Cc qu trnh cng ngh sinh hc c bn Trong

Cng ngh ln men hin i

I. i cng v cng ngh ln

men hin i

1.1. im lch s cng ngh ln men cng nghip

1815: Sn xut dm theo phng php nhanh 1846: Sn xut cng nghip nm men bnh m 1881: Ln men axt lactic nh vi khun 1900 : Thng lc sinh hc x l nc thi th QMCN 1912 : Ln men sn xut axt xitric 1914 : S dng Candida SX thc n gia sc t r ng 1930 (1894): Ln men SXCN ch phm enzym pectinaza 1934 : Sn xut vitamin C, s dng Acetobacter 1940 : Ln men sn xut cng nghip penicillin G

1954 (1959, 1960) : Ln men vi t bo thc vt

1966: S dng t bo bch cu ln men thu Interferon 1980: Ln men thu -interferon nh E. coli ti t hp 1982: Ln men thu SPTCTC (saponin) vi t bo thc vt

+ V bn cht tc nhn sinh hc

Thi k trc 1881: Ln men bng canh trng tp trung 1881 : ln men bng canh trng vi sinh vt thun khit 1940: ln men thun khit, bng chng VSV t bin 1954: ln men thun khit, bng t bo (m) thc vt 1966: ln men thun bng t bo (m) ng vt (34,
37)

1980: ln men thun bng t bo VSV ti t hp gen

+ Kh nng pht trin

Hin ti: mi ch mt s t VSV c ng dng trong SXCN

Tng lai: u th v s a dng sinh hc, nng lc chuyn ho v sinh tng hp, kh nng can thip vo cu trc gen thun li...; tc pht trin v bo ca KH-CN... Ngy cng nhiu hn cc chng VSV c ng dng trong SXCN

Hin ti: mi ng dng CNLMH trn i tng m thc vt, ng vt

Tng lai: CNLMH trn i tng TB gc SX ht ging nhn to

+ V c im cng ngh
Trc XIX: Ln men thng (ym kh, hiu kh gin n) 1815 : ln men hiu kh trn thp m 1846: ln men trong thit b dung tch ln (NMBM) 1935: ln men chm c sc kh v (42) khuy trn c hc

1956: gim st qu trnh ln men (LM metan)

1970: p dng m hnh v ti u ho trong CNLM 1971: Kt ni computer gim st QT ln men 1976: iu khin ti u QT ln men bng computer *** p dng cc gii php cng ngh mi trong khu chun b ln men, x l dch ln men v tinh ch thu sn

1.2. Cng ngh ln men hin i

Quy trnh cng ngh

S dng i tng sinh hc lm tc nhn

(vi sinh vt, t bo m thc vt, t bo m ng vt, enzym...)

Thu sn phm sinh hc c gi tr kinh t cao hay thc hin chuyn i cng ngh nht nh Quy m sn xut hng ho (LM hiu kh, trong thit b V ln, s dng bin chng cao sn, gim st v iu khin nghim ngt cp phn t trong sut qu trnh ln men, t ng ho v iu khin ti u ho qu trnh nh computer, p dng cc k thut chun b ln men, x l dch ln men v tinh ch thu sn phm hiu nng cao...)

1.3. mc ch qu trnh ln men

Thu ton b sinh khi: nhn ging, sn xut

protein n bo

Thu enzym, axit amin, vitamin...

Thu sn phm trao i cht:

cp Bin i cu trc c cht

sn phm trao i

cht s cp; Sn phm trao i cht th (biotransformation)

Cc mc ch khc x l cht thi, thu hi v khai thc

kim loi qu him, VSV ch

th...

1.4.

phn loi qu trnh ln men

Theo c tnh c l mi trng

Ln men trn mi trng rn xp

Ln men vi mi trng lng (LM b mt, LM b su...) Ln men ym kh ln men hiu kh

Theo nhu cu v xy

Theo k thut vn hnh

ln men gin on ln men bn lin tc (cp liu, thu dch ln men...) ln men lin tc Ln men thng Ln men hai pha Ln men nh hng

Theo k thut Iu chnh ng hc qu trnh

1.5. c s xy dng Quy trnh cng ngh

Xy dng tng cng ngh...

