Baocao Chitosan Final

8/13/2019 Baocao Chitosan Final

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B GIO DC V O TOI HC NNG

BO CO TNG KT

TI KHOA HC V CNG NGHCP I HC NNG

NG DNG PHNG PHPSINH HC TNG HPCHITOSAN T PH LIUCH BIN THY SN

M s: 2013-02-53

Ch nhim ti: TS. Bi Xun ng

Nng, 12/2013


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B GIO DC V O TOI HC NNG

BO CO TNG KT

TI KHOA HC V CNG NGH

CP I HC NNG

NG DNG PHNG PHPSINH HC TNG HPCHITOSAN T PH LIUCH BIN THY SN

M s: 2013-02-53

Xc nhn ca c quan ch tr ti Ch nhim ti(k, hv tn, ng du) (k, hv tn)

Nng, 12/2013


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NHNG THNH VIN THAM GIA NGHIN CU TI

TT H v tnn v cng tc v

lnh vc chuyn mn

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KS. Trng Vn Thin

KS. Phm Th Kim Tho

KS. Nguyn Xun Hong

Khoa Ha - Trng i hc Bch Khoa, ihc Nng



N V PHI HP CHNH

Tn n vtrong v ngoi nc

H v tn ngi i din n v

Cng ty c phn XNK Thy sn Mintrung: Cng ty ch bin v xut khu

thy sn Th Quang

Nguyn Anh Tun


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MC LC

MC LC ................................................................................................................................. 4DANH MC BNG BIU ....................................................................................................... 6DANH MC HNH V............................................................................................................. 7CC CH VIT TT ............................................................................................................... 9THNG TIN KT QU NGHIN CU............................................................................... 10INFORMATION ON RESEARCH RESULTS....................................................................... 12M U ................................................................................................................................. 14Chng I - TNG QUAN TI LIU..................................................................................... 15

1.1 Tng quan v cng ngh sn xut chitin/chitosan......................................................... 151.1.1 Gii thiu v chitin ................................................................................................. 151.1.2 Gii thiu v chitosan ............................................................................................. 161.1.3 Tnh cht vt l v tnh cht ha hc ca chitin/chitosan....................................... 171.1.4 Cc phng php thu nhn chitin........................................................................... 191.1.5 Thu nhn chitosan ................................................................................................... 25

1.2 Tng quan v vi khunBacsillus subtilisv enzyme protease ...................................... 301.2.1 Gii thiu v vi khun Bacillus subtilis. ................................................................ 301.2.2 Gii thiu v enzyme protease. ............................................................................... 321.2.3 Mt s nghin cu ng dng Bacillus subtilis ca cc tc gi trong nc............. 37

1.3 Cc lnh vc ng dng ca chitin/chitosan .................................................................... 371.3.1 Trong y hc ............................................................................................................. 381.3.2 Trong x l nc thi v lm trong nc sinh hot................................................ 391.3.3 Trong nng nghip .................................................................................................. 391.3.4 Trong m phm ....................................................................................................... 401.3.5 Trong thc phm ..................................................................................................... 40

Chng 2 -NGUYN LIU V PHNG PHP NGHIN CU..................................... 422.1. Nguyn liu, ha cht v thit b.................................................................................. 42

2.1.1. Nguyn liu: ........................................................................................................... 422.1.2. Ho cht v thit b: .............................................................................................. 43

2.2. Phng php nghin cu .............................................................................................. 432.2.1 nh lng nc ..................................................................................................... 432.2.2 nh lng mui Calci carbonat ............................................................................. 432.2.3 nh lng chitin .................................................................................................... 442.2.4 Phng php xc nh deacetyl ca chitosan.................................................... 442.2.5 Mt s phng php xc nh khc: ....................................................................... 44

2.3 S cc m hnh th nghim (hnh 2.2) ....................................................................... 442.4 X l s liu ................................................................................................................... 46

Chng 3 KT QU V THO LUN ............................................................................. 473.1 Kt qu nghin cu kho st thnh phn ha hc ca v tm, nguyn liu ni tng cv phng php bo qun nguyn liu ni tng c. ............................................................. 47

3.1.1 Thnh phn ha hc ca mt s nguyn liu cha chitin, ph bin thnh ph Nng ................................................................................................................................. 473.1.2 Kt qu xc nh thnh phn ha hc ca nguyn liu ni tng c........................ 483.1.3 Kt qu nghin cu bo qun nguyn liu ni tng c phc v thu hi ch phmenzyme protease ............................................................................................................... 48

3.2 Chn phng php thu hi ch phm enzyme protease t ni tng c, kho st hot ca ch phm enzyme protease thu c. ............................................................................ 49


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3.3. nh hng ca nhit v thi gian bo qun ln hot tnh ca ch phm enzymeprotease tch chit t ni tng c ......................................................................................... 523.4 Cc kt qu nghin cu sn xut chitin/chitosan ng dng enzyme protease tch chitt ni tng c ........................................................................................................................ 54

3.4.1 Kt qu nghin cu phng php x l nguyn liu v tm trc khi loi b

protein: ............................................................................................................................. 553.4.2 Kt qu nghin cu phn ng thy phn protein trong nguyn liu v tm.......... 563.4.3 Kt qu nh gi cht lng chitin c thu nhn bng phng php ng dngenzyme protease t ni tng c ........................................................................................ 593.4.4 nh gi cht lng chitosan sn xut t chitin ng dng ch phm enzyme

protease t ni tng c ..................................................................................................... 603.4.5 Kt qu nghin cu x l cc loi ph thi sinh ra trong qu trnh x l nguyn liuthnh chitin/chitosan ........................................................................................................ 61

3.5 Kt qu nghin cu ng dng vi khun B. subtilis loi b protein trong nguyn liuv tm .................................................................................................................................. 61

3.5.1 Kt qu nghin cu nhm chun b ging vi khun Bacillus subtilis. .................... 613.5.2 Kt qu ng dng Bacillus Subtilis loi b protein trong v tm...................... 65

3.6 So snh cht lng chitosan sn xut theo cc phng php ha hc v ng dng cngngh sinh hc ....................................................................................................................... 703.7 xut s cng ngh sn xut chitin/chitosan ng dng cng ngh sinh hc......... 71

3.6.1 Dy chuyn cng ngh phc hp sn xut chitin/chitosan ng dng ch phmenzyme protease t ni tng c (hnh 3.18) ..................................................................... 713.6.2 Dy chuyn cng ngh sn xut chitin/chitosan ng dng vi khun B.subtilis (hnh3.19) ................................................................................................................................. 72

KT LUN .............................................................................................................................. 74TI LIU THAM KHO ....................................................................................................... 75PH LC................................................................................................................................. 78

Ph lc 1: Phng php pha long thp phn ...................................................................... 78Ph lc 2: Mi trng hot ho ging (pH=6,5)................................................................. 78Ph lc 3: Mi trng kim tra hot lc enzyme................................................................ 78Ph lc 4: Thnh phn thuc nhum Amido-Black............................................................. 78Ph lc 5: Mi trng nhn ging cp 1............................................................................. 78Ph lc 6: Mi trng nhn ging cp 2............................................................................. 79Ph lc 7: Phng php chun phooc-mn .................................................................... 79Ph lc 8: Phng php chun Kjeldahl ......................................................................... 80Ph lc 9: Mi trng kim tra hot lc enzyme................................................................ 81Ph lc 10: Thu nhn chitin/chitosan bng phng php ha hc (mu i chng)........... 81Ph lc 11: Thu nhn ch phm enzyme proteaset ni tng c........................................ 82

Ph lc 12: Nghin cu qu trnh thy phn protein trong v u tm bng ch phmenzyme protease, thu nhn t ni tng c ............................................................................ 82Ph lc 13: Nghin cu qu trnh thy phn protein trong v u tm bng vi sinh vtBacillus subtillis ................................................................................................................... 83Ph lc 14: Cc bc tin hnh nghin cu phng php tch chit chitin s dng vi khun

B. Subtilis ............................................................................................................................. 83


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DANH MC BNG BIU

Bng 3.1 Thnh phn ha hc ca nguyn liu v tm ........................................................... 47Bng 3.2 - Thnh phn ha hc ca nguyn liu ni tng c.................................................. 48Bng 3.3 - Mt s tnh cht ca ch phm protease thu nhn t nguyn liu ti v nguynliu ng lnh ........................................................................................................................... 51Bng 3.4 Mt s ch tiu cht lng ca chitin sn xut bng phng php ng dngenzyme protease t ni tng c ................................................................................................ 60Bng 3.5 Mt s c im cht lng chitosan thu nhn t chitin tch chit t v tm ngdng enzyme protease t ni tng c ....................................................................................... 60Bng 3.6 Mt s c im ca ph thi rn v lng ............................................................. 61Bng 3.7 - Mt khun lc khi nui cy trn mi trng hot ho vi cc pha long khcnhau .......................................................................................................................................... 62Bng 3.8 - ng knh vng thu phn ca cc khun lc..................................................... 63Bng 3.9 - S gim khi lng v tm qua tng cng on................................................... 70Bng 3.10 Mt s ch tiu cht lng ca chitin sn xut bng phng php ng dng vikhunB. subtilis ....................................................................................................................... 70Bng 3.11 So snh mt s c im cht lng ca chitosan sn xut theo phng php hahc v ng dng cng ngh sinh hc ....................................................................................... 71


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DANH MC HNH V

Hnh 1.1: Cng thc cu to ca chitin.................................................................................... 16Hnh 1.2Cng thc cu to ca chitosan ............................................................................. 17Hnh 1.3S tng qut thu nhn chitin bng phng php ha hc................................. 19

