M U

Ngy nay, cng vi s pht trin ca x hi, c bit l trong ngnh cng nghip
thc phm dc phm, nhu cu con ngi c p ng tt hn cc yu t v cuc
sng cng nh sinh hot. c bit, l trong s cung ng cho nhu cu pht trin ca h
thn kinh v tim mch ca con ngi. Vi nhng tin b v khoa hc k thut nht l
s pht trin cng nghip ca con ngi. Cc cuc hi tho, m phn, lin kt trn
ton th gii nhm mc ch pht trin sn phm dng thuc cung cp thnh phn
chc nng cho xng vi vai tr ca chng. Trong qu trnh tm hiu, v mc ch hc
tp, nhm chng ti xin cp n mt sn phm ch bin dng thuc (hoc vin nn)
gi l TRYPTOPHAN. y l tn ca mt loi acid amin thit yu cho h thn kinh
ca con ngi. Ngun cung ng Tryptophan, tuy c th t nhng nguyn liu t nhin,
nhng cung cp cho con ngi mt cch nhanh chng, ph hp v d dng, c
nhng tng sn xut Tryptophan thnh nhng ch phm dng thuc.
Ni dung tm hiu nh sau:
Phn 1: Tng quan
Phn 2: Nguyn liu sn xut Tryptophan.
Phn 3: Quy trnh cng ngh sn xut Tryptophan.
Phn 4: Sn phm.
D nhm chng ti c gng tm hiu, thu thp, tng hp v chn lc ti liu
nhng bi vit s khng trnh khi nhng thiu st ng c. Mong rng vi nhng
thiu st trn s c thy v cc bn gp v chia s cho nhm.
Sau cng, xin c gi li cm n n thy PGS. TS. L Vn Vit Mn, B Mn
Cng ngh Thc phm, trng i Hc Bch Khoa Tp. H Ch Minh truyn t v
to c hi cho chng em cng c, nng cao kin thc.
Tp. H Ch Minh, ngy 05 thng 12 nm 2013
Nhm sinh vin thc hin

MC LC
1

TNG QUAN .........................................................................................................1

1.1

Gii thiu v Tryptophan ...................................................................................1

1.2

Phng php sn xut L-Tryptophan trong cng nghip ..................................2

i

1.2.1

Con ng tng hp tin cht trung gian chorismate .................................3

1.2.2

Sinh tng hp Tryptophan...........................................................................4

NGUYN LIU ......................................................................................................5

2.1

2.1.1

Chn ging vi sinh vt ................................................................................5

2.1.2

c im ca E.coli. ...................................................................................6

2.2

Vi sinh vt ..........................................................................................................5

Mi trng: ........................................................................................................8

2.2.1

Mt r ng ma. ........................................................................................ 8

2.2.2

Nguyn liu ph. ......................................................................................... 9

2.2.2.1

Dch chit nm men. .............................................................................9

2.2.2.2

Nc ...................................................................................................10

2.2.2.3

Tryptone. ............................................................................................ 11

2.2.2.4

Cht chnh pH v trung ha ................ Error! Bookmark not defined.

2.2.2.5

K2HPO4............................................................................................... 12

2.2.2.6

Ngun kim loi vi lng ....................................................................12

QUY TRNH CNG NGH .................................................................................14

3.1

S khi ........................................................................................................14

3.2

Gii thch quy trnh cng ngh.........................................................................16

3.2.1

X l mt r ng ....................................................................................16

3.2.2

Chun b mi trng ln men ...................................................................18

3.2.3

Nhn ging vi sinh vt ..............................................................................19

3.2.4

Tit trng ...................................................................................................21

3.2.5

Ln men. ....................................................................................................22

3.2.6

Ly tm .......................................................................................................26

3.2.7

Kt ta .......................................................................................................27

3.2.8

Ly tm ln 2 .............................................................................................. 28

3.2.9

Ha tan kt ta .......................................................................................... 29

3.2.10 Trao i ion ................................................................................................ 29

3.2.11 C c chn khng .....................................................................................33
3.2.12 Kt tinh ......................................................................................................35
3.2.13 Ly tm .......................................................................................................36
3.2.14 Sy chn khng ......................................................................................... 36
3.2.15 ng gi. ...................................................................................................37
4

SN PHM...........................................................................................................37

THNH TU CNG NGH ...............................................................................38

ii

TI LIU THAM KHO .....................................................................................40

iii

DANH MC CC HNH NH
Hnh 1.1.1: Cng thc cu to Tryptophan ....................................................................1
Hnh 1.1.2: Con ng sinh tng hp tin cht trung gian chorismate.......................... 3
Hnh 1.1.3: Con ng sinh tng hp L-Tryptophan .....................................................4
Hnh 2.1.3.1: Vi khun E.coli ......................................................................................... 6
Hnh 3.1.1: Quy trnh cng ngh sn xut ch phm Tryptophan ................................ 15
Hnh 3.2.1.1: Thit b ni phn ng. ............................................................................17
Hnh 3.2.1.2: Thit b ly tm lm sch mt r. .............................................................. 17
Hnh 3.2.3.1: Thit b phi trn. ...................................................................................18
Hnh 3.2.2.1: My lc n nhit (Personal - 11, TAITE ) ..............................................21
Hnh 3.2.4.1: Thit b tit trng dng bn mng. ......................................................... 22
Hnh 3.2.5.1: Thit b ln men b su. ..........................................................................25
Hnh 3.2.6.1: Thit b ly tm s dng chn xoay dng cn. .........................................27
Hnh 3.2.8.1: Thit b kt tinh bng phng php lm lnh dng tn si. ...................29
Hnh 3.2.7.1: Nguyn l thit b trao i ion. ............................................................... 32
Hnh 3.2.11.1: Thit b c c chn khng ...................................................................34
Hnh 3.2.14.1: Thit b sy chn khng. .......................................................................36
Hnh 4.1: Sn phm Tryptophan dng vin v tinh th ................................................37

iv

DANH MC BNG BIU

Bng 2.2.1.1: Thnh phn cc cht trong mt r ng ma (%) ....................................8

Bng 2.2.1.2: Hm lng vitamin ca r ng (g/1g r ng) .................................9
Bng 2.2.2.1.1: Thnh phn dinh dng trong 100g dch chit nm men ...................10
Bng 2.2.2.2.1: Tiu chun nc dung trong thc phm .............................................10
Bng 4.1: Thnh phn ha hc ca 1 ch phm Tryptophan........................................37

TNG QUAN

1.1 Gii thiu v Tryptophan

Tryptophan l mt trong nhng amino acid khng thay th cn thit cho c th
ca ng vt cng nh con ngi. Tryptophan c cng thc ho hc l C 11H12N2O2, l
mt amino acid d vng, c xp vo nhm acid amin thm (Phenylalanine, Tyrosine
v Tryptophan). Amino acid Tryptophan c hai dng ng phn lp th D v L, trong
L-Tryptophan l amino acid c vai tr quan trng trong cc protein i vi c th
sinh vt.
Cng thc cu to:

Hnh 1.1.1: Cng thc cu to Tryptophan

Tm quan trng ca L-Tryptophan i vi sc kho
Tryptophan l mt acid amin thit yu c hnh thnh t protein trong qu trnh
hot ng ca cc enzyme thy phn protein. Tryptophan ch c hp thu thng qua
thc phm v c th khng th t tng hp c. Trong c th, Tryptophan c
chuyn i thnh 5-HTP, sau c chuyn thnh serotonin (mt cht dn truyn
thn kinh mnh trong no). Serotonin c lin quan n tm trng, gic ng, chn n v
kim sot xung thn kinh. Ngoi ra, Tryptophan cn rt cn thit cho vic sn xut
vitamine B3 v B6, biotine v vitamine C v km to thnh cc enzyme cn thit
cho vic chuyn i.
Tryptophan tng cng th gin v gic ng, gip lm du thn kinh v lo lng,
v lm gim cm gic thm n. Tryptophan gip kim sot hiu ng thi qu tr
em, lm gim cng thng v tng cng hormone tng trng cn thit sn xut
vitamine B6.

