CHOH CHOH CH2OH

H C + H2O

1.6. Cu trc ha hc v tnh cht vt l mt s mui ca glucosaminTrong thc t, mt s dn xut ca glucosamin c s dng lm nguyn liu lm thuc.

1.6.1. Glucosamin hydroclorua.Cng thc cu to:NH2 OHC OH HCl OH OH OH

4

Tn gi khoa hc: 2-amino-2-deoxy-D-glucopyranose hydrochloride; Chitosamine hydrochloride; D-glucosamine hydrochloride; D-(+)-glucosamine hydrochloride. Cng thc phn t: C6H13O5N.HCl Phn t lng: Mglucosamin.HCl = 215.63 Glucosamin hydroclorua l cht rn dng tinh th mu trng, khng mi. im nng chy 190-1940C hoc 3000C Tan c trong nc 0,1g/ml

1.6.2. Glucosamin sulfatCng thc cu to:NH2 OHC OH OH H2SO4 OH OH

5

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http://www.ebook.edu.vn Tn gi khoa hc: 2-Amino-2-deoxy-D-glucose sulfate; D-glucosamine

sulfate. Cng thc phn t: C6H13O5N.H2SO4 Phn t lng: Mglucosamin.H2SO4 = 277,24

1.6.3. Acetyl glucosaminCng thc cu to:NHAc OH OHC OH OH OH

6

Tn gi khoa hc: 2-Acetoamido-2-deoxy-D-glucopyranose; N-acetyl-D(+)-glucosamine; N-acetyl--D-glucosaminide; N-acetylchitosamine. Cng thc phn t: C8H15O6N Phn t lng: Macetylglucosamin = 221,21 im nng chy: 201 - 2100C Tan c trong nc, 0,1g/ml

1.7. Dc l v dc ng hc ca glucosamin v mui ca n [9]Glucosamine l mt aminomonosacharit c thy trong t nhin, nguyn liu tng hp proteoglycan. Glucosamin kch thch t bo sn khp tng tng hp v trng hp nn cu trc proteoglycan bnh thng. Kt qu ca qu trnh trng hp l to ra mucopolysaccharit, thnh phn c bn to nn sn khp. Hnh 5 : au xng ct sng Bnh thng sn khp c cu to ch yu bi nc, collagen v proteoplycan.

Glucosamin ng thi c ch cc enzym ph hy sn khp nh collagenase, phospholipase A2 v gim cc gc t do superoxid ph hy cc t bo sn. Glucosamin cn c tc dng kch thch sinh sn Hnh 6 : Khp niNguyn c Duy Lp CN Ho Dc v Ho Cht BVTV K47 18

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http://www.ebook.edu.vn m lin kt ca xng, gim qu trnh mt canxi ca xng [ 9].

Khi thiu glucosamin th sn c bit l sn khp hng, u gi b hng, cng, to gai xng gy bin dng khp lm hn ch vn ng, dn n bnh vim xng khp pht trin. Do glucosamin lm tng sn xut cht nhy dch khp nn tng nht, kh nng bi trn ca dch khp, v th khng nhng lm gim triu chng ca thoi ha khp (au, kh vn ng) m cn ngn chn qu trnh thoi ha khp, ngn chn bnh tin trin [9]. Thuc tc ng vo c ch sinh bnh ca thoi ha khp, iu tr cc bnh thoi ha xng khp c cp v mn tnh, ci thin chc nng khp v ngn chn bnh tin trin, phc hi cu trc sn khp. Hnh 7 :Cc v tr khp ni

T tui 45 - 50 tr ln, bnh c chiu hng tng (27% tui 60 - 70%, 45% tui 80). i tng nguy c d mc bnh khp nht l ngi gi, ngi bo ph, ngi b chn thng khp, c d tt bm sinh, bnh v chuyn ho, di truyn hoc b xo trn v kch t [1]. Cc mui ca glucosamin c kh nng gii phng v sn sinh mucopolysacharit khuch tn tt vo dch khp, pht huy tt tc dng chng vim khp. Cc dn xut glucosamin thng dng c dng ph bin ngy nay l: Glucosamin hydroclorua. Glucosamin sulfat. Acetyl glucosamin. Trong thc nghim trn th con, ngi ta thy glucosamin ch lm tng thnh phn cc proteoglycan trong cc v tr m sn h cn phi sa cha m khng c tc dng tng t trn phn sn khp bnh thng. Nhng nm gn y, glucosamin c dng rng ri trong iu tr vim khp, thoi ha khp. Tuy nhin, theo mt nghin cu mi ca tin s RonaldNguyn c Duy Lp CN Ho Dc v Ho Cht BVTV K47 19

