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Cng ngh ln men

CDGD: Bi Hng Qun Bin son: Nguyn Minh Hin

Ti liu tham kho Cng ngh vi sinh vt tp 2, 3. PGS-TS Nguyn c Lng Cng ngh vi sinh ng dng, PGS-TS Trn Minh Tm Cng ngh ln men ng dng trong CNTP, Bi i Cng ngh sn xut malt v bia, Hong nh Ha Cng ngh sn xut v kim tra cn etylic, PGS TS Nguyn nh Thng, Nguyn Th Thanh Hng Enzyme vi sinh vt, PGS TS L Ngc T Food microbiology, William C.Frazier Applications of biotechnology to traditional fermented foods (http://www.nap.edu/catalog/1993.html) ..

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Ti liu tham kho (tt) Bamforth C.W. Food, Fermentation and Micro-organisms, Blackwell Publishing, USA, 2005. Hutkins R.W. Microbiology and Technology of Fermented Foods, Blackwell Publishing, USA, 2006. Elmer H. Marth, Applied dairy microbiology, Second edition Springer, Wine microbiology practical and procedures, 2007 Springer, Modern techniques in the microbial ecology of fermented foods, 2008 Elsevier, Handbook of culture media for food microbiology, 2003 The microbiology of safe food, Blackwell Publishing, USA, 2000 Practical food microbiology, 3rd edition, Blackwell Publishing, USA, 2003

NH GI KT QU HC TP CA SV
1. IM QU TRNH (30%)
1.1. KIM TRA 15 PHT (15%) 1.2. BO CO SEMINAR (15%): chn 1 trong 3 phng n sau

1.2.1. Chn 1 bi bo ting Anh, dch sch lin quan ti mn hc c hiu v thuyt trnh power point (4sv/nhm) 1.2.2. Chn 1 bi bo ting Vit lin quan ti mn hc c hiu v thuyt trnh power point (2sv/nhm) 1.2.3. Chn 1 ti c thc hin lin quan ti mn hc c hiu v thuyt trnh power point (4sv/nhm).

2. IM THI KT THC MN HC (70%)

Thi vit t lun

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NI DUNG CHI TIT HC PHN 2 Phn 1. M u www.gbd.edu.vn Phn 2. K thut ln men Phn 3: Cng ngh ln men ng dng
3.1. Ln men ethanol v cc ng dng 3.2. Cng ngh sn xut acid hu c thc phm 3.3. Cng ngh sn xut acid amin 3.4. Cng ngh sn xut sinh khi vi sinh vt 3.5. Cng ngh sn xut polysaccharide t VSV 3.6. Cng ngh enzyme 3.7. Cng ngh sx cc sp ln men truyn thng

PHN 1. M U 1.1. CNG NGH LN MEN 1.1.1. Khi nim v ln men (fermentation)

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1.1.2. Khi nim v CNLM (fermentation technology) 1.2. PHN LOI CC SN PHM LN MEN

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1.1.1. Khi nim v ln men (fermentation)

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(From latin fervere)

1.1.1. Khi nim v ln men (tt)

Quan im ca nh ha sinh hc: ln men l qu trnh sn sinh nng lng. Cc hp cht hu c hot ng vi vai tr va l cht cho, va l cht nhn in t. Ln men l qu trnh ym kh, nng lng c sn xut khng cn c oxy hoc cc cht nhn in t v c khc. Quan im ca Pasteur: 1857, ng cng b qu trnh ln men khng phi cng trnh ca s cht nh nhng nh ha hc ngh m l cng trnh ca s sng. ng a ra khi nim tnh k kh v i kh ca VSV v s ln men l h qu ca cuc sng khng c khng kh Theo ngha m rng: ln men l qu trnh nui cy VSV (c oxy hoc khng c oxy) thu nhn sinh khi, cc sn phm trao i cht, thc hin s chuyn ha c cht.

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1.1.1. Khi nim v ln men (tt) First Fermentation concept, or Pasteur concept in 1857, Fermentation is the transformation process of the sugar to alcohol in presence of "la vie sans l'air" (means life without air).

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Louis Pasteur (27.12.1822 28.9.1895) the father of the Microbiology

1.1.1. Khi nim v ln men (tt) Conclusions of Pasteur from its study of wines: The alcoholic fermentation of grape juice occur only in presence of yeasts. The wine acidification occur in presence of bacteria. When the grape juice is heated the fermentation do not take place. When the wine (the sugar) is heated the acidification do not occur.

Fermentation is the change of the substrate (sugars) by the action of the ferments

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1.1.2. Khi nim v CNLM (fermentation technology)

The fermentation technology is the combined application of the knowledge of process engineering, biochemistry and microbiology for designing or evaluation a fermentation process.
Sn phm
Acid foods (citric, lactic) Alcohol Amino acids Antibiotics Enzymes Pharmaceutical proteins Sn lng (tn/nm) 100,000 1,000,000 10 100,000 10 30,000 0.1 3,000 0.001 - 3

Useful Applications

Bioremediation Environmental monitoring Pollution control

ENVIRONMENT

Yield FARMING Feed stocks Animal health

PHARMACY FOOD

Diagnostics Vaccines Therapeutics

Bioprocess Bioinformatics

Biosensors

Process analysis

TOOLS / TECHNICAL ADVANCES Experimental design Protein engineering

Biochemistry Microbiology

Process engineering Immunology Informatics

SCIENTIFIC KNOWLEDGE
Physiology Statistic

Molecular Biology

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What I should do to do fermentation?

Media preparation Physiology Fermenter preparation Sterilization Adjusting parameters Inoculation Cultivation Taking samples Harvest conservation Kinetics Agitation/Aeration Fermenter types Operation modes Scale-up Medium design Optimization

1.2. PHN LOI CC SN PHM LN MEN T VSV

SINH KHI VSV (biomass): protein n bo (SPC), men bnh m, ging khi ng (starter) CC SP TRAO I CHT SP TC BC 1: acid amin, vitamin, acid citric SP TC BC 2: enzyme VSV, khng sinh

C cht

Development the producer strain

T bo (biomass) SP ln men: ru, acid lactic (ln men k kh)

C cht

Sn phm + T bo (SP TC)

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Phn loi sn phm ln men theo quan im kinh t Bio-products

High value Low Medium value High Low value High product volume product volume (The product volume (Mostly depends on process is relatively (Most of the production
the substrate price)
Antibiotics

expensive and complexity)

Amino acids

some correspond to purification process)

Ethanol

Vitamins Enzymes Vaccines Steroids Hormones Other pharmaceutics

Organic acids Biopolymers Baker yeast Microbial polysaccharides

Biomass Methane Acetone Butane Fructose syrups Feeding products

Cost distribution
High volume and Low value product High investment cost (ph u t cao) High raw material cost (ph nguyn liu cao) High conversion efficiency (hiu qu chuyn i thp) Low recovery cost (Ph quay vng thp) Low margin profit (li nhun thp) Only few regulatory problems (phi theo quy nh php lut) Required not much R&D expenditures (khng tn nhiu tin cho RD Low qualification of the labor force is acceptable (Ngi lao ng c th ko cn trnh chuyn mn cao) Low volume and High value product High investment cost Cost are distributed in all production stages High recovery of the product in the fermentation stage High purification cost High margin profit Too much regulatory and legyslation restrictions, and high plant hygiene Required too much R&D Cost Very high qualification of the labor force is nedeed

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PHN 2. K THUT LN MEN

2.1. VSV TRONG CNG NGH LN MEN TP 2.2. NG HC QU TRNH LN MEN 2.3. PHNG PHP & THIT B LN MEN 2.4. CI TiN QU TRNH LN MEN

2.1. VSV TRONG CNG NGH LN MEN TP

2.1.1. CC YU CU V GING VSV 2.1.2. K THUT TO GING 2.1.3. K THUT NHN GING V SX GING 2.1.4. K THUT KIM TRA GING VSV 2.1.5. K THUT NNG CAO CHT LNG GING 2.1.6. K THUT BO QUN GING

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VSV TRONG CNG NGH LN MEN TP TP ln men vi s tham gia ca VSV trong t nhin (TP ln men truyn thng) TP ln men vi s tham gia ca VSV thun khit (TP ln men cng nghip)

Sx th cng, quy m nh, Sx quy m ln, nng sut cao nng sut khng cao Ch ng cy 1 lng VSV Khng kim sot c vo nguyn liu qu trnh, cht lng cha Kim sot c qu trnh ln n nh v cha ng u. men, cht lng n nh & Mang bn sc m thc, ng u kinh nghim, vn ha ca mi dn tc

2.1.1. Yu cu ging VSV trong CNLM Phi c tc sinh trng v pht trin mnh, thun Phi to ra sn phm c nng sut sinh tng hp cao, cht lng tt Phi c tnh thch nghi nhanh trong iu kin sx CN Phi c kh nng chng chu li VSV tp nhim Phi c kch thc ln, thun tin cho qu trnh lng, lc, tinh ch sau ny. Sn phm sinh khi d tch ra khi mi trng nui cy Chng VSV c bo qun d dng, tn ti cc c tnh trong sut thi gian s dng C kh nng thay i cc c tnh bng k thut di truyn ci thin, nng cao nng sut Khng hoc t to thnh sn phm khng mong mun

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2.1.2. K thut to ging Phn lp trong t nhin

PP

PL trong k sx cng nghip

u im

To ging VSV mi (p dng k thut di truyn)

Nhc

Nguyn tc

PL trong c c cht, c VSV ngun VSV phong Tn thi gian, t nhin phn gii c cht ph a dng hot lc VSV cn thp PL trong Tnh thch nghi cao Hiu qu cao, VSV / sx CN quen vi iu kin sx cng nghip To Tip hp, ti t hp To c ging Yu cu trnh VSV = VSV c c tnh cao, thit KTDT mong mun b hin i

Cc trung tm lu tr ging trn th gii v Vit Nam ABBOTT: Abbott Lab, North Chicago, III.60064, USA ATCC: America Type Culture Collector, 12301, Parklaw Drive Rockvill Md20852, USA HIR: Food and Fermentation Divisio, Hokkatdo Profectural Industrial Research Institute Saporo, Japan FERM: Fermentation Research Institute, Agency of Industrial Science and Technology Ministry of Industrial Trade and Industry, Chiba, Japan VTCC, IMBT, National University, HaNoi, VietNam vtcc@vnu.edu.vn; www.biotechvnu.edu.vn (Ngn hng ging quc gia, HN) Bo tng ging: www.bacteriummuseum.org

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2.1.3. K thut nhn ging Nhn ging trong PTN Nhn ging trong quy m sx ln

PPnhn ging khi ng truyn thng v hin i

Cc yu t nh hng n qu trnh nhn ging Thnh phn cc cht trong MT nhn ging (khng cha cht khng sinh, cht c ch). Penicillin, chloramphenycol c ch sinh trng ca VK lactic. Nng cc cht trong MT nhn ging. Nng ng hoc mui cao tng p thm thu c ch VSV pH: chn pH ca MT nhn ging = pHop ca VSV. pH nh hng trc tip ln b mt t bo tch in khc nhau lm cho hot cc loi enzyme VSV thay i. pH nh hng n s phn ly ca cc cht dinh dng c trong MT Nhit : chn To nhn ging = Toop ca VSV

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2.1. 4. K thut kim tra cht lng ca ging VSV

Quan st i th Quan st vi th Kim tra hot lc ging VSV (thoi ha)

Ging VSV b tp nhim, thoi ha phi phn lp li hoc thay ging khc
Kim tra thun ca ging thng xuyn (b nhim t ng gc) Kh trng MT dinh dng; vi cc MT c bo t cn kh trng trit hn. V d dit bo t Bac. subtilis 1800C/60 - 90 (B nhim trong qt nhn ging) Ging VSV b thoi ha: do tc ng ca mi trng bn trong v tc ng ca nhng sn phm TC do chnh VSV tit ra

2.1.5. K thut nng cao cht lng ging

2.1.5.1. Hun luyn thch nghi ging vi sinh vt Mi VSV u c kh nng thch nghi rt cao vi mi trng. Nhng tc ng mi trng c lp i lp li nhiu ln to nn tnh thch nghi bn vng. Khi to c tnh thch nghi ca VSV phi lun duy tr tc ng mc to ra tnh thch nghi. c im ny khng bn. Yu cu: ngi thc hin phi c tnh kin tr.

Khun lc nm men trn Hansen Agar c hm lng ng 26, 28% (phng php hun luyn ging)

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2.1.5.2. t bin VSV

t bin bng tc nhn vt l ( tia U.V): Tia U.V c s dng rng ri trong cng ngh chn ging VSV. Tia U.V c kh nng to ra cc t bin c cht lng cao hn chng loi ban u hng trm ln. Tia U.V =260nm thng gy ra nhng t bin im cao nht. y l loi bc x khng ion ha v gy ra nhng bin i base cha nit trong gen. t bin bng tc nhn ha hc (khng khng sinh): Dng mi trng c khng sinh tm cc dng t bin b n vng (khng khng sinh). Trn mi trng ny, cc t bo m n cm vi khng sinh s b git cht, ch cn cc t bo t bi n i khng l tn ti

S lng khun lc/ nng khng sinh trong MT nui cy 10-7 (khng b 0,01mg/ml 0,1mg/ml 0,5mg/ml sung khng sinh) Nhm 1 2 3 4 34 58 107 Nhim 8 5 7 Nhim 2 3 5 5 Khng xut hin khun lc

TN tin hnh vi vk E.coli. Nui cy E.coli nng pha long 10 -1 trong cc mi trng b sung khng sinh

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2.1.5.3. Lai ging nm men

Nguyn tc: T 2 ging nm men ging nhau ta c th to ra c ging nm men mi c nhng c tnh ca c 2 loi ban u bng cch cho chng tip xc vi nhau trong iu kin th nghim. Nhc im: T l thnh cng khng cao. S pha trn c tnh di truyn ch trong mt lai nht nh. Cc ging VSV khc nhau th khng th tin hnh qu trnh lai ging c. u im: n gin, d thc hin

Cch thc hin lai ging nm men 1. Cy 2 ging men vo mi trng y M1(mi trng lng). 24h. 2. Cy ging hn hp vo mi trng thch ti thiu M2 v M1 (thch bng). t nht 5 ngy 3. Hai ging nm men lai vi nhau nu trn M2 xut hin khun lc 4. Kim tra ging thu nhn c phi l lng bi th bng cch: Cy khun lc trn mi trng M2 vo mi trng Thch acetat (M3). 48h 5. Lm tiu bn, quan st di knh hin vi, nu thy nang cha 4 bo t (t bo lng bi th) chng t lai thnh cng. 6. Kim tra tnh trng cn quan tm.

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2.1.5.4 S dng k thut di truyn hin i

K thut bin i gen to ra cc sinh vt c tnh trng ch theo mun

Sng bn gen

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2.1.6. K thut bo qun ging VSV

Mc ch: m bo c tnh cht ca ging (duy tr gn nh nguyn vn c tnh ban u ca ging VSV trc lc ct gi) tiu chun cho qu trnh sn xut. Nguyn tc: lm chm qu trnh h hp v trao i cht VSV, ng thi ngn cn s sinh sn ca chng. Phng php: Bc 1: Tin bo qun (thun ha ging). Chn chng VSV iu kin v giai on ti u cho bo qun. Bc 2: Chn phng php thch hp cho bo qun.

2.1.6.1 Bo qun ging trong thch nghing (cy truyn nh k) Nguyn tc: lun i mi t bo, khng gy ra bt thng. Bin php: mi trng ti thiu, nu mi trng giu dinh dng, VSV pht trin nhanh s thoi ha nhanh. u: n gin, d thc hin, t tn km, thch hp quy m nh Nhc im: tn thi gian, thi gian BQ ngn (1- 2 thng) V tnh hun luyn VSV sng iu kin lnh, lm bin i ging VSV ban u. 2.1.6.2. Bo qun ging trong lp du khong Nguyn tc: c ch qu trnh h hp VSV trong c VSV y m kh v hiu kh, hn ch tip xc vi oxy, ngn hin tng mt nc ca mi trng v VSV. Bin php: 1 lp du khong hoc parafin lng u im: n gin, hiu qu cao, thi gian BQ di (1 nm) Nhc im: C ln du

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2.1.6.3. Bo qun ging trong ct, t sy kh

Nguyn tc: S dng t, ct nh nhng gi th mang. Khi m mi trng gim ti thiu, VSV khng pht trin na. (t v ct l mi trng ti thiu)
Cch thc hin: X l t, ct (ry u, ngm trong HCl hoc H2SO4 m c 8-12h. Ra di vi nc cho n pH trung tnh, sy t, ct > 1000C. BQ iu kin v trng.

