1.

Gii thiu
Methionin l mt axit amin khng thay th c pht hin ln u tin bi Mueller.
Hm lng methionin cao c tm thy ht. tht v mt s thc phm thc vt khc.
Mc d vy, ngi v ng vt khng th t tng hp c axit amin ny, do vic b
sung ngun axit amin ny l v cng quan trng. Ngy nay methionin c sn xut
bng phng php tng hp ha hc hoc thy phn protein tuy nhin vic sn xut
theo hai cch ny li rt tn km. Tng hp ha hc to ra mt hn hp gm c D v Lmethionin trong khi thy phn protein to ra mt hn hp phc tp m i hi phi c
qu trnh tch ring methionin. L-methionin c hot tnh sinh hc c th c tng hp
bng phng php enzyme hoc bng cch s dng vi sinh vt ln men chm. V qu
trnh ln men c th tit kim chi ph nn rt c quan tm trong vic nghin cu pht
trin mt quy trnh sn xut thng mai methionin nh vi sinh vt. Bi tiu lun di
y s tm hiu v nh hng ca cc iu kin ln men n vic sinh tng hp
methionin t vi khun Bacillus thuringiensis EC1
2. Chng vi sinh vt
Chng Bacillus thuringiensis EC1 phn lp t du u phng ln men c s dng
trong nghin cu ny. Chng c bo qun trong ng thch nghing 4C.
3. Ln men chm
Chng c hot ha bng cch b sung 1ml ging vo mi trng cha 10g/l pepton;
10g/l cao nm men; 5g/l NaCl iu chnh n pH 7.2 nui trong 24h 30c, lc
160rpm.
Ln men trong bnh 100ml cha 20ml mi trng gm 0,05g/l KH 2HPO4; 0,05g/l
K2HPO4; 0,1g/l MgSO4.7H2O; 0,001g/l MnSO4.4H2O; 0,001g/l FeSO4.7H2O; 20g/l
CaCO3; 20g/l glucose; 10g/l (NH4)2SO4 iu chnh ph 7.2. B sung 1ml chng ging
c hot ha vo trong mi trng vi mt 3.15x106 t bo/ml. Tin hnh ln men
nhit 30C lc 160rpm trong 72h.
4. Phng php phn tch methionin
Methione c xc nh theo phng pho c m t bi Greenstein v Wintz. Dch
ln men c ly tm 1500g trong 15 pht v thu 5ml dch ni. B sung thm 1ml NaOH
1N v 0,1 ml dung dch sodium nitroprusside 10%. Hn hp c trn u v yn
trong 10 pht. Sau b sung thm 2ml glycine 3% lc lin tc trong khong 10 pht.
thm khong 10 pht sau b sung t t 2ml axit orthophosphoric lc u sau
khong 5 pht. Cng mu c o bng quang ph k bc song 540nm. Hm
lng methionin c xc nh da vo th chun.
5. Ti u cc iu kin ln men
5.1.
nh hng ca t l th tch mi trng v th tch bnh ln men v t l cp
ging.
nh hng ca t l th tch mi trng v th tch bnh ln men v t l cp ging trong
vic sinh tng hp methionin bi Bacillus thuringiensis EC1 c kho st bng cch
b sung mi trng cc th tch khc nhau (20,25,30ml) vo bnh ln men 100ml vi
th tch cp ging khc nhau (1 v 2ml)

