Nghiên cứu điều chế Magie lactat bằng phương pháp sinh tổng hợp.pdf

B Y T

TRNG I HC DC H NI

NGUYN TH THU TRANG

NGHIN CU IU CH MAGIE

LACTAT BNG PHNG PHP SINH

TNG HP

KHA LUN TT NGHIP DC S

H NI - 2014

B Y T

TRNG I HC DC H NI

NGUYN TH THU TRANG

NGHIN CU IU CH MAGIE

LACTAT BNG PHNG PHP SINH

TNG HP

KHA LUN TT NGHIP DC S

Ngi hng dn:

1. TS. m Thanh Xun

2. DS. Nguyn Minh Ngc

Ni thc hin:

1. B mn Cng nghip dc

2. Trng i hc Dc H Ni

H NI - 2014

LI CM N

Vi s knh trng v lng bit n su sc, em xin chn thnh gi li

cm n ti c gio TS. m Thanh Xun v DS. Nguyn Minh Ngc,

nhng ngi trc tip hng dn, ng vin v gip em trong sut qu

trnh hon thnh kha lun ny.

Em cng xin gi li cm n chn thnh ti cc thy c gio v cc anh

ch k thut vin b mn Cng nghip dc chia s, ng vin, nhit tnh

gip v to iu kin thun li cho em trong sut qu trnh lm kha lun.

Em rt mong nhng kin ng gp ca thy c v bn b hon

thin hn nghin cu khoa hc ca mnh.

Em xin chn thnh cm n!

H Ni, thng 05 nm 2014

Sinh vin

Nguyn Th Thu Trang

MC LC

LI CM N

MC LC

DANH MC CC K HIU V CH VIT TT

DANH MC CC BNG

DANH MC CC HNH V V TH

T VN ....

Chng 1. TNG QUAN.

1.1. Magie lactat.

1.1.1. c im...

1.1.2. Cng dng.

1.1.3. Mt s sn phm lu hnh trn th trng.

1.2. Cc phng php sn xut magie lactat...

1.2.1. Phng php tng hp ha hc

1.2.2. Phng php vi sinh vt...

1.3. Vi khun sinh axit lactic.

1.3.1. c im nhm vi khun sinh axit lactic

1.3.2. Chi Lactobacillus..

1.3.3. Loi Lactobacillus acidophilus.

1.3.4. Sn phm axit lactic ca vi sinh vt..

1.4. Cc nghin cu v magie actat

Chng 2. I TNG V PHNG PHP NGHIN CU..

2.1. Nguyn vt liu, thit b.

2.1.1. Nguyn vt liu.

2.1.2. Thit b s dng.

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2.2. Ni dung nghin cu..

2.2.1. S b xy dng quy trnh ln men to magie lactat..

2.2.2. Kho st mt s thng s nh hng n qu trnh ln men

2.3. Phng php nghin cu...

2.3.1. Phng php nhn ging.

2.3.2. Phng php ln men sinh tng hp magie lactat....

2.3.3. Phng php tch magie lactat t dch ln men

2.3.4. Phng php nh tnh cc ion lactat v magie..

2.3.5. Phng php sc k lp mng nh tnh magie lactat dihydrat........

2.3.6. Phng php Schoorl Regenbogen nh lng ng

2.3.7. Phng php tnh hiu sut tiu th ng sinh magie lactat..

2.3.8. Phng php tnh ton v x l s liu.

Chng 3. THC NGHIM, KT QU V BN LUN...

3.1. S b xc nh quy trnh n men to magie lactat .

3.1.1. La chn thi im b sung MgCO3 ...

3.1.2. La chn phng php thu sn phm magie lactat..

3.1.3. nh tnh sn phm magie lactat thu c...

3.2. Kho st mt s thng s nh hng n qu trnh n men..

3.2.1. Kho st nh hng ca pH n qu trnh ln men..

3.2.2. Kho st nh hng ca nng glucose.

KT LUN V XUT...

TI LIU THAM KHO

PH LC

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DANH MC CC K HIU V CH VIT TT

ATCC American Type Culture Collection

C% Nng phn trm

DVN Dc in Vit Nam

E. coli Escherichia coli

FAO Food and Agriculture Organization

Glc Glucose

H Hiu sut

L. acidophilus Lactobacillus acidophilus

L. brevis Lactobacillus brevis

L. buchneri Lactobacillus buchneri

L. bulgaricus Lactobacillus bulgaricus

L. casei Lactobacillus casei

L. delbrueckii Lactobacillus delbrueckii

L. fermentum Lactobacillus fermentum

L. leichmenii Lactobacillus leichmenii

L. plantarum Lactobacillus plantarum

S. lactis Sacharomyces lactis

M Khi lng phn t

lch chun

Rf H s di chuyn

x Gi tr trung bnh

v Va

WHO World Health Organization

DANH MC CC BNG

Trang

ng 1.1. Mt s sn phm cha magie lactat trn th trng

Bng 2.1. Cc nguyn liu v ha cht

ng 2.2. Mi trng nhn ging

ng 2.3. Mi trng ln men

Bng 2.4. Cc thit b s dng

Bng 3.1. So snh kt qu tng hp magie lactat v canxi lactat

Bng 3.2. Kt qu tng hp magie lactat bng cch thm MgCO3 ngay

khi bt u qu trnh ln men

Bng 3.3. Kt qu tng hp magie lactat bng cch s dng MgCO3 khi

kt thc qu trnh ln men

Bng 3.4. Kt qu tng hp magie lactat bng cch b sung dn MgCO3

sau mi 24 gi

ng 3.5. Lng sn phm magie lactat thu c bng cc phng

php: ph u lc uchner, li tm thng, li tm lnh

Bng 3.6. Kt qa ln men L. acidophilus thu magie lactat cc pH

Bng 3.7. Kt qu ln men L. acidophilus thu magie lactat vi cc

nng glucose

Bng 3.8. Lng MgCO3 b sung sau mi 24 gi ln men vi nng

glucose 10%, iu chnh pH = 7

Bng 3.9. Lng magie lactat thu c sau 96 gi ln men vi nng


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DANH MC CC HNH V V TH

Trang

Hnh 1.1. L. acidophilus di knh hin vi quang hc

Hnh 1.2. L. acidophilus di knh hin vi in t

Hnh 3.1. Tinh th magie lactat

Hnh 3.2. S quy trnh x l dch ln men

Hnh 3.3. Vng mu lc xut hin khi nh tnh ion lactat

Hnh 3.4. Ta trng xut hin khi thm dung dch NH3 v tan khi

thm dung dch amoni clorid

Hnh 3.5. Dung dch trong sut sau khi thm dung dch amoni clorid

v xut hin ta kt tinh trng khi thm dung dch Na2HPO4

Hnh 3.6. Kt qu chy sc k lp mng: sn phm iu ch c,

vin magie 6, magie lactat iu ch bng phng php tng hp ha

hc (t tri qua phi)

Hnh 3.7. iu so snh hiu sut to magie lactat cc pH

Hnh 3.8. iu so snh hiu sut to magie lactat cc nng

glucose

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1

T VN

Cc sn phm ln men ca vi sinh vt , ang v ngy cng c ng

dng ph bin trong mi lnh vc ca i sng. Trong cc sn phm ca cng

ngh ln men vi sinh vt, axit lactic v mui lactat l nhng sn phm ra i

t rt sm v ng dng rng ri v th Lactobacillus acidophilus c quan

tm nghin cu nhiu. y l mt trong nhng chng vi khun c li v d

nui cy quy m cng nghip [4]. n cnh canxi lactat vn hay c nhc

ti th magie lactat cng l mt mui lactat c tnh ng dng cao trong y dc

phm v mang li li ch kinh t ln [1], [8].

Magie l khong cht cn thit cho s pht trin th lc v tr tu ca

con ngi nhng magie khng c to ra trong c th v cch cung cp

Magie duy nht l thng qua ch dinh dng hng ngy. Vic nghin cu

iu ch cc thuc c magie vn lun l mt vn quan trng v cn thit.

Trong magie lactat c s dng rt ph bin. Ngoi tc dng cha bnh,

magie lactat cn l tin sn phm trong cng nghip ln men sn xut canxi

lactat, axit lactic l nhng sn phm thng dng khng ch s dng trong

ngnh dc m cn trong cc ngnh cng nghip khc nh cng nghip thc

phm, m phm, cng ngh thng tin,[16], [22].

Trn th gii cng nghip ln men vi sinh vt sn xut magie lactat l

mt ngnh cng nghip cn ang pht trin. Vit Nam cng ngh ny cha

pht trin, nhng cng trnh nghin cu v kh nng ln men sinh tng hp

magie lactat cn cha nhiu. Vi mong mun c gp phn vo cc nghin

cu sn xut magie lactat, chng ti la chn ti:

Nghin cu iu ch magie lactat bng phng php sinh tng hp

Vi nhng mc tiu sau:

- S b xc nh quy trnh ln men to magie lactat.

- Kho st mt s thng s nh hng n qu trnh ln men.

2

Chng 1. TNG QUAN

1.1. Magie lactat

1.1.1. c im:

Cng thc tng qut: C6H10MgO6.

Tn khoa hc (theo IUPAC): Magie 2 hydroxyl propanoate.