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http://www.ebook.edu.vn Tallarida thuc trng i Hc Y khoa Temple, Philadelphia (M) cho thy nu

s dng glucosamin n c s khng c hiu qu chng au. Nhng nu kt hp vi mt loi thuc nhm khng vim khng steroid (NSAID) th tc dng chng au, chng vim tng ln rt nhiu. Thc t lm sng cho thy n mang li nhiu u im trong iu tr hn hn cc thuc NSAID. Nhc im ca thuc NSAID l c nhiu tc dng ph, cn khi s dng glucosamin th li rt t tc dng ph. Mt vi d ng khng ng k i vi ngi c c a qu mn cm vi thuc. Trc y glucosamin c xp vo nhm thuc bo v sn (gm c glucosamin, chondroitin v diacerin) hay thuc tc dng chm vi cc bnh vim khp. Hin nay c quan Dc phm Chu u (EMEA) chp nhn xp glucosamin vo danh mc thuc gip ci thin cu trc trong bnh vim khp. Cc loi khc cha c chp nhn v khng p ng c cc yu cu trn lm sng. Vit Nam hin nay cng ang s dng mt s loi thuc c cha glucosamin nh : Lubrex, Lubrex-F, Glucosamin, Glusivac,[9]. Nhng cc thuc ny ch yu vn nhp khu t nc ngoi. c rt nhiu nghin cu th nghim so snh glucosamin vi cc loi thuc NSAID, cho kt qu nh sau [8]: 1. Ci thin triu chng vim khp tng ng vi NSAID trong thi gian ngn v vt tri hn hn nu ung thuc thi gian di.Hnh 8 : Mt s thuc glucosamin hin ang lu by trn th trng Vit Nam

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http://www.ebook.edu.vn 2. Tnh an ton hn hn vi cc loi i t NSAID.

3. Ngi ta dng phi hp glucosamine v NSAID cho kt qu tt hn khi dng n c NSAID trong thi gian ngn. Sau ngng s dng NSAID, tip tc s dng glucosamin th tnh trng ci thin vn tip tc c duy tr theo kiu tuyn tnh. 4. Ngi ta thy nu dng NSAID, nhng ch li gim triu chng cho bnh nhn s nhanh chng mt i ngay sau khi ngng thuc. Ngc li, ngng ung glucosamin tc dng vn tip tc ko di trong nhiu thng sau . 5. Vi nhng bnh nhn tun th phc iu tr dng glucosamin cng di th li ch kinh t cng ln v tnh an ton v hiu qu ca n cng c pht huy. Theo cc nh nghin cu khi b hp thu vo d dy, mui glucosamin sulfat b ion ha hon ton do nng tng i ln axit HCl (pH = 1 - 3) sn c trong d dy. Nh mt tt yu, cc ion glucosamin v cc ion sulfate b trn ln vi mt lng ln ion Cl- v ion H+. Nu kho st hn hp ny v tch loi mui glucosamin ra th li nhn c 99% mui glucosamin hydroclorua do mui dng sulfate b mt ng k do nng rt thp so vi nng rt ln axit HCl c mt trong d dy.

1.8. Mt s quy trnh sn xut chitin, chitosan trn th gii v ti Vit Nam 1.8.1. Trn th gii 1.8.1.1. Quy trnh thy nhit Yamasaki v Nacamichi (Nht bn) [3]Nguyn liu l v cua kh, sch c em kh khong bng HCl 2N trong thi gian l 1 gi (tc gi cho rng hiu qu kh khong c th t c 100%). Sau em ra sch v em lm kh. Tip theo l tin hnh kt hp hai cng on kh protein v deacetyl ng thi trong dung dch NaOH 15N nhit 1500C trong 1 gi, kt qu cho thy protein c tch ra trit v deacetyl t trn 70%. Sau thi gian 1 gi em ra sch v lm kh s thu c chitosan thnh phm.Nguyn c Duy Lp CN Ho Dc v Ho Cht BVTV K47 21

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http://www.ebook.edu.vn Phng php ny c u im l quy trnh n gin 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 cng c nhc im l sn phm chitosan thu c c nht thp, tiu tn nhiu nng lng cho cc khu sn xut.

1.8.1.2. Quy trnh sn xut chitin ca Hackman [3]V tm hm c ra sch v sy kh nhit 1000C; tip theo c kh khong bng HCl 2N vi t l w/v=1/10 nhit phng, sau thi gian 5 gi em ra trung tnh v sy kh 1000C, v em xay nh. Ngm tip trong dung dch HCl 2N vi t l w/v=1/2,5 nhit phng. Sau 48 gi em ly tm thu phn b v em ra trung tnh. Ngm b bt ra trong dung dch NaOH 1N vi t l w/v= 1/2,5 nhit 1000C, sau 42 gi em ly tm thu phn b. Sau li tip tc ngm trong NaOH 1N vi t l v nhit trn, sau 12 gi em ly tm thu phn b. Tip theo ra trung tnh v em lm sch bng cch ly tm vi cc cht theo th t: nc, etanol v ete. Sau lm kh ta c sn phm dng bt mu kem. Vi quy trnh ny th c nhiu cng on tng kh nng kh khong, kh protein song do cng knh, ch thch hp cho i tng l v tm hm, tm m ni v v cua, thi gian thc cc cng on ko di, do quy trnh Hackman ch mang tnh nghin cu th nghim, khng c tnh kh thi nu sn xut i tr, quy trnh mi ch dng li n sn phm l chitin.