Hnh 1.4 - Cc phng n thu nhn chitin ............................................................................... 20t cc ngun nguyn liu khc nhau ........................................................................................ 20Hnh 1.5Kriel ....................................................................................................................... 21Hnh 1.6Phn ng tm tt qu trnh iu ch chitosan........................................................ 26Hnh 1.7 - My sy, to ht loi FL ......................................................................................... 30Hnh 1.8 - Thit b nghin ti kch thc siu nh Cage-Paktor .......................................... 30Hnh 1.9 - Vi khunBacillus subtilissau khi nhum gram..................................................... 31Hnh 1.10 - Hnh nh a khun lc ca vi khunBacillus subtilis......................................... 31Hnh 2.1S khc nhau ca mt s nguyn liu tm, v ca chng dng thu nhnchitin/chitosan .......................................................................................................................... 42Hnh 2.2S m hnh cc bc thc hin trong nghin cu............................................ 46Hnh 3.1 - nh hng ca thi gian bo qun ln ch tiu cht lng ni tng c bo qun nhit -18 -200C ................................................................................................................. 49Hnh 3.2 - Biu s ph thuc hot cc nhm enzyme protease..................................... 50trong cc vng pH khc nhau ................................................................................................... 50Hnh 3.3 - S quy trnh tch chit enzyme protease........................................................... 51t ni tng c ............................................................................................................................ 51Hnh 3.4 - Ch phm enzyme protease tch chit t ni tng c ng lnh............................ 52Hnh 3.5S ph thuc ca hot ca ch phm enzyme protease.................................... 53vo nhit mi trng ........................................................................................................... 53Hnh 3.6 - nh hng ca thi gian bo qun ln hot enzyme protease tch chit t ni

tng c ...................................................................................................................................... 53Hnh 3.7 - nh hng ca thi gian ln kh khong v hm lng cht khong cn litrong v tm ............................................................................................................................. 56Hnh 3.8S ph thuc kh protein trong nguyn liu v tm vo thi gian thy phn. 57Hnh 3.9S ph thuc ca kh protein vo nng ch phm enzyme protease t nitng c ...................................................................................................................................... 58Hnh 3.10 - nh hng ca nhit ln phn ng enzyme thy phn protein trong nguynliu............................................................................................................................................ 59Hnh 3.11 - a khun lc sau khi nhum bi thuc nhum Amido-Black. ........................... 62Sau khi nhum chng ti quan st thy chung quanh cc khun lc xut hin cc vng thu

phn mu sng, nhng vng mi trng cn li c mu xanh m ca thuc nhum nh hnh3.11........................................................................................................................................... 63Hnh 3.12 - Hnh thi vi khunBacillus subtilissoi di knh hin vi................................... 64Hnh 3.13 - Vng thu phn ca vi khunBacillus subtilissau khi nhn ging cp 1........... 64Hnh 3.14 - Vng thu phn ca vi khunBacillus subtilis sau khi nhn ging cp 2........... 64Hnh 3.15S thay i hm lng m ha tan trong mi trng cha vi khunB. Subtilisv v tm nhit 480C ....................................................................................................... 66Hnh 3.16 - Hm lng m ha tan thu c khi thy phn protein trong v tm vi cc t l

b sung ging khc nhau 480C ............................................................................................. 67Hnh 3.17S ph thuc ca qu trnh thy phn protein trong v tm vo nhit ........... 68Hnh 3.18S cng ngh phc hp sn xut chitin/chitosan ng dng ch phm enzyme

protease t ni tng c ............................................................................................................. 72
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Hnh 3.19S cng ngh phc hp sn xut chitin/chitosan ng dng vi khun B. subtilis.................................................................................................................................................. 73


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CC CH VIT TT

Chitin : poly (1,4) - 2 acetamido - 2 deoxy - - D glucose

Chitosan : poly (1,4) -2-amino-2-deoxy--D-glucanKK : Qu trnh kh khongKP : Qu trnh kh proteinKA : Cng on deacetylEDTA : Etilendiamin tetraaxetic axit

G20X : Enzyme proteinase protosubtilina

V tm : u tm + v tmVSV : Vi sinh vtQMAFAM : Quantity of Mesophilic Aerobic and Facultative Anaerobic

Microorganisms


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I HC NNGTRNGI HC BCH KHOA

THNG TIN KT QU NGHIN CU

1. Thng tin chung:

- Tn ti: ng dng phng php sinh hc tng hp chitosan t ph liu chbin thy sn

- M s: 2013-02-53

- Ch nhim: TS. Bi Xun ng

- Thnh vin tham gia: KS. Phm Th Kim Tho, KS.Nguyn Xun Hong, KS.Trng Vn Thin.

- C quan ch tr:Trng i hc Bch Khoai hc Nng

- Thi gian thc hin:t 10/0430/12/2013

2. Mc tiu:

ng dng phng php sinh hc sn xut chitin/chitosan nhm hn ch s

dng ha cht, gy nhim mi trng3. Tnh mi v sng to:

S dng kh nng xc tc phn ng ca enzyme protease trong ni tng c v

enzyme protease ca vi khunB. subtilis loi b protein ra khi chitin trong v tm

4. Tm tt kt qu nghin cu:Trn c s nghin cu qu trnh kh protein trong v tm bng bin php ng

dng cng ngh sinh hc v thc nghim sn xut chitin/chitosan quy m pilot theo

cng ngh mi c nghin cu thu c cc kt qu nh sau:

- Sn phm chitin sn xut ng dng ch phm enzyme protease tch chit t ni

tng c c cht lng tt vi hm lng protein nh hn 0,5%, v ch tiu vi sinh vt

t chun an ton (SanPin 2.3.2.1078-01 LB Nga); chitosan sn xut t loi chitin

ny c deacetyl l 79-89% v nht cao n 18 dl/g ph hp vi cc bo co

khoa hc trong nc v quc t.

- Sn phm chitin sn xut ng dng vi khun B. subtilisc ch tiu cm quanthp, v vi sinh vt tng s tng i cao mc 3,3x105nhng vn nm trong gii


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hn an ton (SanPin 2.3.2.1078-01LB Nga); chitosan sn xut t loi chitin ny c

nht l 18,1 dl/g c cht lng bo m so vi bo co trong nc v quc t.

5. Tn sn phm:

- 2 s dy chuyn cng ngh sn xut chitin/chitosan

- cc sn phm chitin, chitosan v cht mu carotenoid

6. Hiu qu, phng thc chuyn giao kt qu nghin cu v kh nng p dng:

- Hiu qu v cng tc ging dy:

Hng dn thnh cng hai sinh vin lm n tt nghip nghin cu nm hc

2012-2013 l: Trng Quang Thm (08SH) v V Th Ngc Thy (11SHLT).

- Chuyn giao kt qu nghin cu:

Dy chuyn cng ngh sn xut chitin/chitosan ng dng cng ngh sinh hc

c ng ti trn ch trc tuyn: Ch cng ngh v thit b Vit nam.

http://www.techmartvietnam.vn

Ngy thng nmC quan Ch tr

(k, h v tn, ng du)Ch nhim ti

(k, h vtn)
http://www.techmartvietnam.vn/http://www.techmartvietnam.vn/


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DANANG UNIVERSITY OF TECHNOLOGY

UNIVERSITY OF DANANG

INFORMATION ON RESEARCH RESULTS

1. General information:

Project title: Produce chitosan from the wastes of aquatic processes by using bio-

technique

Code number: 2013-02-53

Project Leader: Doctor Bui Xuan Dong

Coordinator: Pham Kim Thao, Nguyen Xuan Hoang, Truong Van Thien

Implementing institution: Danang University of Technology University of

Danang

Duration: from April 10, 2013 to December 30, 2013

2. Objective(s): Produce chitin/ chitosan by bio-technique to eliminate the using of

chemicals to prevent environmental pollution.

3. Creativeness and innovativeness: Applying the catalization of protease in internal

fish organs and protease fromBacillus subtilisto discard protein from chitin in shrimp

shells.

4. Research results:

Base on the using of bio-technique to demineralise the shrimp shells and the

implementation of pilot studies with new technique, we received some given results:

- Chitin product produced by using protease extracted from internal fish organs had

high quality such as protein content was less than 0.5% and the microorganism quota

was obtained the Safety Standard (SanPin 2.3.2.1078-01Russia); Chitosan produced

from these chitin products had deacetylation degree from 79% to 89% and the

viscosity was up to 18 dl/g. The product quality can be compared to products reported

from inside and outside of Vietnam.

Chitin product prepared by Bacillus subtillis fermentation had low perceptible

showing and the total microorganism in the sample was quite high, at 3.3x10-5

but still


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under the Safety Standard (SanPin 2.3.2.1078-01Russia). The viscosity of Chitosan

produced from these chitin products was 18.1dl/g. The product quality can be

compared to products reported from inside and outside of Vietnam.

5. Products:

- Two production lines of Chitin and Chitosan;

- Chitin, chitosan and carotenoid pigment products

6. Effects, transfer alternatives of research results and applicability:- Teaching: Conducting two students to do their final research thesis, including

Truong Quang Thom (08SH) and Vo Thi Ngoc Thuy (11SHLT).

- Hand over the research results: Production lines to produce chitin/ chitsan by

bio-technique were placed in the online market: The Technology and Equipment

Market of Vietnam (http://www.techmartvietnam.vn).

Date:.

IMPLEMENTING INSTITUTION PROJECT LEADER
http://www.techmartvietnam.vn/http://www.techmartvietnam.vn/


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M U

Trong nguyn liu v tm, chitin lin kt rt cht ch vi protein, lipid v cc cht

khong (pha rn). Vic thu nhn chitin t ngun nguyn liu ny xy dng trn cn

bn l a protein v cc cht khong v trng thi lng (pha lng), v t loi b

chng ra khi chitin. sn xut chitin t cc tiu chun cht lng quc t cn loi

b hon ton protein v khong cht, cng nh lipid.

Hin nay phng php ha hcc dng ph bin thu nhn chitin bng cch

s dng cc ha cht m c, tc l x l nguyn liu trong nhng iu kin khc

nghit (mi trng axit mnh, baz mnh, nhit cao v thi gian ko di), thng

dn ti lm gim cht lng chitin thng phm. Cc h hng ca sn phm chitinthng gp khi thu nhn theo phng php ha hc l: bin dng cu trc phn t,

gim nht ca dung dch chitin v chitosan. Bn cnh vic bo qun v s dng

cc ha cht c m c cao m i hi s tip xc trc tip ca cng nhn gy

nhiu quan ngi trong vn bo m an ton v sc khe cho ngi lao ng. Theo

phng php ha hc c th thu nhn c sn phm phn gii protein l hn hp cc

axit amin t do ha ln cng cc cht c hi, cc cht c hi sinh ra do protein b

thy phn su dn n phn gii c cc axit amin nn vic s dng sn phm dch thy

phn gp phi nhiu hn ch, v d khng s dng c trong thc phm hay trong

sn xut thc n chn nui.

Cc hng s dng chitin v dn xut t chitin trong thc t trong nhng nm gn

y khng ngng c m rng dn ti cn tm kim v th nghim cc phng php

mi thu nhn cc polymer sinh hc ny. Mt vn c tnh thi s cn tn ti l

nghin cu phng php x l nguyn liu v tm to iu kin thu hi chitin v ton

b cc hp cht t nhin c hot tnh sinh hc c trong n, bn cnh vic hng ti

pht trin bn vng trn c s gim thiu nhim mi trng.

