TRNG I HC NNG NGHIP H NI KHOA CNG NGH THC PHM

TIU LUN CNG NGH SINH HC THC PHM

ti: Nghin cu quy trnh sn xut cht khng sinh penicillin t vi sinh vt

Ngi hng dn:Ths. Nguyn Th Lm on Nhm sinh vin thc hin: nhm 10

H Ni 2008

MC LC
PHN I - M U PHN II - NI DUNG
I. GII THIU CHUNG V CHT KHNG SINH 1.1.Lch s pht hin cht khng sinh 1.2. nh ngha khng sinh: 1.3. C ch tc dng: 1.4. n v khng sinh:

II. CHT KHNG SINH PENICILLIN

2.1. Lch s pht hin v sn xut penicillin 2.2. Cng thc cu to ca penicillin 2.3. C s cng ngh sinh tng hp penicillin t nm mc III. QUY TRNH SN XUT PENICILLIN T VI SINH VT 3.1. c im chung: 3.2. Chun b ln men 3.3. K thut ln men: 3.3.1. K thut ln men b mt: 3.3.2. K thut ln men chm: 3.4 X l dch ln men v tinh ch thu penicillin t nhin

PHN 3 - KT LUN

PHN I - M U
Cc khng sinh l mt nhm thuc thit yu trong y hc hin i. Nh cc thuc khng sinh m y hc c th loi b c cc dch bnh nguy him nh dch hch, t, thng hn v iu tr hiu qu nhiu loi bnh gy ra bi cc vi khun. i vi cc nc ngho, cc thuc khng sinh li gi mt v tr rt quan trng v cc nc ny do iu kin v sinh yu km v mc sng cn thp nn thng xy ra cc v dch a chy, kit l, nhim khun h hp... Hin nay trn th gii ngi ta pht hin trn 8000 cht khng sinh v mi nm c khong vi trm cht khng sinh mi c pht hin. Trong tng lai chc chn cn c nhiu cht khng sinh khc na cng s c tm ra v a s cc vi sinh vt c kh nng to thnh cht khng sinh c nghin cu cho ti nay u ch thuc v cc chi Streptomyces v Bacillus. K t khi Penicillin c Alexander Fleming pht hin (1929), v c chng minh c tc dng cha bnh (1941), trong hn na th k qua, khng sinh tr thnh mt dc phm thn k sm chim v tr hng u trong lnh vc thuc men th gii, vi nhng kt qu ngy cng mi l, vi nhu cu ngy cng tng v vi lng sn xut ngy cng ln. Hn th na, cnh bn cht Penicillin u n, c thm nhiu loi khng sinh c chit xut t nm, v nhng loi khng sinh tng hp vi danh mc ngy cng di lm cho kho tng khng sinh thm phong ph. Xut pht t thc t , chng ti thc hin ti Nghin cu qu trnh sn xut cht khng sinh penicillin t vi sinh vt nhm gip cho chng ta c th tm hiu v quy trnh sn xut penicillin trong giai on hin nay. Mt khc chng ta c th cng trao i tm ra c nhng u, khuyt im ca quy trnh sn xut ny vi hi vng trong tng lai Vit Nam s c mt nh my sn xut penicillin vi quy m cng nghip.

PHN II - NI DUNG
I. GII THIU CHUNG V CHT KHNG SINH 1.1.Lch s pht hin cht khng sinh S pht trin v vi sinh vt hc ni chung, v vi sinh vt cng nghip ni ring, vi bc ngot lch s l pht minh v i v cht khng sinh ca Alexander Fleming (1982) m ra k nguyn mi trong y hc: khai sinh ra ngnh cng ngh sn xut cht khng sinh v ng dng thuc khng sinh vo iu tr cho con ngi. Thut ng" cht khng sinh" ln u tin c Pasteur v Joubert (1877) s dng m t hin tng km hm kh nng gy bnh ca vi khun Bacillus anthracis trn ng vt nhim bnh nu tim vo cc ng vt ny mt s loi vi khun hiu kh lnh tnh khc. Lin tip sau l nhng pht hin khc ca: Babes (1885) nu ra nh ngha hot tnh khng khun ca mt chng l c tnh tng hp c cc hp cht ho hc c hot tnh km hm cc chng i khng. Nicolle (1907) l ngi u tin pht hin ra hot tnh khng khun ca Bacillus subtilis c lin quan n qu trnh hnh thnh bo t ca loi trc khun ny. Gratia v ng nghip (1925) tch c t nm mc mt ch phm c th s dng iu tr hiu qu cc bnh truyn nhim trn da do cu khun. Mc d vy, trong thc t mi ti nm 1929 thut ng "Cht khng sinh" mi c Alexander Fleming m t mt cch y v chnh thc trong bo co chi tit v penicillin. 1.2. nh ngha khng sinh: Cht khng sinh c hiu l cc cht ho hc xc nh, khng c bn cht enzym, c ngun gc sinh hc (trong ph bin nht l t vi sinh vt), vi c tnh l ngay nng thp (hoc rt thp) c kh nng c ch mnh m hoc tiu dit c cc vi sinh vt gy bnh m vn m bo an ton cho ngi hay ng vt c iu tr.