Bng 1: nh hng ca t l th tch mi trng/th tch bnh ln men v t l cp ging

Kho st trn cho thy vic thng kh l cn thit trong qu trnh chuyn ha to sn
phm ca vi sinh vt. Vi th tch cht lng tng ln trn 20ml s khin nng
methionin gim. Do th tch mi trng ln men chim 20% th tch thit b ln men
l ti u trong ln men chm to methionin. T l cp ging trong qu trnh ln men
cng nh hng n qu trnh chuyn ha sn phm. Nu t l cp ging qu cao hm
lng sinh khi s qu ln c ch chuyn ha hnh thnh sn phm, lm gim lng
oxy ha tan cng nh tng s cch tranh i vi cc cht dinh dng. Nu t l cp
ging thp c th khng sinh khi qua lm gim kh nng chuyn ha to sn
phm. Do cn la chn mt t l cp ging ph hp kh nng cung cp oxy v dinh
dng qua lm tng kh nng chuyn ha to sn phm. Vi kt qu kho st c
trnh by bng 1 c th thy vi t l cp ging 5% hm lng methionin thu c l
cao nht.
5.2.
nh hng ca ngun cacbon
Ngun cacbon khc nhau (glucose, maltose, sucose, lactose) c nh hng khc nhau
n kh nng sinh tng hp methionine ca chng. Theo mt s nghin cu khi s dng
maltose cung cp ngun cacbon th t c hiu qu sn xut methionine tt nht.
Nghin cu ny kho st nh hng ca nng maltose n kh nng sinh tng hp
methionin ca Bacillus thuringiensis EC1. Tin hnh ln men trong bnh 100ml cha
20ml mi trng vi t l cp ging 5%. Thnh phn ln men gm maltose
(20,40,60,80,100g/l); 10g/l (NH4)2SO4 iu chnh ph 7.2. Tin hnh ln men trong 24h
xc nh hm lng methionin thu c.

Bng 2: nh hng ca nng maltose v ammonium sulphate n hm lng

methionin
Nng maltose tng lm tng hm lng sinh khi cng nh kh nng sinh tng hp
methionine tuy nhin nu nng ng qu cao ngc li s c ch kh nng snh tng
hp sn phm. Kt qu khi b sung maltose vi nng 8% s thu c lng
methionine cao nht t 2,49mg/ml (hnh 2).
5.3.
nh hng ca ngun nito
Cc ngun nito khc nhau (ammonium sulphate, ammonium chloride v ure) khc nhau
n kh nng sinh tng hp methionine. Theo mt s nghin cu khi s dng
ammonium sulphate l ngun cung cp nito s t c hiu sut sinh tng hp
methionine cao nht. iu ny c th gii thch do methionine l axit amin cha lu
hunh v th khi cung cp ngun nguyn liu cha lu hunh s lm tng hm lng
methionin. Nghin cu ny kho st nh hng ca nng ammonium sulfate n kh
nng sinh tng hp methionin ca Bacillus thuringiensis EC1. Tin hnh ln men trong
bnh 100ml cha 20ml mi trng vi t l cp ging 5%. Thnh phn ln men gm
20g/l maltose; (NH4)2SO4 (10,20,40,60,80g/l); iu chnh ph 7.2. Tin hnh ln men
trong 24h xc nh hm lng methionin thu c. Nng ammonium sulfate tng
lm gim hiu qu sinh tng hp methionine v ngc li. Khi nng m cao lm
tng p sut thm thu qua nh hng xu n kh nng tng trng v tch ly
methionine. Kt qu hm lng methionin t cao nht khi cung cp ammonium sulfate
vi nng 1% (bng 2)
5.4.
nh hng ca cht kch thch sinh trng
Nghin cu ny kho st nh hng ca cao nm men, peptone, casein v hn hp ca
chng nng 0.1% i vi nng sut methionie to ra bi Bacillus thuringiensis
EC1.

Bng 3: nh hng ca cc cht kch thch tng trng n s tch ly methionie

Kho st cho thy tt c cc cht kch thch sinh trng cng nh hn hp ca chng
u tng cng kh nng sinh tng hp methionine. Vi kt qu c trnh by bng
3 cho thy khi b sung hn hp tt c cc cht kch thch sinh trng ny vi nng
0.1% s cho hm lng sn phm ti a.
5.5.
nh hng ca vitamin
Qu trnh ln men c tin hnh nhng nng khc nhau (0.1; 1; 5; 10mg/ml) ca
riboflavin, pyridoxine, axit nicotinic v thiamine HCl xc nh nh hng ca chng
n vic sn xut methionine bi B. thuringiensis
EC1.

Bng 4: nh hng ca vitamin n hm lng methionine