VSV c nui mi trng thch. ct, t v trng vo ng nghim, lc u, sau rt qua ng nghim khc. Hn kn ming ng u im: thch hp BQ cc ging VSV trong x l mi trng, trong nng nghip (phn bn) khng i hi mc tinh khit cao. S dng bo qun ging VSV to bo t. Thi gian bo qun di (2 nm) Nhc im: khng dng trong sn xut cng ngh thc phm

2.1.6.4 Bo qun ging trong cc ht ng cc

Nguyn tc: s dng ht ng cc nh gi th mang Thng s dng BQ cc nm si v VSV trong thc phm Mc ch: gi VSV trng thi tim sinh. Cch thc hin: Ht ng cc ri, hp chn, nui nm mc trc tip (3 5 ngy), sy t0 < 500C t m W <15%. Nhit bo qun: 15 200C Thi gian bo qun: 2 nm (chu u), 1 nm (Vit nam)

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2.1.6.5 Bo qun ging trong giy lc Nguyn tc: p dng vi VSV c bo t. Ngoi giy lc, c th s dng B.C (bacterium cellulose) Cch thc hin: 1. Chun b giy lc v trng:Ct giy lc 1 3 cm. Cho giy lc ct vo ng nghim, y nt bng, sy 1600 C/ 2h hoc kh trng 1210 C/30. 2. Nui VSV trong mi trng lng n khi to thnh bo t 3. Dng pi pet v trng ht 1 git Vi khun vo giy lc. Sy 400 C dn khi thy ming giy lc kh th chuyn giy lc vo ng nghim. Thi gian BQ: 5 nm

2.1.6.6 Bo qun ging trong gelantine

Cch thc hin: 1. Chun b mi trng: Mi trng N.B b sung 10% gelantin v 5% acid ascorbic. Kh trng 1210C/ 15 2. Chun b ging VSV: nui ging VSV 3. Trn VSV vi mi trng. 4. Dng ng nh git v trng to thnh tng git gelantine nh. Sy kh trong t ht chn khng

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2.1.6.7 Bo qun ging bng phng php lnh ng Nguyn tc: S pht trin ca VSV s b c ch nhit lnh su. Cn s dng cht bo v VSV (glycerin 15%, saccharose 10% + gelantin 10%... Gip VSV khng b cht nhit lnh su Thi gian BQ: -300 C: 9 thng - 400 C: 1 nm - 700 C: 10 nm

2.1.6.8 BQ ging bng pp ng kh (c s dng trong cc ngn hng ging, c quan nghin cu ln)
Nguyn tc: sy nhit thp trong iu kin chn khng, khng nh hng n cht lng ging v c th to ra cc ng ging theo quy m cng nghip. Cch thc hin: ging, nhn ging trong mi trng lng, ki m tra ging (c im sinh ha, sinh l), nu t tiu chun, my ng kh 24h, hn np li. Thi gian bo qun: 20 nm u im: cht lng ging khng i, bo qun nhit thng, thi gian bo qun di Nhc im: chi ph ln

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2.2. NG HC QU TRNH LN MEN (the kinetics of the fermentation processes) 2.2.1. PHNG TRNH MONOD 2.2.2. CC YU T NH HNG N HIU SUT LM

Mc ch nghin cu ng hc Thit k 1 qu trnh ln men mi nh gi hiu qu ca qt ln men ang thc hin Ci tin qu trnh (cht lng, nng sut, gim chi ph) Nng cp hoc gim quy m sx

How to describe a fermentation process? biomass Conversion process of nutrients to products of the microbial metabolism
nutrients cells CO2 H2O Products
CHmOl + aNH3 + bO2 cCHpOnNq + dCHrOsNt + eCO2 + fH2O (1) Substrate Metabolic by
(source of N& O2) (biomass) Rest of the nutrients products or Interest product

Yield is an indicator of the efficiency of the process. From chemical engineering: the Yield is stoichiometry of the reaction.

YX/S: biomass-substrate yield = c Mr(Biomass) / Mr(Substrate) YP/S: product-substrate yield, is calculated through d YP/X: product-biomass yield, is calculated through d/c

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Methods for monitoring fermentation kinetics

Direct estimation of cell number:

Direct methods

Direct estimation of cell mass: Cell dry or wet weight Optical density
Other Nitrogen + required source nutrients

Microscope count Viable plate count Light scattering

Carbon & + Energy source

O 2+

Cells
Measure cell components: Protein DNA and RNA Carbohydrates & Lipids Enzymes

+ Products + CO2 + Heat + H2O Monitor CO2 production: IR analysis Mass spectrometer

Monitor the consumption of the carbon source: Chemical analysis Chromatography

Monitor the consumption of the nitrogen source: Chemical analysis NH3 electrode pH-stat Nitrate electrode

Energy balance Heat evolution

Monitor product formation: Chemical analyses Mass spectrometer pH and viscosity Chromatography (gas and HPLC) Enzyme electrode

Indirect methods

Determine O2 consumption: Paramagnetic O2 analyzer Mass spectrometer Dissolved O2

Monitor the consumption of essential nutrients: Chemical analysis Ion selective electrodes

X: VSV (tb/ml) t: thi gian

P: sn phm (g/L)
=
1 dX X dt

ng cong sinh trng

I. Adaptation (thch nghi) II. Exponential (bng n) III. Stationary (n nh) IV. Accelerated death (suy tn)
Pha thch nghi = 0 Pha bng n Pha n nh = max = 0, do dX / dt = 0

S: c cht (g/L) nh hng ca loi c cht ti Loi c cht Glucose Maltose Maltotriose max (h-1) 0,28 0,22 0,18

: tc to sinh khi ring (Specific growth rate )

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2.2.1.Phng trnh Monod : tc to sinh khi ring (s gia

= max
= max

S Ks + S
S S2 Ks + S + KI

VSV E. coli

tng sinh khi trong 1 n v thi gian (ngy, gi) t 1 n v sinh khi X). = dX/XdT (1/ngy; 1/gi) max: hng s tc sinh trng max [S]: nng c cht
C cht Ks (mg/L) 25 4 20 5 4,5 0,042 0,45 0,7 0,4 120 3

Saccharomyces sp. Glucose Glucose Lactose Aspergillus sp. Candida sp. Pseudomonas sp. Hansenula sp. Glucose Glycerol Oxygen Methanol Methane Methanol Ribose

KS: hng s Monod, l nng c cht khi tc to thnh sinh khi = max/2. KS c x bng thc nghim. KS = f(chng VSV v c cht) .

2.2.1.Phng trnh Monod (tt) Phng trnh Monod: mi lin h gia nng c cht v tc sinh trng ca VSV ngha thc tin ca pt Monod: Tnh c, d on c tc to sinh khi ring () nng c cht (S) nht nh. Hn ch: - Pt Monod khng ng khi [S] qu cao. Khi [S] qu cao th gim do [S] qu cao: to p sut thm thu, c ch VSV. - Pt Monod ch ng trong mt khong gii hn ca [S]

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2.2.2. Cc yu t nh hng n hiu sut ln men 2.2.2.1. nh hng ca ging VSV 2.2.2.2. nh hng ca nng c cht 2.2.2.3. nh hng ca nhit Khi nhit tng, hng s tc phn ng tng, tc phn ng tng. Khi gim nhit , tc phn ng gim, nhng c tnh thun nghch. Nu ta tng nhit tr li vng ti u th hot ng ca enzyme v tc phn ng s tng tr li 2.2.2.4. nh hng ca pH enzyme VSV ch hot ng tt pH nht nh. pH ti u ca mi VSV l khc nhau 2.2.2.5. nh hng ca mt s yu t khc: cht c ch

2.2.2. Cc yu t nh hng n hiu sut ln men (tt)

IN

Internal
Cells

OUT

T pH Medium Agit. Areac. Modo Oper.

External

Cell. Conc. Prod. Conc. Metabolites

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2.3 PHNG PHP V THIT B LN MEN 2.3.1. Chun b mi trng ln men

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2.3.2. Tit trng trong cng nghip ln men 2.3.3. Cc phng php ln men 2.3.4. Cc thit b ln men

2.3.1. Chun b mi trng Nguyn tc thit lp mi trng: - cht v lng -Da trn nhu cu dinh dng ca VSV v: Cc nguyn t c bn (Macroelements: 90-95% dry mass): C, H, N, H, O (Conc: > 10-4 mol/L) Cc nguyn t khong (Microelements): Ca, Mg, Fe, Zn, Mn, Fe, Cu, B, Cr, Mo Yu t sinh trng (Growth factors): vitamin B, Amino acids, hp cht khc (acid bo, acid nucleic) Mi trng lng: cht kh, pH Mi trng rn: m, cht n (tru, rm)
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Cc bc chun b mi trng

2.3.2. Tit trng trong cng nghip ln men

Tit trng khng kh: phng php vi lc, Tit trng/ thanh trng mi trng: -Phng php vt l: nhit , nhit + p sut, vi lc -Phng php ha hc: iu chnh pH, b sung SO2 -Tit trng thit b, h thng ng ng, nh xng: U.V, xng formol, ha cht ty ra

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2.3.3. PHNG PHP LN MEN Phn loi phng php ln men: Theo s pht trin ca VSV trong mi trng: Ln men chm: LM trong cc fermentor vi mi trng lng Ln men b mt: LM trong cc khay vi mi trng lng hay mi trng c c cht rn hay xp Theo nguyn l hot ng ca thit b (Operation mode) Ln men tnh (ln men theo m) (batch culture) Ln men tnh c b sung c cht (fed batch culture) Ln men lin tc (continuous culture) iu kin ln men Ln men hiu kh: thng kh bng h thng trc khuy c sc kh (sx biomass) Ln men k kh (sn xut ru, bia)

Ln men tnh (Ln men theo m- Batch culture)

Culture Media Preparation Air Sterilization Inoculation Fermentation

Ln men tnh c coi l 1 h khp kn do khng Air c tc ng ca cc yu t bn ngoi (tr kh) Harvesting

Cell growth can be expressed as: Dry cell weight (g dry biomass /L culture) Wet cell weight (g wet biomass /L culture) Optical density (O.D) Cell counts on Petri dishes Other parameters used for the characterization of batch cultures Number of generations or duplications Duplication time Yields: Product/Substrate (YP/S); Biomass / substrate (YX/S) Productivity: Volumetric productivity (P); Specific productivity (qP)

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LnX

time
Nng sut =

Pha Lag: di t vi pht ti vi gi. Tlag = f (k mi trng: pH, To, MT nui cy v VSV). Tlag khng to ra sp, nh hng ti tng nng sut ca qt LM T lag

Lng SP mong mun (g/L) Tng thi gian ln men (h)

Clean & decontaminate Load Sterilize

How to minimize the Lag phase ? 1. Correct ratio

(1/5 - 1/10) 2. Inoculum culture medium
Inoculum volume Fermentor volume
=

Culture

Unload

Adaptation Phase Exponential Phase Stationary Phase

Fermentor culture medium the

3. The inoculum should be in Batch culture

exponential phase at moment of inoculation

Mass Balance in batch culture Supposition: Perfect mixing = all parameters in the fermentor are similar at a time in any point For biomass: Biomass in + Biomass grow = Biomass out + Cumulative Biomass In a Batch fermentor: grow = cumulative For substrate: Substrate in = Substrate out + Metabolized substrate + Cumulative substrate In Batch fermentor: 0 = metabolized + cumulative

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Ln men theo m- Batch culture

u im Nhc im

n gin, hn ch c s ly nhim. Thch hp bt u nghin cu 1 sn phm. Cho php chn cc thng s v nghin cu c s tng tc ca chng. X c MT dinh dng, , tc ti u to thnh sp

Nng sut thp. Nng c cht cao c ch VSV. Khng kim sot c tc sinh trng ca VSV. Tnh ng u ca SP khng cao so vi cc PP khc. Chi ph ln (h thng tit trng bng hi nc, nc v sinh thit b, in, din tch, sc lao ng

Khc phc nhc im ca Batch culture bng Fed- Batch Culture v Continuous Culture

Ln men tnh c b sung c cht (Fed-batch culture)

What is it?
Starts with a batch culture which, after a specific time, is fed with fresh medium. The addition of fresh medium can be synchronized with the depletion of the growth-limiting substrate from the starting culture medium.
1 Air

Preparation of the starting culture medium Preparation of the feed culture medium

Fermentor sterilization

Fermentor inoculation

Feed sterilization

Feed addition

Fermentation

Air Harvesting

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Ln men tnh c b sung c cht - Fed-batch culture u im (Advantages) Nhc im (Disadvantages)

Thi gian ca cc k sinh trng ti u c ko di do cc cht c ch b pha long v cc cht dinh dng chnh c chuyn ha nh nhau mi thi im LM Trang thit b t tin, yu cu k thut cao hn pp LM tnh Tch ly cc cht c ch s sinh trng to thnh trong qu trnh TC Accumulation of growthinhibiting metabolites Nu s t bo/th tch mi trng qu ln th c kh nng Possibility of induction of contact inhibition if the number of cells per culture volume is too large

Tng nng sut ca qu trnh do gim 1 s cng on (ra thit b, tit trng, loading v unloading). Sn phm c tnh ng nht cao

Ln men lin tc - Continuous culture

What is it? Starts with a batch culture to which, after a specific period, fresh culture medium is added, and cells plus exhaust medium are removed at a rate that keeps culture conditions constant. When the interval between successive additions and extractions decreases, the culture is named continuous and its volume remains constant. Fresh culture
medium

Preparation of starting culture medium Feed preparation

Fermentor sterilization

Fermentor inoculation

Air

1 Fermentation

Feed sterilization

2 Feed addition

Harvesting

Spent culture medium, cells, Air

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Ln men lin tc - Continuous culture u im Nhc im Dch ln men lun c b sung Nguy c vy nhim cao nn cc cht c ch b pha long Kh nng xut hin t Tng nng sut ca qu trnh do bin cao YX/S, YP/S khng i (= constant) Chi ph thit b t tin iu khin v kim sot c tc So vi cc pp khc, pp c cht chnh vo thit b nn ny cha c nhiu kinh nghim p dng quy duy tr YP/S ti u Tng nng sut ca qu trnh do m cng nghip gim 1 s cng on (ra thit b, tit trng, loading v unloading). Sn phm c tnh ng nht cao

2.3.4. Thit b ln men PHN LOI THIT B LN MEN Theo nguyn l hot ng: fermenter hot ng lin tc, gin on, bn lin tc Theo cu trc thit b: fermenter c cnh khuy (Shaking Tank), khng c cnh khuy Theo hnh dng thit b: Thng (H/ 3); Thp (H/ > 3); ng (dng thp nm ngang, t s dng) Theo nng sut: nh, va, ln Fermenter Types: Shaking Tank, Bubble Column, Airlift, Packed Bed, Fluidized Bed

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Some types of impellers

Anchor

Rake anchor

Baffles

Fermentor: Shaking tank

Mechanical agitation Variety of de impelents Baffles **

Propeller

Rushton Turbine

Palette

Coiled helix

Working volume = (60-70) % of V tank Height Diameter ratio H/D =1; H/D > 1 Heat transfer **

Airlift

CYTOLIFT V= 580 ml H= 38 cm D= 10 cm

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Heat transfer in bioreactors

a) Continuous jacket b) External coiled jacket c) Internal coiled jacket d) Coiled baffles e) With external heat interchanger

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www.gbd.edu.vn

Fermentor ln men bia ti Jangjing Beer. Chp bi BHQ 2007

600 BC
Spontaneous, natural processes

XIX century

XX century
Submerged Aerobic Culture

Submerged Anaerobic & Superficial Aerobic cultures

Cn x nhu cu oxy
CHmOl + aNH3 + bO2 ycCHpOnNq + zCHrOsNt + dCO2 + cH2O

The oxygen demand of the cell and the low solubility of oxygen in water, required a continuous air supply into the fermentor and a continuous transfer of oxygen from the gas phase into the liquid phase

How is the oxygen transfer into the fermentor?

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yO2out, QEA , EA Condenser

Oxygen Mass Balance: O2 in - O2 out = O2 metabolized + O2 cumulative O2 in = yO2 inQAir Air O2 out = yO2 outQEA EA O2 metabolized = qO2XV O2 cumulative = d(CO2V)/dt = VdCO2/dt Assuming: QAir Air = QAE AE

P
manometer

O2 probe

pO2
Flow meter

yO2in, Qair, Air Bioreactor Motor, P 0.2 -Filter

yO2QAir Air = V(qO2X + dCO2/dt)

OTR Oxygen Transfer Rate

But the oxygen transfer in a fermentor finds resistances before to reach the cells

Mass transfer takes place by two basic processes: Convection and Diffusion Therefore, a full treatment of mass transfer requires a fully known flow field. However, a simplified treatment in which the overall mass transfer is schematically divided into different transfer steps is used with good results

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General mechanism of O2 transfer

1. Diffusion of the O2 from the bulk gas to the gas liquid interface 2. Transport across the gas liquid interface 3. Diffusion of the O2 through a relatively stagnant liquid region adjacent to the gas bubble 4. Transport of O2 through the wellmixed liquid to a relatively unmixed liquid region surrounding the cells. 5. Diffusion throught the stagnant region surrounding the cells. 6. Transport from the liquid to the pellet cell aggregate 7. Diffusive transport of oxygen into the pellet 8. Transport across the cell envelope 9. Transport from the cell envelope to the intracellular reaction site. e.g mitochondria
5, 6 and 7 are relevant only for processes in which pellets or cell aggregates appear

2.4. CI TIN QU TRNH LN MEN

How we can improve the YP/X? Strain development approach - Fermentation process optimization

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Phn 3: Cng ngh ln men ng dng

3.1. Ln men ethanol v cc ng dng

www.gbd.edu.vn

3.2. Cng ngh sn xut acid hu c thc phm 3.3. Cng ngh sn xut acid amin 3.4. Cng ngh sn xut sinh khi vi sinh vt 3.5. Cng ngh sn xut polysaccharide t VSV 3.6. Cng ngh enzyme 3.7. Cng ngh sx cc sp ln men truyn thng

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3.1. LN MEN ETHANOL V CC NG DNG

3.1.1. Cng ngh sn xut ethanol 3.1.2. Cng ngh sn xut ru cao 3.1.3. Cng ngh sn xut bia 3.1.4. Cng ngh sn xut ru vang

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3.1.1. Cng ngh sn xut ethanol

3.1.1.1.GII THIU V ETHANOL 3.1.1.2. NGUYN LIU SN XUT ETHANOL 3.1.1.3. PP SN XUT ETHANOL 3.1.1.4. SN XUT ETHANOL BNG PP LN MEN 3.1.1.5. CHNG CT & TINH CH ETHANOL SAU LN MEN 3.1.1.6. SN PHM CA QU TRNH LM ETHANOL 3.1.1.7. ETHANOL GREENFUEL

3.1.1.1. GII THIU V ETHANOL

Cc hnh nh kho c v vic lm ru t khi nn nng nghip xut hin (ngi Xume, 4000 B.C) Tm thy ru trong lng m ca ngi Ai Cp CTHH: C2H5OH, M= 46 Cht lng, trong sut, khng mu, mi v c trng T trng d2020 = 0,79067; d204 = 0,78927 Nhit si: 78,35oC c i vi ngi v VSV

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nh hng ca ETHANOL vi sc khe ngi