1.8.1.3. Quy trnh sn xut chitosan ca Php [3]Nguyn liu (v tm) sch c em i hp chn, phi kh, ta em i xay nh. Kh proteinn bng NaOH 3,5% vi t l w/v=1/10, nhit 650C, sau 2 gi vt ra ra trung tnh, tip ngm trong HCl 1N vi t l w/v=1/10, nhit phng sau 2 gi vt ra tin hnh ty cc cht mu hu c bng aceton vi t l w/v=1/5, nhit phng sau 30 pht vt ra ra sch v ty mu li bng nc javen (NaOCl + NaCl) 0,135%, t l w/v=1/10, nhit phng sau 6 pht vi ra ra trung tnh, thu c chitin sch p. Sau tin hnh deacetyl chitin bng NaOH 40% vi t l w/v=1/4, nhit 850C sau thi gian 4 gi em ra trung tnh, thu c chitosan.Nguyn c Duy Lp CN Ho Dc v Ho Cht BVTV K47 22

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http://www.ebook.edu.vn Quy trnh c u im l thi gian sn xut ngn, sn phm c mu sc p,

sch do c hai bc kh sc t. Tuy nhin NaOCl l mt cht oxy ha mnh, nh hng n mch polyme, do nht sn phm gim r rt. Mt khc aceton c gi tr t tin, tn tht nhiu, gi thnh sn phm s cao. Cha k cc yu t trong an ton sn xut, cng ngh ny kh p dng trong iu kin sn xut nc ta hin nay.

1.8.1.4. Phng php iu ch chitin ca Capozza [17]Cn 149g nguyn liu v tm sch cho vo bnh khuy vi mt my khuy, thm t t 825ml axit HCl 2N vo bnh, thc hin phn ng 40C trong thi gian 48 gi. Sn phm sau qu trnh kh khong c ra sch bng nc n pH = 7. Xc nh hm lng tro 0,4 - 0,5%. Sau sn phm c khuy nhit phng vi 1500ml axit fomic HCOOH 90%, qua m. Hn hp c lc ly tm ly phn b v ra li vi nc nhiu ln cho n khi pH = 7. Sn phm sch sau c ngm ngp trong 2 lt dung dch NaOH 10% v un nng 90 - 1000C trong 2,5 gi. Dung dch c lc v ra sch vi nc n pH = 7, sau sn phm c trng ra li trong ethanol 960 v ether. Sy kh 400C di p sut gim. Khi lng chitin kh sch thu c l 66g. Hiu sut 44,3%.

1.8.2. Ti Vit Nam 1.8.2.1. Quy trnh sn xut chitosan ca Minh Phng [3]Nguyn liu l v tm kh c kh khong bng HCl 6N vi t l w/v=1/2,5, nhit phng, sau 48 gi em ra trung tnh, tip theo un trong NaOH 8% vi t l w/v=1/1,5, nhit 1000C, sau 2 gi kh protein ri em ra trung tnh. Tin hnh ty mu bng KMnO4 1% trong mi trng H2SO4 10%, sau 1 gi em ra sch v kh mu ph bng Na2S2O3 1,5% trong 15 pht, vt ra ra sch thu c chitin. Deacetyl chitin bng NaOH 40% vi t l w/v=1/1, nhit 800C sau 24 gi em ra sch v cui cng thu c chitosan. Sn phm c cht lng kh tt, chitin c mu trng p. Song thi gian cn di, s dng nhiu cht oxy ha d lm nh hng ti nht ca sn phm.

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1.8.2.2. Quy trnh sn xut chitosan Trung tm cao phn t Vin Khoa Hc Vit Nam [3]Nguyn liu l v gh hay v tm sch c kh khong ln 1 bng HCl 4% nhit phng, sau thi gian 24 gi em ra trung tnh lm gim lng NaOH tiu hao cng on sau. Nu trong NaOH 3% nhit 90 - 950C, sau 3 gi em ra trung tnh, tip tc kh khong ln 2 bng HCl nhit phng, sau 24 gi em di ra trung tnh v em nu ln 2 trong NaOH nhit 90 - 950C, sau 3 gi em ra trung tnh. Cui cng nu trong NaOH 40%, ra trung tnh v sy kh thu c chitosan. Sn phm chitosan sn xut theo quy trnh ny c mu sc khng p bng sn phm theo quy trnh ca k s Minh Phng, thi gian thc hin quy trnh ko di, nhiu cng on.

1.8.2.3. Quy trnh sn xut chitin ca X nghip thy sn H Ni [3]Nguyn liu l v tm kh hoc ti c loi b ht tp cht, x l tch khong ln 1 trong HCl 4%, t l w/v=1/2, nhit phng sau 24gi vt ra ra trung tnh. Sau dng NaOH 2% tch protein ln 1 vi t l w/v=1/2,8 nhit 90=950C, sau 3 gi ra v tin hnh kh khong ln 2 cng dng HCl 4%, t l w/v=1/2, nhit phng sau 24 gi em ra trung tnh. tch protein ln 2 cng dng NaOH 2%, w/v=1/2,8, nhit 90=950C, sau 3 gi ra trung tnh v tin hnh kh khong ln 3 cng ging nh ln kh khong trn. Sn phm em lm kh thu c chitin. Chitin thu c c trng cao mc d khng c cng on ty mu. Nhng nhc im l thi gian sn xut ca quy trnh ko di, nng ha cht x l cao kt hp vi thi gian x l di (cng on kh khong) lm ct mch polyme trong mi trng axit dn n nht gim.