Mc tiu ca nghin cu ny l ng dng phng php sinh hc tng hp

chitosan t ph liu ch bin thy sn nhm thu c cc sn phm chnh nh: chitin,

chitosan v cc sn phm ph nh cht mu carotenoid v ch phm enzyme protease

t ni tng c.


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Chng I - TNG QUAN TI LIU

1.1 Tng quan v cng ngh sn xut chitin/chitosan

V tm, gh, cua, mai mc v nhiu loi v gip xc khc chim 15 ti 35% tng

khi lng khai thc, l ngun nguyn liu qu iu ch cc hp cht polymer t

nhinnh aminopolysaccharide (chitin v chitosan), v nhng dn xut ca chng l

cc oligosaccharite v glucosamine, nhng sn phm ngy nay tr nn thit yu

trong cc lnh vc khc nhau. V d:

+ s dng lm cc cht ph gia thc phm, hay thnh phn ca thc phm chc

nng

+ s dng lm cc ch phm y dc ( gim cn, iu ha p huyt, gim

colesteron; cc ch phm chng ung th v cc loi thuc khng th)

+ s dng trong m phm bo v da v tc

+ s dng lm sch nc trong cc m h t nhin v nhn to (keo t v hp

ph cc hn hp c hi, cc loi vi khun, cc kim loi nng)

+ dng trong cc ch phm nng nghip (cc ch phm cha bnh cho ng vt,

ch phm chng li bnh cho thc vt) [21, 27].

1.1.1 Gii thiu v chitin

Chitin phn b rng ri trong t nhin, cng vi xellulo l nhng hp cht hu

c ph bin nht trn b mt tri t. N l ngun ti nguyn di do, c tnh vo

khong 100 nghn t tn/nm. Nhng khc vi xellulo, chitin cn l ngun sinh khi

t nhin t c khai thc.

Chitin ln u tin c Gio s khoa hc t nhin, gim c vn thc vt thuc

Vin hn lm khoa hc ca TP Nensi (Php) Henri Braconnot pht hin nm 1811

khi nghin cu thnh phn ca nm v ng t tn l fungin. Nm 1823 A.Odier

iu ch chitin t cnh b cnh cng v t tn l chitin (Ting Hy-lp. citwu- ngha

l o). Cn chitosan ln u tin pht hin nm 1859 bi nh khoa hc C. Rouget,

cng t thi im ny gii khoa hc bt u nghin cu thc nghim v s dng

nhng polymer ny.


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Nhng cng trnh u tin Nga, lin quan ti vic iu ch chitin c thc

hin di s ch o ca Vin s P. Sorugin nhng nm 1934-1935. Cc th nghim

s dng chitosan c F. Cadov thc hin nm 1941.

Hin nay nhm mc ch pht trin cc nghin cu v chitin/chitosan Nga cHip hi lin bang v chitin (The Russian chitin society: www.chitin.ru), c thnh

lp t nm 2000.

Chitin l mt poly (1,4) - 2 acetamido - 2 deoxy - - D glucose. Cng thc cu

to ca chitin c trnh by trn hnh 1.1

Hnh 1.1: Cng thc cu to ca chitin

Lin kt (1,4) glucoside ca mimt xch cu to nm lch nhau mt gc 1800

to nn mch xon. Lin kt ny km bn, d b ct t bi tc nhn ion H+ca axit.S c mt ca nhm amino trong chitin lm cho chng c nhng c tnh sinh hc

chuyn bit v c th tham gia cc phn ng c trng. Chitin l polymer c nhng

tnh cht xc nh, bao hm c kh nng b phn hy bi vi sinh vt v c hot tnh

sinh hc. Nn chitin cn c quan tm nghin cu nh mt loi vt liu chc nng

c bit mi.

iu khc bit ca chitin so vi cc polysaccharide khc l phn t chitin tch

in dng mnh, n gip cho chitin to ra lin kt vi cc phn t mang in tch m

trn b mt.

Trong mi nguyn liu khc nhau th chitin c s khc bit v thnh phn cu to

v hm lng chitin khc nhau [12, 21, 25].

1.1.2 Gii thiu v chitosan

Chitin c th chuyn ha thnh nhiu dn xut khc nhau, hay hp nht l


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chitosan. Chitosan l poly (1,4) -2-amino-2-deoxy--D-glucan. N c to thnh khi

N-deacetyl ca phn t chitin, c cng thc cu to nh trn hnh 1.2.

Chitosan c mt s u vit so vi chitin, c th k n nh l tnh tan trong nc

v trong dung dch acid. Khi lng phn t chitosan ph thuc vo iu kin x lchitin hay v tm, thng thng t 10 n 1000 kDal. n v phn trm deacety

c xc nh bng mc deacetyl (y l t l ca khi lng glucosamine so vi

s monomer chung trong phn t polymer DD = m(m+n) = Namin/Ntng), thng t

70-90%.

Hnh 1.2Cng thc cu to ca chitosan

1.1.3 Tnh cht vt l v tnh cht ha hc ca chitin/chitosan

Do c trng v cu to nn chitin v chitosan c tnh cht c trng ca nhmamino (-NH) v nhm hydroxyl (-OH). Chng c kh nng lin kt vi nhiu kim loi

nng to nn hp cht gi l chelate. Chitin c c tnh hot ha thp do trong phn t

chitin ch c hai nhm hydroxyl trn mi mt xch (monomer) c th tham gia vo

phn ng ha hc. Chitosan nh c nhm amino t do nn c nng lng t do ln,

to iu kin cho chitosan c th ha tan trong cc dung dch ca cc acid hu cv

mt s acid v c.

Ngoi ra, cc polymer ny cn c th b thy phn do cha cc lin kt glucoside

v lin kt peptide. C th d dng to ra cc nhm a nc bng cch bin tnh

chitin/chitosan, nn m ra kh nng m hnh ha tnh cht ca chng.

Chitin c 3 trng thi tinh th l: , v chitin. Ph bin nht v bn vng

nht l -chitin, trong phn t c cc lin kt i song song, chnh nh cc lin kt

hydro. Phn t v chitin km bn vng hn v t gp hn.

Chitosan cng c th tn ti dng tinh th, s to ra polymer chitosan ph thuc

vo trng thi vt l ca n.


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Chitin/chitosan bn vng di tc dng ca kim, nhng c th b thy phn di

tc dng ca cc acid. Khi b thy phn hon ton bi dung dch acid HCl hoc acid

sunfuric nhit cao chitin v chitosan b thy phn n monosaccharide -D-

glucosamine. iu kin mm hn c th to thnh hn hp cc oligosaccharide.Dung dch HCl thng c dng thy phn tng phn, sn phm to ra trc tip

l glucosamine t cc on oligosaccharide vi kh nng polymer ha 2-4. X l bng

siu m c th lm phn hy chitin v chitosan.

S thy phn lin kt glucoside ca phn t chitin/chitosan di tc dng ca cc

yu t khc nhau (oxy ha, x l nhit) lm t mch polymer ca chng, lm gim

khi lng phn t, tng kh nng ha tan, tnh cht ny thng c ng dng

iu ch dung dch, v sau dng vo cc mc c khc nhau.

S thy phn lin kt amid (peptit) ca chitin to ra chitosan. V nguyn tc

khng c danh gii r rng gia chitin v chitosan. trng thi t nhin chitin lun c

mt s nhm amino t do (5-15%). Thng thng chitosan c cho l sn phm, c

th ha tan trong cc dung dch acid ha c long (nh acid acetic), tng ng vi

deacetyl khng di 55-60%.S thy phn ca cc lin kt peptide trong dung dch

acid v baz hoc di tc dng ca cc enzyme (chitosanase). Mt phng php

deacetyl vi hiu xut cao thng thy phn bng baz mnh. Qu trnh deacetyl lm

bin i cu trc polymer, dn n lmgim khi lng phn t polymer.

Trong t nhin chitin/chitosan b phn gii bi nhiu sinh vt khc nhau, chng c

kh nng sinh enzyme chitinase v chitosanase.

Phn ln enzyme chitinase trong vi sinh vt thy phn lin kt N-acetyl--(1,4)-

glucosamin theo c ch khc nhau. Chitinase trong thc vt bc bao khng c chitin

nh thnh phn cu to. Enzyme chitosanase thy phn chitosan bng c ch ct endo.Nh vy chitosan c th b phn gii di tc ng ca nhiu ch phm enzyme khc

nhau nh glycanase,lipase v protease t nhng ngun khc nhau. Enzyme cellulose

T. viride phn gii chitosan n chitooligosacharide. Enzyme papain xc tc thy

phn chitosan v lm gim nht ca chitosan.

X l chitin bng cc enzyme thy phn m ra kh nng sn xut N-acetyl-D-

glucosamin bng cc enzyme chitinase. Nghin cu nh hng ca nhm N -acetyl ln

qu trnh thy phn vi lysim chng minh rng, deacetyl ti u bng 0,7, khi c


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th thu c cc on oligosaccharide n tetramer.

Oligosaccharide c di polymer t 2-8 ng dng nh ph gia thc phm, thnh

phn ch phm y dc c iu ch bng cch phn gii chitosan bng enzyme

chitosanase t vi khunBacillus 7-M bn pH t 5-11.Chitosan ha tan cng c iu ch bng phng php enzyme nh sau: x l

chitosan vi deacetyl 60-90% bng enzyme chitosanase trong mi trng acid

nhit 30-60 0C. Sn phm thu nhn c, tch ra bng phng php siu m, c

kh nng ha tan trong nc [21,25].

1.1.4 Cc phng php thu nhn chitin

Chitin l polymer khng tan nn khng th thu nhn trc tip t v tm, cua.

thu nhn chitin cn thc hin tun t vic tch protein v cc cht khong, cu trc

nn lp v cng cha chitin, ngha l chuyn protein v khong cht v trng thi ha

tan, v loi b chng.T nhng phng php thu nhn chitin bit chng ta c th

phn chia chng thnh hai nhm phng php c bn: nhm 1 - phng php ha

hc, x l bng acid, kim; nhm 2 - cc phng php cng ngh sinh hc, ng

dng cc ch phm enzyme v cc vi sinh vt phn gii protein.

Hnh 1.3S tng qut thu nhn chitin bng phng php ha hc

a) X l ha hc:


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Phn ln cc phng php thu nhn ca nhm ny xy dng trn mt hoc hai

bc lm sch chitin khi protein v cc hp cht khong - gi l kh protein v kh

khong. Mt s phng php khc c thm bc kh lipid v cc cht khc [13].