1.5. c s xy dng Quy trnh cng ngh

Xy dng tng cng ngh... Tuyn chn tc nhn sinh hc cao sn (VSV)

Xy dng phng n nguyn liu, nghin cu ti u ho thnh phn mi trng ln men Xc nh gii php ging v nhn ging

La chn, thit k thit b, NC iu chnh

QTLM cu trin khai SXCN (scale up) Nghin

S phn b u t
Hnh thnh tng cng ngh

Nghin cu quy m phng th nghim Nghin cu xy dng quy trnh PILOT

Kim tra v sinh v an ton sn

phm

Nghin cu th nghim
quy m bn sn xut

Nng lc tc nhn sinh hc

Nghin cu xy dng v hon thin QTCN

- Vi sinh vt men, nm mc, m t bo - Sinh hc phn t thc vt, ng vt, enzym ... - K thut gen Kh nng tng hp - Ho sinh, ng hc phn ng (chuyn i, phn - dinh dng hc gii...) sn phm v nng - Ho l, ho keo, tng lc tch t SP... hp hu c... - Qu trnh cng ngh - M hnh v ti u ho - iu khin t ng - hon thin SP... Qung b v lu hnh sn phm
Qun l v vn hnh quy trnh

Vi khun, x khun, nm

Quy trnh cng ngh ln men

CN

1.6. cc giai chnh ca QTCNLM

chun b ln men
tuyn chn chng chun b mi trng nhn ging v LM

ln men
Gim st, iu chnh tc pht trin iu chnh qu trnh chuyn ho to sn phm

x l dch LM v tinh ch thu sn phm

Tch sinh khi X l thu sn phm th Tinh ch thu sn phm tinh sch

kh khun mi
trng, thit b... Nhn ging ...

X l thu hi (dung
mi...) X l an ton cht thi

+ s tng qut dy chuyn cng ngh

bo qun ging sx p TN nhn ging nh v trung gian nhn ging sn xut

phng k thut
Phn tch cht lng v gim st QTSX

phn xng ln men

Ngun cp hi, in, nc...

Ln men sn xut
x l dch LM

Nguyn liu

Thanh trng, lm ngui

Phn xng

chun b ln men
chun b, pha ch mi trng

Phn xng x l tch, lm sch... dch ln men

Tinh ch ....

x l an ton cht thi

phn xng ph tr

sn phm th

phn xng
sn phm ph

tinh ch thu sn phm

bao gi, hon thin

Thnh phm
sn phm chnh

II.

Ging v nhn ging trong sn xut

2.1. yu cu chung ca chng cng nghip

1. Nng lc tch t cao sn phm mong mun 2. Pht trin nhanh, tch t sn phm nhanh 3. C chng thun khit: n nh v di truyn, ging c th bo qun lu di; c th nhn ging nhanh, m bo cp ging thng xuyn, n nh cho sn xut 4. Khng c vi ngi, ng vt, thc vt v mi trng sinh thi (nu c th c tnh thp, d x l kh c, hoc mt hot tnh nhanh mi trng ngoi) 5. D tch khi dch ln men khi x l thu sn phm 6. C kh nng x l to bin chng hot tnh cao hn

2.2. cc bc chnh chn chng cng nghip

1. Phn lp, tuyn chn t t nhin 2. X l to bin chng siu tng hp sn phm
3. Chn lc li (phenotyp hay genotyp) 4. Nghin cu c tnh chng v xc nh iu kin ln men ti u quy m phng th nghim on nghin (giai
cu ton din nht, bao gm c tnh chng, ng hc s pht trin, s hnh thnh v tch t sn phm; Nghin cu nh hng ca iu kin mi trng ln men, xc nh gii php cng ngh iu chnh nh hng qu trnh ln men...) 5. Th nghim v chn lc li trn quy m PILOT

6. Th nghim v tuyn chn li trn quy m bn sn xut cng nghip(Kim tra kh nng thch ng vi trang
thit b hin c, ngun cung ng nguyn liu, trnh cng ngh, kh nng p ng ca lc lng sn xut...)