Tryptophan l mt acid amin c hu ht cc loi thc phm: socola, yn mch,

sa, sa chua, ph mai, trng, c, gia cm, tht, vng, u xanh, ht hng dng, ht
b ng, u phng. V Tryptophan cung cp mt phn vitamine B3 cho c th, khi
thiu c th lm tng nguy c thiu ht vitamine B3. Cn c th dn dn mt s bnh
nh: trm cm, mt ng, tm thn phn lit kt hp vi s thiu ht magie gp phn co
tht ng mch tim.
1.2 Phng php sn xut L-Tryptophan trong cng nghip
L-Tryptophan l mt acid amin c tim nng thng mi cao do nhng li ch
i vi sc kho. L-Tryptophan c nghin cu sn xut t nhng nm u ca
th k 20. p ng vi nhu cu tng dn ca th trng, mt lot cc phng php
ho hc v sinh hc c pht trin. Tng hp bng ho hc l phng php u
tin c ng dng vi nguy m cng nghip. Vo cui nhng nm 80, vic sn xut
L-Tryptophan c thc hin thng qua cc phng php ho hc, enzyme v qu
trnh ln men. Hin nay trong cng nghip, vic sn xut amino acid L-Trytophan ch
yu l da trn con ng sinh tng hp Tryptophan vi sinh vt. L-Tryptophan c
sn xut bi cc chng vi khun Corynebacterium glutamicum v Escherichia coli.
Nguyn nhn l do chng ln men vi sinh cho php sn xut c L-Tryptophan t cc
ngun carbon r v c th ti to nh sucrose, glucose hay mt ng. Nhiu nghin
cu nh gy t bin VSV, s dng cng ngh gen ti t hp ADN c tin hnh
nhm nng cao hiu sut sinh tng hp trytophan. Cng nh cc amino acid thm d
vng khc, con ng sinh tng hp Tryptophan bao gm hai giai on: tng hp ra
tin cht trung gian chorismate v sinh tng hp L-Tryptophan.

1.2.1 Con ng tng hp tin cht trung gian chorismate

Hnh 1.1.2: Con ng sinh tng hp tin cht trung gian chorismate
Bc u tin l s ngng t ca phosphoenolpyruvate (l mt glycolytic trung
gian ) vi erythroe-4-phosphate ( mt pentose phosphate ). Kt qu l to ra mt hp
cht 7 carbon m u cho chu i oxy ha tip theo, hp cht ng ny sau b mt
i nhm phosphoryl, thc hin vic ng vng to thnh 3-dehydroquynate. Phn ng
tip theo l s loi i 1 phn t nc to ra 3-dehydroshikimate. S kh
NADPH+H+ thnh NADP+ t 3-dehydroshikimate thnh shikimate. Tip thep
shikimate ly 1 nhm phosphoryl t ATP to thnh shikimate-3-phosphate v ADP.
Mt phn t phosphoenolpyruvate th hai s c s dng ngng t vi shikimate3-phosphate, ti y khi kt hp vi shikimate-3-phosphate n s b mt i nhm
phosphoryl ca n, to thnh 3-enolpyruvyl-shikimate-3-phosphate ri to ra
chorismate . y chnh l tin thn chung ca c 3 acid amin thm. Cc enzyme tham
gia vo cc phn ng gm:
3-deoxy-D-arabino-heptulosonate 7-phosphate synthetase (DAHP synthetase )
3-dehydroquinate synthetase.
Dehydroshikimate reductase.
3

Shikimate kinase.
5-enolpyruvylshikimnate 3-phosphate synthetase.
Chorismate synthetase.
1.2.2 Sinh tng hp Tryptophan

Hnh 1.1.3: Con ng sinh tng hp L-Tryptophan

Chorismate nhn 1 nhm amin t glutamine thy phn mch bn, to thnh
anthranilate v gii phng 1 pyruvate. Sau anthranilate ngng t vi 5phosphoribosyl-1-pyrophosphate (PRPD), mt dng hot ha ca ribose phosphate.
PRPD cng l mt cht trung gian quan trng tng hp histidine, cc nucleotide
purine, v cc nucleotide pyrimidine. Nguyn t C v tr C-1 ca ribose 5- phosphate
lin kt vi nguyn t nit ca anthranilate trong phn ng , v gii phng ra 1
pyrophosphate. Mt na ribose ca phn t phosphoribosylanthranilate chu s sp sp
li to thnh 1-(o-carboxyphenylamino)-1-deoxyribulose 5- phosphate. Sau ngay
lp tc thc hin dehydrate v decarboxylate to thnh. indole-3-glycerol phosphate,
ri thc hin ct 3-glycerol phosphate cho ra indole. Tip theo Indole phn ng vi
serine to thnh Tryptophan. Trong bc cui cng phn ng c xc tc bi
enzym synthetase Tryptophan. Mch bn ca indole-3-glycerol phosphate c ly ra
to thnh glyceraldehyde 3-phosphate v thay th vo l b khung carbon ca
serine. Cc enzym tham gia phn ng gm:
Anrailaie synthetase
Anthranilate-5'-phosphoribosyl-1-pyrophosphatephosphoribosyl transferase (PR
transferase)
N-(5'-phosphoribosyl)-anthranilate isomerase
4

Indoleglycerol phosphate synthetase.

Tryptophan synthetase.

NGUYN LIU

2.1 Vi sinh vt
2.1.1 Chn ging vi sinh vt
Trong thin nhin, c nhiu vi sinh vt c kh nng tng hp Tryptophan, nhng
s loi vi sinh vt c kh nng sinh tng hp nhiu Tryptophan s dng trong qui
m sn xut cng nghip l khng nhiu.

iu kin sng bnh thng, lng acid

amin do chng t tng hp c thng p ng yu cu bn thn nhng trong

iu kin khng bnh thng no lng cc acid amin ny c th vt xa nhu cu
bn thn v tch ly li trong t bo hay thot ra ngoi mi trng. Hin tng ny
c gi l siu tng hp acid amin ca vi sinh vt. thu nhn Tryptophan t vi sinh
vt quy m cng nghip, cn phi tin hnh phn lp ging, tuyn chn, gy t
bin, chn iu kin ti u cho qu trnh sinh tng hp. Chn loi vi sinh vt tch ly
c nhiu acid amin l khu quan trng nht. Nhiu nghin cu xc nh nhiu
loi vi sinh vt tng hp c Tryptophan vi lng khc nhau. Trong s ng k
nht l hai chng Corynebacterium glutamicum v Escherichia coli (E. coli).
Bng cch to ra c cc chng E.coli ti t hp, t bin c kh nng tng hp
Tryptophan cao. Cc tc nhn gy t bin c th l tc nhn vt l nh tia bc x ion
ha hay tc nhn ha hc, s dng cc cht oxy ha mnh nh etylen hoc cc cht
gy ng v phng x mnh. Nh nu trn con ng tng hp L-Tryptophan ca
E.coli thng qua hai giai on. Con ng chung tng hp ra tin cht trung gian
chorismate ( ging nhau c ba con ng tng hp cc amino acid thm d vng) v
con ng tng hp ring to ra Tryptophan.
Nhiu nghin cu c a ra nhm ci to ging E.coli, nng cao hiu sut
sinh tng hp sn phm bt u t nhng nm 1979. ng dng u tin ca cng ngh
DNA ti t hp ging ci tin cho sn xut L - Tryptophan c thc hin vi E. coli
bi Tribe v Pittard (1979). Bo co ch ra vic tng sn lng sinh tng hp sn phm
bng cch khuch i trp operon vi gen trpE. Mt khc, Aiba (1982) xy dng mt
5

chng bin i gen bng cch t hp mt plasmid c cha cc trp operon vi trpE v
gen trpD vo mt vi khun E. coli. Bng cch nui dng vi sinh vt trong mi trng
glucoes b sung lin tc axit anthranilic, h thu c 6,2g/l L-Tryptophan sau 27 gi.
Qu trnh ny ca L-Tryptophan ln men t c hai ng v axit anthranilic c
ci thin hiu qu sn xut cao hn bi Azuma (1993). Vic phn lp mt 6 fluoro
Tryptophan v 8 - azaguanine t chng ti t hp xy dng bi Aiba v cng s
(1982). Trong mt nghin cu khc v vic ti mt t hp E.coli, Chan v cng s
(1993) thu c mt L-Tryptophan tng hp E.coli chng n nh vi vic tch hp ba
bn sao ca trp operon trn ADN v thu c 9.2g/l L-Tryptophan vi hiu sut tng
13% sn lng chuyn i trn ng. Trong qu trnh ny, cc trp operon th hin
c tnh duy tr n nh trong sut qu trnh ln men, ch ra rng vic khuch i gen
trn nhim sc th c th l mt phng php trong cng ngh sn xut Tryptophan.
Mt s phng php tip cn khc ci thin sn xut L-Tryptophan ca chng
E.coli thng qua s khuch i ca cc gen trp operon cng c m t bi Berry
(1996) v Camakaris (1997) [10].
y ta chn gii thiu ging E.coli TRTH0709 ti t hp gen, c cha gen
khuch i trong cc operon (trpEfbr DCBA) v DAHP synthase (aroGfbr), v gen
serine-biosynthetic (serA) trn pBR322, vi s xo b hai gen trpR v tna [10].
2.1.2 c im ca E.coli.