1.8.2.4. Quy trnh sn xut chitosan theo phng php sinh hc kt hp ha hc [3]Vic sn xut chitosan theo phng php sinh hc kt hp ha hc cng thc hin theo cc bc : kh khong, kh protein v deacetyl.

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http://www.ebook.edu.vn Cng on kh khong hiu qu nht v duy nht ch thc hin bng

phng php ha hc. Cng on kh protein v deacetyl c th thay th bng phng php sinh hc, l kh protein bng proteaza v deacetyl bng enzym deacetylaza. V tm c ngm trong HCl 10% t l w/v = 1/10, nhit phng trong thi gian 5gi. Ra sch n pH= 7. Sau kh protein bng papain 13%, t l w/v = 1/5, pH = 5 - 5,5, nhit 70 - 800C trong thi gian 4 gi. Ra sch, ty mu v sy kh 600C thu c chitin kh, trng. Deacetyl chitin bng NaOH 35%, t l w/v = 1/10, 900C trong thi gian 5,5 gi. Ra sch v sy kh thu c chitosan sch. Chitosan thu c c mu sc trng, p, trong v mm mi. Quy trnh papain cho sn phm c nht cao hn cc quy trnh khc. Tuy nhin iu kin kh khn do vic tm mua hoc sn xut enzym deacetylaza nn cng on deacetyl c thc hin bng vic nu NaOH m c.

1.9. Quy trnh sn xut glucosamin hydroclorua (glu.HCl) 1.9.1. Quy trnh sn xut glu.HCl ca Trn Th Luyn [3] kh khong, v tm c ngm trong HCl 10%, t l w/v = 1/10, nhit phng trong thi gian 5 gi, sau vt ra ra sch n pH=7. Sau kh protein kt hp deacetyl ha trong NaOH 40%, t l w/v = 1/10, nhit 95 - 1000C trong thi gian 6,5 gi. Ty mu ra sch, sy kh. Chitosan c un trong HCl 35%, t l w/v = 1/4, nhit 95 - 1000C trong thi gian 4 gi. Sau lc b cn, lm lnh 0 - 20C trong thi gian 2 gi khi kt tinh s xut hin. Lc tch kt tinh, ha tan trong nc ct, kh mu qua than hot tnh, c cn v li thc hin kt tinh. Sau khi kt tinh ln cui (khong 3 ln), tinh th trng, lc ly tinh th em ra li bng cn, sau sy kh 50 - 600C.

1.9.2. Quy trnh sn xut glu.HCl ca nh Rng [14]V tm kh, sch, nghin thnh bt, sau un si nguyn liu vi nc trong 2 gi v gn b protein, sy kh. Nguyn liu kh un trong HCl 5% tch khong, ra sch n pH=7 v sy kh. Kh hon ton protein trong NaOH 5%, un si. Sn phm ra sch n pH=7, sy kh. Chitin thu c c mu trngNguyn c Duy Lp CN Ho Dc v Ho Cht BVTV K47 25

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http://www.ebook.edu.vn pht hng. T chitin chuyn ha thnh glucosamin bng cch thy phn trong

dung dch axit HCl m c 36%, nhit 1000C, ty mu bng than hot tnh, kt tinh v lc. Sy kh, thu c tinh th glucosamin hydroclorua trng. Hiu sut qu trnh 51,4%.

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Chng II : I TNG V PHNG PHP NGHIN CU

2.1. i tng nghin cu*Vt liu: V tm *Ha cht: S dng cc loi ha cht tinh khit nh axit hydroclorua (HCl), natri hydroxit (NaOH), kali permanganat (KMnO4), axit oxalic (C2H2O4), oxi gi (H2O2), axit acetic CH3COOH, ngoi ra cn c cc dung mi khc nh nc ct, methanol, ethanol *Dng c th nghim: Bnh tam gic, bnh cu c nhm, cc thy tinh, cn phn tch, nhit k, thit b o im nng chy, t sy, bnh ht m, thit b gia nhit, khuy t, sinh hn hi lu, dng c lc

2.2. Phng php nghin cu1. S dng cc phng php chit tch ha hc tch chit v tm thu c chitin v deacetyl ha chitin trong dung dch NaOH m c thu c chitosan. 2. Tng hp glucosamin hydroclorua t chitin v chitosan. 3. Kho st mt s yu t nh hng ti phn ng nh: thi gian phn ng, nhit phn ng v nng tc nhn tc nhn phn ng. 4. Kim tra cu trc ca sn phm iu ra thng qua cc loi ph nh : IR,1

H-NMR,

13

C-NMR ti Vin Ha Hc Vin Khoa Hc Vit Nam. So o ph IR trn my FTR IMPACT 410 ca hng Nicolet. o ph 1H NMR v13

snh vi cc s liu ph cng b xc nh cu trc sn phm. -

C NMR trn my Bruker AC-

500MHz trong dung mi D2O.