Thu nhn chitin t nhng ngun nguyn liu khc nhau c s tng qut nhhnh 1.3

Ph thuc vo ngun nguyn liu v cht lng chitin cn thu nhn, cng nh

thu nhn chitosan t chitin m s lng cc bc kh protein v kh khong khc

nhau, trt t c th thay i theo s nh hnh 1.4

Hnh 1.4 - Cc phng n thu nhn chitin

t cc ngun nguyn liu khc nhau

Cc phng n ny th hin vic thu nhn chitin t cc ngun nguyn liu khc

nhau, ph thuc vo yu cu v cht lng sn phm chitin. V d x l v tm, b

cnh cng (lp v mng) ch cn cng on x l kim (x l 1 ln). X l kp (2 ln

x l kim v acid) kh hon ton protein khi chitin. Cc khong cht cn li x

l bng acid khng vt qu 1-3%. Vic lp li bc kh khong c th thu nhn

chitin khng cha tp cht, bc ny cn thc hin vi cc nguyn liu c lp v dy

(nh v cua,) [17].

Vic p dng tun t cc bc KK vKP c mt s li ch v cht lng chitin.

Vic p dng tun t ny nh hng ln cht lng chitin thu nhn c v t chitin

thu nhn chitosan. V d, khi x l nguyn liu cha chitin theo s KK-KP nht

ca ca chitosan thu c cao hn rt nhiuso vi s cng ngh KP-KK.

Hiu sut cc qu trnh KK v KP ph thuc vo nghin nh ca v tm, vcua, ngha l tng din tch tip xc ca nguyn liu vi cc cht ha hc, nhng khi


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nghin nh nguyn liu cng lm hiu sut sn phm gim do tht thot trong qu

trnh x l [18].

Cc thng s ca cng on KP v KK ca cc nghuyn liu khc nhau cng khc

nhau. Theo phng php Hackman [12] v crab c x l bng dung dch HCl 2Ntrong 5h nhit phng, sau hn hp c nghin nh v kh protein bng dung

dch acid NaOH 1000C trong 12h. kh hon ton protein cn thc hin cc cng

on trong 4 ln. Phng php Whisler v BeMiller [12] thc hin kh protein ca

nguyn liu nghin nh trong dung dch NaOH 10% trong thi gian 72 h, kh mu

bng etanol 95%, sau ra bng dung mi hu c, kh protein bng dung dch HCl

37% trong 4h. Kh khong theo phng php Horowits, Rosoman v Blumental [12]

c thc hin trc khi kh protein, nguyn liu x l bng dung dch acid fomic

90% trong 18h nhit phng, sau kh protein bng dung dch NaOH 10% trong

2,5h un nng. Cc nh khoa hc u khng nghin cu nh hng ca trt t cc

bc kh khong, kh protein ln cht lng chitin/chitosan. Nhng sau ny, ngi ta

pht hin trt t KK, KP nh hng n cht lng chitosan thu nhn c.

T cc phng php trn, c mt iu r rng l cc cng on u thc hin

iu kin khc nghit, nguyn liu cha chitin c x l trong dung dch acid v

dung dch kim mt thi gian di v nhit cao kh protein. iu ny dn n

bin dng cu trc, v mt phn kh deacetyl (KA) ca chitin.

Hnh 1.5Kriel

Ti LB Nga t nhng nm 1970-1980 ( Vladivostoc, Murmansk, VNIRO) ng dng cng ngh sn xut chitin v chitosan t v nhuyn th (kriel) v v tm


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(hnh 1.5). Chitin c thu nhn nh kh protein bng dung dch NaOH 1N trong 30

pht nhit 70-800C, sau kh khong bng dung dch HCl 1N trong 30 pht

nhit phng.

tng hiu sut qu trnh kh protein bng dung dch kim nh, cc nh khoahc ng dng cc cht c hot tnh b mt. Phng php thu nhn chitin t v

nhuyn th (kril), c xut bi A.I. Gamzazade cng cng s [22]: v nhuyn th

(krel) c kh protein trong dung dch NaOH 2-5% nhit phng trong 6-8h.

Trong cng on kh protein dng phi hp cc cht hot ng b mt anion (sodium

dodecyl sulfate, mui natri acid aurino), lm gim nng NaOH, bn cnh lm

tng hiu sut kh mu v lipid.

Phng php sn xut chitin t v nhuyn th (kriel) ng dng Ba lan [23] xy

dng trn c s x l s dng nguyn liu ng lnh. Nguyn liu c gi ng,

nghin nh, sau ra sch v kh khong bng dung dch acid HCl 3,5% trong 1,5h

nhit phng. Sau tin hnh loi b lipid v v cht mu bng hn hp gim v

etanol (acetate ester). Kh protein bng dung dch NaOH 3,5% nhit 250C trong

2h. S pht trin cng ngh sn xut chitin cc quc gia ch khc nhau vic chn

ha cht phc v qu trnh KK v KP [14].

n trong cng nghip sn xut chitin t ph liu ch bin tm Matapenaeus

dobsoni. Kh protein c tin hnh trong dung dch NaOH 0,5% un nng trong 30

pht. Phn dung dch c tch khi chitin v tn dng sn xut protein. Bc kh

protein c thc hin ln th hai bng dung dch NaOH 3%. Nguyn liu tip tc

c kh mu bng dung dch hypochloride cha 0,3-0,5% clo lng, sau ra bng

nc v tin hnh kh khong bng dung dch HCl 1,25N nhit phng trong 3h.

Chitin sn xut ra c dng sn xut chitosan.

M ngi ta s dng cng ngh sn xut chitin ca cc nh khoa hc Peniston

K.P. v Jonson E.L. [25]. Nguyn liu cha chitin c nghin nh dng m t ti

kch thc 1,3-1,8 mm v tin hnh KP nhit 65-710C trong thi gian 1-2h trong

dung dch NaOH 0,05-0,2N theo c ch chy ngc.Trong dung dch NaOH trc khi

dng kh protein cho thm 0,1% Na2SO4, kch hat qu trnh thy phn. Sau

tch ra phn dung dch cha protein sn xut protein, phn rn chuyn qua cng

on kh khong bng HCl.


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Tt c cc phng php thng k trn y ch khc nhau trnh t thc hin hai

cng on KK v KP, v cc thng s ca cng on trn cn bn phng php ha

hc.

Kh khong (KK) l cng on quan trng trong sn xut chitin. kh khongxc nh bng nht v cc thng s vt l, ha hc khc, kh khong ca chitin

cng ging nh ca chitosan, tc l mang cng mt ngha. kh khong (kh

CaCO3v Ca3(PO4)2) t nguyn liu cha chitin ngi ta thng dng acid HCl, acid

formic, HNO3v acid sunfuric. Hn ch y l khi x l chitin bng acid c th dn

ti ph hy mt phn cu trc v deacetyl ca chitin, gim nht ca dung dch

chitosan.

C cc phng php khc thu nhn chitin, khng ging nh cc phng php tiu

chun nh trn. l phng php ca Foster v Hackaman [12] thc hin iu

kin mm, khng dn ti lm hng cu trc v deacetyl ca chitin. Tt c cng

ngh ch bao gm 1 bc x l nguyn liu cha chitin trong dung dch etilendiamin

tetraaxetic axit (EDTA). V cua c x l mt thi gian di 2-3 tun trong dung

dch EDTA khi pH = 9. Mt s loi nguyn liu mng nh v tm, nhuyn th (kriel)

sau khi nghin nh ch mt 15 pht x l. Saukhi x l chitin cha 1% tro v 5%

protein.

Peniston K.P. v Jonson E.L. [26] cn c phng php: nghin nh nguyn

liu cha chitin n kch thc 3-6 mm. Sau hn hp cho vo thit b phn ng

cng vi NaOH 0,5-2% trong thi gian 1-4h nhit 50-700C kh protein. Sau

tin hnh theo m hnh KK v KP bng dung dch NaOH 30-50% nhit 120-

1500C. Khi xy ra ng thi qu trnh deacetyl ca chitin chuyn ha CaCO 3v

dng Ca(OH)2theo phng trnh:

nc thi sau cng on ny cha cc thnh phn nh hydroxit natri, acetat

natri va cacbonat natri. Dung dch NaOH c th ti s dng. Hin tng ny hay gp

l hin tng n mn da.

im tn ti chung ca tt c cc phng php trn l x l nguyn liu trong


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iu kin v mi trng khc nghit ca acid v baz, nh vy cn cc thit b bn

vng vi kh nng chng n mn, nh vy lm tng gi thnh sn xut v sn phm.

Ngoi gia khi thit k nh my cn thit k cc khuvc pha ch ha cht, cng nh

bo qun chng, gy nguy hi n an ton sn xut v mi trng. Protein sau khitch khi acid v baz t dch thy phn cha nhiu mui, lm hn ch khi s dng

chng.

b) Tng quan cc qu trnh sinh hc sn xut chitin

ng dng enzyme to iu kin mm x l nguyn liu cha chitin, c th

gp nhiu cng on lm mt nn n gin ha cng ngh. ng thi gim thiu tnh

khc nghit ca mi trng phn ng ln thit b v tng tui th ca chng.

Phng php n gin nht thu nhn chitin l li dng qu trnh t phn hy

di tc dng ca enzyme c trong nguyn liu (qu trnh t gii). Nhng phng

php ny c ng dng sn xut chitin t ph phm kriel [27] v v cua [28]. Qu

trnh kh protein (KP) c tin hnh bng cch nho trn nguyn con kriel vi

nguyn liu cha chitin theo t l 1:2 nhit 500C. Hn hp thu c cn nho trn

lin tc trong 5 gi. chuyn dch protein t nguyn liu vo pha lng t 68%. Kt

qu thu c chitin cha 34% khong cht v 12% protein. Nh vy phng php ny

khng loi b c hon ton protein ra khi chitin, tc l qu trnh KP khng hon

ton v cn cng on ph loi b protein. Ngoi ra, trong hn hp phn ng cha

enzyme chitinase, tc ng ln chitin, lm gim khi lng phn t ca chitin.

T quan im t c hiu sut kh protein cao, mt phng php c tnh

ng dng cao hn l s dng vi khun phn gii protein stam Pseudomanas

maltophilia [29]. Nguyn liu v cua (crab) c kh khong bng dung dchHCl 2N

trong thi gian 2 ngy nhit phng, sau nghin nh n kch thc 0,5 cm.Mi trng nui cy c b sung 0,8 g nguyn liu cha chitin vi so vi 80 ml

dung dch K2HPO40,2%, tip tc cho vo hn hp dung dch HCl iu chnh pH

v pH 7,0. P. maltophiliacy trong ng nghim, lc u n nhit 300C. Cui

qu trnh chitin c ra sch bng nc. Hm lng protein trong chitin gim xung

cn 1%. Phng php kh protein trong v nhuyn th (kriel) ti bng vi khun

Bacillus Subtilis, thy phn protein t 98% sau 36 gi, thu c chitin cha

protein di 1% [30].