Hnh 2.1.3.1: Vi khun E.coli

V c dom in

Bacteria
6

Ng nh ph lum

Proteobacteria

L p cl ss

Gamma Proteobacteria

B (ordo):

Enterobacteriales

f mili

Enterobacteriaceae

Chi (genus):

Escherichia

Lo i (species):

E.coli

Gram m, k kh ty tin, khng sinh bo t.

T bo hnh que di khong 2 m, ng knh 0,5 m, th tch t bo 0,6 0,7

m3.

L vi sinh vt a m, c th sng trong khong nhit 70oC-500oC, nhit

gy cht 600oC- 650oC trong 1 pht

Nhit ti u: 37oC, pH = 4,4 - 8,5

m ti thiu: aw = 0,91
Sinh trng trong iu kin hiu kh hay k kh dng nhiu cp oxy ha kh

khc nhau, bao gm s oxi ha pyruvic acid, formic acid, hydroge, amino acids v s
gim c cht nh oxygen, nitrate, dimethyl sulfoxide v trimethylamine N-oxide.
n in
o

co i c

n c

Ngun cacbon: glucose, lactose, mannitol, maltose, fructose, galactose, sorbitol,

glycerol, acid cinamic.

Ngun nit:
o

V c: ammonia, nitrate, NH4Cl, (NH4)2SO4, (NH4)2-H-citrate.

Hu c: peptone, L-asparagine, adenine, adenosine, agmatine, L- v D-alanine,

aminobutyrate, arginine, aspartate, cytidine, cytosine, glucosamine, glutamine,

glutamate, glycine, ornithine, proline, putrescine, L- v D-serine, threonine.
o

Ngun khong: KH2PO4, K2HPO4, Na2HPO4.12H2O, MgSO4.7H2O, CaCl2,

FeSO4.7H2O, ZnCl2.7H2O, MnSO4, CuSO4.5H2O, Mo7O2.4H2O, Co(NO3).

o

Cc cht sinh trng: Folic acid, Biotin, Pyridoxine (B6), Riboflavin (B2),

Thiamine (B1), Vitamin K, Vitamin B12, Nicotinic acid, Pantothenic acid, thymine.

2.2 Mi trng:
2.2.1 Mt r ng ma.
E.coli c th s dng C t nhiu ngun carbon khc nhau nh glucose, lactose,
mannitol, maltose, fructose,Vi quy m sn xut cng nghip, ta chn mt r ng
do tnh kinh t cao. Theo Elnaz Faghfuri (2013), nguyn liu mt r ng ma l mt
ngun nguyn liu r tin c cha cc tin cht L-Serine v cc cofactor PLP h tr
cho qu trnh ln men, nng cao tnh thng mi. [12]
Mt r ng ma l ph liu cha ng nhiu ng khng kt tinh trong sn
xut ng t ma. Thng thng t l r ng trong sn xut ng ma chim
khong 3-5% trng lng ma. u im ca mt r ng ma l ngun nguyn liu
ph bin, ngun cung cp nhiu, gi thnh r.
Bn 2.2.1.1: Th nh phn cc cht trong mt r ng m

Thnh phn

Mt r t ma

Cht kh (% khi lng mt r)

75 - 83

Sucrose (% khi lng cht kh)

32 - 45

Glucose

5 - 11

Fructose

6 - 15

Cht hu c phi ng
- Cht ni t

-Khng cha ni t

10

Ni t tng

0,4 1,5

Tro

7 11

pH

4,5 6,0

Bn 2 2 1 2:

m lng vit min c r ng g/1g r ng

Hm lng

Vitamin
Thiamin

8,3

Biotin

12,0

Pyridoxine

6,5

Riboflavin

2,5

Acid nicotinic

21,0

Acid folic

0,038

Acid pantothenic

21,4

Tiu chun chn nguyn liu:

-

Cht kh: >= 75%

Hm lng sucrose: 50 51% lng ng

Hm lng N khng t hn 1,4%

S lng vi sinh vt khng qu 15000 cfu/1g nguyn liu.

C th b sung thm ure l ngun cung cp N v supephosphat l ngun cung

cp.

2.2.2 Nguyn liu ph .

2.2.2.1 Dch chit nm men.
Vai tr: Dch chit nm men cha nhiu thnh phn dinh dng nh amino acid,
peptide, carbonhydrate v cc vitamin tan trong nc, c b sung vo mi trng
nui cy cho VSV sinh trng v pht trin

Bng 2.2.2.1.1: Th nh phn dinh dng trong 100g dch chit nm men
Hm lng (g)

Thnh phn
Protein

27,8

Cacbohydrate

11,8

Nc

37

Tro

23,4
Hm lng (mg)

Vitamin

Khong

Hm lng (mg)

Thiamin

9,7

Ca

86

Riboflavin

14,3

Fe

3,7

Vitamin B6

1,3

Mg

180

Folate

1010 mcg

104

Vitamin B12

0,5

2600

Choline

65,1

Na

3600

Zn

2,1

Cu

0,3

2.2.2.2 N c
c s dng pha long mt r, nc s dng phi m bo tiu chun nc
dng trong thc phm.
Bng 2.2.2.2.1: Tiu chun n c dung trong th c phm
Ch tiu

Tiu chun
Ch tiu vt l

Mi v

Khng c

trong (ng Dienert)

100 mL

Mu sc (thang mu coban)

5o

Ch tiu ha hc
pH

6.0 - 7.8

CaO

50 - 100 mg/L

MgO

50 mg/L

Fe2O3

0.3 mg/L

10

MnO

0.2 mg/L

BO43-

1.2 - 2.5 mg/L

SO42-

0.5 mg/L

NH4+

0.1 - 0.3 mg/L

NO2-

khng

NO3-

khng

Pb

0.1 mg/L

As

0.05 mg/L

Cu

2 mg/L

Zn

5 mg/L

0.3 - 0.5 mg/L

Ch tiu vi sinh

Tng s vi sinh vt hiu kh

< 100 cfu/mL

Ch s Coli (s Coli/1 lt nc)

< 20

Chun s Coli (s mL nc c 1Coli)

>50

Vi sinh vt gy bnh

Khng c

2.2.2.3 Tryptone.
Thnh phn b sung cung cp acid amin cho s sinh trng v pht trin ca
E.coli.
Nit tng:

11 12%

Nit amin:

3,5%

Protein:

70%

E.coli:

Khng c

Samonella:

Khng c

pH:

6,5 7,2.

2.2.2.4 Cht chnh pH:

Cht chnh pH trong x l r ng v pH ln men l H2SO4
Cht n nh pH trong qu trnh ln men dung CaCO3
o

CaCO3
tinh khit: 98%

11

Fe2O3 : 0.012%
MgO : 0.2%
Al2O3: 0.04%
SiO2 : 0.02%
Na2O: 0.02%
o

H2SO4
Nng : 93% 1%
SO2 t do : 0.01%
Fe: 0.01 %
Tro: 0.05%

2.2.2.5 K2HPO4.
Cung cp P cho mi trng sinh trng ca VSV cho ging sinh trng v
pht trin bnh thng. Thiu phospho ha tan th sinh trng ca khun ty yu do s
ng ho carbon v nit b chm v hot lc khng sinh thp. Tuy nhin hm lng P
ha tan b sung phi cn nhc do nu tha phospho s tng nhanh tc s dng
hydratcarbon lm cho qu trnh to bo t rt ngn, do c ch s tng hp
tinh khit:

98%

P2O5:

40%

K2O:

53%

Pb:

0,001%

As:

0,0003%

2.2.2.6 Ngun kim loi vi lng

c b sung di dng mui sulfat nhm cung cp vi lng cho s sinh
trng v pht trin ca VSV.
o MnSO4.7H2O
tinh khit:

98%

Mn:

31,8 %

Fe:

0,004%

Pb:

15ppm

As:

5ppm

Cd:

10ppm
12

o MgSO4.7H2O.
tinh khit:

99,5%

Fe:

0,0015%

Pb:

0,0005%

As:

0,0004%

Cd:

10ppm.