Thnh phn: C 35,60%, H 4,98%, Mg 12,01%, O 47,42%.

Khi lng phn t: 202,45g/mol.

Cng thc cu to: gm cation Mg2+ v 2 anion L lactat.

Magie lactat l mui kt tinh ca axit lactic vi ion Mg2+, tn ti trong t

nhin dng dihydrat (C6H10MgO6.2H2O) v trihydrat (C6H10MgO6.3H2O).

Tnh cht: Magie lactat tn ti dng kt tinh mu trng, khng mi, v

cay ng, kh tan trong nc lnh, tan tt trong nc nng (1g bt tan trong

25ml nc lnh; 3,5ml nc nng) v hi tan trong cn 960 [23].

1.1.2. Cng dng

Magie lactat c s dng trong nhiu lnh vc:

Trong Y hc:

- Magie lactat l mt khong cht t nhin cn thit trong c th h tr

chc nng ca h tim mch, thn kinh v tiu ha, n c th dng n hoc

thng thy hn l dng phi hp vi Vitamin B6.

- Magie lactat c s dng phng v iu tr thiu magie, n cng c

s dng iu tr chng nng, kh tiu hoc au bng, h tr trong

chuyn ng ca c v s dng glucose.

3

- B sung magie lactat gip xy dng h c xng tt do magie h tr

trong vic hnh thnh xng v ti hp thu, t duy tr s n nh xng.

- Sn phm b sung magie, phi hp magie lactat v vitamin 6 thng

s dng trong iu tr thiu magie nng, thiu canxi, cc ri lon thn kinh

ngoi bin, cc triu chng lo u km tng thng kh (co git).

- S dng magie lactat c cho l c th gim nhng bin chng c hi

i vi nhng ngi nghin thuc l.

- u im s dng magie lactat b sung so vi cc loi magie khc: cc

loi mui magie trc y c s dng thng c mt s vn v hp thu

dn n sinh kh dng km, s dng mui magie lactat ci thin c vn

ny vi sinh kh dng ln ti 99% [22].

Trong cng ngh thc phm:

- Magie lactat l mt ph gia thc phm dng iu v v x l bt.

- Magie lactat thng c trong cc sn phm b sung nh vitamin, khong

cht b sung v ung [16].

T dc:

- Magie lactat c s dng nh l t dc iu v, iu chnh pH

- Lm t dc trong dp vin: Magie lactat sn xut bng phng php

sy phun c nhit nng chy cao hn, t dnh v bi bm, trn chy cao,

gc ngh thp, c t l gii th ci thin nn rt ph hp vi mc ch dp vin

nn c s dng lm t dc dp vin, t dc trong sn phm m phm: sn

phm chm sc da, chm sc rng ming, [22]

Trong cng nghip:

- Magie lactat l tin sn phm trong cng nghip ln men sn xut axit

lactic, cc mui lactat khc, tiu biu nh canxi lactat. Axit lactic v cc dn

xut ca n c dng rng ri trong cng nghip dc phm, thc phm,

cng nghip dt, tng hp vecni, cht do, nha gia cng bng nhit.

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- Magie lactat cn c s dng tham gia vo ch to cc linh kin

in t, thit b k thut (my tnh xch tay, my tnh bng,) [22]

1.1.3. Mt s sn phm lu hnh trn th trng

ng 1.1. Mt s sn phm cha magie lactat trn th trng

STT Tn bit dc Thnh ph n H ng sn xut

1 Maglek B6

Magie lactat,

Pyridoxin

Lek-AM, Poland Lek-AM,

Ba Lan

2 Magne B6

Sanofi-Aventis, Lithuania,

Sanofi-Aventis, Malta;

Sanofi-Aventis, Ba Lan

3 Magne B6 Stada Stada-VN JV, Vietnam

Stada-VN JV, Vit Nam

4 Magne - B6

Corbire Mekophar, Vit Nam

5 Magnelis B6 Pharmstandard, Lin bang Nga

6 Magvit GlaxoSmithKline, Ba Lan

7 Amfamag B6 Ampharco USA, Vit Nam

8 New - Mag Whan, Hn Quc

9 Magne-B6 Boston Boston Pharma, Vit Nam

10 Magnerot Magie lactat,

Axit orotic Wrwag Pharma, Nga

11 Magnesium recip Magie lactat,

Magie citrat

Recip, Thy in

12 Magnesium NYCOMED, Romania

13 Oromag Theramex, Tunisia

14 Magnegon Takeda Pharma, Thy S

15 Wamag Magie lactat, hn hp mui canxi

Hasco, Ba Lan

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Magnesium

Glycocolle

Lafarge

Magie lactat,

glycin

SERP, Monaco

1.2. Cc phng php sn xut magie lactat

1.2.1. Phng php tng hp ha hc

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Bng cc phng php tng hp ha hc cc nh khoa hc nghin

cu tng hp c magie lactat t cc ngun nguyn liu khc nhau nh

trong sn xut axit lactic [14], [18].

- u im:

+ D thu c magie lactat t nhiu ngun khc nhau nh:

Thy phn halogenid axit t axit cloropropionic.

Kh ha ceton axit bng hydro mi sinh i t axit pyruvic.

Cng hp i nhn vi tc nhn HCN t ngun acetaldehyde [7].

+ Chi ph r [27].

+ Thi gian ngn [27].

- Nhc im: Tng hp magie lactat theo phng php ny thng to

dng mui ca Magie vi axit D (-) lactic v axit racemic D,L - lactic. y l

cc dng gy hi vi sc khe con ngi nn t dng. Vic ci tin k thut

to sn phm t nhin mui ca magie vi axit L (+) lactic vn lun c tch

cc nghin cu [15].

- Phng php c p dng rng ri to magie lactat cho cc ngnh

cng nghip khc: tng hp cht ph gia thc phm, cht do, sn phm khc

[22].

1.2.2. Phng php vi sinh vt

C s ca phng php vi sinh vt l tin hnh ln men trong iu kin

k kh hoc vi hiu kh vi cc chng vi sinh vt sinh axit lactic. Trong qu

trnh chuyn ha cc vi sinh vt nh c h enzym lactat dehydrogenase

chuyn pyruvat thnh axit lactic. Vic b sung cc hp cht magie trung tnh

vo mi trng ln men dn dn hoc ngay t ban u s to ra mui magie

lactat tan trong mi trng ln men. Sau khong thi gian thch hp tin hnh

thu dch ln men, x l dch ln men, kt tinh v tinh ch thu sn phm.

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Thc cht phng php ln men vi sinh vt to magie lactat l qu trnh ln

men to axit lactic.

Phng php sinh tng hp magie lactat t vi sinh vt c nhiu u

im hn so vi phng php tng hp ha hc nh: an ton vi mi trng

do cc d phm ca qu trnh t c hi, d phn hy, nhit sn xut thp,

nng lng sn xut thp, tn dng c cc sn phm tha ca ngnh thc

phm, vy nn gi thnh sn phm r hn, to c sn phm tinh khit hn

[21], [24].

- u im:

+ To sn phm t nhin l mui magie vi axit L (+) lactic [21].

+ Tn dng nguyn liu l cht thi cc ngnh cng nghip khc (cng

nghip sn xut ng, bnh ko, ch bin sa, g, nng lm sn,) [24].

1.3. Vi khun sinh axit lactic

1.3.1. c im nhm vi khun sinh axit lactic

Vi khun sinh lactic gm cc trc khun hay cu khun Gr (+), t l GC

(guanin v cystein) thp, chu c axit, thng khng sinh nha bo, k kh

hoc vi hiu kh, c phn nhm da vo c im chung trong qu trnh

trao i cht v cc c tnh sinh l. an u, cc vi khun ny c tm thy

trong thc vt phn hy v cc sn phm ca axit lactic. im chung ln

nht ca vi khun nhm ny l s ln men hydratcarbon sinh ra axit lactic l

sn phm trao i cht cui cng [3].

Vi khun sinh lactic ch yu thuc 4 chi: Lactobacillus, Pediococcus,

Streptococcus, Leuconostoc; ngoi ra c mt s loi nm thuc chi Rhizopus

[1].

Hnh dng v kch thc ca vi khun sinh lactic rt a dng v phc

tp: hnh cu, hnh oval, hnh que, mc n, mc i hoc mc thnh chui.

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Sinh l ca vi khun lactic kh ging nhau, chng l cc vi khun Gram

(+), khng to bo t, hu nh khng di ng, a nhit, l vi khun ym kh

hoc vi hiu kh, s dng nng lng t phn gii glucid v tit ra axit lactic.

Chng pht trin v tn ti tt pH thp (4,5 6,8), nhit khong 10

500C [1], [3].

Vi khun lactic l vi khun d dng nn mi trng dinh dng tng

i phc tp, giu cht dinh dng, gm: cc vitamin, cc axit amin, cc

peptid ngn Chng c kh nng ng ha nhiu loi ng: glucose,

saccarose, lactose, mantose, mt vi loi cng c kh nng s dng ng

cao phn t [1].