1.3. C ch tc dng: C ch tc dng ln vi sinh vt gy bnh ( hay cc i tng gy bnh khc gi tt l mm bnh) ca mi cht khng sinh thng mang c im ring, ty thuc vo bn cht ca khng sinh ; trong , nhng kiu tc ng thng gp l lm ri lon cu trc thnh t bo, ri lon chc nng iu tit qu trnh vn chuyn vt cht ca mng t bo cht, lm ri lon hay kim to qu trnh sinh tng hp protein, ri lon qu trnh ti bn ADN, hoc tng tc c hiu vi nhng giai on nht nh trong cc chuyn ha trao i cht

Hnh 1. V tr tc dng chnh ca mt s cht khng sinh 1.4. n v khng sinh: Nng lc tch t khng sinh ca chng hay nng cht khng sinh thng c biu th bng mt trong cc n v l : mg/ml, g/ml, hay n v khng sinh UI/ml (hay UI/g, International Unit .

II. CHT KHNG SINH PENICILLIN

2.1. Lch s pht hin v sn xut penicillin

Penicillin c pht hin tnh c vo nm 1928 do Alexander Fleming, khi nhn thy mt hp petri nui Staphylococcus b nhim nm mc Penicillium notatum c xut hin hin tng vng vi khun b tan xung quanh khun lc nm. ng s dng ngay tn ging nm Penicillin t tn cho cht khng sinh ny (1929).

Sau , M trin khai ln men thnh cng penicillin theo phng php ln men b mt (1931). Tuy nhin, cng trong khong thi gian mi n lc nhm tch v tinh ch penicillin t dch ln men u tht bi do khng bo v c hot tnh khng sinh ca ch phm tinh ch v do vn penicillin tm thi b lng qun.

Nm 1938 Oxford, khi tm li cc ti liu khoa hc cng b, Ernst Boris Chain quan tm n pht minh ca Fleming v ng ngh Howara Walter Florey cho tip tc trin khai nghin cu ny. Ngy 25/05/1940 penicillin c th nghim rt thnh cng trn chut. Nm 1942, tuyn chn c chng cng nghip Penicillium chrysogenum NRRL 1951 (1943) v sau c bin chng P. chrysogenum Wis Q - 176 (chng ny c xem l chng gc ca hu ht cc chng cng nghip ang s dng hin nay trn ton th gii ); thnh cng trong vic iu chnh ng hng qu trnh ln men ln men sn xut penicillin G (bng s dng tin cht Phenylacetic, 1944)....

Hnh 2. Cc tc gi gii thng Nobel y hc nm 1945 v cng trnh penicillin Penicillin c xem l loi khng sinh ph rng, c ng dng rng ri trong iu tr v c sn xut ra vi lng ln nht trong s cc cht khng sinh c bit hin nay. Chng tc dng ln hu ht cc vi khun Gram dng v thng c ch nh iu tr trong cc trng hp vim nhim do lin cu khun, t cu khun, th d nh vim mng no, vim tai - mi - hng, vim ph qun, vim phi, lu cu, nhim trng mu...Thi gian u penicillin c ng dng iu tr rt hiu qu. Tuy nhin, ch vi nm sau xut hin cc trng hp khng thuc v hin tng ny ngy cng ph bin hn. V vy 1959, Batchelor v ng nghip tch ra c axit 6-

aminopenicillanic. y l nguyn liu sn xut ra hng lot ch phm penicillin bn tng hp khc nhau. i vi Vit Nam, nm 1946, gio s ng Vn Ng thnh cng trong vic sn xut nc lc penicillin trong mi trng nc ng gp phn ng k vo vic cu cha thng bnh binh v c Bc H thng Hun chng Lao ng hng ba cho thnh tu k diu cha tng ai lm c ny. 2.2. Cng thc cu to ca penicillin Penicillin gm nhiu loi, chng c cu to gn ging nhau, bao gm mt vng thiazolidine, mt vng -lactam, mt nhm amino c gn vi CO 2 v mt mch bn (R). Tt c cc penicillin u l dn sut ca acid 6-aminopenicillanic. S thay th R to nhiu acid amin khc nhau. hu ht cc penicillin u c phn phi di dng mui natrii hoc mui kali.

Hnh 3. Cu to chung ca phn t penicillin Ngy nay trn th gii sn xut ra c trn 500 ch phm penicillin ( trong ch ln men trc tip hai sn phm l penicillin V v penicillin G) v tip tc trin khai sn xut cc ch phm penicillin bn tng hp khc.

Hnh 2: Sn phm penicillin ln men t nhin nh P.chrysogenum 2.3. C s cng ngh sinh tng hp penicillin t nm mc 2.3.1. Lch s tuyn chn chng cng nghip P. chrysogenum

Vo nhng nm u, vic nghin cu sn xut penicillin thng s dng cc chng c hot lc cao thuc loi P. notatum v P. baculatum. Nhng t khi trng i hc Wisconsin (M) phn lp c chng P.chrysogenum c hot tnh cao hn th chng ny dn dn thay th v t khong sau nhng nm 50 ca th k XX n nay tt c cc cng ty sn xut penicillin trn th gii u s dng cc bin chng P.chrysogenum cng nghip. Vic tuyn chn chng cng nghip ln men sn xut penicillin trn nguyn tc cng tri qua su giai on c bn m t trong mc 1.3.1, trong gii php k thut c p dng hiu qu thu nhn bin chng "siu tng hp" penicillin li chnh l cc k thut gy t bin thng nh: x l tia Rn - ghen, x l tia cc tm v to t bin bng ho cht, th d nh Metylbis - amin (metyl -2- -clo- etylamin), N-mustar (tris - -cloetylamin), Sarcrolyzin, HNO2, Dimetylsulfat, 1,2,3,4 -diepoxybutan. 2.3.2. C ch sinh tng hp penicillin nm mc P. chrysogenum : Theo quan im ph bin hin nay, qu trnh sinh tng hp penicillin nm mc P. chrysogenum c th tm tt nh sau: t ba tin cht ban u l

-aminoadipic, cystein v valin s ngng t li

thnh tripeptit -lactam v -( - aminoadipyl) - cysteinyl thiazolidin trao i nhm to thnh vi phenylacetic (hay valin ; tip theo l qu trnh khp mch to vng vng ri

izopenicillin-N;

-aminoadipyl

phenooxyacetic) to thnh sn phm penicillin G (hay penicillin V, xem s tng hp penicillin G trong hnh.