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Cc nh khoa hc nghin cu cc phng php sn xut chitin t v cua, v

gh ng dng cc enzyme khc nhau. Phng php thu nhn chitin [12] bng cch KK

bng HCl 1,4N nhit phng trong 24h. Sau kh protein bng enzyme papain,

pepsin hoc tripsin.Phng php ca Rogozina v cng s [31] th cng on KK v KP c gp

lm mt bng enzyme ngun gc vi sinh vt hot ng trong mi trng acid:

protorizinasevprotavamorinase. Qu trnh xy ra pH 3,0 nhit 35-400C trong

24h. Ch phmenzyme protease c thu hi t nm Aspergillus niger, A. fietidus, A.

orizai.Chitin thu c khng chatro, cha 5-10% protein. C th loi b hon ton

protein cng on cui bng dung dch kim.

Ti LB Nga, trong cng nghip ngi ta sn xut chitin t v nhuyn th (kriel)

bng phng php s dng enzyme proteinase protosubtilina G20X [32] v

pankreatuna [33] hot ng trong mi trng kim.

Cc phng php cng ngh sinh hc thu nhn chitin c nhc im chung l

khng loi b ht protein ra khi chitin ( tinh sch di 95%), c th nh hng xu

n cht lng chitin, v ng dng ca chitin. Ngoi ra, khi la chn ch phm

enzyme cn c bit ch trng n kh nng chng c cha enzyme chitinase, v trong

qu trnh x l nguyn liu nu hot lc chitinase mnh c th dn n ph hng cu

trc ca chitin thnh phm.

1.1.5 Thu nhn chitosan

a) Deacetyl ha (DA) chitin

Dn xut deacetyl ha u tin khi bin tnh chitin l chitosan, l polymer cao

phn t ca glucosamin, ha tan trong dung mi acid hu c v v c long (ngoi tr

acid sunfuric).

im khc bit gia chitin v chitosan l kh nng ha tan ca chitosan trong

dung dch acid, nn c nhiu ng dng trong cng nghip, nng nghip v y dc.

thu nhn chitosan cn kh nhm acetyl t N-acetyl-D-glucosamin (chitin) hay cn gi

l phn ng deacetyl ha (DA). Phn ng iu ch chitosan thu gn nh hnh 1.6.

Phn ng DA i km vi vic ct t lin kt glucosit ca polymer. Nh vy,

chitosan l cht trng hp phn tn theo khi lng phn t - polymer D-glucosamin,


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cha 5-15% cc nhm acetal amid v gn 1% cc nhm c lin kt vi acid amin v

cc peptid.

Hnh 1.6Phn ng tm tt qu trnh iu ch chitosan

B ngoi chitosan c cu trc hnh vy vi ng knh nh hn 10 mm hoc dngbt vi nghin nh khc nhau, c mu trng n mu kem, hay gp chitosan mu

hi ng vng, mu hi nu hoc mu hng, chitosan khng mi. Tnh cht khc ca

chitosan dng kh l tnh dn in v c v ng. Theo c th chitosan thuc lp 4

v khng c.

Qu trnh DA thng c tin hnh trong dung dch kim mnh nhit cao.

Th nghim u tin thu nhn chitosan l lm nng chy chitin vi kim rn 180 0C.

Theo phng phpny thu c chitosan vi deacetyl ti 95%, nhng lm bin

dng mnh cu trc (n 20 n v) ca chitosan.

Ph bin trong k thut ngi ta thc hin qu trnh DA bng dung dch kim 30-

50%, v l mi trng mm hn. C nhiu cch khc nhau bin tnh theo

phng php ny. Thc hin qu trnh DA trong dung dch kim c th t

deacetyl ln n 100% khi thc hin li nhiu ln v t lm bin dng cu trc chitin.

Khi thu nhn chitosan trong nhng iu kin nh trn mt mt l qu trnh DA, ngthi xy ra qu trnh song song l lm hng cu trc chitin, ngha l lm t cc lin


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kt glucosit, dn ti gim khi lng phn t v gim nht ca chitosan.

Trong sn xut chitosan theo phng php trn, tc l cc qu trnh xy ra trong

mi trngkim mnh v nhit cao nn cn s dng cc thit b chuyn dng lm

t hp kim niken hoc ph ngoi bng niken. Thng thng thc hin qu trnhDA s dng cc thit b phn ng lm t thp hp kim chu nhit, c b phn nho

trn v lp v dn hi.

Cng on kh khn, phc tp nht trong cng ngh sn xut chitosan l ti s

dng v x l nc thi l dung dch kim vi nng cao. Trong nhiu trng hp

qu trnh DA thc hin bng dung dch kim 30-50%, mc d trong qu trnh phn

ng nng kimgim, nhng thi ngay nc thi vo h thng l iu khng c

cho php. Cho nn khi thit k nh my sn xut chitosan cn c phng n x l

nc thi v b phn thu nhn protein t trong nc thi.

C hng lot phng php thay th mt phn hay hon ton nc trong qu trnh

DA bng cc dung mi khc nhau hoc cc cht mang nhit. V d trong [34] cht

mang nhit c s dng l paraphin lng, hexan, ru isopropyl. Khi gim mnh

lng nc v dung dch kim trong qu trnh DA, gim s ph hng cu tr c

polymer, deacetyl t 92%.

Phng php Brossignac [12] khng s dng nc, ngi ta thay th nc hon

ton bng etanol vi monoetylenglicol. Khi DA bng KOH nhit 120 0C acetyl

t 83%.

Takeda [34] nghin cu phng php x l chitin trong kh tr hydrocylamin

pH 13 v nhit 1000C trong 10-20h. Hn ch ca phng php ny l deacetyl

thp (70%), thi gian x l di, v lm hng cu trc ca mch polymer.

Phng php deacetyl ca chitin 2-4 ln trong dung dch hydrazine hydrate [25] nhit 120-1500C. Khi hn ch s bin dng cu trc mc thp nht deacetyl

t 90-97%. Ngoi ra, qu trnh DA c th thc hin bng enzyme - deacetylase, c

ngun gc vi sinh vt.

T nhng nghin cu ca cc nh khoa hc nh trn lit k v qu tr nh

deacetyl trong kim c th rt ra mt s nhn nh. Trc ht, bn ca chitin trong

qu trnh DA c l gii bi s c mt ca lin kt hydro gia nhm carboxyl v

nguyn t ni-t ca nhm amid trong cu trc micell. ph v lin kt (lin kt bn


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vng) cn tin hnh nhit cao (100-1600C). Khi tng nhit v gi nng

baz mc thp (30%) deacetyl t gi tr 98%, i khi khi lng phn t gim

lm cho nht ca chitosan cng gim theo. bo ton khi lng phn t trong

qu trnh x l chitin cn gim nhit . Phn ng deacetyl xy ra nhanh nht giu tin trong qu trnh x l, thng t hiu sut 70% khi nng baz l 50%

nhit 1000C. Phn ng chm li gith 5 khi deacetyl t 80%. Nh vy, ko

di qu trnh x l khng lm tng c deacetyl, m cn nh hng n cu trc

ca chitosan.

Cu trc chitin l cu trc mng tinh th nn ha tan v ht nc ca chitin

thp. Kch thc ca chitin sau khi nghin nh hng n ng nht ca sn phm

chitosan sau khi deacetyl. Nghin nh chitin lm tng b mt tip xc ca chitin vi

ha cht t ni phn t. Khi kch thc ht chitin qu ln qu trnh deacetyl xy ra

khng hon ton, lp b mt ca chitin b deacetyl nhiu hn so vi cc lp pha

trong. Khi ha tan trong acid axetic nhng lp mt ngoi to thnh dung dch, lp pha

trong ca ht chitin khng b deacetyl hon ton ch ht nc. Hin tng deacetyl

khng hon ton nh trn ca chitosan lm nh hng n kh nng ng dng ca

chng. Trong trng hp chng ta nghin chitin vi kch thc nh v ng u

th cc ht chitin c deacetyl nh nhau to iu kin thu c sn phm vi

ng nht cao hn.

Mi trng thc hin qu trnh deacetyl nh hng ln n bin dng cu

trc ca chitin, v nh s c mt ca oxy. Cc nh khoa hc thc hin hng lot cc

nghin cu loi b oxy ra khi mi trng phn ng DA. Mt gii php n gin

nht l nn cht nguyn liu, ht ht kh ra khi mi trng phn ng v a kh ni -t

vo ri mi trngkn. Thc hin qu trnh DA trong mi trng nh trn so vi thchin ngoi khng kh l lm tng khi lng phn t v nht ca sn phm

chitosan, khng lm gim deacetyl.

Yu t quan trng trong qu trnh DA l lin tc khuy o hn hp phn ng .

Thc t s dng c thit b c b phn khuy o v khng c b phn khuy o vi

cc nguyn liu polymer khc nhau. Trong trng hp s dng thit b khuy o cn

ch trng n c ca hn hp phn ng, xc nh bng t l gia pha lng v pha

rn.T l khi lng ti u gia chitin v dung dch kim l 1:5 - 1:12, t l ny la


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chn ph thuc vo cht lng chitin. Mt hn hp nh vy d dng khuy o, m

khng cn s dng lng NaOH d tha.

Chitin c cu trc v thnh phn ha hc ging nh cellulose, nn c tnh cht

gn ging nhau. Nh bit, cellulose c tnh ht nc (s trng n) trong cc dungdch kim long. Nhng tnh cht tng t cng c pht hin chitin. V nh, khi

chng ta thc hin 12 ln ng lnh v gi ng chitin, sau cho chitin vo dung

dch kim 15-20%, th xy ra s trng n mnh ca chitin, tan cng tng. S

trng n v ha tan ca chitin trong dung dch kim nhit thp l do s gin

mch ca cu trc chitin, m ra kh nng lm bin tnh v thu nhn polymer vi cht

lng ng nht. Cc nh khoa hc nghin cu phng php thu nhn dung dch

chitin kim tnh bng mt chu k ng lnh v gi ng v sau tin hnh cc phn

ng DA trong mi trng ng nht [26].