S xut s mt vi chng Penicillium cng nghhip hin

nay

2.2. cc bc chnh chn chng cng nghip

* ng thi vi phng php tuyn chn v ci to hot tnh chng trn, k thut to bin chng ti t hp gen c nghin cu v ng dng ngy cng rng ri hn.
+ Tm kim, nghin cu tch dng cc gen qu + Bin np gen tch dng vo chng mang la chn (Escherichia coli, Saccharomyces cerevisiae, Aspergillus niger, A. oryzae...) + Nghin cu iu chnh qu trnh ln men (v x l dch ln men) thu sn phm mi nh bin chng ti t hp

2.2. Cc phng php bo qun ging

* Mc ch Yu cu: CT thun khit, n nh; khng b mt ging, trnh
nhim tp, gim nguy c xut hin t bin ngu nhin...

** Mt s phng php bo qun thng dng + Cy chuyn ln men nh k + bo qun

r tin, d trin khai, vn n nh cht lng, tn nhn cng, d nhim tp, (ph bin hn vi vi khun) ging nhit thp n gin, kinh t, tn nhn cng, vn thoi ho ging ...

+ bo qun ging ch lnh ng

- bo qun thng - bo qun trong du (thch hp bo qu nm si)

+ bo qun ging ch siu lnh - trong glyxerol (-50 - -80oC) Chi ph cao, tn t nhn lc - trong (hay trn) nit lng chi ph cao, c, d trc trc khi khng
N2

+ bo qun ging bng k thut ng kh

u t cao, tn nhn cng, bo qun d dng v di hn, khng thch hp vi nm si khng to bo t

+ bo qun kh (trong t, ct, silicagel, giy... n gin, r tin, tn t nhn

cng

2.2. Cc phng php bo qun ging

+ Hiu qu bo qun
* Khng c gii php ti u cho mi chng; mi ging thng c bo qun song song bng mt s phng php thch ng * Cn c la chn: t l sng st, nhim tp, bin i v di truyn (suy gim hot lc), lng mu, gi tr canh trng cn bo qun, kh nng vn chuyn v cung ng, kh nng hot ho s dng, gi thnh

+ Ni bo qun ging
- Trong cc ngn hng ging vi sinh vt, ti cc vin nghin cu chuyn ngnh, trong cc trng i hc v ngay trong cc phng ging ca cc c s sn xut... - a ch trc tuyn mt vi ngn hng ging ln trn th gii:
American Type Culture Collection, USA www.atcc.org www.wfcc.info

World Federation for Culture Collections, JAPAN

Deutsch Samlung von Microorganismen und Zellkulturen, Deutschland www.dsmz.de

...

2.3. Nhn ging phc v sn xut + Mc ch Yu cu - Cp ging n nh v kp thi cho sn xut - Cp ging v lng,m bo cht lng (k c / hnh thi - nm si), thun khit, ng thi im yu cu ca sn xut + Cy chuyn nhn ging cho sn xut - Hot ho ging v nhn ging phng th nghim - Nhn ging quy m nh - Nhn ging Trung gian

- Nhn ging sn xut

* Mi trng
: khng cn ph hp vi MT ln men SX, nguy c ko di pha thch ng

* Chu k cy chuyn: nguy c nhim tp

- GP ging song song, cp ging trung gian

III.

Mi trng ln men

3.1. Yu cu
+ m bo cung cp cht dinh dng
chng duy tr v pht trin lng sinh khi cn thit

+ Ph hp vi mc tiu cng ngh tp ra sn

phm mong mun

+ m bo cht lng v sinh cng nghip

hn ch v ngn nga nhim tp

+ m bo cc ch tiu kinh t

3.2. Kh khun mi trng

3.2.1. Mc ch yu cu
+ Ngn nga nhim tp, m bo cho qu trnh ln men xy ra vi tc nhn mong mun, m bo nng sut v hiu qu chuyn ho c cht... + Ngn nga suy gim hot tnh sn phm, gim tp cht, hn ch tc ng tiu cc n chng v hiu qu cng ngh p dng

3.2.2. Gii php

+ Thanh trng (s dng nhit cao) + Siu lc + S dng cc tia nng lng (tia cc tm, tia alpha- tia gamma... + S dng tc nhn ho hc + p dng ch ln men c bit (pH, nhit , tc nhn c ch chn lc...)

a/ Thanh trng
* Yu cu
+ Tiu dit vi sinh vt v bo t + Hn ch bin i cht lng: phn ng caramen, phn ng melanoidin, ng thu phn, cc cu t b phn hu bi nhit cao, bin i pH...