Hnh 3. S c ch sinh tng hp penicillin t axit L- - aminoadipic, L-cystein v L-valin Tuy nhin, cng c th n c gii phng ra v tch t trong mi trng (v trong qu trnh ln men sn xut penicillin V bao gi cng pht hin thy trong dch ln men lng ln - aminoadipic dng vng). Nh vy, qu trnh sinh tng hp penicillin, ph thuc vo iu kin ln men c th nht nh, c th xy ra theo su ng hng khc nhau. Do , hiu sut chuyn ho c cht - sn phm cng bin i v ph thuc vo ng hng sinh tng hp tng ng. Theo l thuyt th hiu sut ln men s trong khong 683 - 1544 UI penicillin/g glucoza; song, trong thc t, vi nhng chng c hot tnh sinh tng hp cao nht cng mi ch t khong 200 UI/g glucoza. 2.3.3. Tc ng ca cc thng s cng ngh n qu trnh sinh tng hp penicillin. 2.3.3.1. S pht trin h si v c im hnh thi h si nm: S pht trin h si nm trong qu trnh ln men bao gm: - S tng trng v kch thc h si (tng di si, s ln ln v kch thc, mc phn nhnh ca h si ... )

- S bin thin v s lng khm si nm trong mi trng: Thng thng, s pht trin ny c nh gi qua hai ch tiu l: hm lng sinh khi v tc bin thin hm lng sinh khi trong mi trng. Hai ch tiu ny c th xc nh bng nhiu phng php khc nhau nh: hm lng sinh khi (Sinh khi ti hoc sinh khi kh), mt quang dch ln men, tr lc lc ca dch ln men, hm lng nit, hm lng hydratcacbon, hm lng axit nucleic ... Trong cc phng php trn, c p dng ph bin hn c trong sn xut cng nghip l phng php xc nh qua hm lng sinh khi. Tc pht trin h si nm ph thuc hng lot cc yu t khc nhau trong qu trnh ln men v s tch t penicillin thng xy ra mnh m khi h si pht trin t trng thi cn bng. Trng thi ny c th xc lp c khi ch cung cp va v lin tc lng thc n ti thiu cho nm mc. Thiu thc n, h si nm s t phn, cn nu cung cp qu nhu cu trn, h si s pht trin, nhng khng tch t mnh penicillin m tch t nhiu axit gluconic v axit malic. - c im hnh thi v cu trc h si nm: Trong qu trnh ln men, do nhiu nguyn nhn khc nhau, s lng khm si nm bao gi cng c xu hng tng ln, ngay c trong qu trnh ln men tnh. Trong iu kin ln men c sc kh v khuy trn, do tc dng va p c hc vi cnh khuy v cc chuyn ng dng xoy trong mi trng, mt mt s t gy h si nm xy ra nhiu hn v h si nm bao gi cng c xu hng vn cun li thnh cu trc bi si cun xon, c gi l pellet. Pellet xp (fluffy loose pellets) l dng pellet c phn bn trong h si cun thnh khi chc v mn, lp si pha bn ngoi cun lng lo to thnh cu trc xp hn. Pellet chc v mn (compact smooth pellets) c c im l phn si pha bn trong pellet cun tng i cht ch ra n gn st lp si pha ngoi, lp si pha ngoi cng cng cun chc thnh lp si mn. Pellet rng (hollow pellets) l dng pellet c phn si bn trong b t phn to thnh khong rng, h si pha bn ngoi cun rt cht thnh lp si mn v chc chn. Hiu qu chung ca qu trnh ln men c quan h hu c vi s lng, kch thc v cu trc pellet nm. Trong thc tin sn xut cng nghip, ngi ta thng iu chnh cc thng s cng ngh theo hng u tin to ra dng pellet nh v mn, hn ch to pellet xp v ngn nga hnh thnh cc pellet rng. iu kin cng