13

QUY TRNH CNG NGH

3.1 S khi
Mt r
ng ma

Pha long s b

X l axit
Protein, Cacbohydrat,
H2O, Tro

Ly tm ln 1

Cht chnh
pH, CaCO3
Tryptone

H2O

Chun b mi trng
K2HPO4

Ging VSV

Nhn ging

Tit trng

MnSO4.7H2O
MgSO4.7H2O

Cy ging

Ln men

14

O2

Ly tm ln 2

(NH4)2SO4

Sinh khi

Kt ta

Ly tm ln 3
Dung dch
m phosphate

Ha tan kt ta
Trao i ion

C c chn khng

Kt tinh

Ly tm

Sy chn khng

Bao b

ng gi

Bt
Tryptophan

Hnh 3.1.1: Qu trnh cng ngh sn xut ch phm Tr ptoph n

15

3.2 Gii thch quy trnh cng ngh

3.2.1 X l mt r ng
Mc ch cng ngh
Chun b: chun b mi trng cho qu trnh ln men. Ngoi ra, qu trnh acid
ha mt r cn c tc dng loi b cc cht gy c ch, c hi nh hng n qu trnh
ln men nh: cht mu, cht keo, cc VSV khng c li cho qu trnh ln men, c
bit l vi khun lactic acid acetic lm chua r ng, gim hm lng ng trong mt
r.
Cc bin i ngu n liu
-

Ho hc: gim nng cht kh, chuyn ho ng sucrose thnh glucose v

fructose di s xc tc ca acid; pH gim v 2,8 3,0.

Vt l: gim nht, tng trong, gim khi lng ring ca dung dch

Ho l: tng ho tan cc cht, h keo b ph v, tch pha rn ra khi pha lng.

Vi sinh: vi sinh vt b tiu dit cao v pH thp ( tit trng mi trng).

Phng php thc hin: pha long nc theo t l Vmt r : Vnc = 1:1.
Phng php th c hin v thng s cng ngh

u tin pha long mt r vi nc theo t l Vmt r : Vnc = 1:1.

Lng acid thm vo l 5% khi lng

Mi trng c un nng n nhit n 90-95oC v gi nhit trong

khong 60 pht.

Lm ngui n nhit cn thit, dung dch c a qua thit b ly tm tch

cc tp cht khng tan.
Thit b
Thit b: Ni phn ng: lm bng thp khng r dng tin hnh qu trnh pha

long mt r. Ni phn ng l mt thit b dng xilanh ng c th tch t 0,1

100 m

hoc hn, c o hi. Bn trong thit b c c cu o trn dng tuabin h. Ni phn

ng - my trn c th tho ri hay hn c nh vi cc b phn y elip v np. Trn
thit b c cc khp ni np cc cht ti nhit, chy trn sn phm, ni ng qu
p, ni khp u ng nguyn liu, kho st cht liu, cc khp ni np v thi cht

16

ti nhit v sn phm, van an ton, nhit k. Sau khi np vo thit b mt lng nc

nht nh.Tin hnh un nng mi trng n mt nhit cho bng phng tin
iu chnh t ng.
1- ng ni np cht ti nhit
2- ng chy trn sn phm
3- ng qu p
4- u ni ng np nguyn liu
5- Ca quan st
6- Ca thot cht ti nhit
7- Ca vo cht ti nhit
8- Ca ra ca sn phm
9- Ca thot cht ti nhit
Hnh 3.2.1.1: Thit b ni phn ng.
Thit b ly tm lc: Thit b c dng hnh tr, bn trong c b tr cc thanh chn.
Khi ng c truyn ng cho rto bn trong quay, di tc ng ca lc ly tm cc
cu t khng tan s b vng ra, gi li trn cc thanh chn.
-

ng knh trong ca rto: 1000 mm.

S vng quay ln nht: 1500 vng/pht.

Hnh 3.2.1.2: Thit b l tm l m sch mt r.

17

3.2.2 Chun b mi rng ln men

Mc ch cng ngh
Chun b: b sung cht dinh dng cho vi sinh vt, chun b cho qu trnh ln
men.
Thnh phn b sung cc nguyn liu ph vo mi trng mt r (g.L-1 mi
trng):
-

Dch chit nm men: 0.5% w/w dung dch

Tryptone: 1% w/w dung dch

KH2PO4: 0.188 g.L-1

MgSO4.7H2O: 0.156 g.L-1

MnSO4.7H2O: 0.156 g.L-1

CaCO3: 26 g.L-1
Cc bin i ngu n liu

Vt l: Gim nht

Ha hc: Nng cht kh b gim.

Ha l: Tng ha tan, tng kh nng lin kt ca cc cht. Do nng cao

ng nht ca mi trng.

Hnh 3.2.3.1: Thit b phi tr n.

Thit b v thng s cng ngh
Thit b: thit b phi trn c cnh khuy. Cu to thit b bao gm 1 thng
khuy, bn trong c gn 2 cnh khuy mi cho c t lch tm, thnh thng khuy
t cc thanh chn. Thit b ny dng cho cht lng c nht trung bnh.
18

Thng s cng ngh

Tc khuy: 200 rpm
pH = 6,5 (hiu chnh bng H2SO4).
3.2.3 Nhn ging vi sinh vt
Nhn ging l qu trnh tng dung tch, dch cha sinh khi qua nhiu cp. M i
cp nhn ging thng tng dung khi t 10-15 ln. C lm nh vy cho n khi
ton b dung tch tin hnh qu trnh ln men.
Khi nhn ging giai on phng th nghim, ngi ta s dng cc dng c thy
tinh nh ng nghim, erlen vi cc dung tch 750 ml, 1l, 2l kt hp vi t m, t lc
iu nhit. Do th tch mi trng nh nn vic s dng my lc c th m bo
c s ng nht trong canh trng nui v cung cp y oxy cho s sinh
trng, pht trin ca nhm vi sinh vt hiu kh. Khi nhn ging giai on phn
xng, ngi ta s dng nhng thit b vi dung tch khc nhau: 100l, 500l, 1m3,
3m3, 5m3, 10 m3hoc ln hn ty theo dung tch ca thit b ln men ang s dng ti
nh my.
Mc ch cng ngh:
Chun b ging cho qu trnh ln men. Qu trnh nhn ging gip gia
tng s lng t bo( tng sinh khi), tch ly s lng t bo cn thit
cy ging vo mi trng ln men.
Thnh phn mi trng nhn ging (g/L)
-

Glucose: 70g/L

NH2SO4: 28g/L

KH2PO4: 1g/L

MgSO4.7H2O: 0.5g/L

MnSO4.8H2O: 5mg/L

FeSO4.7H2O: 5mg/L

CaCO3: 30g/L

Dch chit nm men: 2g/L

pH: 7,0

19

Cc bin i ngu n liu

-

Vt l: Gim nht

Ha hc: Nng cht kh b gim.

Ha l: Tng ha tan, tng kh nng lin kt ca cc cht. Do nng cao

ng nht ca mi trng.
Cc u t nh hng:

Nhit : Nu tin hnh nhn ging vi sinh vt nhit thp hoc cao hn
khong nhit ti u (topt = 37oC) th vi sinh vt sinh trng chm hn v hng
s tc sinh trng ca ging ( expo ) s gim xung .

Oxi: vic cung cp oxy cho mi trng l rt cn thit gip vi sinh vt tng
hp nng lng, duy tr cc hot ng trao i cht v tng hp sinh khi.

S khu tr n: S o trn c nhng tc dng nh sau :

Lm tng kh nng tip xc gia cht dinh dng v t bo nm si. C tip xc
gia cht dinh dng vi t bo vi sinh vt mi c s trao i cht. Kh nng tip
xc cng nhiu, kh nng trao i cht cng mnh .
Lm tng kh nng ha tan ca oxy. Cc kh s li lu hn do dng chuyn
ng ca mi trng v nh vy , kh nng ha tan ca oxy t bt kh s cao
hn.