1.3.2. Chi Lactobacillus

L chi ln nht trong cc vi khun sinh lactic vi nhiu chng c

nghin cu v ng dng trong sn xut. Chi Lactobacillus c pht hin vi

hn 125 loi, da vo s chuyn ha glucid trong t bo v sn phm to ra

trong qu trnh ln men, chia thnh 3 nhm chnh [3]:

- Nhm 1: Nhm vi khun ln men ng hnh, sn phm cui cng ca

qu trnh ln men ch yu l axit lactic (90 - 98%), cc sn phm khc ch tn

ti dng vt nh. i din: L. acidophilus, L. bulgaricus, L. delbrueckii

- Nhm 2: Nhm vi khun ln men d hnh khng bt buc, sn phm

ca qu trnh ln men c th l axit lactic, axit acetic, axit formic, ethanol ty

ng trong mi trng. i din: L. plantarum, L. casei, L. leichmenii

- Nhm 3: Nhm vi khun ln men d hnh bt buc, sn phm cui cng

l axit lactic, axit acetic, carbonic, ethanol. i din: L. brevis, L. fermentum,

L. buchneri

1.3.3. Loi Lactobacillus acidophilus

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Loi L. acidophilus thuc gii vi khun, ngnh Fermicutes, lp bacilli,

b lactobacilliales, h lactobacillaceae, ging lactobacillus [3], [6].

Hnh 1.1. L. acidophilus di knh

hin vi quang hc

Hnh 1.2. L. acidophilus di knh

hin vi in t

L. acidophilus l trc khun Gram (+), dng hnh que, mc n, mc

i hoc to chui ngn, khng sinh bo t, khng di ng, phn ng catalase

m tnh; l vi khun a nhit, nhit ti u pht trin l 37 450C, chu

c pH tng i thp (5 6); h hp hiu kh v k kh nhng ch yu l k

kh; c kh nng chuyn ha hydratcarbon cho sn phm l L (+) lactic [3],

[8].

L. acidophilus l probiotic, chng l cc vi khun c li trong h

thng tiu ha gip chng li vi khun c hi gy bnh, gp phn duy tr h vi

khun c li. L. acidophilus c phn lp t dch rut tr em v dch rut b.

Hin nay, L. acidophilus c s dng nhiu trong cc ch phm men tiu

ha nh: Antibio, Lactomin, iu tr cc trng hp ri lon tiu ha

tr mi n dm hoc do dng khng sinh di ngy [9].

L. acidophilus thuc nhm vi khun ln men ng hnh, sn phm cui

cng l axit lactic c ng dng rng ri trong thc ti n nh: bo qun thc

phm, dung dch v sinh ph n (D hng, Lactacyd,).

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1.3.4. Sn phm axit lactic ca vi sinh vt

- Cu trc ca axit lactic

+ Cng thc tng qut: C3H6O3.

+ Khi lng phn t: 90,08.

+ Cng thc cu to:

+ Cc dng ca axit lactic: do trong phn t ca axit lactic c 1 carbon

bt i nn axit lactic c 1 cp i quang: axit D (-) lactic, axit L (+) lactic.

Axit D (-) lactic Axit L (+) lactic

+ Cu trc khng gian:

Axit D (-) lactic Axit L (+) lactic

Axit lactic cn mt dng cha ng thi 2 i quang l hn hp

racemic. Dng axit racemic (DL) lactic thng l kt qu t phng php

tng hp ha hc i t hp cht lactonitril hoc nh hot ng ca enzym DL

lactatdehydrogenase do mt s loi vi sinh vt c kh nng sinh tng hp

axit lactic to thnh [26].

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Cc axit lactic c tnh quang hot khi b nh sng phn cc i qua, cc

dng phn cc c th chuyn thnh dng axit racemic di tc dng ca

enzym racemase t mt vi loi vi khun lactic.

+ Tnh cht ca axit lactic:

Dng dung dch: axit lactic l cht lng trong sut, khng mu, khng

mi, v chua c trng, tan tt trong nc, khng bay hi.

Dng tinh th: d tan p sut kh quyn to cht lng.

pha lng: axit lactic d chuyn sang dng dimer mch thng lactoyl

lactate v polymer mch thng cao hn, khi nhm hydroxyl ca phn t

ny lin kt ester vi nhm carbonyl ca phn t khc. Dng dimer mch

vng lactic cng c th c hnh thnh nu c thc hin un nng ko di

1400C di p sut thp v khi chng ct chng c th b i quay cc.

1.4. Cc nghin cu v magie lactat

Peter Johannes Marie Baets v cc cng s (2013) m t phng

php sn xut mui lactat bng cch ln men t ngun nguyn liu thc vt,

mi trng ln men gm 25% cacbohydrat trong dch chit thc vt, vi khun

sinh lactic ph hp, v Mg(OH)2 thu c 9,5% magie lactat [25].

Jan Van Krieken v cc cng s (2007) s dng mt trong cc hp

cht ca magie l Mg(OH)2, MgO v MgCO3 duy tr mi trng ln men cc

cacbohydrat pH 5 7 vi chng vi khun thch hp, sau thu cc tinh th

magie lactat bng cch kt tinh lactat magie t dch ln men c c c [20].

Gerrit Leendert Nanninga v cc cng s (2005) ch ra rng magie

lactat c th d dng thu c t qu trnh ln men ca cacbohydrat thnh axit

lactic, trong mui magie khng ch c s dng nh mt tc nhn trung

ha, m cn to ra sn phm ca qu trnh ln men l magie lactat. Magie

lactat kt tinh t nc ln men thnh tinh th khi hnh thun di rt tinh

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khit. Do bt k ngun cacbohydrat c th c s dng, thm ch ngun

cacbohydrat tng i th, v d sucrose, tinh bt (lng), r ng [17].

Ben - Yoseph Eliahu, Kogan Leni, Wajc Samuel (2000) ch ra rng,

hu ht cc qu trnh sn xut axit lactic da trn qu trnh ln men ca cc

cacbohydrat bi vi khun [11]. Cc qu trnh ny i hi phi kim sot cht ch

nhit v pH. Mt c im ph bin cho tt c cc qu trnh ln men l

phi trung ha cc axit c tit ra bi cc vi khun trong qu trnh ny. S st

gim pH ty thuc vo loi vi khun c s dng, c th lm hng qu trnh

trao i cht ca vi khun v qu trnh ln men dng li. V vy, ngi ta thng

thm Ca(OH)2 vo dch ln men v thu c canxi lactat, hiu sut ca qu trnh

ny thng thp, tch axit lactic c tinh khit cao vn cn l mt mc tiu

kh. Ngoi ra, vic s dng H2SO4 gii phng axit lactic t canxi lactat sau

to ra canxi sulfat l cht thi rn (thch cao) nh hng ti mi trng. Do vy,

sng ch ra phng php sn xut thn thin mi trng l thng qua

sn xut magie lactat vi cc giai on:

1) Giai on ln men: ng v vi sinh vt c thm vo ln men.

pH iu chnh v 6,5 bng cch thm Mg(OH)2, qu trnh ln men c php

tin hnh trong vi gi nhit t nhit phng v ln n khong 50C.

Dch ln men l hn dch ca vi khun trong dung dch cha magie hay canxi

lactat, cc mui magie hay canxi khc nhau d v phn nh dinh dng cn

d. Sinh khi c tch ra bng cch ly tm..

2) Giai on tinh ch: dch ln men sau khi loi b sinh khi thng cha

khong 5,5% khi lng magie hay canxi lactat, c tnh nh axit lactic.

Nng ny khng kt tinh nhit mi trng xung quanh. Do ,

dch ln men c c trong mt thit b bay hi khong cn khong 15%, lm

lnh, lc hoc ly tm, v ra sch. Ta sau phn ng vi HCl to axit

lactic v dung dch cha mui clorua.

12

Sinh khi tch ra v cc dch ra c ti ch ln men. Cc phn ca

sinh khi m khng ti ch c ra sch bng nc ct thu c t thit b

bay hi, v c s dng nh ph gia thc n cho gia sc hoc kh t.

Aharon Meir Eyal, Rod Fisher (1998) sn xut magie lactat thng qua

vic sinh tng hp axit lactic trong qu trnh ln men ca mt chng vi khun

thuc chi Lactobacillus, loi Lactobacillus delbrueckii, hoc Lactobacillus

acidophilus. Nguyn liu chnh l cc cacbohydrat cng vi cc ngun dinh

dng ph hp. V axit lactic c sinh ra nn vi sinh vt b c ch trong mi

trng axit mnh, pH ca dch ln men thng c duy tr trn 4,5,

thng trong khong 5,0 - 7,0, tt nht trong khong 6,0 - 6,5, mc d qu

trnh ln men thc hin trong pH khong 3,8 - 4,5 vn xy ra. duy tr mc

pH ny, cc cht thm vo phi tan c trong nc, khng c hi cho vi

sinh vt, v phi duy tr c pH ph hp. Vy cc cht ph hp l cc

kim loi kim th, tt hn na l canxi hoc magie, tt nht l nhm

cacbonat, bicacbonat [10].

S. Saha v cc cng s (1995) nghin cu sn xut magie lactat bng

cch b sung MgO, Mg(OH)2 hoc MgCO3 trong qu trnh ln men wey

(lactose) ti pH 4,5 5,03 v kt qu thu c hiu sut cao hn khi so snh

vi phng php to mui canxi [20].