Ung t khng gy nh hng n tim nhng ung nhiu c th lm t lit h thng thn kinh v gy nh hng xu n h thng tun hon mu. Lng ru trong mu: 0.5 - 2 c th gy say 4 c th dn n cht ngi. Hp th nhanh vo thnh d dy (29%) (xy ra nhanh nht khi d dy rng) phn cn li c hp thu rut non. Ethanol c hp thu s c oxy ho 90-98 %. Ngi trng thnh c th oxy ho khong 10 ml/h, ph thuc vo nng cn trong mu. Ung nhiu s gy s hp thu nhanh hn tc oxy ho, dn n gy c. Ethanol khng c oxy ho c th ri c th theo phi v thn, n cng c th c tm thy trong m hi, nc mt, mt, nc ming lng nh.

nh hng ca ETHANOL vi sc khe ngi

Methanol (ru g) l mt cht c cht ngi, gy cho nhng ngi nghin ru tnh trng m hay cht. Methanol dn dn thm vo rut non khi nhm methyl ca aspartame gp enzyme chymotripsin. Mt nh gi ca EPA v trng thi ca methanol cho rng methanol c xem nh mt c cht c tch lu v t l bi tit rt thp. Trong c th, methanol c oxy ho thnh formaldehyt v acid formic; c hai cht chuyn ha ny u l cht c thn kinh cht ngi. Liu lng tiu th cho php khong 7,8mg / ngy

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nh hng ca FURFUROL vi sc khe ngi

L cht c vi h thn kinh, gy nhc u, bun nn. Ung ru c hm lng furfurol cao qu mc cho php gy tch t trong dch ca phi (ph phi). Fufurol c th gy d ng da, nu s d ng pht trin cao, c th gy nga v ni mn da. Tnh trng trn nu tip tc c th gy mt v gic, t li, au u, mt v rng mnh. Tnh trng ko di c th nh hng ti gan. Tip xc lu di vi furfurol c nguy c gy ung th v nguy c i vi sinh sn.

nh hng ca ETHANOL vi VSV Ethanol xm nhp vo mng t bo VSV, gy ng t mt s protein, nh hng n qu trnh TC ca t bo. VSV b cht hoc b c ch

Sn lng ethanol trn TG

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Brasil: sx 0,9-1 triu lt ethanol/ ngy (ng u th gii) 2007, sn lng ethanol ton cu: 55,7 t lt 2015, sn lng ethanol ton cu: 162 t lt (Theo FAO, y ban Kinh t M Latinh (CEPAL) v Ngn hng pht trin Braxin (BNDES)

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TNH HNH SN XUT ETHANOL VN 1898, sn xut cng nghip ru VN. 1962-1965, xut khu ru sang ng u, ch yu l Lin X 1975-1978, nh my ru H Ni sn xut >8 triu lt cn/ nm, 12 triu lt ru mi/ nm (xut khu 6 triu lt/ nm). 1970-1980, xy dng cc x nghip ru a phng (qui m 100 - 500 nghn lt cn/ nm). C nc c gn 300 nh my ru ln nh, tng cng sut 40-80 triu lt/ nm (cha k lng ru dn nu t do). 1997, sn lng ru quc doanh t 17 triu lt/ 1997. Nu k c ca dn th t 264 triu lt). Bnh qun 3,4 lt/ ngi/ nm. 2005, c khong 180 -200 triu lt ru cc loi, trong ru cn t tinh bt chim 30 - 40%.

TNH HNH SN XUT ETHANOL VN

9/2007, Cty Petrosetco k tha thun hp tc thnh lp lin doanh xy dng nh my sx Ethanol vi Tp on Itochu Nht Bn. Nh my d nh t ti KCN Hip Phc - TP.HCM vi cng sut 100 triu lt Ethanol/ nm t sn lt. Vi 1,2 triu tn sn lt xut khu hng nm, Vit Nam c th sn xut c t nht 400 triu lt Ethanol/ nm. Ethanol s dng pha vo xng (p ng 50% nhu cu ca th trng xng Vit Nam)

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VAI TR CA ETHANOL Lnh vc thc phm Lnh vc phi thc phm

Sx ung c cha ethanol (vodka, liqueur, vang) Sn xut acid acetic

Dc hc Y hc: cht st trng Nng lng CN ha hc

3.1.1.2. NGUYN LIU SN XUT ETHANOL NGUYN LIU sx Ethanol theo quy m CN Nguyn liu cha ng: mt r Nguyn liu cha tinh bt: go, khoai m (sn), bp (ng), nc thi cha tinh bt (b khoai m) Nguyn liu cha cellulose (rm, tru, b ma Whey (lactoserum)

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3.1.1.3. PP SN XUT ETHANOL PP HA HC C2H4 + H2O

Xc tc

PP LN MEN Tinh bt/Cellulose

C2H5OH

C6H12O6

VSV

2C2H5OH + 2CO2

1.1.1.4. SN XUT ETHANOL BNG PP LN MEN

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3.1.1.4.1. LM ethanol t nguyn liu cha tinh bt

Ch tiu

Carbohydrate Protein X th Lipid Nc

Thnh phn ho hc ca mt s nguyn liu cha tinh bt trong sx ethanol (%) Khoai ty Ng Sn Go t Tm 12 - 21 68,4 74,74 69,2 42,0 1,2 3,2 8,3 0,205 7,3 5,3 0,5 1,5 4,1 1,11 0,5 22,5 0,1 0,3 5,1 0,41 1,2 2,0 72 -80 12,5 13,12 11 11,5

LM ethanol t nguyn liu cha tinh bt bng pp ln men

PP sx LM truyn thng Amylose Nguyn liu VSV Bnh men c im ca pp n gin, hiu sut thp Phm vi p dng H gia nh

VK lactic Nguyn + mc+ liu cha men tinh bt Mycomalt Ch phm mc + men LM CN

i hi k thut cao Quy m sx khi cy VSV vo CN bnh ln men Tn din tch, cng sc Quy m sx va & CN

Amylase + Yu cu thit b hin Quy m sx men i, hiu sut cao CN

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1,5 2,5%

Quy trnh sx ru theo pp ln men truyn thng

Hnh nh trong sx ru th cng ti VN

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Sn xut ethanol bng pp ln men (tt) Phng php mycomalt: g ng ha v ru ha din ra 2 thit b.

Ch phm VSV (mc) Men S. c

Quy trnh sx ru theo pp mycomalt

Thy phn tinh bt bng malt, ch phm nm mc

Quy trnh sx ru t ng theo pp mycomalt

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Sn xut ethanol bng pp ln men (tt) Phng php amylose: g ng ha v ru ho xy ra gn nh ng thi, trong cng thit b

VSV (VK, mc, men) ng ha, ru ha

Quy trnh sx ru theo pp amylose

Quy trnh sx ethanol theo quy m CN

Thy phn tinh bt bng enzyme

VSV Men Saccharomyces cerevisiae VK Zymononas mobilis VK E.coli ( chuyn gen)

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THIT B NGHIN NGUYN LIU (tinh bt) My nghin ba kiu git nc Nghin trc

Nghin bt mn

Nghin cao p

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Ln men ethanol t TB theo quy m CN

NU NGUYN LIU H HA - GELATINISATION Mc ch Ph v mng t bo ca ht tinh bt Chuyn ho tinh bt sng thnh tinh bt chn Chuyn tinh bt thnh dng ha tan Thng s k thut Nhit nu = f (nguyn liu v kch thc ca ht tinh bt) Phng php Gin on Bn lin tuc Lin tc

LN MEN ETHANOL T TB - THY PHN TB = enzyme

DCH HA

NG HA

Chuyn tbt thnh ng, cung cp c cht cho qt ln men Quyt nh phn ln hiu sut thu hi ru.

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LN MEN ETHANOL T TB THNG S K THUT CA MI TRNG LN MEN Nng cht kh: 16 18% Hm lng ng kh: 90 -100 g/L Khong (Nit, Mg, P) Chnh pH: pHop 4,5 - 5 Dch ln men 12oP 16oP 18oP Dch ln men t sn Dch LM t r ng Yu cu Nit trong dch ln men (nit hu c, v c) 140 -150 mg/L 200 mg/L > 280 mg/L 300 mg/L 150 200 mg/L

THNG S K THUT TI CTY RU BNH TY Trnh t cng vic Xung bt: 1080 L nc + 300kg go (xay 1mm) + 445g CaCl2 + 283mL Termamyl Nng nhit Gi nhit Nng nhit Gi nhit, cho vo 120mL H2SO4 To (oC) 32 32 83 83 83 95 95 pht 10 40 50 15 30 30

Bm v h nhit dch nu bng corbin sang 95 58 thng ng ha, m bo To thng ng ha l 58oC Cho 32g San super, o trn, gi nhit dch khng lm mt mu iod, h nhit Bm sang thng ln men, + 50g ure 56 58 56 30

60 40

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THNG S K THUT TI CTY RU BNH TY Trnh t cng vic Ln men: t l men 8 10% Kim tra mu sau 4 ngy ln men: Bal, chua Kt thc ln men: Bal <0; chua < 2,5mg/L; ru > 10%v/v Lc bng my ly tm Chng ct (600mm Hg) Lc lnh ru thnh phm 5oC To (oC) pht

68 - 72

3.1.1.4.2. LM ethanol t nguyn liu cha ng (mt r)

Ch tiu Nng lng th (MJ/kg VCK) VCK (%) ng kh (%) Saccharose (g/100g VCK) Glucose (g/100g VCK) Fructose (g/100g VCK) Nit (g/100g VCK) Protein th (%) Khong (g/100g VCK) Ca (g/100g VCK) Mg (g/100g VCK) K (g/100g VCK) P (mg/100g VCK) Na (mg/kg VCK) S (g/100g VCK) Cu (g/kg VCK) Fe (mg/kg VCK) Mn (mg/kg VCK) Zn (mg/kg VCK)

Mt r sx cng nghip c 9,47 75,9 34,6 23,3 5,1 6,2 0,46 2,87 11,7 0,48 0,32 1,94 47 661 0,3 1,58 97 52 3,8 VN 9,87 76,7 39 21,2 8,7 8,47 0,29 1,80 6,2 0,44 0,18 0,92 30 75 0,33 3,01 417 52 12,6

THNH PHN HA HC CA MT R

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Quy trnh sx ethanol t mt r

LN MEN ETHANOL T MT R

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LN MEN ETHANOL T MT R

3.1.1.4.3. LM ethanol t nguyn liu cha CELLULOSE

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CELLULOSE

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LN MEN ETHANOL T CELLULOSE - THY PHN

Tc nhn thy phn Acid Ch phm nm mc Ch phm enzyne cellulase (thy phn lin kt 1,4beta-D-glycosidic trong cellulose, lichenin v beta-Dglucans) Kt hp acid v enzyme

Thy phn cellulose bng enzyme

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Thy phn cellulose v hemicellulose bng mc Trichoderma reseii

S quy trnh LM ethanol t nguyn liu cha CELLULOSE

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3.1.1.5. CHNG CT (DISTILLATION) & TINH CH (RETIFICATION) ETHANOL SAU LN MEN Qu trnh thu ethanol v tp cht d bay hi t dch ln men

Chng ct: thu sn phm cn th (raw spirit) 82 87% Tinh ch: thu sn phm cn tinh luyn 94 98% Kt hp chng ct & tinh ch: s dng nhiu thp chng ct

CHNG CT ETHANOL LIN TC (continuous rectifier )

U: thit b thu hi nhit

B: ct tinh luyn C, D: b phn ngng t R: b phn lm lnh sp E: my xc nh v cn

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Thit b chng ct ti nh my ru Bc Kinh TQ

Dy chuyn ng chai ti nh my ru Bc Kinh TQ

3.1.1.6. CC SN PHM CA QU TRNH LM ETHANOL

Sn phm: C2H5OH Aldehydes: by oxidation of alcohol Acids: by oxidation of aldehydes Fusel Oil: by conversion of free amino acids in water to higher alcohols Easters: by esterification of alcohols and acids Volatile sulphur compounds: by combination of sulphate and sulphur with amino acids

Sn phm ph Men CO2 Hm ru

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CC SN PHM CA QU TRNH LN MEN ETHANOL

CH TIU K THUT CA ETHANOL Nng ru 15oC Acid hu c (tnh theo mg acetic/l ) Furfurol Metanol Aldehyde tng s (mg/L) Ru cao phn t (mg/L) Este (mg/L) Hm lng kim loi ng ( Cu mg/l ) Xyanhydric ( HCN mg/l cn 100o ) (vi cn sx t khoai m > 96,2 < 15 0 0 <4 <4 < 30 < 30 <50

SX ETHANOL BNG PP LN MEN

CC SN PHM PH CA QU TRNH LN MEN ETHANOL B MEN

HNG S DNG MEN Men bnh m SPC (Thc n cho gia sc) Cao nm men (yeast extract)

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CC SN PHM PH CA QU TRNH LN MEN ETHANOL HM RU Thnh phn Cht kh (%) Protein th (% tng cht kh) Lipid Cellulose Cht chit phi nit Tro Dch hm t nguyn liu Khoai ty 6 27 2,7 8,1 49,9 12,6 Khoai m 3,3 14,2 1,8 2,8 74,2 6,7

HNG S DNG HM RU Thc n cho gia sc Nguyn liu lm phn vi sinh Nguyn liu sx vitamin bng VSV

CC SN PHM PH CA QU TRNH LN MEN ETHANOL CO2

Quy trnh x l CO2 Ra nc

KMnO4/ K2Cr2O7

Sn phm

B sung vo bia lon, nc ngt c gas, Champagne Nui to

Oxi ha Tch nc

Nn ha lng

X l bng than hot tnh, silicagel Tch du

Lm ngui

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SX ETHANOL BNG PP LN MEN HIU SUT THU HI ETHANOL

3.1.1.7. ETHANOL - GREENFUEL

Nhin liu sinh hc

Nhin liu truyn thng ang cn kit

Mt r, bp, ph phm nng nghip, cellulose

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CHNH SCH PHT TRIN GREENFUEL

Nguyn liu EU M Php n Bp, c ci ng, ma, sn Bp C ci ng Cy Jatropha, Pongamia Nhin liu sinh hc/ tng lng xng du Nm 2010: 5,7% Nm 2012: 24 t lt Nm 2010: 7% Nm 2012: 5,% Tng 12 triu tn nhin liu sinh hc/nm Nm 2010: 3 triu lt

Trung Quc Cy la min Philippines Thi Lan Brasil Ma, Cy la min Ma, sn Ma

Production of bioethanol from sugar cane or sugar beet

This is the simplest of all the processes for producing bioethanol by fermentation. Sugar cane has an energy production per hectare that is substantially higher than the other feedstocks considered here, but the process conversion efficiency is only about 0.35-0.40 GJ bioethanol per GJ feedstock. The sugar cane residue or bagasse can be burned to generate electricity, producing about 0.08 GJ electricity per GJ feedstock.

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Production of bioethanol from corn using wet or dry milling

Wet milling, several types of residues are produced; dry milling produces only one type of animal feed product. The starches were broken down into C6 sugars. The current conversion efficiency of both process routes is about 0.55 GJ of ethanol per GJ wheat (USDA 2002). The processes are well established but there is some limited scope for efficiency improvements.

Production of Bioethanol from wheat from malting and fermentation

The process is similar and very slow. The wheat is first crushed or milled. In its passive form, malting is a process by which under controlled conditions of temperature and humidity, enzymes present in the wheat break down starches into C6 sugars. These sugars are washed out of the wheat with water, whilst the leftover residue can be sold for animal feed. Fermentation at 32-35oC; pH 5.2. Ethanol is produced at 10-15% concentration and the solution is distilled to produce ethanol at higher concentrations.

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Production of bioethanol from wood or straw by acid hydrolysis and fermentation

It requires the production of ethanol from both C5 and C6 sugars. This process is technically feasible but is complex and expensive and there are few industrial examples. Ongoing research and development in the US aims to address cost issues and develop a more efficient process. This is thought by many to be a step on the way to the eventual goal of an enzyme hydrolysis process

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SC water: supercritical water

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9 FUEL ICONS Unleaded Petrol Super Unleaded High Octane Diesel Bio-Ethanol Bio-Diesel CNG LPG Leaded Four Star.

3.1.2. Cng ngh sn xut ru cao 3.1.2.1. SX RU RUM T MA

www.gbd.edu.vn

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Husks All the discarded husks of the sugar cane. The first step in making rum is to crush the cane to get out all the juiceyou can see they make a lot of rum here at River's.

SX RU RUM T MA

After all the sugar cane juice is squeezed out, the next step in the process of making rum is to boil it down in giant kettles (ex: copper kettles) - like the ones you see here.

SX RU RUM T MA

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After the sugar cane juice is boiled down in the coppers it has to ferment for a couple of days.

SX RU RUM T MA

Condenser The final step is the condenser, where the alcohol gets seperated out thru coils and a cooling process. What comes out is the final product- pure white rum.

SX RU RUM T MT R

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SX RU RUM T MA

Whisky: The difference between rum and whisky manufacture begins with the starting point. Whisky starts with barley that must be malted before it is converted from a starch to a sugar that can be fermented. After fermentation, it is also distilled usually using a pot still or single column. Cognac: The sugar that is fermented to make cognac, come from grapes. As is the French style, only grapes grown in a particular area in France can be used. First the grapes are fermented into wine and this is distilled in special stills to yield cognac. Brandy is made the same way but because the grapes arent grown in the Cognac region, it can not be called cognac. All spirits are aged in a special barrel prior to blending and bottling.