Tm li, tt c cc phng php DA bit c ng dng trong sn xut

chitosan quy m cng nghip u thc hin nhit cao v mi trng kim

mnh. Ngi ta nghin cu phng php DA lnh [27], cc phn ng xy ra

nhit phng (khng thp hn 20-220C) s dng cc thit b lm t thp khng r

hoc vt liu polymer. Nng NaOH gim xung 30-40% vi t l chitin: dung dch

kim l 1:10 - 1:15. iu kin mm nh trn c th thu c chitosan vi nht v

deacetyl cao.

b) Sy chitosan. Nghin v bo qun.

Sau khi deacetyl v ra n pH trung ha sn phm chitosan c kh nng hydrat

ha mnh, chitosan trng n cha hn 70% nc.


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Hnh 1.7 - My sy, to ht loi FL

bo qun chitosan cn sy nhit 50-550C. Khi sy nhit cao chitosan

b ng tng, ti mu v gim ha tan, gim kh nng ng dng ca chitosan. Ch

sy tt nht ca chitosan thc hin trong my sy si phn tng 50 0C. Chitosan

vi khi lng phn t thp v tan trong nc th phi sy phun. Chitosan kh c

m 8-10% (hnh 1.7).

ng dng chitosan trong y dc lm bao bc thuc v lm vin cn nghin

nh chitosan n kch thc 100-200 mkM. Chitosan cn gi cu trc tinh th ca

chitin nn kh nghin nh, nn thu c chitosan dng bt cn nghin nh, sau

ct, mi v p trong my phay hay my nghin bi (hnh 1.8).

Hnh 1.8 - Thit b nghin ti kch thc siu nh Cage-Paktor

Kh khn nht trong vic nghin chitosan l sy khng ng quy cch hoc

chitosan b sng ha, trong nhng trng hp ny chitosan c bn cao, rt kh

nghin nh.Trong qu trnh bo qun chitosan, quan st di nh sng thy mu ca chitosan

bin i n mu nu v gim ha tan. Cho nn cn bo qun chitosan trong bao b

kn, khng cho nh sng i qua (v d, trong hp, ti, bao) trong cc phng kn, kh

ro v nhit thng.

1.2 Tng quan v vi khun Bacsil lus subtil isv enzyme protease

1.2.1 Gii thiu v vi khun Bacillus subtilis.


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Vi khun Bacillus subtilis thuc gii Bacteria, ngnh Firmicuter, lp Bacili, b

Bacillales, h Bacillaceae, chi Bacillus, loi subtilis.

Hnh 1.9 - Vi khun Bacillus subtili ssau khi nhum gram

Hnh 1.10 - Hnh nh a khun lc ca vi khun Bacillus subtili s

Nm 1835, vi khun ny c tm ra v t tn l Vibrio subtilisbi Christian

Gottfried Ehrenberg.n nm 1872 c Ferdinand Cohn i thnh Bacillus subtilis.

Bacillus subtilis l loi trc khun gram dng, c hnh que ng ring l hoc

kt thnh chui hoc thnh si v c kh nng sinh ngoi bo t. Ngoi bo t c

hnh thnh khi gp cc iu kin bt li ca mi trng nh thiu ht cc thnh phn

dinh dng, nhit qu cao, hoc cc sn phm ph to thnh trong mi trng c

ch hot ng ca vi khun, ... Bo t c th tn ti trong mi trng mt thi gian
http://en.wikipedia.org/wiki/Christian_Gottfried_Ehrenberghttp://en.wikipedia.org/wiki/Christian_Gottfried_Ehrenberghttp://en.wikipedia.org/wiki/Christian_Gottfried_Ehrenberghttp://en.wikipedia.org/wiki/Ferdinand_Cohnhttp://en.wikipedia.org/wiki/Ferdinand_Cohnhttp://en.wikipedia.org/wiki/Christian_Gottfried_Ehrenberghttp://en.wikipedia.org/wiki/Christian_Gottfried_Ehrenberg


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kh di v khi gp iu kin thun li tr li, n s ny mm v hnh thnh nn t bo

vi khun mi.

Vi khunBacillus subtilisc hot tnh proteaza v amilaza, c kh nng phn hu

nhiu dng hp cht hu c c bit l protein v tinh bt.Trc y, ngi ta ngh rng y l loi trc khun hiu kh bt buc, nhng

iu khng chnh xc. Trong iu kin ym kh vi khun Bacillus subtilisvn pht

trin, mc d c km hn trong iu kin hiu kh. Nhit thch hp cho chng pht

trin l 35 450C (mi trng nui cy k 370C) v nhit ti a l 600C. Mi

trng c pH di 4,5 chng ngng pht trin [4].

Vi khun ny c tm thy ph bin trong t nhin, nhiu nht trn c kh, v

vy cn c gi l trc khun c kh.

Trn y l hnh nh ca vi khunBacillus subtilisc soi di knh hin vi sau

khi nhum gram v hnh nh khn lc thu nhn c sau khi nui cy (hnh 1.9).

Khun lc ca vi khun c mu trng c, mp vin rng ca, b mt khun lc

khng nhn bng m nhn v c nhng np gp nh (hnh 1.10).

1.2.2 Gii thiu v enzyme protease.

a) Lch s pht trin

Enzyme protease l nhm enzyme xc tc cho cc qu trnh thy phn lin kt

peptide trong cc peptide hoc protein. Trong cc protease, cc enzyme ca h tiu

ho c pht hin sm hn c. T th k 18, nh bc hc ngi Php Recomur

pht hin trong dch d dy ca chim n tht c kh nng tiu ho tht. Nm 1836,

Schwam quan st c hot ng phn giiprotein ca dch v. Tuy nhin, mi n

30 nm sau mi tch c enzyme ny. Nm 1859, Corvisart tch c tripxin t

dch ty, l protease u tin nhn c dng ch phm nhng ch phm ny vn

cn cha nhu protein v cc tp cht khc. Nm 1862, Danilevxki dng phng php

hp ph trn colodion tch c tripxin vi amylaza tu tng. Phng php hp th

la chn ca Danilevxki c ngha ln trong nghin cu tinh ch enzyme cng nh

protein. Nm 1861 Brunke cng tch c cc pepxin t dch d dy ch dng

tng i tinh khit. Nm 1872, Hommarster tch c ch phm chymozin

(renin).


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Cc protease thc vt c pht hin mun hn. Nm 1874, Group Besanez cng

b nhn c protease t ht ca mt loi cy h u ...

Cc protease ca vi sinh vt c ch nghin cu nhiu t nm 1950 mc d t

nm 1918 1919 Waksman pht hin c kh nng phn gii ca x khun.Trong hn mi nm nay, s cng trnh nghin cu v protease vi sinh vt ang tng

ln ng k nhiu hn cc cng trnh nghin cu protease v ng vt v thc vt [9].

b) Tnh cht ca enzyme protease

Protease l cc enzyme xc tc s thu phn lin kt peptide (CO-NH) trong phn

t protein v cc c cht tng t. Nhiu protease cng c kh nng thu phn lin

kt este v vn chuyn axit amin.

Di tc dng ca protease, protein b thu phn theo s sau:

Protein pepton polypeptit peptit axit amin

y l qu trnh thu phn tng i phc tp v c s tham gia ca nhiu loi

protease khc nhau gm hai loi chnh l exoprotease v endoprotease.

c) Phn loi enzyme protease

phn loi protease, ta da vo 2 cch sau:

Cch 1: Da vo trung tm hot ng:

Theo phn loi quc t cc enzyme thuc nhm ny chia thnh 4 phn nhm:

Aminopeptidase: xc tc s thu phn lin kt peptit u Nit ca mch

polypeptide; Cacboxypeptidase: xc tc s thu phn lin kt peptide u cacbon

ca mch polypeptide. C hai phn nhm enzyme trn u l cc exo-peptidase;

Dipeptihydrolase: xc tc s thu phn cc lin kt dipeptide; Proteinase: xc tc s

thu phn cc lin kt peptide ni mch [2].

Cch 2 : Da vo c ch thu phn, protease c phn thnh hai nhmchnh:

Exoprotease: nhng enzyme thu phn cc lin kt u mt ca chui mch

polypeptide; Endoprotease: nhng enzyme xc tc thu phn cc lin kt peptide

gia mch polypeptide.

Ngi ta cng phn loi cc protease theo vng pH hot ng ti thch: protease

axit, trung tnh, v kim tnh. Tuy nhin s phn loi ny ch c ngha thc dng

khng tht s chnh xc v pH hot ng ti thch ca enzyme cn ph thuc vo bn


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cht c cht v nhiu yu t khc na [2].

d) Ngun thu enzyme proteaza

Protease thu nhn t thc vt: chng ta c th thu mt lng ln protease t thc

vt. Tuy nhin vic thu nhn protease t thc vt gp nhiu vn : phi c t trngtrt, ph thuc vo iu kin thi tit kh hu, t ai, phi tri qua mt thi gian di...

Tuy vy, mt s cc protein thu nhn t thc vt c cht lng rt tt nh Papain,

Bromelain, Ficin ...

Protein thu nhn t ng vt: Cc protein c ngun gc t ng vt nh tripxin,

chymotrypxin, pepxin... c th thu c vi s lng tinh khit. Tuy nhin vic sn

xut n phi ph thuc vo vt nui cn git m, cc iu kin nng nghip...

Protease c ngun gc t vi sinh vt: Kh nng khng p ng c nhu cu trn

th gii ca enzyme ng vt v thc vt dn n s quan tm ngy cng tng ca

enzyme vi sinh vt. Vi sinh vt l ngun cung cp enzyme tuyt vi do tnh a dng

sinh ho v tnh nhy cm v k thut di truyn ca chng. Protease vi sinh vt chim

khong 40% tng lng enzyme bn trn th gii [2], chng c nhiu u im hn so

vi protease ca ng vt v thc vt do cc tnh cht ca chng ph hp vi kh

nng ng dng cng ngh sinh hc. Cc protease c th c thu nhn t vi khun,

nm v x khun.

e) Cc yu t nh hng n qu trnh sinh tng hp protease t visinh vt

Qu trnh tng hp enzyme ni chung cng nh tng hp protease vi sinh vt

ni ring cng chu nh hng ca nhiu yu t khc nhau nh: m mi trng,

nhit nui, pH mi trng, thng kh, thnh phn mi trng

nh hng ca nhit :Nhit nh hng n s sinh trng pht trin, kh

nng sinh tng hp enzyme ca vi sinh vt cng nh tnh cht ca enzyme c tnghp. Ty tng vi sinh vt, nhit thch hp c khc nhau: cc loi nm mc pht

trin thch hp nhit 22-320C, cn cc vi khun nhit cao hn 36-550C. Ni

chung a s vi sinh vt sinh tng hp enzyme khng bn vi nhit v b km hm

nhanh chng nhit cao hn nhit thch hp

nh hng ca pH mi trng: Khi dng phng php nui cy b mt, pH mi

trng t nh hng n qu trnh sinh tng hp enzyme vi sinh vt. Hn na pH

mi trng hu nh khng thay i trong qu trnh pht trin ca vi sinh vt. i vi


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nm mc pH thch hp cho qu trnh sinh tng hp protease vo khong 6 - 6,5. Cc

vi khun pht trin tt v to thnh nhiu enzyme pH gn trung tnh 6,6 - 7,4.