Bode Harold Eli v cc cng s (1969) a ra quy trnh tinh ch

magie lactat t mt hn hp chit axit lactic th vi r ng, sa v cc loi

ru. un nng hn hp, loi b ta canxi phytate, thm magie sulfat kt

ta ion canxi v chuyn i cc canxi lactat, magie lactat; lc loi b cc mui

canxi khng ha tan, lm mt; sau lc, v magie lactat kt tinh; lc, thu

tinh th c tinh khit cao l magie lactat, ra vi nc lnh v sy kh [12].

Lesniak Wladyslaw, Przemysl Refment (1964) dng MgCO3 lm tc

nhn trung ha axit lactic khi ln men mt ng v ng trng [19].

13

Ngoi cc nghin cu v sinh tng hp, magie lactat cn c tng hp

bng phng php ha hc dng lm nguyn liu trong cng ngh thc phm, cc

tc gi Novinyuk Lyudmila v Kykin Mikhail m t quy trnh sn xut magie

lactat t axit lactic v MgO cng nh iu kin kt tinh v tinh ch ca n [27].

Mc d magie lactat v ang c s dng rng ri trong nhiu lnh

vc nhng hin ti Vit Nam cha c nghin cu cng b no lin quan

hay quy trnh sn xut nn y cng l l do m chng ti thc hin ti

ny.

14

Chng 2. I TNG V PHNG PHP NGHIN CU

2.1. Nguyn vt liu, thit b

2.1.1. Nguyn vt liu

Chng ging: Lactobacillus acidophilus ATCC 4356 dng ng kh

(ngun gc: Vin Kim nghim).

Nguyn liu v ha cht

Bng 2.1. Cc nguyn liu v ha cht

Nguyn iu, ha cht

Ngun gc Nguyn iu, ha

cht Ngun gc

Glucose Trung Quc NaOH Trung Quc

Sa bt Vit Nam NaK(C4H4O6) Trung Quc

Cao nm men Merk, c H2SO4 98% Trung Quc

(NH4)2SO4 Trung Quc Na2S2O3 Trung Quc

KH2PO4 Trung Quc KMnO4 Trung Quc

MnSO4.H2O Trung Quc Br2 Trung Quc

FeSO4.7H2O Trung Quc Isopropanol Vit Nam

MgSO4.7H2O Trung Quc Methanol Vit Nam

MgCO3 Trung Quc HCl Trung Quc

CuSO4.5H2O Trung Quc NH3 Vit Nam

Axit oxalic Trung Quc Br2 Trung Quc

CuSO4.5H2O Trung Quc CuSO4.5H2O Trung Quc

NaK(C4H4O6) Trung Quc HCl Trung Quc

Na2S2O3 Trung Quc Isopropanol Vit Nam

HCl Vit Nam Methanol Vit Nam

Bn mng silica gel G

c NH3 Vit Nam

15

Mi trng nui cy s dng

Mi trng nhn ging

ng 2.2. Mi trng nhn ging

Thnh ph n Khi ng

Glucose 4,0g

Sa bt 1,0g

Cao nm men 0,4g

(NH4)2SO4 0,2g

KH2PO4 0,25g

MnSO4.H2O 0,001g

FeSO4.7H2O 0,001g

MgSO4.7H2O 0,03g

Nc my v 100ml

iu chnh pH = 6,3 6,5

Mi trng ln men

ng 2.3. Mi trng ln men

Thnh ph n Khi ng

Glucose 7,0g

Sa bt 2,6g

Cao nm men 1,3g

(NH4)2SO4 0,2g

KH2PO4 0,25g

MnSO4.H2O 0,001g

FeSO4.7H2O 0,001g

MgSO4.7H2O 0,03g

Nc my v 100ml

iu chnh pH = 6,3 6,5

Cc dung dch s dng

Cc dung dch c pha theo hng dn ca DVN IV:

Dung dch axit oxalic 10%: Ha tan 10g axit oxalic trong nc v thm

nc va 100 ml.

Dung dch Schoorl A: Cn chnh xc 34,66g CuSO4.5H2O, ha tan

trong nc axit ha bng 2-3 git H2SO4 long. Thm nc va 500ml.

Dung dch Schoorl : Cn 173g mui kp Natri kali tactrat

NaK(C4H4O6) v 50g NaOH, ha tan trong 300ml H2O, ngui, thm H2O

va 500ml.

Dung dch H2SO4 25%: ong chnh xc 71ml H2SO4 98% pha vi

chnh xc 500ml H2O, lm ngui.

16

Dung dch Na2S2O3 0,1N: Cn chnh xc 25,0g Na2S2O3 v 0,2g

Na2CO3 ha tan trong nc ct khng c CO2, thm H2O va 1000ml.

Dung dch HCl 0,01M: Pha long 2,4 ml axit hydrocloric vi nc va

100 ml.

2.1.2. Thit b s dng

Bng 2.4. Cc thit b s dng

Thit b, dng c Ngun gc, xut x

Cn k thut Satorius, c

Cn phn tch Satorius, c

Ni hp ALP, Nht

T cy Sanyo, Nht

T m Memmert, c

T sy Daihan, Hn Quc

My li tm Hettich, c

My li tm lnh Sigma Laborzentrifugen GmbH, c

Bp cch thy HHS, Trung Quc

T lnh Daewoo, Hn Quc

L vi sng Daewoo, Hn Quc

a petri, buret, bnh nh mc, pipet, ng nghim, u cn, dng c

pha ch,

2.2. Ni dung nghin cu

2.2.1. S b xy dng quy trnh ln men to magie lactat

La chn thi im b sung magie cacbonat

- Bt u qu trnh ln men.

- Trong qu trnh ln men.

17

- Kt thc qu trnh ln men.

La chn phng php thu sn phm magie lactat

- Lc nng bng ph u lc Buchner.

- Li tm thng.

- Li tm lnh.

nh tnh sn phm magie lactat thu c

- nh tnh ion Mg2+.

- nh tnh ion lactat.

- Sc k lp mng.

2.2.2. Kho st mt s thng s nh hng n qu trnh ln men

Kho st nh hng ca pH: 6 8.

Kho st nh hng ca nng glucose: 7 12%.

2.3. Phng php nghin cu

2.3.1. Phng php nhn ging:

Mi trng nhn ging (bng 2.2) sau khi c tit trng 0,6 atm

trong 20 pht ngui xung 40 450C s cy chng L. acidophilus. em

trong t m nhit 370C. Sau 24 gi nui cy c ging cn thit.

Nu ging c gi nhit thp (trong t lnh) lu ngy th phi

a ging v trng thi hot ng bng cch trong t m nhit 370C

trong 2 - 3 ngy ri tin hnh cc bc nh trn [7].

2.3.2. Phng php ln men sinh tng hp magie lactat:

Chun b 100ml mi trng ln men (bng 2.3) vi cc thnh phn v t

l nghin cu vo cc bnh nn 250ml. Cc thnh phn yu cu tit trng

ring bit th pha vo cc bnh nn khc nhau. Tit trng mi trng 0,6

atm trong 20 pht, ngui n nhit khong 40 450C, trn ln cc thnh

18

phn tit trng ring. Sau cy ging vo cc bnh ny trong t cy v trng

vi t l ging l 10%. Mi th nghim nghin cu lm 3 bnh song song.

Sau khi cy ging, ln men tnh trong t m nhit 370C, thnh

thong lc. B sung MgCO3 to magie lactat. Thu ly dch ln men kt

tinh magie lactate [7].

2.3.3. Phng php tch magie lactat t dch ln men:

Dch ln men c un nng cch thy n nhit 800C v gi

nhit trong 20 pht dit t bo. Lc nng loi ta protein v cc

thnh phn khng tan. C cch thy cn th tch. Kt tinh lnh 4 100C

qua m trong t lnh. Thu tinh th, ra 2 ln bng nc ct lnh. Sn

phm mu trng c sy kh nhit 400C trong 24h. Cn lng sn

phm thu c [7].

2.3.4. Phng php nh tnh cc ion lactat v magie:

Th nh tnh ion lactat: Ha tan khong 20mg ch phm trong 5ml

nc, thm 1 ml nc brom v 0,5ml dung dch H2SO4 1M. un cch thy

n khi mt mu, thnh thong khuy bng a thy tinh. Thm 4g amoni

sulfat, trn u. Thm tng git theo thnh ng nghim 0,2ml dung dch natri

nitroprusiat 10% trong H2SO4 1M (khng trn) v 1ml amoniac m c.

yn 30 pht s c vng mu lc gia b mt 2 cht lng [13].

Th nh tnh ion magie: Ha tan 15mg ch phm trong 2ml nc.

Thm 1ml dung dch NH3 6M, to ta trng. Ta trng tan khi thm 1ml dung

dch amoni clorid. Thm 1ml dung dch Na2HPO4 9% c ta kt tinh trng

[13].

2.3.5. Phng php sc k lp mng nh tnh magie lactat dihydrat:

19

Bn mng: Silica gel G.

Dung mi khai trin: Isopropanol - nc - amoniac m c (85 : 20 : 1).

Dung mi ha tan: Dung dch HCl 0,01M - methanol (70 : 30).

Dung dch th: Ha tan lng bt vin (vin Magie 6 - Mekophar) tng

ng khong 0,2g magie lactat dihydrat trong dung mi ha tan nhit

70C n 80C, lm ngui v thm dung mi ha tan va 10 ml. Lc.