ngh tng ng vi mc tiu trn thng p dng l : t l cy ging 10%, vi mt dch ging (2-10).1011 bo t /m3; phi hp iu chnh gia sc kh v khuy trn m bo cung cp oxy ha tan d so vi nhu cu tng ng vi thi im ln men, v to ra pellet mn v nh (kch thc pellet thch hp nht khong 0,2 0,5mm), trong iu kin cn i vi nhu cu tit kim mc tiu tn nng lng do khuy trn. 2.3.3.2. c tnh nhit ng ca dch ln men: Trong cc thit b ln men dung tch ln c sc kh v khut trn, thc t khng th xc lp c s ng u ti khp cc vng th tch lm vic ca thit b. Ti cc vng chy ri (vng gn cnh khuy), tc trao i nhit, tc chuyn khi xy ra mnh m hn. Cn ti cc vng chy mng (vng st thnh thit b, vng gn cc ng xon trao i nhit, vng km hiu qu hay vng cht ca thit b) tc chuyn khi hay tc truyn nhit cng gim i. Ngoi ra, ti nhng khu vc nht nh ca thit b c th xut hin vng xoy cc b hay cc dng chy th cp lm thiu ht v hm lng oxy ha tan. Cc yu t nu trn y s tc ng trc tip n nng lc sinh tng hp ca chng, hiu qu chuyn ha to sn phm v hiu qu kinh t chung ca ton qu trnh ln men. Thc t thng chn ch khuy trn d trn mc yu cu. 2.3.3.3. Thnh phn mi trng ln men: Mi trng c s ln men penicillin, vo thi k u trong nhng nm 40 50, l mi trng lactoza - nc chit ng, vi thnh phn chnh nu trong bng 2.1. Ngun c cht chnh: l lactoza c th c thay th tng phn hoc ton b bng cc c cht khc nh: cc loi ng hexoza, ng pentoza, disaccarit, dextrin hay thay th bng du thc vt. Trong cc c cht nu trn, hiu qu cao hn c vn l glucoza. Ngoi ra, khi s dng du thc vt lm cht ph bt phi xt n hiu ng nm mc s dng mt phn du thc vt lm ngun cung cp thc n cacbon, tnh ton iu chnh nng glucoza trong mi trng ln men (v c s cn tr qu trnh chuyn khi do nh hng ca du ph bt). Ngun cung cp thc n nit: c th s dng l bt u tng, bt ht bng, cc loi du cm. Nhu cu v thc n nit cng c th c p ng bng cch cung cp lin tc (NH4)2SO4, nhng duy tr nng thp, khong 250 - 340g/l (nu d tha hiu qu sinh tng hp penicillin s gim, nu thiu s xy ra hin tng t phn h si) .

Hm lng cc cht khong b sung: c tnh ton, ph thuc vo lng dch chit ng s dng; pH mi trng c iu chnh trc khi thanh trng, sau trong sut qu trnh ln men c gim st cht ch v iu chnh theo yu cu cng ngh. Nng tin cht to nhnh:Trong qu trnh sinh tng hp penicillin, vic kt gn mch nhnh ca phn t penicillin khng mang tnh c hiu cht ch. Nh vy, nu duy tr nng tin cht to nhnh cn thit phenylacetat (hoc phenooxyacetat) s cho php thu nhn ch yu mt loi penicillin G trong dch ln men (hoc penicillin V). Theo l thuyt, nhu cu v phenylaceta l 0,47g/gam penicillin G (hoc phenooxyacetat l 0,50g/gam penicillin V ). Cn ch c hai cu t trn thc cht u gy c cho nm nn ngi ta thng la chn gii php b sung lin tc cu t ny v khng ch cht ch nng theo yu cu, khng lm suy gim nng lc ln men ca chng sn xut.

2.3.3.4. iu kin tin hnh ln men: Nhit l thng s c nh hng ln n s pht trin ca nm mc, kh nng sinh tng hp v nng lc tch t penicillin ca chng. Nhn chung nm mc pht trin thun li hn di nhit khong 300C. Tuy nhin, di nhit ny tc phn hu penicillin cng xy ra mnh m. Trong thc t, giai on nhn

ging sn xut ngi ta thng nhn di nhit 30 0C; sang giai on ln men thng p dng mt trong hai ch nhit l : Ln men mt di nhit : Thng duy tr nhit trong sut qu trnh ln men di nhit 25 - 270C. Ln men hai ch nhit : Giai on ln men bt u tin hnh 30 0C cho n khi h si pht trin t yu cu v hm lng sinh khi th iu chnh nhit sang ch ln men penicillin di nhit 22 - 250C (c cng ngh iu chnh xung 22 - 230C, gi nhit ny tip hai ngy ri chuyn sang ln men tip 250C cho n khi kt thc qu trnh ln men). pH mi trng thun li cho s pht trin h si v cho qu trnh sinh tng hp penicillin thng dao ng trong khong pH = 6,2 - 7,4. Tuy nhin iu kin pH cao xu hng phn hu penicillin cng tng ln. V vy, trong sn xut pH mi trng thng c khng ch cht ch gi tr la chn trong khong pH = 6,2 6,8. Nng oxy ho tan v cng khuy trn dch ln men: Vi nhiu chng nm mc, nng oxy ha tan thun li cho qu trnh sinh tng hp penicillin dao ng quanh mc 30% nng oxy bo ha. Nng CO2 trong dch ln men mc nht nh cng cn thit cho qu trnh ny mm ca bo t nm mc; tuy nhin nu nng CO 2 qu cao s lm cn tr qu trnh hp thu v chuyn ho c cht ca chng, ngha lm lm cn tr qu trnh sinh tng hp penicillin. 2.3.3.5. S tch t v phn hu penicillin: Trong qu trnh ln men, do nhiu nguyn nhn khc nhau, trong c nh hng ca nng penicillin tch t trong mi trng ngy cng tng, lm cho nng lc sinh tng hp penicillin ca chng c xu hng gim dn theo thi gian ln men. ng thi, ph thuc vo nhit v pH mi trng, mt phn lng penicillin tch t cng b phn hu theo thi gian. Nhm gim tn tht trn, ngay sau khi kt thc qu trnh ln men cn x l thu sn phm sm hoc c gii php h thp nhanh nhit dch ln men. III. QUY TRNH SN XUT PENICILLIN T VI SINH VT 3.1. c im chung: Cng ngh ln men sn xut penicillin mang nt c th ring ca tng c s sn xut v cc thng tin ny rt hn ch cung cp cng khai, ngay mi bng sng