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http://www.ebook.edu.vn 5. Kim tra, o mt s thng s nh nhit nng chy trn my Boetius

MK (c). 6. Xc nh m bng phng php sy n khi lng khng i. 7. Phong php xc nh hm lng cht khng tan. a) Nguyn l: Chitosan tan c trong axit acetic long, cn chitin v cc tp cht khc thi khng tan. b) Tin hnh : Cn chnh xc a gam chitosan ho tan trong dung dch axit acetic 1%, khuy o 60 pht cho chitosan tan ht ri lc, ra cn bng axit acetic long, sau ra li bng nc ct v em sy kh n khi lng khng i. Hm lng cht khng tan c tnh theo cng thc:X (%) = A B * 100(%) a

Trong : A : khi lng phu lc + giy + tp cht sau khi sy (g). B : khi lng phu lc + giy lc trc khi sy (g). a : khi lng chitosan s dng (g). X : Hm lng cht khng tan (%). 8. Phng php xc nh hiu sut qu trnh bng phng php trng lng.

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Chng III : THC NGHIM V KT QU

3.1.Quy trnh iu ch chitin, chitosan v glucosamin hydroclorua (glu.HCl)V tm sch Loi Khong Ngm trong HCl 10%, Ts = 12 gi, T0 = t0 phng

Ra trung tnh Loi Protein un trong NaOH 3%, Ts = 3,5 - 4 gi, T0 = 90 - 950C

Ra trung tnh

1.Ngm trong hn hp KMnO4 + C2H2O4. Hoc 2.Ngm trong H2O2 1%. Ra sch, sy kh T0 = 600C Chitin trng sch

un trong NaOH 50%, Ts = 4 gi, T0=110 - 1200C Ra trung tnh, sy kh Chitosan un trong HCl 36%, Ts= 4 gi, T0 = 90-95% Ty mu, lc, sy kh Glucosamin hydroclorua

S 3.1: Quy trnh iu ch chitin, chitosan v glu.HCl t v tmNguyn c Duy Lp CN Ho Dc v Ho Cht BVTV K47 29

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3.2. iu ch chitin t v tmCn 40g v tm kh cho vo cc 1lt, sau cho t t 200ml HCl 10% vo v ngp ton b v tm, nhn thy c hin tng si bt mnh chng t c phn ng xy ra, kim tra pH=1 - 2 l c, ngm trong khong 12 gi ( qua m) v nhit phng, thnh thong li khuy cho phn ng xy ra nhanh hn (n khi khng thy bt, kh thot ra, th pH vn cn axit). Sau 12 gi, vt v tm ra v ra li bng nc thng n pH=7, sau ra bng nc ct. Lc ny v tm c mu hng nht v mm do c loi ht cc tp cht v c. V tm thu c ro bt nc sau cho vo bnh cu 500ml, ng thi cho NaOH 3% vo cho n khi ngp hon ton v tm v kim tra pH= 1112 l t yu cu. un nhit 90 - 950C trong khong 3,5 - 4 gi, lc un c lp sinh hn trnh hin tng tro bt ra ngoi v mi kh chu. Sau khi phn ng kt thc em sn phm i ra bng nc thng n pH=7, sau trng li bng nc ct. Sn phm thu c c mu trng pht hng. Cht mu (astaxanthin) c trong chitin c loi b theo 2 phng php: 1. Cho 10ml dung dch KMnO4 1% vo trong bnh ng chitin trn, sau trn u vi nhau, trong thi gian 1 gi nhit phng. Sau 1 gi, ra sch sn phm, nhn thy sn phm c mu tm en chng t vn cn nhiu KMnO4 cha c ra sch. loi ht KMnO4 ta dng 50ml axit Oxalic HOOC-COOH 1%, nhn thy nu nhit phng th kh loi ht mu tm, ta em un nng nhit 50 - 600C th mu mt hon ton, ra sch thu c sn phm c mu trng p. 2. Ngm ngp chitin bng 60ml H2O2 1%, qua m nhit phng. Sn phm sau khi ngm tuy mu bt nhiu nhng vn cn thy hi pht hng, ra sch bng nc, trng li bng nc ct. Sy kh sn phm nhit 600C, thu c chitin trng sch. Hiu sut qu trnh iu ch chitin c tnh theo cng thc sau:

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Nghin cu iu ch glucosamin t v tmH1 (%) = m2 *100% m1


Trong : m1: s gam ca v tm ban u (g). m2: s gam chitin thu c (g). H1(%) : hiu sut ca qu trnh (%). Kt qu thu c nh bng 3.1: Bng 3.1. Hm lng chitin trong v tm STT 1 2 3 4 5 V tm (g) 40 40 40 40 40 Chitin (g) 10,87 13,67 12,27 14,33 13,09 Hiu sut (%) 27,18 32,73 30,68 35,83 34,65

3.3.iu ch chitosan bng cch deacetyl chitinT chitin iu ch trn, cn lng chnh xc 10g chitin cho vo bnh cu 500ml, sau cho 350ml dung dch NaOH 50% cho n ngp hon ton chitin. un nhit 110 - 1200C, c hi lu trnh bay hi dung mi, bay mi kh chu, thnh thong c khuy cho phn ng xy ra nhanh, thi gian un l 4 gi. Sn phm sau phn ng c ra sch n pH=7, ra li bng nc ct, sy kh. Sn phm thu c l chitosan c mu trng. Hiu sut qu trnh iu ch chitosan c tnh theo cng thc:H 2 (%) = m4 *100% m3