Thi gi n: Theo l thuyt, vo thi im u ca giai on n nh, s lng t

bo thu c trong canh trng l cao nht v chng c hot tnh sinh l n nh.
Do , ta kt thc qu trnh nhn ging thu nhn sinh khi vo thi im ny
t c hiu qu k thut v kinh t cao nht. Thng thng thi gian nhn
ging t 30 36 gi.
Thit b v thng s cng ngh

Nhn ging giai on phng th nghim:

Sau khi chun b mi trng, tin hnh tit trng mi trng 121 0C trong 20

pht. Mi trng sau khi tit trng c lc u v lm ngui n nhit phng.

Ging c nui lin trong mi trng c lm ngui, v gi nhit phng.

20

Hnh 3.2.2.1: M lc n nhit Person l - 11, TAITE )

o

Nhn ging giai on phn xng:

S dng thit b nhn ging hnh tr ng c cnh khuy , b phn sc kh v c

lp v o iu nhit.
3.2.4 Tit trng
Mc ch cng ngh
Ch bin: tiu dit ton b vi sinh vt trong mi trng m t lm bin i thnh
phn mi trng
Cc bin i ngu n liu
-

Sinh hc: vi sinh vt b c ch tiu dit.

Vt l: To gradient nhit . C s thay i v th tch, khi lng, t trng.

Ha hc: thay i tc cc phn ng ha hc.

Ha l: c s bc hi nc, ng t protein.

Ha sinh: enzym b v hot do Protein b bin tnh.

Thit b v thng s cng ngh
Thit b: Qu trnh tit trng c th s dng thit b trao i nhit dng bn mng.
Thit b gm nhng tm bn c t st vo nhau. dy ca cc tm bn rt

mng v trn b mt ca chng c cc khe li lm nhm mc ch lm tng h s v

b mt truyn nhit. M i tm bng s c 4 l ti bn gc v h thng cc ng rnh
trn khp b mt to s chy ri v tng din tch truyn nhit.

21

Khi ghp cc tm bn li vi nhau trn b khung ca thit b s to nn h thng

ng dn vo v ra cho mi trng v cht ti nhit chuyn ng xen k nhau. Ti
y mi trng s c gia nhit gin tip bi cc tc nhn nhit. Mu kho st c th
chuyn ng cng chiu hay ngc chiu. Ty thuc vo iu kin c th, cc nh sn
xut s b tr h thng nhng ng dn thch hp.

Hnh 3.2.4.1: Thit b tit trng dng bn mng.

Thng s cng ngh
iu kin tit trng 120oC trong 10 pht
3.2.5 Ln men.
Mc ch cng ngh
Khai thc: chuyn ha c cht thnh Tryptophan.
Cc bin i ngu n liu
-

Ha sinh: din ra cc phn ng vi s xc tc ca cc enzim.

Sinh hc: s tng sinh khi ca t bo vi sinh vt do tiu th cht dinh dng ca
mi trng.

Vt l: nhit tng do s trao i cht, sinh nng lng ca vi sinh vt.

Ha l: Kh CO2 sinh ra ha tan vo pha lng pH thay i do c acid sinh ra,

protein b bin tnh to kt ta lm cho dch ln men c hn.

22

Cc u t nh hng
o

Mi trng ln men:

Hm lng cht kh: Cc cht ha tan trong mi trng s to nn mt p lc

thm thu. Hm lng cc cht ha tan trong mi trng cng ln th gi tr p
lc thm thu cng cao. Khi hm lng cc cht ha tan trong mi trng qu
cao, mng t bo cht khng th duy tr s cn bng p lc thm thu c na,
do phi xc nh hm lng cht kh ti u cho mi trng trc khi ln men.

Thnh phn ha hc: t l cc cht trong mi trng phi cn i pH ti u cho

s pht trin ca vi khun.

pH mi trng: pH c nh hng nhiu n tch t ch phm tryptophan sau ln

men nn trong thc t sn xut, ngi ta thng s dng acid hoc ammoniac
iu chnh cho pH lun trong khong ti u.

Vi khun: chng s dng, lng ging cy, trng thi sinh l.

iu kin ln men:

Lng ging cy: Nu lng ging cy qu t, thi gian ln men ko di. Cc vi

sinh vt tp nhim trong mi trng ln men d pht trin v c th gy h hng
sn phm. Nu lng ging cy qu nhiu, thi gian ln men c th rt ngn,
nhng hm lng cc sn phm ph to thnh s thay i v gy nh hng n
cht lng thnh phm, hn na, vic tng lng ging cy s lm tng chi ph
cho qu trnh nhn ging trong sn xut.

T l ging cy: 2 5%.

Nhit : Khi nhit thp, thng khng gy cht vi sinh vt ngay m n tc

ng ln kh nng chuyn ha cc hp cht, lm c ch hot ng cc h
enzyme, lm thay i kh nng trao i cht ca chng, v th lm vi sinh vt mt
kh nng pht trin v sinh sn. Nhiu trng hp vi sinh vt s b cht. V th
qu trnh ln men s b ko di.

Khi nhit qu cao, thng gy cht vi sinh vt. Tuy nhin hot tnh trao i
cht ca vi sinh vt b gim v cc cu t mn cm vi nhit b tn tht.

Trong qu trnh ln men, mt phn nng lng do vi sinh vt chuyn ha s c

thi vo mi trng di dng nhit nng, dn n lm tng nhit dch ln

23

men, do quy m cng nghip, thit b ln men c b phn hiu chnh nhit
n nh hot tnh cho ging trong sut qu trnh ln men
o

Thi gian ln men:

Trong sn xut, xc nh thi im kt thc qu trnh ln men thng qua vic ly
mu nh gi kim tra mt s ch tiu ha l, vi sinh v cm quan (thng 2 4
ngy).
Cung cp oxy:
Trong mi trng lng, t bo vi sinh vt phn tn trong dch th, s tip xc ca
t bo vi oxy phi thng qua mi trng nui cy. Do , khuy v sc kh l
bin php tt oxy ca khng kh c th ha tan vo canh trng. Lng oxy
ha tan vo mi trng phi b p cho s pht trin ca vi sinh vt, ng
thi phi d ra t 1 3mg/L.
Mc ha tan ca oxy ph thuc vo cc yu t: nhit , nng cht v
phng php sc kh, trong nhit cao th oxy ha tan km, cn nng
cht t l nghch vi kh nng ha tan ca oxy.
Mt s cht khi ha tan vo mi trng s nh hng xu n kh nng ha tan
ca oxy, v d nh trong dung dch c nng cht kh l 13%, ha tan oxy
s gim 85%, v nu ch b 2% bt giy hoc xc t bo cht vo dung dch th
ha tan ca oxy s gim n 85 90%. Ngc li, c mt s cht khi ha tan
vo dung dch li lm tng ha tan ca oxy, l cc ester, ru, acid hu c
v cc hp cht ho nc khc.
Khi nui cy nm men thu nhn ch phm tryptophan, lng khng kh cn
sc vo 1m3 mi trng vo khong 30 40m3/m3gi. Vi s pht trin ca k
thut sc kh, tiu hao khng kh cho 1m3 mi trnggi s gim dn.Chiu
hng pht trin hin nay khng phi l gim lng khng kh cho 1m3 mi
trng m ngi ta tng nng cht dinh dng tng ng vi lng oxy
ha tan t sinh khi cao v do tng nng sut ca thit b.
Thit b v thng s cng ngh:
Thit b: thng ln men.
Thit b c dng hnh tr ng, c ch to t vt liu thp khng r. Bn trong

thit b c h thng cnh khuy v cc u d nhit , pH, theo di trc tip cc

24

thng s cng ngh trong qu trnh ln men. H thng sc kh c b tr trn y

thit b cung cp lng oxy cn thit cho vi sinh vt hiu kh.
Xung quanh thit b l lp v o cho tc nhn iu nhit n nh nhit canh
trng trong qu trnh ln men. Phn trn np thit b c cc ca vi nhiu chc nng
khc nhau: ca thng cnh khuy gn vi motor, ca np ging, ca vo v ra cho
khng kh, ca np cht ph bt, ca np cht iu chnh pH, Ca np mi trng
v tho canh trng ra khi thit b thng c b tr phn y.
Thit b c thit k dng v trng: kn v c th chu c p lc cao, c th
dng hi v trng trc tip v v sinh thit b.
Thng s cng ngh
-

Nhit : 32 39oC .

Khng kh cn sc vo mi trng: 30 40m3/m3gi. Vi nng O2 25%.

pH = 6,5, thi gian ln men 30

36 gi.