ch thng cng ch gii hn nhng cng on nht nh; v vy rt kh a ra c cng ngh tng qut chung. Theo cng ngh ln men ca hng Gist-Brocades (H Lan), ton b dy chuyn sn xut thuc khng sinh penicillin c th phn chia lm bn cng on chnh nh sau (xem s hnh 2.8) Ln men sn xut penicillin t nhin (thng thu penicillin V hoc penicillin G) . X l dch ln men tinh ch thu bn thnh phm penicillin t nhin. Sn xut cc penicillin bn tng hp (t nguyn liu penicillin t nhin) Pha ch cc loi thuc khng sinh penicillin thng mi

Hnh 4. S dy chuyn sn xut penicillin (theo Gist-Brocades Copr. (H Lan)) 3.2. Chun b ln men - Ging, bo qun v nhn ging cho sn xut: Ging dng ln men penicillin l P.chrysogenum, y l loi nm si bo t h. Khi mi pht hin trong mi trng c, chng to ra hai dng khun ty: khun ty kh sinh v khun ty dinh dng mu trng. Sau khi nui cy mt ngy, khun ty bt u chuyn sang mu xanh xm v nh bo t bt u xut hin. Thi gian ny xut hin mt t bo t trn t tin bo t nm trong cc nh bo t. Cc bo t ln lt c to thnh theo thi gian nui cy v cui cng th mu ca nm penicillium sm hn.

Ging cng nghip P.chrysogenum c bo qun lu di dng ng kh, bo qun siu lnh hoc bo qun trong nit lng. Ging t mi trng bo qun c cy chuyn ra trn mi trng thch hp hot ho v nui thu bo t. Dch huyn ph bo t thu t hp petri c cy chuyn tip sang mi trng bnh tam gic, ri sang thit b phn ging nh, qua thit b nhn ging trung gian ... v cui cng l trn thit b nhn ging sn xut. Yu cu quan trng ca ca cng on nhn ging l phi m bo cung cp lng ging cn thit, vi hot lc cao, cht lng m bo ng thi im cho cc cng on nhn ging k tip v cui cng l cung cp lng ging t cc yu cu k thut cho ln men sn xut. Trong thc tin, m bo cho qu trnh ln men thun li ngi ta thng tnh ton lng ging cp sao cho mt ging trong dch ln men ban u khong 1 - 5.109 bo t / m3. - Chun b mi trng ln men v thit b: Chun b mi trng ln men: Cn ong, pha ch ring r cc thnh phn mi trng ln men trong cc thng cha ph hp Thanh trng gin on 1210C ( hay thanh trng lin tc khong 140-1460C) hoc lc qua cc vt liu siu lc ri mi bm vo thng ln men. Nu c tnh cng ngh ca thit b ln men cho php, c th pha ch ri thanh trng ng thi dch ln men trong cng mt thit b. Tt c cc cu t b

sung vo mi trng ln men u phi c x l kh khun trc v sau b sung theo ch vn hnh v khun.

Thit b ln men: Phi c v khun trc khi a vo s dng. Thng

thanh trng bng hi qu nhit 2,5 3,0 at trong thi gian 3 gi. ng thi kh khun nghim ngt tt c cc h thng ng dn, khp ni, van, phin lc v tt c cc thit b ph tr khc.Trong qu trnh ln men luun c gng duy tr p sut d trong thit b nhm hn ch ri ro do nhim tp. Khng kh thng c kh khun s b bng nn on nhit, sau qua mng lc v khun hay mng siu lc . - Chun b mi trng nhn ging

Chun b mi trng nhn ging: lm mi trng nhn ging ngi ta cng

chun b nh mi trng ln men nhng chng khng cha lactose (nu c ch cha mt lng rt nh), mt s khong cht v tin khong cht. Mt khc thnh phn mi trng nhn ging cn c tnh ton m bo cung cp ngun thc n C, N, cc cht khong v cc thnh phn khc, m bo cho s hnh thnh v pht trin thun li ca pellet. Sau khi lm m mi trng n m nht nh, ngi ta s phn phi vo chng vo cc dng c thy tinh (chai thy tinh hay cc bnh tam gic) vi khi lng bng 1/5 hay 1/6 dung tch ca dng c, y nt bng v em thanh trng 121oC (0,5 at) trong 30 pht 3.3. K thut ln men: 3.3.1. K thut ln men b mt: p dng t lu, hin nay hu nh khng cn c trin khai trong sn xut ln na. Gm 2 phng php: * Ln men trn nguyn liu rn (cm m, cm ng c b sung ng lactose) * Ln men trn b mt mi trng lng tnh (ph bin s dng mi trng c bn lactose - nc chit ng)..

Qu trnh nhn ging

Qu trnh nhn ging bt u t ging c trong ng nghim. Trong cc nh my, mi ln cy truyn ging, ngi ta thng cy lm 3 ng. Mt ng dng kim tra trc khi sn xut, mt ng dng sn xut v mt ng dng bo qun.