Dung dch i chiu: Ha tan 0,2g magie lactat dihydrat chun trong dung

mi ha tan nhit 70C n 80C, lm ngui v thm dung mi ha tan

va 10 ml. Lc.

Tin hnh: Chm ring bit ln bn mng 10l mi dung dch. Trin khai sc

k n khi dung mi i khong 10cm, ly bn mng ra, kh ngoi khng

kh, phun dung dch kali pemanganat 1%, quan st bng mt thng. Vt

chnh thu c trn sc k ca dung dch th phi tng ng v v tr v

mu sc vi vt chnh trn sc k ca dung dch i chiu [2].

i lng c trng cho mc di chuyn ca cht phn tch l h s di

chuyn Rf c tnh bng t l gia khong dch chuyn ca cht th v

khong dch chuyn ca dung mi:

Trong :

a: khong cch t im xut pht n tm ca vt mu th, tnh bng cm.

b: khong cch t im xut pht n mc dung mi o trn cng ng i

ca vt, tnh bng cm.

Rf: Ch c gi tr t 0 n l.

2.3.6. Phng php Schoorl Regenbogen nh lng ng

20

Nguyn l: Xc nh lng ng tiu th v hiu sut chuyn ha

ng sinh lactat, nh lng ng khi im v ng kt thc.

Thuc th Schoorl A v thuc th Schoorl B cho phc hp mu xanh

ca Cu2+, nhm cha aldehyd ceton kh Cu2+ thnh Cu2O. Lng Cu2+

d

trong dung dch cho tc dng vi mt lng KI d trong mi trng axit s

gii phng I2. nh lng I2 gii phng bng Na2S2O3 0,1N. Tin hnh song

song vi mu trng. Hiu s gia s mililit dung dch Na2S2O3 0,1N tiu th

cho mu trng (V0) v mu th (V) cho ta s mililit Na2S2O3 0,1N tng ng

vi lng ng tiu th cn xc nh. Tra bng tm c lng ng .

Phng trnh c trng ca phng php:

Cu2+

+ 2OH- Cu(OH)2

Cu(OH)2 + 2[OOCCHOHCHOHCOO]2-

[Cu(C4H4O6)2]2-

+ 2OH-

2[Cu(C4H4O6)2]2-

+ RCHO + 5OH- Cu2O + RCOO- + 3H2O + 4C4H4O6

2-

2Cu2+

+ 4I- 2CuI + I2

I2 + Na2S2O3 2NaI + Na2S4O6

Phng php tin hnh:

Ly chnh xc 1ml dch mi trng ln men ban u khi cha cy

ging (hoc dch ln men khi kt thc), cho vo 0,5ml dung dch axit

oxalic 5%. Lc loi ta qua ph u lc Buchner. Ra ph u bng 5 10ml nc

ct. Thm vo dch lc chnh xc 10ml dung dch Schoorl A v 10ml dung

dch Schoorl . Thm nc ct t tng th tch 50ml. un si 2 pht 10

giy, lm ngui v nhit phng, thm 3g KI lc tan hon ton, b sung

10ml dung dch H2SO4 25%. Lp tc gii phng I2 cho dung dch mu nu.

Chun I2 gii phng ra bng dung dch Na2S2O3 0,1N vi ch th h tinh

bt 2%.

Mu trng lm tng t nhng thay dch ln men bng nc ct.

21

2.3.7. Phng php tnh hiu sut tiu th ng sinh magie lactat

Phng trnh phn ng tng qut ca qu trnh:

C6H12O6 2CH3CHOHCOOH (CH3CHOHCOO)2Mg

Mglucose = 180

Mmagie lactat = 238 (tnh cho dng dihydrat)

T kt qu nh lng ng trong mu khi im v kt thc, tra bng ta

c nng % ng trong 2 mu, t tnh c lng ng tiu th.

Cng thc tnh hiu sut tiu th ng:

Trong :

H: hiu sut (%).

m: s gam magie lactat/100ml dch ln men.

a: C% glucose khi im.

b: C% glucose kt thc.

2.3.8. Phng php tnh ton v x l s liu

- S dng phn mm exel tnh ton v v cc biu .

- Cng thc tnh lch chun.

22

Chng 3. THC NGHIM, KT QU V BN LUN

3.3. S b xc nh quy trnh n men to magie lactat

Mc tiu: La chn c quy trnh ln men to magie lactat t ngun

hydratcacbon, chng ln men l L. acidophilus v tin hnh kim tra khng

nh sn phm to thnh l magie lactat.

3.3.1. La chn thi im b sung MgCO3

Theo nghin cu ca Aharon Meir Eyal, Rod Fisher, sn xut magie lactat

thng qua vic sinh tng hp axit lactic trong qu trnh ln men. Nguyn liu

chnh l cc cacbohydrat cng vi cc ngun dinh dng ph hp 10]. Gerrit

Leendert Nanninga v cc cng s ch ra rng magie lactat c th d dng thu

c t qu trnh ln men ca cacbohydrat thnh axit lactic, trong mui

magie khng ch c s dng nh mt tc nhn trung ha, m cn to ra sn

phm ca qu trnh ln men l magie lactat [17].

Chng ti tin hnh ln men theo phng php nu mc 2.3.2, s

dng MgCO3 vo cc thi im khc nhau:

Bt u qu trnh ln men (i chiu vi s dng CaCO3).

Kt thc qu trnh ln men.

Trong qu trnh ln men.

nh gi nh hng ca thi im s dng MgCO3 n kh nng to

magie lactat la chn cch thch hp p dng cho cc th nghim tip theo.

S dng MgCO3 ngay khi bt u qu trnh ln men:

Theo nghin cu ca L Th Thu Hin, canxi lactat c to ra bng

cch thm 5g CaCO3 vo mi trng ngay khi bt u qu trnh ln men, c

cy chng L. acidophilus [5]. Magie v canxi l 2 kim loi cng thuc nhm

kim loi kim th nn tin hnh th nghim ln men theo phng php nu

23

mc 2.3.2, so snh vic to thnh magie lactat t MgCO3 v canxi lactat t

CaCO3.

Tin hnh: 2 th nghim

- Ln men theo phng php ln men nu mc 2.3.2.

- Ngay khi bt u qu trnh ln men: Th nghim 1: Thm 5g MgCO3.

Th nghim 2: Thm 5g CaCO3.

- Sau 96 gi ln men, thu dch ln men, x l dch ln men thu sn phm

theo phng php nu mc 2.3.3, cn lng sn phm, nh lng ng

theo phng php Schoorl Regenbogen nu mc 2.3.6.

Kt qu thc nghim c th hin bng 3.1.

Bng 3.1. So snh kt qu tng hp magie lactat v canxi lactat

Cht b sung

Cc tiu ch MgCO3 CaCO3

Lng thm vo (g) 5 5

pH bt u ~ 10 ~ 6

pH kt thc ~ 10 ~ 5

Lng ng kt thc (%) 6,93 0,31

Lng sn phm Khng thu c sn phm 3,21g

Nhn xt: cng iu kin ln men, th nghim thm 5g CaCO3 thu

c 3,21g sn phm, lng glucose c tiu th gn nh hon ton ph hp

vi kt qu nghin cu ca L Th Thu Hin [5] trong khi th nghim thm 5g

MgCO3 khng thu c sn phm, glucose hu nh khng c tiu th. S d

c s khc nhau ny do khi thm 5g CaCO3 pH mi trng vn l 6, y l pH

thch hp cho s sinh trng v pht trin ca L. acidophilus. Khi thm 5g

MgCO3 mi trng ln men t pH = 10, pH ny L. acidophilus b c ch,

khng ng ha c glucose to axit lactic. Nh vy, MgCO3 thm vo

thi im ban u nh hng n pH mi trng ln men nn nh hng kh

nng ng ha glucose ca vi khun L. acidophilus.

24

Tip tc tin hnh vi cch b sung MgCO3 ngay khi bt u qu trnh

ln men nhng vi khi lng gim dn kho st nh hng ca khi

lng MgCO3 thm vo ngay khi bt u qu trnh ln men c nh hng nh

th no ti pH mi trng ln men dn ti vic nh hng kh nng ng ha

glucose ca vi khun L. acidophilus.

Tin hnh: 4 th nghim

- Ln men theo phng php ln men nu mc 2.3.2.

- Ngay khi bt u qu trnh ln men: Thm vo mi bnh ln men lng

MgCO3 ln lt l 0,5g, 1g, 2g, 3g.



theo phng php Schoorl Regenbogen nu mc 2.3.6.


Bng 3.2. Kt qu tng hp magie lactat bng cch thm MgCO3 ngay khi bt

u qu trnh ln men

Nhn xt: Vi cch thm MgCO3 (0,5 3g) ngay khi bt u qu trnh

ln men u khng thu c hoc thu c rt t sn phm, lng glucose hu

nh khng c tiu th. Mc d khi lng MgCO3 thm vo gim t 3

xung 0,5g, pH mi trng gim , nhng vn t pH = 8 khi ch thm 0,5g

MgCO3 y vn thuc khong pH c ch L. acidophilus. Vy MgCO3 thm

Th nghim

Cc tiu ch 1 2 3 4

Lng MgCO3 thm vo (g) 0,5 1 2 3

pH bt u ~ 8 ~ 8 ~ 9 ~ 9

pH kt thc ~ 7 ~ 7 ~ 9 ~ 9

Lng ng kt thc (%) 6,56 6,54 6,85 6,88

Lng sn phm Thu c rt t sn

phm

Khng thu c sn

phm

25

vo thi im ban u nh hng nhiu n pH mi trng nn nh hng

kh nng ng ha glucose to axit lactic ca vi khun. MgCO3 c pH = 8

10 [23] l tc nhn to ra mi trng ln men c pH = 8 10 c ch s pht

trin v ng ha glucose ca vi khun L. acidophilus trong khi pH ti u ca

vi khun ny l pH = 5,5 6,5, kt qu ny ph hp vi cng b ca Aharon

Meir Eyal, Rod Fisher [10].