Khi dng phng php b su, pH mi trng c nh hng rt ln, nhiu khi c

vai tr quyt nh i vi s tch lu protease trong mi trng. ng lu l pH mitrng thng c th thay i sau khi kh trng v trong qu trnh pht trin ca vi

sinh vt. pH ban u ca mi trng thch hp cho s pht trin ca cc vi sinh vt

khc nhau khng ging nhau. i vi cc nm mc, pH ny vo khong 3,8 5,6, cn

i vi vi khun l 6,2 7,4 [2].

nh hng ca thng kh ca mi trng: Tu theo tnh cht ca tng loi vi

sinh vt khc nhau v sn phm cn thu nhn chng m s nh hng ca yu t

ny l khc nhau. Trong mt s trng hp, thiu oxy tuy kim hm s sinh trng

ca vi sinh vt nhng li lm tng qu trnh sinh tng hp protease v c th ngc

li.

thng kh ca mi trng c nh hng ln n qu trnh tng hp protease.

Tuy nhin nh hng ny c khc nhau tu theo vi sinh vt. Trong mt s trng hp,

thiu oxy tuyc kim hm s sinh trng ca vi sinh vt nhng li lm tng qu trnh

tng hp protease. Lng oxy thch hp cho qu trnh tng hp protease cc vi sinh

vt khc nhau l khng ging nhau (Aleeva 1973-Aravina v cng s nm 1976).

Ngay c i vi mt s vi sinh vt nht nh, s hiu kh cng nh hng khc nhau

n qu trnh tng hp cc protease khc nhau (Crivova v tp th nm 1977). Trong

mt s trng hp, s hiu kh mnh li km hm tng hp protease.

i vi nm mc, ch sc kh thch hp 10-12m3khng kh v trng (c nhit

khng cao qu 400C) trn 1m3mi trng trong 1h vi thi gian nui 68-73h. Vi

thi gian nui ngn hn trong cc thng ln men ging (48h) v trong cc thng lnmen sn xut (48-52h) th lng khng kh cn sc vo mi trng nui

Asp.oryzaephi l 30m3/m3mi trng/gi i vi thng nhn ging v m3/m3mi

trng/ gi cho thng sn xut. Khi nuiBacillus subtilistrong cc thng nhn ging

c cnh khuy lm vic lin tc v sc khng kh v trng vi lng l 40 m3/m3mi

trng/1h. V nui vi khun ny trong cc thng ln men trong sn xut (trong 48h

370C) c cnh khuy lm vic lin tc v sc khng kh v trng vi lng 60 m 3/m3

mi trng/h.


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nh hng ca thnh phn mi trng: Thnh phn mi trng nh hng rt ln

n kh nng sinh tng hp enzyme. tng lng enzyme trong mi trng cn la

chn ngun C, N, mui khong thch hp, chn t l nng thch hp gia cc thnh

phn ny v c bit l cn s dng cc cht cm ng v protease l enzyme cm ng.i vi mt vi sinh vt nht nh, iu kin thch hp cho qu trnh sinh tng hp

cc enzyme khc nhau th khng ging nhau. Nm vng cc qui lut nh hng ca

cc yu t ny c th iu khin vi sinh vt sinh tng hp nh hng mt loi

enzyme xc nh.

* nh hng ca ngun cacbon: Ngun cacbon thng dng nui cy vi sinh

vt thng dng l cc glucid: monosacarit, disacarit v c polysacarit. Cc cht ny

nh hng khc nhau n s sinh tng hp enzyme ni chung cng nh tng hp

protease vi sinh vt. c tnh tc dng ca ngun cacbon tu thuc vo bn cht ho

hc ca chng v c tnh sinh l ca vi sinh vt.

Ngun cacbon v nit t nhin thng dng trong mi trng nui cy l bt m,

bt u tng, cm v nc chit ca chng.

Trong s monosacarit, glucose nh hng rt khc nhau n qu trnh sinh tng

hp enzyme ni chung cng nh sinh tng hp protease.

* nh hng ca ngun nit: Cc cht c th dng lm ngun nit trong mi

trng nui cy vi sinh vt c th l cc cht hu c (protein v cc sn phm thu

phn ca chng) hoc cc mui v c (mui amn, mui nitrat). Nhiu vi sinh vt cn

c th s dng c nit ca HNO3, HNO2. Nng dng nit trong mi trng c nh

hng r rt v c quy lut n qu trnh tng hp protease ngoi bo vi sinh vt.

Ngun nit hu c thng c nh hng tt n qu trnh sinh tng hp protease

v chng c vai tr nh cht cm ng ca qu trnh ny. Cc ngun nit hu cthng dng l: cc loi bt khc nhau (c gluten), nc chit ca cm, nm men

t phn gii, pepton, protein

Ngoi vic chn ngun v lng cacbon, nit thch hp cn cn phi chi chn t

l thch hp gia C : N. Bn cnh photpho v lu hunh cng c nh hng quan

trng n qu trnh sinh tng hp protease.

Ngoi cc yu t k trn cc nguyn t vi lng (Steiner 1961), NaCl (Kovaleva

v tp th 1977), vitamin (Emxva 1975) v mt s yu t khc (Aravina v tp th


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1977) cng c nh hng n lng protease trong mi trng nui cy vi sinh vt.

1.2.3 Mt s nghin cu ng dng Bacillus subtilis ca cc tc gi trong nc

Nm 1997, TS. L c Mnh tin hnh nghin cu: Nghin cu thu nhn v

ng dng -amylase trong cng nghip sn xut bia dngBacillus subtilisCNTP-B-

08 c kh nng sinh tng hp -amylase cao, hot ca n t mi trng nui cy

b mt l 2610SKB/g thay th mt malt i mch bng cc loi nguyn liu sn c

trong nc nh: ng 30%, go 30%, i mch 40%, do gi thnh sn phm h.

Nm 2004, tc gi V Ngc Bi c cng trnh Nghin cu qu trnh thu

phn protein tBacillus subtilisS5 trong iu kin nhit l 50oC, pH t nhin, t l

ph phm 0,3% v nng mui thch hp l 3%.

Nm 1998, tc gi L c Mnh c nghin cu Nghin cu thu nhn v bo

qun protease t ch phm lnmen b mt ca vi khun Bacillus subtilis. Protease

ca vi khun hot ng mnh nht nhit 42 48oC, nu nhit cao hn hoc

thp hn th protease s b mt hot ng k ch cn li khong 40%, pH thch hp

cho protease hot ng l 6,4 7. Protease ca vi khun kh bn vi nhit , nhit

50o

C, hot ca protease khng gim sau 6 gi. nhit cao hn th hot protease gim dn theo thi gian v c bit nhit 70oC hot ca protease

gim rt nhanh.

Nm 2004, Th.S Th Bch Thu v TS. Trn Th X thc hin ti

Nghin cu mt s nh hng ln kh nng sinh protease caBacillus subtilis. Kt

qu ca nghin cu cho thy ngun cacbon v nit c tc dng kch thch cao nht l

tinh bt tan v casein. Thnh phn nui cy chng Bacillus subtillis trong phng th

nghim bao gm: Pepton 1%, cao tht 0,3%, mui 0,5%, tinh bt tan 1,75%, cao nm

0,1%, ph ban u l 6,5 v nhit nui cy thch hp nht ng vi mi trng ti u

Bacillus subtillissinh protease mnh nht l 35oC.

1.3 Cc lnh vc ng dng ca chitin/chitosan

Chitin v chitosan c th d dng b vi khun phn hy, tc l chng cng thn

thin vi mi trng hn cc loi polymer tng hp. Hin nay chitosan c ng


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dng trong rt nhiu lnh vc nh: y hc, thc phm, nng nghip, mi trng

1.3.1 Trong y hc

Chitin v chitosan l nguyn liu sn xut thuc iu tr bng, vim lot d dy,

h cholesterol, tr bo ph, gim au, chng ng t mu, tng sc khng, cha

xng khp v chng c c bnh ung th.

Tc nhn h cholesterol: Chitosan c chc nng h cholesterol trong rut ng

vt. Ngi ta tin hnh th nghim vi th v thy khi c cho n thc n giu

cholesterol 0.9% trong 39 ngy, lng cholesterol huyt thanh tng t 79 ln 650 mg/

trn 1kg th trng. Trong trng hp vi khu phn n nh trn nhng c b sung 2%

chitosan, lng cholesterol huyt thanh ch tng khong 300 mg/1kg th trng, trong

lng cholesterol c ch (HDL cholesterol) gim khng ng k. V t lu, mt

s chuyn gia Trung tm Huyt hc thuc Vin Hn lm Y hc Nga cng pht

hin chitosan c th ngn chn s pht trin ca chng nhi mu c tim v bnh t

qu

iu tr bo ph: Khi vo ng tiu ha, chitosan c kh nng bao cc ht cu

bo v ko chng thi ra ngoi theo (ng vt khng tiu ha chitosan) nh nc ng dng lm thuc gim bo. Nc ta hin nay c thuc Chitozan gim

bo.

Cc vt liu y sinh hc v dc phm: chitin v cc dn xut c s dng nh

nhng vt liu y sinh hc hay vt liu bao gi cc loi thuc tan chm. Film

chitosan bao thuc cng c cng dng nh cc dng con nhng thng phm v c

s dng nh vt liu dng cy gii phng chm cc loi thuc chng ung th.

Chitosan cng c a vo cng thc cc loi thuc ung lm gia tng s hp thu

ca thuc vo mu.

Vt liu v vt thng: Cc vt thng m ng, thc vt c th c bao bng

mt tm mng hay mt ming xp chitin v chitosan dng bng hoc dng bt mn.

Cc vt thng cng c th c tr liu bng cc dung dch hay kem chitin v

chitosan. Kt qu l s pht trin ca cc t bo vng m b thng c kch thch,

chitinase v lysozyme c tng cng dn n mau lnh vt thng v hn ch

nhim trng. Ti cuc chin Iraq va qua, M cng s dng loi bng cu thng


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kiu mi, k thut cao, c thnh phn cu to bi cht chitosan. So vi cc loi bng

thng, tc cm mu, tnh st khun v thi gian lnh m khi s dng loi bng

ny c hiu qu hn gp nhiu ln.