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3.1.3. Cng ngh sn xut bia

3.1.3.1. GII THIU V BIA
www.gbd.edu.vn

3.1.3.2. NGUYN LIU SN XUT BIA 3.1.3.3. VSV TRONG QU TRNH LN MEN BIA 3.1.3.4. CNG NGH LN MEN BIA SN PHM

3.1.3.1. GII THIU V BIA - LCH S PHT TRIN 7000 B.C, Ngi Babylon (a ra vn t lut php quy nh v vic bn ru bia) pha ch c 20 loi bia. 3000 B.C, ngi Ai Cp c i pht minh ra ng ht ung bia cn ln cn v la m 500 400 B.C, bia l sn phm dnh cho ngi lao ng 768: Baviere s dng hoa houblon (trc kia dng tho mc to v ng cho bia) 1516: lut sx bia u tin c p dng (bia nu t malt i mch, hoa houblon v nc 1857: Pasteur nghin cu qu trnh ln men ethanol, thanh trng bia 63oC/ 20 30 1883: Hansen v ging men thun khit

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GII THIU V BIA Sn lng bia Sn lng bia trn th gii: >120 t lt/nm Lng bia tiu th: CHLB c 170L/ngi/nm VN: 5 10 L/ngi/nm

Babylonia's table (6000 BC): Describe the beer preparation

GII THIU V BIA

Lut sx bia Cng ngh Sn phm

Nguyn liu
c: 100% malt i mch Php: s dng 30% th liu VN: 2030% go

Ph gia

B sung caramen B sung acid

Men ging khng c t bo cht

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GII THIU V BIA - CC HNG LN SX BIA TRN TH GII

Anherue busch (M) Heineken (H Lan) Sn lng bia trn th gii: Miller (M) >120 t lt/nm Kirin (Nht) Lng bia tiu th: Fosters (c) CHLB c: 170L/ngi/nm Danone (Php) Brahma (Brasile) VN: 5 10 L/ngi/nm Guiness (Anh) (bia en) SAB (Nam Phi) Pilsen (Tip) Carlsberg (an Mch) (men chm)

PHN LOI BIA THEO NH SX

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PHN LOI BIA THEO NH SX

PHN LOI BIA THEO MU SC VNG EN

www.gbd.edu.vn XANH

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GII THIU V BIA

Weizen

Bia l sn phm ca qu trnh ln men ethanol t dch nha, khng qua chng ct
Bia en Munich

3.1.3.2. NGUYN LIU SN XUT BIA

Nguyn liu chnh: malt ai mch Th liu: nguyn liu giu glucid Hoa Houblon Nc

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NGUYN LIU SN XUT BIA - MALT I MCH Ht i mch N y mm Ht malt

Hot ho v tch lu h enzyme (amilase, protase)

Nguyn liu chnh sn xut cc loi bia. i mch ny mm v sy n m bt buc

H enzyme trong ht malt s lm nhim v ct cc hp cht cao phn t trong ni nh ca ht thnh cc cht c trng lng phn t thp hn (dextrin, acid amin, pepton, ng n) ho tan vo nc tr thnh cht chit ca dch ng.

NGUYN LIU SN XUT BIA - MALT I MCH

Ch tiu cht lng malt Mu sc Vng rm Mi v chit sut Hot lc enz m pH nc malt Tg ng ha Thm > 785 > 285 oWK < 4,5 5,8 6,3 10 15 Ch tiu cht lng malt (tt) Mu nc malt 2,8 3,5 oEBC Pr tng s Pr ha tan Ch s Kolback nht xp C ht >2,5mm C ht <2,2mm 9 13 % Min 4,5 % 42 48% M 1,6 cp Min 84% >92% <1,6%

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NGUYN LIU SN XUT BIA - TH LIU TH LIU CHA TINH BT: ht i mch, go, tm (VN), bp tch phi (chu u), khoai m, khoai ty, tinh bt
nguyn liu giu glucid, thay th mt phn hoc ton b malt i mch trong sx bia

TH LIU TH LIU CHA NG:

saccharose, syrus thu phn tinh bt (maltose, glucose)

NGUYN LIU SN XUT BIA - TH LIU

www.gbd.edu.vn

U IM H gi thnh sn phm Ci thin mt vi tnh cht ca sn phm (tng bn ca keo) To ra chng loi bia c cc mc cht lng khc nhau

NHC IM nh hng cho qu trnh nu (do th liu c t hoc ko c enzyme) nh hng ti qu trnh ln men (Protein ha tan trong dch nha gim, khng cn i thnh phn dinh dng)

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NGUYN LIU SN XUT BIA - TH LIU TH LIU S DNG TI VN - GO cu trc ht tinh bt cht, nhit h ha cao, kh ng ha Bia Heineken: 100% malt i mch Bia Tiger: 15% go Bia Si Gn: 20% go TH LIU S DNG TI CHU U BP Tch phi loi cht bo Cht bo ph hy h bt, nh hng n cht lng cm quan

NGUYN LIU SN XUT BIA - HOA HOUBLON Nguyn liu khng th thay th trong cng ngh sx bia To v ng du, hng th m c trng Tng kh nng to v gi bt Tng bn keo v tnh n nh sinh hc ca sn phm. Trong sx bia ch s dng hoa ci cha th phn (c ht lupulin) do hoa c v hoa ci th phn c hm lng cht mi v cht to v thp (khng c ht lupulin)

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NGUYN LIU SN XUT BIA - HOA HOUBLON HOUBLON Houblon to hng 3,85 5,14% acid Ch tiu Mi v Alpha acid m Hoa vin Thm c trng, khng mi l 8 0,3 <10 Houblon to v ng 8,62 9,31% acid Hoa cao Thm c trng, khng mi l 30 2

NGUYN LIU SN XUT BIA - HOA HOUBLON Thnh phn ha hc c trong hoa houblon (% khi lng cht kh) m 10 -11 Alpha acid 2 -12 Beta acid 2 - 10 Tinh du (> 200 cht nh terpene, ester, 0,5 2 ketone, hp cht cha lu hunh) Polyphenol 2 -5 Protein 12 18 Lipide 2-4 Cellulose 40 50 Pectin 12 Tro 1-2

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nh gi cht lng hoa houplon Ch s Mu hoa Mu ht lupulin Mi Loi 1 Vng ng nh Thm d chu, c trng Loi 2 Vng, vng sm Thm, khng c mi tp cht Cnh hoa c th b rch (1,5cm), c chm c ph <3% <1 % >12 % <11 % <3 % Loi 3 Vng xanh Vng sm Hi nng Rch nhiu, chm c ph nhiu <9% <5 % >10 % >12 % <13 % Vng vng ng Vng lc

Cnh hoa To u chc, khng rch Tp cht Hoa bnh Hoa ng Tro m <1,75% 0% <10 % <15 % <13 %

NGUYN LIU SN XUT BIA - HOA HOUBLON

Hoa ti: W 75 80%, VN khng s dng.

Cnh hoa kh: W8 10%

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Hoa kh (W 4 -6%) Nghin Ry (2-8mm) Bt hoa kh Bao gi (chn khng, CO2, N2) Bo qun Hoa dng ht, vin: W 4 6%, bt hoa kh (c th thm 20% bentonote SiO2 ri to thnh vin Hoa cao trch ly; dng st c hm lng polyphenol thp, lm gim bn keo

NGUYN LIU SN XUT BIA NC Quyt nh n cht lng bia Chim 90 95% trong bia YU CU V NC TRONG SN XUT BIA YU CU CM QUAN: trong, khng c mi v l YU CU VI SINH: khng c E.coli v Streptococci/100 ml nc. YU CU HO L pH 7.0 cng 2-4mg/L kim <50mg CaC03/L Cht kh <1500mg/L Sulphate < 500/L Clorute 250 300mg/L Nitrate <30 mg/L

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3.1.3.3. VSV TRONG LN MEN BIA

MEN NI (S. cerevisiae) Nhit ln men Kh nng ln men Kh nng s dng ng 3C rafinose Kh nng lng khi ln men kt thc 10 - 25 MEN CHM (S. carlsbergensis) 0 - 10

Mnh, xy ra trn b Mnh, xy ra trong mt mi trng lng mt 33% Cc tb kt chm, chui to thnh lp dy ni ln b mt mt, bia trong chm 100% Cc tb kt chm, chui lng xung y thng ln men rt nhanh, bia t trong nhanh hn

3.1.3.4. CNG NGH SX BIA

Malt ai mach The lieu

Tach tap chat

Tap chat

Nghien

Nc

Nau dch nha

Loc

Ba malt

Houblon

un soi vi hoa houblon

Tach ba houblon

Ba houblon

vo trung

Lam lanh

, bao hoa O

Nam men

Nhan giong

Len men chnh

Sinh khoi nam men

Len men phu

Can

X ly lam trong bia

Can

CO

Bao hoa CO

, tang tr

Chai

/ lon

Chiet chai

/ lon

Thanh trung

San pham bia chai / lon

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CNG NGH SN XUT BIA

3.1.3.4.1. Sx bia NGHIN Mc ch NGHIN Tng b mt tip xc vi nc Nc xm nhp vo cc thnh phn trong ni nh nhanh hn Cc yu t nh hng n qu trnh nghin m Cu trc ht tinh bt Thit b Phng php nghin Nghin th liu: go Nguyn tc: nghin go cng mn cng tt

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Sn phm nghin malt: v tru, ht tm ln, tm nh, bt. Mc nghin MALT Nghin th: 20 25% (280mL/100g malt) Nghin mn: 50 60% bt (210mL/100g malt) Nghin rt mn: 85 90% bt (200mL/100g malt) Mc nghin nh hng ti trch ly, thi gian lc b Yu cu v mc nghin Ch tiu Nghin trc (lc bng ni lc) Tru 15 18 Ht tm ln 12 15 Ht tm nh 30 35 Bt 25 - 35 malt (% khi lng) Nghin ba (lc bng thit b lc khung bn) 9 12 12 15 30 35 40 - 45

3.1.3.4.2. Sx bia NU DCH NHA Trch ly cc cht chit c Mthp (ng, acid amin, vitamine, khong) t malt i mch v th liu vo nc Thy phn mt s cht c Mln thnh cc cht c Mnh (c cht cho nm men) Cc phn ng xy ra trong qu trnh nu dch nha Thy phn Protein: to thnh polypeptide, peptide (endoenzyme), acid amin (exoenzyme) Thy phn tinh bt: to thnh dextrin, oligosaccharide (alpha amilase), maltose (beta amilase) Thy phn hemicellulose: to thnh beta glucan mch ngn Thy phn mt s cht hu c c cha acid phosphoric: to thnh acid phosphoric

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3.1.3.4.2. Sx bia NU DCH NHA (tt) THNG S K THUT T l malt: nc = 1:4 1:5 T l th liu: nc = 1:1,5 2 pH: 5,4 5,6 (chnh pH 1 ln) Thc hin cc im dng trong qu trnh nu Thit b nu c cnh khuy

CNG NGH SX BIA Nh my Bia LIDA 500kg nguyn liu/m thu c 3100 3300L dch ln men. T l malt: go = 3/7 (130 kg go: 370 kg malt). Ni go: 130 kg go + 520- 650 lit nc + 200g CaCl2. Dng H3PO4 chnh pH 5,3 -5,4. 40oC/15. Trong 15 tng t 40oC n 72oC, 72oC/20. Trong 10 tng n 83oC, 83oC/5. Trong 5, h xung 72oC, 72oC/20. Trong 20 tng ln 100oC, 100oC/15. Trong 5, h xung 65 67oC Ni malt: 370 kg malt + 1480 1850 lit nc + 300g CaCl2. Dng H3PO4 chnh pH 5,3 -5,4. Khi nu go c 60 th nu malt. 40oC/20. Trong 15, Tng n 50oC, 50oC/20. Trong 10 tng n 65 67oC .

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Ni malt v go u 65 67oC. Trn 2 ni lm mt. Duy tr 67oC/30. Trong 15, tng ti 72oC, 72oC/15. Trong 15, tng ti 76oC, 76oC/15. Tng thi gian nu go v malt l 210. Sau lc dch v ra b (40 50). Dch lc c un si trong 120. Trong thi gian un si, cho hoa houblon 3 ln. Thi gian cho hoa: ln 1 sau 10 un, ln 2 sau 30, ln 3 sau 75. B sung 250mL caramen chnh mu dch ng. + 2g ZnCl2 b sung nguyn t vi lng cho men. Sau 120 un, chuyn qua lng 25 tch cn, cc cht khng ha tan nh xc hoa, cht kt ta Lm lnh nhanh. Chuyn dch vo tank cy men trc

3.1.3.4.3. Sx bia Lc dch nha Mc ch Tch b b malt Ra b malt Phng php Ra b malt bng nc nng Toop = 78 80oC

Cc yu t nh hng n qu trnh lc Mc nghin malt Nhit nht Thit b lc tch b malt: thng lc hnh tr y bng (thng s dng) Cu to thit b: phc tp (1 y gi l mt sng v h thng cnh o trn, dao gt b, h thng tho b malt)

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3.1.3.4.3. Sx bia Lc dch nha (tt)

So snh hiu qu s dng thit b lc Ch tiu Ni lc Lc khung bn Mng lc B malt Vi coton Chiu cao mng lc 30 60cm Tc lc 0,5 4,5m/s 1 2m/s Thi gian lc 60 110 20 50; P 0,3 bar Lng nc ra Tn nhiu nc Thi gian ra b Thi gian tho b Tn tht cht chit m b Mc c gii ha v t ng ha 60 120, ra ti a 3 ln 30 40 pht Thp Cao hn T t 90 140. P 0,4 1,2 bar Tho b t ng Cao hn Thp Tn nhn cng

3.1.3.4.4. Sx bia un si dch nha vi hoa Houplon MC CH Chit cht ng t hoa houplon (alpha acid) vo dch nha, ng phn ha mt s cht ng V hot enzyme, ng t Protein km bn nhit iu chnh nng cht kh trong dch nha To thnh mt s cht c li cho sn phm Tch mt s hp cht d bay hi nh hng xu n cht lng sn phm

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3.1.3.4.4. Sx bia un si dch nha vi hoa Houplon (tt) LNG HOA HOUPLON B SUNG (Tnh theo acid) 7 20 g acid/hL THI GIAN UN SI: 60 120 PHNG PHP B SUNG HOA Mt ln: sau khi dch nha si Hai ln: sau khi dch nha si + 30 trc khi kt thc qu trnh nu Ba ln: sau khi dch nha si + 30 trc khi kt thc qu trnh nu + 15 trc khi kt thc qu trnh nu

3.1.3.4.5. Sx bia Tch b hoa Houplon

Nguyn l: Bm dch nha vo thit b hnh tr ng, dung dch chuyn ng theo ng xon c. Dng lc ly tm lng (lng xoy tm)

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Thnh phn dch nha pH Nng cht kh Glucose Fructose Saccharose Maltose Trisaccharide (maltotriose, raffinose) Dextrin Beta glucan v gum Nit tng Nit amin t do (FAN) Cc cht khc (acid nucleic, vitamine, khong, polyphenol) 5,2 5,4 10 13oPt 5 15 g/L 0,5 2 g/L 2 6g/L 45 65g/L / / 200 800mg/L 500 1.100mg/L 100 250mg/L /

3.1.3.4.6. Sx bia Lm ngui v np oxy vo dch nha MC CH H nhit dch nha, chun b cho ln men Cung cp oxy cn thit cho sinh trng ca men YU CU Lm ngui nhanh, thit b kn Np Oxy (khng kh) v trng, lng oxy ha tan (6 8mg oxy/lt dch nha, P = 1atm, To = To ln men

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3.1.3.4.7. Sx bia NHN GING MEN & LN MEN Nhn ging 2 giai on PTN: thch nghing, cy vo ng 10ml nc nha+ pepton 12h Cy qua bnh tam gic 150 170mL dch, 12h. Cy qua bnh tam gic 1,5L, 24h Chuyn vo tank 1 (V tank 1: 200L, V dch ng: 150L), c cung cp oxi trong tank, Nui 48h. Chuyn tank 1 vo tank 2 (V tank 2: 1000L, V dch 800L, nui 48h, 20 25oC Bm men t tank 2 vo Tank cha dch ln men (V 12.000L)

3.1.3.4.7. Sx bia NHN GING MEN & LN MEN (tt) MC CH LN MEN: Chuyn dch nha thnh bia Ln men chnh Chuyn ha ng thnh ethanol, CO2 v cc sn phm ph, sp trung gian Tc ln men din ra nhanh, cc cht cn (protein) v t bo men cn st li lng dn xung y, bia trong dn Ln men ng cn li, bo ha CO2 cho bia ti, n nh hng v, cht lng, trong cho sn phm, tng gi tr cm quan

Ln men ph

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3.1.3.4.7. Sx bia NHN GING MEN & LN MEN (tt) Ln men chnh Primary fermentation THNG S K THUT 0,5L men/hL dch nha hay 10 25.106 tb/mL dch nha To= 13 15oC Thi gian: 7 9 ngy Nng cht kh cn 1,15 0,2% kt thc ln men chnh CC YU T NH HNG N LN MEN CHNH Mi trng ln men (dch nha): pH, nng cht kh, thnh phn ha hc Nm men iu kin ln men: To,

3.1.3.4.7. Sx bia NHN GING MEN & LN MEN (tt) Ln men Ln men ph Secondary fermentation THNG S K THUT LM PH 4 8.106 t bo men/mL dch nha To= 0 5oC Thi gian: 7 30 ngy (ph thuc vo tng loi bia) Thi gian ln men ph quyt nh hm lng diacetyl trong bia, cng nh mc chn ca bia. Lin tc tch nm men cht v cc kt ta ra khi thit b trong qu trnh ln men ph

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Hnh nh thit b ln men bia

3.1.3.4.8. Sx bia Lm trong bia MC CH Tch trit cc phn t rn lng, khuych tn trong bia Tng trong, Lm n nh thnh phn c hc Tng bn keo v bn sinh hc cho sn phm YU CU Nhit lm trong: 1 2oC THIT B LC Thit b lc khung bn: r, d vn hnh, bi v VSV d xm nhp Thit b lc ly tm, lc da, lc nn.