Song song , ngi ta chun b mt bnh tam gic dung tch 200 250 ml v chun b 50 g mi trng nh phn trnh by trn. Mi trng c thanh trng v lm ngui n 30oC. 10ml thanh trng v lm ngui vo ng ging, dng que thy tinh nh cho bo t ha trn vi nc. Bng bin php v trng (thc hin trong cc t nui cy v trng) chuyn ton b vo bnh tam gic trn lc cho tht u v chuyn chng sang t m 30 37oC. Nui trong iu kin ny chi n khi bo t nm xut hin v pht trin u khp mi trng. Ta gi qu trnh thc hin nh trn l qu trnh nhn ging cp 1. C ln lt thc hin tip ta c ging cp 2, cp 3cho n khi 5 10% ging cho sn xut. Mi mt cp nhn ging t cp ny sang cp khc, khi lng mi trng tng t 10 15 ln.

Qu trnh ln men
i vi phng php ln men trn nguyn liu rn (cm m, cm ng c b sung ng lactose), khi mi trng c kh trng v lm ngui n 30oC, tin hnh trn ging vo vi t l t 5 10%. Cc khay c xp chng ln nhau trn nhng gi c mt khong cch nht nh thong kh v thong nhit. Qu trnh ln men ko di 6 7 ngy nhit 24 28oC. u im ca phng php ny l ng lactose c nm mc ng ha chm nn khng xy ra hin tng d tha ng trong t bo, cn dch nc chit ng cung cp cho nm mc ngun thc n nit, cc cht khong v cc cht sinh trng, trong phenylalanin khi b thy phn s to thnh phenylacetic cung cp tin cht to mch nhnh cho phn t penicillin. Khi ln men trong mi trng lng, p dng cng ngh b sung lin tc phenylacetic vo mi trng ln men, hm lng b sung ph thuc pH mi trng thng l 0,2-0,8 kg phenylacetic/m3 dch ln men. Dung dch ln men sau khi c kh trng s c phn phi vo cc khay c kch thc ging cc khay nui cy b mt vi mi trng bn rn. y cc khay ny khng c c l v phi cha mi trng lng. Chiu cao ca dung dch mi trng trong cc khay l 3 4 cm. ngi ta cng tin hnh ln men trong khong thi gian 6 7 ngy nhit ln men l 24 28oC. Tin hnh ln men trong iu kin mi trng lng ny, lng penicillin G c tng hp tng r rt cn hm lng cc penicillin khc cng gim i. hn ch qu trnh oxy ha tin cht, thng phi b sung vo mi trng mt lng nh axit axetic. Trong k thut ln men lng gin on khng iu chnh pH mi trng

thng tng nh, sau tng i n nh v vo cui qu trnh ln men thng trong khong pH = 6,8 7.4. Khi s dng c cht chnh l lactose, ngi ta xc nh c penicillin ch c tng hp v tch t mnh m trong mi trng khi nm mc s dng ng ny v khi lactose c du hiu cn kit th si nm cng bt u t phn. V vy ngi ta thng kt thc qu trnh ln men vo thi im sp ht ng lactose. 3.3.2. K thut ln men chm: K thut len men chm l k thut c p dng trong hu ht cc c c sn xut penicillin cng nghip hin nay v thng c vn hnh theo phng php ln men bn lin tc, gm phng n ln men gin on theo m c b sung lin tc (hay bn lin tc) mt hay mt vi cu t kt hp vi phng n tun hon li mt phn h si ca m ln men trc (hoc khng)

Qu trnh nhn ging

Trong qu trnh ln men chm ngi ta nhn ging trong mi trng lng. Mc ch ca qu trnh nhn ging l thu nhn c s lng t bo cao( thng tnh tng lng t bo/ml). Qu trnh nhn ging c bt u bng vic chuyn ging t ng nghim sang nhng bnh tam gic cha sn mi trng nhn ging. Ngi ta thng nhn ging vo cc bnh ln men dung tch t 1 lt cho n hng ngn lt. Nhit trong qu trnh nhn ging duy tr khong 26 1oC v thi gian nhn ging mi cp khong 72 gi.

Qu trnh ln men
Qu trnh ln men trong mi trng lng bng phng php ln men chm sn xut penicillin c vn hnh theo phng php ln men hai pha: - Pha th nht nui thu sinh khi trong khong 2 3 ngy. Trong pha ny h si pht trin rt mnh v cc cht dinh dng d ng ha s c t bo hp th rt mnh, tc sinh sn ca nm xy ra rt nhanh, s to thnh penicillin mi bt u. - Pha th hai ln mn thu sn phm. pha ny h si pht trin chm li, pH tng dn v t n gi tr khong 7 7,5. Trong pha ny penicillin c to thnh vi mc cc i. Trong hu ht cc trng hp, khi ln men, ngi ta thay th phn ln (hoc hon ton) ng lactose bng ng glucose. Lng glucose ny c th c b

sung lin tc hay bn lin tc nhng phi gim st cht ch nng glucose trong sut qu trnh vn hnh pha duy tr nng glucose lun mc thch hp nhm va gi khi lng h si n nh, va m bo sinh tng hp nhiu penicillin. Trong thc tin, trnh xy ra thiu ht nht thi glucose , ngi ta c th kt hp b sung mt lng nh ng lactose (khi , nu cha b sung kp glucose th nm mc s t iu chnh s dng ng lactose nn khng xy ra hin tng t phn h si). Ngoi ngun nit trong nc chit ng, ngi ta thng s dng phi hp (NH4)2SO4 va cung cp thc n N v S, va s dng iu chnh pH trong qu trnh ln men (pH dch len men ban u thng c iu chnh v khong pH = 6,5 6,8 bng dung dch NaOH hoc H 3PO4); nng NH4
+