Hnh 3.2.5.1: Thit b ln men b su.

1 - ng c, 2 - H p gim tc, 3 - Kh p ni, 4 - bi, 5 - Vng bt kn, 6 - Trc, 7 Thnh thit b, 8 - My khuy tr n tuabin, 9 - B tr o i nhit dng ng xon, 10 Kh p ni, 11 - ng np khng kh, 12 - My tr n kiu cnh qut, 13 - B si b t, 14 -

25

My khuy dng vit, 15 - , 16 - Kh p tho, 17 o, 18 - Kh p np liu, 19 Kh p tho khng kh.

X l thit b vi H2SO4 v HNO3 long, thng kh trong 24 gi. Tit trng thit
b bng hi nc (0,2 bar), lm ngui bng tc nhn lnh thng qua v o.
3.2.6 Ly tm
Dch sau ln men c cha Tryptophan, sinh khi E. Coli, c cht st, cc cht
khong v mt s sn phm ph trong qu trnh ln men.
Mc ch cng ngh
-

Khai thc: Phn ring huyn ph, thu nhn dch ln men. Qu trnh c thc
hin nhm tch b xc vi sinh vt v mt phn tp cht khi dung dch sau ln
men.

Chun b: Loi b sinh khi vi sinh vt sau qu trnh ln men, chun b cho qu
trnh trao i ion thu nhn Tryptophan.
Cc bin i ngu n liu

Ha hc: hu nh khng lm thay i thnh phn ha hc, nhng nng cc

cht b thay i. Tryptophan cng b tn tht trong qu trnh ny.

Vt l: thay i nht, khi lng gim.

Ha l: c s tch pha, pha lng v pha rn.

Sinh hc: hu ht vi sinh vt b tch.

Thit b v thng s cng ngh

Thit b: s dng thit b ly tm c chn xoay dng cn. Cu to thit b bao gm

mt chn xoay dng cn quay quanh 1 trc thng ng. Tt c c t trong v
c nh. Nguyn liu i vo y ca chn xoay. Di tc dng ca lc ly tm, cc
huyn ph s di chuyn v thnh ca chn xoay v bm trn . Phn dung dch
sch di chuyn ln pha trn v chy trn ra ngoi v. Khi b dy thit b c
ngng hot ng, lp b c tch ra bng dao v tho ra ngoi bng ca x y
ca chn xoay. c th tho b c lin tc, chn xoay c khot l vi
ng knh t 0,75 - 2mm, ty thuc vo kch thc ca huyn ph. Khi dng
b c tho ra lin tc qua cc l ny.

26

Hnh 3.2.6.1: Thit b l tm s dng chn xo

dng cn.

Thng s cng ngh

Tc quay 10.000

15.000 vng/pht, m phn rn tch ra: 30

50%.

3.2.7 Kt ta
Mc ch cng ngh
-

Khai thc: tch thnh phn tryptophan ra khi dung dch th sau khi ln men, thu
tryptophan di dng kt ta.

Chun b: chun b cho qu trnh trao i ion

Cc bin i ngu n liu

Ha l: s kt ta v tch pha. Tryptophan chuyn t dng lng sang dng rn.

Ha hc: hm lng tryptophan ha tan trong dung dch gim.

Phng php th c hin
Dch tryptophan th thu c sau qu trnh ly tm, tch sinh khi v tp cht

c thm vo mi trng dung dch (NH4)2SO4 70% theo t l gia th tch mi

trng v th tch cht lm kt ta l 1:4. Trong qu trnh thm vo, h n hp c
khuy vi tc 1000rpm. Qu trnh trn nhm mc ch a (NH4)2SO4 bo ha vo
tng nhanh hiu sut qu trnh kt ta tch tryptophan ra khi dung dch th ([14],
p279)
Thit b v thng s cng ngh
Thit b: thit b thng hnh tr, lm bng thp khng r. Thit b c trc khuy
theo ng tm c truyn ng bng ng c. H thng c iu khin tc cnh
khuy to chuyn ng quay thch hp. Nguyn liu c nhp vo pha trn v
tho ra di y.
27

Thng s cng ngh

Nhit 4oC, mui (NH4)2SO4 70%, thi gian

30 pht

Hnh 3.2.7.1 Thit b kt tinh bng phng php l m lnh dng tn

si.
3.2.8 Ly tm ln 2
Mc ch cng ngh
Khai thc: thu nhn kt ta.
Cc bin i ngu n li
-

Ha hc: thay i hm lng cht kh v cht ha tan trong 2 pha rn lng.

Vt l: s thay i v th tch, khi lng ring, nhit ca dung dch.

Ha l: c s tch pha, pha lng v pha rn.

Thit b v thng s cng ngh
Thit b: s dng thit b ly tm c chn xoay dng cn. S chnh lch khi
lng ring ca 2 pha rn lng: chnh lch nhiu th qu trnh phn ring s d
dng hn. Tc quay ca roto: Tc quay cng nhanh th qu trnh ly
tm cng thun li.

28

Hnh 3.2.8.1 Thit b kt tinh bng phng php l m lnh dng tn si.
3.2.9 Ha tan kt ta
Mc ch cng ngh
Chun b: chun b cho qu trnh trao i ion.
Cc bin i ngu n liu
-

Ha hc: Nng dung dch tryptophan gim do mt cc cht ha tan.

Ha l: tryptophan chuyn t dng rn sang dng lng.

Thit b v thng s cng ngh

Thit b: dng thit b dng hnh tr c cnh khuy.

Dung dch m: dung dch m phosphate 0,05M, pH = 7.

3.2.10 Trao i ion

Nguyn l
Sc k trao i ion da vo s tng tc gia in tch ca cc acid amine trong
mu v cc nhm tch in c c nh trn b mt trao i. Acid amine c gn
vo cc b mt nh cc lin kt tnh in. S bn vng ca lin kt c xc nh bi
tr s in tch ca acid amine, m tr s ny li ph thuc vo pH ca dung dch, nng
mui (ca dung dch m).
Ti bt k mt im pH no tr im ng in, cc acid amine u c mang mt
in tch tng ng vi im pH . Da vo in tch thc ca chng ti mt im
pH nht nh, ta c th phn tch acid amine.
Trong phng php ny, pha tnh l nhng ht mang sn mt in tch nht nh,
nhng ht ny s tng tc vi cc phn t (protein) mang in tch tri du vi
chng. C th, nu ht mang in m (nh ct carboxymethyl-cellulose (CM29

cellulose)), tin trnh c gi l sc k trao i ion dng, th s tng tc vi nhng

phn t mang in tch dng. Ngc li, nu ht mang in tch dng (nh ct
diethylaminoethyl-cellulose (DEAE-cellulose)), gi l sc k trao i ion m, th
tng tc vi phn t mang in tch m.
V th, nhng protein cng du vi ct s chy ra khi ct trong khi nhng
protein tri du b gi li ct. phng thch nhng acid amine ny, c th dng mt
dung dch m phn hp ph c pH khc hoc thm mt loi ion khc c lc ion
ln hn. Khi , phn t acid amine s b y ra khi cht trao i ion. Khi tin hnh
phn hp ph, thng ngi ta thm vo cc ion Na+ v Cl-, trong NaCl c nng
tng dn theo bc thang hay theo gradient. Cc phn t acid amine no c in tch
tng s nh th s b y ra trc do lc lin kt vi cht trao i ion yu. Cn nhng
acid amine no c lin kt vi ionit ln hn th s b y ra bng mt lc ion ca mui
ln hn. Nh vy, bng cch ny, chng ta c th tch c tng phn cc loi acid
amine. Vic tch tng phn c la chn tt nht l khi tng dn nng cc ion thay
th. Nh nng ion ca mui tng dn (gradient) ngi ta c th rt ra t ct cc loi
acid amine khc nhau. C th thu nhn dch chit acid amine sau khi qua ct bng my
thu phn on t ng. Ngoi vic dng mui NaCl cho cc ion Na+ v Cl-, ngi ta
c th dng cc loi mui khc nh KCl, Na3PO4, ta tng nng ion ca pha ng
(dung dch ra gii) (s dng gradient, nhng ion ny s thay th phn t acid amine
tng tc vi cc ht mang in tch.
V d, trong sc k trao i ion m, ta thm mui natri clorua trong dung dch
tch gii bi v ion Cl- s tranh bm vo ct vi cc acid amine c in tch m, do ,
nhng acid amine mang in tch m c phng thch ra ngoi ct ln lt theo
ln v in tch. in tch ca acid amine ph thuc vo pH ca mi trng:
-

Nu pH ln hn pI th acid amine tch in m

Nu pH nh hn pI th acid amine tch in dng

Nu pH bng pI th acid amine khng tch in.