* Cc yu t nh hng n hiu qu thanh trng: Lng

vi sinh vt trong mu, kh nng chu nhit ca cc chng, c tnh c l ca mu, h s ri ro nhim tp cho php, iu kin cng ngh kh thi, chi ph vn hnh ...

* Ch thanh trng:

A - B - C CTT = t

- A Thi gian gia nhit mu n nhit thanh trng, pht - B Thi gian gi mu nhit thanh trng, pht - C Thi gian lm ngui mu xung nhit phng, pht - T Nhit thanh trng, oC, oF, (oK)

Thanh trng mi trng + Thanh trng gin on

110 - 121oC, 15-30 pht

+ Thanh trng lin tc

140 - 146oC, 2-3 pht

Gi nhit

Bay hi
Gi nhit Gia nhit

Gia nhit
Gia nhit thanh trng

Lm lnh

Lm lnh

a/ Lc v khun mi trng * C ch hot ng : Chn cn, hp ph, bc cu, ng

khi qun tnh, tng tc nhm, tng tc tnh in...

* Dng cu trc lc
+ Mng siu lc
Axetat xenlulo, Polyetylenphthalat; nilon...; dy 70-150m, mao qun < 10 m (microfiltration 0,02-10 m, ultrafiltration 0,001-0,02 m, hyperultrafiltration <0,001 m); rng 60-85%

+ Lc thng
- Bng thu tinh, bng, si khong, xenlulo, aming... - 0,5-100m, b dy ln - x l khng kh (200C/m3): 0,15m/s, 1C/1000m3, 10m3/pht - 100 h

3.3. Kh khun Thit b v thit b ph tr

3.3.1. Mc ch yu cu
+ m bo cho qu trnh ln men xy ra vi tc nhn mong mun, ngn nga nhim tp, m bo hiu qu nng sut v hiu qu chuyn ho c cht... + Ngn nga suy gim hot tnh sn phm, gp phn lm gim tp cht hay gim thiu ri ro pht sinh thm trong x l thu sn phm + Gii php kh thi, ng b cho c thit b v h thng thit b ph tr v mc pht sinh do u t, vn hnh v bo tr chp nhn c

3.3.2. Gii php

+ Nhit cao, ho cht... + Thanh trng ti ch. (Clean in Place, In Situ Sterilyzation)
CIP: Ra sch bng nc 1. V sinh bng dung dch kim long

Trng li bng nc
2. V sinh bng dung dch axt long Trng li bng nc 3. Thanh trng bng dung dch cht st khun (Trng li bng nc)

IV. ln men sn xut

S nguyn l qu trnh ln men

Tch bt Gia nhit kh khun iu nhit Gim st v iu chnh cc thng s cng ngh: Si, Pi, to, pH, O2, ...

Lc kh thi

Thanh trng

Khng kh

Khng kh sch

Khuy trn

4.1. ng hc qu trnh ln men

4.1.1. Qu trnh ln men gin on
+ Qua bn giai on: thch ng, pht trin lu tin, cn bng, dit vong + Hin tng ng cong sinh trng kp, khi c nhiu ngun dinh
Hm lng sinh khi (BM)

Nng sn phm (TC th cp)

RNA/Unit of BM Mt t bo

dng khc nhau

+ Sn phm TC s cp v SP TC th cp
Nng c cht Thi gian ln men

4.1.2. Qu trnh ln men bn lin tc

+ c im c trng: ko di pha lu tin hay pha cn bng (hoc dit vong) + ng cong sinh trng kp do thay i t ngt iu kin ln men

4.1. ng hc qu trnh ln men

4.1.1. Qu trnh ln men gin on 4.1.2. Qu trnh ln men bn lin tc 4.1.3. Ln men lin tc

vng cn
bng ng Nng sut

vng bt
n nh

vng
ra tri Nng c cht

k pha long =

V vo (ra) V lm vic TB
Mt t bo H s pha long MT

4.1.4. c im sinh thi pellet nm si

+ pellet xp + pellet + pellet rng

mn + Tc ng qua li gia cu trc pellet vi cc thng s cng ngh

4.2. Kim tra gim st cc thng s cng ngh

4.2.1. Nng t bo vi sinh vt

4.2.1.1. o mt quang
A/ o c 90o/ xuyn

- c tnh quang hc ph thuc kch thc, nng cc cu t...