Trong : m3: khi lng chitin tham gia phn ng (g). m4 : khi lng sn phm chitosan thu c(g). H2(%) : hiu sut ca qu trnh (%). Kt qu thu c bng 3.2:

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http://www.ebook.edu.vn Bng 3.2. Hiu sut iu ch chitosan t chitin

STT 1 2 3 4 5

Chitin (g) 10 10 10 10 10

Chitosan (g) 6,33 6,56 7,23 7,0 7,47

Hiu sut (%) 63,3 65,6 72,3 70,0 74,7

Tinh ch chitosanCn 5g chitosan cho vo cc thy tinh 1lt, sau cho 300ml axit acetic CH3COOH 1%, khuy nhit thng trong thi gian 30 pht thu c dung dch trong sut. Dung dch c em i lc b cn bn. Sau cho 30 ml NaOH 10% vo, khuy u trong 10 pht thu c dung dch keo. Lc ly kt ta keo v ra 3 ln bng methanol (5ml/ln). Cho kt ta keo bo bnh cu 250ml, thm 140ml CH3OH v un nhit 60 - 650C trong thi gian 5 gi. Lc v ra bng aceton 2 ln, sau sy kh 400C. Thu c 4,2g chitosan tinh khit. Hiu sut 84%.

3.4.iu ch glu.HCl 3.4.1.iu ch glu.HCl t chitinCn 5g chitin kh ( ct nh) cho vo bnh cu 250ml, sau thm 40ml HCl 36%. un cch thy c lp sinh hn hi lu, khuy u v duy tr nhit khong 90 - 950C. Khi nhit hn hp trong bnh phn ng t 65 - 700C th chitin bt u tan rt nhanh to thnh dung dch mu nu en. un trong thi gian 4gi. Kt thc thi gian phn ng, sn phm c lc nng loi b cn bn, sau dch lc c ty mu bng 1g than hot tnh, giai on ny c hn hp c un nng n 900C, sau duy tr nhit 50 - 600C trong thi gian 30 pht.

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http://www.ebook.edu.vn Lc b than hot tnh thu c dung dch c mu xanh nht trong sut,

ngui thu c dung dch c mu vng rm. Dung dch ngui lnh nhit 0 - 40C, thi gian kt tinh l 12 gi ( qua m). Tinh th glu.HCl c tch ra bng phng php lc, sau ra li sn phm thu c bng cn 960. Sn phm c sy kh nhit 600C th thu c tinh th glu.HCl kh trng p. Dch lc cn li sau khi lc kt tinh c c bt dung mi bng ct chn khng nhit 60 - 650C, n khi th tch bng 1/3 th tch em c ban u, kt tinh v lc ly kt tinh, ra bng cn 960, sy kh. Hiu sut ca qu trnh iu ch glu.HCl t chitin c tnh theo cng thc sau:H 3 (%) = m6 + m7 *100% m5

Trong : m5: khi lng chitin ban u (g). m6 : khi lng glu.HCl kt tinh ln th nht (g). m7: khi lng glu.HCl kt tinh ln th hai (g). H3(%): hiu sut ca qu trnh (%). Kt qu thu c bng 3.3: Bng 3.3. Hiu sut iu ch glucosamin hydroclorua t chitin STT 1 2 3 Chitin (g) 5 5 5 Glu.HCl (g) 3,12 3,14 3,10 Hiu sut (%) 62,4 62,8 62,0

3.4.2.iu ch glu.HCl t chitosanCn 5 gam chitosan cho vo bnh cu 250ml, thm t t 40ml axit HCl 36% vo bnh, sau thc hin cc thao tc tng t nh phn 3.4.1 trn. Tnh thi gian phn ng khi chitosan bt u tan vo dung dch. un c 1 gi th nhn thy dung dch c nht cao, un tip n 3 gi th thu c dung dch c qunh. Kt thc thi gian phn ng, cho thm 20ml nc ct voNguyn c Duy Lp CN Ho Dc v Ho Cht BVTV K47 33

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http://www.ebook.edu.vn pha long dung dch ri em lc ly dung dch nc ci. Dung dch c mu

nu en c ty mu bng than hot tnh. Do c pha long dung dch, lng nc a vo tng i nhiu nn c kt tinh cn phi c bt dung mi n th tch 1/5 th tch dung dch ban u. Sau khi c, dung dch c ngui lnh nhit 0 - 40C qua m. Nhn thy c rt t tinh th glu.HCl c to thnh, hu nh l khng ng k. Lc ly kt tinh, ra li bng cn, sy kh. Hiu sut ca qu trnh iu ch glu.HCl t chitin c tnh theo cng thc sau:H 4 (%) = m9 * 100% m8