B sung MgCO3 khi kt thc qu trnh ln men.

Theo nghin cu ca Novinyuk Lyudmila v Kykin Mikhail, magie

lactat c to thnh t phn ng ha hc gia axit lactic do vi sinh vt sinh

ra v MgCO3 [27]. Chng ti tin hnh ln men cc cacbohydrat thnh axit

lactic bng chng L. acidophilus, khi kt thc qu trnh ln men b sung

MgCO3 to magie lactat.

Tin hnh:

- Ln men theo phng php nu mc 2.3.2.

- Sau 96 gi kt thc qu trnh ln men, thm MgCO3 iu chnh pH =

7, thu dch ln men, x l dch ln men thu sn phm theo phng php

nu mc 2.3.3, cn lng sn phm, nh lng ng theo phng php

Schoorl Regenbogen nu mc 2.3.6.

- Lp li th nghim 3 ln.


Bng 3.3. Kt qu tng hp magie lactat bng cch s dng MgCO3 khi kt

thc qu trnh ln men

Th nghim

Cc tiu ch 1 2 3

pH kt thc ~ 4 ~ 4 ~ 4

Lng MgCO3 b sung (g) 2,24 1,99 2,03

Lng ng kt thc (%) 2,87 3,04 2,92

Lng sn phm Khng thu c sn phm

26

Nhn xt: Cc th nghim hu nh khng thu c sn phm, lng

glucose vn c tiu th, pH gim chng t vi khun vn ng ha glucose

to axit lactic. Nhng khng thu c sn phm, nh vy khng c phn

ng ha hc xy ra gia axit lactic sinh ra vi MgCO3 hoc c phn ng xy

ra nhng qu yu, khng to sn phm thu c theo phng php

ang nghin cu.

Theo nghin cu ca Aharon Meir Eyal, Rod Fisher, trong qu trnh ln

men axit lactic c sinh ra nn vi sinh vt b c ch trong mi trng axit

mnh, vy nn trong qu trnh ln men phi thm cht duy tr c pH

ph hp [10]. Khi tin hnh theo cch ny trong 96 gi ln men, chng ti

khng thm cht trung ha axit nn mi trng axit mnh, L. acidophilus b

c ch kh nng sinh trng v to axit lactic, v th khi kt thc qu trnh ln

men, lng axit lactic qu t khng phn ng to nhiu magie lactat.

B sung MgCO3 sau mi 24 gi trong qu trnh ln men, iu

chnh pH = 7

Theo nghin cu ca Aharon Meir Eyal, Rod Fisher, axit lactic c

sinh ra nn vi sinh vt b c ch trong mi trng axit mnh. Trong qu trnh

ln men phi thm cht duy tr c pH ph hp. Cc cht ph hp l

cc kim loi kim th, tt hn na l canxi hoc magie, tt nht l nhm

cacbonat, bicacbonat [10]. Gerrit Leendert Nanninga v cc cng s ch ra

rng MgCO3 thm vo trong qu trnh ln men ca cacbohydrat thnh axit

lactic nh mt tc nhn trung ha [17].

V vy chng ti tin hnh th nghim ln men theo phng php mc

2.3.2 b sung MgCO3 dn dn vo cc thi im 24, 48, 72, 96 gi, duy tr

pH = 7.

Tin hnh:

- Ln men theo phng php nu mc 2.3.2.

27

- Sau mi 24 gi b sung MgCO3, iu chnh pH = 7.



theo phng php Schoorl Regenbogen nu mc 2.3.6, tnh ton v x

l s liu theo phng php nu mc 2.3.8.

- Lp li th nghim 3 ln.

Kt qu thc nghim th hin bng 3.4.

Bng 3.4. Kt qu tng hp magie lactat bng cch b sung dn MgCO3 sau

mi 24 gi

Th nghim

Cc tiu ch

Thi im (gi) Tng MgCO3

b sung (g)

Lng ng tiu th

(%)

Sn phm

(g) 0 24 48 72 96

1

pH 6 5 4 5 6

4,18 100% 3,07 MgCO3 b sung (g)

0 1,1 1,43 1,11 0,54

2

pH 6 4 4 4 6

3,97 100% 2,81 MgCO3 b sung (g)

0 1,31 1,31 1,05 0,3

3

pH 6 5 4 4 6

4,23 100% 2,90 MgCO3 b sung (g)

0 1,03 1,44 1,4 0,36

Trung bnh 4,127 100% 2,93

Nhn xt: Vi phng php b sung MgCO3 sau mi 24 gi v iu

chnh pH = 7 trong sut qu trnh ln men u thu c sn phm vi khi

lng trung bnh l 2,93g, lch chun 0,108.

Trong qu trnh ln men, MgCO3 b sung khng ch c s dng nh

mt tc nhn trung ha, m cn to ra sn phm ca qu trnh ln men l

28

magie lactat, ph hp vi kt qu nghin cu ca Gerrit Leendert Nanninga

v cc cng s 17].

Kt lun: Cch b sung dn MgCO3 sau mi 24 gi, iu chnh pH =

7 l thch hp nht, la chn phng php ny cho cc th nghim tip theo.

3.3.2. La chn phng php thu sn phm magie lactat

Mc tiu: La chn cch thu tinh th kt tinh magie lactat thch hp

nht t dch ln men.

Ben - Yoseph Eliahu, Kogan Leni, Wajc Samuel ch ra rng giai

on tinh ch: dch ln men sau khi loi b sinh khi thng cha khong

5,5% khi lng magie hay canxi lactat, c tnh nh axit lactic. Nng

ny khng kt tinh nhit mi trng xung quanh. Do , dch ln

men c c cn khong 15%, lm lnh, lc hoc ly tm, v ra sch [11].

Tin hnh cc cch thu sn phm khc nhau la chn c cch ti

u nht thu sn phm ph hp vi tnh cht ca magie lactat mc 1.1.1.

Tin hnh:

- Tin hnh ln men theo phng php nu mc 2.3.2 v b sung

dn dn MgCO3 sau mi 24 gi, iu chnh pH = 7.

- Sau 96 gi ln men, x l dch ln men theo phng php nu mc

2.3.3, trong cc phng php thu tinh th sau khi kt tinh c nghin

cu ln lt l:

+ Lc ht qua ph u Buchner, ng knh giy lc 0,45 mcm;

+ Li tm nhit thng (4000 vng / 5 pht);

+ Li tm lnh 20C (8000 vng / 10 pht).

- Lm song song 3 bnh th nghim.

Cn sn phm, so snh lng sn phm thu c mi phng php, nhn

xt v rt ra kt lun.

29

Kt qu thu magie lactat c th hin trong bng 3.5.

ng 3.5. Lng sn phm magie lactat thu c bng cc phng php:

ph u lc uchner, li tm thng, li tm lnh

Phng php

thu tinh th

Giai on

Phu c

Buchner

Li tm

thng Li tm nh

Lc nng Ph u Buchner Ph u Buchner Ph u Buchner

Kt tinh T lnh T lnh T lnh

Thu sn phm Ph u Buchner Li tm thng Li tm lnh

Ra ta Nc lnh

+ Ph u Buchner

Nc lnh +

Li tm thng

Nc lnh +

Li tm lnh

Sy T sy T sy T sy

Lng sn phm (g) 0,46 1,41 3,08

Nhn xt: Vi phng php lc ht qua ph u uchner, lng sn phm

thu c rt t do tinh th qu nh v mn nn sn phm tht thot gn ht qua

giy lc.

Phng php li tm gip hn ch s tht thot sn phm qua giy lc

do mn ca tinh th, lng sn phm thu c nhiu hn so vi phng

php lc ht qua ph u uchner. Tuy nhin khi tin hnh li tm, nhit tng

lm tht thot mt phn sn phm vo dch lc v nc ra, do tan ca

magie lactat tng ln khi tng nhit , ph hp vi tnh cht ca magie lactat

[23].

Phng php li tm lnh gip khc phc c s tht thot sn phm

do tng nhit khi li tm, lng sn phm thu c t nhiu nht trong cc

phng php nghin cu.

Sn phm thu c dng tinh th khi hnh thun di mu trng, khng

mi. Kt qu ny ng nh cng b ca Gerrit Leendert Nanninga v cc

cng s, magie lactat kt tinh thu c dng tinh th khi hnh thun di. Do

30

s kt tinh chm, nn khng c tp cht kt tinh cng vi cc tinh th magie

lactat v hnh thnh cc tinh th vi mt hnh dng xc nh r rng. V vy,

bc kt tinh vn l mt bc lc hiu qu. Magie lactat thu c rt tinh

khit [17].