thng

khng ch trong khong 0,3 0,4 kg/m3 dch ln men. Cht ph bt thng s dng l cc loi du bo nh: m ln, du u tng, du vng, du cmTin cht to nhnh phenylacetic trong ln men sn xut penicillin G (hoc phenooxyacetic trong ln men sn xut penicillin V) c b sung lin tc (hoc b sung gin on lm nhiu ln) trong sut thi gian pha ln men penicillin, duy tr nng trong khong 0,1 1,0 kg/m3 dch (nu t qu nm mc s tng hp ng thi nhiu penicillin khc, nu nhiu qu s gy c cho nm v tng cng thc y qu trnh hydroxyl ha sn phm penicillin). Nhit ln men pha u khng ch 30 oC, sau sang pha sau gi 22 25oC. Tc sc kh v khuy trn c iu chnh duy tr nng oxy ha tan trong dch trong khong 30%. Trong iu kin trn thi gian ln men mi m thng ko di khong 144 180 gi. Kt thc qu trnh ln men ngi ta c gng lc sm dch ln men, lm lnh ri chuyn sang cng on trch ly v tinh ch thu penicillin. * c im v thit b ln men chm: Qu trnh ln men sn xut penicillin ngy nay ch yu c tin hnh trong thit b ln men chm ch to bng nhm thp chu n mn CT2 vi khuy trn kiu tuc-bin (gm nhiu tng cnh khuy), kt hp b tr h vch dn dng trong thng). Cng sut khuy trn tiu hao c thit k khong 3kW/m 3/h. Khng kh nn v khun c cp vo qua h ng phn phi kiu vng xoy hay kiu r qut c l lp t st di y (hay pha di cnh tuc-bin). Bn trong thit b c lp t nhiu tng ng trao i nhit kiu vng xon kt hp ng thi vi trao i nhit qua thnh thit b hai lp v, m bo iu nhit hiu qu trong sut qu trnh ln men. Dung tch thit b ph bin trong khong 150 300m 3, h s y thng chn khong 80%V (ph thuc vo k thut v thit b ph bt). Thit b nhn sn xut ging c dung tch khong 10%V thit b len men, c thit k tng

t v thng c ghp cng vi thit b ln men. Ton b thit b ln men sn xut, thit b nhn ging ln v h thng trang thit b ph tr c thit k v lp t m bo c th v sinh v thao tc vn hnh theo ch v khun cao (tt nht nn b tr sao cho c thap dng ch thanh trng ng thi cho ton b h thit b ny). Cc thng s kim tra qu trnh ln men bao gm: pH mi trng, nng oxy ha tan, nhit , hm lng sinh khi v tc bin thin lng sinh khi, s lng, kch thc v cu trc pellet, nng cc cu t c cht, nng penicillin, thnh phn kh thi v cc ch tiu kim tra v vi sinh vt. Vic gim st v iu chnh qu trnh ln men c xy dng trn c s khai thc c hai kiu tng tc trc tuyn (online control) v tng tc khng phn hi theo quy lut (offline control), ph thuc vo kh nng p ng ca h thit b hin c. ng thi xu hng my tnh ha trong kim tra v gim st qu trnh ln men ang dn chim u th trong sn xut cng nghip.

Hnh 5. S h ln men dng cho sn xut penicillin. * Hiu qu kinh t chung ca qu trnh ln men chm Nng lc sinh tng hp v tch t penicillin trong dch ln men l kt qu ca mi tng tc ng thi ca hng lot yu t cng ngh nh: hot tnh sinh tng hp ca chng, cng ngh ln men p dng, cht lng nguyn liu, c tnh thit b v nng lc p ng cc yu cu cng ngh ca thit b, ch gim st v iu chnh cc thng s cng ngh, nng lc v k nng vn hnh ca cng nhn.... Vi ngun c cht chnh l glucoza v ln men theo phng php chm, h s phn b nguyn liu d tnh khong 25% glucoza c nm mc s dng tng hp h si, 65% ng c s dng duy tr s sng st ca h si, cn li ch khong 10% c nm mc s dng tng hp penicillin. H s s dng thc n nit v lu

hunh tng hp penicillin tng ng l 20% v 80%. Nng penicillin G trong dch ln men nhng nm 80 - 90 ca th k XX t khong 80.000 UI/ml (tng ng nng sut khong 40 - 50 kg penicillin G/ m3 dch ln men ). 3.4 X l dch ln men v tinh ch thu penicillin t nhin C ba phng php thu nhn v tinh ch penicillin t mi trng nui cy, l:

Trch ly bng dung mi hu c Hp ph Trao i ion

Trong ba phng php trn th phng php trch ly bng dung mi hu c c s dng nhiu hn c. phng php ny da trn nhng u im sau:

Mui ca penicillin rt d tan trong nc

Acid penicillic rt d tan trong dung mi hu c Cng on x l dch ln men v tinh ch thu penicillin t nhin c tm tt trong s hnh 2.9 , bao gm cc cng on chnh sau y:

Hnh 6. S tm tt cng on x l dch ln men thu penicillin t nhin 3.4.1. Lc dch ln men :