Trong : m8 : khi lng chitosan ban u (g). m9 : khi lng glucosamin thu c (g). H4(%) : Hiu sut qu trnh (%). Kt qu thu c bng 3.4: Bng 3.4. Hiu sut iu ch glucosamin hydroclorua t chitosan STT 1 2 3 Chitosan (g) 5 5 5 Glu.HCl (g) 0,5 0.35 0.6 Hiu sut (%) 10,0 7,0 12,0

3.4.3. Tinh ch glu.HClCn 2g glu.HCl cho vo bnh cu 100ml, thm dn 25ml cn 960, un si, c khuy nhit 75 - 800C. Sau khi hn hp si, cho t t nc ct ti thiu vo, cho n khi glu.HCl tan ht (vn duy tr nhit si), lng nc ct ti thiu l 5,5 - 6ml cho 2g glu.HCl. un tip 60 pht, lc nng. kt tinh li nhit 0 - 40C trong thi gian 4 gi. Tinh th c lc v ra bng cn 960 (2 ln x 3ml), sy kh nhit 600C thu c glu.HCl tinh khit. Dung dch nc ci em c bt dung mi v

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http://www.ebook.edu.vn kt tinh li thu thm glu.HCl. Sn phm thu c c nhit nng chy 192 -

1940C. Cu trc ca glu.HCl iu ch ra c o cc loi ph IR, 1H-NMR,13

C-NMR. Ph IR c cc vch c trng sau:

(OHO)

3354,44(cm-1);

(N-H)

3289.88(cm-1); (C-H) 2852,33-2945,58(cm-1); (OH phng) 1422,37(cm-1); (OH khngphng)

1248,95(cm-1); (C-O) 1185,36(cm-1); (C-N) 1029,28(cm-1). -D-glucosamin hydroclorua6OH

4HO HO

5 3

O

2

1OH

NH2.HCl

Ph 1H-NMR (500MHz, D2O, : ppm) 5,42 (d, J = 3,5Hz, 1H, H1); 3,3 (dd, J = 3,5Hz; 10,5Hz, 1H, H2); 3,5 3,9 (m, 4H, H3, H4, H5, H6). Ph 13C-NMR (125MHz, D2O, : ppm) 89,6 (C1); 54,7 (C2); 70,0 (C3); 70,2 (C4); 72,0 (C5); 60,8 (C6). -D-glucosamin hydroclorua4HO HO

6

OH

5 2 3

O OH

1

NH2.HCl

Ph 1H-NMR (500MHz, D2O, : ppm) 4,93 (d, J = 8,5Hz, 1H, H1); 2,98 (dd, J = 8,5Hz; 10,5Hz,1H, H2); 3,2 3,9 (m, 4H, H3, H4, H5, H6). Ph 13C-NMR (125MHz, D2O, : ppm) 93,1 (C1); 57,2 (C2); 72,5 (C3); 76,6 (C4); 70,2 (C5); 60,9 (C6).

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Hnh 9: Ph IR glucosamin hydroclorua

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Hnh 10 : Ph 1H-NMR glucosamin hydrocloruaNguyn c Duy Lp CN Ho Dc v Ho Cht BVTV K47 37

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Hnh 11 : Ph 13C-NMR glucosamin hydrocloruaNguyn c Duy Lp CN Ho Dc v Ho Cht BVTV K47 38

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3.6. Kho st nh hng ca thi gian n hiu sut iu ch glu.HCl t chitinTin hnh phn ng nh mc 3.4.1 lm trong bnh cu 250ml (5 ln) vi mi ln 5g chitin, 40ml HCl 36%, nhit phn ng 90 - 950C nhng thc hin vi iu kin thi gian khc nhau: Ln 1: thi gian phn ng 1gi. Ln 2: thi gian phn ng 2 gi. Ln 3: thi gian phn ng 3 gi. Ln 4: thi gian phn ng 4 gi. Ln 5: thi gian phn ng 5 gi. Kt qu thu c c trnh by trong bng 3.5: Bng 3.5: nh hng ca thi gian n hiu sut ca phn ng: Ts (h) Chitin(g) Glu.HCl(g) H(%) 1 5 2,93 58,6 2 5 2,97 59,4 3 5 3,05 61,0 4 5 3,14 62,8 5 5 3,11 62,2

3.7. Kho st nh hng ca nhit n hiu sut iu ch glu.HCl t chitinTin hnh phn ng nh mc 3.4.1 lm trong bnh cu 250ml (3 ln) vi mi ln 5g chitin, 40ml HCl 36%, thi gian phn ng 4 gi nhng thc hin vi iu kin nhng khong nhit khc nhau. Ln 1: khong nhit 25 - 300C (nhit phng). Ln 2: khong nhit 55 - 600C. Ln 3: khong nhit 90 - 950C. Kt qu thu c c trnh by trong bng 3.6:

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Bng 3.6 : nh hng ca nhit n hiu sut phn ng: T0 (0C) Chitin(g) Glu.HCl(g) Hiu sut (%) 25 - 30 5 55 - 60 5 1,83 36,6 90 - 95 5 3,14 62,8

- : khng xc nh. 3.8. Kho st nh hng ca nng axit HCl n hiu sut iu ch glu.HCl t chitinTin hnh phn ng nh mc 3.4.1 lm trong bnh cu 250ml (3 ln) vi ln 5g chitin, thi gian phn ng 4gi, nhit phn ng 90 - 950C nhng thc hin trong iu kin nng axit hydrocloric khc nhau:

Ln 1: Nng axit HCl 10%. Ln 2: Nng axit HCl 20%. Ln 3: Nng axit HCl 36%.Kt qu thu c c trnh by trong bng 3.7: Bng 3.7.nh hng ca nng axit HCl n hiu sut phn ng. HCl (%) Chitin (g) Glu.HCl (g) Hiu sut (%) 10 5 20 5 36 5 3,14 62,8

- : khng xc nh.