Hnh 3.1. Tinh th magie lactat

Kt lun: Phng php li tm lnh (20C, 8000 vng / 10 pht) cho kt

qu thu sn phm magie lactat tt nht trong cc phng php nghin cu,

c la chn tin hnh cc th nghim tip theo.

31

Hnh 3.2. S quy trnh x l dch ln men

3.3.3. nh tnh sn phm magie lactat thu c

Mc tiu: Kim tra sn phm to thnh c phi magie lactat khng.

Sn phm to thnh l magie lactat c cu to bi ion Mg2+ v ion

lactat nn tin hnh cc phn ng ha hc c trng xc nh s c mt ca 2

ion. ng thi, tin hnh chy sc k lp mng i chiu gia 3 mu: sn

phm iu ch c bng phng php sinh tng hp, magie lactat tng hp

bng phng php ha hc v magie lactat trong thnh phn thuc ang lu

hnh (Magie B6). T khng nh sn phm iu ch c c phi l magie

lactat.

Dch ln men un nng

Lc nng

C

Kt tinh

Li tm lnh

Sy

800C/ 20 pht

Ph u Buchner

40C/ 24 gi

20C/ 8000 vng/ 10 pht

400C/ 24 gi

Sn phm th

32

Th nh tnh ion lactat:

Tin hnh theo phng php nh tnh ion lactat nu mc 2.3.4.

Kt qu thc nghim c th hin hnh 3.3.

Hnh 3.3. Vng mu lc xut hin khi nh tnh ion lactat

Nhn xt: Kt qu nh tnh c vng mu lc xut hin gia b mt

phn cch 2 cht lng nh trong DVN IV [2] m t.

Kt lun: C phn ng ca ion lactat.

Th nh tnh ion Mg2+:

Tin hnh theo phng php nh tnh Mg2+ nu mc 2.3.4.

Kt qu thc nghim c th hin hnh 3.4 3.5.

Hnh 3.4. Ta trng xut hin khi

thm dung dch NH3 v tan khi

thm dung dch amoni clorid

Hnh 3.5. Dung dch trong sut sau

khi thm dung dch amoni clorid v

xut hin ta kt tinh trng khi thm

dung dch Na2HPO4

33

Nhn xt: Kt qu thu c ng nh m t trong DVN IV [2].

Kt lun: C phn ng ca ion Mg2+.

nh tnh magie lactat dihydrat bng sc k lp mng

Tin hnh: Trin khai sc k lp mng theo phng php nu mc

2.3.5 vi 3 mu: sn phm iu ch c, vin magie B6, magie lactat iu

ch bng phng php tng hp ha hc.

Kt qu thc nghim c th hin hnh 3.6.

Hnh 3.6. Kt qu chy sc k lp mng: sn phm iu ch c,

vin magie 6, magie lactat iu ch bng phng php tng hp

ha hc (t tri qua phi)

H s lu Rf ca:

- Sn phm:

- Thuc:

- Sn phm tng hp ha hc:

Rf1 = 6,7 / 10 = 0,67

Rf2 = 6,6 / 10 = 0,66

Rf3 = 6,7 / 10 = 0,67

Nhn xt: Vt chnh thu c trn sc k ca dung dch th tng ng

v v tr v mu sc vi vt chnh trn sc k ca dung dch i chiu.

Kt lun: Sn phm iu ch c l magie lactat

34

Vy cch b sung dn MgCO3 sau mi 24 gi, iu chnh pH = 7 trong

sut qu trnh ln men kt hp phng php li tm lnh thu tinh th t dch

ln men s thu c nhiu sn phm magie lactat nht, la chn phng php

ny cho cc kho st tip theo. Sn phm thu c c khng inh l magie

lactat nh nh tnh theo tiu chun Dc in Anh 2009 v DVN IV.

3.4. Kho st mt s thng s nh hng n qu trnh n men

3.4.1. Kho st nh hng ca pH n qu trnh ln men

Mc tiu: La chn pH thch hp iu chnh sau mi 24 gi trong qu

trnh ln men cho hiu sut sinh magie lactat cao.

Jan Van Krieken v cc cng s s dng mt trong cc hp cht ca

magie l Mg(OH)2, MgO v MgCO3 duy tr mi trng ln men cc cacbohydrat

pH 5 7 vi chng vi khun thch hp, sau thu cc tinh th magie lactat

bng cch kt tinh t dch ln men c c c [19].

Chng ti tin hnh kho st ti cc pH 6, 7, 8 nh gi nh hng ca

pH ti qu trnh ln men v to magie lactat.

Tin hnh: 3 th nghim

- Ln men theo phng php nu mc 2.3.2 v b sung dn dn

MgCO3 sau mi 24 gi, 3 th nghim ln lt iu chnh pH bng 6, 7, 8.


theo phng php nu mc 2.3.3 v quy trnh hnh 3.2, cn lng sn

phm, nh lng ng theo phng php Schoorl Regenbogen nu

mc 2.3.6, tnh hiu sut theo phng php nu mc 2.3.7.


35

Bng 3.6. Kt qa ln men L. acidophilus thu magie lactat cc pH

pH duy tr

Cc tiu ch 6 7 8

Lng MgCO3 b sung (g) 3,27 4,18 3,92

Sn phm magie lactat (g) 0,4 3,07 0,58

Lng ng kt thc (%) 0,56 0 2,87

Hiu sut (%) 5,09 36,85 12,79

Hnh 3.7. Biu so snh hiu sut to magie lactat cc pH

Nhn xt: Vi cng mt lng ng khi im, khi b sung MgCO3

sau mi 24 gi, iu chnh pH = 7 l iu kin thch hp nht to sn phm

magie lactat, vi khun tiu th ht ng v to lng sn phm ln, hiu sut

t 36,85%. Khi iu chnh pH mi trng v 6, v kim ca MgCO3 cao

nn lng b sung khng c nhiu nh khi iu chnh v pH = 7, cho d

lng ng tiu th gn ht (cn 0,56%) nhng y khng phi mi trng

thch hp vi khun sn sinh axit lactic, lng sn phm magie lactat thu

c khng ng k, hiu sut thp nht ch t 5,09%. Cn khi mi trng

05

10152025303540

Hi

u s

u

t (%

)

pH 6 pH 7 pH 8

pH

36

c iu chnh v pH = 8, lng MgCO3 thm vo tng i nhiu v pH

ny gn vi kim vn c ca MgCO3, nhng sn phm thu c khng

nhiu v lng ng sau qu trnh ln men cn d nhiu (2,87% so vi khi

im l 7%), do pH = 5,5 6,5 thch hp cho L. acidophilus, ti pH = 8 vi

khun b c ch qu trnh sinh trng v to sn phm, v th hiu sut cng

ch t 12,79%. Kt qu ny ph hp vi cng b ca Aharon Meir Eyal, Rod

Fisher sn xut magie lactat thng qua vic sinh tng hp axit lactic. Trong

qu trnh ln men axit lactic c sinh ra nn vi sinh vt b c ch trong mi

trng axit mnh, pH ca dch ln men thng c duy tr trn 4,5,

thng trong khong 5,0 - 7,0, tt nht trong khong 6,0 - 6,5, mc d qu

trnh ln men thc hin trong pH khong 3,8 - 4,5 vn xy ra 10].

Kt lun: pH thch hp nht cho qu trnh ln men l 7.

3.4.2. Kho st nh hng ca nng glucose

Mc tiu: La chn nng glucose thch hp cho hiu sut sinh

magie lactat cao.

Tin hnh:

- Ln men theo phng php mc 2.3.2 v b sung dn dn MgCO3 sau

mi 24 gi, vi 5 nng glucose 7%, 9%, 10%, 11%, 12%.


theo phng php nu mc 2.3.3 v quy trnh hnh 3.2. Cn lng sn


mc 2.3.6, tnh hiu sut theo phng php nu mc 2.3.7.

Kt qu thc nghim c trnh by trong bng 3.7

37

Bng 3.7. Kt qu ln men L.acidophilus thu magie lactat vi cc nng

glucose

Lng glucose (g) Cc tiu ch

7 9 10 11 12

Lng MgCO3 b sung (g) 4,18 4,63 5,89 6,01 6,07

Lng ng kt thc (%) 0 0 0 0,37 0,91

Sn phm magie lactat(g) 3,07 4,29 6,17 6,28 6,44

Hiu sut (%) 36,85 38,17 50,17 47,35 45,99

Hnh 3.8. Biu so snh hiu sut to magie lactat cc nng glucose

Nhn xt: Kt qu kho st cho thy chng L. acidophilus s dng cho

hiu sut sinh magie lactat kh cao khi s dng nng glucose hp l.

nng glucose 10%, L. acidophilus cho hiu sut sinh magie lactat cao

nht l 50,17% v sau 96 gi ln men vi khun tiu th ht ng. nng

ng thp hn (7% - 9%) vi khun tiu th ht ng nhng hiu sut sinh

magie lactat thp hn. nng ng 11% v 12%, lng MgCO3 b sung

c kh nhiu tuy nhin sau 96 gi ln men vi khun cha s dng ht

ng (lng ng cn d l 0,37% v 0,91%), hiu sut cng khng cao

hn khi tin hnh nng 10% glucose.