- Sai s ln: t bo cht, cn c hc, bt kh, kch thc thit b * Mt quang OD: I vo / I xuyn , s dng nh sng n sc, o nhiu v tr, o nng thp hay pha long o khi nng cao...

Phn x 180o

Khc x 90o

b/ o ph hp ph/pht x

- NADH+, NADPH+: hp ph 460nm, pht x 340-360cm - nh hng: tnh trng pht trin, cng trao i cht, nng cht tan trong mi trng (Glucoza cao), bt kh
c/ S dng thit b m qua khe hp

4.2. Kim tra gim st cc thng s cng ngh

4.2.1. Nng t bo vi sinh vt

4.2.1.1. o mt quang

4.2.1.2. o nhit lng

- Phng php Twin-Type: s dng thit b i chng - Phng php Different: s dng b o cch nhit cc b V thiu dinh dng O2,.... - Phng php Head-Flux: o gin tip qua nng lng tiu hao ca thit b n nhit chung ca h thng

4.2.1.3. Phng php xc nh hm lng sinh khi (lc v cn) 4.2.1.4. Phng php o nht dch ln men (s dng thit b mao qun) 4.2.1.4. Phng php o in tr dn v tr khng (s dng thit b mao qun) 4.2.1.4. Phng php o in th (s dng 2 in cc hydro, 1 c mng cch ly) 4.2.1.4. Phng php khc (to phc vi protein, qua c cht chuyn ho hay sn phm to
thnh, ph cng hng t ht nhn...)

4.2. Kim tra gim st cc thng s cng ngh

4.2.2. nhit
- Nhit k thu ngn: giao tip thun li, chnh xc, kh o xa, d v..... - Nhit k in tr - Cc dng khc ....
(Pt100: 100 Ohm 0oC - 123,4 Ohm 60oC, 0,5oC - 20-130oC)

4.2.3. pH mi trng
- in cc kp Ag-Ag
2,3 R T Un = F

U = Uo + log (H+)
chun b li

- in tr bin i theo thi gian, p khi thanh trng

4.2.4. nng xy ho tan

- Anode: Zn; Pb - Catode: Au; Ag - Cn bng li sau khi thanh trng
4.2.5.

lu lng vo - ra

(chnh p qua thit b, l lng k phao, in t trng...)

4.2.6. Cc thng s khc (p sut, khi lng lm vic trong thit b, nht dch, in
nng tiu th, kin sot to bt...)

4.3. iu chnh qu trnh ln men

4.2.1. Cng

c iu chnh: Bm, qut, van, TB in... l C

4.2.2. Nguyn

+ Dng iu chnh:
C theo quy lut, C trc tuyn, C theo dng phi hp

+ Kiu tng tc:

4.4. Thit b ln men cng nghip

4.4.1. Characteristics + Volume: depends on the process and it operation. + Operating mode: batch, semi-continuous, continuous operation + Typical types: aerated and stirred fermentor from stainless steel, large cylinder form closed at the top and bottom and with external cooling jacket; equiped with sparger, impeller and fitted with various pipers and valves

4.4. Thit b ln men cng nghip

4.4.2. Dng thi

aerated and sterred tank multi impeller non aeration

aerated and sterred tank

agitation

4.4. Thit b ln men cng nghip

4.4.3. Technological characteristics of the fermentor

- Main part of the complex system, where the strain growth and product formation occur. - Made from rolled steels with rough surface, spot of electric soldering, connection plugs etc. difficult of sterilization - Unequal conditions at any volume locations, because of rheological properties of the broth (appearing primary flow, secondary flow,uneffective working zones etc during agitation)

4.5. Nhim tp trong sn xut cng nghip

4.5.1. Khi nim v ln men thun khit v nhim tp 4.5.2. Nguyn nhn nhim tp 4.5.3. c im ca nhim tp 4.5.4. nh hng ca nhim tp 5.5.5. Kh nng pht hin nhim tp

4.5.6. Cc gii php hn ch v ngn nga nhim tp

5. Product recovery Dowstream processing

5. X l dch ln men thu sn phm

5.1. i cng 5.2. Phn ly tch cc phn t rn (hoc sinh khi) 5.3. Cc gii php x thu sn phm ni bo