Mc ch cng ngh
Khai thc: bng phng php trao i ion, ta thu c Tryptophan tinh khit hn,

nn mc ch ca qu trnh trao i ion l khai thc.

30

Cc bin i ngu n liu

-

Vt l: khi ta cho cc ht nha trao i ion vo trong mt dung dch, chng s

trng n v gia tng th tch. Km theo l s solvate ha xy ra. Cc cu t
tch in trong mu lng s th ch cho cc ion trn pha rn, ngc li cc ion
trn pha rn s dch chuyn vo mu lng. ng thi cc phn t dung mi v
cht tan s dch chuyn vo bn trong cu trc xp ca cc ht nha theo nguyn
tc thm thu.

Ha hc: lm thay i thnh phn ha hc trc v sau x l.

Ha l: mt s cu t khng tch in s b hp ph trong mao dn ca cc ht

nha.

Ha sinh v sinh hc: trao i ion khng lm bin i cc qu trnh ha sinh v

sinh hc.
Cc yu t nh hng

Nhit : khi tng nhit th s khuch tn ca cc cu t trong dung dch tng

ln. Tuy nhin, khi nhit cao c th gy ra nhng phn ng ph khng mong
mu ng thi lm tng chi ph nng lng cho qu trnh x l.

Thi gian: y l mt thng s quan trng. Nu thi gian x l ca mu vi ionit

qu ngn th phn ng trao i ion xy ra khng hon ton.

y ta cn ch

n tnh cht cn bng ca ionit.

Thit b v thng s cng ngh
Thit b: Thit b c dng hnh tr ng, y cu v c ch to bng thp
khng r. Pha trn y l tm li . Ngi ta s cho cc ht nha trao i ion ln
tm li ny. Khi cho ionit tip xc vi dung dch, cc ht nha s trng n. Chiu
cao ca lp ht nha trong qu trnh hot ng thng dao ng t 1 - 2m.

pha

di tm li ngi ta thng cho cc cht mang dng hnh cu. Vai tr ca cc cht
mang ny l hn ch s xo trn ca cc cu t trong dng ra v bo ton
gradient nng trong qu trnh hot ng ca thit b. Di cng ti ca thot l mt
tm li lc ngn cn s cun tri cc ht nha trao i ion ra khi thit b.

pha

bn trn cc ht nha l ng phn phi mu nguyn liu. Nguyn liu s c bm

vo thit b qua ng phn phi. Ti y, nguyn liu s c phn b u trn hnh
trn ca mt ct thn tr ri chy xung bn di. Sn phm c ly ra ti ca y.
31

Ngi ta thng s dng qu trnh trao i ion gn cu t cn thu nhn ln

ct ionit. Nh vy, khi cho mu nguyn liu qua thit b trao i ion th sn phm cn
thu nhn s b gi li trn ct. Sau , ngi ta dng mt dung dch m c pH hoc
lc ion thch hp tch sn phm ra khi ct. Dung dch ra gii cng s cho vo ct
qua b phn phn phi ri di chuyn theo chiu t trn xung di. Ty theo bn cht
ca cc ion gn trn ht nha m ngi ta s chn dung dch ti sinh ph hp.

Hnh 3.2.7.1: Ngu n l thit b tr o i ion.

1. Tm l i cc ionit, 2 t nh

tr o i ion, 3 Cht m ng l m

gi , 4 L i l c, 5 - ng phn phi ngu n liu, 6 Cm bin

nh mc dch lng trong thit b, 7 C np ngu n liu cn x l,
8 C tho sn phm, 9 C tho n c v sinh. S dng ch
ch ngc .
Thng s cng ngh:
-

Tryptophan thu nhn t canh trng sau ln men c pI = 5,89. Chn pH dung
dch m l 5.4 th tryptophan s tch in dng . Khi ta thc hin trao i
cation

Nha trao i ion HZ-001 c thng s sau:

Nha trao i anion: Styrene - DVB

Dung dch m s dng l m phosphate 0,21M Na+ c pH = 5,4.

Dung dch ra gii l h n hp gm m phosphate 0,05M c pH = 7 v dung

dch NaCl 1,5 - 1M (t l th tch 1:1).

32

Nhit : 25-300C.

Sau qu trnh trao i ion: hiu sut thu hi tryptophan t 70-75%.

3.2.11 C c chn khng

Mc ch cng ngh
-

Khai thc: nng cao nng cht kh cc sn phm bng phng php bay hi
nc.

Chun b: tng hm lng cht kh, chun b cho qu trnh sy phun.

Cc bin i ngu n liu

Vt l:

Nng cht ha tan tng, m gim.

Nhit , nht, khi lng ring tng.

Th tch, khi lng gim.

H s truyn nhit gim.

Ha l:

Keo t protit.

S bc hi nc.

Sinh hc: hn ch hot ng ca vi sinh vt do nng cht kh cao, mt s loi

vi sinh vt c th b tiu dit do nhit kh cao.

Cc u t nh hng
-

Yu t nh hng n tc truyn nhit: s chnh lch nhit hi bo ha

(tc nhn gia nhit) v nhit si ca nguyn liu; hin tng cn bm b mt
trao i nhit, hin tng mng bin, nht ca dung dch.

Yu t nh hng n hiu qu kinh t ca qu trnh c c:

Hin tng to bt: trong qu trnh c c s xut hin nhng bong bng hi, nu
dung dch cha cc yu t lm bn h bt th s to ra mt lp bt trn b mt
dung dch trong thit b lm gim tc .
Tn tht cht kh do hi th: hi th thot ra khi thit b c th li cun theo
mt s cu t dng sng m lm tn tht cht kh ca dung dch.

33

Thit b v thng s cng ngh

Thit b: Thit b gm 3 phn c bn: bung t ngoi, bung bc v thit b
ngng t.
o

Bung t: Bn trong c nhiu ng c lp t cnh nhau to thnh mt chm

ng. Hai u ca chm ng c c nh bi 2 v ng v ton b chng c bao bc

bi mt lp v o. Hi nc c a vo bn trong lp v o gia nhit, cn cht
lng c a vo khong khng gian gia cc ng. Cht lng c vo phn trn
nh bung t v qua h thng phn phi lng chng s c phn phi mt cch u
n mt trong ca ng, chy xung to thnh mt mng mng. Qu trnh bc hi s
din ra ti y nh nhit c cung cp bi hi nc. Hi nc ngng t v chy
xung mt ngoi ca ng. Cht lng c c c phn ln s c tho ra y
bung t, mt phn b hi nc li cun i qua bung bc.
o

Bung bc: Bung bc thng c ng knh ln, l ni phn ly h n hp hi v

dung dch c c. Hi th c ht sang thit b ngng t, ti y c th b tr h

thng thu hi cc cht thm d bay hi. Sau khi tch hi, phn cht lng ny cng
c tho ra cng vi phn lng c c c ly ra bung t ri tip tc c
x l tip qu trnh sau. Cn hi s ri khi b phn tch hi ti nh.
o

Thit b ngng t: a nc vo tip xc trc tip vi dng hi, c thit b ht

chn khng i vi nhng hi khng ngng.

Hnh 3.2.11.1: 1 Thit b c c chn khng; 2 Thit b c c chn khng

nhiu ni
34

Thng s cng ngh

-

Nng sau khi c c t t 40 50%.

Nhit c c: 55 - 60oC.

Thi gian c c: 1 2 gi.

p sut c c chn khng: 0,09 - 0,098 Mpa.

3.2.12 Kt tinh
Mc ch cng ngh
Khai thc: thu nhn tryptophan dng rn.
Cc bin i ngu n liu
-

Vt l: nng cht kh trong pha lng thay i, nht tng.

Ha l: c s hnh thnh pha r n do hin tng kt tinh.

Ha hc: Nng cht kh tng.

Thit b v thng s cng ngh

Thit b: thit b kt tinh bng phng php lm lnh dng tng si. Dung dch

c lm lnh n nhit dung dch t nng bo ha. Khi t bo ha th b

sung thm mm tinh th qu trnh kt tinh din ra d dng. t c tinh
khit ca sn phm cng cao th sau khi kt tinh ngi ta s ha tan tinh th trong cn
(3 - 4 ln th tch) ri cho kt tinh v lm i lm l i nhiu ln, cc tinh th s tch
khi tp cht. Qu trnh kt tinh c th tn hao ti 25 30% tryptophan.
o

Thng s cng ngh: nhit 10 12oC.