Mc ch: Penicillin l sn phm ln men ngoi bo. V vy, ngay sau khi kt thc qu trnh ln men ngi ta thng tin hnh lc ngay gim tn hao do phn hu penicillin v gim bt kh khn khi tinh ch, do cc tp cht to ra khi h si nm t phn. Thit b lc: ph bin l thit b lc ht kiu bng ti hoc kiu thng quay. Thng thng, ngi ta ch cn lc mt ln ri lm lnh dch ngay chuyn sang cng on tip theo. Ch trong nhng trng hp rt c bit mi cn phi x l kt ta mt phn protein v lc li dch ln th hai. Hin tng t phn h si nm thng ko theo hu qu lm cho dch kh lc hn. Thu hi sinh khi nm: Phn sinh khi nm c ra sch, sy kh v s dng ch bin thc n gia sc. 3.4.2. Trch ly : Penicillin thng c trch ly dng axt ra khi dch lc bng dung mi amylacetat hoc butylacetat pH = 2,0 - 2,5, nhit 0 - 30C. Nhm hn ch lng penicillin b phn hu, qu trnh trch ly c thc hin trong thi gian rt ngn trong thit b trch ly ngc dng lin tc kiu ly tm nhiu tng cnh. ng thi, trong thi gian trch ly cn gim st cht ch cc thng s cng ngh nh: nhit pH, v khun.... hn ch tn tht do phn hu penicillin. Dch ln men sau khi lc c bm trn ng thi vi dung dch H 2SO4 hoc H3PO4 long c b sung thm cht chng to nh v bm song song cng vi dung mi trch ly vo trong thit b. T l dch lc: dung mi thng chn trong khong 4 - 10V dch lc /1V dung mi. Trong mt s cng ngh, nhm ci thin cht lng sn phm, ngi ta c th p dng phng php trch ly hai ln dung mi, vi ln u trch ly penicillin bng amylacetat hoc butylacetat; tip theo penicillin li c trch ly ngc sang dung dch m pH = 7,2 - 7,5, thng l dung dch KOH long hoc dung dch NaHCO 3; sau penicillin li c trch ly sang dung mi ln th 2, vi lng dung mi t hn.

Hnh 7. S cng ngh trch ly 2 ln dung mi tinh ch penicillin 3.4.3. Ty mu : ty mu v loi b mt s tp cht khc, ngi ta thng b sung trc tip cht hp ph vo dung mi cha penicillin sau trch ly, s dng ph bin nht l than hot tnh. Sau than hot tnh c tch v ra li bng s dng thit b lc ht bng ti hoc thit b lc ht kiu thng quay. Phn than sau lc c a i chng thu hi dung mi v x l hon nguyn, phc v cho cc m sau. 3.4.4. Kt tinh, lc, ra v sy thu penicillin t nhin: Vic kt tinh penicillin V hay penicillin G di dng mui c th c thc hin rt n gin, bng cch b sung trc tip vo dung mi sau khi ty mu mt lng nh kali acetat (hay natri acetat) hoc ngi ta trch ly li sang dung dch KOH long (hay NaOH long), tin hnh c chn khng nhit thp, sau b sung BuOH penicillin t kt tinh. Cc thng s cng ngh c nh hng ln n hiu qa kt tinh l : nng penicillin, nng mui acetat, pH dung mi hay pH dung dch c c, nhit kt tinh ... Sau khi kt tinh, tinh th penicillin c lc tch bng my lc ht thng quay. m bo tinh khit cao hn, c th tin hnh ha tan v kt tinh li penicillin. Khi sn phm t tinh sch theo yu cu, thng tinh khit khng di 99,5%, chng c lc tnh tinh th; tip theo ra v lm kh s b bng dung mi k nc nh izopropanol hay butylalcohl; ht chn khng tch dung mi trn my lc bng ti ri sy bng khng kh nng n dng sn phm bt mui penicillin. Sn phm ny, mt phn c s dng trc tip pha ch thuc

khng sinh penicillin; cn li, phn ln c s dng lm nguyn liu phc v cho vic sn xut cc sn phm penicillin v cephalosporin bn tng hp khc. Ngoi ra, sn xut ra cc sn phm penicillin c tinh khit rt cao, ngi ta cn phi s dng phi hp thm mt s gii php cng ngh khc nh phng php phn tn tnh in. nguyn tc ca phng php ny l s dng mt hiu in th cao (ln n 25 kV) to nhng vi git ca dung dch cha penicillin.

PHN 3 - KT LUN

Penicillin l lai khng sinh u tin c dng iu vim nhim ngi. Khm ph v penicillin t mt pht hin tnh c nhng c ngha cc k to ln i vi nhn lai. Penicillin thng thng c sn xut bng phng php ln men a kh chm v c chit xut pH thp t dch ln men s dng butyl axetat hoc kerosen. Do tnh khng n nh ca penicillin pH thp, quy trnh chit xut cn thc hin nhit thp, song c ti 10-15% sn phm b tht thot khi s dng quy trnh cng nghip hin nay. Ngoi nng sut tng i thp, cc phng php sn xut truyn thng c chi ph cao, tiu th nhiu nng lng thu hi butyl axetat t cc cht l quy trnh i hi nhiu cng on. Vi nhng vn nu ra trn, chng ti hi vng rng em n cho cc bn nhng kin thc c bn v cng ngh sn xut penicillin trong quy m cng nghip. V kin thc cn hn hp nn c rt nhiu sai st mong c v cc bn cng ng gp kin bi tiu lun ca chng ti hon chnh hn. Qua y chng ti xin c chn thnh cm n c Nguyn Th Lm on gip chng ti hon thnh tiu lun ny.