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3.1.3.4.8. Sx bia Lm trong bia (tt) Cc cht lm trong Cho thm mt hay nhiu cht lm trong vo bia (khng b bt buc phi cng b nh l mt thnh phn ca bia) Vit Nam, thng s dng thit b lc khung bn, b sung cht tr lc diatomid

Lc khung bn

3.1.3.4.9. Sx bia Bo ha CO2 Hm lng CO2 trong bia non: 4-4,5g/l. Nu khng t tiu chun: b sung thm CO2 MC CH Chun ha hm lng CO2 cho bia chai v bia lon YU CU Nhit : 0 1oC P = 0,15 -0,2 MPa

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3.1.3.4.10. Sx bia Chit v thanh trng bia MC CH Ko di thi gian BQ sn phm D vn chuyn v s dng YU CU Nhit : 0 1oC P = 0,15 -0,2 MPa

Quy trnh cng ngh phn xng chit

3.1.3.4.10. Sx bia Cht lng sn phm Ch tiu cm quan Mu sc trong bt Mi V Yu cu Vng rm sng Trong sut Bt trng, mn lu tan Thm c trng ng du, m , c hu, c trng

Ch tiu vi sinh Yu cu Vi khun k kh 0 VKHK <1cfu/100mL Men st <100cfu/100mL

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Ch tiu ha l Cht ha tan ban u (% khi lng) ng st (%) cn pH chua (mL NaOH/100mL bia) mu (EBC) CO2 (g/L) bn ca bt (giy) Oxi ng Diacetyl

Yu cu 10,2 0,2 1,8 2,2 4,3 0,2 4,0 4,2 1,6 0,2 7,0 7,2 4,0 4,5 150 250 < 3ppm 20oBU < 0,1ppm

Sa hnh nh my bia ti Bc Kinh - TQ

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3.1.4. Cng ngh sn xut ru vang

www.gbd.edu.vn

3.2. Cng ngh sn xut acid hu c thc phm 3.2.1. Sx acid lactic 3.2.2. Sx acid acetic 3.2.3. Sx acid citric

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3.2.1. Sx acid lactic

3.2.1.1. GII THIU V ACID LACTIC 3.2.1.2. VAI TR CA ACID LACTIC 3.2.1.3. C CHT SN XUT ACID LACTIC 3.2.1.4. VSV LN MEN ACID LACTIC 3.2.1.5. CNG NGH SN XUT ACID LACTIC 3.2.1.6. THU NHN V TINH SCH SN PHM

3.2.1.1. GII THIU V ACID LACTIC 1780, Carl Wilhelm Scheele (nh ho hc Thy in) tch acid lactic t sa b chua. 1881, sx theo quy m cng nghip nh Frmy (nh khoa hc Php) sx acid lactic bng pp ln men C3H6O3 CH3-CHOH-COOH M= 98,08; khng mu, mi nh, ht m cao, To si = 122oC, tan 17oC 2 dng ng phn D- v L+. Dng ng phn sinh hc L+ c trong c th ngi Ph thuc vo tinh sch ca acid lactic p dng vo cc lnh vc khc nhau

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D-acid lactic

L-acid lactic

T l D:L = 50:50 (Hn hp raxemic (k hiu l DL-acid lactic) Raxemic c dng lng, tan trong nc, cn, khng tan trong CHCl3, To si = 122oC, nhit nng chy 16,8oC Raxemic ch c c khi tin hnh tng hp hu c
(dng L c trong c th ngi)

3.2.1.2. VAI TR CA ACID LACTIC

LACTIC TRONG LNH VC THC PHM LACTIC TRONG LNH VC PHI THC PHM Sn lng acid lactic c tnh khong 50 ngn tn/nm, khong 2/3 sn lng c sn xut bng pp ln men

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3.2.1.2. VAI TR CA ACID LACTIC (tt) trong thc phm Tht gia sc, gia cm, c (Meat, Poultry & Fish) ung (Beverages) Rau ngm gim (Pickled vegetables) Salad v nc st (Salads & dressings) Bnh ko (Confectionery) Cc sn phm t sa (Dairy) Baked Goods Savory Flavors

3.2.1.2. VAI TR CA ACID LACTIC (tt) LNH VC PHI THC PHM Dc phm (Pharmaceutical), m phm (Comestics) Vt liu sinh hc (Biomaterials) Mt s ngnh k thut (Technical) Cht ty ra (Detergents) Thc n cho gia sc (Animal Feed) Cht do t phn hy (biodegradable plastics) Nha PLA (Poly lactic acid) chu c 175oC, thch hp lm chai rt nng, khay s dng trong l vi ba, cc loi si v thit b in t chu nhit

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3.2.1.3. C CHT SN XUT ACID LACTIC

Glucose, sucrose, lactose Mt r ng Huyt thanh sa Nguyn liu cha tinh bt (ng, khoai ty, nc thi cha tinh bt)

3.2.1.3. C CHT SN XUT ACID LACTIC (tt)

Pha long mt r (1mt r : 3 nc)

X L MT R

Than hot tnh Mt r x l mu

H2O

5% H2SO4 (w/w)

Mt r x l h keo Dch trong

H2O Khong Na2CO3/NaO H

Mi trng ln men lactic

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3.2.1.3. C CHT SN XUT ACID LACTIC (tt) X L NGUYN LIU CHA TINH BT
Nguyn liu cha tinh bt Thy phn Nguyn liu cha cc ng n gin
Nit CaCO3

Mi trng ln men lactic

3.2.1.4. VSV LN MEN ACID LACTIC 1857, Pasteur kt lun VK khun lactic ln men sa 1878, Joseph Lister phn lp c VK lactic t tn l Bacterium lactic nay gi l Streptococcus lactic. VK lactic thuc h Lactobacterium, khc nhau v hnh dng, sinh l v kh nng ln men VK lactic thuc nhm k kh khng bt buc VK lactic chia lm 2 nhm: -VK lactic ng hnh (cu khun v trc khun): sn sinh 85-95% acid lactic. -VK lactic d hnh: sn sinh 50% acid lactic v ru. Trong cng nghip ln men lactic s dng VK lactic ng hnh

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3.2.1.4. VSV LN MEN ACID LACTIC (tt)

Cc VSV chnh s dng trong cng nghip sx acid lactic hin nay: Lactobacillus casei. Streptococcus thermophilus. Lactobacillus acidophilus. Lactobacillus bulgaricus. Lactobacillus delbrueckii Chng c th s dng tinh bt: L. amylophilus, L. amylovorus

C ch chuyn ha glucose ca VK lactic ng hnh

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3.2.1.4. VSV LN MEN ACID LACTIC (tt)

VK Lb. casei Top 3539oC pHop c im sinh ha 5 7 +: Glu, Fructose, Lactose, Galactose LM chm: Maltose, Saccha., -: Manitol, Dextrin 5,4 +: Glu, Lactose, Galactose, -: Maltose, Manitol, Dextrin 5,5 6,5 4,6 5,4 +: glucose, fructose, galactose, maltose +: Glu, fructose, galactose, maltose -: lactose 5,8 +: Glu, fructose, galactose, 6,0 maltose SP D acid lactic

Lb. bulgaricus Lb. acidophilus Lb. delbrueckii

4548oC 4550oC 35 45oC

L acid lactic D, L acid lactic L acid lactic L acid lactic

Strep. 43 thermophilus 46oC

3.2.1.4. VSV LN MEN ACID LACTIC (tt)

Strep. thermophilus

Lactobacillus bulgaricus

Lb. acidophilus Lb. delbrueckii (2 (2 7 0,4 0,4 0,8m) Dng k thut bc x t ngoi to Lac. delbrueckii t bin. Chng c tc pht trin mnh hn, pha lag ngn hn, nng sut v hiu sut sinh acid lactic ln hn

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3.2.1.4. VSV LN MEN ACID LACTIC (tt) Nhu cu dinh dng ca VK lactic - Carbon: cu trc t bo v cung cp nng lng - Nit: nit c trong MT (cc acid amin), b sung thm peptone, cao tht, cao nm men, casein - Vitamine: VK lactic t c kh nng tng hp vitamine. Chng cn cc vitamine nh riboflavin, thiamin, acid pantotenoic, nicotinic, biotine -Cc cht hu c: + Base cha nit: adenine, guanine, uraxine, + Acid hu c: acid citric, acid acetic, + Acid amin: L- asparagine, L-glutamine -Khong: P, S, Mg, Ca, Mn, Fe, K, Na Mn2+, Mg2+, Fe2+ c tc ng tch cc ln s pht trin v sinh ra acid lactic ca VK Lactobacillus

3.2.1.5. CNG NGH SN XUT ACID LACTIC

Nguyn liu X l Mi trng ln men VK lactic 3-5%v/v Ln men lactic Mui lactac Thu nhn lactic Acid lactic

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Commercial lactic acid is produced naturally by fermentation of carbohydrates such as glucose, sucrose, or lactose.

Sn xut L-Lactic Acid (lactide) bng PP ln men trc tip khoai ty Hokkaido

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Sago Industry: PLA (Poly Lactic Acid) production incorporated with Sago

3.2.1.5. CNG NGH SN XUT ACID LACTIC (tt)

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GALACTIC, sx L(+) lactic theo pp Nh my B&G, sx lactic ln nht LM ln th 2 trn TG, xut khu > Chu TQ, cung cp cho th 90% sn phm cho > 50 nc. trng thc n gia sc v sx PLA (Poly-Lactic Acid)

3.2.1.5. CNG NGH SN XUT ACID LACTIC (tt) THNG S K THUT Mi trng ln men 10 15oBx pH = f (pHop ca VK s dng ln men) VSV S dng chng VK lactic ng hnh T l ging 3-5% (v/v), mt t bo 106 tb/ml iu kin ln men To = f (Top ca VK s dng ln men) Fermenter c cnh khuy v thi kh Duy tr pHop trong sut qu trnh ln men

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3.2.1.6. THU NHN V TINH SCH LACTIC Cng ngh truyn thng Cng ngh hin i
PP thm tch in &trao i ion PP tch pha Dch sau ln men (lactate canci) Lactic un dch n 8080-90oC
CaCO3

Thit b to kt ta Ca2+lactate (ln 2) C c dch lc Dch lc

Kt ta

Chnh pHdch 6.5

35h

Lc khung bn
7070-80oC

Dch lc
sinh khi

Thit b to kt ta Ca2+lactate (ln 1)

Tinh sch lactic theo cng ngh truyn thng

Dch sau ln men (lactate canci)

Na2CO3

Chnh pHdch 6.5

35h

Lc
Na2CO3
sinh khi

Dch trong

C c n 40 70% Wban u Chnh pHdch 6.5 Thit b tch pha

(c trialkyamin & CO2 (P= 75pSi)

Thm tch in Na+ lactate (lng)

Tb C c chn khng Thm tch in trch ly Dung dch acid lactic Ct trao i ion
PP thm tch in & trao i ion

Pha hu c (cht hp th lactic) + Pha ha tan (Na2CO3 + H2CO3)

80 240oC; 2 100mmHg

Chng ct chn khng Lactic tinh khit (99%)

PP tch pha

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SN PHM ACID LACTIC Nng acid lactic 95% Chloride 0.1% Cyanide 5mg/kg Kim loi nng (ch) 10mg/kg St 10mg/kg. Cn 0.1% Sulfate 0.25%

3.2.2. Sx acid acetic

3.2.2.1. GII THIU V ACID ACETIC 3.2.2.2. VAI TR CA ACID ACETIC 3.2.2.3. C CHT SN XUT ACID ACETIC 3.2.2.4. VSV LN MEN ACID ACETIC 3.2.2.5. CNG NGH SN XUT ACID ACETIC 3.2.2.6. THU NHN V TINH SCH SN PHM
www.gbd.edu.vn

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3.2.2.1. GII THIU V ACID ACETIC (ETHANOIC ACID) C2H4O2 CH3-COOH L carboxylic acid n gin nht v cu to M= 60,05 To to tinh th = 16,7oC n mn nhiu kim loi, gy bng da Gim n (acetic 4 8%)

3.2.2.2. VAI TR CA ACID ACETIC

ACETIC TRONG LNH VC THC PHM ACETIC TRONG LNH VC PHI THC PHM Nhu cu v acetic acid khong 6.5 triu tn/nm (Mt/a). Khong 4 Mt/a c sn xut t cng nghip ha du hoc t cc ngun sinh hc. Tnh ti 2006, VN nhp khu acid acetic 100%

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3.2.2.2. VAI TR CA ACID ACETIC (tt) VAI TR CA ACETIC TRONG THC PHM S dng lm cht ph gia thc phm (E260) Gim n

Nhiu nc quy nh, acetic acid dng lm gim phi c sx bng pp ln men. Mi v v ca gim ph thuc vo nguyn liu v cch sx.

3.2.2.2. VAI TR CA ACID ACETIC (tt) TRONG LNH VC PHI THC PHM CN ha cht: s dng nhiu trong thuc n Sx polyethylene terephthalate (s dng trong nc gi i kht ng chai) Sx cellulose acetate: s dng trong phim chp hnh Sx polyvinyl acetate: dng trong keo dn g S dng lm dung mi pha ha cht Sx vinyl acetate monomer dng trong sn Cng nghip ch bin cao su Tng hp cht do t si Trong gia nh: acetic long lm sch cn bn.

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3.2.2.3. C CHT SN XUT ACID ACETIC

Nguyn liu cha tinh bt Cellulose (s dng Clostridium lentocellum SG6 nghin cu ca H Osmania, n ) Nguyn liu cha ng Cn, ru vang

3.2.2.4. VSV LN MEN ACID ACETIC

VK hiu kh Sau 12h, sinh khi tng 17 triu ln Chuyn ha ru thnh acid acetic

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3.2.2.5. CNG NGH SN XUT ACID ACETIC PP HA HC (3) Methanol carbonization Butane oxidation Acetaldehyde oxidation PP SINH HC

3.2.2.5. CNG NGH SN XUT ACID ACETIC (tt) PP HA HC Cho ti 1960s, acetic acid c sx ch yu bng pp oxi ha cc ngun hydrocarbon. 1960s, PP sx acetic quy m cng nghip (methanol carbonization). 1970s, Monsanto ci tin pp ny, gim chi ph ph sx. Hin nay, s dng pp methanol carbonization, kt hp methanol v carbon monoxide to thnh acetic acid. Lng acetic sx t pp ny chim 80% tng sn lng

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CATIVA acetic acid plants in Hull, UK (left), and Chongqing, China (right)

At the heart of this success is BP's proprietary and trademarked CATIVA process for acetic acid manufacture, which, with a market share of more than 25 per cent of the 10 million tones of acetic acid produced globally each year, can justifiably claim to be leader of the pack.

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3.2.2.5. CNG NGH SN XUT ACID ACETIC (tt) PP SINH HC Pasteur pht hin ra VK Acetobacter ln men gim Ngi Php a ra cng ngh sx gim (pp ln men chm) Thi k chin tranh th gii th 2, ngi c ci tin v a ra cng ngh ln men nhanh.

3.5.2.5.1. Phng php ln men chm (PP Orleans)

3.5.2.5.2. Phng php ln men nhanh 3.5.2.5.3. Phng php ln men chm 3.5.2.5.4. Phng php kt hp

3.2.2.5. CNG NGH SN XUT ACID ACETIC (tt)

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3.2.2.5. CNG NGH SN XUT ACID ACETIC (tt)

S sn xut gim go bng pp ln men acetic

Gim n Cc yu t nh hng

Mi trng dinh dng Gim: B sung vo mi trng ban u acid ho mi trng, ngn chn s pht trin vi sinh vt c hi, b sung lng t bo nht nh. Ru ethylic: c s dng nh l mt c cht, t 2 10%V, > 10% s lm gim nng sut ln men. Cc cht C, N, P v cc nguyn t vi lng: cn b sung thm mt s mui khong nh: K2HPO4, KH2PO4, MgSO4.7H2O, FeCl3.6H2O, (NH4)2SO4, (NH4)2HPO4 ty theo nhu cu c th ca tng loi. B sung thm vitamin v cc cht kch thch sinh trng nh: glucoza, cao nm men, pepton...

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Gim n Cc yu t nh hng

nh hng ca oxy Cn lng khng kh ln cho qu trnh ln men gim. oxy ho ht 1 kg ru khan cn 2,3 m3 khng kh (theo l thuyt), hay oxy ho ht 1 mol ru cn 1mol O2. Qua thc t nhiu tc gi cho thy lng oxy cn thit phi ln gp i lng oxy ho theo l thuyt.

Gim n Cc yu t nh hng

nh hng ca nhit Acetobacter thuc nhm a m, Toop: 25 32oC nhit thp: qu trnh ln men gim xy ra chm. nhit cao: c ch s hot ng v n mc no s nh ch s sinh sn ca t bo lm tng tn tht cho vi khun v lm gim hiu sut qu trnh ln men do s bay hi acid acetic v ru etylic. nh hng ca tc nhn sinh hc (Ln gim, b gim, rui gim

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Gim n Cc h hng thng gp

Cc h hng thng gp (Gim b c, gim chua) Do oxy ho ho hc (oxy ho ru thnh CO2 v H2O) Do oxy ho sinh hc: tc nhn l nm men Comdida mycoderma, Acetobacter xylium, Acetobacter aceti, ln gim (2), b gim, rui gim (3) gy ra. Khc phc (1,3): v sinh thit b ln men sch, thanh trng Pasteur dch nhit 60 -70oC ri cho ln men li, cho thm vo K2S2O5 vi liu lng 5 15gam/100 lt gim kt hp vi thanh trng Pasteur tng bn ca gim, y kn thit b Khc phc (2): cho 1 2% NaCl vo gim thnh phm. Sau 1 2 ngy ln gim cht, lc hoc a nhit ln n 40 500C ri lc loi b ln gim.

3.5.2.5.1. PP LN MEN CHM (PHP - PP Orleans) Xut x: t 1670 vng trng nho ni ting ti Php (dch vang nho trong nhng thng g h np dn dn b c, c mng trng ph trn b mt cha VK Acetobacter orleaneuse v tr nn chua). Hin nay ch cn mt vi nc chm pht trin cn s dng phng php ny, trong c VN

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3.5.2.5.1. PP LN MEN CHM (PHP - PP Orleans) (tt) Nguyn liu: dch nho Ln men trong thng g si 250 300 lit V dch ln men: 1/2 - 2/3 V thng Mi trng ln men: 12 14oBx VSV: Ace. orleaneuse Thi gian ln men: 10 20 ngy 5 tun Hm lng acid acetic t c: 5 -6%

Thng ln men acetic theo pp Orleans (150 L)

3.5.2.5.1. PP LN MEN CHM (PHP - PP Orleans) (tt) Nhc im Thi gian ln men di Hm lng acid acetic thp Nng sut thp, kh c gii ha, hin i ha Tn din tch Sau 20 ngy, acetic b oxi ha (ha hc v sinh hc)

u im D thc hin Thit b khng phc tp Sn phm thm

Khc phc s oxi ha acetic Trong dd acetic phi cn 5% cn Thanh trng Pasteur B sung 0,2% NaCl tiu dit ln dm

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3.5.2.5.2. PP LN MEN NHANH (C) Xut x: Shiizenbachi tm ra nm 1923. Nguyn tc: to ra b mt tip xc ln hn gia VK Acetobacter vi khng kh oxy ha ru bng cch s dng vt liu xp (v bo, li ng xp trong thit b hnh tr, cn, nn ct) lm cht mang. Yu cu cht mang: c b mt tip xc ring ln, th tch t do ln, khi lng ring b, bn c hc cao, r tin, d kim, phi c nhm, xp vi sinh vt bm cht. u im: Thi gian ln men nhanh 5 6 ngy. Nhc im: Hiu sut kinh t cha cao (ru v acid l cht d bay hi), thit b phc tp, kh iu chnh thit b thng kh.