Hnh 3.2.12 Thit b kt tinh bng phng php l m lnh dng tn si.

35

3.2.13 Ly tm
Mc ch cng ngh
Khai thc: Qu trnh ly tm nhm tch tinh th tryptophan ra kh\i dung dch
bng lc ly tm.
Cc bin i ngu n liu
Ha l: xy ra s tch pha, cc tnh cht ha l trong m i pha b thay i: t
trng, nht,
Thit b v thng s cng ngh
Thit b: Dng phng php ly tm tch tinh th tryptophan kt tinh trong
dung dch.
Thng s cng ngh: tc quay 10.000

15.000 vng/pht.

3.2.14 Sy chn khng

Mc ch cng ngh
Bo qun: gim hm m ca nc, ko di thi gian bo qun sn phm.
Cc bin i ngu n liu
Vt l: nhit tng, khi lng sn phm gim.
Ha l: hm m gim, nc chuyn t pha lng sang pha hi,
Thit b v thng s cng ngh
Thit b: Dng phng php ly tm tch tinh th tryptophan kt tinh trong
dung dch.

Hnh 3.2.14.1 Thit b s chn khng.

o

Thng s cng ngh: nhit 50oC.

36

3.2.15 n

i.

Mc ch cng ngh
Bo qun v hon thin.
Qu trnh ng gi gip n nh cht lng sn phm trong qu trnh bo qun,
ng thi vic phn phi sn phm s c d dng hn.
Cc bin i ngu n liu
Qu trnh rt sn phm vo bao b khng gy ra nhng bin i ng k v cht
lng sn phm.
Thit b v thng s cng ngh
S dng h thng thit b ng gi theo nguyn tc lin tc v t ng ha. Ty
theo yu cu ca nh sn xut m kch thc bao b s thay i.

SN PHM

Hnh 4.1: Sn phm Tr ptoph n dng vin v tinh th.

Sn phm: Ch phm L-Tryptophan (Standard: AJI92 & USP30).
- Hnh dng: dng tinh th mu trng
Bn 4 1 Th nh phn h h c c 1 ch phm Tr ptoph n.
Hm lng
-30,5 - -32,8

Thnh phn
Gc quay phn cc
37

Clo (Cl):
Mui amoni (NH4+)
Sulfat (SO42-)
St (Fe)
Kim loi nng:
Ch (Pb)
Asen (As)
Cd
Thy ngn (Hg)
Arsenic (AS2O3)
Cc amino acid khc
Tn tht trong qu trnh sy:
Hm lng tro:

0,02% (max)
0,02% (max)
0,02% (max)
20ppm (max)
10ppm (max)
2ppm (max)
1ppm (max)
0,1 ppm (max)
1 ppm (max)
chim t l nh sao cho phng php
hynh quang khng nhn bit c
0,20% (max)
0,10% (max)

THNH TU CNG NGH

Sn xut Tr ptoph n t ADN ti t hp

Nghin cu ni v phng php sn xut L-Tryptophan bng cng ngh ln men

v cp ring v phng php ln men L-Tryptophan s dng Coryneform
glutamic acid v vi khun ti t hp gen.

Khi L-Tryptophan c sn xut bng ln men, vi sinh vt t bin nhn to

c s dng sn xut Tryptophan v cc ging nguyn thy kh sn xut
Tryptophan bn ngoi t bo.

C th s dng nhng t bo t bin t cc ging nh Brevi bacterium,

Microbacterium hay Coryne bacterium, chng chu c 5-methyltryptophan.
Corynebacterium th cn Tyrosine v Phenyl alanine pht trin v chng chu
c cht khng Phenyl alanine. Bacillus t bin th chu c 5fluorotryptophan. Brevi bacterium t bin th chu c 5-methyltryptophan v
m-fluorotryptophan.

Mt khc mt vi bo co gn y cn cp n k thut ti t hp gen khc t

k thut t bin nhn to c cp trn. V d nh trong bo co ca (Appl
Environ, Microbiol, 1979), t bin t trng ca Escherichia coli cha plasmid
c gen trp-E472 sn xut khong 1,3 g/l L-Tryptophan.
38

Bn cnh , trong Proceedings of 1980 Annual Meeting of the Society of

Fermentation Technology, Japan, E. coli t bin cha plasmis c nhm hot
ng (operons) Tryptophan sn xut c 360 mg/l L-Tryptophan.

Mc d 1 vi nh sn xut bit n t bin vi khun Coryneform trong

glutamic acid cp trn, nhng khng c du hiu no cho thy cc nh sn
xut s dng Coryneform bacteria nh trn dng n c tim nng ln trong
vic sn xut L-Tryptophan.

i tng ca nghin cu l cung cp phng php mi sn xut LTryptophan bng ln men s dng Coryneform bacteria c ti t hp gen.

Phng php sn xut L-Tryptophan bng canh trng vi sinh vt v sau

thu nhn L-Tryptophan tch ly c t canh trng lng, vi sinh vt thu nhn
bi thm mt on gen c cha plasmis c kh nng t ti to t bo
Coryneform-bacteria ri sau kt hp plasmis vo trong DNA ca t bo nhn
ph thuc vo Coryneform-bacteria.

on gen s uc chuyn t DNA t bo cho vo DNA t bo nhn ri plasmis

c kt hp khng cht ch thu nhn c t Coryneform-bacteria v i hi t
nht mt lng L-Tryptophan cho s sinh trng m khi s dng vi sinh vt t
bin khng cn b sung L-Tryptophan.

Ngoi ra ngi ta cn s dng E.coli t bin gen bng k thut ti t hp khi

on gen ca E.coli s c xy dng li. Kt qu cho thy L-Tryptophan tng t
46,8% so vi ging vi sinh vt ban u, gim hm lng di-acetate 2,5g/L.

39

TI LIU THAM KHO

[1]. L Vn Vit Mn v Cng s, Cng ngh ch bin th c phm, NXB i
Hc Quc Gia Tp.HCM, 2010.
[2]. L Vn Vit Mn, Cng ngh sn xut cc loi sn phm t sa v thc
ung - tp 1, NXB i Hc Quc Gia Tp.HCM, 2011.
[3]. Bi i, Cng ngh ln men ng dng trong cng ngh th c phm, NXB
i Hc Quc Gia Tp.HCM, 2005.
[4]. Lng c Phm, Gio trnh Cng ngh ln men, NXB Gio Dc, 2010.
[5]. ng Th Anh o, K thut bao b th c phm, NXB i Hc Quc Gia
Tp.HCM, 2012.
[6]. V Vn Bang - V B Minh, Qu trnh v thit b cng ngh ha h c &
th c phm - tp 3: Truyn khi, NXB i hc quc gia Tp.HCM, 2007.
[7]. L Ngc T (ch bin), Ha Sinh Cng Nghip, NXB Khoa Hc v K
Thut, 2005.
[8]. Nguyn c Lng, Cng ngh vi sinh - Tp 2: Vi sinh vt h c cng
nghip, NXB i Hc Quc gia Tp. HCM, 2006.
[9]. Arun S. Mujumda, Handbook of Industrial Drying, p 215 - 254, Lodz,
Poland, 2006.
[10]. Kalidas Shetty, Gopinadhan Paliyath, Anthony Pometto, Robert E. Levin Food Biotechnology - Taylor & Francis Group LLC (2006).
[11]. Masato Ikeda - Towards bacterial strains overproducing L-tryptophan and
other aromatics by metabolic engineering - Appl Microbiol Biotechnol (2006).
[12]. Faghfuri, Jamshid Fooladi, Shayesteh sepehr, Seyedeh Zahra MoosaviNejad - Tryptophan Production by Whole Cells of Escherichia coli Based on
Iranian Sugar Beet Molasses - Jundishapur J Microbiol (2013).
[13]. Nguyn Th Hin (ch bin) v cng s, Cng ngh sn xut enz m, protein
v ng dng, NXB Gio Dc Vit Nam, 2012.
[14]. Nguyn c Lng (ch bin) v cng s, Cng ngh enz me, NXB i
Hc Quc gia Tp. H Ch Minh, 2004.

40