0

10

20

30

40

50

60

Hi

u s

u

t (%

)

Glc 7% Glc 9% Glc 10% Glc 11% Glc 12%

Nng Glc (%)

38

Kt lun: Nng glucose thch hp nht cho qu trnh ln men l 10%.

Qua cc th nghim kho st nh hng ca pH v nng glucose,

chng ti nhn thy nng glucose 10% v iu chnh pH = 7 l thch hp

nht i vi quy trnh xy dng. Chng ti tin hnh th nghim vi nng

glucose 10% v iu chnh pH = 7.

Tin hnh:

- Ln men theo phng php mc 2.3.2 vi nng glucose 10%, v b

sung dn dn MgCO3 sau mi 24 gi, iu chnh pH = 7.


theo phng php nu mc 2.3.3, quy trnh hnh 3.2. Cn lng sn


mc 2.3.6, tnh hiu sut theo phng php nu mc 2.3.7, tnh ton v

x l s liu theo phng php nu mc 2.3.8.

Lp li th nghim 3 ln. Kt qu c th hin bng 3.8 3.9.

Bng 3.8. Lng MgCO3 b sung sau mi 24 gi ln men vi nng


Th nghim

Thi im (gi)

0 24 48 72 96

1 pH 6 4 3 3 5

MgCO3 b sung (g) 0 1,52 1,70 1,65 1,04

2 pH 6 4 4 4 5

MgCO3 b sung (g) 0 1,58 1,50 1,48 1,18

3 pH 6 3 4 3 5

MgCO3 b sung (g) 0 1,67 1,44 1,69 1,22

39

Bng 3.9. Lng magie lactat thu c sau 96 gi ln men vi nng


Th nghim 1 2 3 Trung bnh

Lng MgCO3 b sung (g) 5,91 5,74 6,02 5,89

Lng ng kt thc (%) 0 0 0 0

Sn phm magie lactat (g) 6,01 5,97 6,14 6,04

Hiu sut (%) 48,88 49,62 49,93 49,48

Vi quy trnh b sung dn dn MgCO3 sau mi 24 gi, iu chnh pH =

7, lng magie lactat to c trung bnh l 6,04g, lch chun 0,073

lng ng c tiu th trit , hiu sut qu trnh tng i n nh (trung

bnh 49,48%, lch chun 0,440). Vi phng php ny hiu sut to magie

lactat thu c cao hn so vi cng b ca Peter Johannes Marie Baets v cc

cng s khi sn xut mui lactat bng cch ln men t ngun nguyn liu thc

vt, mi trng ln men gm 25% cacbohydrat trong dch chit thc vt, vi

khun sinh lactic ph hp, v Mg(OH)2 thu c 9,5% magie lactat [25].

Vy nng glucose 10% v iu chnh pH = 7 l iu kin thch hp

nht cho to magie lactat bng phng php b sung dn MgCO3 sau mi 24

gi ln men.

40

KT LUN V XUT

1. Kt lun

Sau thi gian thc hin ti thu c mt s kt qu nh sau:

- s b xy dng c quy trnh ln men to magie lactat: b sung

dn MgCO3 sau mi 24 gi, iu chnh pH = 7 trong sut qu trnh ln men s

thu c nhiu sn phm magie lactat nht. ng thi, hon thin quy trnh

tch magie lactat t dch ln men bng phng php li tm lnh thu c

nhiu sn phm magie lactat nht. Sn phm c nh tnh xc nh l magie

lactat.

- kho st c 2 thng s nh hng n qu trnh ln men: pH v

nng glucose. Nhn thy vi nng glucose 10% v iu chnh pH = 7 l

thch hp nht cho qu trnh to sn phm magie lactat v hiu sut to sn

phm l cao nht, t 49,48%.

2. xut

ti c tnh ng dng trong thc t, chng ti xin a ra mt s xut

nh sau:

- Tin hnh nghin cu thm v nh hng ca cc yu t: nhit , p

sut, loi ng, loi sa, ngun hydratcacbon, hon thin quy trnh v

nng cao hiu sut qu trnh ln men sinh tng hp magie lactat bng chng

L. acidophilus.

- M rng nghin cu v vic s dng cc ngun nguyn liu r tin hn

phc v nng cp quy trnh sinh tng hp magie lactat ln quy m ln.

- Tip tc nghin cu qu trnh ln men sinh tng hp magie lactat bng

cc chng vi khun khc nng cao hiu sut nh Lactobacillus delbrueckii.

TI LIU THAM KHO

Ting Vit

1. Kiu Hu nh (1999), Gio trnh vi sinh vt hc cng nghip, NXB

Khoa hc k thut, tr. 113 228.

2. B Y t (2009), Dc in Vit Nam IV, Nxb Y hc, H Ni.

3. Nguy n Ln Dng v cng s (1976), Mt s phng php nghin cu

vi sinh vt, tp 2, NXB Khoa hc k thut, H Ni.

4. T Minh Gii (1996), Ln men sinh tng hp calci lactate, Kha lun

dc s, trng i hc Dc H Ni.

5. L Th Thu Hin (2012), Nghin cu c nh vi khun Lactobacillus

acidophilus ATCC 4356 sn xut calci lactate, Lun vn thc s dc hc,

Trng i hc Dc H Ni.

6. L Xun Honh (2006), Phn lp, tuyn chn, phn loi mt s chng

Lactobacillus acidophilus sinh acid lactic mnh v nhy cm vi vitamin

B12, Lun vn thc s dc hc, trng i hc Dc H Ni.

7. Kiu Th Hng (1999), Ti u ha qu trnh ln men sinh tng hp

calci lactate, Lun vn tt nghip thc s dc hc, Trng i hc Dc

H Ni.

8. L Gia Hy (2006), Vi sinh vt hc i cng (tp II), Vin cng ngh

sinh hc, Vin khoa hc v cng ngh Vit Nam, H Ni.

9. Nguy n c Lng (1996), Cng ngh vi sinh vt, tp 3. Thc phm

ln men truyn thng, Trng i hc k thut TP HCM.

Ting Anh

10. Aharon Meir Eyal, Rod Fisher (1998), A process for the recovery of

lactic acid, WO 1998037050.

11. Ben-Yoseph Eliahu , Kogan Leni , Wajc Samuel (1999), Process for

preparing lactic acid, WO 2000017378 A3

12. Bode Harold Eli (1969), High purity magnesium lactate from

steepwater US3429777

13. British Pharmacopeial Commission (2009), British Pharmacopeia

2009, Stationery Office.

14. Demi C.G, S.C Prescettle (1959), The production of lactic acid by

fermentation, Ind. Micr, New York, page. 304 331.

15. Dominguw R.M (1998), Optimization of the L lactic acid production

as feedstock for the polymers formation, Polymer preprints, American

Chemical society, Division of polymer chemistry No.1, Vol 39, page. 282

283.

16. FAO/ WHO (1989), Report of the 15th session of the codex committee

on food additives, the United Nations.

17. Gerrit Leendert Nanninga (2005), Process for the preparation of lactic

acid or lactate from a magnesium lactate comprising medium,

WO 2005123647 A1

18. Hagerdal B.H (1997), L lactic acid production from whole wheat

flour hydrolysate using strains of lactobacillus and lotococci, Enzyme and

microbial technology, Vol. 20, page. 301 307.

19. Jan Van Krieken, el al (2007), Method for preparing an organic

amine-lactic acid complex, CN 101426755 B

20. Jan Van Krieken, el al (2007), Process for the preparation of lactic

acid or lactate from a magnesium lactate comprising medium

US 7705180 B2

21. Litchefield J.H (1996), Microbiological production of acid lactic,

Advances in applied microbiology, vol.12.

22. Marcus J.A.W. Vorage (2009), Metal lactate powder and method for

preparation, US 8337867 B2

23. Merck Index, 13th Edition, Merck & Co, Whitehouse Station, New

Jersey, USA 2001.

24. Moon S.H and Tsai S.P (1998), An intergrated bioconversion process

for production of L Lactic acid from starchy potato feed stocks, Applied

biochemistry and biotechnology, vol. 70 72.

25. Peter Johannes Marie Baets, el al (2013), Process for the fermentative

production of lactic acid from a plant extract the presence of a caustic

magnesium salt, WO 2013/087901 A1

26. Schelef L.A (1994), Antimicrobial effects of lactates, A review, J. of

food protection, vol.57, page. 445 450.

Ting Nga

27. , . ., , . ., , . ., & , . .

(2009), .

. , 2110194.

Ph lc: Bng xc nh ng theo phng php Schoor Regenbogen

Tiu th Na2S2O3 0,1N (ml)

Glucose C6H12O6 (mg)

Tiu th Na2S2O3 0,1N (ml)

Glucose C6H12O6 (mg)

1

2

3

4

5

6

7

8

9

10

11

12

3,2

6,3

9,4

12,6

15,9

19,2

22,4

25,6

28,9

32,3

35,7

39,0

3,1

3,1

3,2

3,3

3,3

3,2

3,2

3,3

3,4

3,4

3,3

3,4

13

14

15

16

17

18

19

20

21

22

23

24

25

42,4

45,8

49,3

52,8

56,3

59,8

63,3

66,9

70,7

74,5

78,5

82,6

86,6

3,4

3,5

3,5

3,5

3,5

3,5

3,6

3,8

3,8

4,0

4,1

4,0

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