3.5.2.5.2. PP LN MEN NHANH (C) (tt) B1.Thanh trng cht mang B2. Gn VSV vo cht mang B3. Cho dch ln men tip xc vi VSV c nh

Nhc im Thi kh lm oxi ha ru v acetic

u im Thi gian ln men nhanh Hm lng acid acetic cao Thit b n gin t tn din tch

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3.5.2.5.2. PP LN MEN NHANH (C) (tt) Thng ln men g, hnh tr, H=2,5 6m; =1,2 3m; H= 2 C h thng thi kh t di ln trn MT ln men: Cn, 0,1 0,5% ng, P, N, K VSV: Ace. xylinum gn vo cht mang (mt ca, li bp) Tgian LM: 8 - 10 ngy Hm lng acid acetic t c: 8 -10% Thit b ln men gim (Vinegar Generator)

3.5.2.5.3. PP LN MEN CHM (Submerged Process) H thng ln men acetic c gi l acetator. Cho dung dch ln men vo thit b v thi kh rt mnh. Khi trong dung dch ln men s to ra th huyn ph v dung dch ln men. Hai th ny lun trn ln vo nhau v nh vy qu trnh oxy ho xy ra rt mnh lit. u im: hiu sut chuyn ho c khi t c 98 99%.

Thit b ln men chm

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3.5.2.5.3. PP LN MEN KT HP

Cu to h thng ln men: phn trn l lp m (lp cht mang cha VSV), lp gia l 1 thng cha dung dch sau khi ln men phn trn chy xung, di cng l h thng thi kh mnh, kh s c thi qua phn dung dch ny ri chuyn ngc ln phn trn (ging thit b ln men chm). u im: hiu sut ln men rt cao, nng acid acetic thu c thng nm trong khong 10 -12%. Nhc im: o thit b yu cu qu phc tp, cng knh. o ging vi khun phi ph hp

Bnh ln men hi lu, c sc kh ti PTN Vi Sinh H NL. VK Acetobacter aceti c c nh trn cht mang l b ma. C cht l dung dch nc xoi

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NNG CAO NNG ACETIC TRONG PP LN MEN PP CHNG CT BNG NHIT PP CHNG CT BNG MUI S dung mui c kh nng phn ly mnh (CaCl2 hoc CH3COONa). Hiu sut thu c s cao PP CHNG CT TRCH LY Chn cu t phn ly c bay hi nh hn cu t c trong hn hp cn chng ct. cu t phn ly + cu t c trong dung dch to thnh hn hp kh bay hi v thot ra di y thp chng ct PP CHNG CT CHN KHNG

3.2.3. Sx acid citric

Citric Acid Production - Recovery Based on Precipitation

C-Source
S-104 S-105 S-120

S-107 S-108

Water 1
S-106 S-103 S-102 PFLN-101 / PF-101 S-109 Plate & Frame Filtration HSRL-101 / ST-101 Heat Sterilization S-117 S-113 AFLN-102 / AF-102 S-119 Air Filtration

BLND-101 / V-101 Blending

Biomass

Nutrients

INX-101 / C-101 Ion Exchange

S-112
S-111 S-121

Water 2
STRG-101 / V-103 S-110 HSRL-102 / ST-102 Heat Sterilization BLND-103 / V-104 Blending FRMN-101 / F-101 Fermentation Storage Tank

S-101

RVFL-101 / RVF-101 Rotary Vacuum Filtration

Air
S-115 AFLN-101 / AF-101 S-118 Air Filtration CMPS-101 / G-101 CF Compressor

S-123 S-133 S-141 S-137 S-128 S-129

Sulfuric Acid
S-125

Gypsum
S-122

S-131

Product

S-139 S-126

Lime
S-124 RXN-101 / R-101 Product Precipitation

S-140 RDRNG-101 / RDR-101 Rotary Drying

S-138 RVFL-104 / RVF-104

S-134 RVFL-103 / RVF-103 CCRN-101 / R-103 Cont. Crystallization Rotary Vacuum Filtration RXN-102 / R-102 Gypsum Formation S-130

Rotary Vacuum Filtration

S-127 RVFL-102 / RVF-102

Rotary Vacuum Filtration

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3.2.3. Sx acid citric chi ph sx

Elements Raw material Depreciation and Maintenance Salaries Operation supply Lab./QA Waste treatment Aux. facilities Total $/kg 0,40 1,09 0,27 0,00 0,04 0,13 0,30 2,22
5,8% 1,8% 0,1% 12,0% 49,0% 13,5%

$/ao 3 949 000 10 866 000 2 668 000 19 000 400 000 1 290 000 3 003 000 22 195 000
17,8%

% 17,79 48,96 12,02 0,09 1,80 5,81 13,53 100,00

Materias primas Depreciacin Salarios Sum de operacin Lab/Aseg. Calidad Tratam. de residual Fac. auxiliares

Costo de produccin

3.3. Cng ngh sn xut acid amin 3.3.1. Sx Lysine 3.3.2. Sx Glutamic

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3.3. Cng ngh sn xut acid amin NG DNG CA ACID AMIN Thc phm Ha hc Y h c Cc lnh vc khc CC PP SX ACID AMIN THY PHN PROTEIN TNG HP HA HC CHUYN HA SINH HC LN MEN

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3.3.1. Sx Lysine
3.3.1.1. GII THIU V LYSINE 3.3.1.2. C CHT SN XUT LYSINE 3.3.1.3. VSV LN MEN LYSINE 3.3.1.4. LN MEN LYSINE 3.3.1.5. THU NHN V TINH SCH LYSINE

3.3.1.1. GII THIU V LYSINE L mt trong 8 aa khng thay th C nhiu trong tht, trng, sa, u nnh D b phn hy nhit cao C vai tr trong tng hp enzyme, hocmon, khng th, gip tng sc khng Yu cu: 1g/ngy/ngi ng dng: thm vo khu phn n ca tr em gip tr n ngon ming, hp th dinh dng tt, pht trin chiu cao; cht ph gia thc n; thc phm chc nng, dch truyn amin, b sung vo thc n gia sc

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3.3.1.1. GII THIU V LYSINE (tt) LYSINE (2,6-diaminohexanoic acid) ( amino acid) C6H14N2O2, M= 146.188, cu hnh L v D CH2 CH2 CH2 CH2 CH COOH | | NH2 NH2 1985, Nht Bn sx lysine qui m ln bng Corynebacterium glutamicum Tng sn lng trn TG: 1triu tn/nm

3.3.1.2. C CHT SN XUT LYSINE C cht: mt r, methanol, glucose. MT ln men Glucose: 50g (10 12%) tinh bt ng ( loi bo): 20 g Ngun nit: ure, cc loi mui amon hoc nc amoniac: (NH4)2SO4: 25g; Urea: 1 g Khong: mui Photpho 0,008 0,02mg/l; KH2PO4: 2.5g MgSO4.7H2O: 0.75g (0,03 0,5%) biotin 7,5g/l; treonin 40mg/l duy tr pH = 7.0 bng NaOH 2M Nhit : 28 300C.

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3.3.1.4. VSV LN MEN LYSINE Micrococcus glutamicum Brevibacterium flavum Brevibacterium lactofermentum Corynebacterium acetophilum Corynebacterium glutamicum Gleocladium sp Ustilago maydis Cc vi khun tham gia tng hp lysine u c kh nng ng ho glucose, fructose, maltose, saccharose. Khng c kh nng ng ho lactose, rafinose, pentose

3.3.1.4. VSV LN MEN LYSINE (tt) VSV Cyronebacterium glutacium b-6 C.glutacium H-8241 C.glutamicum C.lactofermentum AJ 12592 Brevibacterium lactofermentum AJ 12937 B.lactofermetum Bacillus methanolicus C cht R ng 32oC/48h Y (g/L) 100

10%Sucrose 32oC/72h 48 18%Glucose 27oC/70h 60 Glucose 31,5oC/48h 11,8 Glucose 31,5oC/58h 120,5

Glucose Methanol

32oC/72h 32oC/48h

48,8

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3.3.1.4. VSV LN MEN LYSINE (tt)

Sinh khi & cc sp lin quan trong sx lysine bng C. glutamicum ATCC 13287 theo thi gian khi nui cy theo m (mmol/mol) Sn phm Biomass Lysine Valine Alanine Glycine Glutamate 5.8 h 86.1 0.0 0.0 0.0 8.3 6.9 h 68.1 191.0 0.0 0.0 0.0 8.1 h 55.5 181.9 8.6 3.1 1.7 9.2 h 56.2 184.0 15.2 17.4 3.9 0.0

0.0 0.0 0.0 Corynebacterium glutamicum

SX Lysine t Methanol bng Bacillus methanolicus MG3: cht t bin Thi kh: 5,5L/pht Duy tr pH 7,1 (b sung NH4OH 8N) To: 50oC 1M KH2PO4: 0,1M MgCl2: 0,01M CaCl2 = 100: 10: 1 Duy tr nng Methanol trong bnh phn ng: 100mM

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3.3.1.4. LN MEN LYSINE Cng ngh sinh hc LM LLysine c ci thin khng ngng v s tin b v k thut to ra s lng ln lysine p ng nhu cu ngy nay Thit b ln men c cnh khuy v thi kh lin tc Qu trnh gm hai pha: 18 24 h u (Pha to thnh sinh khi) + 40 60h sau (Pha to thnh lysine)

Lysine C. glutamicum

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Asp: Aspactat Asp-P: beta Aspactyl phosphate ASA: Aspactate beta Sinialdehyde DAP: Dihydropicolinasyntetase AK: aspartokinase Lys: Lysine

Hom: Homogerin Thr: Threonin Ile: Ilexin Met: Methionine HSD: homoserine dehydrogenase

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Pilot-Scale Batch Production of High Lysine Content Animal Feed Suppliment (20h/m)

Pilot-Scale Continuous Production of High Lysine Content Animal Feed Suppliment

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Variant pathways for the synthesis of m-DAP/lysine in plants and bacteria. m-DAP/lysine biosynthesis genes present in chlamydiae are boxed

McCoy A. J. et.al. PNAS 2006;103:17909-17914

2006 by National Academy of Sciences

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3.3.1.6. THU NHN V TINH SCH LYSINE ly tm dch ln men, loi sinh khi v cht khng tan Dch sau khi ly tm qua h thng trao i ion cationit Ra ct nhiu ln cho ht mu v nh hp ph bng 0,5 5 % dung dch amoniac un nng dch cha lysine loi amoniac H pH 4,9 5 bng HCl, (to monochlohydrat lysine) C chn khng nng cht kh t 30 50% Kt tinh 10 120C(tinh th mu vng nht) Ly tm thu nhn tinh th Dch sau khi ly tm s ti kt tinh thu nhn trit lysine c trong dch ln men Ha tan trong cn, kt tinh li ( tinh sch cao) Sn phm: sch 97%; m: 0,7%; tro 0,3%

3.3.2. Sx Glutamic
3.3.2.1. GII THIU V GLUTAMIC 3.3.2.2. C CHT SN XUT GLUTAMIC 3.3.2.3. VSV LN MEN GLUTAMIC 3.3.2.4. LN MEN GLUTAMIC 3.3.2.5. CC YU T NH HNG 3.3.2.5. THU NHN V TINH SCH GLUTAMIC

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3.3.2.1. GII THIU V GLUTAMIC M = 147,13 To nng chy: 247 249oC Tan hon ton trong nc Khng tan trong cn, eter v mt s dung mi

3.3.2.1. GII THIU V GLUTAMIC (tt) VAI TR CA ACID GLUTAMIC Trong y hc v dc hc: nng thp, acid glutamic l cht an thn, thuc tr nhc u, c tc dng b no Trong thc phm: mui ca acid glutamic (glutamate) l cht iu v l tng M chnh (bt ngt) c s dng ti hu ht cc nc trn TG Sn lng khong 1,6 triu tn/nm. Cc dng sn phm: cht iu v 621, ht nm, bt ngt cnh to, bt ngt xay nh Hnh thc s dng: thc phm ch bin tm p bt ngt, b sung trc tip vo thc phm

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3.3.2.1. GII THIU V GLUTAMIC (tt) 1860, Ritthaussen, c, x thnh phn cc pr ng vt, cc a.a trong c a glutamic Woff, c, x trng lng phn t, cu trc v cc hng s vt l ca cc a.a 1889, Kikunae Ikeda (Nht) tch a. glutamic v iu ch natri glutamate t rong bin 21/4/1909, ng ng k patent s 9440 ti Anh: sx cht to v 1920, ngi Trung Quc cng tm c cng ngh sx glutamate

3.3.2.1. GII THIU V GLUTAMIC (tt) M chnh t nhin To Formate Ch xanh Mc C chua S Ng Khoai ty To Mg/100g 2240 1206 668 146 140 132 130 102 102 M chnh t nhin Bp ci N m u tng Khoai lang Tm Hn C rt Sa m Sa b Mg/100g 100 67 66 60 43 41 33 22 2

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3.3.2.1. GII THIU V GLUTAMIC (tt)

Bt ngt - cht iu v E621. Bt ngt c v Umami - l mt trong 5 v chnh m con ngi vn cm nhn trong ba n hng ngy cng vi chua, ngt, mn, ng. "iu v: c php s dng trong thc phm gip iu ha, lm tng v ngon ca mn n, khng cung cp dinh dng Cht iu v c php s dng: E621, E627 (disodium inosinate) v E631 (disodium guanylate) 627 v 631 ("siu bt ngt) c "ngt" cao hn nhiu so vi 621.

3.3.2.1. GII THIU V GLUTAMIC (tt) 1970s, JECFA - y ban hn hp v Ph gia thc phm ca T chc Y t th gii (WHO) v T chc Lng nng (FAO) nh gi tnh an ton ca m chnh v a ra quy nh v lng s dng hng ngy: 0-120mg/kg th trng 1987, Hi ngh ln th 31 ca JECFA nh gi li an ton ca m chnh v a ra kt lun liu dng khng xc nh Ro cn ngn Glutamate xm nhp vo no tr: Ro cn rut Ro cn nhau thai Ro cn no Tr em tiu ha m chnh ging ngi ln nn khng tm thy mi nguy hi no i vi tr em

n bt ngt c hi khng?

Tr em n bt ngt c hi khng?

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3.3.2.2. C CHT SN XUT GLUTAMIC Nguyn liu cha tinh bt: sn, ng, go Nguyn liu cha ng: mt r, dch tinh bt thy phn. Khi s dng r ng phi s dng cht khng biotin kim sot sinh trng ca VSV

3.3.2.2. C CHT SN XUT GLUTAMIC (tt)

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3.3.2.2. C CHT SN XUT GLUTAMIC (tt) MT 1 Mt r: 20%; Urea: 2%; K2HPO4: 0,05%; MgSO4.7H2O: 0,03%; CaCO3: 1%; pH: 6,8 7,8 MT2 Saccharose: 8,5 - 10% Urea: 0,5% K2HPO4: 0,1% MgSO4.7H2O: 0,1% ZnSO4: 0,1% Dch chit bp: 0,25% (nhn t sinh trng) pH 7 - 8

3.3.2.3. VSV LN MEN GLUTAMIC Brevibacterium Micrococcus Arthrobacter Brevibacterium flavum Corynebacterium glutamicum Corynebacterium glutamicum Ging t nhin: nng sut 0,2g/L Ging chuyn gen: nng sut 135 g/L (nm 1960) Ajinomoto: s dng ging siu tng hp: 170 180 g/L

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3.3.2.4. PHNG PHP SN XUT GLUTAMIC

3.3.2.4.1. PP HA HC: (thy phn protein t bt m, 3.3.2.4.2. PP SINH HC: (ln men)

3.3.2.4.1. PP HA HC

Sx glutamic v bt ngt bng pp ha hc

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3.3.2.4.2. PP SINH HC: (ln men) PHNG PHP LN MEN Ln men tnh Ln men tnh c b sung c cht (khng thng dng) Ln men tnh (V = 100m3) T l ging: 5 6% To: 28 30oC (c iu nhit) pH 7 - 8 Sc kh: 40 85mg O2/L.min. nu khng sc kh th sn phm to ra lactate. Thi gian: 40 60g Sn phm: 100 150 g/L

1957, Nht sx glutamic theo quy m cng nghip bng pp ln men

3.3.2.4.2. PP SINH HC (tt)

Men ging l Corynebacterium glutamicum

Sx glutamic v bt ngt bng pp ln men t nguyn liu tinh bt

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Sx glutamic v bt ngt bng pp ln men t glucose

FBP: Fructose 1-6 Biphosphate GAP: Glyceraldehyde 3 phosphate PEP: phospho enolpyruvate AcCoA: acetyl coenzyme A IsoCit: IsoCitrate KG: alpha ketoglutanate Glu: glutamate Suc: Succinate Fum: Fumarate Oxa: oxaloacetate

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3.3.2.4.2. PP SINH HC (tt)

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3.3.2.4.2. PP SINH HC (tt)

3.3.2.4.2. PP SINH HC (tt)

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3.3.2.5.YU T NH HNG TRONG PP LN MEN

Cung cp oxy: (hiu kh bt buc)

pHop = 6,8 8 iu chnh pH bng NH4+ (ure, nc NH3, kh NH3, NH4Cl) Toop: 26 37oC Thc t: Giai on u: 30-32oC Giai on sau: 36-37oC