5.4. Cc gii php x l lm giu cu t tch

5.5. Tch v tinh ch thu sn phm tinh sch

5.1. General features:

+ Nature of product: - intracellular, extracellulare or membrane-adherent product - low concentration and relatively inactivation

+ Requirement of recovery operation: bioactive product with desired or acceptably clean level; cheap, simple and reliable operation, acceptable yield + Remarkable: any treatment step: gainable
expectation, but increase total cost and reduce efficiency

5.1. General features:

5.2. Separation of solid particles (or biomass)

+ Flotation + Flocculation + Sedimentation + Filtration

+ Centrifugation
+ + Aims: a well clarified supernatant, solid with maximum dryness and contained operation Preferable: filtration for mycelial broths; but centrifugation for cell debris, bacterial and yeast broths

Feed Effluent

Centrifugation

+ Filtration
- Double step depth filtration (clarified and sterile filtration) - Crossflow filtration (microfiltration - 0.02-10 m), ultrafiltration - 0.001-0.02m), hyperultrafiltration (less than 0.001m - osmosis effect)

Suspension out Sterile effluent in

Sterile filtration

clarified effluent Suspension

clarified filtration

Washing port

Effluent Ultrafilter membrane

Concentrate

Hyperultrafiltration

Clarify filtration

5.3. Treatment for release of intracellulare products

Degradation of cell membrane (or changing permeability): enzyme treatment, cell wall autolyse etc.)

- Destroying cell membrane: ultrasonic treatment, High presure homogenerator, glass bed mill etc.

5.4. Enrichment of desired compounds

Evaporation (vacuum evap.)

Extraction (simple or using reverse centrifugal extractor)

Precipitation (fractional precipitation)

- Membrane processes: ultrafiltration, reverse osmosic, etc.

Structure of centrifugal extractor

5.5. Separation and purification of desired products Adsoption and dis-adsoption (fractional precipitation)

- Chromatographies (gel chromatography, ion exchange chromatography, affinity chromatography etc.) Fractional precipitation Crossflow filtration (ultra- and hyperultrafiltration)

Disolve and re-pricipitation (fractional precipitation)

Precipitation, filtration and dry-rise Freeze drying (lyophylization) etc.

* These methodes can be selected and applied sequently in range, dependent from nature of separative components

6. c im ring bit trong mt vi trng hp ln men

6.1. Ln men s dng cc bin chng ti t hp gen + Sn phm: Vaxin ti t hp, dc phm ti t hp (insullin, interferon); enzym, protein, cc cu t phc v chun on + u th: r, t c, tnh c hiu cao, nng sut cao, kh nng ti sn xut cao, gim chi ph kim tra th nghim an ton... + c im: l sn phm TC s cp (SD cm ng promoter; LM gin on, bn lin tc) + Ch : Thoi ho chng (phn ly Plasmid trong phn bo..., vn s dng khng sinh cm ng Plasmid...)

6. c im ring bit trong mt vi trng hp ln men

6.2. Ln men vi canh trng t bo ng vt + c im: t bo d v, i hi cao v iu kin mi trng nui, kh nng phn chia gii hn... + Nguyn l to dng t bo v hn (Cell line): Dng enzym (Trypxin) x l tch thu t bo, x l thu t bo tinh sch, x l lai to dng t bo v hn, chn lc li thu dng hot tnh, bo qun dng TB. 6.2. LM vi t bo thc vt:

+ Nui cy m:
+ Nui cy t bo trn: TV m sn t bo ln men x l...

6. c im ring bit trong mt vi trng hp ln men

6.1. Ln men s dng cc bin chng ti t hp gen 6.1. Ln men vi canh trng t bo ng vt

c s tm kim pht hin sn phm mi

bn tng hp mi trn c s sn phm bit

tng hp ho hc t sn phm c hot tnh sinh hc bit phi hp tng hp ho hc - sinh hc

tm kim, khai thc sn xut sn phm mi phn lp, tuyn chn chng mi t t nhin

iu chnh QT TC ca chng sinh tng hp sn phm mi

p dng cng ngh tin tin khai thc c chng qu hot lc thp

thit k sn phm mi
s dng chng ti t hp gen