Cha bnh khp: in hnh trn th trng dc hin nay l loi thuc cha khplm t v tm c tn Glucosamin ang c thnh hnh trn ton th gii. So vi sn

phm cng loi th Glucosamin c u th hn, do sn xut t ngun v tm t nhin

nn sn phm t gy phn ng ph, khng c hi v khng b ri lon tiu ho cho

ngi bnh (iu ny c ngha rt quan trng). Nc M tiu th c hn 1 t

vin nang Glucosamin. Nhng nm gn y, loi thuc cha khp ny cn c ph

cp rng nhiu nc trong c Vit Nam. Nc ta cng c thuc Glusivac c tr

thoi ha khp [19].

1.3.2 Trong x l nc thi v lm trong nc sinh hot

Dng chitosan hpth cc ion kim loi nng gy nhim mi trng sinh thi

nh Cu, Hg, Cr, Pb, Ni, Cd.

Chitosan cationit to cc phc hp a in ly vi nhng polymer polyanionic v

to phc hp chelate vi cc ion kim loi to kt ta. Cc phn ng ny c ngdng lm trong nc thi nhim. Vo nm 1975, mui chitosan acetate ln u

tin c mt cng ty Nht gii thiu nh mt tc nhn cationit t nhin ng t

v loi cc cht thi trong nc cng. Hin nay h thng ny vn cn c s dng

x l nc thi sinh hot, ti s dng nc thi (v d nc h bi), thu hi cc

protein v khong t nc thi cng nghip, bao bc cc ht bo, phn lp cc cht c

hot tnh sinh hc trong nc tiu v tch cc cht c ni bo t dung dch long.

Chitosan cngc s dng nh mt cht hp ph tch cc ng v phng x nguy

him t nc nhim v thu hi uranium t nc bin v nc ngt [27].

1.3.3 Trong nng nghip

Lm phn bn rau sch: Chitin v chitosan ch yu c bn dng bt, dng

ming hay dng dung dch vo t nng nghip hay mi trng nui cy lng. Ngoi

ra dung dch chitosan c th c phun ln l cy.


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X l ht ging: ht ging c th c nhng vo dung dch chitosan long, bao

ph b mt bng mt mng chitin hay chitosan mng hay bt mn. Khi c bao ph

nh vy, hot ng ca chitinase ca ht c tng cng trong giai on ny mm.

S tng cng chitinase trong ht s lm gia tng kh nng t v sinh hc ca ht quavic nhim VSV, kt qu l lm cho cy trng pht trin tt.

1.3.4 Trong m phm

Chitosan l nguyn liu sn xut kem dng da, kem chng tia t ngoi

Cc mui hu c ca chitosan phn t thp ha tan trong ethanol long v c

s dng nh mt thnh phn ca keo xt tc.

CM-chitin v HP-chitosan cationic ha tan trong nc v bn trong mt khong

pH rng, chng c s dng nh mt thnh phn ca m phm chm sc da.

Chitosan, CM-chitosan, HP-chitosan c chc nng to m cho da, ngn cn s hy

hoi c hc tc. c tnh gi m tng ng vi dung dch propylenglycol 20% v

dung dch hyaluronic long. Nhng dn xut trn ca chitosan c th ngn chn s

nhim khun trn da v hot ha t bo da dn n s ngn chn s lo ha ca da

[19, 27].

1.3.5 Trong thc phm

Hin nay chitosan c s dng rt ph bin trong sn xut thc phm. C th s

dng thu ht nc v bo (qu trnh nh ha), kt hp vi thuc nhum (qu trnh

ng ha), qu trnh ng c. Chitosan cng c chng minh l c kh nng to

dng mng mng s dng nh l nhng lp mng mng hoc nhng lp bao khng

c hi (c th n c). Mng bao Chitosan c th ci thin kh nng bo qun cc

loi thc phm d b thi ra bng cch gim lng khng kh bn trong bao gi cng

nh gim qu trnh thot hi nc.

C th nhng trc tip thc phm vo dung dch chitosan pha sn ri kh, to

thnh mt lp mng mng t nhin trn b mt sn phm: trng, tht c, rau qu, gi

, bnh go, nhng ci bp trc khi lm kim chi

Hoc cng c th to thnh mng trc ri mi cho sn phm vo: bnh m, xc


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xch

Hoc cho chitosan trc tip vo sn phm dng lng: x l nc qu, lm trong

gim, bo qun tu h, ng ha sa, kem v mayonaise, bo qun m[12, 19, 24].


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Chng 2 - NGUYN LIU V PHNG PHP NGHIN CU

2.1. Nguyn liu, hacht v thit b

2.1.1. Nguyn liu:

Nguyn liu c s dng bao gm:

a) Chng vi khun Bacillus subtilis c phn lp v gi ging ti phng th

nghim CNSH - Khoa Ho - trng i hc Bch Khoa - i hc Nng;

b) V u tm c thu nhn t cng ty c phn XNK Thy sn Min trung:

Cng ty ch bin v xut khu thy sn Th Quang;

Ph phm sau khi thu nhn t nh my ch bin thy sn c sy cho kh rn

nhit 400C ri nghin nh v bo qun lm ngun nguyn liu thu nhn chitin, v

sau t chitin thu nhn chitosan.

Trong nghin cu ny nguyn liu v tm c thu nhn t hai loi tm ph bin

trong ch bin trn a bn TP Nng hin nay l tm s (tiger shrimp Penaeus

monodon Fabricius) v tn th chn trng (White Shrimp - Penaeus vannamei). Cc

th nghim c tin hnh trn hn hp 2 loi u v tm ny.

a b

Hnh 2.1S khc nhau ca mt s nguyn liu tm, v ca chng dng

thu nhn chitin/chitosan

a)Tm s; b) tm th chn trng


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c) Ni tng c thu nhn ch phm enzyme protease c thu gom t cng ty c

phn XNK Thy sn Min trung: Cng ty ch bin v xut khu thy sn Th Quang.

Ni tng c sau khi c thu nhn t nh my v c ra sch, x l bng nc

mui 5%, sau c bo qun ng lnh nhit -220

C phc v thu nhn chphm enzyme protease.

2.1.2. Ho cht v thit b:

Ho cht chnh:

- Cc ho cht tinh khit nh : HCl m c, Foocmol 40%, cn tuyt i, KOH

0,01N, H2SO4

- Cc ch th mu : thimolphtalein, phenolphtalein

- Pepton, casein, cao tht, cao nm

- V cc ha cht phc v cc th nghim phn tch khc.

Thit b:

- T n nhit, my lc, thit b tit trng, t lnh ng, t cy v trng.

- Thit b li tm

- Thit b c c chn khng- Thit b phn tch m Kjeldahl.

- V cc thit b thngdng trong phng th nghim CNSH khc.

2.2. Phng php nghin cu

2.2.1 nh lng nc

V tm c sy 1200C trong 3 gi, n khi lng khng i. ngui, sau

cn xc nh lng nc theo cng thc:

2.2.2 nh lng mui Calci carbonat

Mui calci carbonat trong v tm c nh lng bng cch ngm v tm

c xay nh v sy kh trong dung dch HCl 10% trong 6 gi, nhit phng. Sau

lc, ly nc ci dng acid H2SO430% kt ta Ca

2+

trong dung dch. T trnglng CaSO4kt ta v sy kh suy ra hm lng calci carbonat


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2.2.3 nh lng chitin

Sauk hi loi b calci carbonat v protein, chitin c un nng vi dung dch

K2CO3 m c 1600C chitin khng tan. Lc, ra sch, sy 1000C, thu c

chitin.

2.2.4 Phng php xc nh deacetyl ca chitosan

n v phn trm deacety trong sn phm chitosan c xc nh bng mc

deacetyl (y l t l ca khi lng glucosamine so vi s monomer chung trong

phn t polymer DD = m(m+n) = Namin/Ntng), thng t 70-90%.

2.2.5 Mt s phng php xc nh khc:

-Hm lng lipid (%), protein (%), tro (%) v hm lng nc (%) c xc nh

theo GOST 7636-85 (LB Nga)

-Hm lng khong c phn tch theo chun ca AOAC (1990)

- Phng php xc nh N-formon theo Sorensen

- Hot enzyme, v ch phm enzyme protease xc nh theo phng phpAnson ci tin

- Phng php xc nh QMAFAM (CFU/g) theo GOST 10444.15 94 (LB Nga)

- kh khong trong v tm c xc nh theo cng thc KK=[(mK1-

mK2)/mK1]x100%; mk1, mk2-hm lng cht khong banu v sau khi kh khong

trong v tm .

- kh protein trong v tm c xc nh theo cng thc KK=[(mP1-

mP2)/mP1]x100%; mP1, mP2-hm lng protein ban u v sau khi kh protein trong v

tm.

- deacety trong sn phm chitosan c xc nh theo t l ca khi lng

glucosamine so vi s monomer chung trong phn t polymer DD = m(m+n) =

Namin/Ntng), thng t 70-90%; nht ca chitosan c xc nh trn nht k

Roto Brookfield

2.3 S cc m hnh th nghim (hnh 2.2)


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Tng quan cc nghin cu trong v ngoi nc vchitin/chitosan

Tnh cht vtl v ha hc

cachitin/chitosan

Cc phngphp thu nhnchitin: ha hc,

sinh hc

Phng phpthu nhnchitosan

Cc lnh vcng dng

chitin/chitosa

n

ng dng vikhun B. subtilis

trong sn xutchitin

Xy dng cc m hnh th nghim gii quyt cc vn cn nghin cu ca ti

Nghin cu thnh phnha hc ca v u tm

Nghin cu thnh phn hahc v hot enzyme ca ni

Nghin cu mi trng nui cyv kh nng sinh protease ca

Tch chit enzyme protease tni tng c

Kh mu ca v u tm

Kh khong ca v u tm

Nghin cu kh protein ca u v tm

Kh protein bng ch phm enzyme protease,tch chit t ni tng c

Kh protein bng ch phm enzymeprotease

Kh khong ln 2

Chitin Chitosannh gi cht lng

chitin

nh gi chtlng chitosan


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Hnh 2.2S m hnh cc bc thc hin trong nghin cu

2.4 X l s liu

S liu trong nghin cu ny l trung bnh ca ba ln phn tch. Kt qu c

phn tch thng k bng phn mm Minitab Professional v16.1.0.0. . Gi tr ca p

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Baocao Chitosan Final