Thiu oxy: sn phm ch yu l acid lactic Tha oxy: sn phm ch yu l acid alpha xetoglutavic PP cung cp oxy Sc khng kh v trng kt hp vi khuy trn (150 vng/pht) S dng cht ph bt trong qu trnh ln men

3.3.2.5.YU T NH HNG TRONG PP LN MEN (tt) Cht kch thch sinh trng biotin (2 5g/l) Ngun cung cp biotin: cao ng, mt r Khng nh hng n kh nng tng hp glutamic nh hng n tnh th m thu ca mng t bo, gip glutamic thot ra khi tb khuych tn vo mi trng Tha biotin: sinh tng hp acid glutamic t Tha biotin + sc kh t: sn phm ch yu l alanin v acid lactic Khc phc: b sung 0,15% Tween 60, penicillin 5UI/mL, km hm s pht trin ca VK trong mt giu biotin ng thi tng kh nng tng hp acid glutamic

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3.3.2.6. THU NHN V TINH SCH GLUTAMIC

Cc phng php kt tinh glutamic t mi trng nui cy PP im ng in PP to hydrochloxit ca acid glutamic PP dng dung mi hu c PP trao i ion PP chuyn acid glutamic thnh cc mui kim loi

3.3.2.6. THU NHN V TINH SCH GLUTAMIC (tt)

PP to hydrochloxit ca acid glutamic

Dch MT sau ln men C c 50 60oC Ty mu

pH 3.2 To 15oC

PP d thc hin Hiu sut thu nhn glutamic t 60%

HCl

Kt tinh glutamic

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3.3.2.6. THU NHN V TINH SCH GLUTAMIC (tt)

PP trao i ion

Dch MT sau ln men Thit b t tin Ct trao i cation (gi li tp cht) Ct trao i anion (gi li glutamic) Dung dch m Ra ct anion Hiu sut thu nhn glutamic cao

3.3.2.6. THU NHN V TINH SCH GLUTAMIC (tt) M chnh non (t) Tinh th glutamic
Nc

Ly tm Ty mu Kt tinh
Mm

Na2CO3/ NaOH

Trung ha HCl
Na2S + C2H2O4

C c n 30,2 30,5 Be
65oC

pH 6,8

Kh Fe2+, Ca2+

Ty mu
60oC

Than hot tnh

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3.3.2.6. THU NHN V TINH SCH GLUTAMIC (tt)

Sn phm bt ngt: Hiu sut thu hi: 70% - 80% khi lng glutamic Cc ch tiu cht lng ca bt ngt: Tinh th ln, ng u, m Lng gluNa: 98 -99% m: <0,5% NaCl <0,5% Cc tp cht cn li khng cha asen, kim loi v hp cht canxi

SN XUT BT NGT TI VN Canh trng sau ln men + NaOH Glutamate Natri

Tinh sch

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3.4. CNG NGH SN XUT SINH KHI VSV 3.4.1. k c bn sx sinh khi VSV 3.4.2. Sn xut sinh khi nm men 3.4.3. Sn xut SPC single protein cell

3.4.1. iu kin c bn trong k thut sx sinh khi VSV

Ging VSV

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Chn phng php thch hp cho qu trnh nui cy Nguyn liu cho sn xut Thit b v phng php thu nhn, tinh sch ph hp

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3.4.2. SN XUT SINH KHI NM MEN

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3.4.2.1. Nguyn liu sx sinh khi nm men 3.4.2.2. Cng ngh sn xut sinh khi nm men 3.4.2.3. Cc sn phm sinh khi t nm men

3.4.2. SN XUT SINH KHI NM MEN 3.4.2.1. Nguyn liu sx sinh khi nm men

Mt r (r ng) Nc: t tiu chun nc cp thnh ph Nguyn liu cha nit: ure, D.A.P Nguyn liu cha photpho: P2O5

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3.4.2.1. Nguyn liu sx sinh khi nm men MT R

MT R
SACCHAROSE BIOTIN MU SM H KEO

38 42%, C cht pht trin sinh khi

Kch thch s sinh trng ca nm men

Cn tr s ha tan ca oxy

nh hng ti qt sinh sn ca men Ngn qu xm oxy cn trnh nhp

X L MU V H KEO TRONG MT R
PP NNG Mt r 80oBx+ H2SO4 (1 tn mt r : 3,5 kg H2SO4 ) Khuy lin tc 85oC/6h Khuy lin tc 28 32oC /24h PP LNH

Ly tm hoc lng Dch trong Pha ch mi trng nui men

2 4% ng; 0,15 0,25% urea; 0,25 0,35% D.A.P

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3.4.2.2. Cng ngh sn xut sinh khi nm men

Mt r ng
Sac. cerevisiae
H2SO4 Urea, D.A.P Thi kh

X l mu v h keo To mi trng nui cy Ln men

28 32oC 16 22h Men kem 3- 5%

Nhn ging

Thu sinh khi Thu nhn sp trong TB Ly tm, Ra nc Sy

Men paste

TP cho ngi

Men kh

Diagrammatic Representation of Commercial Production of Single Cell from Hydrocarbons

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CC THNG S TRONG THIT B LN MEN Fermenter c dng thng: H = 2 Fermenter cn c cnh khuy v h thng cung cp kh. Tc khuy: 50 300 vng/pht Kch thc l thng kh = 10 ln kch thc t bo u khng kh vo phi gn mng lc kh. u khng kh ra phi cho kh qua dung dch KI 10% ri lc bng bng thy tinh trnh kh ra c hi du diezen khi chy bng my nn. T l Khi lng khng kh: Khi lng dung dch: Thi gian = 1:1:1 (m3 oxy: m3 th tch: 1 pht)

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3.4.2.3. Cc sn phm sinh khi t nm men MEN BNH M - STARTER Men kem (Men lng) Hot tnh mnh Thi gian BQ ngn BQ lnh Thng mi kh Men p: m =75 % Hot tnh gn bng men kem Thi gian BQ: 10 20 ngy BQ lnh D vn chuyn, thng mi ha c Men kh: m < 10% Thi gian BQ lu. D vn chuyn D thng mi ha Hot tnh khng mnh bng men kem, men p. Men d cht do tc ng nhit khi sy

3.4.2.3. Cc sn phm sinh khi t nm men THC N GIA SC

Sinh khi nm men Sinh khi nm men lm thc n gia sc lm men bnh m Quy trnh cng ngh Ging nhau Mc v sinh Yu cu cao Khng yu cu cao Thi im thu nhn u phase cn bng Cui phase cn bng sinh khi

Ch tiu

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3.4.3. Sn xut SCP single cell protein

3.4.3.1. Thut ng SCP 3.4.3.2. L do sx SCP 3.4.3.3. Nguyn liu sx SCP 3.4.3.4. SCP t vi khun 3.4.3.5. SCP t nm men 3.4.3.6. SCP t nm mc 3.4.3.7. SCP t nm qu th 3.4.3.8. SCP t to

3.4.3.1. Thut ng SCP Thut ng SCP c dng ln u tin ni v cc dng Pr thu nhn t nm men (c th VSV n bo) Protein Protein a bo Protein V Protein TV Protein n bo Protein c ngun gc t VSV
- Nm men - Spirulina

- Casein - Globulin

SCP

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3.4.3.2. L do sx SCP Nhu cu Protein: 70 100g Pr/ ngi/ngy Thiu Protein HNG GII QUYT (5) 1. Nng nghip 2. Chn nui 3. Khai thc bin 4. Tng hp ha hc 5. Sn xut v s dng SPC
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Sn xut Pr t nm men: Sac. cerevisiae, Candidas utilis, Tropicalis utilis Sn xut Pr t to: Chlorella, Spirulina platensis, Slenedemus Quy m sx: quy m ln, c li ch kinh t cao

3.4.3.2. L do sx SCP (tt) U IM CA SCP Hm lng Pr: SCP cao hn Pr V v Pr TV Cht lng Pr: SCP tng ng Pr V, cao hn Pr TV VSV dng nguyn liu r tin Sx SCP khng ph thuc vo kh hu, iu kin a l C th sn xut theo quy m cng nghip NHC IM SCP Khng ph hp vi 1 s tnh cht thc phm: mu, mi, c l C mt s t bo VSV c th ngi khng th hp thu

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3.4.3.3. Nguyn liu sx SCP whey, sulphate waste liquors, hydrocarbon waste from the pet petroleum industry, and the vats used to produce alcoholic beverages. The production process involves growth of the organisms in large fermenting tanks with forced aeration for vigorous cell-growth. Manufacture process used by British Petroleum Industry for single-cell protein from hydrocarbons is represented in Fig. 17.7. :
VSV VK Hygrogenomonas, Cellulomonas, Pseudomonas Candida spp., Saccharomyces fragilis, Torula sp., Rhodotorula spp Aspergillus terreus, Trichoderma reesei, Penicillium spp. Scenedesmus acutues, Scenedesmus acutues, Spirulina, Chlorella Nguyn liu Hydrocarbon wastes from petroleum industry. Whey, ethyl alcohol, starches, n-paraffins, sulphite waste, etc. Paper-pulp, starches, coffee wastes, straw, bassage, etc. culture ponds

Nm men

Nm mc To

3.4.3.4. SCP t vi khun Cc ging thng c s dng u c kh nng ng ha cc alkan (C6 C18) Phi tinh sch v ch bin lm thc n cho ngi
C cht Metan Hydocacbua (du t m) VSV Methylomonas methanica Methylococens capsulatus Pseudomonas, Corynebacterium Mycobacterium Brevibacterium

Nui Mycobacterium trn 1kg metan thu c 1kg sinh khi c hm lng Pr 60 70%; Gl 20 40%; L 2 4%; khong 2-6%, cc vitamin, c bit l B12

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3.4.3.5. SCP t nm men 3.4.3.5.1. c im SCP t men

Hm lng Pr ln: 60 65% Pr nm men l Pr hon ho, tng ng Pr tht Khng cha c t Nga: sx trng c hi nhn to t Pr nm men Anh, Php: b sung 40% Pr nm men vo nc chm Maggi

Candidas utilis

Trong chin tranh th gii 2, c sx Candidas utilis nh 1 ngun thc phm thay th cho Pr. SCP t Can. utilis c FDA cho php s dng. c b sung trong thc phm, nhiu nc trn th gii s dng n trong CBTP

3.4.3.5.2. PP thu nhn cc sn phm trong t bo nm men lm thc phm cho ngi PP ha hc PP vt l PP sinh hc PP kt hp

PP HA HC Thy phn bng acid hoc kim Ph hy a.a trong t bo, gi m gi tr dinh dng Sn phm thu c khi s dng kim: 50% a.a dng L + 50% a.a dng D (c th ngi khng hp thu c) Thit b phi chu c acid v kim c hi

PP VT L Sock nhit: ng lnh sinh kh i VSV -20oC -40oC/2-4h ri a ngay v 40 45oC. Lm sock nhit 3 ln lin tip s ph v c 100% t bo S dng nng lng: l viba, hng ngoi, t ngoi Nghin: t s dng khi sn xut ln

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3.4.3.5.2. PP thu nhn cc sn phm trong t bo nm men lm thc phm cho ngi (tt)
PP SINH HC Thy phn bng enzyme ngoi bo v ni bo C ch s dng enzyme ngoi bo: glucanase hoc dng hn hp cellulase ph v thnh t bo C ch s dng enzyme ni bo (t phn): to k cho enzyme c sn trong t bo t phn hy tb bng cch iu chnh to, pH Thng s k thut trong qu trnh t phn T l nc: sinh khi men paste= 1,8 -2 lit : 1kg Duy tr pH 4,5 5 Duy tr To = 48 52oC Thi gian: 10 -12h Sau qu trnh t phn, sy thu bt Pr m c (60% Pr). B sung bt Pr vi cc sn phm thc phm khc

3.4.3.6. SCP t nm mc
www.gbd.edu.vn Nm si thng sinh trng chm hn nm men.

Thi gian th h: khong 10h Hm lng Pr trong nm si thp: khong 30% Cc loi nm dng sn xut SPC trong thc phm: - Kt hp Trichoderma viridea v Sac. cerevisiae - Kt hp Trichoderma viridea v Candida utilis -Kt hp Endomycopsis v Candida utilis D tch sinh khi, hng v c bit

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3.4.3.7. SCP t nm c qu th Cc loi nm c qu th: nm m, nm mo, nm rm, Morchella (Mor. Crassique, Mor. esculata, Mor. Hortesis nui Morchella rt tn km v d b tp nhim) Cung cp 30 50% Pr Kh cng nghip ha Mi trng nui cy r tin: rm, r, cy mc, mt ca, ph thi, v chui, v c ph, r ng SPC t nm ln sx nhiu : c 3000 tn/nm, i Loan, Indonexia

Mor. esculata

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Cc hnh nh trng nm ti Bc Kinh -TQ

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Cc hnh nh trng nm ti Bc Kinh -TQ

Cc hnh nh trng nm ti Bc KinhTQ - Mr Qun

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VSV t dng quang nng Cn cung cp nh sng, mi trng nui su 20 -30cm. T tng hp cht hu c Tng sinh khi nhanh. Kch thc ln, thun li cho vic thu nhn Khng b VSV khc tn cng.

3.4.3.8. SCP t to iu kin sinh trng, c im SPC t to pht trin ca to Hm lng Pr cao: 70 75%
cht kh Pr to l Pr hon ho, cht lng cao Khng c c t nguy him tn ti trong sinh khi to

To Spirulina

1963, Brandily tm thy ti h Tchad (Chu Phi) 1968, hi ngh quc t (Thy in) bn v li ch v cch nghin cu sn xut to Spirulina lm thc n cho ngi, c bit l tr s sinh, vn ng vin 1980, cng b rng ri cc gi tr dinh dng v chc nng sinh hc ca Spirulina Php, M, Nht, Canada, Mehico, i Loan 1985, Spirulina c a vo VN.

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To Chlorella

Sng trong nc ngt

Chlorella (to n bo, lc tiu cu)

Quy trnh xn xut SPC t to Chlorella

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Quy trnh xn xut SPC t to Spirulina

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Spirulina c nui trong ao su 30 cm

1ha b mt thu nhn 10 15 tn to/nm

Thnh t bo Hnh thi TB Pr (%) Glucid (%) Lipid (%) Tro (%) Khong (Ca, P, Fe, Na, Cl, Mg, Mn, K, Vitamin (B3, B12, B6, B1, E, caroten, acid folic) Cc acid amin thit yu Net protein utilization - N.P.U

Spirulina Mng 5m *2mm, xon 60 - 73 4,7 - 6 7-8 4-5 6 Nhiu Nhiu 80-85%

Chlorella Dy Trn 40 -50 25 35 10 -15 10 -34 4-5 t t hn Thp hn

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c im nui cy pH To nh hng ca chu k sng ti PP thu hoch

Spirulina Chlorella Ni trn b mt Khng khuy s chm pHop = 8,5 9 To op = 35 t b chi phi n gin pHop = 5,5 7

To op = 35
B chi phi Phc tp

TNH HNH SX V S DNG TO TRN TG Cc b tc Chu Phi v Chu M la tinh s dng Spirulina lm nc chm, nu cho, ch bin thc n. B sung Pr cho ngi v gia sc, thc phm chc nng X l mi trng Cc nc sn xut to theo quy m cng nghip: M, Mehico, i Loan, Nga, Tip, c, Bungari, Nht, Myanma, Thi Lan, n , TQ.

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TNH HNH SX V S DNG TO TI VN 1985-1995, TS. Nguyn Hu Thc nghin cu ti "Cng nghip nui trng v s dng to Spirulina". Bs Nguyn Th Kim Hng (HCM) "Nghin cu sx v s dng thc n c to Spirulina trong dinh dng iu tr Mt s a phng nui trng to Spirulilla: Vnh Ho (Bnh Thun), Chu Ct, Lng Sng (Thun Hi), Su i Ngh (ng Nai), c Min (c Lc). Cng ngh: nui to trn cc b nng xi mng s dng kh CO2, ngun CO2 ly t cc nh my bia, cn, runn ha lng vo bnh cha.

SPIRULINA FARM: Production, harvesting, drying and conditioning of micro algae spirulina, located in Guanacaste, the only one in C.A., 5 hectares land or 12.35 acres, culture size is 2000m2, production of around 3.5 tons per year with capacity to 5 tons, 8 productions pools of 250m2 each covered with greenhouses, full working equipments, independent home of 130m2, 60 meters deep well and more. International clients from Germany / Switzerland / France, classification as one of the best production and product over the world. USD $954,000

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D n SEVAS nui to n

Ao xi mng nui to Komatipalli, South India D n SEVAS

Spirulina Packets: (Powder) 100 gms - Rs. 200 ($ 5) One Kilo - Rs. 2000 ($ 50) Spirulina Tablets: 100 Gms - Rs. 250 ($ 6.5) One Kilo - Rs. 2500 ($ 65) 25 % of total amount will go to SAVAS Spirulina project

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B sung sn phm to Chlorella vo sn phm to Spirulina lm tng kh nng o thi kim loi nng khi c th.

150 vin/hp,70.000/hp; 15 vin/ngy

186.000 VND/ hp

3.5. Cng ngh sn xut polysaccharide t VSV

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www.gbd.edu.vn

3.6. Cng ngh enzyme 3.6.1. Cc khi nim c bn 3.6.2. Ngun thu nhn ch phm enzyme 3.6.3. Sn xut enz t VSV 3.6.4. C nh enz

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3.6.1. Cc khi nim c bn Enzyme: cht xc tc sinh hc v n c tt c cc t bo sinh vt Tnh cht enzyme - c hiu - Kh nng xc tc cao - Ch hot ng Top, pHop Ch phm enzyme: sp thng mi hay hng ha, sp c hot tnh enz

Sn phm enzyme C 1 enz vt tri so vi cc enz khc T hp nhiu enz

Pht hin thy > 2000 enz. 200 ch phm enz c s dng nhiu Cc sn phm c ng dng r rng km theo khuyn co v cch s dng. Cc ch phm ph bin: ch phm enz thy phn: protease, amylase, pectinase.