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Chng IV : KT QU V BN LUN

4.1. iu ch chitin t v tm 4.1.1. Qu trnh kh khongTrong v tm, thnh phn khong ch yu l mui CaCO3, MgCO3 v rt t Ca3(PO4)2. Nn ngi ta thng dng cc loi axit nh HCl, H2SO4 kh khong. Khi kh khong, nu dng HCl th cho hiu qu cao hn. Nu dng H2SO4 s to mui kh tan nn t s dng. Phn ng ca HCl kh khong nh sau: MgCO3 + 2HCl = MgCl2 + CO2 + H2O. CaCO3 + 2HCl = CaCl2 + CO2 + H2O. Ca3(PO4)2 + 6HCl = 3CaCl2 + 2H3PO4. Trong qu trnh ra th mui Cl- to thnh c ra tri, nng axit HCl c nh hng ln n cht lng ca chitosan thnh phm, ng thi n nh hng ln ti thi gian v hiu qu kh khong. Nu nng HCl cao s rt ngn c thi gian kh khong nhng s lm ct mch do c hin tng thy phn cc lin kt -(1-4) glucozit to ra cc polyme c trng lng phn t trung bnh thp, c khi b thy phn trit n glucosamin. Ngc li nu nng HCl qu thp th qu trnh kh khong s khng trit v thi gian x l ko di nh hng ti cht lng ca sn phm. T l nguyn liu v dung dch axit HCl cng nh hng ti hiu qu kh khong. V vy, y chng ti chn dung dch HCl 10% vi t l chitin/HCl l w/v = 1/5 v thi gian kh khong l 12 gi (qua m) m bo qu trnh kh khong xy ra hon ton. Sau khi kh khong chng ti tin hnh ra trung tnh, cng on ny c tc dng ra tri ht cc mui, axit d tan trong nc. Qu trnh ra kt thc khi dch ra cho pH=7.

4.1.2. Qu trnh loi b proteinSau khi c v tm loi khong, chng ti tin hnh loi b hon ton protein bng dung dch NaOH 3%, protein b kim thy phn thnh cc amin t

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http://www.ebook.edu.vn do tan v c loi ra theo quy trnh ra tri. Lng NaOH 3% cho vo n khi

ngp ton b v tm v kim tra pH = 11 - 12 l c m bo vic loi b protein c hon ton. un nhit 90 - 950C trong khong 3,5 - 4 gi (trong qu trnh un lu vn tro dung mi do to bt nhiu v mi bay ra kh chu). Sn phm sau khi un c ra sch bng nc thng v nc ct n pH = 7. Tip chng ti tin hnh ra trung tnh, nhm mc ch ra tri ht cc mui natri, cc amin t do v NaOH d. Sy kh 600C, thu c chitin th.

4.1.3. Qu trnh ty mu (loi b astaxanthin)Chitin th thu c c mu hng nht do cn sc t astaxanthin. Do chitin n nh vi cc cht oxy ho nh thuc tm (KMnO4), oxy gi (H2O2), nc javen (NaOCl+NaCl), Na2S2O3, CH3COCH3,li dng tnh cht ny chng ti s dng KMnO4, H2O2 kh mu cho chitin. Cch 1: Dng hn hp KMnO4 1% + C2H2O4 1% ty mu. Trc tin chng ti cho chitin th (phn lp t 40g v tm) vo cc thu tinh, sau cho dung dch KMnO4 1% (10ml) vo, khuy o sao cho ton b chitin u ngm dung dch KMnO4 1%, nhit phng trong thi gian 1 gi. Hn hp c ra sch, sn phm chitin lc ny c mu tm ca KMnO4. loi mu tm chng ti s dng 50ml axit oxalic 1% ((COOH)2), tng nhanh kh nng ty mu chng ti un nng hn hp dung dch nhit 50 - 600C n khi chitin c mu trng hon ton. Theo cch ny mt s tc gi rng sn phm ca phn ng c to ra Mn2+, m trong chitin - chitosan c nhm NHCOCH3 v nhm NH2, do cc nhm ny c th to phc vi Mn2+ gy ra hin tng khu mch lm nh hng n phn ng chuyn ho tip theo. Cch 2: Dng H2O2 1% ty mu. Chitin th (phn lp t 40g v tm) cho vo cc thu tinh, sau cho 60ml dung dch H2O2 1% vo ngp ton b chitin, qua m nhit phng. Chitin thu c c mu trng pht hng. Chng ti dng H2O2 vi nng

do an glucosamin pp hoa hoc 9124

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