3.6.1. Cc khi nim c bn (tt) Enz ngoi bo (exoenzyme): chim a s, vsv tng hp enz ri y ra ngoi t bo cht Enz ni bo (endoenzyme): vsv tng hp enz ri gi li trong t bo cht Enz bn th (constitutive enzyme): vsv tng hp enz, m khng ph thuc vo iu kin mi trng v iu kin nui cy Enz cm ng (inductive enzyme): thng l enz ni bo. Cm ng ph thuc vo thnh phn thc n trong mi trng. Khi mi trng c cht cm ng, vsv ch sinh tng hp nhng g cn thit, vi hm lng cho n s dng

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3.6.1. Cc khi nim c bn (tt) N V HOT CA ENZYME IU: 1 n v hot IU l lng enzyme cn thit : xc tc chuyn ha 1 mol c cht hoc xc tc to thnh 1 mol sn phm trong 1 pht cc iu kin xc nh (pH, nhit , nng , c cht) Mt n v hot ca Termamyl 120L ( amylase) l lng enzyme cn thu phn 5,26 g tinh bt trong mt gi (cht nn l tinh bt ho tan, Canxi 0,0043M trong 720 37oC ti pH 5,6) Lng enz xc tc cng thp th hot tnh enz cng cao

3.6.2. Ngun thu nhn ch phm enzyme VSV: cc VSV ny c xp loi l VSV an ton GRAS (generally recognized as safe) Thc vt ng vt

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3.6.3. Sn xut enz t VSV 3.6.3.1. Cc VSV s dng trong sn xut enzyme 3.6.3.2. Quy trnh sx enz t VSV 3.6.3.2.1. Thu nhn sinh khi th 3.6.3.2.2. Ph v t bo 3.6.3.2.3. C c v bc u tinh sch enz 3.6.3.2.4. Tinh sch enz 3.6.3.2.5. B sung cht bo v, n nh enz 3.6.3.3. Cc yu t nh hng ti sx enz t VSV 3.6.3.4. nh gi cht lng ch phm enz

3.6.3.1. Cc VSV s dng trong sn xut enzyme

Chng loi: Hot tnh Gii php ci thin hot tnh Thi gian thu nhn Gi thnh Kh nng pht trin cng ngh

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3.6.3.1. Cc VSV s dng trong sn xut enzyme (tt)

Enzyme VSV Substrate Use Amylases Asp. Wheat Starch hydrolysis to dextrin and sugars oryzae bran mainly in alcoholic fermentation, food preparations & fabric; in preparing adhesives; in designing textiles; in clarifying fruit juices; in manufacturing medicines. Enzyme Microorganism Substrate Use Pectinase Aspergillus, Wheat In clarification, pressing, Penicillium sp. bran filtration and concentration of fruit juices and wines; in hydrolyzing pectin in the retting of flex for the manufacture of line in coffee-bean fermentation.

3.6.3.1. Cc VSV s dng trong sn xut enzyme (tt)

Enzyme VSV Substrate Use Invertase Sac. Sugarbeet In hydrolyzing sucrose to glucose cerevisiae and levulose; in producing non crystallization syrups from sucrose which is partially hydrolysed by this enzyme; in candy making; in producing artificial honey. Enzyme Microorganism Substrate Use Cellulase Trichoderma Cellulose Digestive add; in cellulose reesii hydrolysis to cellobiose; in cheese making. Lipase Rhizopus sp. Wheat Digestive add; bran in hydrolyzing fat into fatty acid and glycerol.

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3.6.3.1. Cc VSV s dng trong sn xut enzyme (tt)

Enzyme VSV Substrate Use

Proteases Asp. niger, Wheat Bacillus spp. bran

In leather processing; ion food processing; in manufacture of liquid glue;in degumminga of silk; in clarification of beer protein haze and as an adjunct to soap for cleaning in laundries; in tenderizing meat; in recovery of silver from spent film (photography).

Protease c s dng nhiu nht. Protease dng trong nc tng, nc mm phi chu c acid, mui. Protease dng trong bt git phi chu c kim

3.6.3.1. Cc VSV s dng trong sn xut enzyme (tt)

Enzyme Microorganism Substrate Use Takadiastase Aspergillus spp. Wheat Strach dextrinization bran and designing of textiles; bread supplement; in production of syrups; in digestive aid Rennin Bacillus spp. Cheese production. (via genetic engineering)

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3.6.3.2. Quy trnh sx enz t VSV

3.6.3.2. Quy trnh sx enz t VSV (tt)

How are enzymes manufactured?

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VSV Ngun enz ni bo

Ln men

3.6.3.2. Quy trnh sx enz t VSV (tt) Ngun enz ngoi bo

Thu nhn tb (Ly tm/lc) Ph v tb (ng ha)

Loi b tb (Ly tm/lc)

Loi b vch, mnh v tb (Ly tm/lc) C c (Siu lc/ta) B sung cht bo v, cht n nh
Sy phun Enz lng Enz bt

3.6.3.2. Quy trnh sx enz t VSV (tt) LN MEN B MT (VN) (Solid state fermentation) u: u t t

PP LN MEN

LN MEN B SU (TG) (Submerged fermentation) u: Lng c cht st thp Nhc: din tch ln Canh trng thu nhn c T ng ha, c gii ha l t hp ca nhiu enzyme Khng cn lao ng th cng p dng ph bin Thu nhn 1 enzyme vt tr i so vi cc enzyme khc Hot tnh enzyme cao (tnh theo hm lng c cht trong mi trng hoc theo g cht kh ca canh trng) Fermenter 5L

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Thu nhn sinh khi enz th

Enz ngoi bo Enz ni bo Ta

X l dch ln men Thu sinh khi Ph v t bo Loi b vch, mnh v tb

C c v bc u tinh sch enz

Siu lc Thm tch

Tinh sch enz = k thut sc k

C c

3.6.3.2. Quy trnh sx enz t VSV (tt)

3.6.3.2.1. Thu nhn sinh khi enzyme th

CANH TRNG LNG Enz ngoi bo: lc, ly tm, thu phn lng, dch enz th l canh trng lng Enz ni bo: ly canh trng , ly tm, lc thu sinh khi, ph v mng t bo trch ly enzyme. Ly tm, lc, dch lng l enzyme th

CANH TRNG RN (thu nhn enz ni bo, p dng cho nm mc v x khun) Ly canh trng em nghin ph v thnh t bo (nu canh trng ti xp th khng cn nghin), trch ly bng nc mui 0,9%, lc, ly phn lng.

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3.6.3.2.2. Ph v t bo PP PH V THNH T BO VSV VT L: nghin bng bi thy tinh 0.2 0.3mm, nn p lc HA HC: trn tb vi cc cht oxi ha kh mnh nh benzen, hexan, to thnh l thng trn mng tb Nhc im: cc dung mi hu c, cht ty ra c th lm v hot, bin tnh enz SINH HC: t phn, enz HA SINH KT HP

3.6.3.2.3. Loi b vch, mnh v tb 1.Tch acid nucleic v lipid: S dng nuclease tch nucleic. Tch lipid bng ct lc nhi vi hoc bng thy tinh c xp nh 2. Ly tm: 30.000 v/p trong 45 u: thch hp tch enz khi tb, mnh v tb Nhc: chi ph cao 3. Tch dch 2 lp 4. Lc Lc su: depth filter: gi tb, mnh v tb trn b mt mng v su bn trong mng Lc mng: gi tb, mnh v tb trn b mt mng. 0.2 - 10m Lc mng r, thao tc n gin nhng p nn, bt l lc, thng mng lc. C th dng mng nhiu lp, l lc gim dn

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3.6.3.2.3. Loi b vch, mnh v tb (tt)

Th t xp mng trong thit b lc mng (kch thc l mng lc t ngoi vo trong 5, 1, 0.45m)

Polymer polyethylence 3.6.3.2.3. Loi b vch, mnh v tb (tt) glycol (PEG)

Dextran Phase PEG cha enz

Phase dextran cha tb, mnh v PP tch dch 2 lp t s dng trong cng nghip

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3.6.3.2.4. C c v bc u tinh sch enzyme Dung mi hu c (ethanol, propanol, acetone) lm lch cn bng tng tc, gi m ha tan ca Pr. Tin hnh 4 10oC. Vdung mi/ Vdd enz = (2.5 3)/1 Mui (Na2SO4, (NH4)2SO4, NaCl) + nc lm mt lp v hydrate ca Pr. Cc Pr lin kt vi nhau, kt ta. Tch mui = pp thm tch Ta pI: ti pI, Pr c ha tan thp, hydrate ha ca Pr min lm tng tng tc gia cc Pr, dn n to ta

Ta

Polymer: c s dng rng ri. PEG MW 6000, 12000 t l 5 -15% w/v. Tin hnh 37oC. C ch ging dung mi hu c

3.6.3.2.4. C c v bc u tinh sch enz (tt) Thm tch: qt siu lc tch cc LMW (thnh phn m, peptid, mui, etylic, a.a) ra khi dd Pr. Tin hnh loi mui v lc trc khi thm tch. Lin tc b sung dung mi sch vo dd Pr ly nhng phn t c LMW ra khi dch nh qt thm thu. tch nhng cht tch in ra khi nhng cht khng tch in v ngc li

Thm tch

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3.6.3.2.4. C c v bc u tinh sch enz (tt) C dch enz = cht trao i ion C dch enz = siu lc

3.6.3.2.4. C c v bc u tinh sch enz (tt) C dch enz = cht trao i ion Nguyn l: iu chnh pH v lc ion to ta cha Pr gn kt vi cht trao i ion mang du ngc vi Pr. Ly tm tch ta, gii phng Pr khi cht trao i ion, ra cht trao i ion ti s dng Cht trao i ion (cht mang) Diethyaminoethyl (DEAE) Quaternary ammonium (Q) Quaternary aminonium (QAE) Carboxymethyl (CM) Methyl sulphonate (S) Sulphopropyl (SP) Kiu trao i ion Trao i anion Trao i anion Trao i anion Trao i cation Trao i cation Trao i cation

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3.6.3.2.4. C c v bc u tinh sch enz (tt) C dch enz = siu lc u: khng lm mt hot tnh Pr, hiu sut tch cao. Nhc: d b bt l lc Mng lc c 1 20nm tch Pr c MW t 1 300kDa. Vt liu mng siu lc: ch to t mng cellulose acetate hoc cellulose nitrate, polyvinyl, polycarbonate.

3.6.3.2.5. Tinh sch enz Tinh sch enzyme = k thut sc k

K thut sc k da trn tng tc ca Pr vi cht mang phn tch

Sc k lc gel (gel filtration) Sc k trao i ion (ion exchange chromatography) Sc k i lc (affined chromatography) in di (electrophoresis)

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Nguyn tc hot ng ca cc sc k thng dng K thut sc k Bn cht Sc k trao i ion Khc bit v in tch trn mt Pr gi tr pH quy nh Sc k lc gel Khc bit v hnh dng v MW ca phn t Sc k i lc Da vo tng tc c hiu gia Pr v ligand tng thch Sc k tng tc Khc bit v tnh k nc trn b mt k nc Pr Sc k ng in Tch theo im ng in ca tng phn t Pr Sc k trn nn Tng tc tng hp gia Pr v cht hydroxyapatide mang Ca-phosphate

3.6.3.2.6. B sung cht bo v, cht n nh Cc cht bo v: propylene glycol, glycerol, sorbitol. Dch enz c c: thng b sung thm ammoniumsulfate, sodium sulfate, sodium chloride to ta hoc tinh th enz. gi enz dng huyn ph b sung silicon dioxide

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3.6.3.3. Cc yu t nh hng ti sx enz t VSV CC YU T NH HNG TI QU TRNH LN MEN PP B MT M CHIU CAO MI TRNG NHIT CUNG CP OXY pH THI GIAN THIT B

3.6.3.4. nh gi cht lng ch phm enz

NH GI CHT LNG CH PHM ENZYME Hot tnh xc tc: s n v hot trong 1g/1mL ch phm Hot tnh ca E = s n v hot /g (mL) n v hot tnh ca enz: s lng enz c kh nng xc tc c cht/n v thi gian S n v hot t l thun vi hot tnh enz v tinh sch ca enz tinh sch: s n v hot c trong 1mg Pr ca ch phm (tnh theo Pr v bn cht ca enz l Pr) Cc enz k thut l sn phm th, hot tnh enz = 2 -5%Pr tng s. PP Cc thng s c nh Theo di 1 Thi gian Bin thin Nng enz ca S hay P 2 Lng S mt i (hay lng P to thnh) Thi gian Nng E 3 Thi gian Nng E Lng S mt i (hay lng P to thnh)

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3.6.4. C nh enzyme 3.6.4.1. Gii thiu chung v c nh enz 3.6.4.2. Cht mang gn enz 3.6.4.3. Phng php c nh enz 3.6.4.4. Cc dng bnh phn ng v enz c nh 3.6.4.5. Cc ng dng ca enz c nh 3.6.4.6. C nh enz v c nh t bo VSV

3.6.4.1. Gii thiu chung v c nh enz u im Khi nim: gn enz ln 1 cht mang tr ha hc, to enz khng tan trong nc Ti s dng nhiu ln (60 70 ln) m hot tnh enz gim khng ng k Khng ln enz trong sn phm C th gn ng thi nhiu enz ln cht mang thc hin chui phn ng. T c th xy dng m hnh h a enz trong t bo nghin cu C th c gii ha, t ng ha C th hi lu lp li phn ng nu sn phm cha t yu cu

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3.6.4.2. Cht mang gn enzyme Cht mang hu c Tng hp ha hc: bn vi tc ng MT v VSV Polymer: poly-acrylamid, poly-styren, poly-acetate Cht mang v c

bn vi tc ng MT, kh gn

Cc oxit kim loi T nhin: khng bn vi tc ng (Al, Mg, Ti), cao lanh, than hot MT v VSV tnh, si bng Dn xut cellulose (si bng, d m thy tinh bo, li ng), dn xut chitin, chitosan, collagen, tinh bt, alginate, oligosaccharide (dextran)

3.6.4.2. Cht mang gn enzyme (tt) Yu cu ca cht mang gn enzyme Gn cht vi enz, khng c tch ra khi enz trong khi phn ng D tm, gi r Khng lm thay i hot tnh ca enz Bn trong mi trng

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3.6.4.3. Phng php c nh enzyme PP vt l (lin kt vt l) s hp ph, to khun gel, bao bc mng bn thm PP ha hc (lin kt ng ha tr gia enz v cht mang)

3.6.4.3. Phng php c nh enz (tt) - PP vt l PP hp ph: trn cht hp ph vi enz. Enz hp ph trn b mt cht mang nh lin kt (ion, k nc, tnh in, Vander Walls) u: n gin, s dng nhiu cht mang khc nhau) Nhc: khng bn, nu thay i pH, To t ngt s gii hp ph (tch enz ra) PP vi ti (micro capsul): gn v nht enz trong mng bn th m. C th nht a h enz trong vi ti. ng dng: vi ti urease cha bnh thn, catalase cha co c. Gn vi ti enz vo cc c quan khng tng hp c enz, vi ti khng i theo mu.

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3.6.4.3. Phng php c nh enz (tt) - PP ha hc Gn cht mang qua cht hot ha trung gian (2 giai on). PP s dng ph bin. Thng s dng vi cht mang v c nh si thy tinh, si silicate G1: hot ha cht mang G2: Gn cht mang hot ha vi enz
Cht mang + Cht hot ha Cht mang Cht hot ha
+

Cht mang Cht hot ha Enz Enz

Cht mang Cht hot ha

3.6.4.4. Cc dng bnh phn ng v enzyme c nh Enzyme c nh l lng huyn ph Enzyme c nh trn mng mng, xp nhiu lp Enzyme gn trn si cellulose (xp cc si thng ng, b si) Enzyme c nh gn trn mng mng, kch thc ln, cun xon Lp enzyme c nh c chiu cao ng k, dy

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3.6.4.4. Cc dng bnh phn ng v enzyme c nh (tt) Thit b (Bioreactor) Thp hnh tr, h>> , h ln c thi gian phn ng gia c cht S vi enzyme E ng dn c cht nh, c van y tho sn phm, c th iu chnh V Cu to: Bnh c 2 lp v, gia c nc iu chnh nhit phn ng. ng hi lu ln nh thp.

3.6.4.5. ng dng ca enzyme c nh Sn xut fructose bng enzyme glucose isomerase c nh Sn xut L-acid amin Sn xut sa bo philactose Urease c nh dng vi ti cha bnh thn, phn gii urea S dng enzyme glucose oxydase nh lng nhanh glucose trong dch sinh hc Enzyme pectinase c nh

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3.6.4.5. ng dng ca enz c nh (tt)

Tinh bt amilase Dextrin MW nh amilase GA Glucose

Glucoseisomerase

Glucoseisomerase c tch t x khun Actinomyces Cht mang: silicrom (dn xut cellulose) Cht hot ha: glutaraldehyte

Fructose Tinh sch Dung dch giu fructose

3.6.4.6. C nh enz v c nh t bo VSV

C NH T BO VSV Khng phi tch chit enz, khng tn cng sc Cc enz hot ng trong mi trng t bo (dng t nhin), thun li, ph hp hn enz c tch chit ra Tn dng c a h enz trong t bo C kh ha, t ng ha, sn xut lin tc Ph hot ng ca enz trong t bo thng kh rng Lng enz c th tng ln khi t bo sng

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3.6.4.6. C nh enz v c nh t bo VSV (tt) Enzyme c nh T bo VSV c nh u gn ln cht mang C th ti s dng Cc cht mang dng cho enz c nh u c th dng cho c nh t bo VSV Thit b c th tng t nhau Cht mang gn trc tip Cht mang khng gn trc tip vi enz hoc gn ln cht vi enz m gn vi mng t bo hot ha) VSV. Enz trng thi t do, c th dch chuyn trong t bo Khng cn cung cp Cn cung cp dinh dng dinh dng nui enz nui t bo

ng dng ca c nh t bo VSV trong sx gim n

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3.7. Cng ngh sx cc sp ln men truyn thng

www.gbd.edu.vn

Hm ru ti Dynasty

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